Assessment of Regulatory Compliance Testing for Vitamin D in Infant Formula-Impact of Delegated Regulation (EU) 2019/828.

BACKGROUND: Since the publication of Standard Method Performance Requirements (SMPR®) for vitamin D in infant formula (SMPR 2011.004) by AOAC INTERNATIONAL, revised vitamin D limits have been recommended by the European Food Safety Authority (EFSA) for infant formula and adopted in Commission Delegated Regulation (EU) 2019/828. The vitamin D range introduced, 2-2.5 µg/100 kcal, is significantly narrower than previous limits specified by Codex Standard 72-1981 and requires lower method reproducibility metrics to adequately assess regulatory compliance. The narrower limits for vitamin D present a significant challenge for current-generation reference analytical methods that comply with SMPR 2011.004. OBJECTIVE: We evaluate the impact of Delegated Regulation (EU) 2019/828 on the demonstrated performance of AOAC Method 2016.05/ISO 20636:2018 to assess the likelihood that vitamin D results produced by the method would be found outside the EU limits when testing infant formula that is compliant as manufactured. METHODS: AOAC Method 2016.05/ISO 20636:2018, specifically data generated during multi-laboratory study, was used as a basis for statistical evaluation of the impact of the narrower EU vitamin D limits. RESULTS: The review of AOAC Method 2016.05/ISO 20636:2018 method performance against the vitamin D regulatory range introduced in (EU) 2019/828 indicates methods capable of performing in alignment with SMPR 2011.004 are likely to produce results that fail to meet EU requirements. CONCLUSIONS: Our assessment illustrates the high probability that a well-manufactured product with vitamin D levels within the EU regulatory range would fail to meet the regulatory requirements due to analytical method variability when tested using fit-for-purpose methods. Further, required method performance cannot be expected with the future development of new methods. To avoid this, consideration should be given to aligning proposed regulatory limits with method performance metrics of current-generation compendial methods. HIGHLIGHTS: Current, state-of-the-art methods cannot consistently verify infant formula product compliance for vitamin D in accordance with (EU) 2019/828.

Simultaneous determination of vitamins D2 and D3 by electrospray ionization LC/MS/MS in infant formula and adult nutritionals: First Action 2012.11.

A method was developed for the analysis of vitamins D2 and D3 in a variety of nutritional products. To extract vitamins D2 and D3 from products containing substantial amounts of fat, a saponification with alcoholic potassium hydroxide is required to release the vitamin D. Trideuterium-labeled vitamin D is added to the sample prior to saponification, and quantitation is achieved using linear regression of the ratio of peak response for 2H3-D and vitamin D. Acceptable linearity was achieved between 0.6 and 27 microg/100 g with a correlation requirement of >0.999. The method detection limit of 0.02 microg/100 g was verified by spiking placebo products carried through the saponification and extraction steps of the method. At the quantitation limit (0.12 microg/100 g), the signal was easily distinguished from the background. Vitamin D3 spike recoveries ranged from 107 to 119% at the low level and 104 to 116% at the high-level spike. Vitamin D2 recoveries were 105 to 116% and 91 to 110% for the low- and high-level spikes, respectively. SRM 1849a has a certified concentration of 11.1 +/- 1.7 microg/100 g; using this standard reference material, the range of 9.4 to 12.8 microg/100 g was met on each of the 6 days. Method repeatability, determined in 12 vitamin D3 product matrixes over 6 days, ranged from 3.9 to 48%. The adult nutrition-milk protein sample was the most notable; it failed within-day, as well as day-to-day, precision requirements. There was no attempt to optimize the sample preparation to accommodate any problem matrix.

Simultaneous determination of vitamins D2 and D3 by LC-MS/MS in infant formula and adult nutritionals: First Action 2011.13.

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held on June 29, 2011, an Expert Review Panel (ERP) on behalf of AOAC INTERNATIONAL adopted the method "Simultaneous Determination of Vitamins D2 and D3 by LC-MS/MS in Infant Formula and Adult Nutritionals" as an AOAC Official First Action method. Vitamins D2 and D3 are extracted from the sample using pentane-ether; the extract is collected and dried under nitrogen. Vitamin D is separated from interfering compounds using UPLC, and quantitated using tandem mass spectrometry (MS/MS). Preliminary data showed the intermediate precision ranged from 3.34-8.05% and an accuracy range of 98.5-111% over the samples tested for vitamin D3. For vitamin D2, the intermediate precision ranged from 2.37-5.45% and accuracy ranged from 96.4-104% over the four matrixes evaluated. The analytical range for the method is bounded by the concentrations of the working standards, 21-270 ng/mL, and is equivalent to 0.168-2.16 mcg/100 g in ready-to-feed product. The practical method quantitation limit is 0.168 mcg/100 g product with method detection limit of 60 ng/100 g product. The ERP reviewed the data and determined that the performance characteristics of the method met the standard method performance requirements, and therefore the method was granted First Action status.