GC/MS/MS Dioxin and Dioxin-like PCBs in

Feed and Food

Jessica Westland, LSAG Applications Chemist

What is a Dioxin?

• The term “dioxins” refers to a group of chemically and structurally related halogenated aromatic hydrocarbons

• 75 polychlorinated dibenzo-p-dioxin (PCDD) • 135 polychlorinated dibenzofuran (PCDF) congeners

• The toxicity of individual dioxin and furan congeners differs considerably • Only those substituted in each of the 2-, 3-, 7- and 8-positions (17 congeners) of the two aromatic rings

are of toxicological concern • Exhibit a similar toxicological profile, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) the most

toxic congener

• Polychlorinated biphenyls (PCBs) are chlorinated aromatic hydrocarbons, which

are synthesized by direct chlorination of biphenyl • PCBs can be divided into different groups according to their biochemical and

toxicological properties • Non-ortho and mono-ortho substituted PCBs show toxicological properties that are similar to dioxins –

termed “dioxin-like PCBs”

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Why are Dioxins Toxic?

Dioxins and dioxin-like PCBs are fat soluble and tend to bio-accumulate in body fat, both in animals and humans, accumulating through the food chain These 2 groups of toxic and environmentally persistent chemicals have effects on human health that include: • Dermal toxicity • Immunotoxicity • Reproductive effects and teratogenicity • Endocrine disrupting effects • Carcinogenicity International studies have concluded that around 95% of human exposure occurs through consumption of food of animal origin, with meat, dairy products and fish being the main source

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Dioxins and Dioxin-like PCBs in Feed and Food

This GC/MS/MS Analysis is for Dioxin and Dioxin-like PCBs in Feed and Food • Developed in accordance with the Commission Regulations (EU):

• No 589/2014 & No 709/2014

• Sample preparation holds as a high priority within dioxin regulations, but is not presented

Previously, the use of a High Res Mass Spectrometry (HRMS) was needed to confirm and quantify dioxins, due to the ability to identify, confirm, and quantitate the trace levels of dioxins

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GC/MS/MS is now a confirmatory technique in Dioxin/Furan/PCB analysis! European Market for Dioxin analysis in [Animal] Feed and Foodstuffs

Commission Regulation (EU) No 589/2014 (of 2 June 2014) laying down methods of sampling and analysis for the control of levels of dioxins, dioxin-like PCBs and non-dioxin-like PCBs in certain foodstuffs and repealing Regulation (EU) No 252/2012

Commission Regulation (EU) No 709/2014 (of 20 June 2014)

amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls in feed

In force as of June 20th 2014

Except from EU No 589/2014

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Sample Preparation Critical and Important

The most frequently used methods for the determination of PCDD/PCDF and DL-PCB in foodstuffs and animal feed combine fat extraction (i.e. Soxhlet) with cleanup steps using different column chromatographies (i.e. silica gel coated with sulphuric acid, florisil, alumina, and active carbon) Manual dioxin sample preparation is tedious and comprehensive; multicolumn automated systems have been made to automate dioxin sample extraction to reduce analysis times and to attempt to reduce costs The final extract is collected as two separate fractions: 1. PCDDs/PCDFs and the NO-PCB congeners 2. MO-PCB congeners and the NDL-PCB congeners Along with the analysis of the native compounds: • 13C-ISTDs are required for each individual standard for accurate identification and quantitation • Surrogates are also added prior to extraction to correct for analyte recovery

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GC/MS/MS Dioxin Analysis Configuration Analysis utilizes: • New 7010 MS/MS – Increased sensitivity • Same GC method for both sample fractions – Increased productivity • MMI – Flexibility with injection techniques/volumes • GC column – Validated for dioxin analysis • RT locked MRM transitions – Optimized & validated in a European Dioxin Reference Lab

Simple straight forward configuration, already accepted by EU Dioxin Labs!

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7010 High-Efficiency EI Source

MS sensitivity depends on the number of ions measured

This new ultra-efficient EI source maximizes the number of ions that are created and transferred out of the source body and into the quadrupole analyzer Advantages: • Increased response and better precision at

all levels • Lower detection limits • More precise ion ratios and better qualitative

information

GC Method Parameters

GC Conditions Column DB 5MSUI 60 m x 0.25 mmID x 0.25 µm Injection port liner 2mm id dimpled splitless liner, UI Injection mode Cold-splitless (compressed air/CO2 cooled MMI) Injection volume 1 µL

Inlet temperature program 60 °C 0.31 min

600 °C/min 330 °C 5 min Carrier gas He, constant flow 0.700 mL/min

Oven program

60 °C 1 min 30 °C/min 270 °C 1 min 2 °C/min 310 °C 0 min 5 °C/min 350 °C 0.5 min

MS transfer line temperature 350 °C

GC Conditions same for both fractions! increase in productivity and more efficient data analysis

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In addition to decreasing detection limits, the new EI source enables the use of lower-volume injections, allowing the scale down of sample prep and a reduction of GC maintenance.

7010 MS Parameters

MS set points Electron Energy 70 eV Tune eihs.tune.xml EM gain 10 MS1 resolution Unit MS2 resolution Unit

Collision Cell 1.5 mL/min N2 4 mL/min He

Quant/Qual transitions Fraction Specific Dwell times Fraction Specific Collision energies Optimized Source temperature 350 °C Quad temperatures 150 °C

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Unit mass resolution allows for sufficient resolution to separate two peaks one mass unit apart As well as the ability to minimize possible interferences on the analytes of interest

Dioxins/Furans – Chromatogram Excellent separation of the difficult hexa-dioxin/furan isomers GC Column – DB5MS UI (60m x 0.25mm x 0.25 µm)

2x10

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+ MRM (333.9 -> 269.9) Dioxin_CS5_001.D

Counts (%) vs. Acquisition Time (min)

16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 30.5

Penta

Hepta

Octa

Tetra

Level CS5 (TCDD = 2.0 ppb; 13C12 = 100 ppb) 1x10

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+ MRM (333.9 -> 269.9) Dioxin_CS5_001.D

Counts (%) vs. Acquisition Time (min)

21.3 21.35 21.4 21.45 21.5 21.55 21.6 21.65 21.7 21.75 21.8 21.85 21.9 21.95 22 22.05 22.1 22.15 22.2 22.25 22.3 22.35 22.4 22.45 22.5 22.55 22.6 22.65 22.7 22.75 22.8 22.85 22.9 22.95 23 23.05 23.1 23.15

Hexa

Resolution = 0.64 Peak to Peak Valley = 14.1%

Resolution = 0.69 Peak to Peak Valley = 23.5%

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PCBs – Chromatogram Key separation between the difficult mono-ortho substituted PCBs 123 & 118 is achieved on same method parameters as the dioxin method! GC Column – DB 5MS UI (60m x 0.25mm x 0.25 µm)

2x10

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PCB – 138: + MRM (359.8 -> 289.9) PCB_CS4_check.D

Counts (%) vs. Acquisition Time (min)

12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2 14.4 14.6 14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2

28

52

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81 &77

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& 11

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126 167 15

6 &

157

180

169 189

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PCB – 138: + MRM (359.8 -> 289.9) PCB_CS4_check.D

Counts (%) vs. Acquisition Time (min)

14.76 14.78 14.8 14.82 14.84 14.86 14.88 14.9 14.92 14.94 14.96 14.98 15 15.02 15.04 15.06 15.08 15.1 15.12 15.14 15.16 15.18 15.2 15.22 15.24 15.26 15.28 15.3 15.32

123 & 118

Resolution= 0.57

Level CS4 (PCB 123/118 = 10/50 ppb; 13C12 = 10 ppb)

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1x10

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PCB – 169: + MRM (359.9 -> 289.9) PCDD_fraction_001.D

18.34718.925

22.01721.494

30.37423.029

24.685

22.564 30.08226.778

14.387

16.178

26.001

15.702

16.45215.340

15.063

16.976 20.199

12 23 34 45 56 67 78 89 910 1011 1112

Counts (%) vs. Acquisition Time (min)

14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 30.5

5 17

19.5 20 20.5

16.1

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isition Time (min)

15 15

2x10

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PCB – 189: + MRM (395.8 -> 325.9) PCB_fraction_001.D

12.068

12.469

15.06113.796

15.504

16.222

18.132

15.70015.339

16.977 17.68920.199

12 23 34 45 56 67 78 89 910

Counts (%) vs. Acquisition Time (min)

11.8 12 12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2 14.4 14.6 14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4

Both fractions are consolidated to report the analysis of one sample • EU regulation compounds • Separation of key compounds

• STD01 – Hexa-dioxin/furan-isomers • STD02 – PCBs 123 & 118

Sample Fraction Analysis

Fraction 01 Dioxins/Furans & NO–PCB

22 cmpds

Fraction 02 MO– & NDL– PCBs

14 cmpds

Blue – PCBs Black – Dioxins/Furans

Example Chromatograms

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7010 MS/MS Improved Linearity Dioxin/Furan R2

2378-TCDF 1.0000

2378-TCDD 1.0000

12378-PeCDF 1.0000

23478-PeCDF 1.0000

12378-PeCDD 1.0000

123478-HxCDF 1.0000

123678-HxCDF 1.0000

234678-HxCDF 1.0000

123478-HxCDD 1.0000

123678-HxCDD 1.0000

123789-HxCDD 1.0000

123789-HxCDF 1.0000

1234678-HpCDF 1.0000

1234678-HpCDD 1.0000

1234789-HpCDF 1.0000

OCDD 1.0000

OCDF 0.9998

PCB R2

PCB – 28 1.0000

PCB – 52 1.0000

PCB – 101 1.0000

PCB – 81 1.0000

PCB – 77 1.0000

PCB – 123 0.9974

PCB – 118 0.9998

PCB – 114 0.9978

PCB – 105 0.9999

PCB – 153 0.9999

PCB – 138 1.0000

PCB – 126 1.0000

PCB – 167 1.0000

PCB – 156 1.0000

PCB – 157 1.0000

PCB – 180 1.0000

PCB – 169 1.0000

PCB – 189 1.0000

y = 0.0122x + 0.0075 R² = 1

y = 0.0112x + 0.0117 R² = 1

y = 0.0112x + 0.0042 R² = 1

y = 0.0182x + 0.0172 R² = 1

0.0000

2.0000

4.0000

6.0000

8.0000

10.0000

12.0000

14.0000

16.0000

18.0000

20.0000

0 200 400 600 800 1000 1200

Rel

ativ

e R

espo

nse

Concentration (pg/µL)

Selected Hexa-Furan Calibrations

123478-HxCDF 123678-HxCDF 234678-HxCDF 123789-HxCDF

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Recovery Experiment Sample (spike at 0.79pg-TEQ/g fat)

Compound REP_1 REP_2 REP_3 REP_4 REP_5 REP_6 RSD% PCB 81 2.230 2.228 1.902 1.899 1.958 2.024 7.5 PCB 77 5.568 5.602 5.590 5.611 5.630 5.528 0.6 PCB 126 1.677 1.684 1.687 1.751 1.684 1.490 5.3 PCB 169 1.415 1.387 1.341 1.396 1.397 1.351 2.1 2378-TCDF 0.082 0.088 0.083 0.087 0.095 0.084 5.6 2378-TCDD 0.086 0.076 0.077 0.076 0.071 0.084 7.2 12378-PeCDF 0.083 0.087 0.090 0.085 0.087 0.106 9.0 23478-PeCDF 0.077 0.080 0.078 0.085 0.083 0.089 5.7 12378-PeCDD 0.081 0.067 0.091 0.082 0.078 0.078 9.8 123478-HxCDF 0.086 0.081 0.081 0.089 0.086 0.104 9.8 123678-HxCDF 0.080 0.075 0.085 0.078 0.090 0.089 7.4 234678-HxCDF 0.076 0.061 0.076 0.075 0.071 0.069 7.9 123478-HxCDD 0.069 0.079 0.070 0.073 0.069 0.067 6.1 123678-HxCDD 0.093 0.101 0.089 0.078 0.090 0.100 9.2 123789-HxCDD 0.067 0.069 0.072 0.062 0.057 0.067 8.3 123789-HxCDF 0.076 0.085 0.084 0.094 0.084 0.074 8.5 1234678-HpCDF 0.386 0.361 0.391 0.388 0.415 0.451 7.8 1234678-HpCDD 0.121 0.119 0.122 0.121 0.149 0.122 9.2 1234789-HpCDF 0.025 0.025 0.025 0.027 0.029 0.028 7.0 OCDD 0.481 0.461 0.484 0.475 0.486 0.486 2.0 OCDF 0.166 0.160 0.157 0.174 0.174 0.182 5.7

Average RSD: 6.7

Instrumental limit of quantitation (iLOQ) – a ‘performance LOQ’ iLOQ = 10xStdDev (10 replicate injections @ lowest calibration point)

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PCB StdDev RSD % MDL iLOQ (fg)

PCB – 28 0.012 2.40 0.033 122.6

PCB – 52 0.009 1.91 0.025 93.8

PCB – 101 0.008 1.57 0.022 76.8

PCB – 81 0.002 1.71 0.006 17.4

PCB – 77 0.001 1.41 0.003 14.8

PCB – 123 0.012 1.43 0.033 120.2

PCB – 118 0.005 16.11 0.014 50.3

PCB – 114 0.298 19.44 0.824 2977.1

PCB – 105 0.003 9.89 0.008 31.9

PCB – 153 0.013 1.97 0.036 128.5

PCB – 138 0.006 1.17 0.017 56.6

PCB – 126 0.005 5.43 0.014 54.8

PCB – 167 0.002 2.11 0.006 21.6

PCB – 156 0.004 4.24 0.011 43.0

PCB – 157 0.004 3.49 0.011 36.7

PCB – 180 0.006 1.24 0.017 61.3

PCB – 169 0.002 2.12 0.006 22.3

PCB – 189 0.003 3.13 0.008 32.3

Dioxin/Furan StdDev RSD % MDL iLOQ (fg)

2378-TCDF 0.003 4.92 0.008 28.1

2378-TCDD 0.003 4.28 0.008 25.2

12378-PeCDF 0.007 2.39 0.019 66.3

23478-PeCDF 0.008 2.98 0.022 83.7

12378-PeCDD 0.011 3.91 0.030 108.2

123478-HxCDF 0.009 3.33 0.025 93.3

123678-HxCDF 0.007 2.58 0.019 73.0

234678-HxCDF 0.008 2.71 0.022 76.1

123478-HxCDD 0.013 4.83 0.036 132.2

123678-HxCDD 0.013 4.40 0.036 126.1

123789-HxCDD 0.014 4.92 0.039 143.8

123789-HxCDF 0.006 2.24 0.017 63.0

1234678-HpCDF 0.007 2.54 0.019 72.4

1234678-HpCDD 0.010 3.37 0.028 100.5

1234789-HpCDF 0.016 5.44 0.044 157.1

OCDD 0.022 3.69 0.061 217.0

OCDF 0.017 3.04 0.047 170.3

Ion Ratios @ lowest calibration point – iLOQ

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Dioxin/Furan AVG StdDev %RSD (±15%)

2378-TCDF 1.10 0.07 5.90

2378-TCDD 0.95 0.04 4.49

12378-PeCDF 0.80 0.02 2.90

23478-PeCDF 0.79 0.02 2.71

12378-PeCDD 0.79 0.05 5.83

123478-HxCDF 0.64 0.03 4.25

123678-HxCDF 0.63 0.03 4.80

234678-HxCDF 0.64 0.04 5.43

123478-HxCDD 0.57 0.05 9.42

123678-HxCDD 0.60 0.06 9.61

123789-HxCDD 0.59 0.03 5.63

123789-HxCDF 0.63 0.02 2.63

1234678-HpCDF 0.79 0.04 4.51

1234678-HpCDD 1.21 0.06 4.87

1234789-HpCDF 1.24 0.05 3.71

OCDD 0.99 0.04 4.15

OCDF 1.07 0.03 3.20

PCB AVG StdDev %RSD (±20%)

PCB – 28 0.63 0.01 0.89

PCB – 52 0.64 0.01 1.34

PCB – 101 1.11 0.02 1.56

PCB – 81 0.64 0.01 1.60

PCB – 77 0.65 0.01 2.01

PCB – 123 0.86 0.08 9.24

PCB – 118 0.96 0.03 3.34

PCB – 114 0.86 0.09 10.41

PCB – 153 0.79 0.02 2.96

PCB – 105 0.95 0.01 0.78

PCB – 138 0.78 0.01 1.33

PCB – 126 1.00 0.09 8.53

PCB – 167 0.78 0.02 2.25

PCB – 156 0.81 0.04 4.90

PCB – 157 0.81 0.06 7.30

PCB – 180 0.64 0.01 2.05

PCB – 169 0.80 0.03 3.59

PCB – 189 0.64 0.02 3.83

7010 MS/MS Instrument Detection Limit (IDLRSD) in fg

CMPD RRF 10 reps (CS1) %RSD IDLRSD (fg)

2378-TCDF 1.180 4.92 6.8 2378-TCDD 1.258 4.28 5.9 12378-PeCDF 1.206 2.39 16.5 23478-PeCDF 0.961 2.98 20.6 12378-PeCDD 1.080 3.91 27.0 123478-HxCDF 1.278 3.33 23.0 123678-HxCDF 1.194 2.58 17.8 234678-HxCDF 1.171 2.71 18.7 123478-HxCDD 1.184 4.83 33.4 123678-HxCDD 1.183 4.40 30.4 123789-HxCDD 1.178 4.92 34.0 123789-HxCDF 1.906 2.24 15.5 1234678-HpCDF 1.183 2.54 17.6 1234678-HpCDD 1.171 3.37 23.3 1234789-HpCDF 0.875 5.44 37.6 OCDD 1.391 3.69 51.0 OCDF 1.963 3.04 42.0

IDLRSD = tα,n-1 x RSD x c 100

tα,n-1 = t value (coefficient) at the level of α with the sample size of n-1 c = concentration of the std sample injected

CMPD RRF 10 reps (CS1) %RSD IDLRSD (fg)

PCB – 28 1.077 2.40 33.2 PCB – 52 1.465 1.91 26.3 PCB – 101 1.276 1.57 21.6 PCB – 77 1.024 1.71 4.7 PCB – 81 1.040 1.41 3.9 PCB – 118 0.620 1.43 19.8 PCB – 123 2.854 16.11 44.5 PCB – 105 0.671 19.44 53.7 PCB – 114 3.316 9.89 27.3 PCB – 153 0.883 1.97 27.3 PCB – 138 1.402 1.17 16.2 PCB – 126 1.061 5.43 15.0 PCB – 167 1.168 2.11 5.8 PCB – 156 1.053 4.24 11.7 PCB – 157 1.025 3.49 9.6 PCB – 180 0.930 1.24 17.2 PCB – 169 1.228 2.12 5.9 PCB – 189 1.095 3.13 8.7

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Comparison of New High-Efficiency Source to Previous Extractor Ion Source • Linearity & Calibration

• MDLs

• LODs

• LOQs

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7010 MS/MS Dioxin & Furan Linearity/Calibration

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Dioxin/Furan Calibration Range (pg/µL) R2 Average RF StdDev RF %RSD

2378-TCDF 0.05 – 200 1.0000 1.180 0.076 6.43

2378-TCDD 0.05 – 200 1.0000 1.258 0.071 5.65

12378-PeCDF 0.25 – 1000 1.0000 1.206 0.043 3.55

23478-PeCDF 0.25 – 1000 1.0000 0.961 0.036 3.70

12378-PeCDD 0.25 – 1000 1.0000 1.080 0.047 4.32

123478-HxCDF 0.25 – 1000 1.0000 1.278 0.060 4.67

123678-HxCDF 0.25 – 1000 1.0000 1.194 0.059 4.91

234678-HxCDF 0.25 – 1000 1.0000 1.171 0.048 4.13

123478-HxCDD 0.25 – 1000 1.0000 1.184 0.052 4.38

123678-HxCDD 0.25 – 1000 1.0000 1.183 0.073 6.13

123789-HxCDD 0.25 – 1000 1.0000 1.178 0.058 4.90

123789-HxCDF 0.25 – 1000 1.0000 1.906 0.063 3.30

1234678-HpCDF 0.25 – 1000 1.0000 1.183 0.063 5.33

1234678-HpCDD 0.25 – 1000 1.0000 1.171 0.103 8.82

1234789-HpCDF 0.25 – 1000 1.0000 0.875 0.034 3.92

OCDD 0.5 – 2000 1.0000 1.391 0.162 11.68

OCDF 0.5 – 2000 0.9998 1.963 0.233 11.89

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Dioxin/Furan Calibration Range (pg/µL) R2 Average RF StdDev RF %RSD

2378-TCDF 0.1 – 200 1.0000 0.937 0.273 29.09

2378-TCDD 0.1 – 200 1.0000 0.913 0.371 40.58

12378-PeCDF 0.5 – 1000 1.0000 1.118 0.078 6.99

23478-PeCDF 0.5 – 1000 1.0000 1.052 0.084 7.95

12378-PeCDD 0.5 – 1000 1.0000 0.988 0.191 19.38

123478-HxCDF 0.5 – 1000 1.0000 1.202 0.082 6.84

123678-HxCDF 0.5 – 1000 1.0000 1.106 0.028 2.49

234678-HxCDF 0.5 – 1000 1.0000 1.150 0.036 3.09

123478-HxCDD 0.5 – 1000 1.0000 1.093 0.079 7.26

123678-HxCDD 0.5 – 1000 1.0000 1.031 0.302 29.27

123789-HxCDD 0.5 – 1000 0.9986 0.877 0.320 36.42

123789-HxCDF 0.5 – 1000 1.0000 1.786 0.087 4.89

1234678-HpCDF 0.5 – 1000 1.0000 1.101 0.051 4.62

1234678-HpCDD 0.5 – 1000 0.9999 1.055 0.048 4.52

1234789-HpCDF 0.5 – 1000 1.0000 0.821 0.065 7.89

OCDD 0.5 – 1000 1.0000 1.044 0.234 22.46

OCDF 0.5 – 1000 1.0000 1.482 0.465 31.38

Previous MS/MS Dioxin & Furan Linearity/Calibration

Comparison – TCDD @ 500 fg/µL

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Dioxin/Furan RRF StdDev RSD % MDL (fg) LOD (fg) iLOQ (fg)

7000C 7010 7000C 7010 7000C 7010 7000C 7010 7000C 7010 7000C 7010

2378-TCDF 1.201 0.855 0.033 0.013 31.55 3.61 93 37 97 39 329 131

2378-TCDD 1.273 0.905 0.042 0.022 43.89 6.01 118 61 112 58 418 216

12378-PeCDF 1.216 0.829 0.061 0.074 6.56 4.31 172 209 170 206 610 741

23478-PeCDF 0.970 0.763 0.230 0.076 30.50 3.85 649 216 803 267 2302 764

12378-PeCDD 1.091 0.695 0.102 0.067 18.78 4.18 287 190 318 210 1017 673

123478-HxCDF 1.293 0.828 0.038 0.095 4.64 5.89 106 269 102 258 376 954

123678-HxCDF 1.210 0.771 0.085 0.099 8.48 6.13 240 279 250 291 850 989

234678-HxCDF 1.184 0.764 0.137 0.068 18.91 4.14 388 190 405 199 1374 675

123478-HxCDD 1.195 0.724 0.168 0.077 23.37 5.03 475 217 498 228 1682 770

123678-HxCDD 1.205 0.756 0.119 0.077 13.81 4.81 335 217 340 220 1187 769

123789-HxCDD 1.194 0.699 0.083 0.064 15.79 4.32 234 181 249 192 830 641

123789-HxCDF 1.923 1.170 0.126 0.066 16.93 4.33 355 188 228 121 1259 665

1234678-HpCDF 1.197 0.754 0.092 0.102 9.97 6.41 258 288 267 298 915 1022

1234678-HpCDD 1.197 0.717 0.124 0.086 17.28 5.60 349 242 370 256 1236 858

1234789-HpCDF 0.886 0.549 0.184 0.080 27.17 5.08 518 225 715 310 1836 796

OCDD 1.426 0.408 0.387 0.156 30.27 5.32 1093 440 2014 811 3874 1559

OCDF 1.997 0.599 0.370 0.192 20.28 6.31 1043 543 1234 642 3698 1925

Comparison – TCDD @ 250 fg/µL

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Dioxin/Furan RRF StdDev RSD % MDL (fg) LOD (fg) iLOQ (fg)

7000C 7010 7000C 7010 7000C 7010 7000C 7010 7000C 7010 7000C 7010

2378-TCDF 1.201 0.812 0.018 0.015 46.06 8.64 50 42 52 44 176 149

2378-TCDD 1.273 0.878 0.016 0.015 55.31 8.37 45 41 42 39 158 146

12378-PeCDF 1.216 0.808 0.036 0.067 8.63 8.01 102 189 101 187 363 671

23478-PeCDF 0.970 0.733 0.075 0.061 29.73 6.43 211 173 261 214 749 613

12378-PeCDD 1.091 0.677 0.107 0.068 50.23 8.63 301 191 334 212 1067 676

123478-HxCDF 1.293 0.797 0.060 0.065 14.69 8.36 169 184 162 176 598 651

123678-HxCDF 1.210 0.745 0.070 0.068 15.08 8.68 196 191 205 199 696 677

234678-HxCDF 1.184 0.740 0.068 0.035 16.98 4.44 192 99 200 103 680 350

123478-HxCDD 1.195 0.691 0.149 0.060 45.28 8.18 420 169 441 177 1489 597

123678-HxCDD 1.205 0.738 0.118 0.064 30.64 8.24 332 181 338 184 1178 642

123789-HxCDD 1.194 0.653 0.087 0.068 38.12 9.78 245 191 261 203 870 677

123789-HxCDF 1.923 1.139 0.057 0.053 14.45 7.15 161 151 104 97 572 534

1234678-HpCDF 1.197 0.724 0.067 0.061 17.34 8.03 188 173 195 179 668 615

1234678-HpCDD 1.197 0.698 0.116 0.062 41.97 8.35 326 175 345 186 1155 622

1234789-HpCDF 0.886 0.544 0.074 0.064 32.96 8.30 208 182 287 251 738 644

OCDD 1.426 0.399 0.155 0.116 26.11 8.11 438 328 807 604 1552 1163

OCDF 1.997 0.593 0.138 0.153 19.19 10.15 390 433 461 512 1382 1535

Dioxins and Dioxin-like PCBs in Feed and Food Reporting New efforts were made in generating supplemental software and a customized report that can calculate mandatory reporting values and combine both dioxin sample fractions into one report

Not all of the work flow is automated. The complex calculations for the report are automated, but need to be executed.

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Upper/Middle/Lower Bounds: PCDD/F and DLPCBs

& NDLPCBs

RT: Compound’s Retention time from the Acquisition Method.

LOQ: Compound’s (calculated) LOQ value

WHO-TEF: Designated Toxicity Values

Conc: Calculated Conc. By MassHunter. Value is being pulled from the Calc. Conc. Column

TEF Conc: Calculated value

Customized Report!

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Customized reporting software makes the complicated calculations automated and provides a detailed report for customers!

Combines both sample fraction runs into ONE consolidated report!

Note: The report does not calculate any recoveries. Recoveries will need to be calculated from “surrogate standards”, these standards are added to the standard before sample prep. The addition of an internal standard is usually added to the sample right before injection to account for instrument fluctuation.

2x10

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2378-TCDD: + MRM (321.9 -> 258.9) TCDD_5fg_009.D Smooth

Counts (%) vs. Acquisition Time (min)

16.05 16.1 16.15 16.2 16.25 16.3 16.35 16.4 16.45 16.5 16.55 16.6 16.65 16.7 16.75 16.8 16.85

7010 MS/MS Instrument Detection Limit (IDLRSD) in fg

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IDLRSD = tα,n-1 x RSD x c 100

tα,n-1 = t value (coefficient) at the level of α with the sample size of n-1 c = concentration of the std sample injected

CMPD RRF 10 reps (5 fg/µL) %RSD IDLRSD (fg)

2378-TCDF 1.239 8.99 1.3 2378-TCDD 1.379 11.33 1.6 12378-PeCDF 1.247 3.96 2.8 23478-PeCDF 1.072 4.15 2.9 12378-PeCDD 1.120 4.29 3.0 123478-HxCDF 1.300 7.81 5.5 123678-HxCDF 1.185 6.56 4.6 234678-HxCDF 1.247 4.53 3.2 123478-HxCDD 1.213 8.02 5.7 123678-HxCDD 1.175 9.76 6.9 123789-HxCDD 1.277 6.88 4.8 123789-HxCDF 1.908 6.70 4.7 1234678-HpCDF 1.274 5.15 3.6 1234678-HpCDD 1.278 7.79 5.5 1234789-HpCDF 0.977 7.93 5.6 OCDD 0.761 7.56 10.7 OCDF 1.064 5.16 7.3

Average S/N = 3.282 2378-TCDD

Advantages of 7010 Dioxin Analysis Provides a complete workflow

• Based on application developed, optimized and validated in a European Dioxin Reference Lab

• Multi Mode Inlet offers a wider range of injection techniques and injection volumes to maximize detection limits

• Same GC parameters for Dioxin and Dioxin-like PCB fractions to increase productivity - Retention time locked time segment boundaries

• MRM transitions based on application developed, optimized and validated in a European Dioxin Reference Lab

• Increased sensitivity with the 7010 GC/MS/MS! • Report combining results from Dioxin and Dioxin-like PCB fractions and

automatically performing complex calculations required by the EU regulations

Cost Effective Alternative to High Res Mass Spec • Lower instrumental purchase cost • Lower cost of real estate • Lower operational cost from lower training requirements and lower maintenance costs

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Feature Elements for GC/MS/MS Dioxin Analysis

• Compliant with new European Union Commission Regulations No 589/2014 and No 709/2014 which allows GC/MS/MS use as a confirmatory method for analysis of certain foodstuffs

• Sensitivity of the new 7010 GC/MS/MS detector meets detection requirements of the European regulations and is 10x more sensitive

• Solution for the complete workflow uses RTL and MRM Transitions Based on application developed, optimized and validated in a European Dioxin Reference Lab

• Customized Report combines results from Dioxin and Dioxin-like PCB fractions and automatically performs complex calculations required by the EU regulations

• Cost Effective alternative to High Res Mass Spec systems

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Real World Samples

• Unknown A

• Unknown B

• Unknown C1

• Unknown C2

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Cpd 34: OCDF: +EI MRM CID@40.0 (443.7 -> 380.8) PCDD_Dx_Unk_A_11-28-14_05.D Noise (RMS) = 81.39; SNR (30.411min) = 3.3

14.310

16.122

16.471

18.887

18.307

22.004

21.355

23.02624.688

26.80522.553

30.419

26.019

30.131

* 14 921 20 120 23 554 28 78325 200 27 56226 515

12 23 34 45 56 67 78 89 910

Counts vs. Acquisition Time (min)

14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 30.5

Real Sample (UNK A)

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Cpd 34: OCDF: +EI MRM CID@40.0 (443.7 -> 380.8) PCDD_Dx_Unk_B_11-28-14_06.D Noise (RMS) = 168.55; SNR (36.638min) = 1.2

14.305

16.122

16.475

18.887

18.303

22.004

21.35523.022

24.688

26.805

22.557

30.414

26.019

30.118

15.583 20 37119 609 23 80218 033 27 58425 12124 328

12 23 34 45 56 67 78 89 910

Counts vs. Acquisition Time (min)

14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 30.5

Real Sample (UNK B)

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Cpd 34: OCDF: +EI MRM CID@40.0 (443.7 -> 380.8) PCDD_Dx_C1_11-28-14_07.D Noise (RMS) = 127.93; SNR (36.914min) = 1.5

16.117

18.887

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21.99930.414

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23.022 24.688

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19.758 20.936 23.96723.414 28.08926.40325.324

12 23 34 45 56 67 78 89 910

Counts vs. Acquisition Time (min)

16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 30.5

Real Sample (C1)

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Cpd 43: 13C-PCB-157(MO): +EI MRM CID@30.0 (371.9 -> 301.9) PCB_C2_11-28-14_12.D

11.969

15.635

12.374

14.990

13.714

18.089

17.64415.44516.92616.165

20.171

14 415 16 37015 27813 55112 854 16 701 18 47717 157

12 23 34 45 56 67 78 89 910 1011 1112 1213

Counts vs. Acquisition Time (min)

12 12.2 12.4 12.6 12.8 13 13.2 13.4 13.6 13.8 14 14.2 14.4 14.6 14.8 15 15.2 15.4 15.6 15.8 16 16.2 16.4 16.6 16.8 17 17.2 17.4 17.6 17.8 18 18.2 18.4 18.6 18.8 19 19.2 19.4 19.6 19.8 20 20.2 20.4

Real Sample (C2)

Questions… Thank you for your attention

Dioxin Solution Homepage

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Appendix

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7010 MS/MS PCB Linearity/Calibration PCB Calibration Range

(pg/µL) R2 Average RF StdDev RF %RSD

PCB – 28 0.5 – 1000 1.0000 1.077 0.093 8.62

PCB – 52 0.5 – 1000 1.0000 1.465 0.120 8.21

PCB – 101 0.5 – 1000 1.0000 1.276 0.101 7.93

PCB – 81 0.1 – 200 1.0000 1.024 0.030 2.89

PCB – 77 0.1 – 200 1.0000 1.040 0.029 2.79

PCB – 123 0.1 – 200 0.9974 0.620 0.440 70.96

PCB – 118 0.5 – 1000 0.9998 2.854 1.930 67.65

PCB – 114 0.1 – 200 0.9978 0.671 0.217 32.34

PCB – 105 0.1 – 200 0.9999 3.316 2.355 71.02

PCB – 153 0.5 – 1000 0.9999 0.883 0.430 48.67

PCB – 138 0.5 – 1000 1.0000 1.402 0.118 8.40

PCB – 126 0.1 – 200 1.0000 1.061 0.027 2.51

PCB – 167 0.1 – 200 1.0000 1.168 0.018 1.57

PCB – 156 0.1 – 200 1.0000 1.053 0.020 1.91

PCB – 157 0.1 – 200 1.0000 1.025 0.014 1.32

PCB – 180 0.5 – 1000 1.0000 0.930 0.073 7.84

PCB – 169 0.1 – 200 1.0000 1.228 0.018 1.50

PCB – 189 0.1 – 200 1.0000 1.095 0.025 2.25

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