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SPECIAL

2nd Revised and Expanded Edition

Intelligent Automated Sample Preparation

Effi cient LC/MStrace analysis

Water Analysis

PFCs in Water & Waste Water

Food Safety

Pesticides, Afl atoxins, Antibiotics & Fungicides

Drug Monitoring

Drugs of abuse in Blood and Urine

P FCs are creations of organic synthe-sis, developed in the R&D laborato-ry, produced in signifi cant amounts

and ubiquitous in the environment. PFC surfactants are synthesized from carboxyl-ic acids or sulfonic acids with chain lengths of four to ten carbon atoms by substitut-ing hydrogen atoms with fl uorine atoms. Strictly speaking, PFCs can be divided into two groups: Perfl uorinated alkylsulfonates (PFAS), among which perfl uorooct anesul-fonate (PFOS) is the most widely known compound, and perfl uorinated carboxylic acids (PFCA), whose most famous repre-sentative is perfl uorooctanoic acid (PFOA). Experts are of the opinion that PFCs only have limited toxicity. As to long term effects, there is no consensus even though PFCs have been reported as having cancer promoting properties and though the USEPA considers PFOA a “probable human carcinogen”. In fact, mainly the effects of PFOA and PFOS have been investigated while those of other

PFCs have been less extensively studied. The German Commission for Drinking Water lists an upper concentration limit of 1 µg/L.

Intelligent Automated Sample Preparation

Fast, accurate, and highly sensitive LC/MS determination of PFCs

The determination of Perfl uorinated Compounds (PFCs) in water fol-lowing the recognized international standard method (ISO/DIS 25101) is performed based on solid phase Extraction combined with HPLC-MS/MS determination. Application chemists have recently shown that if sample preparation and PFC determination is fully automated; the analysis can be performed more productively – and with signifi cantly improved performance.

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Published by GERSTEL GmbH & Co. KG Eberhard-Gerstel-Platz 1 45473 Mülheim an der Ruhr Germany

Editorial Director Guido Deußing ScienceCommunicationNeuss, Germany [email protected]

Translation and editingKaj Petersen [email protected]

Scientifi c advisory boardEike Kleine-Benne, Ph.D. [email protected] Lerch, Ph.D. [email protected] Reimold, [email protected]

Contact [email protected]

Design Paura Design, Hagen, Germany www.paura.de

GERSTEL Solutions Worldwide LC/MS Special

Perfl uorooctanesulfonate (PFOS)

Perfl uorooctanoic acid (PFOA)

HPLC/MS(/MS) Applications:

PFCs in water & waste water ........... 2

Drugs of abuse in blood and urine ... 5

Malachite Green in farmed fi sh ........ 8

Pesticides in fruit and vegetables .... 9

Afl atoxins in food ........................... 12

Chloramphenicol in fi sh and meat ............................. 14

2 GERSTEL Solutions Worldwide – LC/MS Special

Overlay MRM traces of eight different sample preparations of a waste water sample spiked with 0.5 ng/mL. Excellent reproducibility is obtained for all compounds: PFPeA (blue), PFHxA (brown), PFHpA (purple), PFOA (green), PFNA (red), PFOS (black), PFDA (yellow)

PFC calibration curves covering the range from 5 to 500 ng/mL based on external standard calibration.

PFPeA 0,9998

PFHxA 0,9996

PFHpA 0,9997

PFOA 0,9966

PFNA 0,9993

PFDA 0,9991

PFOS 0,9992

35

30

25

20

15

10

5

0

-5

Millions

Correlation Coeffi cient

0 100 200 300 400 500 600

3000

2500

2000

1500

1000

500

0

Thousands

PFOS Spike 5 ng/mL

PFOA Spike 5 ng/mL

PFPeA Spike 5 ng/mL

mean SDEV RSD %

PFOS 526056,3 17686,2 3,4

PFOA 2466710,5 24314,9

PFPeA 232213,1 3689,1 1,6

Reproducibility of the method for PFOS, PFOA, PFPeA.


Developing an automated SPE-HPLC-MS/MS methodThe automated method presented in this work is based on the current ISO standard method: “ISO 25101:2009 Water quality – Determination of perfl uorooctanesulfona-te (PFOS) and perfl uorooctanoate (PFOA) – Method for unfi ltered samples using solid phase extraction and liquid chromatogra-phy/mass spectrometry”. While the ISO me-thod focuses on PFOS and PFOA, our goal for the project reported here was to develop a simple and rugged automated SPE-HPLC-MS/MS method for the determination of a wider list of PFCs in water and sludge. Automating the SPE process eliminates extensive and tedious manual sample pre-paration steps that are known sources of errors, for example when cartridges run dry or when sample matrix restricts the fl ow of liquids through the cartridge.

The benefi ts of automation are mani-fold: Higher recovery, improved reproduc-ibility, higher sample throughput, increased fl exibility and reduced exposure of labo-ratory staff to potentially hazardous sol-vents. Automated SPE can be performed using two different set-ups: The SPE sys-tem can be coupled directly to the HPLC-MS/MS instrument, enabling direct sample introduction of the extracts and fully auto-mated operation from SPE to LC-MS/MS. Alternatively, the SPE system can be oper-ated as a sample preparation workstation separate from the analysis instruments. The WorkStation set-up provides the fl exibility of choosing between different techniques or different instruments for the sample anal-

ysis in order to meet individual requirements.

Instrumentation and Methods River water and waste water samples that had been spiked with PFCs at differ-ent concentration levels were analyzed. Method development as well as the en-suing analysis was performed using an LC-MS/MS system from Agilent Tech-nologies (1200 Series LC and 6400 Series Triple Quad LC/MS) combined with a GERSTEL SPE system. Two SPE systems were used, one based on the single rail MultiPurpose Sampler and one based on the dual rail MPS PrepStation, which offers additional liquid handling capabilities. The following compounds were determined us-ing automated SPE-HPLC-MS/MS:

Perfl uorodecanoic acid (PFDA)•

Perfl uorononanoic acid (PFNA)•

Perfl uorooctanoic acid (PFOA) •

Perfl uorooctanesulfonate (PFOS) •

Perfl uoroheptanoic acid (PFHpA) •

Perfl uorohexanoic acid (PFHxA) •

Perfl uoropentanoic acid • (PFPeA)

Isotopically labeled standards are used for quantitation in the ISO 25101 standard. In this work, perfl uorobutanoic acid was used since this compound was not found in the samples.

HPLC ParametersHPLC System: Agilent 1200 SLHPLC COlumn: Maisch Reprosil C18HD, 50 x 2.1 mm, 3 µmFlow: 0.3 mL/minEluent: Ammonium acetate/ Methanol (MeOH)Gradient: 0 min 20 % MeOH 10 min 100 % MeOH 14 min 100 % MeOH 15 min 20 % MeOHAnalysis time: 25 minInjection volume: 2 µL

MS ParametersMSD: Agilent 6410 Triple QuadrupoleIonization mode: ESI neg.N2 Temperature: 350 °CN2 Flow: 10 L/minMS Mode: MRM (multiple reaction monitoring)Mass transfers: PFDA 513->469 m/z PFNA 463->419 m/z

PFOA 413->369 m/z PFOS 499->99 m/z PFHpA 363->319 m/z PFHxA 313->269 m/z PFPeA 263->219 m/z

The SPE-LC/MS/MS System used for this work. The dual rail MPS enables additional automation of liquid handling steps performed during sample preparation.

3GERSTEL Solutions Worldwide – LC/MS Special

SPE MethodThe GERSTEL MPS and GERSTEL Prep-Station (Dual rail MPS) are both multi-functional autosamplers and sample prep-aration robots for GC/MS and LC/MS.

In addition to automated SPE using stan-dard cartridge formats, practically all stan-dard sample preparation techniques used for LC/MS and GC/MS can be performed auto-matically. This provides the user with signi-fi cant added value: Method development tasks can be performed in a highly fl exi-ble manner and routine analysis chores handled effi ciently and productively with minimal intervention. Among other bene-fi ts, the SPE systems are controlled through the MAESTRO software or integrated with the Agilent ChemStation and MassHunter. Just one sequence table is required to op-erate the entire system from SPE through liquid sample prep and sample introduc-tion to GC/MS or LC/MS analysis. Every-thing is performed using just a few mouse-clicks. The PrepAhead function enables the system to plan ahead delivering time-opti-mized sample preparation. Samples are pre-pared just-in-time for introduction exactly when the LC/MS system becomes ready af-ter the previous run, ensuring that the LC/MS is utilized to its fullest capacity. In this work, the sample preparation steps were completed in around 25 minutes.

Results and DiscussionThe automated SPE-LC-MS/MS method presented in this work resulted in excellent calibration, sensitivity, recovery and repro-ducibility results. System performance was productive, reliable and rugged. Automated SPE is performed by the MPS or MPS Prep-Station using commercially available stan-dard volume cartridges. This means that existing manually performed SPE meth-ods can easily be transferred to the auto-mated system as is shown in the case of the ISO 25101 method. For every sample, a new cartridge is used eliminating the risk of cross contamination. Thanks to the PrepA-head function, LC/MS analysis of the cur-rent sample and SPE of the next sample in the sequence table are performed simulta-neously, ensuring maximum productivity. For the PFC method, sample preparation took 25 minutes to complete. Apart from the time required to prepare the fi rst sam-ple for introduction, the LC/MS system is never slowed down by the sample prepara-tion. Calibration based on samples spiked with 5 - 500 ng/mL of PFCs produced ex-cellent linearity and reproducibility. Rela-tive standard deviations were between 1 and 3 % depending on the compound. Follow-ing 2.5-fold enrichment of the SPE eluate, limits of quantitation were 0.5 ng/mL. Au-tomated enrichment/concentration of the SPE eluate up to a factor 100 is possible and this would lead to a further signifi cant re-duction in the limit of quantitation.

The sample is transferred to a 20 mL vial and placed in the GERSTEL SPE system.

The cartridge is conditioned using 2 mL MeOH/NH3, 2 mL MeOH and 2 mL H2O

10 mL sample is added to the cartridge

Cartridge is dried for 1 min with a fl ow of N2

2 mL acetate buffer is added to the cartridge

Cartridge is dried for 1 min with a fl ow of N2

Elution with 2 mL MeOH and 2 mL NH3/MeOH

Introduction of the eluate to the HPLC-MS/MS system

Optional: Evaporative concentration of the eluate under a fl ow of N2 at a preset temp.

This diagram shows the individual steps in the SPE process as performed by the MPS- or MPS PrepStation based SPE systems. Standard format cartridges of the type Oasis WAX 150 mg 6cc were used.


Just like the members of the infamous „dirty dozen“, i.e. the persistent organic pollutants (POPs), PFCs are ubiquitous. Mainly through industrial waste and waste water as well as through sludge, they end up in the environment. Without exaggeration, it is safe to say that PFCs are found in surface-, run-off- and ground water across planet Earth. They subsequently accumulate in the food chain, found in the livers of polar bears as well as in human blood.

Conclusion and outlookThe automated SPE-LC/MS/MS method for determination of PFCs in water and sludge proved its worth in all aspects. The SPE process is performed using positive displacement pumping of sample and elu-ent, reducing matrix and restriction varia-tion effects in the SPE cartridge in a simple and effi cient manner. The automated meth-od produces results of signifi cantly higher quality than the ISO 25101 method in terms of both sensitivity and reproducibility, es-pecially when analyzing diffi cult samples such as waste water.

The results of our work indicate that the automated method described in this work would be highly suitable for the determi-nation of PFCs in other matrices such as urine and blood.

An Automated Weighing Option is avail-able for the GERSTEL MultiPurpose Sampler (MPS) based on a Sartorius ME laboratory balance. Standard autosam-pler vials are placed in the balance by the MPS and liquid samples, standards or diluents added, weighed and regis-tered separately. For each sample, mul-tiple liquid additions can be defi ned by mouse-click in the MAESTRO Software. Results are automatically transferred to pre-defi ned Microsoft Excel tables for convenient processing. The MPS Weighing Option simplifi es the labo-ratory workfl ow and reduces the risk of operator error for improved conve-nience and productivity.

Weighing Option

Automated weighingof liquid additions



Automated Sample Preparation

Doing Drugs ?

Comprehensive Analysis of Drugs of Abuse in Blood and Urine with Automated Disposable Pipette Extraction and HPLC/MS/MS.

I n order to analyze biological specimens for drugs and their metabolites, it is nec-essary to perform sample preparation to

eliminate matrix interference. Solid-phase extraction is generally the preferred sample preparation technique, in this study Dis-posable Pipette Extraction (DPX) was uti-lized. DPX is a novel dispersive solid-phase extraction technique that uses loosely con-tained sorbent in a disposable pipette tip. The sample is aspirated into the tip where it is actively mixed with the sorbent and forms a suspension. The main advantages of the DPX technology are that the extrac-tion is very rapid, minimal solvent waste is generated, and the entire process can be fully automated including introduction of the extract to the chromatographic system.

The GERSTEL MPS autosampler performs DPX extractions in approximately 5 min-utes using reversed phase (DPX-RP) or cat-ion exchange (DPX-CX) sorbent materi-al. For chemical analysis of target drugs, GC/MS or HPLC/MS/MS are generally the preferred techniques. The advantage of LC/MS/MS is that chemical derivatiza-tion of the analytes is not required, mak-ing sample preparation simpler and less time consuming. In addition, highly effi -cient ionization, in combination with tan-dem mass spectrometry results in high sen-sitivity and selectivity. This study focused on performing automated extraction of re-duced sample volumes coupled with LC/MS/MS to provide high throughput analy-sis “one sample at a time”. The sample prep-

aration time was decreased suffi ciently to allow the extraction of a sample during the chromatographic analysis of the previous sample in the sequence.

ExperimentalInstrumentation. Sample extraction and introduction was automated using a GERSTEL MPS dual rail PrepStation with DPX option (GERSTEL).

Analysis conditions LCMobile Phase: A - 4.5 mM Ammonium acetate B - MethanolGradient: Initial 90 % A / 10 % B 2 min 85 % A / 15 % B 3 min 65 % A / 35 % B 4 min 50 % A / 50 % B 6 min 35 % A / 65 % B 8 min 90 % A / 10 % B (4 min)Flowrate: 350 µL/minColumn: 2.1 mm x 30 mm, 3.5 µm, Eclipse XDB C18 (Agilent)Inj. volume: 10 µL

Analysis conditions MSPositive ion mode, Single reaction monitoringRun time: 10 minCapillary: 3 kVExtractor: 2.81 VSource Temp.: 130 °CDesolvation Temp.: 391 °C

Compound M + H Dwell Cone Time Voltage [m/z] [ms] [V]

d3-Oxymorphone 305 50 30Oxymorphone 302 50 30Morphine 286 50 30Hydromorphone 286 50 30d3-Oxycodone 319 50 30Oxycodone 316 50 306-MAM 328 50 30Codeine 300 50 30Hydrocodone 300 50 30

Sample preparation. All opiate standards were obtained from Cerilliant (Round Rock, TX). A 10 ppm stock solution was prepared in methanol for all sample forti-fi cations. Two internal standards were used, d3-Oxymorphone for quantitation of Oxy-morphone and d3-Oxycodone for all oth-er opiates. All solvents used were of HPLC grade or higher.



stated at 70°C for 2 hours, and then cooled to room temperature. To precipitate pro-teins, 250 µL of acetonitrile was added to the hydrolyzed urine and the sample was vortex mixed and centrifuged. The super-natant was decanted into a clean labeled sample tube. 200 µL of 0.1 M HCl was add-ed to the sample solution, and the sample tube was placed on the MPS sample tray for automated DPX extraction.

Extraction. A GERSTEL MPS dual rail PrepStation was set up with 1 mL DPX-CX tips (DPX Labs, LLC, Columbia, SC) for extraction of drugs from blood and hydrolyzed urine. The following automa-tion method was used: 250 µL of 30% ac-etonitrile/water was slowly added through the top of the DPX tip at a rate of 50 µL/s to wet the sorbent. The sample was then aspi-rated into the DPX tip at a rate of 90 µL/s and mixed with the sorbent by drawing in an additional 2 mL of air. After a 30 s equil-ibration time to allow analyte binding, the resulting solution was dispensed to waste. To wash off excess matrix, a 500 µL wash of 10% acetonitrile/water was added to the sorbent material through the top of the DPX tip and dispensed to waste followed by an additional wash using 500 µL of ac-etone. For elution of the analytes, 700 µL of 78/20/2 (v/v) of methylene chloride/iso-

Blood sample preparation. A 250 µL blood sample was spiked at the specifi ed concen-tration with the stock opiates mix. To pre-cipitate proteins, 500 µL of acetonitrile was added and the solution was vortex mixed and centrifuged. The supernatant was de-canted into a clean labeled sample tube. 100 µL of 0.1 M HCl was added to the so-lution, and the sample tube was placed on the MPS 2 sample tray for automated DPX extraction.

Urine sample preparation. 50 µL of 0.6 M sodium acetate buffer (pH = 4) and 10 µL of ß-glucuronidase was added to a 200 µL sample of urine. The solution was thermo-

Automated DPX process

propanol/ammonium hydroxide was add-ed to the sorbent material through the top of the DPX tip and dispensed directly into a clean HPLC vial. All eluents were dried and reconstituted with 100 µL of metha-nol and 400 µL of 4.5mM ammonium ac-etate before injection.

Results and Discussion The DPX-CX extractions were readily per-formed using the GERSTEL MPS dual rail PrepStation. These DPX tips are ideal for basic drugs due to their mixed-mode cat-ion exchange and reversed phase charac-teristics. The entire extraction process took approximately 5 minutes per sample. Be-cause a basic eluent is used with the cat-ion exchange sorbent, the eluents had to be solvent exchanged into the HPLC mo-bile phase. The extract was dried in about 4 minutes using low heat and nitrogen gas fl ow. All HPLC/MS spectra were collected using single reaction monitoring (SRM) because under the HPLC conditions used we were unable to generate quality daugh-ter ions for the opiate drugs using multi-ple reaction monitoring (MRM). Although SRM MS analysis may not provide the best sensitivity for the analysis of these drugs at low concentrations, this study focused on the automated DPX extraction and the utility of this automated sample prepara-

4 Extracted analytes are eluted using a suitable solvent, which is added from above for most effi cient elution. The eluate is collected in a vial for subsequent sample introduction to LC/MS or GC/MS.

The total time required for extraction in the examples shown in this article was always less than 6 minutes. Sample preparation and GC/MS or LC/MS determination can be performed in parallel for best possible throughput and system utilization.

All steps are performed automatically by the MPS.If needed, the sorbent is conditioned with solvent prior to the extraction process.

1 Sample is drawn into the pipette tip for direct contact with the solid phase sorbent. There is no contact between the sample and the syringe used to aspirate the sample and therefore no risk of cross contamination.

2 Air is drawn into the pipette tip from below through the frit. Turbulent air bubble mixing creates a suspension of sorbent in the sample, ensuring optimal contact, highly effi cient extraction, and high recovery.

3 The extracted sample is discharged, typically after 30 seconds.

If needed, the sorbent can be washed to remove unwanted residue.



Extracted ion chromatogram of a DPX extract of whole blood spiked with 100 ppb of the opiate mix and with 400 ppb of the internal standards (d3-oxymorphone and d3-oxycodone) in whole blood. Even when performing SRM MS analysis, the sensitivity is more than suffi cient, demonstrating the high ionization effi ciency of the electrospray system. Most importantly, no matrix effect or ion suppression was observed, showing that sample extraction and cleanup with DPX is well suited for the analysis. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydromorphone, (5) d3-oxycodone, (6) oxycodone,(7) 6-MAM, (8) codeine, and (9) hydrocodone.

tion for HPLC/MS analysis of opiates. A rapid resolution HPLC column was cho-sen to generate chromatographic data in less than 10 minutes.

ConclusionAutomated DPX extraction of opiates from biological specimens can be performed successfully using the GERSTEL MPS du-al rail PrepStation. In the work presented here, the total extraction time was 5 min-utes, additionally 4 minutes were required for evaporation and solvent exchange. The total sample preparation time was less than the chromatographic run time, which means that the next sample can be prepared

Overlay total ion LC/MS chromatograms of DPX extracts of a blank blood sample and of a matrix-matched sample, both spiked at 0.5 ppm. The overlay shows that interferences are negligible

Recovery and % RSD for opiates (400 ppb) extracted from whole blood.

Extracted ion chromatogram of a DPX extract of whole blood spiked with 400 ppb of opiates. The chromatogram is free from interferences, the opiates were extracted reproducibly and with high recoveries. It is noteworthy that this blood sample was only 0.25 mL. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydromorphone, (5) d3-oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, and (9) hydrocodone.

Recovery and %RSD for opiates (500 ppb) extracted from urine.

Analysis of urine spiked with 500 ppb of opiates. Again, extracts were free from interfer-ences. No matrix effect or ion suppression for opiates was seen. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydromorphone, (5) d3- oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, and (9) hydrocodone.

while separation of the current sample is in progress. Whenever the LC/MS/MS sys-tem has fi nished a run, the next sample is ready to be introduced ensuring the high-est possible throughput. Additionally, “just in time” sample preparation helps to ensure that the prepared sample is not kept in the autosampler for a long time prior to being analyzed, reducing the risk of analyte deg-radation and helping to maintain sample integrity. The DPX-CX tips work very well for extraction of opiates, recoveries were in the range from 60 to 85% with RSD’s be-low 6%. Future work will focus on deter-mining the lower limits of detection and quantitation using tandem mass spectrom-

etry with multiple reaction monitoring. Al-so, automated DPX combined with HPLC/MS/MS will be optimized for other drugs and metabolites.

Sparkle T. Ellison, William E. Brewer, Stephen L. MorganDepartment of Chemistry and Biochemis-try, University of SouthCarolina, 631 Sumter Street, Columbia, SC 29208, USA

Fred D. FosterGerstel, Inc., 701 Digital Dr. Suite J,Linthicum, MD 21090, USA


Quality control and food safety

Trout Malachite Green

Norbert Helle, Ph.D. and Martina Bohlje (TeLA GmbH, Bremerhaven), Jürgen Wendt, Ph.D. (Agilent Technologies, Waldbronn), Frederick D. Foster (GERSTEL, Inc., Balti-more, USA), Carlos Gil (GERSTEL GmbH & Co. KG, Mülheim an der Ruhr)

T he triphenyl methane dye malachite green (MG) is highly effi cient in bat-tling fungi, bacteria and various single

cell parasites. MG, however, is under sus-picion for being a human carcinogen and for causing damage to genetic material if it reaches the human organism through con-sumption of contaminated foods.

Malachite green (MG) is traditionally administered as a fungicide in aquaculture, either as treatment or to prevent infections. Once inside the fi sh organism, MG is metab-olized and reduced to leucomalachite green (LMG) which accumulates in fatty tissue. Fish that are contaminated with MG or LMG should not be consumed since they pose a health risk. In 2003, the EU Commission set threshold value of 2 µg/kg as the upper con-centration limit for MG and LMG.

A fish filet sample was homogenized with a water/acetonitrile mixture, extract-ed, centrifuged and the supernatant collect-ed. The extraction procedure was repeated twice. The extracts were subsequently com-bined and concentrated before being taken up in a mixture of water and ethanol. Sam-ple clean-up was performed using automat-ed SPE in a GERSTEL MultiPurpose Sam-pler (MPS).

The MPS was integrated in an Agi-lent 1100 LC/MS Iontrap System, consist-ing of a binary pump, a thermostated col-umn compartment, a Diode Array Detec-tor and an XCT+ Iontrap-MS. The LC/IT-MS was used in Electron Spray Ionization (ESI), positive ion mode. The injection vol-ume used for all determinations was 5 µL. The separation was performed on a Zor-bax SB-C18 column (50 x 2.1 mm, 1.8 µm) with a fl ow rate of 0.6 mL/min in gradi-ent mode (Eluate A: 0.1 % formic acid, el-uate B: acetonitrile). The column was kept at 50 °C. The complete system control, in-cluding sample preparation, sample intro-duction, LC/MS analysis and data handling, was performed using the GERSTEL MAE-STRO Software integrated with the Agilent Technologies ChemStation Software (Rev. A10.03).

Malachite green (MG) and its metabo-lite leucomalachite green (LMG) are easi-ly ionized using Electron Spray Ionization (ESI) in positive ion mode. MG differs from LMG in that it forms a doubly charged ion (m/z 166) in addition to the single charged molecular ion [M+H]+. This is due to the non-planar sterical organization of the cen-tral carbon in the leuco form. In MS2 mode, the MG-precursor ion forms a product ion (m/z 313), while the doubly charged LMG precursor also forms a doubly charged frag-ment. The transition can be used for high-ly sensitive determination of LMG. Using these transitions, limits of determination of 0.5 µg/kg for MG and 0.05 µg/kg for LMG can be achieved.

Even though malachite green (MG) is banned as a veterinary pharmaceutical for animals used for human consumption, authorities regularly fi nd residues of this toxic compound or

its metabolites during routine checks of fi sh farms. Scientists from GERSTEL, TeLA and Agilent Technologies have succeeded in improving detection limits and in auto-

mating sample preparation for the determination of MG and its metabolite leucoma-lachite green (LMG) in fi sh products using automated SPE coupled with LC/Iontrap-MS.

Automated SPE directly coupled with the LC/MS system provides recoveries as high as 90 % and excellent reproducibili-ty for the SPE step. Additionally, automat-ed SPE reduces the time required for sam-ple preparation by 50 % compared with the manual procedure.

The described automated SPE/LC/IT-MS system enables automated sam-ple cleaning and sample preparation fol-lowed directly by injection and analysis of the generated extracts. The sample prep-aration method is easily adapted to indi-vidual requirements by selecting the de-sired steps from a simple menu by mouse-click. The entire method including sam-ple introduction, LC/MS analysis and da-ta handling steps is performed using one integrated method and one sequence ta-

ble from within the Agilent Technologies ChemStation Software.

The sample clean-up steps ensure the removal of interfering matrix residue leading to signifi cantly bet-ter signal to noise ratios and improved detection limits for MG and LMG in the MS system. The method is rug-ged and stable. RSDs range from 3.4 % to 5.3 % while recoveries are in the range from 89.5 % to 90.3 %.

Mass spectra of malachite green (MG)and leucomalachite green (LMG)

MS2 spectra of MG and LMG Calibration curve

for leucomalachite green

Leucomalachite green (LMG)Leucomalachite green (LMG)

Malachite green (MG) Malachite green (MG)


Trout Malachite Green

Norbert Helle, Ph.D. and Martina Bohlje

Even though malachite green (MG) is banned as a veterinary pharmaceutical for animals used for human consumption, authorities regularly fi nd residues of this toxic compound or

its metabolites during routine checks of fi sh farms. Scientists from GERSTEL, TeLA and Agilent Technologies have succeeded in improving detection limits and in auto-

mating sample preparation for the determination of MG and its metabolite leucoma-lachite green (LMG) in fi sh products using automated SPE coupled with LC/Iontrap-MS.


Pesticide analysis EZ

Application specialists from TeLA GmbH have developed a new method that dramatically simplifi es LC/MS determination of pesticide levels, providing high-quality results independent of the sample matrix type and complexity.

Norbert Helle, Ph.D. and Meike Baden, TeLa GmbH, Bremerhaven, Germany


P esticides, fungicides and herbicides are needed in order to provide an adequate supply of food to the ever-growing hu-

man population across the world. The other side of the coin is that residues of these types of compounds in foods cannot be allowed to endanger or affect the health of the con-sumer. To ensure that foods do not endanger us, maximum acceptable levels, sometimes referred to as tolerated levels, have been es-tablished for individual compounds accord-ing to the current state of scientifi c knowl-edge. If these levels are exceeded, it would be illegal to market the contaminated product in Europe. Corresponding laws were estab-lished by the EU. This legal basis must be, or, in some cases, already has been, adapted into National law by EU member states.

In Germany, details on maximum ac-ceptable levels of residues can be found in the German LFGB, acronym for the com-pendium of laws governing Food, Feed and various consumer products. As an aside, the term “consumer products” in this con-text spans a great variety of products rang-ing from packaging that comes into con-tact with food, feed or personal care prod-ucts to personal care products themselves, such as cosmetics, tooth paste or sham-poo or other personal care items that make more than brief contact with skin or mu-cous membranes. Rules, unless properly en-forced, are of course worth less than the pro-verbial paper they were printed on. In oth-er words trust is fi ne, but we should verify and if needed take corrective action to en-sure compliance and best possible consum-er safety. This requires a network of reliable laboratories, which is not a trivial matter, as can be seen later in this text.

World-wide, around 700 pesticides are in use, very few of which can be legally used throughout Europe. Vari-ous compound classes have been established, but even these can cover a wide range of polarities, making it diffi -

cult to develop a fast, all encompassing ana-lysis method.

Still, effective multi-residue methods are in use for the determination of pesti-cides, helping to ensure food safety. When fruit and vegetables are analyzed for pes-ticide residues, often several pesticides are found. The effects on human health have only been documented for very few of these compounds or compound groups.

Tracking down pesticides using GC/MS and LC/MSClassical pesticide analysis relied on gas chromatography (GC) using an electron capture detector (ECD) or a nitrogen phos-phorous detector (NPD). The most wide-ly used detector today is the mass selective detector (MS).

In Germany, the analytes that are main-ly in focus are those listed in the DFG S19 method, a multi-residue method for the determination of pesticides in food, which enjoys Europe-wide recognition. The anal-ysis of the 270 compounds listed in the S19 method does, however, require signifi cant sample preparation including a gel chroma-tography clean-up step to separate analytes from the matrix.

Different analysis techniques are used for different types of pesticides. Liquid chro-matography (LC) combined with a mass selec-tive detec-tor (MS) is used to de-

termine polar to moderately apolar com-pounds. Gas chromatography (GC), most often in combination with a mass selective detector (MS) covers apolar to moderately polar compounds. As can be seen from this description, there is some overlap between the techniques. Recently a new multi-resi-due method for the determination of pesti-cide levels in fruits and vegetables was pre-sented (QuEChERS: Quick, Easy, Cheap, Ef-fective, Rugged & Safe) [*]. Compared to previous methods, the QuEChERS sample preparation steps are much less time-con-suming, enabling the preparation of 8 sam-ples in less than 30 minutes. QuEChERS is a sample preparation method well suited for both GC, GC/MS and LC/MS analysis. The QuEChERS sample preparation steps are listed below.

The main benefi t of this sample prepara-tion method is that the overall analysis is less time-consuming and less error-prone than more traditional approaches. Unfortunate-ly, extracts obtained following this proce-dure often have a high matrix content, which causes chromatographic problems for GC analysis due to residue build-up in the liner unless an automated liner exchange system such as the GERSTEL ALEX is used. (Cf.: GERSTEL Solutions Worldwide Magazine No. 5 p. 18)

When the sample matrix no longer matters

Fully automated Sample clean-up and Pesticide Screening with Agilent 6410 LC/MS QQQ online SPE System, Agilent ordering Number: 5990-3866EN

Fully automated Sample


QuEChERS method:

Weigh 10 g of sample –> Add 10 ml of Acetonitrile (AcN)Shake vigorously 1 min –> Add 4 g MgSO4 and 1 gNaClShake vigorously 1 min –> Add internal standard solutionShake 30 sec and centrifuge –> Take Aliquot of supernatant –> Add MgSO4 and sorbentShake 30 sec and centrifuge –> Take Aliquot of supernatant –> inject to GC-MS and LC-MS

The results obtained using QuEChERS sample preparation are comparable to those reached using the S19 method. The QuECh-ERS method is much faster, requires much less sample preparation, covers a wider range of analytes and is more readily automated. In addition, much smaller volumes of part-ly toxic organic solvents are required, com-pared with other currently used methods for determining pesticides in fruit and veg-etables. In addition to the fi nancial bene-fi ts of a much higher laboratory through-put, the cost of materials at around one Eu-ro per sample is relatively low.

The limits of QuEChERS are encoun-tered whenever samples with more complex matrices need to be analyzed, such as gar-lic, onion, artichoke or avocado with much higher fat content. This can lead to problems with interferences, that can especially infl u-ence quantifi cation unless further clean-up steps are performed.To enable reliable and rugged analysis inde-pendent of the sample matrix, we looked for a similarly effective alternative sample prep-

aration procedure. We found that automat-ed solid phase extraction (SPE) based on the GERSTEL MultiPurpose Sampler (MPS) provided an excellent solution. The GER-STEL SPE, we have previously used success-fully for a number of applications, includ-ing afl atoxins, chloramphenicol and mala-chite green in foods. In summary, we can re-port that our automated SPE-LC-MS/MS-ESI multi-residue method reduces the num-ber of manual steps required to a minimum while increasing laboratory throughput. The results are solid and reproducible combined with high sensitivity and good limits of de-termination.

Instrumental requirements The GERSTEL SPE was fi tted with an injec-tion valve; sample introduction to the Agi-lent LC 1200 was performed directly by the SPE system; detection was performed using an Agilent 6410 MS/MS Triple Quad instru-ment. Sample Preparation: 15 mL of an ace-tonitrile/water mixture (80:20) was added to a fi ve gram sample of fruit or vegetable for extraction. The SPE cartridge (M&N C-18ec, 6 mL, 1 g) was conditioned using 10 mL methanol (MeOH) and 10 mL water. All steps in the sample preparation proce-dure, including sample introduction were fully automated.

5 mL sample was added to the cartridge, which was subsequently rinsed with 5 mL water. Analytes were then eluted using an acetonitrile/water mixture added at a fl ow rate of 600µL/min. In contrast to most man-ual SPE methods, the liquid is not aspirated through the cartridge under vacuum, rath-er it is added under positive pressure using a syringe. This means that fl ows, and there-fore also the elution speed, are accurately controlled and results more reproducible. This holds true even when sample matrix changes the restriction across the cartridge. The eluate was concentrated for six minutes at 50 °C and the residual analytes taken up in 5 mL of a acetonitrile/formic acid mix-ture (30:70).Sample introduction and analyte separa-tion: 20 µL of the cleaned-up extract was introduced directly to the LC/MS-MS Sys-tem. The temperature of the column (Zor-baxXDB C-18 100x2.1 mm, 1.8 µm rapid resolution) was set to 50 °C; fl ow rate: 0.5 mL/min resulting in a column head pressure of approximately 420 bar. A solvent mixture of 5mM formic acid (A) and acetonitrile (B) was used as mobile phase based on the fol-lowing gradient programming: 0 min (20 % B); 5 min (20 % B), 30 min (90 % B). Detection: Analytes were detected with pos-itive Electron Spray Ionization (ESI) using the electron spray ion source or, alternative-ly, the Agilent Multimode ion source. Our experiments clearly showed that the Mul-timode source provided signifi cantly lower detection limits for some pesticides than the ESI source. For other compounds, howev-

er, a lower response was obtained than with the ESI ion source. The settings for the ion source were optimized for the fl ow and el-uent used. The following parameters were used: N2 temperature: 340 °C; carrier gas fl ow (N2): 9 L/min; nebulizer pressure: 30 psi. The triple quadrupole instrument was operated in MRM mode, with 5 different time segments, monitoring two transitions for each pesticide. In each segment 40 to 50 analytes were monitored.

The proof of the puddingWhen using the QuEChERS method, it is necessary to adapt the clean-up steps to the sample at hand. It has been clearly shown that for “uncomplicated” matrices, such as lettuce or cucumber, additional clean-up steps are not required following the ace-tonitrile/water extraction. For complex ma-trices that contain fat and other challenging matrix components, further clean-up steps are of course needed. For this purpose we used the GERSTEL SPE system.

Raw sample extracts were automatical-ly loaded onto standard SPE cartridges and cleaned. A new cartridge was used for every sample to eliminate cross-contamination. Macherey-Nagel cartridges containing C18 reversed phase material were found to pro-duce excellent, reliable results.

Automated SPE clean-up as described in this article took around 20 minutes to complete. Apart from the fi rst sample, the SPE process was performed during LC/MS or GC/MS analysis of the preceding sample, ensuring that the SPE step was performed without increasing the overall analysis time. Once the fi rst sample had been prepared for analysis, the LC/MS or GC/MS system never had to wait idly for the next sample.

An LC 1200 Rapid Resolution HPLC sys-tem from Agilent Technologies was used for the analysis. In order to achieve good sepa-ration combined with method ruggedness, the conscious decision was made to only seek a moderate reduction of the analysis time. The total analysis time required to de-termine around 140 compounds was in the order of 35 minutes. This time period was more than suffi cient to prepare the following sample for just-in-time sample introduction to the LC/MS system.

Sample clean-up using SPE contributes not only to the ruggedness of the method, it also improves reproducibility and linearity, among other things. To illustrate this, a bell pepper sample was spiked with a pesticide mixture and analyzed. Following SPE clean-up, retention times and peak areas of the an-alytes showed excellent reproducibility. The linearity was excellent, both for polar com-pounds like Carbendazim und Thiabenda-zole as well as for apolar pesticides like Di-azinon und Pirimiphosmethyl.

Orange oil samples were cleaned up us-ing a slightly modifi ed SPE method. The ef-fi ciency of SPE clean-up is illustrated by the fact that the intense yellow color of the sam-


Calibration curves for nine pesticides, determined using the TeLA GmbH SPE-LC-MS/MS pesticide multi-residue method.


ple was transferred to the cartridge while the resulting extract was a clear and colorless liq-uid. Recovery for the various compounds in this diffi cult matrix ranged from 70 to 90 % while recoveries from fruit and vegeta-ble samples were mainly in the range from 80 to 100 %. It is worth noting that the Zor-bax SB-C18 Rapid Resolution columns used provided excellent peak symmetry.

One fi nal comment: Every method must prove its worth in practice. The test, as al-ways, is in the analysis of real world samples. To prove the validity of our method, we took part in a Europe-wide round robin with 46 participating laboratories. A vegetable sam-ple (zucchini) had to be analyzed for 185 dif-ferent pesticide residues. Out of 46 labora-tories, TeLA GmbH was among the 12 that managed to correctly identify and quantify the analytes thus meeting the round robin requirements and passing the test.

128 of the 185 pesticides were deter-mined using our SPE-LC-MS/MS pesticide multi-residue method. 90 of the 185 pesti-cides were determined using a GC/MS sys-tem (GC 6890 / MSD 5973, both from Agi-lent Technologies) in combination with the GERSTEL MultiPurpose Sampler (MPS) us-ing a Retention Time Locking (RTL) meth-od.

[*] M. Anastassiades, S. Lehotay, D. Stajnbaher and F. Schenck: Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int 86 (2) (2003) 412-31.

Overlay medium polarity sections of 8 different chromato-grams: 8 separate sample preparations and injections of a bell pepper sample spiked with a standard mix-ture of pesticides, 100 ng/mL each. The peaks shown are for the pesticides Terbutylazin, Cyprodinil, Prochloraz, Flusilazol and Fenoxy-carb, all showing good reproducibility.

Carbendazim

ThiabendazolDetermination of polar and apolar pesticides respectively in orange oil. Overlay chromatograms covering 9 different concentrations are shown.


In LC/MS, we work towards the mutually irreconcil-able goals of achieving the perfect LC separation and combining it with the most effi cient ionization and lowest achievable MS detection limits for our analytes. The LC separation may require a certain pH and polarity range of the eluent, while analyte ionization in the LC/MS ionization source requires yet another pH, a different buffer – or even deriva-tization of the analyte for best possible effi ciency or optimized spectral information. How to optimize both? Well the logical answer is to take the effl u-ent from the perfect LC separation and then opti-mize it for MS analysis. This task is easily possi-ble when you add the GERSTEL LC/MS Effl uent Optimizer (LEO) module to your LC/MS/MS sys-tem. Application examples show sensitivity gains of up to a factor of 40 by simply adding a salt solu-tion to the LC effl uent and/or changing its pH. The LEO module is quickly and easily installed in your LC/MS system. A solvent mixture, buffer solution

or reagent is then added to the effl uent ensuring that the LC separation can be performed under optimal conditions while also enabling maximum yield in the MS ionization pro-cess. Whether you are looking to perform pH adjustment or post-column derivatiza-tion, for method develop-ment or routine analysis, when you use LEO and the GERSTEL MAESTRO soft-ware you can easily and ef-fi ciently control all param-eters as part of the overall method. Just one sequence table controls the entire system from sample preparation through LC separation and effl uent optimiza-tion to MS analysis. It is all done at the click of a mouse.

GERSTEL LC/MS Effl uent Optimizer (LEO)

Optimized LC separation andMS detection – get the best of both worlds

or reagent is then added to the effl uent ensuring that the LC separation can be performed under optimal conditions while also enabling maximum yield in the MS ionization pro-cess. Whether you are looking to perform pH adjustment

method. Just one sequence table controls the entire system from sample preparation through LC separation and effl uent optimiza-tion to MS analysis. It is all done at the

MS detection – get the best of both worlds


Fast and reliable answers regarding afl atoxins in foods

If you are going to determine the concentration of mycotoxins in foods, in pharmaceutical products, or in raw materials used in their production, you will probably rely on Solid Phase Ex-traction (SPE) combined with LC/MS analysis. This approach ensures that detection limits will be lower than the maximum

concentrations allowed by law. While long-established manual SPE procedures may leave little room for further optimization,

automation of the process can provide laboratories with more reli-able results in less than half the time.

The well-stocked cheese counter may seem to tell a different story, but who-ever consumes moldy foods – other

than mold cheese – is putting his or her health at risk. This is due to mycotoxins: Toxins that are created as metabolites by certain molds. Mycotoxins can lead to acute illness as well as chronic ailments, caused by carcinogenic, mutagenic and hormone active properties that are especially harm-ful to infants and toddlers.

To date, more than 300 mycotoxins, formed by approximately 250 mold types, have been found. For food safety purposes, however, only a few mycotoxins are of im-portance, such as those of the genus asper-gillus flavus and aspergillus parasiticus. These molds thrive, especially under hu-mid-warm conditions, on oily and starchy seeds such as peanuts, walnuts, hazelnuts, pistachios, almonds, fi gs, coco, grains, rice, corn and soy, as well as on dried fruits and spices.

High concentrations of a group of my-cotoxins called afl atoxins have been found, for example, in pistachios, fi gs and cereals. Afl atoxins are among the most potent hu-man carcinogens found in plants. The afl a-toxins B1, B2, G1, G2 and M1, produced by Aspergillus fl avus and Aspergillus parasiti-cus, belong to the most potent mycotoxins that exist. Afl atoxin B1 poses the greatest hazard of all due to its carcinogenic prop-erties. Because of the extreme toxicity of afl atoxins, EU legislation specifi es very low acceptable daily intakes and low maximum residue limits.

The risk of acute poisoning through high mycotoxin concentrations is relatively low in most of the developed world thanks to the overall good food quality. In Afri-ca and parts of Asia, things can be quite

different: Conditions for growing, storing and transporting agricultural products are frequently bad, resulting in moldy peanut or corn products. Consumption of moldy products regularly results in acute and even fatal afl atoxin poisoning. According to liter-ature references, the lethal dose for an adult is 1 to 10 mg per kilogram body weight.

Pervasive food contamination resulted in government regulation Mycotoxin contamination of food and feed is a global problem. The UN Food and Agri-cultural Organisation (FAO) estimates that up to 25% of the world’s food production is contaminated with mycotoxins. Approxi-mately 20% of the EU‘s cereal harvest con-tains detectable amounts of mycotoxins.

Due to the health risk posed by molds and due to their universal presence in cer-tain food products, maximum concentra-tion values for mycotoxins have been es-tablished in the range of a few micrograms per kilogram (µg/kg): For peanuts, indehis-cent fruits (mainly nuts), dried fruits and grain intended for direct consumption or for use in food products, maximum allow-able concentrations of 2 µg/kg afl atoxin B1 or 4 µg/kg total of B1, B2, G1 and G2 apply. The concentration of afl atoxin M1 in milk is not allowed to exceed 0.05 µg/kg. Regula-tions limit the acceptable quantity in foods for infants and toddlers to 0.05 µg/kg afl a-toxin B1, and 0.025 µg/kg M1.

Faster results and lower detection limitsThe method of choice for reliable and sen-sitive determination of afl atoxins is Solid Phase Extraction (SPE) or immuno-affi n-ity cartridges, combined with LC/MS anal-ysis. This approach ensures that detection

Health and Food Safety

Aflatoxin B1

OO

O

OO

O

O

OCH3

Aflatoxin G1Aflatoxin G1

OCH3

O

OOOO

O

O

OO

Aflatoxin G2Aflatoxin G2

OCH3

O

OO

O

OO OOO

O

Aflatoxin B2

OCH3O

OO

O

OOOO O

Chemical structure

of afl atoxins

Afl atoxins are a group of more than 20 different fl uorescent heterocyclic com-pounds, consisting of a dihydro or tet-rahydrofuran unit connected with a substituted coumarin ring. The toxico-logically relevant compounds are Afl a-toxins B1, B2, G1 and G2, with B1 oc-curring most frequently. Afl atoxin M1, found for example in milk and milk products, is a metabolite of Afl atoxin B1. M1 is formed in humans or in ani-mals when they have consumed food or feed contaminated with B1. The toxicity of Afl atoxin M1 is comparable to Afl atoxin B1; however, M1 is signif-

icantly less carcinogenic than B1.

BrBrominated aflatoxin B1ominated aflatoxin B1O O

O

O

O O

O

HH3C

CH3

Br

BrBrominated aflatoxin G1ominated aflatoxin G1 O O

O

O

O O

OO

H3C

CH3

Br



Monobrominated aflatoxins exhibit longer retention times than the non-brominated compounds, resulting in better separation of the four afl atoxins and reduced interference from matrix components (fi g. above). Deri-vatization of afl atoxins B1 and G1 results in signifi cantly improved MS responses combined with characteristic bromine patterns in the mass spectra (fi g. left): The detection limits for the examined afl atoxins are below 0.01 µg/kg.

limits will be lower than the maximum con-centrations allowed by law.

Norbert Helle, Ph.D., food safety analy-sis expert and owner of TeLA GmbH, a Ger-man contract laboratory based in Bremer-haven, explains the background behind some of his recent work on the determi-nation of Afl atoxins in foods: „Established sample preparation methods used in LC/MS determination of afl atoxin levels pro-vide only limited scope for optimization, but reliable and useful analysis results can be obtained in less than half the time if the SPE process is automated. In the case of afl atoxin determination, manual process-ing requires in the order of 4 hours for eight samples. The GERSTEL SPE requires only 80 to 95 minutes to prepare the same num-ber of samples, according to Norbert Helle.

„All steps from standard addition and derivatization through Solid Phase Extrac-tion to LC/MS analysis are fully automat-ed”, says the applications expert, while add-ing: “Software-controlled parallel process-ing of sample preparation and analysis en-sures that there is only negligible analyte decomposition. The preparation steps for each and every sample are performed at ex-actly the same point in time prior to anal-ysis. The GERSTEL SPE system provides on-time sample prep for best possible re-sults”.

Mr. Helle has developed an LC/MS method for the determination of B1, B2, G1 and G2 afl atoxins in foods such as pis-tachios, bell pepper seasoning and various fruits. Following clean-up on an SPE affi n-ity column, the two afl atoxin compounds

Further information: www.gerstel.com (application note 6 / 2007)

PrepBuilder method steps used for the automated SPE sample preparation described in this article. All steps are selected by mouse-click from a menu and added to the list.

GERSTEL MultiPurpose Sampler MPS with SPE option

MOVE

ADD 1

ADD 2

SPE-SHIFT

ADD 3

ADD 3

ADD 3

WAIT

SPE-SHIFT

MOVE

MOVE


with an isolated, non-conjugated, double bond, B1 and G1, are brominated by stir-ring the extract with a 3 % solution of bro-mine in chloroform. The mass spectra in-dicate that bromination results only in the formation of 1-methoxy-2-bromo-substi-tuted compounds. Under the chosen exper-imental conditions, dibromo-substituted afl atoxins are not detected. The 1-methoxy-2-bromo-substituted compounds show longer retention times in reversed phase chromatography than the non-brominat-ed species, resulting in baseline separation for the four afl atoxins with minimal inter-ference from residual matrix. Additional-

ly, the derivatized compounds yield signif-icantly better MS responses and the char-acteristic bromine pattern in the mass spec-tra provides improved differentiation from background signals and thus a better signal to noise ratio. These combined advantages enable the system to reach detection limits below 0.01 µg/kg for the afl atoxins.

MOVE

ADD 1

ADD 2

SPE-SHIFT

ADD 3

ADD 3

ADD 3

WAIT

SPE-SHIFT

MOVE

MOVE


Analyzing Chloramphenicolin half the time normally required

As many users will tell you, performing manual Solid Phase Extraction (SPE) requires a lot of time - and strong nerves when insuffi cient recovery and bad reproducibility are experienced. It can be hard to discern what went wrong: Was conditioning adequate? Did the cartridge run dry in an unguarded moment? Was the eluent fl ow rate too high? Did cartridges get mixed up? If only cartridges could speak to us. If the SPE process can be reliably automated, such questions will not arise in the fi rst place.The automated GERSTEL SPE System is based on standard cartridges making it easy to transfer and automate existing methods. Fully automated liquid handling and exact timing of all processes make SPE a more relaxing and much more effi cient activity.

Automated SPE option for the MultiPurpose Sampler (MPS)

based on standard SPE cartridges

T he antibiotic Chloramphenicol (CAP) is banned for use in food products of animal origin such as meat and fi sh that

are imported to Europe. CAP is a known hu-man carcinogenic, suspected of causing ge-netic damage in human cells as well as irre-versible damage to the blood-forming cells of the bone marrow.

The determina-tion of CAP is usu-ally performed by LC/MS. The sensi-tivity of the method depends greatly on sample preparation. A high matrix load can result in incor-rect quantification of CAP even when highly selective LC-MS/MS methods are used. The same is the case for many other analytes in the areas of food, pharmaceu-tical or environmen-tal analysis.

SPE is the sample preparation technique of choice for many such samples, separating analytes from the matrix prior to LC or GC determination. Manual SPE methods have serious drawbacks. A lot of time and patience is needed, recovery and reproducibility can be subject to extreme deviations. This large-ly depends on the experience of the user and on how meticulously each step is performed. If the SPE process and all associated liquid handling steps are automated, the process be-comes much more reliable and effi cient.

A GERSTEL MPS with Automated SPE Option coupled to an LC/MS system was used for the determination of chlorampheni-col (CAP) in food products of animal ori-gin. Manual SPE using standard cartridges can provide good results under tightly con-trolled conditions.

MPS with the Automated SPE Option provides slightly better results than manual SPE performed by an experienced and di-ligent technician. For CAP determinations

using the MPS, standard deviations were 2.0 % compared with 2.2 % for manual SPE. The recovery was 92 % on the MPS compa-red with 90 % for manual SPE. The MPS provided slight, but clear improvements over the best achievable manual results and, mo-re importantly, a big improvement in pro-ductivity.

Fragmentation spectrum of CAP

257.0

fragment

parent321.0

Prawn sample,spiked with 0.01 µg/kg CAP

1 pg CAP

2 4 6 8 10 min

Chloramphenicol

NO2 CH CH OHCH2

OH NH CO CHCI2

Thousands500

400

300

200 MPS

Recovery: 92 %

Relative Standard Deviation: 2.0 %

Manual

Relative Standard Deviation: 2.2 %

Recovery: 90 %

CAP manual SPE

CAP MPS

1 2 3 4 5 6 7 8

Chromatogram of transfer 321.0 – 257.0; detection of a CAP residue of 0.01 µg/kg in a prawn sample following automated SPE on the GERSTEL MultiPurpose Sampler MPS 2.

Recovery and reproducibility of chloramphenicoldetermination in prawn meat with manual andautomated sample preparation, respectively.

Further information: www.gerstel.com (application note 7 / 2006)



GERSTEL MAESTRO software

• Stand-Alone operation or integrated in the Agilent ChemStation or MassHunter Software

• One sequence table operates the entire system including LC/MS or GC/MS

• Sample Prep by Mouse-Click using the PrepBuilder functions

• Scheduler for easy planning• PrepAhead / Multiple Sample Overlap: Automated

overlapping of sample preparation and analysis for maximum throughput

• Priority samples can be added to the system at any point in the analysis sequence

• LOG fi le and Service LOG fi le functions ensure traceability

• Automated E-mail notifi cation if the sequence is stopped

• Control of up to 4 systems from one PC• Real-time monitoring of all modules and parameters • Interactive on-line help function Sample Prep by Mouse-Click

The MultiPurpose Sampler (MPS) is an autosampler and sam-ple preparation robot for GC and LC. Sample preparation steps are performed during the analysis of the preceding sam-ple for best possible system utilization and highest sample throughput. Sample preparation steps are performed in a con-trolled and highly accurate and reproducible manner for best possible results. Every step is selected by mouse-click from a pull-down menu in the MAESTRO software and added to the overall GC/MS or LC/MS method. Available sample prep techniques are:

• Solid Phase Extraction (SPE)• Disposable Pipette Extraction (DPX) • Internal standard addition • Weighing, Sonication, Centrifugation• Derivatization • Extraction and dilution • Heating, conditioning and mixing • Twister Back Extraction (TBE) • Automated Liner EXchange (ALEX) • Automated Twister desorption and analysis (SBSE)• Solid Phase Micro Extraction (SPME) • Thermal Desorption and Thermal Extraction (TDS/TDU) • Dynamic Headspace (DHS) • Multi Column Switching (MCS)

MAESTRO Software enables Sample Prep by Mouse-Click. All sample preparation steps are conveniently and easily selected from a drop down menu and added to the method. Example:

ADDAdd solvent, internal standard or reagent

MOVEMove the vial or cartridge

MIXAgitate or stir and incubate the sample at a set temperature

INJECTIntroduce an aliquot of the sample to the GC or LC system

MAESTRO Software enables Sample Prep by Mouse-Click. All sample preparation steps are conveniently and easily selected from a drop

MAESTRO Software enables Sample Prep by

Intelligent Sample Preparationby Mouse-Click (Part I)

Next generation software for automated sample pre paration and sample introduction. MAESTRO optimizes performance and throughput of GERSTEL modules and systems.



GERSTEL GmbH & Co. KGEberhard-Gerstel-Platz 145473 Mülheim an der RuhrGermany

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GERSTEL GmbH & Co. KGEberhard-Gerstel-Platz 145473 Mülheim an der RuhrGermany

+49 208 - 7 65 03-0+49 208 - 7 65 03 33

[email protected]

GERSTEL, Inc.Caton Research Center1510 Caton Center Drive,Suite HBaltimore, MD 21227USA

+1 410 - 247 5885+1 410 - 247 5887

[email protected]

G L O B A L A N A L Y T I C A L S O L U T I O N S

Subject to change. GERSTEL®, GRAPHPACK® and TWISTER® are registered trademarks of GERSTEL GmbH & Co. KG.Printed in Germany · 0208b · © Copyright by GERSTEL GmbH & Co. KG

GERSTEL K.K.2-13-18 Nakane, Meguro-ku152-0031 TokyoDai-Hyaku Seimei ToritsudaiEkimae Bldg 2FJapan

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[email protected]

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[email protected]

Ask us how GERSTEL technology can benefi t youAsk us how GERSTEL technology can benefi t you

Ultrasonic Bath improved sample preparation

Bar Code Reader tracking and ID verifi cation of samples and extracts

Heated Agitator highly controlled reactions such as analyte derivatization

Weighing Option automated weighing of liquids and liquid additions

Centrifuge separation of matrix and extract

And more ... You name it!

Intelligent AutomatedSample Preparation (Part II) for LC/MS

www.gerstel.com

Additional techniques, now available from the leader in automated sample preparation:

gerstel solutions lc/ms special issuems parameters msd: agilent 6410 triple quadrupole ionization...

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