AOAC SPIFAN Stakeholder Panel Meeting Book (March 14, 2019)

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GB

Comment leading to result difference

InternationalOfficialMethod (OM) AOAC2016.05, ISO 20636:2018 VitaminD2 andVitamin D3 Infant Formula andAdultNutritionals

GB 5009.82-2016 (method 3) VitaminD2 and VitaminD3

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Method number

Analyte Scope

Different detection products lead todifferentmatrix effects. OMmethod using d6-vitaminD2 and d6-vitaminD3 as internal standardof vitaminD2 and vitaminD3 ; GBmethod using d3-vitaminD2 and d3-vitaminD3 as internal standardof vitaminD2 and vitaminD3 ; VitaminD becomes vitaminD-PTAD adduct after the reaction ofOM method; VitaminD did not change during the saponification ofGBmethod.

Food (forVitamin D2 andVitaminD3 )

Comment lead to result difference

Method Principle

Samples are saponified athigh temperature then lipid soluble components are extracted into isooctane. Aportion of the isooctane layer is transferred,washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is added toderivatise vitaminD to form a highmolecularmass, easily ionisable adduct. The vitaminD-adduct is then re extracted into a small volume of acetonitrile and analysed by reversed-phase liquid chromatography. Detection is bymass spectrometry usingmultiple reactionmonitoring (MRM). Stable isotope labelled d6-vitaminD2 and d6-vitaminD3 internal standards are used forquantitation to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies.

After adding the isotope internal standard of vitaminD2 and vitaminD3 to the sample, it is saponifiedby potassium hydroxide ethanol solution (the starch sample is firstdigested with amylase),extracted, purified by silica gel solid phase extraction column, concentrated, and then reversed efficiently. Liquid chromatography C18 column separation, tandem mass spectrometry detection, internal standardmethod quantitative.

VitaminD3:Codex STAN72-1981 Minimum 1 njg/100kcal, 0.25njg/100kJ Maximum 2.5njg/100kcal, 0.6 njg/100kJ GUL - Minimum 0.65 njg/100ml liquid IF Minimum 5.85 njg/100gpowder IF

VitaminD: CodexGB 10765-2010

Regulatoryminimum andmaximum limits

Minimum 1.05 njg/100kcal, 0.25 njg/100kJ Maximum 2.51njg/100kcal, 0.60njg/100kJ CodexGB 10767 -2010 andGB 10769-2010 Minimum 1.05 njg/100kcal, 0.25 njg/100kJ Maximum 3.14njg/100kcal, 0.75njg/100kJ Liquid chromatography-tandemmass spectrometry (LC-MS/MS)

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Liquid chromatography-tandemmass spectrometry (LC-MS/MS)

Analysis Technology

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Calibration curve concentration range Five-point calibration curve: 0.4–50ng/mL

Six-point calibration curve: 0.0njg/L 澝 20.0njg/L 澝 50.0njg/L 澝 100njg/L 澝 150njg/L 澝 200njg/L 澞 Calibration curve isdifferent.

Chromatography: Reversed Phase Liquid Chromatography column =C18 column 1.8njm, 2.1mmȜ mm, or equivalent; binary gradient(A:0.05% formic acid -5mmol/L ammonium formatesolution;mobile phase B:0.05% formic acid -5mmol/L ammonium formatemethanol solution; Mass Spectrometry: ElectrosparyIonisationwithMultiple ReactionMonitoring ionizationmode: ESI+; detection:multiple rwactionmonitoring (MRM) VD2

Equipment Parameters

Chromatography: Reversed Phase Liquid Chromatography column =Phenomenex Kinetex C18 2.6njm, 2.1mm x 50mm, or equivalent binary gradient (A=0.1% formic acid,B= 100%methanol) Mass Spectrometry: Electrospary IonisationwithMultiple ReactionMonitoring ionizationmode = ESI+ detection:multiple rwactionmonitoring (MRM)

Mobile phase, quantifier ion and qualifier ion are different. The analyte is the vitaminD-PTAD adduct, and the internal standard ion is the d6-vitamin D-PTAD adduct of theOMmethod; The analyte is the vitaminD, and the internal standard ion is the d3-vitamin D of theGBmethod.

analyteTXDQWLILHULRQ ମ analyteTXDOLILHULRQ ମ LQWHUQDOVWDQGDUGTXDQWLILHULRQ ମ LQWHUQDOVWDQGDUGTXDOLILHULRQ ମ VD3 analyteTXDQWLILHULRQ ମ analyteTXDOLILHULRQ ମ LQWHUQDOVWDQGDUGTXDQWLILHULRQ ମ LQWHUQDOVWDQGDUGTXDOLILHULRQ ମ

DQDO\WHTXDQWLILHULRQ ମ DQDO\WHTXDOLILHULRQ ମ LQWHUQDOVWDQGDUGTXDQWLILHULRQ ମ LQWHUQDOVWDQGDUGTXDOLILHULRQ ମ

Preliminary conclusion AOAC2016.05, ISO 20636:2018 and GB 5009.82-2016 (method 3) will probably give different results for the same sample.

20PHWKRGDGGLQJ njJPL[HG internal standards; *%PHWKRGDGGLQJ njJPL[HG internal standards.

Calibration Technique

Stable Isotope Internal Standardisation: VitaminD2-d3 and VitaminD3-d3mixed standard at a concentration of 1 njJP/ Added amount = 0.1mL.

Stable Isotope Internal Standardisation: 2H6-Vitamin D2 and 2H6-Vitamin D3mixed standard at a concentration of 1 njJP/$GGHGDPRXQW P/ Samplesweighed in grams to four decimal places. Powder sample 2g; Liquid Sample:10mL; 5HFRQVWLWXWHGVDPSOH JVDPSOHLVGLVVROYHGDQGPL[HGLQ P/RIZDWHUDQGWKHQ 10gof the above reconstituted sample isweighed. VitaminD is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification (ethanolic pyrogallol solution, internal standard, potassium hydroxide VROXWLRQ q C/1hr; 2. Extraction (isooctane) 3.Derivitisation PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) 4.Back extraction (acetonitrile) 5. Inject on LC-MS

Sampleweight

Samplesweighed in grams to three decimal places Solid samples: 2g ; (accurate to 0.01g)

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Sample preparation procedure

Sample preparation A certain number of sampleswere condensed, crushed and homogenized according to the requirements, and then stored in the sample bottle. The sampleswere stored in the light- proof refrigeration and determined as soon as possible. Sample treatment Note: the process should avoidultraviolet light and avoid light as far as possible. Procedure Because vitaminD is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification ((ethanolicVcsolution, internal standard, potassium hydroxide VROXWLRQ q C/30min;);

OMmethod using saponification conditions at 70 q C for 1h; GBmethod using saponification conditions at 80 q C for 30min. OMmethod using 4-phenyl-1,2,4- triazoline-3,5-dione as aderivatization reagent of the derivatization reaction; GBmethod using ascorbic acid as an antioxidant during the saponification.

2.Extract (Petroleum ether) 3.Back extraction (methanol) 4. Inject on LC-MS

njJ JWRRQHGHFLPDOSODFH VitaminD2: LOD (powders)= 0.12njg/100g LOQ (powders)= 0.15njg/100g, LOD (liquids)= 0.013njg/100g LOQ (liquids)= 0.016njg/100g VitaminD3: LOD (powders)= 0.16njg/100g LOQ (powders)= 0.25njg/100g, LOD (liquids)= 0.018njg/100g LOQ (liquids)= 0.028njg/100g Repeatability: 5.8%RSD Reproducibility: 12.7%RSD VitaminD3: Repeatability: 1.9–5.1%RSD Reproducibility: 6.4–12.4%RSD

njJ J 7KHFDOFXODWLRQUHVXOWVUHWDLQ YDOLGGLJLWV

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Final reportable results

LOD& LOQ

VitaminD2: LOD= 1njg/100g LOQ = 3njg/100g, VitaminD3: LOD= 0.2njg/100g LOQ = 0.6njg/100g.

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Precison (Repeatbility, Reproducibility) VitaminD2:

repeatability VD2:1.66%RSD VD3:2.05%RSD Reproducibility

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VD2:1.05-2.60%RSD VD3:2.81-6.03%RSD

Conclusion: AOAC2016.05, IS+R[-17]C[1]+R[-7]C:R[1]C[3]+R[-9]C:R[1]C[3]+R[-12]C:R[1]C[3]+R[-17]C[1]+R[-7]C:R[1]C[3]+R[-13]C:R[1]C[3]+R[-17]+R[-18]C:R[1]C[3]

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)USVGXOYUT UL

ISO 14892:2002, IDF 177:2002 and GB 5009.82-2016 (4)

Comment leading to result difference

GB

InternationalOfficialMethod (OM) ISO 14892:2002, IDF 177:2002

Method number

GB 5009.82-2016 (method 4) VitaminD2 orVitaminD3

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Analyte

VitaminD2 orVitaminD3 Skimmedmilk powder

Different detection products lead to differentmatrix effects

Comment lead to result difference

Scope

Formula food (forVitamin D2 orVitamin D3 )

The vitaminD2 or vitaminD3 in the sample is saponified by potassium hydroxide ethanol solution (the starch sample is first digestedwith amylase), extracted, purified, concentrated, and then semi-prepared by normal phase high performance liquid chromatography. Separation by phase chromatography on C18 column chromatography, detection byUV ordiode array detector, internal standardmethod (or external standard method). For the determination of vitaminD2, vitaminD3 can be used as an internal standard; if vitaminD3 is determined, vitaminD2 can be used as an internal standard.

Method Principle

The test sample is saponified and extracted. VitaminD is separated from impurities by a semi-preparative clean-up using normal-phase HPLC.The vitaminD from the clean-up column is collected. The content is determined using reverse-phase HPLCwithUVdetection. VitaminD2 is used as an internal standard in the determination of vitaminD3 and vice versa. The internal standard is added to each test portion prior to saponification.

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Regulatoryminimum and maximum limits

VitaminD: CodexGB 10765-2010

Minimum 1.05 njg/100kcal, 0.25 njg/100kJ Maximum 2.51njg/100kcal, 0.60njg/100kJ CodexGB 10767 -2010 andGB 10769-2010 Minimum 1.05 njg/100kcal, 0.25 njg/100kJ Maximum 3.14njg/100kcal, 0.75njg/100kJ

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Analysis Technology

High performance liquid chromatography-ultraviolet detection (HPLC-UV)

High performance liquid chromatography-ultraviolet detection (HPLC-UV)

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OMmethod using single point calibration for quantification ; GBmethod using six-point calibration curve for quantification. OMmethod using acetonitrile/ propan-2-ol/water, 90/7/3 as mobile phase; GBmethod usingmethanol /water, 95/5 asmobile phase. 20PHWKRGDGGLQJ njJ internal standards; *%PHWKRGDGGLQJ njJ LQWHUQDO standards.

Calibration curve concentration range

Six-point calibration curve:0.05njg/mL 澝 0.10njg/mL 澝 0.20njg/mL 澝 0.40njg/mL 澝 0.60njg/mL 澝 1.00njg/mL

Single-point calibration: 0.2njg/mL

Reversed Phase Liquid Chromatography: column =C18 5njm, 4.6mm x 250mm isocratic (acetonitrile/propan-2-ol/water, 90/7/3)

Equipment Parameters

Reversed Phase Liquid Chromatography: column =C18 5njm, 4.6mm Ȝ mm isocratic (methanol/water, 95/5)

UltravioletDetection: wavelength: 265nm

UltravioletDetection: wavelength: 264nm

Calibration Technique

Internal Standardisation: VitaminD2 is the internal standard for vitaminD3 analyssi(and vice versa). 7KHFRQFHQWUDWLRQRIYLWDPLQ' LV njJP/$GGHGDPRXQW P/ Samplesweighed in grams to four decimal places. Powder sample 2g; Liquid Sample:10mL; 5HFRQVWLWXWHGVDPSOH JVDPSOHLVGLVVROYHGDQGPL[HGLQ P/RIZDWHU and then 10gof the above reconstituted sample isweighed.

nternal Standardisation: VitaminD2 is the internal standard for vitaminD3 analyssi (and vice versa). 7KHFRQFHQWUDWLRQRIYLWDPLQ' LV njJP/$GGHGDPRXQW P/

Preliminary conclusion : GB 5009.82-2016 (method 4) and ISO 14892:2002, IDF 177:2002 will probably give similar results for the same sample.

Sampleweight

Samplesweighed in grams to three decimal places Solid sample: 5-10 g; Liquid sample: 50g ; (accurate to 0.01g)

Sampleweight isdifferent.

Sample preparation procedure

Sample preparation A certain number of sampleswere condensed, crushed and homogenized according to the requirements, and then stored in the sample bottle. The sampleswere stored in the light-proof refrigeration and determined as The process should avoidultraviolet light and avoid light as far as possible. If the sample contains only vitaminD3, vitaminD2 can be used as an internal standard.If only contains vitaminD2, can use vitaminD3 as the internal standard; otherwise,with external standardquantitative, but need to verify the recovery rate canmeet the detection requirements. Procedure Because vitaminD is sensitive to light, perform all steps under UV-shielded lighting: 1.Saponification ((ethanolic Vc solution, internal standard, potassium K\GUR[LGHVROXWLRQ q C/30min;); 2.Extract (Petroleum ether) 3.Concentrate 4.Back extraction (methanol) 5. Inject onHPLC soon aspossible. Sample treatment

VitaminD is sensitive to light, perform all steps under UV-shielded lighting: 1. Saponification (ethanol, sodium ascorbate, internal standard,potassium K\GUR[LGHVROXWLRQVWHDPEDWK PLQV 2.Extract: Light petroleum; 3.Concentrate: final volume of 2mL light petroleum 4.Normall phase semi-prep chromatography 5. Inject onHPLC

The saponification temperature(100 q C) of OM method is higher than theGB method(80 q C).

Final reportable results

njJJWRWZRGHFLPDOSODFHV

njJ J7KHFDOFXODWLRQUHVXOWVUHWDLQ YDOLGGLJLWV

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LOD& LOQ

When the sample volume is 10 g, VitaminD2 orVitaminD3

VitaminD2 andVitamin D3: LOD= 2.5njg/100g LOQ=10njg/100g (from scope)

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/2' njJ J /24 njJ J

Repeatability VD2:7.63%RSD VD3:3.57%RSD Reproducibility

Precison (Repeatbility, Reproducibility)

VitaminD2 andVitamin D3: Repeatability:14% RSD Reproducibility: 17% RSD

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VD2:2.72-8.10%RSD VD3:1.00-8.19%RSD

Conclusion: GB 5009.82-2016 (method 4) and ISO 14892:2002, IDF 177:2002will probably give similar results for the same sample. It should be noted that ISO 14892:2002, IDF 177:2002, is probably based on EN 12821 and also the validation data from thismethod. EN 12821 is validated for:Margarine,milk powder,milk, liquid IF, cooking oil, IF, fish oil. ISO 14892:2002, IDF 177:2002 has probably thewrong precision data, should bedata from EN 12821

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