GC/MS/MS Dioxin and Dioxin-like PCBs in Feed and Food · GC/MS/MS is now a confirmatory technique...
Transcript of GC/MS/MS Dioxin and Dioxin-like PCBs in Feed and Food · GC/MS/MS is now a confirmatory technique...
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
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126 167 15
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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
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19.5 20 20.5
16.1
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isition Time (min)
15 15
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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
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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.
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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 [email protected] (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 [email protected] (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 [email protected] (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
18.303
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 [email protected] (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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