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May 2015
Volume 28 Number 5
www.chromatographyonline.com
SAMPLE PREPARATION
PERSPECTIVES
New sample prep instruments
LC TROUBLESHOOTING
Calibration problemsGC CONNECTIONS
Review of new GC products on the market
Up In SmokeAn improved GC–MS method for
cigarette smoke characterization
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LC•GC Europe May 2015
Editorial Policy:
All articles submitted to LC•GC Europe
are subject to a peer-review process in association
with the magazine’s Editorial Advisory Board.
Cover:
Original materials: Cordula Damm/EyeEm/Getty
Images
Features265 Large-Volume Injection LC–MS–MS Methods for Aqueous
Samples and Organic Extracts
B. McKay Allred, Matthew J. Perkins, and Jennifer A. Field
Environmental sample analysis by large-volume injection (LVI) in
combination with liquid chromatography–tandem mass spectrometry
(LC–MS–MS) is described for polar and nonpolar analytes in both
aqueous samples and organic extracts.
272 Simultaneous Determination of Methotrexate and
Sulphasalazine in Plasma by HPLC–DAD
Siji Joseph, Sreelakshmy Menon, and Smriti Khera
A simple and robust high performance liquid chromatography–
diode array detection (HPLC–DAD) method for the simultaneous
determination of methotrexate (MTX) and sulphasalazine (SSZ)
from plasma is presented.
Columns278 LC TROUBLESHOOTING
Calibration Problems — A Case Study
John W. Dolan
Unexpected results from calibration standards create confusion in a
clinical liquid chromatography (LC) method.
282 GC CONNECTIONS
New Gas Chromatography Products, 2014–2015
John V. Hinshaw
John Hinshaw presents his annual review of new developments in the
fi eld of gas chromatography seen at Pittcon and other venues in the
past 12 months.
290 SAMPLE PREPARATION PERSPECTIVES
New Sample Preparation Products and Accessories at Pittcon 2015
Douglas E. Raynie
This yearly report on new products introduced at Pittcon (or in the
preceding year) covers sample preparation instruments.
Departments294 Products
298 Events
COVER STORY258 An Improved GC–MS Method for
Cigarette Smoke Characterization
Using a Novel Cold Trap, Dual
Column, and Cryofocusing System
J.R. Crudo, E. Rouget, and M. Rotach
The development of a novel trapping
system and a modifi ed GC–MS layout
(using dual chromatographic columns
and cryogenic focusing devices) has
enabled a major improvement in the
chromatographic separation of volatile
and semivolatile compounds in cigarette
smoke. This improvement has led to the
potential for identifying compounds which
are usually masked by the solvent peak.
May | 2015
Volume 28 Number 5
252
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©2015 Waters Corporation. Waters and The Science of What’s Possible are registered trademarks of Waters Corporation.
In the hands of leading researchers and clinicians,
scientific technologies from Waters are making a true
difference in the fight against cancer.
PHARMACEUTICAL n HEALTH SCIENCES n FOOD n ENVIRONMENTAL n CHEMICAL MATERIALS
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LC•GC Europe May 2015254
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Attila FelingerProfessor of Chemistry, Department of
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Joseph L. GlajchMomenta Pharmaceuticals, Cambridge,
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Jun HaginakaSchool of Pharmacy and Pharmaceutical
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Luigi MondelloDipartimento Farmaco-chimico, Facoltà
di Farmacia, Università di Messina,
Messina, Italy
Peter MyersDepartment of Chemistry,
University of Liverpool, Liverpool, UK
Janusz PawliszynDepartment of Chemistry, University of
Waterloo, Ontario, Canada
Colin PooleWayne State University, Detroit,
Michigan, USA
Fred E. RegnierDepartment of Biochemistry, Purdue
University, West Lafayette, Indiana, USA
Harald RitchieTrajan Scientific and Medical. Milton
Keynes, UK
Pat SandraResearch Institute for Chromatography,
Kortrijk, Belgium
Peter SchoenmakersDepartment of Chemical Engineering,
Universiteit van Amsterdam, Amsterdam,
The Netherlands
Robert ShellieAustralian Centre for Research on
Separation Science (ACROSS), University
of Tasmania, Hobart, Australia
Yvan Vander HeydenVrije Universiteit Brussel,
Brussels, Belgium
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LC•GC Europe May 2015256
LCGC ONLINE
Selected highlights of digital content from LCGC Europe and LCGC North America:
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LCGC TVGiorgia Greco on HILIC–MS
HILIC was introduced more than
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HILIC can be hyphenated with atmospheric pressure
chemical ionization MS. Watch Here: goo.gl/aZCDlb
THE COLUMN — DIGITAL MAGAZINEVolume 11 Issue 7
LCGC ’s global digital magazine The
Column is published on-line twice a
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QUESTIONS OF QUALITYLife Cycle Risk Assessment of
HPLC InstrumentsWhat does risk assessment in the context of the life cycle of a HPLC instrument really mean? This instalment looked at problems with an operational liquid chromatograph to see if they could be picked up in the performance qualification (PQ) or prevented in the operational
qualification (OQ). Read Here: goo.gl/4dycxa
QUICK TUTORIALPragmatic Rules for GC Column Selection
Choosing the correct column selectivity and
dimensions is fundamental to successful gas
chromatography method development. This
short tutorial from LCGC ’s CHROMacademy
introduces some simple rules for selecting the
optimum gas chromatography column.
Read Here: goo.gl/6jRgF9
CURRENT TRENDS IN MSLC–MS–MS Screening of 24 Synthetic
and Natural Cannabinoids
LC–MS and GC–MS are limited to
screening known species and are
therefore one step behind the designer
drugs market. This LC–MS–MS
method addresses this by providing
accurate masses for all detected
species, thus allowing post analysis
identification of initially untargeted
compounds. Read Here: goo.gl/WvW23V
INTERVIEWFinding Answers
with Foodomics
The Column spoke to Miguel Herrero
of the Institute of Food Science
Research (CIAL-CSIC) at the Spanish
National Research Council, in
Madrid, Spain, about his research
in foodomics-based approaches,
the evolution of food analysis, and the
benefits of 2D LC in this field. Read Here: goo.gl/3LQ0MV
THE LCGC BLOGHow to Get the Most Out
of Your First Conference
Experience
This time of year feels like the
beginning of conference season
to LCGC blogger Kevin Schug.
He shares his advice on how to
get the most out of conferences,
especially for those attending
your first major conference. Read Here: goo.gl/glJvCA
Photo Credit: Stephen Morris/Getty
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Cigarette smoke is a complex matrix containing between
4000 and 5300 different identified compounds (1,2,3).
When performing gas chromatography–mass spectrometry
(GC–MS) analysis on cigarette smoke extracts, scientists are
usually confronted with two main difficulties: The first is the
limited chromatographic space, which leads to unresolved
peaks, and the second is the risk of chemical interactions
between the sample and the collection solvent.
Cigarette smoke is known to consist of two distinct parts: the
particulate phase, which can be trapped using a glass fibre
pad filter (as described in ISO 4387 [4] and ISO 22634 [5]),
and the volatile phase, which can be collected in sampling
bags (6,7) or by impingers containing a trapping solvent either
at room temperature (8) or cooled (9,10). A further trapping
method has been detailed using a cryogenic instrument (11),
with a liquid extraction after the smoking run. However, all of
these trapping methods have drawbacks: the glass fibre trap
does not allow the collection of volatile compounds; and the
trapping in sampling bags leads to rapid decomposition of
unstable species, which requires time‑dependent handling
to avoid significant analytical variations. All of the described
trapping methods using solvents result in sample dilution,
which has the potential for chemical interactions between the
trapped compounds and the solvent.
On the chromatographic side, the choice of column
is a critical factor because the samples usually contain
compounds that cover a wide range of volatility and polarity.
Column selection is therefore usually a compromise between
suitability and optimization for a large variety of compounds.
The objective of this project was firstly to develop a
solvent‑free trapping tool for cigarette smoke that is efficient
and simple to use, and secondly to improve separation by
using two different chromatographic columns.
A glassware device was developed to trap cigarette
smoke at a low temperature in a vial compatible with the
headspace sampler. The GC–MS instrument was modified
to improve the chromatographic separation by the addition
of two cryo‑traps, each preceding a chromatographic
column of different polarity (adapted to the type of chemical
compounds), which significantly increased the total peak
capacity of the analysis.
Accordingly, several further technical modifications
were applied to allow the simultaneous use of two
chromatographic columns for the analysis of the samples,
which led to the development of a new alternative analytical
platform described in this article.
ExperimentalThe reference cigarettes 3R4F were supplied by the
University of Kentucky and conditioned according to
the ISO 3402 standard (12) (at least 48 h at 60% relative
humidity and 22 °C). They were inserted into the cigarette
An Improved GC–MS Method for Cigarette Smoke Characterization Using a Novel Cold Trap, Dual Column, and Cryofocusing SystemJ.R. Crudo, E. Rouget, and M. Rotach, Philip Morris International R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.
Cigarette smoke is a highly complex matrix and presents analytical difficulties for the analyst performing compound identifcation by gas chromatography analysis coupled with mass spectrometric detection (GC–MS). The development of a novel trapping system and a modifed GC–MS layout (using dual chromatographic columns and cryogenic focusing devices) has improved the chromatographic separation of volatile and semivolatile compounds found in cigarette smoke. This improvement has led to the potential to identify compounds usually masked by the solvent peak. This approach has also reduced the amount of peak overlapping by increasing the chromatographic peak capacity with the use of two capillary columns chosen for their analytical specifcity.
KEY POINTS• Compounds were trapped at low temperature without
solvent, avoiding dilution, preventing chemical
interaction with solvent and allowing determination of
very volatile species, which are usually masked by the
solvent peak.
• Compounds were sorted by volatility and directed
to chromatographic columns well adapted for their
separation.
• Improvement of the characterization of cigarette smoke
was achieved.
Ph
oto
Cre
dit: C
ord
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es
LC•GC Europe May 2015258
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holder (Figure 1, part 8 and 9) and were lit with an electric
lighter. Eight puffs were taken following the ISO 3308
standard (13) (one puff per minute, puff volume: 35 mL, puff
duration: 2 s, puff shape: bell curve) using a puff generator
developed in‑house. The cigarette smoke was collected
using two traps mounted sequentially (Figure 1, parts 2
and 8). The first one, a standard glass fibre filter according
to the ISO 3308 standard (part 8 in Figure 1) (Borgwaldt),
collected the particulate phase of the aerosol. The second
trap, a custom‑made glass vial (Figure 2) cooled to a
temperature of ‑150 °C using a flow of liquid nitrogen
regulated by an electronic controller, collected the volatile
compounds representing the vapour phase.
The custom‑designed vial had a Vigreux column‑like
internal geometry to increase flow turbulence and to
maximize contact between the vial surface and the aerosol.
The external dimensions of the vial corresponded to the
model used for headspace analyses with the Turbomatrix
40 Trap (PerkinElmer) headspace sampling device.
To prevent contamination from the laboratory, a glass
tube filled with activated charcoal (part 7 in Figure 1) was
connected to the switch valve (part 6 in Figure 1). Between
each puff, the valve was switched to let pressure equilibrate
and avoid any back flush through the glass fibre filter and
the cigarette.
At the end of each smoking run, the vial was capped with
a Teflon cap. The glass filter contained in the filter holder
was transferred into a separate standard headspace vial
and capped with a Teflon cap.
This trapping mode enabled the analysis of compounds with
a large range of volatility without liquid extraction or dilution.
The use of a headspace sampler gave the opportunity to
sample the volatile and highly volatile compounds, without
extracting the compounds with low volatility.
Headspace Analysis: Headspace sampling was able
to capture a fraction of the headspace volume onto a
headspace trap prior to thermal desorption and injection onto
the chromatographic system via a heated transfer line. The
headspace trap was loaded with three different sorbents:
Tenax GR (PerkinElmer), Carbotrap and Carboxen (Supelco).
The choice of sorbents was made based upon internal
(unpublished) and external studies (14), which demonstrated
that no universal sorbent was adequate to effectively trap
and desorb the compounds of interest belonging to various
chemical classes, and that a mixture of sorbents was required.
After each smoking run, trapped semivolatile (glass fibre
filter in standard headspace vial) and volatile (custom‑made
headspace vial) fractions were submitted for headspace
extraction. The instrument conditions were optimized for
each fraction to ensure the effective transfer of trapped
smoke constituents. During the release phase, vapour
pressure was a key parameter influencing both the diversity
and quantity of compounds extracted (15); fine‑tuning was
required to avoid any segregation of compounds as a result
of differing volatilities. The headspace volumes generated
from each fraction were trapped concomitantly onto the
same headspace trap, and the retained compounds were
then released for injection into the chromatographic system
by thermal desorption at 210 °C.
The GC–MS system (Perkin Elmer Clarus 500) was
modified by the addition of two cryogenic systems (termed
cryo‑traps), developed and manufactured internally at
Philip Morris International for this device (Figure 3), and two
electronically actuated valves (4 ports gas valve, 300 psi,
1/16’’, N9302813, PerkinElmer, 8 ports gas valve, 300 psi,
1/16’’, N9302815, PerkinElmer), which were installed in the
oven to connect the two different chromatographic columns
LC•GC Europe May 2015260
Crudo et al.
87
6
3
41
2
5
9
Figure 1: HS‑cold trap trapping device: 1: glass cooling mantel; 2: vial; 3: glass head connection; 4: nitrogen evacuation pipe; 5: pipe connection to the puff generator; 6: 3‑way valve; 7: adsorption tube; 8: glass fibre filter holder; 9: cigarette holder.
2
Figure 2: Drawing of the custom‑designed vial.
4
53
2 1
76
Figure 3: Technical scheme of a cryo‑trap: 1: Metal trap formed by two cylinders separated by isolation material; 2: heating device; 3: electrical connection and type K thermocouple; 4: entrance of the capillary column coming from the valve; 5: exit for N2; 6: exit of the capillary column towards the GC; 7: liquid nitrogen entry. The trap is located on the GC, replacing the classical injectors.
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(1st column: 30 m × 0.32 mm, 1‑μm Agilent DB‑WAXetr;
2nd column: 30 m × 0.32 mm, 20‑μm Agilent HP‑PLOT/Q).
The two cryogenic systems, cooled using liquid nitrogen,
enabled the cryo‑focusing of compounds (at a temperature
of ‑80 °C for the one located before the DB‑WAXetr column
and at ‑120 °C for the one located before the HP‑PLOT/Q
column) before introduction to the respective columns. The
two switching valves were controlled by the GC–MS events
software. These modifications enabled a two‑dimensional
analysis of cigarette smoke fractions by GC–MS.
The analytical process was initiated by the operator.
LabVIEW software (National Instruments) then conveyed
this start signal to the GC and to the cryo‑traps, and was
therefore used as a master controller for the analytical
system. The two valves, V1 and V2, were controlled
indirectly via the GC programme sequence.
Improved Gas Chromatography, with Dual Columns and Cryogenic Systems Each analytical run was split in four successive phases to
allow adequate separation of compounds in the two cryogenic
traps and in the two chromatographic columns.
Phase 1: Sample Introduction and Cryo-Focusing
(Figure 4): Following desorption of the headspace trap, which
contained headspace constituents from both semivolatile and
volatile fraction collections, eluted compounds were focused
at ‑80 °C in the cryo‑trap positioned in front of the first column.
Highly volatile compounds, which were not retained at this
temperature, passed directly through the first chromatographic
column. These were trapped by the second cryo‑trap cooled
at ‑120 °C, located in front of the second chromatographic
column, which was maintained at ‑120 °C. These two
different trapping temperatures led to a split of very volatiles
compounds from volatiles, to elute each group of compound
selectively on the two dedicated chromatographic columns.
Phase 2: Release from Cryo-Trap #1 and Elution onto
the First Column: The first cryo‑trap was rapidly heated to
200 °C and a start signal was sent by the software to the
GC–MS system to start the chromatographic run. All
compounds trapped in cryo‑trap #1 were released and
introduced onto the WAX column. Analytes with limited affinity
for the column eluted rapidly and were trapped in the second
cryo‑trap. The remaining analytes continued to be separated
on the WAX column by their different retention behaviours.
Phase 3: Analysis of Semivolatile Compounds: Valve
V2 was switched (by the GC–MS) to isolate cryo‑trap #2
(maintained at ‑120 °C) and the PLOT Q column. This
switch connected the exit of the WAXetr column to the mass
spectrometer and enabled the analysis of the semivolatile
compounds remaining in the chromatographic column.
A first heat cycle of a dedicated double heat cycle GC
oven temperature programme was used with the following
parameters: initial temperature: 41 °C during 5 min, followed
by a temperature increase at a rate of 5 °C/min, up to 250 °C.
Phase 4: Release from Cryo-Trap #2 and Analysis of
Volatile Compounds: After chromatographic analysis of
the semivolatile compounds using the WAX column (30 min
run time), the oven was cooled to 60 °C using CO2 to speed
the process. For the release of the volatiles, valve V1 was
switched to isolate the WAX column and valve V2 was
switched back to direct the carrier gas through cryo‑trap #2,
the PLOT Q column, and into the mass spectrometer. The
volatile compounds were then introduced onto the PLOT
Q column by rapidly heating cryo‑trap #2 to 150 °C and
chromatographically separated using the second heat cycle
of the GC oven temperature programme with the following
parameters: 60 °C during 7.2 min, followed by a temperature
increase at a rate of 7 °C/min, up to 250 °C, final temperature
maintained during 9 min.
At the end of the analysis, the valve V1 was switched
again to allow the carrier gas to pass through both columns
and therefore flush the system. This step was done with
the oven heated to its maximum temperature, and to allow
the valve to be ready for the next run because it was not
possible to switch valve V1 after the end of the GC run.
Data acquisition in scan mode was initiated 15 min after
the start of each run because no meaningful data were
generated during the trapping phase for cryo‑trap #2.
A second pause in acquisition was also made between
46.8 min and 54 min, during the cooling phase of the oven
before the second temperature gradient started.
To compare the described approach with a classical
analysis, the cigarette 3R4F (supplied by University
of Kentucky) was conditioned according to ISO 3402
standard and smoked using the ISO smoking regime
(as described in ISO 3308 standard, 1 puff per min, puff
LC•GC Europe May 2015262
Crudo et al.
Transfer Line
PLOTColumn
GC oven
WAXColumn
1
1
2
3X
X
X4
2
345
6
78
V2
V1
MS
Highly volatile compounds
Volatile compounds
Semivolatile compounds
Less volatile compounds
Transfer LineHead spaceautosampler
CRY
O-T
RA
P 1
-80o
C
CRY
O-T
RA
P 2
-120
oC
Liquid N2 Liquid N2
Figure 4: Phase 1.
Scan EI+
TIC
1.04e6
1 2 3100
4.17 9.17 14.17 19.17 24.17
Time (min)
29.17 34.17 39.17 44.17
%
0
5 7 8
6
4
Figure 5: Chromatogram of mainstream smoke from the reference cigarette (3R4F supplied by the University of Kentucky). Ten cigarettes were smoked under ISO smoking regime and trapped on a glass fibre filter followed by three impingers containing each 10 mL ethyl acetate cooled at
‑78 °C. One microlitre of sample was injected on‑column in a DB‑FFAP column (30 m × 0.25 mm, 0.25‑μm). Analytes: (1) isoprene, (2) solvent, (3) toluene, (4) limonene, (5) nicotine, (6) neophytadiene, (7) triacetin, and (8) glycerin.
ES610530_LCE0515_262.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
duration: 2 s, puff volume 35 mL, puff profile: bell shape).
The particulate phase of the smoke of 10 cigarettes was
trapped using a glass fibre filter and the gas phase was
bubbled through cooled (‑78 °C) ethyl acetate in three
small impingers mounted in series. The glass fibre filter
was extracted together with the solvent contained in the
impingers and was analyzed by GC–MS in scan mode
following an internal GC–MS method.
Results and DiscussionA typical total ion chromatogram (TIC) for the classical
analysis is presented in Figure 5. One drawback of this
technique is linked to the solvent, which prevents the
detection of coeluting compounds (volatile compounds).
Another issue is compound coelution, which can lead to
uncertainty during the peak identification process.
The innovative aspect of the new platform lies in the
use of a dedicated column selected specifically for
the volatility of each group of compounds. Using this
set‑up, the chromatogram showed first the semivolatile
compounds eluted on the 30 m × 0.32 mm, 1‑μm
column (Figure 6) and later the volatile ones eluted on
the 30 m × 0.32 mm, 20‑μm column (Figure 7). The
split between semivolatile and volatile compounds was
handled using two specific chromatographic columns with
dedicated oven programmes, allowing an improvement in
the chromatography.
A clear improvement in selectivity was apparent when
using the cold trap coupled with the headspace sampling
compared with the traditional analysis. Peaks are properly
resolved and can be identified with improved certainty. In
addition, the second part of the chromatogram (starting
at approximately 53 min) allowed the identification of
numerous volatile compounds that are masked by the
solvent peak when using conventional GC–MS analysis. For
example, Figure 7 demonstrates that compounds such as
chloromethane, HCN, acetaldehyde, ethanol, acetonitrile,
propionaldehyde, and acetone were clearly separated.
263www.chromatographyonline.com
Crudo et al.
21.6
14.4
7.2
016.52 18.03 19.54 21.05 22.56 24.07 25.58 27.09 28.60 30.11 31.63 33.14 34.65 36.16 37.67 39.18 40.69 42.20 43.71 45.22 46.73
Time (min)
1 2
3
5
7
8
9
10
11 14
13
12
15 16 17
6
4
Figure 6: Chromatogram of the semivolatile part of the mainstream smoke from one single reference cigarette (3R4F, supplied by the University of Kentucky) after separation on a DB‑WAXetr column. Analytes: (1) ethylbenzene, (2) limonene, (3) styrene, (4) acetol, (5) 2‑cyclopentene‑1‑one, (6) acetic acid, (7) Propanoic acid, (8) propylene glycol, (9) 2‑furanmethanol, (10) solanone, (11) nicotine, (12) neophytadiene, (13) phenol, (14) triacetin, (15) myosmine, (16) glycerin, and (17) nicotyrine.
Chromatography is what we do and who we are.We are an independent, international, and diverse team of employee-owners not bound
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countries and six continents with unrivaled Plus 1 service, applications, and expertise. From LC
and GC columns to sample prep, reference standards to accessories, Restek is your frst and
best choice for chromatography.
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ES610528_LCE0515_263.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan
Furthermore, the sensitivity of the technique allows the
analysis of cigarette mainstream smoke from a single
cigarette, therefore reducing the number of cigarettes
required, while increasing the level of information gained.
The automation of the analytical process ensured
reproducible retention times across several different runs
(Figure 8) and displayed very volatile compounds on the
30 m × 0.32 mm, 20‑μm column. The same reproducibility
in retention times was observed on the 30 m × 0.32 mm,
1‑μm column.
ConclusionThe platform for trapping and analysis allowed the
characterization of cigarette smoke in an efficient and
simple way. The trapping device is an improvement
compared to classical liquid trapping because of the
absence of solvent, which reduces sample preparation,
prevents any chemical interaction between solvent and
compounds of interest, and allows determination of
chemical species which are usually masked by the solvent
peak. In addition, no dilution is performed during sample
preparation; therefore, the analysis can be performed
on a single cigarette. Improved sensitivity and resolution
enables a better and more comprehensive characterization
of cigarette mainstream smoke.
The method was focused on qualitative aspects to
facilitate compound identification, rather than to quantify
them. Because compounds may have very different
vapour pressures under the chosen conditions, headspace
sampling would not be suitable for direct quantification of
samples.
The improved chromatographic analysis allowed
an easier identification of compounds because of the
improved peak capacity provided by the hyphenation of
the two columns, which led to a better peak separation.
Retention times were reproducible because the complete
process was fully automated. This reproducibility
also allows the creation of custom libraries to improve
identification certainty.
References(1) R.R. Baker, in Tobacco: production, chemistry and technology, D.L.
Davis and M.T. Nielsen, Eds. (Blackwell Science Ltd, Oxford, 1999),
p. 398–439.
(2) M.F. Borgerding, J.A. Bodnar, H.L. Chung, et al., Food and
Chemical Toxicology 36, 169–182 (1997).
(3) T. Perfetti and A. Rodgman, Beiträge zur Tabakforschung
International 24(5), (May 2011).
(4) International Organization for Standardization (ISO): ISO 4387:2000,
Cigarettes ‑‑ Determination of total and nicotine‑free dry particulate
matter using a routine analytical smoking machine. Geneva,
Switzerland.
(5) International Organization for Standardization (ISO): ISO
22634:2008, Cigarettes ‑‑ Determination of benzo[a]pyrene in
cigarette mainstream smoke ‑‑ Method using gas chromatography–
mass spectrometry. Geneva, Switzerland.
(6) Ji‑Zhou Dong, J. Neil Glass, and Serban C. Moldoveanu, Journal of
Microcolumn Separations 12(3), 142–152 (2000).
(7) J.Z. Dong and S.M. Debusk, Chromatographia 71(3/4), (2010).
(8) N. Mottier, F. Jeanneret, and M. Rotach, Journal of AOAC
International 93(3), (2010).
(9) Health Canada: Determination of Ammonia in Mainstream Tobacco
Smoke, Official Method T‑101 (1999) http://laws‑lois.justice.gc.ca/
eng/regulations/SOR‑2000‑273/page‑14.html
(10) Health Canada: Determination of Selected Carbonyls in Mainstream
Tobacco Smoke, Official Method T‑104 (1999) http://laws‑lois.
justice.gc.ca/eng/regulations/SOR‑2000‑273/page‑14.html
(11) N. Plata, I. Hofer, S. Roudier, and J.P. Schaller, Journal of Aerosol
Science 37(12), 1871–1875, (2006).
(12) International Organization for Standardization (ISO): ISO 3402:1999,
Tobacco and tobacco products ‑‑ Atmosphere for conditioning and
testing. Geneva, Switzerland.
(13) International Organization for Standardization (ISO): ISO 3308:2000,
Routine Analytical Smoking Machine ‑‑ Definition and Standard
Conditions. Geneva, Switzerland.
(14) M. Schneider and K.‑U. Goss, Analytical Chemistry 81, 3017–3021
(2009).
(15) B. Kolb and L.S. Ettre, Static Headspace-Gas Chromatography:
Theory And Practice (Wiley, UK, 2nd ed., 2006).
Jean-René Crudo is an associate scientist at Philip
Morris International (Research and Development,
Neuchâtel, Switzerland). His analytical focus is on
the development and application of GC–MS methods
focusing on the qualitative and quantitative analysis of
complex matrices.
Emmanuel Rouget is a scientist at Philip Morris
International (Research and Development, Neuchâtel,
Switzerland). His main research interests are related to
the development and application of GC–MS techniques
concerning qualitative and quantitative analysis of
molecules.
Michel Rotach is a senior scientist at Philip Morris
International (Research and Development, Neuchâtel,
Switzerland). His main activities involve providing
scientific advice to sustain the development of Reduced
Risk Product (RRPs), perform data analysis, and provide
scientific guidance.
LC•GC Europe May 2015264
Crudo et al.
23.5
15.6
7.8
0
54.96 56.66 58.35 60.05 61.75 63.44 65.14 66.84 68.53 70.23 71.92 73.62 75.32 77.01 78.71 80.41 82.11 83.81 85.50 87.20 88.89
Time (min)
1 2
3
57
8
9
10
11
1413
12
15
16
17
18
19
21
22
20
6
4
Figure 7: Chromatogram of the volatile part of the mainstream smoke from one single reference cigarette (3R4F, supplied by the University of Kentucky) after separation on the HP‑PLOT/Q column. Analytes: (1) chloromethan, (2) HCN, (3) acetaldehyde, (4) 2‑methyl‑1‑propene, (5) 1‑3, butadiene, (6) 2‑ butene, (7) ethanol, (8) acetonitrile, (9) 2‑ butyne, (10) furan, (11) propionaldehyde, (12) acetone, (13) acrylonitrile, (14) propanenitrile, (15) metacrolein, (16) methylfuran, (17) mix of butyraldehyde and methyl ethyl ketone, (18) benzene, (19) 2‑pentanone, (20) 2,3‑pentanedione, (21) dimethyl disulphide, and (22) toluene.
75.15 76.15 77.15 78.15
TIC
9.58e9
79.15 80.15 81.15
Time (min)
100
%
2
Figure 8: Overlay of the volatile part of three chromatograms from reference cigarettes (3R4F, supplied by the University of Kentucky).
ES610520_LCE0515_264.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan
Environmental sample analysis by large-volume injection
(LVI) in combination with liquid chromatography–tandem
mass spectrometry (LC–MS–MS) is the direct introduction
of large sample volumes (for example, 900–1800 µL) into
an LC system for separation and subsequent detection. The
primary advantages of LVI compared to traditional off-line or
on-line sample preparation techniques, such as solid-phase
extraction (SPE) or liquid–liquid extraction (LLE), include
decreased sample preparation, greater analyte mass
introduced for detection (that is, increased sensitivity),
and less solvent and solid waste. LVI is well-suited for the
concentration and separation of water-soluble analytes
on reversed-phase guard and analytical columns (1),
and has also been extended to organic extracts (2).
Successful combination of LVI with LC–MS–MS requires an
understanding of analyte properties (polarity, stability, and
so on) and sample properties (salinity, particulate matter,
matrix components, and more) (3,4). The unique elements
of several applications are discussed here to highlight
the various capabilities and limitations of LVI and are
divided between aqueous and organic solvent injections.
Finally, a set of loading strategies designed to capture
nonionic analytes from large volumes of organic extracts
on reversed-phase columns is described and experimental
components are listed.
ExperimentalAn Agilent 1100 high performance liquid chromatography
(HPLC) system, upgraded with a 900-µL analytical head
and an extended-seat capillary sample loop, was used for
all methods described below.
Aqueous Samples: Direct InjectionPast examples of aqueous LVI include the analysis
of neurotoxins in drinking water, pharmaceuticals in
wastewater, and corrosion inhibitors in surface water (4).
Here, two LVI methods are presented with a focus on the
important technical features required to inject 1800 µL
onto C18 guard and analytical columns. The LVI methods
highlighted involve the direct injection of raw wastewater
for the determination of illicit drugs (5) and seawater with
25% isopropanol for the determination of the surfactant
components in the oil dispersant Corexit (Nalco) (6).
Before injection, raw wastewater is centrifuged to remove
particulate matter that can potentially clog columns and
other LC components (5). Filtration is a commonly used
alternative, but its suitability is method- and analyte-specific
because filter membranes can serve as a potential sink or
source of analyte contamination (4,7). SPE, however, is not
limited by particulate matter and has been used to analyze
both the aqueous and particulate bound fraction (8). After
centrifugation, the raw wastewater sample is added to
an autosampler vial and spiked with isotopically labelled
internal standards.
The six-port valve connections in the LC autosampler
(Figure 1) are labelled as either mainpass (solid red
channel) or bypass mode (dashed blue channel). The
mobile phase in mainpass mode flows sequentially through
Large-Volume Injection LC–MS–MS Methods for Aqueous Samples and Organic ExtractsB. McKay Allred1, Matthew J. Perkins2, and Jennifer A. Field2, 1Department of Chemistry, Oregon State University, Corvallis,
Oregon, USA, 2Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA.
Environmental sample analysis by large-volume injection (LVI) in combination with liquid chromatography–tandem mass spectrometry (LC–MS–MS) is described for polar and nonpolar analytes in both aqueous samples and organic extracts. LVI is the direct introduction of large sample volumes (for example, 900–1800 µL) into the LC system for separation and subsequent detection. The practical benefts of LVI include faster sample preparation times, lower detection limits, and minimal waste generation.
KEY POINTS
• Environmental sample analysis by LVI in combination
with LC–MS–MS is described for polar and nonpolar
analytes in both aqueous samples and organic
extracts.
• The technique offers several advantages over
traditional small-volume injection and off-line sample
cleanup techniques without diminished method
performance.
• Benefits include faster sample preparation times,
lower detection limits, and minimal waste generation.
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265www.chromatographyonline.com
ES610635_LCE0515_265.pgs 04.30.2015 17:11 ADV blackyellowmagentacyan
the analytical head, sample loop, needle, extended-seat
capillary sample loop, and then onto the columns. Bypass
mode directs the flow directly onto the column. Initially, the
LC system is operated in bypass mode so that the needle
may be inserted in the sample vial and the first 900-µL
volume is withdrawn. The needle is returned to its seat and
the 900 µL of wastewater is then ejected into a 1400-µL
extended-seat capillary sample loop, which acts as a
storage device during the withdrawal of the second 900 µL.
Afterwards, the six-port valve is switched to mainpass
mode and the cumulative 1800 µL of wastewater sample is
chased by the mobile phase out of the sample loops and
onto C18 guard and analytical columns. After the sample is
out of the sample loops, the six-port valve switches back to
bypass mode. If left in mainpass mode, the gradient would
travel through all of the mainpass tubing before reaching
the column, requiring that the total run time be extended by
several minutes.
Both the wastewater sample and initial mobile-phase
solvents are aqueous with characteristically low eluotropic
strengths on reversed-phase columns, such that
methamphetamine and other illicit drugs and biomarkers are
focused on the head of the C18 guard column. The guard
column protects the analytical column and is preemptively
replaced every 50 sample injections. This is ultimately more
cost and time effective than using individual SPE cartridges
for sample cleanup and analytical column protection.
After loading analytes onto the C18 guard and analytical
columns, a traditional reversed-phase gradient is used to
separate and elute all analytes.
Neither reversed-phase LVI or SPE can selectively remove
matrix components that are coeluted with the analytes
of interest. With LVI, a postcolumn divert valve can be
precisely controlled to divert column effluent containing
sample matrix components away from the detector. The
control of divert valve timing is achieved with greater
precision than the analogous wash steps used in SPE
and can send all unfavourable effluent to waste. As will be
discussed below, the postcolumn divert valve is of even
greater importance with the direct injection of seawater.
An LVI-based method was developed by Place and
colleagues (6) using the direct injection of seawater to
quantify the surfactant components of Corexit: dioctyl
sulphosuccinate (DOSS), Tween 80, Tween 85, and Span
80. Liquid standards of sorbitan monooleate (Span 80;
purity: 70.5%), sorbitan monooleate polyethoxylate (Tween
80; purity: 74%), and sorbitan monooleate polyethoxylate
(Tween 85; purity: 67%) were obtained from Sigma
Aldrich. The direct injection of 1800 µL followed similar
steps as described above for wastewater with two crucial
adjustments. Open seawater does not require centrifugation
and filtration was shown to remove the analytes of interest
from solution. In addition, the Corexit surfactants undergo
hydrophobic exclusion from aqueous solutions and exhibit
significant loss (>50%) to polypropylene or glass container
walls within 8 h of sampling at ambient temperature. To
prevent analyte loss before analysis, seawater is diluted
with isopropanol at the time of collection to give a final
composition of 1:3 isopropanol–seawater. Injections of
1800 µL of the isopropanol-stabilized matrix resulted in
well-resolved peaks for anionic and nonionic surfactant
classes. At higher additions of isopropanol, there is no
improvement of analyte stability in aqueous samples and
peak shape begins to suffer.
Direct injection of seawater into the LC–MS–MS system
is possible without damaging the mass spectrometer
source by using the divert valve to direct the nonvolatile
salts contained in seawater to waste. The 1800-µL injection
volume (3.6 min loading at 0.5 mL/min) is followed by a
5.9-min “wash” under the primary mobile-phase conditions
(5% acetonitrile and 0.5 mM ammonium acetate in water).
This wash is diverted to waste and is sufficient to flush the
nonvolatile salts from the column and away from the mass
spectrometer before elution of the analytes of interest. The
time (5.9 min) used to wash the salts from the seawater
from the columns was evaluated by testing effluent fractions
collected at 30-s intervals with the addition of 100 µL of 1 M
silver nitrate–0.6 M nitric acid. The presence of chloride
in the collected effluent was determined by the formation
of a white silver chloride precipitate, visible in aqueous
solutions containing more than 0.1% seawater. No silver
chloride was observed in the effluent 7.5 min after injection
and a precautionary period of 2 min was added to extend
the loading and wash period to 9.5 min. For this application,
guard columns are replaced approximately every 100
injections and the analytical column was replaced after
approximately 1500 injections. Analysis over two years and
more than 5000 injections did not lead to salt accumulation
or corrosion in the LC–MS–MS system.
Organic Extracts: Orthogonal ChromatographySample treatment to create organic extracts is desirable
if analytes are unstable in aqueous solution, because
of hydrolysis or hydrophobic exclusion, or if the sample
matrices (for example, landfill leachate) are sufficiently
complex that significant sample cleanup is required before
injection. In addition, some analytes do not focus well
on reversed-phase columns, even under high aqueous
conditions, and require a different retention mechanism. For
example, per- and polyfluorinated alkyl substances (PFAS)
present in landfill leachate are commonly concentrated
by SPE before injection (8) because of leachate matrix
complexity. Direct aqueous injection for PFAS analysis is
additionally disadvantageous because polar PFAS are not
retained well on reversed-phase columns and the more
hydrophobic PFAS are lost from aqueous matrices onto
autosampler vials and to the air–water interface. The two
Analytical
head
C18 analytical column
MS–MS
Waste
Waste
Pump
Mainpass
Bypass
C18 guard
Six-port valve
Figure 1: Basic LVI configuration for 1800-µL injections
onto a reversed-phase system. The six-port valve and
injection assembly was adapted from Agilent autosampler
schematics (16).
LC•GC Europe May 2015266
Allred et al.
ES610634_LCE0515_266.pgs 04.30.2015 17:11 ADV blackyellowmagentacyan
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new strategies discussed here for the LVI of PFAS in an organic solvent extract from leachate include orthogonal chromatography (9) and on-line extract dilution.
In-line orthogonal chromatography was developed for PFAS in ethyl acetate–trifluoroethanol extracts of landfill leachates (9). The orthogonal columns system consisted of two zirconium-modified diol (Zr-diol) guard columns (6 µm, 150 Å, 12.5 mm × 4.6 mm, Agilent) placed in-line with a Zorbax Eclipse Plus C18 analytical column (3.5 µm, 95 Å, 75 mm × 4.6 mm, Agilent) (Figure 2). The system proved capable of capturing 70 anionic PFAS from 900-µL injections of the 1200-µL ethyl acetate–trifluoroethanol extract, which is 75% of the extract volume generated.
The PFAS are captured by the Zr-diol columns through interactions between the anionic PFAS and the zirconium d-orbitals during the 900-µL injection of the ethyl acetate–trifluoroethanol extract. The trapped PFAS are then eluted from the Zr-diol guard columns by a mobile phase containing 50% methanol and 5 mM ammonium acetate that then refocuses the majority of analytes on the C18 analytical column. PFAS are then separated by a traditional reversed-phase gradient with an increasing percentage of organic solvent (Figure 3). Without LVI, additional sample preparation steps like solvent transfer and blow-down are necessary to introduce sufficient analyte mass in the small injection volumes typically used (such as 10–20 µL). Moreover, an injection of 10–20 µL of an extract concentrated into about 100 µL is, at most, 20% of the total extract volume generated. Comparatively, LVI capitalizes on the time, effort, and labour costs expended during the creation of organic extracts by analyzing a greater percentage, while also eliminating additional steps that increase the possibility of analyte loss.
Organic Extracts: DilutionOrthogonal chromatography (as described above) is not capable of capturing nonionic PFAS from a large volume of organic extract. Dilution of organic extracts with water is one approach to perform LVI for the analysis of nonionics in organic extracts (for example, pesticides in vegetable [10] or soil extracts [11]). The traditional use of small-volume injections with organic solvent extracts of high eluotropic strength achieve focusing of analytes on reversed-phase columns because of dilution of the small organic extract volume with the larger volume of initial aqueous mobile phase. In the case of PFAS in landfill leachates, an eightfold dilution with water would be needed to retain the most water-soluble, nonionic PFAS on C18 columns (for example,
MeFBSA). For comparable levels of sensitivity using off-line dilution, the 900-µL extract, injected previously, would become at least 7.1 mL and require multiple large autosampler vials, larger extended sample loops, and a longer injection programme. However, as previously mentioned, many PFAS can partition out of the bulk solution under high aqueous conditions. Therefore, on-line dilution strategies are explored here.
The objective of the following dilution-based LVI method is to use the ethyl acetate–trifluoroethanol leachate extracts and the orthogonal Zr-diol–C18 column system for the analysis of nonionic PFAS. Thus, the first alternative to off-line dilution was to inject 900 µL in small volume “packets” segmented by defined volumes (determined by flow rate and time) of the initial aqueous mobile phase. To minimize the overhead time required by the autosampler transport assembly to inject multiple small volumes, one 900-µL volume was drawn up into the sample loop and loaded onto the column in small packets by switching back and forth between mainpass and bypass mode (Figure 2). The volume of packets is determined by the flow rate and time spent in mainpass mode. The flow rate while loading packets onto the Zr-diol guard columns and C18 analytical column was 2 mL/min and the times spent in mainpass and bypass mode were programmed as 0.01 and 0.3 min, respectively (Figure 4). Mainpass and bypass were switched back and forth 18 times over the first 7.5 min where the six-port valve was left in mainpass mode for 1 min the last time. At 8.3 min the gradient was started in bypass mode and the flow rate was decreased to 0.5 mL/min.
The solvents in the mainpass assembly are pressurized when the six-port valve is in mainpass mode. But when in bypass mode, small volumes are lost to the waste line on the six-port valve as the mainpass assembly solvents depressurize and expand. Consequently, the six-port valve leading to waste (Figure 2) was plugged with a blanking nut to avoid losing small volumes of sample while switching between mainpass and bypass mode. Because the waste line was plugged, the 900 µL of mainpass solvent normally ejected to waste in preparation for drawing up sample were instead programmed to eject into an empty sample vial.
120
Time (min)
Resp
on
se (
%)
100
80
60
40
20
00 5
1 2 3 4 5 6
10 15 20 25 30 35
Figure 3: Typical leachate chromatogram for select PFAS using the Zr-diol guard column configuration. Peaks: 1 = PFBA, 2 = PFHxS, 3 = PFOS, 4 = MeFBSAA, 5 = 6:2 DiPAP, 6 = PFHxDA.
Analytical head
Pump
Six-port valve
Waste
Waste
MS–MSC18 analytical
column
MainpassBypass
Zr-diol guard
Zr-diol guard
Figure 2: Schematic of configuration for PFAS analysis in leachate extracts.
LC•GC Europe May 2015268
Allred et al.
ES610639_LCE0515_268.pgs 04.30.2015 17:12 ADV blackyellowmagentacyan
When samples are loaded using this on-line,
packet-dilution configuration, both ionic and nonionic PFAS
are captured and separated from landfill extracts (Figure 4).
The most water-soluble ionic (PFBA) and nonionic
(MeFBSA) PFAS are successfully retained after 18 packet
injections of leachate extract.
The LC pressure isotherm (Figure 4) is an important
tool to aid method development. Pure and mixed solvents
have different viscosities and therefore exert different
corresponding back pressures that can be used to track
gradient and sample delivery. The sawtooth pressure profile
during the first 7.5 min is evidence of extract solvent packets
mixing with the aqueous mobile phase in the Zr-diol guard
columns. While programmed as 0.01 and 0.3 min, the actual
times spent in mainpass and bypass mode were calculated
using the average frequency of packet mixing seen in the
pressure isotherm and were closer to 0.028 and 0.35 min,
respectively. So at 2 mL/min the average packet was
56 µL with 700 µL of aqueous mobile phase on either side.
The volume of mobile phase on both sides of the organic
packets effectively dilutes the extract solvent inside the
Zr-diol guard columns, thus reducing the eluotropic strength
of the sample solvent so that focusing of the nonionic PFAS
occurs on the C18 analytical column. The Zr-diol guard
0 5 10 15 20 25
250 120
100
80
60
40
20
0
200
150
100
50
0
Pre
ssu
re (
bar)
Resp
on
se (
%)
Time (min)
Pressure
Response
1 2 3 4
Figure 4: Pressure isotherm and chromatogram for
mainpass and bypass packet injections. Landfill leachate
peaks for ionic PFAS: 1 = PFBA, 4 = PFOcDA. Nonionic
PFAS peaks: 2 = MeFBSA, 3 = EtFOSA.
C18guard
Pump
C18 analyticalcolumn
Six-port valve
MS–MS
Waste
Waste
MainpassBypass
At-column dilutionhardware changes
Analyticalhead
Figure 5: Divided-flow at-column dilution schematic. The
tubing internal diameters for tubing 1 and 2 were changed
from 0.175 mm to 0.125 and 0.76 mm, respectively.
Allred et al.
Hamilton Bonaduz AG
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columns were more effective mixing chambers than other
guard columns (such as amino propyl, C18) that required
smaller packet volumes to focus nonionic PFAS on the head
of the C18 analytical column. After loading nonionic PFAS
the analytes were eluted from the Zr-diol guard columns and
C18 analytical column separately; the anionic PFAS were
focused on the Zr-diol columns and nonionic PFAS were
focused on the C18 column.
The second alternative strategy is to trap nonionic PFAS
from an organic extract on reversed-phase columns using
a variation of at-column dilution that divides the flow from a
single pump. At-column dilution typically uses two pumps
that either pushes the sample at a slow flow rate to the tee
junction or pumps a weak solvent at a high flow rate that
dilutes the sample when the two flows combine at the tee
(12). The amount of sample dilution is a function of the two
flow rates. At-column dilution has been applied to load
larger sample volumes onto columns during preparative LC
(13) or analytical LC (for example, pesticides in river water
using on-line SPE with LC–MS–MS) (14). The advantage
of divided-flow at-column dilution is that a second pump
does not need to be purchased and at-column dilution is
achieved using only two tee junctions and tubing of different
internal diameters (Figure 5).
Chromatographic separation was accomplished using a
Zorbax Eclipse Plus C18 guard column (12.5 mm × 4.6 mm,
5-µm Agilent) and a Zorbax Eclipse Plus C18 analytical
column (75 mm × 4.6 mm, 3.5-µm Agilent). The internal
diameter of the tubing between the tee and the autosampler
six-port valve (Figure 5) was decreased from 0.175 to
0.125 mm. The internal diameter of the tubing connecting
the two tees was 0.175 mm and the internal diameter of
the 3-cm length of tubing on the outflow of the second tee
and before the C18 guard column was large (0.76 mm) to
ensure complete mixing of the two recombined flowpaths.
Mobile-phase A consisted of 3% methanol in deionized
water and mobile phase B consisted of 100% methanol
and 10 mM ammonium acetate. The initial aqueous mobile
phase flow rate was 3 mL/min for 3.5 min, during which
time the 900-µL leachate extract was loaded onto the C18
guard column. The C18 guard column effluent was sent to
waste for the first 3.5 min to keep the pressure low during
the high flow rate. After 3.5 min the flow rate dropped to
0.5 mL/min and the guard column effluent was directed
onto the analytical column. The gradient was then increased
to 100% over the next 10 min and held for 5 min. PFAS
were detected using a Quattro Premier XE MS–MS system
(Waters Corporation), operated in multireaction monitoring
mode.
Adequate dilution of the organic extract before the C18
guard column is evidenced by nonionic peak retention and
symmetry (Figure 6). More polar PFAS (such as PFBA) were
not retained using only a C18 guard column. To capture
anionic PFAS additional guard columns with orthogonal
retention mechanisms (for example, Zr-diol) can be added
before or after the C18 guard column with minimal increases
to the back pressure.
While in mainpass mode the flow was divided at the first
tee following the pump (Figure 5), with less mobile phase
being directed through the sample loop. The reduced
flow of mobile phase in the sample loop slowly moved the
sample volume to where it combined with the rest of the
flow at the second tee. The pressure isotherm (Figure 6)
increased 1 min after switching to mainpass mode when
the diluted 900-µL organic solvent extract first reached the
guard column. After 3.5 min the entire sample had passed
through the guard column and the C18 guard column
was once again filled with the aqueous mobile phase.
For 900 µL to pass through the guard column in under
2.5 min the flow rate through the autosampler was at least
0.36 mL/min, which corresponds to a 8.3-fold dilution when
combined at the second tee with the rest of the 3 mL/min.
Reducing the internal diameter of the tubing leading to
the six-port valve from the first tee from 0.175 to 0.125 mm
effectively reduced the percent flow to the six-port valve
from 20% to 12% to achieve the eightfold dilution necessary
for nonionic PFAS retention.
The incidental introduction of foreign matter can adversely
change the internal diameter and by extension the percent
flow to the six-port valve will also change (15). Should the
internal diameter change significantly, samples may not
be sufficiently diluted or may take longer to load onto the
column. The risk of foreign matter introduction can be
reduced by centrifuging or filtering the samples and mobile
phases. Any change of the flow division at the first tee will
be evident in the pressure isotherm profile, and clogged
tubing can be cleared with extended flushing (15) or easily
replaced; the divided-flow at-column dilution tubing used is
relatively short (<5 cm) and therefore inexpensive. Overall,
divided-flow at-column dilution has a simple, more universal
configuration compared to “packet injections,” and it exerts
lower back pressures, is faster, and can be used to inject
more than 900 µL by using the extending capillary (Figure 1)
used in the wastewater and seawater methods described
above.
ConclusionLVI combined with LC–MS–MS offers several advantages
over traditional small-volume injection and off-line
sample cleanup techniques without diminished method
performance. Direct injection LVI often requires little to no
sample preparation and reduces solid and solvent waste
generation associated with traditional off-line sample
preparation. The injection of large sample volumes is
analogous to the concentration step of SPE and increases
1
0 2 4 6 8 10 12 14
2 3 4
70 120
Pressure
Response
100
80
60
40
20
0
60
50
40
30
20
10
0
Pre
ssu
re (
bar)
Resp
on
se (
%)
Time (min)
Figure 6: Divided-flow at-column dilution pressure isotherm
and chromatogram. Peaks 1 = PFHxS, 2 = MeFBSA,
3 = EtFOSA, 4 = PFOcDA.
LC•GC Europe May 2015270
Allred et al.
ES610637_LCE0515_270.pgs 04.30.2015 17:11 ADV blackyellowmagentacyan
the sensitivity of a given analysis by
introducing more mass of the analytes
of interest to the detector. Matrix
components can be separated from
the analytes of interest with greater
specificity using LVI because of the
control afforded by gradient elution
and waste divert valves. Centrifugation
or filtration is necessary before the
direct injection of aqueous samples
with suspended particulate matter,
whereas some SPE-based methods
simultaneously analyze the dissolved
and particulate phases.
When matrix complexity or analyte
stability require the generation of
an organic extract, LVI capitalizes
on the time, effort, and labour costs
expended by injecting a larger
percentage of the total extract
volume. LVI of organic extracts also
shortens sample preparation without
a loss in sensitivity by eliminating
additional steps associated with small
volume injections (such as solvent
evaporation) and the corresponding
opportunities for analyte loss. Direct
injection of organic extracts onto
orthogonal columns was sensitive
and robust, but was limited to
ionic analytes. On-line dilution of
organic extracts enables both ionic
and nonionic analyte retention on
reversed-phase columns and is
preferable to off-line dilution for
analytes unstable in aqueous solution.
Divided-flow at-column dilution
required only one pump, tee junctions,
and tubing and efficiently focused
anionic and nonionic analytes onto a
reversed-phased system.
References(1) T. Reemtsma, L. Alder, and U. Banasiak, J.
Chromatogr. A 1271, 95–104 (2013).
(2) W.J. Backe, T.C. Day, and J.A. Field,
Environ. Sci. Technol. 47, 5226–5234
(2013).
(3) W.J. Backe and J.A. Field, Environ. Sci.
Technol. 46, 6750–6758 (2012).
(4) F. Busetti, W.J. Backe, N. Bendixen, U.
Maier, B. Place, W. Giger, and J.A. Field,
Anal. Bioanal. Chem. 402, 175–186 (2012).
(5) A.C. Chiaia, C. Banta-Green, and J. Field,
Environ. Sci. Technol. 42, 8841–8848
(2008).
(6) B.J. Place, M.J. Perkins, E. Sinclair, A.L.
Barsamian, P.R. Blakemore, and J.A.
Field, Deep Sea Research II (http://dx.doi.
org/10.1016/j.dsr2.2014.01.015i) (2014).
(7) J.W. Martin, K. Kannan, U. Berger, P.D.
Voogt, J. Field, J. Franklin, J.P. Giesy, T.
Harner, D.C.G. Muir, B. Scott, M. Kaiser,
U. Järnberg, K.C. Jones, S.A. Mabury,
H. Schroeder, M. Simcik, C. Sottani, B.V.
Bavel, A. Kärrman, G. Lindström, and
S.V. Leeuwen, Environ. Sci. Technol. 38,
248A–255A (2004).
(8) J.P. Benskin, M.G. Ikonomou, M.B.
Woudneh, and J.R. Cosgrove, J.
Chromatogr. A 1247, 165–170 (2012).
(9) B.M. Allred, J.R. Lang, M.A. Barlaz, and
J.A. Field, J. Chromatogr. A1359, 202–211
(2014).
(10) A.C. Hogenboom, M.P. Hofman, S.J. Kok,
W.M.A. Niessen, and U.A.T. Brinkman, J.
Chromatogr. A 892, 379–390 (2000).
(11) I. Rybar, R. Gora, and M. Hutta, J. Sep. Sci.
30, 3164–3173 (2007).
(12) “At-Column Dilution Application Notes,”
Waters Corp., Milford, Massachusetts, USA,
2003, pp. 1–22.
(13) K.F. Blom, J. Comb. Chem. 4, 295–301
(2002).
(14) H. Sasaki, J. Yonekubo, and K. Hayakawa,
Anal. Sci. 22, 835–840 (2006).
(15) J.W. Dolan, LCGC North Am. 19, 478–482
(2001).
(16) Agilent Technologies, Agilent 1100
Series Standard Micro and Preparative
Autosamplers, Agilent Technologies,
Germany, 2001, pp. 205–206.
B. McKay Allred is with the
Department of Chemistry and Matthew
J. Perkins and Jennifer A. Field are
with the Department of Environmental
and Molecular Toxicology, all at Oregon
State University, in Corvallis, Oregon,
USA. Direct correspondence to:
jennifer.field@oregonstate.edu
www.chromatographyonline.com
Allred et al.
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ES610638_LCE0515_271.pgs 04.30.2015 17:11 ADV blackyellowmagentacyan
N
N
N
OO
O
OH
HO
NH
S
Methotrexate (MTX, Figure 1) is considered an “anchor
drug” in the treatment of rheumatoid arthritis and is among
the most effective disease-modifying antirheumatic drugs
(DMARDs) with less toxicity and better tolerability. However,
MTX alone is often not able to fully control disease activity.
In fact, MTX is used in combination with other nonbiological
DMARDs by 99% of all rheumatologists (1–3). In a 2009
review and meta-analysis by Katchamart and colleagues
(2), results from 19 clinical trials on the efficacy and toxicity
of various nonbiological DMARDs in combination with
MTX, or of MTX alone were compared. It was noted in this
review that several of the combination drug trials (5 out of
19) were recorded evaluating the combination of MTX and
sulphasalazine (SSZ, Figure 1). It has also been reported
elsewhere that the MTX and SSZ combination therapy is
frequently used, and it is inexpensive and efficacious for
the treatment of rheumatoid arthritis (1–6).
The drug concentration of MTX is currently monitored
in clinical practice for the adjustment of treatment (7). The
clinical monitoring of the plasma concentration of SSZ
as well as the patient’s liver function is also performed
for the detection of any immune-allergenic reaction (8).
Additionally, both MTX and SSZ are highly protein bound
(50% for MTX, and 99% for SSZ) and thus are likely to
displace each other through interactions with plasma
proteins (9,10). They also have a common mechanism of
action, that is, inhibition of folate-dependent enzymes (6).
Therefore, the simultaneous monitoring of plasma levels of
MTX and SSZ when used in combination may have both
clinical relevance as well as help further pharmacokinetic
research into any synergistic effects.
The typical oral therapeutic dose of MTX used in
rheumatoid arthritis ranges from 7.5 to 25 mg per week
(2,3,7). Oral doses of SSZ are usually 500 mg twice
daily, but may be increased to 3 g per day depending
on response. However, only 2–13% of this oral dose
reaches the systemic circulation in unchanged form.
Thus, the highest SSZ plasma concentration found in
healthy volunteers ranges from 6 µg/mL to 32 μg/mL
(8). Many methods for the chromatographic analysis of
MTX have been published, and their relative merits have
been reviewed extensively by Rubino and colleagues
(7). Most of these published methods (50 out of 72) used
UV detection for the determination of MTX and the most
commonly used wavelength was between 303 and 313 nm
(in 42 out of 50 assays). Similarly, there are several studies
about the determination of SSZ and its metabolites for
clinical monitoring of its pharmacologic effects in different
biological matrices. Most of these analyses have been
carried out by high performance liquid chromatography
(HPLC) (8). However, to our knowledge, no methods
currently exist for the simultaneous determination of MTX
and SSZ.
Simultaneous Determination of Methotrexate and Sulphasalazine in Plasma by HPLC–DADSiji Joseph1, Sreelakshmy Menon1, and Smriti Khera2, 1Life Science Center, Agilent Technologies India Pvt. Ltd., Bangalore, India, 2Life Science and Diagnostics Group, Agilent Technologies, Inc., Santa Clara, California, USA.
Here we describe a simple and robust high performance liquid chromatography–diode array detection (HPLC–DAD) method for the simultaneous determination of methotrexate (MTX) and sulphasalazine (SSZ) from plasma. MTX and SSZ are used in combination for the treatment of rheumatoid arthritis. Using two detector wavelengths, 304 nm for MTX and 358 nm for SSZ, we were able to selectively quantitate both analytes during the same chromatographic run. The method was validated using quality control samples for critical analytical performance criteria of recovery, reproducibility, selectivity, accuracy, and precision.
KEY POINTS• A simple and robust high performance liquid
chromatography–diode array detection (HPLC–
DAD) method for the simultaneous determination of
methotrexate (MTX) and sulphasalazine (SSZ) from
plasma is described.
• The method is more sensitive, allows a wider
calibration range, and can be performed using a
much smaller (200 µL) volume of plasma sample than
previously reported UV methods for the bioanalysis of
MTX.
• The method was validated consistently with
FDA guidance, using quality control samples for
critical analytical performance criteria of recovery,
reproducibility, selectivity, accuracy, and precision.
Ph
oto
Cre
dit: C
ou
rte
sy o
f th
e A
uth
ors
LC•GC Europe May 2015272
ES610529_LCE0515_272.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
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ES610686_LCE0515_273_FP.pgs 04.30.2015 19:56 ADV blackyellowmagentacyan
Here, we describe a simple HPLC–diode-array detection
(DAD) method for the simultaneous determination of MTX
and SSZ in plasma. Using two detector wavelengths we
were able to selectively quantitate both analytes during the
same chromatographic run. The chromatographic method
was optimized for selectivity between the two analytes as
well as from background matrix interferences. We have
also compared two sample preparation strategies: off-line
solid-phase extraction (SPE) and protein precipitation.
Although the SPE approach allowed for more-sensitive
detection and a wider calibration range, protein
precipitation is also discussed in brief because of its
simple and inexpensive nature. The bioanalytical method
developed here was validated for the critical analytical
performance criteria recovery, reproducibility, linearity,
sensitivity, selectivity, accuracy, and precision, and those
results are also presented here.
Experimental SectionChemicals and Reagents: MTX, SSZ, methanol
(Chromasolv grade), blank human plasma, ammonium
hydroxide, formic acid, and ammonium acetate used in this
study were all obtained from Sigma-Aldrich India. Milli-Q
grade water (Millipore Corporation) was used throughout
the work. All chemicals used otherwise were of analytical
grade.
Instrumentation: Chromatographic analysis was carried
out using an Agilent 1290 HPLC–DAD system comprising a
binary pump, an autosampler equipped with a thermostat,
a thermostated column compartment, and a diode-array
detector equipped with a 60-mm Max-Light flow cell
(Agilent Technologies). It was operated with ChemStation
Openlab CDS Software Version C.01.05 (Agilent
Technologies).
Calibration Standards: Stock solutions of 2000-ng/µL
MTX and SSZ were prepared by dissolving each compound
in 100:0.1 (v/v) methanol–ammonium hydroxide. A 200-ng/
µL plasma stock solution of a combination of MTX and SSZ
was prepared by spiking the appropriate volume of each
stock solution into plasma. Serial dilutions from this plasma
stock solution were made using blank plasma to achieve
11 concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 5.0,
10.0, 50.0, and 100 ng/µL. Next, 200-µL aliquots of each of
these plasma calibration standard solutions were extracted
using protein precipitation or SPE as described below to
prepare the calibration standards.
Quality Control Samples: Three sets of quality control
(QC) samples were prepared by spiking stock solutions of
MTX and SSZ into plasma to yield 0.8-, 8.0-, and 80-ng/
µL concentrations to be used as low, middle, and high QC
samples, respectively. QC samples were analyzed in five
replicates each.
Extraction from Human Plasma: Two sample preparation
techniques were evaluated for the extraction of each
Table 1: Accuracy values for each linearity level of MTX and SSZ.
Concentration (ng/µL)Accuracy
MTX SSZ
0.02 85 NA
0.05 95.6 NA
0.1 102 107.1
0.2 101 90
0.5 101.2 86.4
1 99.7 104.5
5 101.4 88.8
10 105.2 89.1
50 106.3 94.4
100 98.4 101.6
NA = not available
H2N NH
2N
NN
N
N
N
O
O
OH
OHO
MTX
Formula
Mol. mass
Formula
Mol. massC
20H
22N
8O
5
C18
H14
N4O
5S
454.44 g/mol 398.39 g/mol
H
H
HO
OH
O
SSZ
N
N
NN
O O
S
Resolution: 3.9 Resolution: 2.5
Resolution: 0.9 Resolution: 2.5
MTX
MTX
SSZ
SSZ
5 6 7
654 7
Time (min)
Time (min)
2
1
0
-1
2
Ab
sorb
an
ce (
mA
U)
3.5
7
7.2
67.2
87.4
27.4
2
3.6
23.6
1
Ab
sorb
an
ce (
mA
U)
3.3
2
1
0
25
Ab
sorb
an
ce (
mA
U)
3.6
1
7.2
7
20
15
10
5
0
2 4 6 8 10 12 14
Time (min)
Figure 1: Structure of methotrexate (MTX) and sulphasalazine
(SSZ).
Figure 2: HPLC chromatogram of standard plasma solution
12.5 ng/µL of MTX and SSZ using SPE (top trace) and protein
precipitation (bottom trace) at 304 nm.
Figure 3: Elution profile of standard plasma solution of 2 ng/µL
of MTX (retention time = 3.6 min) and SSZ (7.2 min) using SPE
at 304 nm overlaid with blank plasma chromatogram.
LC•GC Europe May 2015274
Khera et al.
ES610551_LCE0515_274.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
calibration standard from plasma, namely, protein
precipitation and SPE. To perform protein precipitation on
a 200-µL plasma sample, double volume of 1:4 (v/v) 0.4 M
zinc sulphate–methanol solution was used. After adding
the precipitating reagent and vortexing for 45 s, the mixture
was centrifuged at 10,000 rpm for 5 min and the resulting
supernatant was used for liquid chromatography (LC)
analyses.
For SPE, 100-mg, 3-mL Bond Elut-C18 cartridges
(Agilent Technologies) were used for the extraction
of analytes from plasma. The SPE cartridges were
preconditioned with 3 × 1 mL of methanol–0.1% ammonium
hydroxide, followed by 3 mL of purified (Milli-Q, Millipore)
water. Then, 200 µL of each plasma standard solution
was loaded onto the SPE cartridge under gentle vacuum.
The cartridges were then washed three times with 1 mL
of mobile-phase A under vacuum for 5 min to near
dryness. The analytes were eluted from SPE cartridges
with 2 × 0.5 mL of methanol–0.1% ammonium hydroxide.
Eluted fractions were concentrated to dryness using a
concentrator (Eppendorf) and further reconstituted with
200 µL of 100:100:0.1 (v/v/v) methanol–water–ammonium
hydroxide.
HPLC–DAD Method: The separation was performed
using a 150 mm × 3.0 mm, 2.7-µm Poroshell Extend-C18
column (Agilent Technologies) that was maintained at 30 °C
using a column oven. Mobile-phase A was water–10 mM
ammonium acetate–0.1% formic acid and mobile-phase
B was methanol–10 mM ammonium acetate –0.1%
formic acid. The mobile phase was freshly prepared and
degassed before use. The chromatographic gradient
consisted of elution with 10–95–95–10% mobile-phase B
in mobile-phase A at 0–10–15–15.1 min at a flow rate of
0.8 mL/min. The column was equilibrated for 5 min using
the initial mobile-phase ratio before every injection. An
injection volume of 5 µL was used for all sample injections
and all injections were preceded with a 10-s needle wash
with methanol–0.1% ammonium hydroxide. The diode-array
detector monitored two UV wavelengths, 304 nm and
358 nm, that corresponded to the absorbance maxima of
MTX and SSZ, respectively.
Results and DiscussionOptimization of Plasma Extraction: The extraction
efficiency for protein precipitation versus SPE was
evaluated by comparing the area under the curve (AUC) of
the analyte peaks from the plasma calibration standards
with the AUC observed for standard solutions at a
concentration of 12.5 ng/µL. The extraction efficiencies
for MTX using protein precipitation and SPE were 17%
and 50%, respectively. The extraction efficiencies for
SSZ using protein precipitation versus SPE were 53%
and 78%, respectively. Thus, the extraction efficiency
proved to be significantly higher using SPE compared with
Table 2: QC sample results summarizing mean recoveries, accuracies, %CV of accuracies, retention time (Rt) RSD, and AUC RSD.
QCTarget
(ng/µL)
MTX (n = 5) SSZ (n = 5)
Mean
Recovery
(ng/µL)
Accuracy
(%)CV (%)
Rt RSD
(%)
AUC
RSD (%)
Mean
Recovery
(ng/µL)
Accuracy
(%)CV (%)
Rt RSD
(%)
AUC RSD
(%)
Low QC 0.8 0.70 87.7 1.02 0.03 0.26 0.82 102.2 0.86 0.09 0.34
Middle QC 8.0 8.1 101.1 0.26 0.03 0.25 7.5 94.2 0.28 0.02 0.41
High QC 80.0 75.5 94.3 0.29 0.03 0.29 78.1 97.6 0.29 0.02 0.29
4
3
2
1
0
4
3
2
1
0
2
Ab
sorb
an
ce (
mA
U)
Ab
sorb
an
ce (
mA
U)
3.6
5 M
TX
3.6
5
3 4 5
Time (min)
Rt of MTX
6 7
2 3 4 5
Time (min)
6 7
12
8
4
0
12
5 7
Rt of SSZ
9 11
5 7 9 11
Ab
sorb
an
ce (
mA
U)
SSZ
Ab
sorb
an
ce (
mA
U)
8
4
0
Time (min)
Time (min)
Figure 4: Elution profile of blank human plasma (top trace)
and plasma spiked with MTX at the LLOQ (bottom trace)
extracted with SPE.
Figure 5: Elution profile of blank human plasma (top trace)
and plasma spiked with SSZ at the LLOQ (bottom trace)
extracted with SPE.
275www.chromatographyonline.com
Khera et al.
ES610550_LCE0515_275.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
protein precipitation as expected. Additionally, a detailed
inspection of the chromatographic data over baseline
evidenced a better resolution of the analytes from matrix
interferences using SPE (Figure 2). A matrix peak (retention
time = 3.57 min) eluted just before MTX while using protein
precipitation was found to be completely eliminated with
SPE.
Therefore, SPE allowed better extraction efficiency, better
resolution, and thus more sensitive detection across a
wider calibration range. However, because of its simple and
inexpensive nature, protein precipitation may be used when
the sensitivity requirements of the bioanalytical method are
within the range observed here for protein precipitation.
Chromatographic Method Development: A C18
column was found highly retentive for the analytes, and
the best separation was achieved by using a gradient
of mobile-phase B in mobile-phase A as described
in the experimental section. The use of a low organic
mobile-phase concentration (10%) at the beginning of the
gradient resulted in good resolution of the analytes from
the initial polar matrix background. Also the late-eluted
analyte SSZ peak was well resolved from late-eluted matrix
interferences using a higher organic content (95%) at
the end of the gradient run. Within a run time of 15 min,
baseline separation of the target analytes from each other,
as well as from matrix peaks, was accomplished using
these chromatographic conditions (Figure 3).
Method Validation:
Method Selectivity: The demonstration of selectivity for
the analytes in the presence of matrix interferences
is of high interest for a bioanalytical method. In this
study, the selectivity of the method for the two analytes,
MTX and SSZ, during the chromatographic elution was
examined across the elution window at the lower limit
of quantification (LLOQ). The LLOQ was established as
the lowest concentration on the calibration curve. At the
LLOQ, the analyte response was at least five times higher
compared to the blank response. Figures 4 and 5 provide
chromatograms of MTX and SSZ calibration standards at
the LLOQ, respectively. Minor interferences were observed;
however, these did not impact peak integration and
quantitation of analytes at LLOQ.
Method Linearity: A study of method linearity was
performed by constructing calibration curves consistent
with the Food and Drug Administration’s (FDA) draft
guidance for bioanalytical method validation (11) across
several concentration levels including the LLOQ and
upper limit of quantification (ULOQ) in five replicates.
Calibration curves were constructed for peak area
versus concentration and it was observed that the area
response was linearly and correctly regressed over a wide
concentration range. The linear dynamic range for the
current bioanalytical method is 0.02–100 ng/µL for MTX
and 0.1–100 ng/µL for SSZ. The coefficient of correlation
(R2) was greater than 0.998 in each case. The calibration
curves for both analytes are shown in Figure 6. Observed
accuracy values for each linearity level for MTX and SSZ
are summarized in Table 1. The accuracy of the method at
the LLOQ was 85% for MTX and 107.1% for SSZ. Consistent
with the FDA guidelines, the observed precision standard
deviation at LLOQ was below 20% and for all other
concentration levels including the ULOQ, the precision
standard deviation observed was below 15%.
Limits of Detection and LLOQ: The limits of detection
(LOD) and LLOQ were determined at signal-to-noise ratios
(S/N) at or greater than 3 and 10 for both MTX and SSZ,
respectively. Thus, the LOD and LLOQ for MTX were 0.01
and 0.02 ng/µL with S/N values of 6 and 10, respectively.
For SSZ the LOD and LLOQ were 0.05 and 0.1 ng/µL
with S/N values of 8 and 18, respectively. Figure 7 shows
a typical chromatogram for MTX at the LLOQ overlaid
with blank traces in replicates. The chromatographic
reproducibility at the LLOQ was verified by replicate
injections. The percent of the coefficient of variation (%CV)
of AUC and retention time at the LLOQ were 0.02% and
1.46%, respectively, for MTX, and 0.01% and 0.79%,
respectively, for SSZ.
Analyte Recovery: Analyte recovery was measured
consistent with the FDA guidance for bioanalytical method
validation (12). Thus, three sets of QC samples for each
analyte were analyzed in five replicates. The three sets
of QC samples were selected in such a way that the
concentration range covers the lower, mid, and high region
of the calibration curve. The analyte recoveries from QC
samples were calculated using linearity equations of each
of the analytes. These results were then compared with
theoretical concentrations. The resultant mean recoveries
and %CV are given in Table 2.
Method Reproducibility: The reproducibility of the method
MTX
y = 107.33x +41.177R2 = 0.9986
y = 100.35x -119.77R2 = 0.9987
MTX, Range: 0.02–100 ng/µL
0.02-1ng/µL
0.1-1ng/µL
150
100
1200
1000
8000
60004000
20000
50
Ab
sorb
an
ce (
mA
U)
Ab
sorb
an
ce (
mA
U)
120010008000600040002000
-20000
Ab
sorb
an
ce (
mA
U)
Ab
sorb
an
ce (
mA
U)
0.5
SSZ
0
100
50
0
0
0 0.2 0.4 0.6 0.8 1
0 20 40 60 80 100
1Concentration (ng/µL)
Concentration (ng/µL)
Concentration (ng/µL)
0 20 40 60 80 100Concentration (ng/µL)
SSZ, Range: 0.1–100 ng/µL
1.5
0.5
Ab
sorb
an
ce (
mA
U)
0
3 3.2 3.4 3.6 3.8 4 4.2
Time (min)
3.6
5
1
Figure 6: Linearity curves for MTX (top) and SSZ (bottom). A
zoomed-in view for lower linearity levels is also shown.
Figure 7: Chromatogram for MTX at the LLOQ overlaid with
blank traces in duplicates.
LC•GC Europe May 2015276
Khera et al.
ES610552_LCE0515_276.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
was determined in accordance with the FDA draft guidance
for bioanalytical method validation (11) by measuring the
accuracy and precision of the method across three QC
samples in the calibration range and using five replicates
at each concentration. The results for intraday precision
and accuracy in plasma quality control samples for MTX
and SSZ are summarized in Table 2. The calculated relative
standard deviation for retention time and area was found
to be within 0.1% and 0.4%, respectively. These results
promise high reproducibility of the method.
ConclusionsA combination regimen of low-dose MTX and high-dose
SSZ is frequently used to combat rheumatoid arthritis.
Therefore, the simultaneous monitoring of plasma levels
of MTX and SSZ has clinical relevance, and also may
aid further pharmacokinetic research into any synergistic
effects. Here, we described a simple, sensitive, and robust
HPLC–DAD method for the simultaneous determination
of MTX and SSZ in plasma. The method is linear within
biologically relevant concentration ranges of 0.02–100 ng/µL
(0.04–220 µM) for MTX and 0.1–100 ng/µL (0.25–251 µM)
for SSZ with a R2 coefficient greater than 0.998 for each.
The method has an LLOQ of 0.02 ng/µL for MTX and an
LLOQ of 0.1 ng/µL for SSZ. In addition, this method to our
knowledge is more sensitive, allows a wider calibration
range, and can be performed using a much smaller
(200 µL) volume of plasma sample than previously reported
UV methods for the bioanalysis of MTX. The method was
validated consistently with FDA guidance, using quality
control samples for critical analytical performance criteria
of recovery, reproducibility, selectivity, accuracy, and
precision.
References(1) J.R. O’Dell, R. Leff, G. Paulsen, C. Haire, J. Mallek, P.J. Eckhoff,
A. Fernandez, K. Blakely, S. Wees, J. Stoner, S. Hadley, J. Felt, W.
Palmer, P. Waytz, M. Churchill, L. Klassen, and G. Moore, Arthritis
Rheum. 46, 1164–1170 (2002).
(2) W. Katchamart, J. Trudeau, and V. Phumethum, Ann. Rheum. Dis.
68, 1105–1112 (2009).
(3) F.M.P. Meier, M. Frerix, W. Hermann, and U. Müller-Ladner,
Immunotherapy 5, 955–974 (2013).
(4) S. Sadray, S. Rezaee, and S. Rezakhah, J. Chromatogr. B 787,
293–302 (2003).
(5) H.A. Capell, R. Madhok, D.R. Porter, R.A. L. Munro, I.B. McInnes,
J.A. Hunter, M. Steven, A. Zoma, E. Morrison, M. Sambrook, F.W.
Poon, R. Hampson, F. McDonald, A. Tierney, N. Henderson, and I.
Ford, Ann. Rheum. Dis. 66, 235–241 (2007).
(6) H.M. James, D. Gillis , P. Hissaria, S. Lester, A.A. Somogyi, L.G.
Cleland, and S.M. Proudman, J. Rheumatol. 35, 562–571 (2008).
(7) F.M. Rubino, J. Chromatogr. B 764, 217–254 (2001).
(8) N. Pastor-Navarro, E. Gallego-Iglesias, A. Maquieira, and R.
Puchades, Anal. Chim. Acta 583, 377–383 (2007).
(9) M.S. Elmasry, I.S. Blagbrough, M.G. Rowan, H.M. Saleh, A.A. Kheir,
and P.J. Rogers, J. Pharmaceut. Biomed. Anal. 54, 646–652 (2011).
(10) U. Klotz, Clin. Pharmacokinet. 10, 285–302 (1985).
(11) US Food and Drug Administration, Guidance for Industry:
Bioanalytical Method Validation, Bio Pharmaceutics Revision 1,
(U.S. Department of Health and Human Services, Food and Drug
Administration; Center for Drug Evaluation and Research [CDER],
Center for Veterinary Medicine [CVM], Rockville, Maryland, USA,
2013).
Siji Joseph and Sreelakshmy Menon are with the Life Science
Center at Agilent Technologies India Pvt. Ltd., in Bangalore, India.
Smriti Khera is with the Life Science and Diagnostics Group at
Agilent Technologies, Inc., in Santa Clara, California, USA. Direct
correspondence to: Smriti_khera@agilent.com
277www.chromatographyonline.com
Khera et al.
ES610553_LCE0515_277.pgs 04.30.2015 13:22 ADV blackyellowmagentacyan
LC•GC Europe May 2015278
LC TROUBLESHOOTING
Recently, I received an inquiry from
a reader regarding a problem he
encountered with a routine liquid
chromatography (LC) method in his
clinical laboratory. He had prepared
a fresh calibration standard (check
sample) for the analyte of interest
(I’ll call it “X” to keep the reader’s
laboratory anonymous), yet when he
assayed a blank sample spiked with
160 ppm of X, he found an indicated
400 ppm. This was puzzling and not
a problem normally encountered,
so he sent the sample to another
laboratory that was analyzing the same
compound by gas chromatography
(GC), and their results showed that
the spiked sample indeed contained
160 ppm of X. At this point he
contacted me to help figure out what
was happening. As we look at possible
causes for and solutions to this
problem, we can use this as a specific
example to which we can apply
general troubleshooting principles.
BackgroundBefore we get further, let’s take a look
at the method, which is designed for
the analysis of X in serum. Samples
are prepared by taking an aliquot of
serum, adding an aliquot of internal
standard (IS), and a small amount
of hydrochloric acid to acidify it. The
solution is vortexed to mix, then an
aliquot of dichloromethane is added,
the solution is vortexed again, and
then centrifuged to separate the two
phases. The dichloromethane phase
is removed, evaporated to dryness,
and reconstituted in the injection
solvent. The separation conditions
comprise a reversed-phase column
(size, stationary phase, and flow
rate were not mentioned) with an
isocratic mobile phase of acetonitrile,
water, and trifluoroacetic acid.
Ultraviolet (UV) detection is used.
The chromatographic conditions give
typical retention times of 9 min (IS)
and 12 min (X), and the chromatogram
is normally free of any other peaks.
Calibration standards are prepared
by spiking a stock solution of X into
serum at 40, 120, and 160 ppm; these
spiked calibrators are then extracted
in the same manner as samples. A
three-point calibration curve is run
and if the regression is acceptable,
this calibration curve is used for three
months. With each batch of samples, a
single injection of blank serum spiked
to 160 ppm is made as a system
suitability test; if this check sample
assays at 160 ppm, the system is
deemed stable and samples are run.
The method had been running
acceptably until he ran out of the
1000 ppm stock of X used for spiking
the check sample. When the new
stock was prepared, the problem of
a 400 ppm assay for the 160 ppm
sample appeared.
Consider the PossibilitiesIn a case like this, I like to divide the
issue up into several possible problem
areas, then see how many of these
possibilities I can eliminate with the
data at hand. This helps to focus my
attention on the source of the problem
so that it can be investigated further,
if necessary, and corrected. We can
broadly, and somewhat arbitrarily,
divide the possible problem areas into
chemistry, hardware, sample-related,
and calibration. Let’s look at each of
these in more detail.
Chemistry: By chemistry, here I
mean the chromatographically related
chemical influences. These are the
nature of the sample, the column,
the mobile phase, and the column
temperature. We can quickly eliminate
these as the likely sources of the
problem. If the column chemistry,
mobile-phase chemistry, or column
temperature had changed, we would
expect a shift in retention for X and
the IS, but this was not observed. The
sample chemistry, or identity, is unlikely
to have changed, because the check
sample had no apparent retention
problems in either the LC or GC assay.
Hardware: LC system hardware
could malfunction in terms of flow
rate, injection problems, or detection.
The flow rate must be correct or the
retention times would shift for both
X and the IS. It is possible that the
autosampler is not working properly,
but this is unlikely to cause the
noted problem, because any volume
error in the autosampler would be
compensated by the use of the IS. The
purpose of the IS is to add it early in
the sample preparation process so that
any loss of sample volume or injection
error would not matter, because it
is the ratio of X/IS that is used in the
calibration process, not the absolute
response of either compound.
Problems related to the detector are
a possible source of error, and should
be checked. Two obvious possibilities
are that the wrong wavelength was
selected or that there is something
wrong with the detector lamp. The
response of X and the IS would be
Calibration Problems — A Case StudyJohn W. Dolan, LC Troubleshooting Editor.
Unexpected results from calibration standards create confusion in a clinical liquid chromatography (LC) method.
ES610496_LCE0515_278.pgs 04.30.2015 13:20 ADV blackyellowmagentacyan
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LC•GC Europe May 2015280
LC TROUBLESHOOTING
expected to change if the detection
wavelength was changed, and a
change in the relative response of
X and the IS would be likely. This
would generate a different X/IS ratio
for a given concentration, which in
turn would change the assay value
for X in the check sample. A change
in lamp energy as the detector lamp
aged could also cause a change in
response, and although I would expect
that such intensity would affect X and
the IS similarly, that is not a certainty.
The proper wavelength should be
verified and the lamp energy should
be compared to normal values to
determine if either of these items could
be the problem source.
Sample-Related Problems: We know
that the identity of the sample is correct
and that the standard was made at
the proper concentration because the
sample assayed at 160 ppm by GC.
The reader did not state if a new batch
of IS stock was made at the same time,
but if we consider the method, either
the new batch of IS was made correctly
or the old one was still good and was
used. The check sample is made by
spiking serum, and serum would never
be injected directly, so it follows that
the check sample was spiked with IS
and extracted in the normal manner.
One of the reasons for adding IS is to
account for the inevitable changes in
sample volume that take place during
sample preparation.
Let’s review the sample cleanup
procedure: 300 µL of serum is
combined with 50 µL of IS and 200 µL
of dilute hydrochloric acid (550 µL
total), centrifuged, and extracted
with 600 µL of dichloromethane.
All of X and the IS should transfer
into the dichloromethane, so the
concentration of X and IS is 550/600
of its concentration in the original
diluted serum. Next, 400 µL of
the dichloromethane is removed,
evaporated to dryness, and
reconstituted in 50 µL of methanol.
This concentrates the dichloromethane
extract by 400/50 or eightfold. With
the extraction, evaporation, and
reconstitution steps, there will be
inevitable volumetric errors introduced,
which is why the IS is added — the
same losses of X and IS should occur,
so the X/IS ratio should stay constant.
All this leads me to conclude that the
GC method would be very unlikely to
give an assay value of 160 ppm of X
by an external standard method, even
if the results were adjusted for the
theoretical changes in concentration.
Instead, I conclude that the IS method
was used for GC, as well, and because
the assay was as expected, it tells
me that the check sample was made
correctly, even though it doesn’t assay
properly by the LC method. The bottom
line here is that it is unlikely that the
current problem lies with the sample or
sample preparation.
Calibration: At this point we’ve
eliminated chemistry problems,
hardware problems (assuming the
detector wavelength is set correctly
and the detector lamp is in acceptable
condition), and sample-related
problems. This leaves calibration
problems as the most likely problem
source (assuming that we haven’t
overlooked something else obvious,
which is always a possibility).
My initial interaction with the reader
simply indicated that the check sample
did not assay correctly by LC, but gave
the expected answer by GC. When
I requested more information about
the method, I learned of the practice
of calibrating every three months and
using the system suitability check
sample to verify that the method
was working properly. Although the
rules are a bit different in the clinical
laboratory industry, this goes strongly
against the analysis of the same drugs
in serum or plasma to support drug
development in the pharmaceutical
industry. The latter techniques fall
under guidelines from the United
States Food and Drug Administration
(FDA). The FDA’s “Guidance for
Industry: Bioanalytical Method
Validation” (1) discusses validation
of methods for the analysis of small
molecular weight drugs in plasma
and other tissues (generally called
“bioanalytical” methods, as opposed to
methods for the analysis of biological
compounds). In this document in the
section titled “Application Of Validated
Method To Routine Drug Analysis”
(pp. 13–14), it is stated:
A calibration curve should be
generated for each analyte to
assay samples in each analytical
run and should be used to
calculate the concentration of the
analyte in the unknown samples
in the run . . . . The calibration
(standard) curve should cover
the expected unknown sample
concentration range in addition
to a calibrator sample at LLOQ
[lower limit of quantification].
It goes on to say:
Once the analytical method has
been validated for routine use, its
accuracy and precision should
be monitored regularly to ensure
that the method continues to
perform satisfactorily. To achieve
this objective, a number of
QC [quality control] samples
prepared separately should be
analyzed with processed test
samples at intervals based on
the total number of samples. . . .
The QC samples in duplicate at
three concentrations . . .
Additionally, it is noted:
A matrix-based standard curve
should consist of a minimum of
six standard points, excluding
blanks (either single or replicate),
covering the entire range.
This says that the calibration curve
should be run with each batch of
samples, not once every three months.
The calibration curve should cover the
expected sample concentration range,
and include the LLOQ. Furthermore,
QC samples should be run at three
concentrations that fall within the
range of sample concentrations. These
guidelines also make good sense from
an analytical chemistry standpoint.
There are just too many potential
problems that can occur which might
cause the calibration curve to be
different on different days. I have
been involved with research and
development (R&D) studies where
the reference standards were so rare
and valuable that it was not possible
to run them every day, but a surrogate
standard was found to verify that the
original calibration was still adequate.
That may seem to align with the current
problem, but in fact the drug X and
its IS are very common compounds
that can be purchased in reference
standard grade for reasonable prices,
so it is hard to justify trimonthly
calibration on economic grounds.
The fact that the check sample was
formulated at 160 ppm and verified
by GC underlines the probability that
ES610495_LCE0515_280.pgs 04.30.2015 13:20 ADV blackmagentacyan
www.chromatographyonline.com
batch by about 2.5 h. It may be very
easy to compensate for this increase
in run time by increasing the flow rate;
with an isocratic run, the separation
should not be affected by the flow rate.
The pressure would rise in proportion
to the increase in flow rate, but it is
fairly rare with conventional LC runs
that pressure is a limiting condition, so
the added pressure is unlikely to be an
issue.
ConclusionsWe have used a specific example of
a method problem to illustrate how to
break down the problem into several
potential problem sources. Most of
these sources could be eliminated by
careful consideration of the method
and how the results deviated from
the expected ones. This left us with
two likely problem sources. First, a
problem with the detector wavelength
setting or detector lamp energy.
These could be quickly checked by
examining the instrument. The second
potential problem source was that the
instrument response to X or the IS had
drifted between the time the original
calibration curve was run and the
problem was noted.
The recommended solution was
to first check for detector problems,
and second rerun the calibration
curve. A more permanent fix to the
problem would be to change the
method to comply better with current
FDA guidelines and general analytical
chemistry practices of running
calibrators contemporaneously with
samples.
References(1) United Stated Food and Drug
Administration, Guidance for Industry:
Bioanalytical Method Validation (FDA,
Rockville, Maryland, USA, 2001).
“LC Troubleshooting” Editor John
Dolan has been writing “LC
Troubleshooting” for LCGC for more
than 30 years. One of the industry’s
most respected professionals, John is
currently the Vice President of and a
principal instructor for LC Resources
in Lafayette, California, USA. He is
also a member of LCGC Europe’s
editorial advisory board. Direct
correspondence about this column via
e-mail to John.Dolan@LCResources.
com. To contact the editor-in-chief,
Alasdair Matheson, please e-mail:
amatheson@advanstar.com
the source of the problem lies with the
calibration curve. My best guess is that
something in the LC system has drifted
over time, most likely the detector
response (or an improper wavelength
setting), and has caused the current
response to the X/IS ratio to be much
larger than it was when the calibration
curve was run originally.
What Now?I recommend that the proper
wavelength setting and detector
lamp performance be verified before
proceeding. After these are found to
be satisfactory, I would generate a
new calibration curve using freshly
prepared standards of X spiked into
blank serum and extracted normally. I
believe that the check sample will now
assay correctly, closing the loop on
identifying the problem source.
Technically, the check sample has
done exactly what it was intended
for — it has alerted the operator to a
problem with the assay before valuable
patient samples were run. However, I
would modify the method to comply
more with the industry standard of
the FDA guidelines (1). This would
require running a calibration curve,
containing samples with at least six
concentrations, each day with each
batch of samples run. In addition, a
set of check samples, or QCs, should
be prepared and included in each
sample batch to show that during
the analysis, the method gives the
expected results for samples of known
concentration. There will be some
documentation required to make these
changes, but the method reliability
will be much improved and should
justify this extra work. The quality of the
results produced should improve as
well. Finally, should the laboratory be
audited by a regulatory agency, there
will be much less likelihood of negative
findings by the auditors.
In terms of day-to-day added work,
there should be only a small impact on
the total batch run time for a potentially
large improvement in data quality. The
calibration and check samples can be
quickly spiked with known amounts
of X and extracted with QC samples
and samples to be analyzed. A total
of six calibrators and six QC samples
(duplicates at three concentrations)
would add 12 samples to the day’s
run. At a 12-min retention time for X,
this would increase the run time for the
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LC•GC Europe May 2015282
GC CONNECTIONS
From 8–12 March 2015 the Pittsburgh
Conference on Analytical Chemistry
and Applied Spectroscopy (Pittcon)
returned to the Morial Convention
Center in New Orleans, Louisiana,
USA, for its 66th annual meeting.
This was the first time since 2008
that the conference had taken
place in New Orleans, and the city
welcomed the 14,272 registered
attendees with open arms, brass
bands, and beignets. There were 919
exhibitors in 1690 booths, and this
year 90 countries were represented.
Although I did not attend many of
the technical sessions, they were
of as high a quality and as well
attended as in previous years. Of
particular interest to the readers
of LCGC was the half-day session
devoted to the presentation of the
2015 LCGC Lifetime Achievement
in Chromatography Award to Jack
Kirkland (Advanced Materials
Technology) and the LCGC Emerging
Leader Award in Chromatography to
Caroline West (Université d’Orléans,
France). Please see the February
2015 issue of LCGC North America
(1) for more information about this
year’s awards.
Pittcon will head to Atlanta in
2016, where conferees will enjoy a
second consecutive year of Southern
hospitality. In 2017, the conference
returns to Chicago.
This annual instalment reviews
gas chromatography (GC)
instrumentation, columns, and
accessories shown at this year’s
Pittcon or introduced during the
previous year. For a review of
new products in other areas of
chromatography, columns, and
related accessories, please see the
additional coverage in the April issue
as well as this issue of LCGC Europe
(2–4), which are also available
on-line at LCGC ’s website.
The information presented here is
based on manufacturers’ replies to
questionnaires, as well as additional
information from manufacturers’
press releases, websites, and
product literature about the past
year’s products, and not on actual
use or experience of the author.
During Pittcon, I took time to stroll
around the convention aisles and see
some of the new products firsthand
as well as discover a number of
items that weren’t covered by the
questionnaires. Every effort has
been made to collect accurate
information, but because of the
preliminary nature of some of the
material, LCGC Europe cannot be
responsible for errors or omissions.
This column instalment cannot be
considered to be a complete record
of all new GC products introduced
this year at Pittcon or elsewhere
because not all manufacturers chose
to respond to the questionnaire or
attend the conference, nor is all of
the submitted information necessarily
included here because of the limited
available space and the editors’
judgment as to its suitability.
Gas Chromatography in 2014–2015
Gas chromatography again
displayed renewed vigour in the past
year, which certainly was evident
at the 2015 Pittcon conference.
Comprehensive GC×GC continues
to yield significant advances, in
particular when combined with mass
spectrometry (MS) detection. MS
detectors for GC alone experienced
no fewer than six new or enhanced
New Gas Chromatography Products, 2014–2015 John V. Hinshaw, GC Connections Editor.
In this instalment, John Hinshaw reviews gas chromatography (GC) instruments, columns, and
accessories that were newly on display at the Pittsburgh Conference in New Orleans, Louisiana, USA,
during March 2015, or were introduced to the marketplace in the preceding year.
Table 1: Companies introducing new
GC products.
Company Name
Agilent Technologies
AFP
Baseline Mocon
DANI
Defiant Technologies
Gow-Mac
Ionicon
JEOL
LECO
Phemonenex
Qmicro
Restek
SGE
Shimadzu
Thermo Scientific
VICI
New Orleans welcomed the 14,272 registered attendees with open arms, brass bands, and beignets. There were 919 exhibitors in 1690 booths, and this year 90 countries were represented.
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is about to change. An exciting new chapter in GC-MS is about to open, with the superior resolving
power, mass accuracy and sensitivity that only Thermo Scientifc™ Orbitrap™ technology can deliver.
in GC-MS
A new chapter
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LC•GC Europe May 2015284
GC CONNECTIONS
Table 2: New GC instruments.
Product Company Description
7010 Triple-Quad
GC–MS system
with improved EI
source
Agilent Agilent’s 7010 triple-quadrupole GC–MS system features a redesigned high-efficiency electron
ionization (EI) source that enables attogram detection limits. The system’s new EI source creates
more than 20 times as many ions as the current generation of EI sources, according to Agilent,
to deliver an instrument detection limit (IDL) of 0.5 fg OFN. The new EI source technology is also
available to current and future 7000 C owners in the form of an upgrade package. Specifications:
mode of operation: EI standard, CI optional; ion source temperature: 150–350 °C; dual filaments
for EI; electron energy: 10–300 eV; mass filters: proprietary monolithic hyperbolic gold-coated
quadrupole; mass axis stability: less than ± 0.10 u over 24 h (10–40 °C); quadrupole temperature:
106–200 °C; mass range: m/z 10–1050; resolution: selectable, 0.4–4.0 Da, custom tune; scan rate:
up to 6250 u/s; detector: triple-Axis HED-EM with extended-life EM and dynamically ramped-iris;
MRM speed: 800 transitions/s; minimum MRM dwell: 0.5 ms; collision cell: linear hexapole; collision
cell gas: nitrogen with helium quench gas; collision energy: selectable up to 60 eV.
7200B Q-TOF
GC–MS system
Agilent The Agilent 7200B Series Q-TOF GC–MS system with Agilent MassHunter software replaces the
company’s 7200A offering and provides an improved mass accuracy specification of less than
3 ppm over an extended mass range, with acquisition rates up to 50 Hz. The system must be
combined with the high performance Agilent 7890B gas chromatograph. 7200B specifications:
EI (high sensitivity extraction source), PCI, and NCI ionization mode as standard; ion source
temperatures: 106–350 °C; electron energy: 10–200 eV; removable ion source without breaking
vacuum through an isolation valve; dual filaments for EI source, single filament for CI source; quad
isolation mass range (m/z) 20–1050; resolution (full width at half height) settable from 0.4 to 4.0 Da;
dynamic range (electronic) greater than 105; quadrupole mass axis stability less than ±0.10 Da
over 24 h (10−40 °C); quadrupole temperature: 100–200 °C; collision cell: linear hexapole, nitrogen
collision cell gas; collision energy: selectable up to 60 eV; ion extraction and mirror: two-stage
second-order corrected; TOF flight pathlength: 2 m; microchannel plate/scintillator/PMT detector;
TOF mass range (m/z): 25−1700, extended 15−3000; TOF detector sampling rate ADC: 32 Gbits/s;
autotune or manual tuning; spectra acquisition rate: 1−50 spectra/s; EI instrument detection limit of
240 fg or less of OFN.
AccuTOF-GCx
time-of-flight
GC–MS system
JEOL The fourth-generation AccuTOF-GCx features high sensitivity (S/N > 300 at OFN 1 pg/µL) and
offers improved resolution, accuracy, and sensitivity, while retaining the power and flexibility of
the company’s previous models. The GCx offers both powerful chromatographic separation and
high-resolution mass spectra in combination with comprehensive 2D GC (GC×GC) using the Zoex
thermal modulator. Specifications: mass resolution: 10,000 (FWHM); mass range: 4–6000 (m/z);
data acquisition speed: up to 4 GS/s; spectrum acquisition speed: up to 16,000 spectra/s; spectrum
recording speed: up to 50 spectra/s; sensitivity: 1 pg octafluoronaphthalene (OFN) S/N ≥300.
AQMAlert Ozone
Precursor system
Baseline
Mocon
The company’s AQMAlert multiple-GC field system combines two Series 9100 GC systems with a
Series 9300 Preconcentrator into a photochemical assessment monitoring station (PAMS). The first
GC system uses flame ionization detection (FID) for light hydrocarbon detection; the second GC
system incorporates photoionization detection (PID) for the remaining components. The optional
preconcentrator is a dual-tube desorption system that allows lower detection limits.
Gas
chromatography
cartridge
QMicro The new Qmicro gas chromatography cartridge is based on an innovative micro gas analysis
platform with integrated injector and thermal conductivity detection systems plus columns,
backflush to detector, and temperature programming, all packaged in a small palm-size
oven. Backflush enables protection of sensitive columns by minimizing exposure to harmful
gas components — such as carbon dioxide and water on a molecular sieve 5A column —
thus increasing lifetime. Backflush to detector functionality allows quantification of the total
backflushed sample peak. This enables fast analysis of total C6+ or C9+ content of a natural
gas. The cartridge is based on silicon chips made by MEMS microtechnology and micro
assembly technologies, for virtual zero dead volumes and microscopic small flow channels.
The cartridge is intended for OEM partners and system integrators to integrate micro GC
technology for fast, small, and reliable analyses into instruments and systems.
GCMS-TQ8040 Shimadzu Shimadzu’s GCMS-TQ8040 triple-quadrupole GC–MS system includes the following features:
Smart MRM (multiple reaction monitoring), which can combine over 400 compounds into a single
MRM method without losses in sensitivity or selectivity; MRM analysis at up to 800 transitions/s;
high-speed scanning control at 20,000 u/s; an MRM optimization tool that automatically determines
optimum transitions and collision energies for all compounds in a single sequence; an off-axis
design that eliminates neutral noise; UFsweeper technology that accelerates ions out of the
collision cell to eliminate crosstalk; an automatic adjustment of retention times (AART) function
that updates retention times in both the acquisition and data processing methods after column
maintenance, without changing chromatographic conditions or requiring multiple injections of
standards. The system also has Shimadzu’s Smart Database Series software to create MRM and
scan-MRM methods automatically, and a scan-MRM mode that simultaneously acquires accurate
library-searchable mass spectra and low-level MRM quantitation in a single analysis.
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LC•GC Europe May 2015286
GC CONNECTIONS
(EI) source. Both of these offerings
work with the company’s gas
chromatographs. Also in the GC–MS
area, the AccuTOF-GCx TOF GC–
MS system from JEOL works with
comprehensive two dimensional (2D)
GC to make a powerful GC×GC–MS
analyzer. LECO displayed its newest
GC×GC–MS system, the Pegasus
GC-HRT 4D. Also coming in with a
new product in this area, Shimadzu
introduced the GCMS-TQ8040
product introductions. Advances
in fast mini- and micro-sized GC
systems were evident, too, as well as
a nice assortment of valves, fittings,
syringes, gas accessories, and
columns.
In the instrument system area,
Agilent Technologies introduced
the 7200B quadrupole time-of-flight
(QTOF) GC–MS system plus the
7010 Triple-Quad GC–MS system
with an improved electron ionization
triple-quadrupole GC–MS system.
Finally, Thermo Scientific had
their TSQ Duo triple-quadrupole
GC–MS–MS system on-hand.
Please see Table 2 for the details
and specifications supplied by the
manufacturers for these high-end
hyphenated GC instruments.
The AQMAlert Ozone Precursor
system from Baseline Mocon
combines two of the company’s
model 9100 GC systems and
Table 2: New GC instruments (continued).
Product Company Description
Pegasus
GC-HRT 4D
LECO LECO’s newest GC×GC–MS system combines the company’s Pegasus GC×GC system
with high resolution TOF MS and ChromaTOF-HRT software, which uses high-resolution
deconvolution (HRD) for component detection, National Institute of Standards and
Technology (NIST), and Accurate Mass Library searches, pseudomolecular ions (via
chemical ionization), retention time matching, isotope patterns, and mass accuracy of
deconvoluted fragments within a complete package for data acquisition, processing, and
reporting. According to LECO, the system can produce resolution up to 50,000 FWHM,
mass accuracies less than 1 ppm, and acquisition rates up to 200 spectra/s.
Series 8100
Programmable
GC system
Gow-Mac Gow-Mac’s new Series 8100 GC system is a custom, application-specific system
configurable for research, industrial, laboratory, academic, and quality assurance
(QA) and quality control (QC) environments. The system accommodates up to two
independently controlled detectors that can be operated either individually, in series, or
in parallel depending on the ordered configuration. Detection currently available includes
TCD and FID. Features of the instrument include an ambient plus 5 °C to 450 °C operating
temperature; independently programmed and controlled temperatures at injection
ports, detectors, and column oven; the column oven accommodates up to five packed,
wide-bore capillary, or capillary columns; a column oven temperature programming rate
of 0.1 °C to 40 °C /min in 1 °C increments; an oven cooling rate of 350 °C to 75 °C in
5 min; method storage of three internal methods and an infinite number external methods;
manual differential flow controllers or pressure regulators (detector dependent); and
direct on-column (direct packed–capillary or split–splitless) or gas sample valve injection
methods; and a full array of optional output capabilities that include analog outputs of
0–1 V, 0–1 mV, and 0–10 V VDC, or digital outputs to RS-232, USB, and ethernet utilizing
MODBUS and PROFINET (read only) communication protocols.
TOCAM Defiant
Technologies
The TOCAM miniature GC-based toxic organic chemical monitor for airborne volatile
organic compounds (VOC) includes a microconcentrator, micro-GC column, and two
miniature photoionization detectors for rapid screening as well as detailed analysis of
trapped and desorbed compounds of interest. The first detector responds directly to
desorbed compounds. High levels can trigger a detailed GC analysis onto the second
detector. The portable or mountable instrument features a 10.6-eV detector lamp, a 2.5-m
or 4.6-m GC column, and operates from a 9–2 VDC AC wall adapter.
TSQ Duo
triple-quadrupole
GC–MS–MS
system
Thermo
Scientific
The Thermo Scientific TSQ Duo triple-quadrupole GC–MS–MS bridges from
single-quadrupole full scan and SIM methods to the high selectivity and sensitivity of
SRM (selected ion monitoring) methods. The system operates in both single and triple
quadrupole modes with automatic selected reaction monitoring (AutoSRM) software for
method development and optimization. A selected ion monitoring bridge method migration
tool easily and accurately migrates existing methods to either SIM or SRM methods.
The system uses Thermo Scientific Dionex Chromeleon chromatography data system
software. Other system specifications include a mass analyzer with quadrupole scanning
up to 20,000 u/s; heated, off-axis ion guide for noise reduction and solid, homogeneous,
noncoated, maintenance-free quadrupole rods; automatic tuning down to 0.4 u; selectable
SRM resolution settings in method at autotune value, 0.7, 1.5, and 2.5 u; Thermo Scientific
DynaMax XR detection system, with off-axis 10 kV dynode, discrete dynode electron
multiplier and electrometer, linear range of greater than 107 (0–68 μA); a collision energy
range of 0–60 eV; a mass range of 1.2–1100 u; and scanning capabilities of up to
20,000 u/s with the ability to acquire more than 97 scans/s in FS when scanning over a
range of 125 µ; 1.0 ms minimum SRM dwell times, and up to 300 SRM transitions/s.
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www.chromatographyonline.com
a preconcentrator into a photochemical assessment
monitoring station. From Gow-Mac, the Series 8100
programmable GC system represents a new customizable
application-specific laboratory system with multiple inlets,
detectors, and other options. In miniature and micro GC
systems, Defiant Technologies showed the TOCAM toxic
organic chemical monitor, based on a microconcentrator
and micro-GC column for rapid screening and more
detailed analyses. A new entry to micro-GC, QMicro brought
examples of its GC cartridge with integrated sampling valve,
detector, and backflushing options, to be made available to
original equipment manufacturers (OEMs).
Table 3 lists new GC accessories such as autosamplers,
detectors, and more. From DANI, the Peakblade 77
GC×GC modulator is a liquid-nitrogen-free device with
rapid and programmable thermal modulation. Two fast GC
accessories were shown at Pittcon: the fast GC conversion
kit from VICI that integrates the company’s resistively heated
columns and controller with Agilent GC systems, and a fast
GC add-on for Ionicon’s PTR-TOF gas analyzer. Analytical
Flow Products (AFP) introduced a modular multipurpose
valve oven, a miniature version of the company’s multiport
valve, and a new design for zero-dead-volume fittings.
Shimadzu introduced two accessory products: the
AOC-6000 autosampler, which automates calibration
sample preparation, and the ECD-2010 Exceed
electron-capture detector. SGE introduced a new version of
its Diamond headspace syringe line. The EZGC Software
Suite Online from Restek has been expanded with some
additional calculation and translation capabilities.
Only two companies submitted information about new
GC columns, as shown in Table 4. Restek has two new
columns, the Rt-Silica BOND porous-layer open-tubular
(PLOT) column for permanent gas separations and the
Rxi-1301Sil MS column, which targets solvent analyses
with MS detection. Phenomenex introduced a two-column
set that consists of the company’s ZB-CLPesticides-1 and
ZB-CLPesticides-2 columns and is targeted for multiple
polychlorinated biphenyl (PCB) US Environmental Protection
Agency (EPA) methods that use electron-capture detection.
Acknowledgements I would like to thank the manufacturers and distributors that
kindly furnished the requested information, which allowed
a timely report on new product introductions over the past
year. For those manufacturers who did not receive a “New
Products” questionnaire this year and would like to receive
one and be considered for early inclusion into the 2015–2016
new GC and related product introductions review, please
send the name of the primary company contact, the mailing
address, fax number, and e-mail address to Laura Bush,
Editorial Director, LCGC Europe, lbush@advanstar.com, with
the subject line “2016 New GC Products”. The questionnaire
will be sent out in December 2015.
Pittcon will head to Atlanta in 2016, where conferees will enjoy a second consecutive year of Southern hospitality. In 2017, the conference returns to Chicago.
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LC•GC Europe May 2015288
GC CONNECTIONS
Table 3: New GC accessories.
Product Company Description
5-mL Diamond
headspace
syringe
SGE SGE enhanced its line of Diamond headspace syringes with a 5-mL model. These syringes
incorporate features for headspace sampling such as a unique “energized” plunger tip
that eliminates dead volume at the end of the plunger stroke and is compatible with a wide
range of solvents and a gas-tight seal that is not affected by temperature cycling up to at
least 150 °C.
AOC-6000 Shimadzu The Shimadzu AOC-6000 system is capable of exchanging syringe modules automatically
between liquid injection, headspace injection, or solid-phase microextraction (SPME)
injection. The autosampler features automated pretreatment with automatic syringe
exchange (10 μL to 1000 μL) and mixing functions that automate the preparation of
calibration curve samples, addition of internal standard substances, and sample dilution.
The device performs sample pretreatment and analysis in parallel so that no time is lost in
the continuous analysis of samples requiring headspace sampling or other time-consuming
pretreatments.
ECD-2010
Exceed
Shimadzu The Shimadzu ECD-2010 Exceed system uses contact-free technology in which the sample gas
flow makes as little contact as possible with the collector electrode or the 63Ni radiation source,
which greatly improves the the ECD cell durability by reducing deposition of sample residue on
the detecor and radiation source. The detector has a limit of detection of 4.0 fg/s and a dynamic
range of 105 for γ-BHC.
EZGC
software suite
Restek Restek has upgraded its EZGC software suite to include a method translator, flow calculator,
and chromatogram modeler. The software application is available in an on-line version. For
new methods, the chromatogram creator uses a database of thousands of compounds to
accurately predict the best column and conditions for a wide range of applications. For
optimization and troubleshooting of established methods, the method translator and flow
calculator helps analysts easily change column formats and carrier gases for faster analyses
or improved capacity, and also calculates splitless hold times for applications using splitless
injections.
Fast GC
conversion kit
for HP6890
and Agilent
7890 systems
VICI This conversion kit from VICI includes the parts and tools to convert HP6890 or Agilent 7890
instruments to fast GC systems. An adapter plate relocates the detector next to the injector to
make room for the high speed components. The kit enables use of VICI’s resistively heated
columns that are wrapped with nickel wire and heated by applying a low-voltage current.
Column temperature is controlled by regulating the amount of current; a small fan provides rapid
cooling of the low-mass column to near-ambient temperatures. The company’s fast temperature
programmer (a separate item) provides precise temperature programming with rapid heating
and cooling in an eight-state profile at up to 1200 °C/min with a 5-m long or 500 °C/min with a
15-m-long nickel-wire or nickel-clad resistively heated column.
fastGC
add-on for
PTR-TOFMS
series
Ionicon Ionicon PTR-TOF systems are capable of measuring trace gas samples in real-time with a
high mass resolving power. The new fastGC module adds an optional chemical preseparation
step before the analysis. The module consists of a short GC column with an advanced heating
concept for ultrafast heating and equally fast cooling rates that makes the preseparation step
nearly real time. The fastGC module is integrated with the PTR-TOF and the normal sample
gas inlet is used, which allows researchers to perform real-time measurements and add fastGC
runs at time points of interest for enhanced separation and identification. Winner of the Gases
& Instrumentation International Magazine’s 2015 Golden Gas Award in the gas chromatography
category.
IMOv
Intelligent
Modular oven
AFP The IMOv Intelligent Modular oven is a modular GC oven system offering flexibility in the
number of column ovens and amount of space for valves and other accessories. The valve
oven is independent from the column, with the choice of one to three column ovens. The valve
oven has a preperforated bottom plate to accommodate AFP’s valves and comes with custom
tube brackets for easy routing. All electronics such as the company’s IVD intelligent valve
driver, electronic relays, electronic pressure control (EPC), flow meters, and communications
ports are accessible from the back of the controlled temperature zone. The system also
features a built-in leak detection system and has software drivers for various third-party
instruments.
IPAPS:
Intelligent
Plasma
Assisted
Purifier system
AFP The Intelligent Plasma Assisted Purifier system from AFP is a gas purifier that uses a plasma to
increase the lifetime for the heated gettering alloy. The purifier features an end-of-life monitoring
system, impurities overload detection, and a capacity of 1 L/min. The device is suitable for gas
purification, zero gas generation for calibration of on-line analyzers, mass spectrometers, and
GC detectors such as plasma emission, helium ionization, discharge ionization, flame ionization,
pulsed-discharge ionization, and electron capture. The purifier can deliver a total outlet impurity
level of less than 1 ppb, with 99.9999% grade inlet gas. It supports AFP’s IMOv and other
accessories through a serial communications link.
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GC CONNECTIONS
References
(1) M. L’Heureux, LCGC North Am. 33(2),
108–121 (2015).
(2) M. Swartz, LCGC Europe 28(4),
232–243 (2015).
(3) M.W. Dong, LCGC Europe 28(4),
223–231 (2015).
(4) D.E. Raynie, LCGC Europe 28 (5), In
Press (2015).
John V. Hinshaw is a senior scientist
at Serveron Corporation in Beaverton,
Oregon, USA, and is a member of the
LCGC Europe editorial advisory board.
Direct correspondence about this
column should be addressed
to “GC Connections”, LCGC Europe,
Honeycomb West, Chester Business
Park, Chester, CH4 9QH, UK, or e-mail
the editor-in-chief, Alasdair Matheson,
at amatheson@advanstar.com
Table 3: New GC accessories (continued).
Product Company Description
LipSeal fitting AFP AFP introduced a new patented fitting concept that provides two levels of sealing, requires
a much lower torque, and permits an increased number of remakes. Ultrafine-pitch threads
transfer the sealing force to the ferrule and an antifriction and antigalling coating is applied
to the front and the threaded portions of the nut, which reduces the friction by a factor of at
least 10 and eliminates rotation of the ferrule when the nut is tightened. Gold-plated ferrules
optimize sealing performance, and a fine lip is added on the bottom of the detail that creates
a metal-to-metal seal between the tube end and fitting body with no dead volume. The front
portion of the ferrule is longer to occupy more of the empty volume.
MRV: Mini
rotary valve
AFP AFP introduced a smaller form factor rotary valve that includes features from the company’s
larger valve series such as a double stopper to prevent side loading of the rotor, which results
in a longer lifetime; two dowel pins to lock the stator body in place to prevent rotation; and a
treated rotor to enable the use of the high temperature version at ambient temperature without
damaging the valve. The MRV works with the company’s Mini Pneumatic Actuator.
PeakBlade 77
GC×GC
modulation
system
DANI The DANI PeakBlade 77 GC×GC modulator is able to reach down to 77 K without using
liquid nitrogen, which opens up analysis of the most demanding molecules and allows fast
chromatography with sharp peaks. The modulator cuts chromatographic peaks to unfold complex
sample composition. Driven by a cryocooler system, a cool tip connects to the cryofocusing area
of the GC×GC system by heat conduction to allow an effective focusing process into the GC oven.
The system features an innovative heater design that allows ultrafast vaporization of cryofocused
molecules resulting in an ultranarrow peak bandwidth. The modulator can achieve as much as
450 °C difference between temperature of the cold tip and the GC oven when the oven is at
350–400 °C. The system is capable of the analysis of compounds down to C3. The modulator can
use different vaporization temperatures to adapt to different compounds, programmable during
the run. It is possible to change modulation frequency for target peaks or in timed intervals to
better select sensitivity versus peak-cutting. The modulator is available as part of the DANI Master
GC×GC/TOF-MS system or can be sold to support other GC and GC–MS systems on the market.
Table 4: New GC columns.
Product Company Description
Rt-Silica BOND Restek The Rt-Silica BOND column is aimed at analysis of permanent gases, chlorofluorocarbons
(CFC), light hydrocarbons, and sulphur analyses. The column has the retention and
capacity of a traditional PLOT column, but the company’s manufacturing process for this
column nearly eliminates particle release, which minimizes detector spikes, maintenance,
and downtime. The silica phase easily stands up to water, eliminating the need for long
bakeouts after injections of water-contaminated samples. Each column is individually
tested with C4 hydrocarbons, including methyl acetylene and 1,3-butadiene, both of which
are extremely sensitive to changes in inertness and selectivity.
Rxi-1301Sil MS Restek The Rxi-1301Sil MS column targets solvent analyses, glycols by MS, and EPA Method
551. It features an arylene-stabilized cyano phase, combining retention and selectivity
for volatiles with the highest maximum temperature and lowest bleed of any cyano
column. This allows for reliable MS analyses for volatiles and polar compounds, fast
elution of less-volatile analytes for faster cycle times, as well as improved robustness.
ZB-CLPesticides-1
and
ZB-CLPesticides-2
column set
Phenomenex Phenomenex’s new columns are intended for application-specific analysis of chlorinated
pesticides, herbicides, and polychlorinated biphenyls (PCB) by GC–ECD. The columns
provide baseline separation of all analytes of interest in under 10 min and are available
as a two-column kit. The column set supports seven EPA methods (8081, 8082, 8151,
504, 505, 508, and 552) without changing columns, which reduces down-time by
eliminating column installations otherwise needed when changing methods. The columns
have temperature limits of 320 °C isothermal and 340 °C programmed temperature. The
ZB-CLPesticides-1 column is available with film thicknesses of 0.25, 0.32, and 0.50 µm,
and the ZB-CLPesticides-2 column is available with 0.20-, 0.25-, 0.42-, and 0.50-µm films.
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LC•GC Europe May 2015290
SAMPLE PREPARATION
PERSPECTIVES
As expected, the new products
introduced in the past year in the
area of chromatographic sample
preparation, while somewhat limited,
mirror the current development in
the field. That is, a few systems were
developed to automate or streamline
the sample preparation process;
new sorptive phases and formats,
including QuEChERS (quick, easy,
cheap, effective, rugged, and safe),
were developed; and accessories and
other stepwise advances in the field
were noted. In late 2014, the LCGC
editorial staff submitted a survey to
vendors of sample preparation products.
Responses to this survey are compiled in
this review. In addition, a keyword search
using the terms “sample preparation”
and “extraction” was conducted for
exhibitors at Pittcon 2015; then each
of these vendors was visited. While
attempts were made to be as inclusive
as possible, we apologize for any
oversight.
Hollow-Fibre Microextraction
Perhaps the highlight among new sample
preparation products is an unheralded
introduction by one of the smallest
vendors. Biomics, Inc., brought forth
devices for hollow‑fibre microextraction
(HFME) at Pittcon 2015. HFME has been
developed for quite some time (more
than a decade) and is performed in a
variety of configurations; for example, it
was reviewed in a 2010 “Sample Prep
Perspectives” column (1). Biomics claims
that their hollow‑fibre product, available in
single‑vial and 96‑well formats, is the only
commercially available HFME product.
Regardless of the validity of this claim,
such products are certainly scarce and
this development by Biomics should drive
the acceptance of the technique. Figure 1
shows an example of the format of a
96‑well plate HFME device. The HFME
approach should work for the isolation of
environmental, pharmaceutical, food, and
nutraceutical samples.
Systems
Several sample preparation systems were
introduced in the past year, typically with
multisample capabilities and generally in
the bioanalytical realm.
Similar to the HFME product
introduction above, Phenomenex
expanded its offerings with the Novum
Simplified Liquid Extraction (SLE)
product line. Available in both cartridge
and 96‑well plate formats, these liquid
extraction products are designed to
replace conventional liquid–liquid
extraction (LLE) in bioanalytical, food
safety, and environmental testing.
In the suggested protocol with the
Novum product, a sample is diluted
with buffer solution and added to the
SLE medium, and after a brief soaking
period, elution with ethyl acetate or
dichloromethane follows — the process
is completed within about 15 min. The
Extrahera system by Biotage supports
both supported liquid extraction and
solid‑phase extraction (SPE), in either
column (1, 3, and 6 mL) or plate formats.
The Extrahera system can also be
used in protein‑crashing applications
and uses positive pressure for more
reproducible flow. Added automation to
the Fotector Plus automated SPE system
(Reeko Instrument USA) provides
capacity to run 48 samples continuously
with positive pressure sampling and
elution modes.
Keeping with developments in the
bioanalytical area, the ECO2Chrom
flash chromatograph from Applied
Separations uses liquid carbon dioxide
to reduce organic solvent use and
lower the analyte concentration time.
The high diffusivity of the mobile phase
allows smaller particle sizes to be used,
allowing for greater efficiency or faster
analysis times for the same efficiency as
with liquid organic solvents. This flash
chromatography system accommodates
multiple sample introduction formats
with time‑ or peak‑triggered fraction
collection. Meanwhile, wet or dry
homogenization of biological samples
can be performed with the Biotage
Bead Ruptor 24. The bead mill uses
24 2‑mL tubes, 12 7‑mL tubes, or
six 30‑mL tubes simultaneously. The
SiliCycle MiniBlock is a general purpose
system that allows flow‑through parallel
processing of chemical reactions,
including derivatizations, peptide
synthesis, and screening, with resin
agitation and washing. The system
operates over a temperature range from
‑20 °C to 120 °C with capacities ranging
from six 40‑mL vials to 48 4‑mL tubes.
Other significant introductions in the
area of sample preparation systems
were updates or product extensions,
especially in systems for environmental
analysis. The Pickering Laboratories
DEXTech system uses columns
with different formats for sample
cleanup in the analysis of dioxins and
polychlorinated biphenyls. Meanwhile,
Horizon Technologies added plungers
for greater flexibility to the SmartPrep
Extractor automated SPE system
and Environmental Express added
chemistries to its SimpleDist system
New Sample Preparation Products and Accessories at Pittcon 2015Douglas E. Raynie, Sample Preparation Perspectives Editor.
This yearly report on new products introduced at Pittcon (or in the preceding year) covers sample preparation instruments.
ES610519_LCE0515_290.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan
291www.chromatographyonline.com
SAMPLE PREPARATION PERSPECTIVES
Table 1: New sorbent products.
Company Product Format Notes
SiliCycle SiliaQuick QuEChERS
Salt packets with centrifuge tubes for performing QuEChERS method
Available as MgSO4 with primary secondary amine (PSA), carbon black, or C18.
UCT, Inc. Styre Screen HL DVB
SPE cartridges
Cross‑linked divinylbenzene for extraction of acidic, basic, polar, and nonpolar compounds with greater loading capacity than silica‑based phases.
Enviro‑Clean QuEChERS
Salt packets with centrifuge tubes for performing QuEChERS method
Salt ratios optimized for biological samples with limited volumes. Protein precipitation not required for blood samples.
Separation Methods Technologies
SMT MEB Bulk packings
Methyl (1% carbon load), ethyl (2% carbon load), and butyl (4% carbon load) silica‑based phases, 35–50 μm particles, 60‑ or 150‑Å pores. Selective for polar and nonpolar pharmaceuticals, natural products, and very hydrophobic proteins and biomolecules.
Bonna‑Agela Technologies
Cleanert PEP‑2
96‑well, modular microplates
Five phases available:• PVB: functionalized vinyl pyrrolidone
and urea to retain most acidic, basic, and neutral polar compounds without adjusting pH. Design for small sample amounts, resulting in one‑third less evaporation time and reconstitution solvent.
• PWCX: combines weak cation-exchange
and reversed phases using carboxylate radical functional group for improved retention of basic analytes.
• PWAX: combines weak anion-exchange
and reversed phases on polymer support with amino functional group.
• PAX: quaternary ammonium base
functional group with reversed‑phase and strong anion‑exchange modes. Stable from 0–14 pH range.
• PCX: sulpho-functional group
with reversed‑phase and strong cation‑exchange modes. Stable thoughout the pH 0–14 range.
Thermo Scientific
ASE Prep Sorbent cartridges
6‑mL cartridges with 500‑mg resin
Four resins available, designed for cleanup of extracts following accelerated solvent extraction:• Florisil for adsorption of polar
compounds.• Alumina acid for anion exchange and
adsorption of polar compounds.• Alumina base for cation exchange and
adsorption of polar compounds.• Alumina neutral for adsorption of polar
compounds, capable of anion or cation exchange with pH adjustment.
ES610524_LCE0515_291.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan
LC•GC Europe May 2015292
SAMPLE PREPARATION PERSPECTIVES
for the distillation of phenols. The
Omni‑Sampler Plus sample handling
system from Entech Instruments
updated cryogenic preconcentration
for volatile organic compounds onto
glass beads, with mild temperatures
(60–100 °C) for the transfer of C2–
C24 compounds. The Omni‑Sampler
Plus sample handling system has
multiple modes for the determination of
volatile analytes, including headspace
sampling, thermal desorption,
porous cartridge microextraction (a
high‑capacity version of solid‑phase
microextraction [SPME]), and on‑column
trapping. For water analysis, the 4100
Water/Soil Sample Handler from OI
Analytical automates sample handling
and processing in collaboration with the
company’s Eclipse 4660 purge‑and‑trap
concentrator.
Sorbents
Various sorbents, in cartridges or as
bulk phases, have been introduced
in the past year. These phases are
designed for SPE, including dispersive
SPE (dSPE) approaches such as the
QuEChERS method, high sample
capability via polymer supports, and
selectivity in sample cleanup. These
sorbent products are summarized in
Table 1.
Accessories and Other Products
Several other sample preparation
products were recently introduced
to the market. Most notably, Supelco
continues to develop its SPME product
line in the area of biocompatible
SPME. This product extension is more
compatible with biological analyses
such as the direct sampling of small
animals like mice, as well as dried
blood spot analysis, 96‑well plates,
and other microsampling situations.
Since gas chromatography (GC) and
derivatization reactions for GC are
often considered mature technologies,
it is somewhat surprising to see a
new derivatization reagent from Regis
Technologies. N‑Methyl‑N‑(trimethylsilyl)
trifluoroacetamide (MSTFA) with 1%
trimethylsilyl chloride is also marketed
by other vendors for the silylation of
hindered hydroxyl groups that do not
ordinarily react with MSTFA, along with
secondary amines, amides, carboxyls,
and steroids. Thermo Scientific
addresses an expanding number
of application areas for accelerated
solvent extraction (ASE), particularly
polymers, with the offering of ASE
extraction thimbles for samples that
melt at the operating temperatures
used in ASE. The goal is to prevent
the plugging of filters and tubing by
fine particles by using cellulose or
glass fibre filters. The GlycoWorks
RapiFluor‑MS N‑Glycan kit from Waters
is a 96‑well plate product based on
hydrophilic interaction chromatography.
The GlycoWorks kit is used for the
sample preparation of N‑linked glycans
released following rapid deglycosylation
and labelling to provide enhanced
sensitivity for both fluorescence and
mass spectrometric determination.
Sample analysis of glycoproteins can
be completed in less than 1 h. Finally,
J.G. Finneran Associates marketed a
vial loader for 96‑well plates with insert
vial sizes ranging from 350‑μL glass
vials to volumes of 2 mL.
Conclusions
With this review of new product
offerings in the field of chromatographic
sample preparation, the natural
question is: “What’s next?” Based on
this year’s offerings and advancements
in the field, it is anticipated that
commercial developments in the
current year will address several issues.
Sorbent‑based sample preparation
will continue to see significant
commercialization in several areas.
QuEChERS will remain a growing
area and the end of patent protection
for SPME will bring new competitors
to the field and new areas such as
biocompatible SPME and SPME
designed for liquid chromatography
applications. Other advancements
will accommodate serial or parallel
sample processing for increased
throughput. Bioanalytical and food
safety applications will drive these
developments.
References(1) L. Zhao, H.K. Lee, and R.E. Majors, LCGC
North Am. 28(8), 580–591 (2010).
(2) G. Borijijan, Y. Li, J. Gao, and J.J. Bao, J.
Sep. Sci. 37, 1155–1161 (2014).
“Sample Prep Perspectives” editor
Douglas E. Raynie is an Associate
Research Professor at South
Dakota State University, USA. His
research interests include green
chemistry, alternative solvents,
sample preparation, high resolution
chromatography, and bioprocessing in
supercritical fluids. He earned his PhD
in 1990 at Brigham Young University
under the direction of Milton L. Lee.
To contact the editor‑in‑chief, Alasdair
Matheson, please e‑mail: amatheson@
advanstar.com
(a)
(b)(d)
(c)
12
3
4
5
(a)
(b)(d)
(c)
12
3
4
5
(a)
(b)(d)
(c)
12
3
4
5
(a)
(b)(d)
(c)
12
3
4
5
Figure 1: Hollow‑fibre microextraction in a 96‑well format: (a) the plastic base, (b) the attachment of hollow fibre to the plastic base, (c) the 96‑well plate, and (d) an expanded view of the hollow‑fibre device. 1 = hollow‑fibre attachment tip, 2 = donor phase collection tip, 3 = acceptor phase, 4 = donor phase, 5 = hollow fibre. Adapted with permission from reference 2.
ES610522_LCE0515_292.pgs 04.30.2015 13:21 ADV blackyellowmagentacyan
Measurement of trace organics?
Markes International leads the world in analytical thermal desorption (TD) and associated air sampling
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ES610685_LCE0515_293_FP.pgs 04.30.2015 19:57 ADV blackyellowmagentacyan
LC•GC Europe May 2015294
PRODUCTS
2015 GC catalogue
MEGA has presented
its 2015 GC Products
Catalogue. The edition
presents all of the
MEGA GC columns
ranging from the most
commonly used phases
to custom and proprietary
application-specif c
stationary phases. The
catalogue also features the
MEGA-DEX chiral columns
line and the Metal columns for high-temperature GC.
www.mega.mi.it
MEGA s.n.c., Milan, Italy.
2d LC system
The Agilent 1290 Inf nity 2D LC
is a one vendor platform that
allows users to switch between
one-dimensional UHPLC
and the chromatographic
power of heart-cutting,
multiple heart-cutting, and
comprehensive 2D LC.
According to the company, this
instrument achieves ultrahigh peak capacity with a single
2D LC analysis and allows setup of new methods within
seconds.
www.agilent.com/chem/infi nity-2d-lc
Agilent technologies, California, usA.
Automated sample preparation
The PAL RTC system
with robotic tool change
from CTC Analytics
AG automates sample
preparation and sample
injection. According
to the company, it
improves productivity by
automatically changing between different syringes, liquid
injection, headspace, and SPME without the need for
manual intervention. This enables workf ows like liquid–
liquid extraction or SPE to be fully automated.
www.palsystem.com
CtC Analytics AG, Zwingen, switzerland.
Molecular imaging system
The Full Spectrum Molecular Imaging
System from Waters combines advanced
MS imaging technologies to provide
comprehensive large and small molecule
distribution information, according to
the company. Integrating MALDI, DESI,
and ion mobility into a single system
delivers a level of detail and molecular
information beyond any individual
imaging technique. The SYNAPT
HDMS-based system expands imaging
capabilities for the study of molecular distribution in f elds such
as proteomics, metabolomics, cell and tissue biology, research
pathology, and histology.
www.waters.com/msimaging
Waters, Massachusetts, usA.
Meat reference materials
Six new meat mixture
reference materials are
available from LGC to
help protect consumers
from food fraud. The meat
mixtures enable food testing
laboratories to assess the
quality of their measurements
and ensure detection of
substitutions in meat products
at low levels. According to the company, the materials have been
tested by DNA sequencing, a PCR-based and immunoassay
method, to conf rm the expected meat species in samples and
absence of species cross-contamination. Limit of detection is
below 1% of one meat species in the presence of another.
www.lgcstandards.com
LGC, teddington, Middlesex, uK.
HILIC HPLC columns
SeQuant ZIC-HILIC HPLC
columns are offered by
Merck Millipore. HILIC is a
chromatographic technique
for separation of polar and
hydrophilic compounds.
HILIC is a normal-phase
(NPLC) type of separation
but uses reversed-phase
type eluents. It is the method
of choice for separating
charged and neutral hydrophilic compounds, such as acids,
bases, ions, peptides, metabolites, and sugars.
www.merckmillipore.com/chromatography
Merck Millipore, darmstadt, Germany.
ES610658_LCE0515_294.pgs 04.30.2015 17:17 ADV blackyellowmagentacyan
295www.chromatographyonline.com
ProduCts
thermal desorption tube
cleaner
Markes’ TC-20 tube conditioner
allows up to 20 sorbent-packed
TD tubes to be simultaneously
cleaned at elevated temperatures.
According to the company, the
TC-20 frees up instrument time to
run samples rather than condition
tubes, providing a rapid return on
investment.
www.markes.com
Markes International,
Llantrisant, uK.
uHPLC sEC–MALs detector
Wyatt has launched μDAWN,
a multi-angle light scattering
(MALS) detector that can
reportedly be coupled to any
UHPLC system to determine
absolute molecular weights and
sizes of polymers, peptides,
proteins, or other biopolymers
directly. To accommodate
narrow peaks in UHPLC, the light scattering f ow cell volume has
been reduced from 63 μL to 10 μL. To minimize interdetector
mixing, band broadening is under 7 μL.
www.wyatt.com
Wyatt technology, California, usA.
Evaporative light-scattering
detector
Sedere has introduced the
Sedex 90LT, a low-temperature
evaporative light-scattering
detector (LT-ELSDTM). According
to the company, the detector
provides ppb level sensitivity, four
orders of magnitude dynamic
range, direct linearity on the full
range, excellent efficiency, and
response consistency. The detector
reportedly suits every application in
LC and SFC.
www.sedere.com
sedere sAs, Alfortville, France.
Please submit new product press releases
and catalogue information to
Bethany degg at bdegg@advanstar.com
uHPLC system
As a new UHPLC system
designed for enhanced
levels of performance,
productivity, and usability,
the Thermo Scientif c
Vanquish can be used
standalone or with the
latest mass spectrometer. It
features a clean design that
combines the ruggedness
of an integrated system
with the f exibility and serviceability of a modular system,
according to the company.
www.thermofi sher.com/vanquish
thermo Fisher scientifi c, California, usA.
HPLC
The classif ed directory from LC•GC Europe
To advertise in this section call Elizabeth McLean
today at +44 (0) 1244 629 315 or email
emclean@advanstar.com to reserve your space
Supplies
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ES610659_LCE0515_295.pgs 04.30.2015 17:17 ADV blackyellowmagentacyan
LC•GC Europe May 2015296
ProduCts
sPE system
The EconoTrace SPE system from
FMS is a parallel SPE system that uses
positive pressure pumping for precise
and accurate delivery of the sample,
conditioning, washing, and elution
solvents. Up to 8 extractions can run
simultaneously. The system uses any
size or type SPE cartridge. According
the company, the system is an economic
solution for consistent, reproducible,
high throughput extractions for
pharmaceutical, food, and water
matrices.
www.fms-inc.com
FMs, Inc., Massachusetts, usA.
Ion chromatography modules
Cecil Instruments has
introduced the IonQuest
ion chromatography
system, which offers
modular and automated
ion chromatography. The
low-drift and ultra-low noise
conductivity detector may
be purchased separately
for use with third party
LC systems. The use of
PowerStream software
helps to achieve ease of use and 21 CFR part 11 compliance.
www.cecilinstruments.com/ionquest-1.html
Cecil Instruments Limited, Cambridge, uK.
dried blood spot autosampler
Flow-through desorption
(FTD) is the concept on
which the new Dried Blood
Spot Autosampler from
Spark Holland is based.
Farewell to punching! Using
a spot recognition camera,
DBS cards are accurately
positioned in a high pressure
clamp for direct, fow-through
desorption of bloodspots into
the analytical system. The company report that the system
provides maximum sensitivity and a fully automated
workfow for DBS analysis.
www.sparkholland.com
spark Holland B.V., Emmen, the Netherlands.
Forensic analysis kits
UCT, LLC has created complete
forensic analytical kits to perform
toxicology extractions and
analyses. The kits contain buffer
reagents, SPE columns, HPLC
columns, hydrolyzing reagents,
and stock drug standards. CoA’s
for all standards as well as
recommended extraction and
analytical procedures are included.
According to the company, these
kits are ideal for start-up labs, labs
developing a new method, or converting from GC–MS to
LC–MS.
www.unitedchem.com
uCt, LLC, Pennsylvania, usA.
sFE–sFC–Ms system
Shimadzu’s Nexera UC
unifed chromatography
system is reportedly
the world’s frst-ever
unifed and fully
automated instrument
combining supercritical
fuid extraction (SFE)
with supercritical fuid
chromatography (SFC). The SFE–SFC–MS platform
merges quick and easy on-line sample preparation with
advanced chromatographic analysis and high sensitivity
detection.
www.shimadzu.eu
shimadzu Europa GmbH, duisburg, Germany.
GC–Ms columns
The Restek Rxi-1301Sil MS
GC columns offer cyano
phase selectivity along with
high thermal stability, which
ensure dependable, accurate
MS results and increased
uptime. The company report
that the columns can improve
the performance of existing
methods for solvents, glycols,
and other polar compounds.
www.restek.com/catalog/view/41734
restek, Bellefonte, Pennsylvania, usA.
ES610670_LCE0515_296.pgs 04.30.2015 17:47 ADV blackyellowmagentacyan
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LC•GC Europe May 2015298
EVENT NEWS
21–25 June 2015 42nd International Symposium on
High Performance Liquid Phase
Separations and Related Techniques
(HPLC 2015)
International Conference Centre,
Geneva, Switzerland
Tel: +41 22 839 84 84
E-mail: chairman@hplc2015.org
Website: www.hplc2015-Geneva.org
28 June–1 July 2015Recent Developments in
Pharmaceutical Analysis
University of Perugia, Perugia, Italy
Tel: +39 0755855131
E-mail: symposium@rdpa2015.com
Website: rdpa2015.chimfarm.unipg.it
30 June–3 July 201521st International Symposium on
Separation Science
Grand Hotel Union, Ljubljana, Slovenia
Tel: +386 1 477 0265
E-mail: info@isss2015.si
Website: www.isss2015.si
3–6 August 20155th International Network of
Environmental Forensics (INEF)
Victoria University College, Toronto,
Canada
E-mail: inef2015@gmail.com
Website: www.inef2015.com
23–28 August 201535th International Symposium on
Halogenated Persistent Organic
Pollutants (Dioxin 2015)
Hotel Maksoud Plaza, Sao Paulo, Brazil
Tel: +55 11 3056 6000
E-mail: dioxin2015@acquacon.com.br
Website: www.dioxin2015.org
18–19 November 2015PEFTEC International Conference
and Exhibition for Petrochemical
Analysis
Antwerp Exhibition Centre,
Antwerp, Belgium
Tel: +44 1727 858840
E-mail: info@peftec.com
Website: www.peftec.com
31st Montreux Symposium on LC–MS and MS–MS
The 31st Montreux Symposium on
LC–MS and MS–MS will be hosted
by the International Association
of Environmental Analytical
Chemistry (IAEAC) at the Aldershof
Convention & Exhibition Centre,
Berlin-Aldershof, Germany, on
4–6 November 2015. The Montreux
Symposium series was first established in 1980 by the IAEAC, with the symposium
historically alternating location between Montreux, Switzerland, and the USA.
This year it will be held in Berlin-Aldershof, the location of the science campus of
Humboldt-Universität and of Germany’s leading science and technology park.
The conference symposium will focus on the most recent developments in
liquid chromatography coupled to mass spectrometry (LC–MS) and tandem
mass spectrometry (MS–MS) in the three fields of metabolomics, lipidomics,
and glycomics. These fields were selected because, aside from genomics and
proteomics, the work in these areas is reshaping our understanding of biological
processes and the development of therapies for diseases.
This symposium will bring together scientists working with up-to-date
bioanalytical tools, particularly using separation techniques such as gas and
liquid chromatography, electrophoresis, and ion mobility in combination with
spectroscopy and mass spectrometry. They will join with enabling companies
from those fields, who will have the opportunity to display the latest developments
in all areas of MS and the separation sciences. The organizers promise first-rate
presentations, posters presenting up-to-date research, and an exhibition
highlighting new scientific instrumentation and technical information.
E-mail: analytik@chemie.hu-berlin.de • Website: www.lcms-montreux2015.de
7th International Symposium on Recent Advances in Food Analysis (RAFA 2015)
The 7th International Symposium on Recent Advances
in Food Analysis (RAFA 2015) will take place at the
Clarion Congress Hotel, Prague, Czech Republic, on
3–6 November 2015. The RAFA 2015 symposium will provide
an overview of the current state-of-the-art on analytical
and bioanalytical food quality, safety control strategies,
and introduce the challenges for novel approaches in this
field. The programme will be tailored to provide networking
opportunities as well as exploring the latest results from the
food analysis community. Presentations will be given by leading scientists
through keynote lectures and contributed oral and poster presentations
covering the topics of general food analysis issues and food quality/safety.
The conference programme will also be accompanied by several satellite
events including workshops on novel analytical strategies and interactive
seminars. An exhibition of recently introduced instrumentation in food analysis
and other valuable equipment will be available during the symposium. Vendor
seminars will also be organized to introduce recent developments and scientific
strategies for advanced food quality and safety control.
Young scientists are encouraged to present their scientific work, with a
number of RAFA 2015 Student Travel Grants available. The prestigious
RAFA Poster Award will also be given for the best poster presentation by a
young scientist, along with other sponsored poster awards. The deadline for
registration at a reduced fee and submission of an abstract for oral presentation
is 31 July 2015; deadline for submission of an abstract for poster presentation is
31 August 2015.
E-mail: rafa2015@vscht.cz • Website: www.rafa2015.eu
EVENT NEWS
Imag
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298LC•GC Europe May 2015
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SPME, SBSE,
Thermal Desorption
Headspace, Dynamic Headspace
(DHS) and PYRO
Automated Liner Exchange
(ALEX)
Example: MPS Dual Head WorkStation with
Centrifuge, Filtration, and Vortex (mVORX) options
Example: ALEX-GC-MS/MS-System
for QuEChERS, Metabolomics ….
ES610683_LCE0515_CV4_FP.pgs 04.30.2015 19:56 ADV blackyellowmagentacyan
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