AOAC SPIFAN Stakeholder Panel Meeting Book (September 7, 2019)

AOAC 2016.05 Method

Principle Samples are saponified at high temperature, and then lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl1,2,4-triazoline-3,5-dione (PTAD) is added to derivatize vitamin D to form a high- molecular-mass, easily ionisable adduct. The vitamin D adduct is subsequently extracted into a small volume of acetonitrile and analyzed by

high temperature saponification

solvent extraction

solvent wash

solvent transfer

reversed-phase liquid chromatography (RPLC). Detection is by tandem mass spectrometry (MS/MS) using multiple reaction monitoring (MRM). Stable isotope- labelled (SIL) d6 -vitamin D 2 and d6 -vitamin D 3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies [2].

PTAD derivitisation

LC-MS analysis

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133 rd Annual Meeting & Exposition | Denver, Colorado | September 6-12, 2019

Response - CEN TC 275 WG9

Recommendation In recent WG 9 meeting (July 2019), the EU Regulation was discussed in the context of fitness-for-purpose of analytical standards for vitamin D, particular AOAC 2016.05/ISO 20636 and made the following recommendation [4]: Recommendation 173 – Fitness for purpose analytical standards vitamin D WG 9 is concerned about EU legislation (EU 2019/828, modifying EU

Regulation 2016/127) on the regulatory limits of vitamin D in infant formula, in relation to the fitness-for-purpose of analytical standards on determination of vitamin D (e. g. EN 12821 and ISO 20636). Given the precision of these methods, there is a high risk of failing to demonstrate compliance to the regulation. The probability of an analytical measurement out of regulatory specifications for a compliant product is high DSSUR[LPDWHO\ XQOHVV PHDVXUHPHQW XQFHUWDLQW\ LV WDNHQ LQWR DFFRXQW

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133 rd Annual Meeting & Exposition | Denver, Colorado | September 6-12, 2019