AOAC SPIFAN Stakeholder Panel (August 25, 2018)

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Type C: Example International method & GB comparison for vitamin D in infant formula & dairy

International Official Method

Method number

AOAC 2016.05(ISO 20636)

ISO 14892 (IDF 177)

GB 5009.82-2016 (method 3)

GB 5009.82-2016 (method 4)

Analyte Scope

Vitamin D

Fortified milk powders, infant formulas, and adult/pediatric nutritional formulas. (forVitamin D2 and Vitamin D3 ) Samples are saponified at high temperature; then lipid-soluble componentsare extracted into isooctane. A portion of the isooctane layer is transferredand washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is added to derivatize vitamin D to form a high-molecular-mass,easily ionizable adduct. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by MS using multiple reaction monitoring (MRM). Stable isotope-labeled (SIL) d6-vitamin D2 and d6-vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies.

Dried skimmedmilk

Food (forVitamin D2 and Vitamin D3 )

Formula food (forVitamin D2 or Vitamin D3 )

The vitamin D2 or vitamin D3 in the sample is saponified by potassium hydroxide ethanol solution (the starch sample is first digested with amylase), extracted, purified, concentrated,and then semi-preparedby normal phase high performance liquid chromatography. Separation by phase chromatographyon C18 column chromatography, detection by UV or diode arraydetector, internal standard method (or external standard method).For the determinationof vitamin D2, vitamin D3 can be used as an internal standard; if vitamin D3 is determined,vitamin D2 can be used as an internal standard.

The test sample is saponified and extracted. Vitamin D is separated from impurities by a semi-preparative clean-up using normal-phaseHPLC. Thevitamin D from the clean-up column is collected. Thecontent is determinedusing reverse- phase HPLC with UV detection. Vitamin D2 is used as an internal standard in the determinationof vitamin D3 and vice versa. The internal standard is added to each test portion prior to saponification.

After adding the isotope internal standardof vitamin D2 and vitamin D3 to the sample, it is saponified by potassium hydroxide ethanol solution (the starch sample is first digested with amylase),extracted,purified by silica gel solid phase extraction column, concentrated,and then reversed efficiently. Liquid chromatographyC18 column separation, tandem mass spectrometrydetection, internal standard methodquantitative.

Method Principle

Analysis Technology

LC-MS/MS

HPLC

LC-MS

HPLC

D2 and D3 are internal standard solutions at a concentration of 1 ȝJ P/ Add amount:1mLD3 or D2; Theexternal standard method needs to verify that the recovery rate meets the requirements.

Calibration Technique D2-[2H6] and D3-[2H6] mixed isotope internal standard at a concentration of 1 ȝJ P/ Add amount:0.5ml

D2 and D3 are internal standard solutions, the concentration ' LV ȝJ P/ Add amount:4mL

D2-[2H3] and D3-[2H3] mixed isotope internal standard, FRQFHQWUDWLRQ ȝJ P/ $GG DPRXQW ȝ/

Reconstituted sample: weigh 50g and dissolve it in 100mL 60°C-80°C hot water,and weigh 20g of the above complex solution; (accurate to 0.01g)

Powder sample: 1.8-2.2g; Liquid sample: 10.0 mL; Reconstitutedsample: 19.0- 21.0g sample is dissolved and mixed in 80mLof water, and then 9.5-10.5g of the above reconstituted sample is weighed; (accurate to 0.01g)

Sample weighing

Solid samples: 2g ; (accurate to 0.01g)

Solid sample: 5-10 g; Liquid sample: 50g ; (accurate to 0.01g)

Because vitamin D is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification : Saponification solution:Starch-containing sample need to be added with amylase Saponification temperature: 80 Ԩ ; Saponification time: 30min; 2.Extract: centrifugation 3.Purify 4. Inject on LC-MS

Because vitamin D is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification; Saponification solution: Pyrogallol ethanol solution, internal standard,potassium hydroxide solution; Saponification temperature: 70 Ԩ ; Saponification time: 1h; 2.Extract: H[WUDFWLRQĺFHQWULIXJDWLRQĺGHULYDWL]DWLRQ 3.Purify 4. Inject on LC-MS

Because vitamin D is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification : Condensation reflux in a boiling water bath (steam bath) for30 min; 2.Extract: H[WUDFWLRQĺZDVKLQJ 3.Concentrate 4.Purify 5. Inject on HPLC

Because vitamin D is sensitive to light, perform all steps under UV- shielded lighting: 1.Saponification : Saponification solution:Starch- containing sample need to be added with amylase Saponification temperature:80 Ԩ ; Saponification time: 30min; 2.Extract: H[WUDFWLRQĺZDVKLQJ 4.Purify 5. Inject on HPLC

Sample preparation Procedure

Final reportable results ȝJ J

ȝJ J

When the sample volume is 2 g,vitamin D2 㸸 LOD-1 ȝJ J 㸹 LOQ- ȝJ J 㸹 vitamin D3 㸸 LOD- ȝJ /100g 㸹 LOQ- ȝJ J

When the sample volume is 10 g,vitamin D2 or vitamin D3 㸸 LOD- ȝJ J 㸹 LOQ- ȝJ J 㸹

LOD & LOQ

The absolute differencebetween two independent single test results, obtained using the samemethodon identical test material in the same laboratoryby the sameoperatorusing the sameequipmentwithin a short interval of time, will in not more than 5 % of cases be greater than 14 % (relative) of the arithmeticmeanof the two results.

Precison (Repeatbility, Reproducibility)

The absolute differencebetween two independent determinations obtained under repeatability conditions shall not exceed 15% of the arithmeticmean

Type C: Example International Method/GB comparison for vitamin D in infant formula and dairy

Conclusion: Possible differences between OM/ISO/GB results (i.e due to Subsampling representativeness , internal standard, Isotope internal standard 㸪 and/or sample preparation/equipment 㸧

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