A new gold standard for Dioxin analysisA combined approach ; automated efficient clean-up together with a new Triple Quadrupole GC-MS/MS

Haruhiko Miyagawa, GCMS Product ManagerAnalytical &Measuring Instruments DivisionSHIMADZU Cooperation DIOXIN 2016

Recent demands for Dioxin analysis in food/feed

2

Dioxins S3, Smart Solution Systems

Reliable results • Easier and faster operation • Automated procedure• Lower running cost

AnalysisAnalysis

Benefits of the system

3

Sample Preparation Extraction Purification Concentration Measurement

FattyAcid

Fat

…

1 day > 2 days 0.5 day> 1 day

>2hr >3hrs

Analysis of12 samples

BUCHI SpeedExtractor E-914 / E-916

4

>10x Faster extraction• Extracts the samples at elevated temperature

and pressure

High throughput• Parallel extraction of up to 6 samples

Less solvent use• Sample volumes 10-120 mL, no limitation for

large sample size

Reliable and efficient• Direct sample transfer between extraction and

concentration • Extract collection glassware is compatible to

evaporation instruments from BUCHI

MIURA GO-HT series

5

Consumables

Silver Nitrate – Si columnSulfuric Acid – Si columnCarbon columnAlOx column

Total Solvents 110mL

required Solvents Hex, Tol

Cost Sol(¥120/euro) ¥221 (€2)

Final fraction volume 1 ~ 1.5 mL

Max. Sample Weight 3 g fat

Max. Capacity 6 samples

Basic Time 94 mins

No Cross-contaminationReliable Results!Washing the system is not necessary!

Less solventNo DCM use

Small fraction

Less solventNo DCM use

Small fraction

Automated & shorter time

process

Automated & shorter time

process

Ultra-high sensitivity for the fg quantitative analysis

Durable hardware for routine work

Easy to use, simple operation and maintenance

6

Shimadzu GCMS-TQ8050

A new GC-MSMS, our latest model!

Foods : Pork meat, Egg, Salmon, Olive oil from the market, 3g fat collected.

Standard Reference Material :Cod liver oil (EDF-5462, EDF-5463 from CIL), 1g fat collected.

Sample were extracted by SpeedExtractor (BUCHI) and purified by GO-HT (MIURA)

Obtain 1.3mL Dioxin fraction and 1.0mL PCB fraction, concentrated to 20uL and 50uL.

7

Samples

8

GC-MS/MS conditionsGCColumn： DB-5MS (60 m, 0.25 mm I.D., df =0.25 μm) Glass Insert： Custom SkyLiner, Splitless (Restek)Injection Volume： 2 μLInjection Mode： SplitlessInjection Temp： 280 ℃Column Oven Temp. program：150 ℃ (1 min)→(20 ℃/min) → 220 ℃ → (2 ℃/min) → 260 ℃(3 min) → (5 ℃/min) → 320 ℃(8.5min)Flow Control Mode： Constant Linear Velocity 29.4 cm/sec

MS Ionization Mode： EI Acq.Mode： MRM modeIonization Volt.： 70 eVEmission current： 150 μATransision, CE： refer to Smart Env. DatabaseLoop Time: 1 sec (Target: 0.8 sec., ISTD: 0.2 sec.)Pressure of CID gas: 150 kPaMS resolution: Q1 Unit, Q3 UnitDetector volt.: 2.1 kVCalibration range： conc. 0.05 ppb ~ 2 ppb

New RegulationFood Commission Regulation

No 589/2014

30.50 30.75 31.00 31.25 31.50 31.75

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x10,000)

375.80>312.90373.80>310.90

1,2,3,4,7,8- HxCDF

1,2,3,6,7,8- HxCDF

Instrument performance check

9

compoundRRF at conc. (ppb) LOQ LOD

0.05 0.1 0.2 3σ 10σ2,3,7,8-TeCDD 1.06 1.17 1.26 0.025 0.082 1,2,3,7,8-PeCDD 1.16 1.00 1.09 0.008 0.028 1,2,3,4,7,8-HxCDD 1.15 1.07 1.07 0.013 0.045 1,2,3,6,7,8-HxCDD 1.21 1.07 1.10 0.017 0.058 1,2,3,7,8,9-HxCDD 1.03 0.99 1.07 0.023 0.078 1,2,3,4,6,7,8-HpCDD 1.23 1.24 1.08 0.017 0.057 OCDD 1.05 1.08 1.21 0.044 0.146 2,3,7,8-TeCDF 1.25 1.20 1.12 0.023 0.077 1,2,3,7,8-PeCDF 0.96 1.13 1.07 0.021 0.068 2,3,4,7,8-PeCDF 1.06 1.09 1.03 0.019 0.064 1,2,3,4,7,8-HxCDF 1.13 1.08 1.06 0.020 0.068 1,2,3,6,7,8-HxCDF 1.06 1.12 1.05 0.008 0.028 2,3,4,6,7,8-HxCDF 1.08 1.18 1.07 0.024 0.080 1,2,3,7,8,9-HxCDF 1.00 0.93 0.94 0.031 0.102 1,2,3,4,6,7,8-HpCDF 1.34 1.15 1.17 0.018 0.060 1,2,3,4,7,8,9-HpCDF 1.07 1.07 1.01 0.012 0.042 OCDF 1.09 1.14 1.12 0.029 0.098

20.0 21.0

5000

10000

15000

20000

25000 321.90>258.90319.90>256.90

25.0 26.0 27.0

5000

10000

15000

20000

25000353.90>290.90355.90>292.90

31 0 32 0

5000

10000

15000

20000

25000

30000

391.80>328.80389.80>326.90

2,3,7,8-TeCDD

1,2,3,7,8-PeCDD

1,2,3,4,7,8-HxCDD

0.05ppbStandards15 injectionsStandards15 injections

10

Stress test : Stability check

Standards15 injectionsStandards15 injections

Pig fat15 injections

Compound NameInjection number Mean

valueConc(ppb)

%RSD6 7 8 31 47 63 76 89 99

2,3,7,8-TeCDD 0.52 0.53 0.48 0.52 0.50 0.50 0.46 0.50 0.48 0.50 0.50 4.40 1,2,3,7,8-PeCDD 0.47 0.51 0.51 0.50 0.47 0.51 0.49 0.50 0.44 0.50 0.50 3.51 1,2,3,4,7,8-HxCDD 0.93 1.05 1.13 1.21 1.14 1.04 1.21 1.11 1.11 1.10 1.00 8.65 1,2,3,6,7,8-HxCDD 1.14 1.09 0.95 1.08 1.09 1.05 1.03 1.05 1.17 1.06 1.00 5.41 1,2,3,7,8,9-HxCDD 1.03 1.03 0.97 1.09 0.98 1.03 1.09 1.14 1.05 1.04 1.00 5.57 1,2,3,4,6,7,8-HpCDD 0.97 0.98 1.19 1.11 1.07 1.12 1.15 1.06 1.02 1.08 1.00 7.17 OCDD 2.64 2.75 2.84 3.04 2.78 2.73 2.74 2.78 2.73 2.79 3.00 4.17 2,3,7,8-TeCDF 0.54 0.51 0.56 0.55 0.55 0.52 0.56 0.57 0.55 0.55 0.50 4.12 1,2,3,7,8-PeCDF 0.49 0.51 0.53 0.52 0.51 0.52 0.51 0.55 0.49 0.52 0.50 3.26 2,3,4,7,8-PeCDF 0.55 0.54 0.49 0.51 0.51 0.46 0.55 0.52 0.50 0.52 0.50 5.89 1,2,3,4,7,8-HxCDF 1.24 1.03 1.15 1.11 1.16 1.11 1.10 1.08 1.17 1.12 1.00 5.42 1,2,3,6,7,8-HxCDF 0.99 1.03 1.05 1.23 1.05 1.09 1.20 1.14 1.09 1.10 1.00 7.55 2,3,4,6,7,8-HxCDF 1.09 1.11 1.14 1.15 1.13 1.12 1.14 1.12 1.13 1.12 1.00 1.72 1,2,3,7,8,9-HxCDF 1.07 1.01 1.05 1.15 1.02 1.10 1.08 1.03 1.15 1.06 1.00 4.30 1,2,3,4,6,7,8-HpCDF 0.99 1.16 0.96 1.07 1.05 0.99 1.14 1.08 1.15 1.05 1.00 6.95 1,2,3,4,7,8,9-HpCDF 1.08 1.10 1.02 1.12 1.40 1.14 1.19 1.05 1.16 1.14 1.00 10.35 OCDF 2.96 2.64 3.02 3.05 2.79 3.01 2.94 2.93 3.11 2.92 3.00 4.77

Olive15 injections

Egg15 injections

Salmon15 injections

EDF-5462EDF-5463

11

Analysis of the food samples (pg/g)

Compound NamePig fat Oliveoil Egg fat Salmon

GC-MS/MS GCHRMS GC-MS/MS GCHRMS GC-MS/MS GCHRMS GC-MS/MS GCHRMS2,3,7,8-TeCDD ND ND ND ND ND ND ND ND1,2,3,7,8-PeCDD ND ND ND ND ND ND ND ND1,2,3,4,7,8-HxCDD ND ND ND ND ND ND ND ND1,2,3,6,7,8-HxCDD ND ND ND ND ND ND ND ND1,2,3,7,8,9-HxCDD ND ND ND ND ND (0.3) ND ND1,2,3,4,6,7,8-HpCDD ND ND ND ND 0.6 (0.5) ND (0.2)OCDD 1.0 (1.1) ND ND 3.0 3.6 ND (0.5)2,3,7,8-TeCDF ND ND ND ND (0.2) (0.3) (0.3) (0.4)1,2,3,7,8-PeCDF ND ND ND ND ND (0.2) ND ND2,3,4,7,8-PeCDF ND ND ND ND (0.1) (0.2) (0.2) (0.3)1,2,3,4,7,8-HxCDF ND ND ND ND ND ND ND ND1,2,3,6,7,8-HxCDF ND ND ND ND ND ND ND ND1,2,3,7,8,9-HxCDF ND ND ND ND ND ND ND ND2,3,4,6,7,8-HxCDF ND ND ND ND ND ND ND ND1,2,3,4,6,7,8-HpCDF ND ND ND ND ND ND ND ND1,2,3,4,7,8,9-HpCDF ND ND ND ND ND ND ND NDOCDF ND ND ND ND ND ND ND ND

Evaluation of quantitative values

( ) : LOD < value < LOQ

Pig fat15 injections

Olive15 injections

Egg15 injections

Salmon15 injections

20.0 21.0

0.0

2.5

5.0

7.5

(x1,000)

305.90>242.95303.90>240.95

2,3,7,8-TeCDF(in Egg fat)

39.0 40.0 41.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5(x10,000)

459.70>396.70457.70>394.70

OCDD(in Egg fat)

12

Evaluation of quantitative values Quantitative values of EDF-5462Comparison of the individual dioxin concentrations using GC-HRMS and GC-MS/MS.

Compounds NameMean value %RSD(n=3)

GC-MS/MS GCHRMS GC-MS/MS GCHRMS2,3,7,8-TeCDD 16.71 16.25 7.38 5.89 1,2,3,7,8-PeCDD 39.74 43.25 4.66 6.37 1,2,3,4,7,8-HxCDD 43.75 40.25 5.71 3.73 1,2,3,6,7,8-HxCDD 37.59 39.25 5.94 5.65 1,2,3,7,8,9-HxCDD 49.44 43.25 5.32 3.95 1,2,3,4,6,7,8-HpCDD 40.83 40.50 6.30 7.41 OCDD 120.95 110.00 5.87 0.00 2,3,7,8-TeCDF *1069.94 1425.00 3.26 3.51 1,2,3,7,8-PeCDF 355.86 387.50 4.03 3.87 2,3,4,7,8-PeCDF 41.44 40.00 4.50 5.40 1,2,3,4,7,8-HxCDF 45.45 41.00 6.60 1.99 1,2,3,6,7,8-HxCDF 46.63 41.50 2.74 5.74 2,3,4,6,7,8-HxCDF 37.52 41.00 4.50 1.99 1,2,3,7,8,9-HxCDF 44.73 39.25 5.38 1.27 1,2,3,4,6,7,8-HpCDF 41.74 41.00 5.47 3.45 1,2,3,4,7,8,9-HpCDF 38.36 43.00 7.17 4.25 OCDF 82.26 76.00 5.41 1.86

*Peak was saturated

EDF-5462EDF-5463

Dioxins S3, Smart Solution Systems contribute

Speed of the sample preparation

Reduction of running cost

Reliability of the results

13

Summary

New RegulationFood Commission Regulation

No 589/2014

Shimadzu Europe

Future work

More evaluationsFaster GC analysis

14

48 min.(Column:DB-5MS 60 m))

31 min.(Column:DB-5MS 30 m)

19.0 19.5 20.0 20.5 21.0

100000

200000

300000

400000

500000

600000

700000

800000

900000375.80>312.90373.80>310.90

Peak separation of 1,2,3,4,7,8- HxCDF to 1,2,3,6,7,8-HxCDF is satisfactory to the criteria.

New RegulationFood Commission Regulation

No 589/2014Feed Commission Regulation

(EC) No 709/2014

Thank you for your attention

15