Meeting the challenges of EU

regulations with up-to-date

confirmatory solutions

Lunch Seminar at the

34th International Symposium on Halogenated

Persistent Organic Pollutants (Dioxin 2014)

Validation of a triple quadrupole GC-MS/MS

technology for detection, quantification and

confirmation of low level dioxins in feed

and food samples

Cristian Cojocariu, PhD

ThermoFisher Scientific, Runcorn, UK

3

Overview

• Thermo Scientific™ TSQ 8000™ Evo triple quadrupole GC-MS was used to

detect and confirm low level of PCDD/Fs in sample extracts, taking into account

the EU Commission Regulation 589/2014 and the new Regulation No. 709/2014

• A thorough validation of the TSQ 8000 Evo GC-MS/MS for dioxin confirmation

was performed.

• Instrument sensitivity, linearity, chromatography, LOD/LOQs and precision of

measurements were assessed.

• Data acquired using the GC-MS/MS was compared with the GC-HRMS data

acquired for the same samples and measurements uncertainties were

assessed.

4

EXPERIMENTAL

5

TSQ 8000 Evo GC-MS System

• State of the art triple quadrupole GC-MS system introduced at ASMS 2014

• Fast collision cell instrument with enhanced velocity optics with EvoCell technology

to provide high SRM transition speeds, precision and sensitivity

• ExtractaBrite™ ion source is heated throughout ensuring high matrix tolerance

• Ion source fully removable, hot, under vacuum when cleaning is necessary or

swapping with a spare; no vent to clean your instrument

6

GC & MS Conditions

Thermo Scientific™ TRACE™ 1310 GC Parameters

Column TG-5 SIL MS, 60 m x 0.25 x 0.25

Injection Volume (mL): 2

Liner SSL single taper 4mm ID 78.5mm

Inlet (°C): 260

Inlet Module and Mode: splitless

Carrier Gas, (mL/min): He, 1.2

Oven Temperature Program time (min): 33

TSQ 8000 Evo Mass Spectrometer Parameters

Transfer line (°C): 280

Ionization type: EI

Ion source(°C): 300

Electron energy (eV): 40

Acquisition Mode: SRM

Q1 Peak Width (AMU): 0.7

Q3 Peak Width (AMU): 0.7

GC

MS

7

Samples

• Wellington Lab standards used in the experiments described below:

EPA1613 calibration set TF-TCDD-MXD

Compound Concentration (fg/µL)

1368-TCDD 10

1379-TCDD 25

1378-TCDD 100

1478-TCDD 250

1234-TCDD 500

2378-TCDD 1000

8

Samples

• Matrix samples (previously analysed on a GC-HRMS instrument) provided by EU-RL, Freiburg

• Samples chosen were naturally contaminated with low level of PCDD/Fs.

• Used to assess and validate the LOQs in matrix samples as well as to check precision of total dioxin

content at low level.

• Extraction and clean-up was carried out in accordance with the existing methods for food and feed

either automatically (PowerPrep™ SPE) or partly manual/partly automated (GPC, multi-layer silica,

Florisil®, carbon).

Mixed animal fat Fish meal Egg fat 11 Egg fat 2

Weighted sample: 10.0 g fat 60.2 g sample (= 10.0 g fat) 0.52 g fat 0.51 g fat

Lipid content [%]: n/a 16.6 n/a n/a

Moisture content [%]: n/a 5.3 n/a n/a

Final volume: 40 µl 40 µl 10 µl 10 µl 13C Internal standard 13C Internal standard 13C Internal standard 13C Internal standard

2378 TCDF 50 50 10 10

12378 PCDF 50 50 10 10

23478-PCDF 200 200 40 40

123478 HxCDF 100 100 20 20

123678 HxCDF 100 100 20 20

234678 HxCDF 50 50 10 10

123789 HxCDF 50 50 10 10

1234678 HpCDF 100 100 20 20

1234789 HpCDF 50 50 10 10

OCDF 100 100 20 20

2378 TCDD 50 50 10 10

12378 PCDD 100 100 20 20

123478 HxCDD 100 100 20 20

123678 HxCDD 200 200 40 40

123789 HxCDD 50 50 10 10

1234678 HpCDD 200 200 40 40

OCDD 600 600 120 120 13C Recovery standard 13C Recovery standard 13C Recovery standard 13C Recovery standard

1234-TCDD 50 50 10 10

9

Data Acquisition and Data Processing

• Data were acquired using timed-Selected Reaction Monitoring

(timed-SRM).

• A minimum of 12 points/chromatographic peak were acquired.

• Data processing was performed with Thermo Scientific™ TargetQuan

3.1 software.

10

Linearity of Response

• Linearity assessed by determining the average RF %RSD values from a six point calibration

curve measured at the beginning and at the end of the sample batch.

• Values represent duplicate measurements of each calibration point, measured at the beginning

and end of a batch.

Compound

Concentration

range (pg/µL)

Average

RF stdev RF %RSD

2378-TCDF 0.1 - 40 1.0 0.04 3.7

2378-TCDD 0.1 - 40 1.4 0.02 1.4

12378-PeCDF 0.5 - 200 1.0 0.01 0.9

23478-PeCDF 0.5 - 200 1.0 0.02 1.8

12378-PeCDD 0.5 - 200 1.2 0.03 2.5

123478-HxCDF 0.5 - 200 1.0 0.01 0.8

123678-HxCDF 0.5 - 200 1.0 0.01 1.3

234678-HxCDF 0.5 - 200 1.0 0.01 1.3

123478-HxCDD 0.5 - 200 0.9 0.01 0.7

123678-HxCDD 0.5 - 200 1.1 0.01 1.3

123789-HxCDD 0.5 - 200 1.0 0.01 0.7

123789-HxCDF 0.5 - 200 1.0 0.05 4.5

1234678-HpCDF 0.5 - 200 1.1 0.01 0.9

1234678-HpCDD 0.5 - 200 1.1 0.01 1.0

1234789-HpCDF 0.5 - 200 1.1 0.02 1.4

OCDD 1.0 - 400 1.5 0.02 1.6

OCDF 1.0 - 400 1.1 0.02 1.4

11

Determination of Limit of Quantification

• LOQ calculated from CSL x5 and CSL x10 dilution and taking into account the ion

ratio, response factors and precision limits.

• LOQ for each native compound was calculated taking into account the student’s-t

critical values for the corresponding degrees of freedom (99% confidence).

Compound pg/µL

2378-TCDF 0.01

2378-TCDD 0.01

12378-PeCDF 0.02

23478-PeCDF 0.02

12378-PeCDD 0.02

123478-HxCDF 0.04

123678-HxCDF 0.04

234678-HxCDF 0.03

123478-HxCDD 0.06

123678-HxCDD 0.03

123789-HxCDD 0.04

123789-HxCDF 0.04

1234678-HpCDF 0.02

1234678-HpCDD 0.05

1234789-HpCDF 0.02

OCDD 0.05

OCDF 0.03

12

Sensitivity test: stepped standard

• Overlayed SRM chromatograms (quan ion) of six TCDD congeners in the stepped

standard.

• Concentrations represents ‘on column’ amount.

RT: 13.37 - 14.94 SM: 3B

13.4 13.5 13.6 13.7 13.8 13.9 14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

10014.78

14.57

14.30

14.15

13.6513.52 14.8614.38 14.4113.76 14.6713.53

14.56

NL: 8.47E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_11

NL: 8.33E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_03

NL: 8.49E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_05

NL: 8.25E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_06

NL: 8.35E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_07

NL: 8.38E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_08

NL: 8.53E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_09

NL: 8.48E4

TIC F: + c EI SRM ms2 321.890@cid20.00 [258.925-258.935] MS 19May2014_10

1368-TCDD10 fg

IR % = 9

1379-TCDD25 fg

IR % = 6

1378-TCDD100 fg

IR % = 7

1478-TCDD250 fg

IR % = 6

2378-TCDD1000 fgIR % = 4

1234-TCDD500 fg

IR % = 11

Inj. No. Concentration

10 fg 25 fg 100 fg 250 fg 500 fg 1000 fg

1 3129 7011 25940 60219 87635 239216

2 3045 6209 25998 59108 85789 240162

3 3047 7055 27425 58262 87578 237274

4 2637 7440 25734 59335 87079 233655

5 2678 6376 27805 58583 83897 234470

6 2964 6513 27097 58902 85373 238554

7 2828 6931 26347 59936 86844 242740

8 2699 6579 25033 55519 79471 232038

mean 2878.4 6764.3 26422.4 58733.0 85458.3 237263.6

STDEV 192.5 411.7 940.7 1452.1 2730.3 3625.3

% RSD 6.7 6.1 3.6 2.5 3.2 1.5

13

DETERMINATION OF DIOXINS IN SAMPLE EXTRACTS

14

Quantification of Dioxins in the Sample Extracts

• The egg, animal fat and fish meal samples were analyzed for their dioxin

content.

• The calculated concentration of each individual dioxin congener

(pg TEQ/g fat or ng/kg %dw) was compared with the values obtained

from the GC-HRMS.

15

Chromatography & Selectivity 2378-TCDD in Sample Extracts

95 fg 28 fg 98 fg

fish meal mixed animal fat egg

16

Egg Samples - Comparison with GC-HRMS

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

WH

O p

g TE

Q/g

fat

GC-HRMS

GC-MS/MS egg 0.52g

GC-MS/MS egg 0.51 g

17

Fish Meal - Comparison with GC-HRMS

18

Ion Ratio Stability: IR Egg Sample vs. Theoretical IR

19

Ion Ratio Stability: IR Egg Sample vs. IR CS3

20

Precision of Measurements: Sample Extracts

• Obtaining valid estimates of PCDD/Fs concentration in sample extracts

is critical.

• Precision measurement of the total dioxin content (as WHO TEQ pg/g

or ng/kg) for a sequence of n=13 repeat injections of the mixed animal

fat and the fish meal samples

21

Fish Meal: Precision of Total Dioxin Content (n=13)

22

Mixed Animal Fat: Precision of Total Dioxin Content (n=13)

23

Comparison with GC-HRMS

Total dioxin content of sample extracts

• Average values of replicate injections (n) with corresponding standard

deviations (in brackets) are given.

Sample type WHO-PCDD/F-TEQ-ub

GC-HRMS GC-MS/MS Deviation %

egg (n=2) 3.39 pg/g 3.27 (0.03) -3.5

fish meal (n=13) 0.26 ng/kg % dw 0.25 (0.01) -2.2

mixed animal fat (n=13) 0.83 pg/g 0.85 (0.04) 2.8

24

Conclusions

• Excellent selectivity and sensitivity was achieved

(ex: 28 fg 2378-TCDD absolute amount on column)

• Precision measurements of the total dioxin content (WHO-PCDD/F-TEQ-ub)

were obtained for two low level contaminated samples

(fish meal and mixed animal fat) with %RSD values <6% (n=13).

• Deviation of the total dioxin content (WHO-PCDD/F-TEQ-ub) from the

GC-HRMS data was analyzed within the ±20% for all the samples.

• The data shows excellent agreement between the results obtained using the

TSQ 8000 Evo GC-MS/MS and those obtained using GC-HRMS.

• TSQ 8000 Evo triple quadrupole system is a sensitive and robust system

suitable for the quantification and confirmation of low level dioxins and furans in

food and feed matrices.

25

Acknowledgements

26

Thank You for Your Attention!

Stay connected with us

Twitter

@ChromSolutions

Chromatography Solutions Blog

http://chromblog.thermoscientific.com/blog

YouTube

http://www.youtube.com/ChromSolutions

Facebook

http://www.facebook.com/Chromatography

Solutions

Pinterest

http://pinterest.com/chromsolutions/

Economic POPs Dave Hope, Patrick Pond, Wasana Mudalige, Jose del

Pozo, Matt Wright Pacific Rim Laboratories Inc.

Presented at Thermo Scientific Vendor Seminar

Madrid, Spain September 2,2014

Outline

• Cost of Analysis • Saving time on extraction - Accelerated

Solvent Extraction (ASE) • Saving time and money on clean-up -

Cape Technologies Clean-up Kits • Is GC-MS/MS technology ready for POPs –

Thermo Scientific™ TSQ™ 8000 system

Economic Costs – POPs Lab

• Laboratory – $1-3M or higher if budget allows

• Instrument – $150-400k up to $250-600k depending on options

• Staffing (2-10) – $20-50 per hour ($40-100k per annum)

• Soft costs – Chemicals 7-10% revenue – Consumables 5-10% revenue – Service contracts/R&M 5-7% revenue or 5-10% instrument cost – Staff Training 6 weeks to 6 months or $5-35k

Cost of Analysis

• Sample Extraction (batch of 10 samples) – Water: liquid/liquid sep funnel 6-8 h – Soil/tissue: soxhlet 6-8 h – Tissue: acid digestion 12 h – @ $20 per hour with 10 sample batch $16-24 – @ $20 per hour with 6 sample batch $25-40 – @ $20 per hour with 2 sample batch $50-75

Sample clean-up

• Clean-up steps (4-8 h each) – Acid or base washes – Acid/base silica gel – Basic Alumina – Florisil (separate dioxins and PCBs) – Carbon

• Soft costs – Preparing reagents – Cleaning reusable glassware – 2 h per day – Re-analyzing cross contaminated samples

Simplified Clean-up for Food and Environmental Sample • Cape Technologies tandem acid silica gel / carbon

columns – Originally designed as clean-up kit for EPA Method 4025 (Screening for PCDD/F

by Immunoassay) – quick and cheap – 4-6 h to complete 12 samples, <$20/sample disposable

columns, $500 hardware – Columns are disposable, therefore reduces PCB carryover from improper washing

of reusable columns (not to mention the labor savings) – Separates dioxins from PCBs – Separates dl PCBs from interfering PCBs with high LOC – Yang et al (MOE Ontario) developed method to separate PCB, PBDE and PCDD/F

(Dioxin 2010)

Cape Technologies Clean-up Kit

• Glass column, 15 mm x 27 cm, loaded with 5 g acid (30%) silica gel (63-200 µm)

• 25 mm column available with 14 or 28 g acid (50%) silica (32-63 µm)

• Each column individually sealed, wrapped in boxes of 12

• Carbon soxhlet extracted with Toluene prior to packing in Teflon column (one end flat and the other end bevelled for easy identification of flow direction)

• 150 mg of 2% carbon in FEP Teflon

Columning Procedure

• Pre-rinse carbon column – 10 mL toluene – 10 mL DCM – 30 mL hexane

• Attached carbon column to acid silica gel – bevelled side down

• Load sample in 1-2 mL hexane • Elute 30 mL hexane (F1) –

pressurize to 10 psi (1-2 mL/min) • Remove carbon and attach to

empty glass column • Elute 6 mL 1:1 toluene/hexane

(F2) • Reverse carbon column flow • Elute 30 mL toluene (F3)

F3 – PCDD/F

• All 17 congeners found in F3 • Spike recoveries for TeCDD/F-

HpCDD/F were 93-107% with RSD<12%

• Recovery of OCDD was 85% and OCDF 49%

• There may be some loss of non-2,3,7,8 congeners (especially TeCDD/F)

• Cut point between F2/F3 tight for TeCDD/F (F2 max volume 6 mL of 50:50 toluene/hexane)

Accelerated Solvent Extraction (ASE) • Has been widely used for soils and tissues in conventional analyses

(ppm/ppb) • Cells and lines subject to trace contamination and carry over at ultra-trace

levels • Recently given an Thermo Scientific™ Dionex™ ASE™ 350 Accelerated

Solvent Extractor to play with complete with 100 mL Thermo Scientific™ Dionium™ cells

• Thermo Fisher Scientific has application notes for tissue samples using max 1 g lipid

• PRL methodology requires 5-10 g lipid for lowest DLs • Initial problems with packing cells – critical not to

– over pack them – SOLVENT LEAKAGE WARNING – under pack them – VAPOUR PRESSURE WARNING

• Instrument lines and cells required thorough cleaning after installation

ASE 350 System Extraction Conditions Extraction Solvent: Hexane/Acetone 3:1 Temperature: 100 °C Pressure: 1600 psi Heat Time: 5 min Static Time: 5 min Flush Volume: 70% Purge Time: 120 s Static Cycles: 3 Total Prep Time: 15 min per sample Total Extraction Time: 25 min per sample

Butter (n=5) Units: ng/kg (pg/g) Control Average Spike Recovery RSD

2378-TCDD <0.03 <0.03 20 91% 15%

12378-PeCDD <0.033 <0.033 100 94% 5%

123478-HxCDD 0.15 0.20 100 82% 14%

123678-HxCDD 0.56 0.60 100 87% 8%

123789-HxCDD 0.18 0.25 100 90% 3%

1234678-HpCDD 1.21 1.69 100 96% 7%

OCDD 0.88 1.83 200 95% 2%

2378-TCDF <0.03 <0.03 100 94% 11%

12378-PeCDF <0.039 0.07 100 92% 5%

23478-PeCDF <0.039 <0.039 100 90% 8%

123478-HxCDF 0.16 0.24 100 82% 14%

123678-HxCDF 0.12 0.12 100 86% 9%

123789-HxCDF <0.056 <0.056 100 93% 5%

234678-HxCDF 0.11 0.20 100 80% 16%

1234678-HpCDF <0.052 <0.052 100 88% 12%

1234789-HpCDF <0.085 <0.085 100 87% 9%

OCDF <0.13 0.20 200 66% 6%

Use 6.25 g of butter (80% lipid) mixed with 10 g diatomaceous earth Add internal standards Pack 100 mL Dionium cell with • 30 mm cellulose filter • 10 g Dionex ASE Prep CR Na+

form (sulphonated divinyl benzene/styrene copolymer)

• Add sample mix • Top with diatomaceous earth to

10 mm from top of cell Cape Technologies column clean-up

Butter PCB data ng/kg Control

Average (n=3)

Spike Recovery

PCB 28 <2 12.8 73%

PCB 52 5.1 19.1 92%

PCB 101 <2 <2 116%

PCB 153 61.7 58.4 112%

PCB 138 39.6 48.7 109%

PCB 180 25.6 22.0 99%

PCB 81 <1 <1 103%

PCB 77 <1 <1 97%

PCB 123 <1 <1 88%

PCB 118 51.1 41.7 86%

PCB 114 <1 <1 103%

PCB 105 <1 <1 99%

PCB 126 <1 <1 96%

PCB 167 <1 <1 103%

PCB 156 5.8 4.1 110%

PCB 157 <1 <1 105%

PCB 169 <1 <1 95%

PCB 189 <1 <1 82%

Horse Meat

Control (n=1)

Average (n=5) RSD

2,3,7,8-TCDD 0.27 0.18 21% 1,2,3,7,8-PeCDD 1.0 0.94 22% 1,2,3,4,7,8-HxCDD 1.8 1.7 22% 1,2,3,6,7,8-HxCDD 3.8 4.2 9% 1,2,3,7,8,9-HxCDD 0.57 0.42 31% 1,2,3,4,6,7,8-HpCDD 33 30 4% OCDD 58 54 8% 2,3,7,8-TCDF 0.075 0.11 65% 1,2,3,7,8-PeCDF 0.087 0.093 90% 2,3,4,7,8-PeCDF 0.36 0.32 38% 1,2,3,4,7,8-HxCDF 0.29 0.27 25% 1,2,3,6,7,8-HxCDF 0.46 0.49 20% 1,2,3,7,8,9-HxCDF 0.091 0.085 133% 2,3,4,6,7,8-HxCDF 0.20 0.24 53% 1,2,3,4,6,7,8-HpCDF 1.97 1.92 9% 1,2,3,4,7,8,9-HpCDF 0.076 0.11 69% OCDF 0.39 0.30 55% TEQ 2.47 2.29

PCB 81 <0.5 <0.5 PCB 77 <0.5 <0.5 PCB 123 21 8.8 22% PCB 118 714 401 5% PCB 114 21 11 30% PCB 105_127 265 150 3% PCB 126 16 9.2 30% PCB 167 61 55 3% PCB 156 91 97 3% PCB 157 24 22 11% PCB 169 <0.5 <0.5 PCB 189 14 21 12%

Total TEQ 4.15 3.24

5 g fat processed through ASE and Cape Technologies clean-up and analyzed on Thermo Scientific™ DFS™ High Resolution GC/MS System. Control sample processed through our standard 3N HCl digestion

Can economy model TSQ 8000 GC/MS run PCDD/F • Benchtop instrument • More affordable • Increased uptime • Easier maintenance • Lower electrical usage

So easy anyone can learn???

• It helps if you know how to type!

• Monitor M-COCl – No chlorinated

diphenylether interference

• EPA 1613b calibration, with CS-Lo and 0.2 x CS-Lo

TSQ 8000 GC/MS Calibration 20 fg – 200 pg

20 fg/uL to 200 pg/uL TCDD

Data Comparison ASE – Cape Tech – TSQ 8000 MS • Ten vials of CARP-2 (ground whole carp reference material) from

NRC Canada • Reference concentrations for PCDD/F and select PCBs (several

congeners in excess of 100 ug/kg) • Entire contents of vial (approx 10 g) extracted using ASE 350 • Cape Technologies tandem acid silica gel / carbon clean-up • Analysis by DFS HRMS System • Analysis by TSQ 8000 Triple Quadrupole GC-MS

CARP-2 results

DFS (n=10) TSQ 8000 (n=7) Reference Values Average RSD Average RSD

ng/kg ng/kg ng/kg 2378-TCDD 6.7-8.1 7.74 5% 8.18 6% 12378-PeCDD 4.0-6.6 4.65 13% 5.09 14% 123478-HxCDD 1.3-1.9 1.56 12% 1.56 11% 123678-HxCDD 5.0-6.6 5.66 10% 5.23 13% 123789-HxCDD 0.66-0.90 0.71 32% 0.60 13% 1234678-HpCDD 5.5-7.3 6.77 7% 5.75 14% OCDD 7.7-11.1 8.71 10% 8.80 9%

2378-TCDF 16.6-19.8 18.5 6% 18.3 8% 12378-PeCDF 5.3-5.9 5.97 14% 6.13 8% 23478-PeCDF 15.2 8% 15.4 14% 123478-HxCDF 4.04 17% 3.88 18% 123678-HxCDF 2.56 19% 2.48 15% 234678-HxCDF 1.27 32% 1.37 34% 123789-HxCDF 0.27 38% 0.22 69% 1234678-HpCDF 4.42 11% 3.96 17% 1234789-HpCDF <1 0.74 17% OCDF <1 1.29 17%

7.7

11.1

0

2

4

6

8

10

12

ng/k

g CARP-2 Comparison – TSQ 8000 GC-MS vs DFS HRMS

Conclusions

• Cape Technologies provides a simple, inexpensive clean-up for dioxins and PCBs

• ASE can be made clean and effective for food samples at ppq levels

• TSQ 8000 GC-MS system has the sensitivity and specificity to analyze PCDD/F from food extracts.