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.

The Challenge of Measuring Sweet Taste in Food Ingredients and Products for Regulatory Compliance: A Scientific Opinion.

The Codex Alimentarius Commission, a central part of the joint Food and Agricultural Organization/World Health Organizations Food Standards Program, adopts internationally recognized standards, guidelines, and code of practices that help ensure safety, quality, and fairness of food trade globally. Although Codex standards are not regulations per se, regulatory authorities around the world may benchmark against these standards or introduce them into regulations within their countries. Recently, the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) initiated a draft revision to the Codex standard for follow-up formula (FUF), a drink/product (with added nutrients) for young children, to include requirements for limiting or measuring the amount of sweet taste contributed by carbohydrates in a product. Stakeholders from multiple food and beverage manufacturers expressed concern about the subjectivity of sweetness and challenges with objective measurement for verifying regulatory compliance. It is a requirement that Codex standards include a reference to a suitable method of analysis for verifying compliance with the standard. In response, AOAC INTERNATIONAL formed the Ad Hoc Expert Panel on Sweetness in November 2020 to review human perception of sweet taste, assess the landscape of internationally recognized analytical and sensory methods for measuring sweet taste in food ingredients and products, deliver recommendations to Codex regarding verification of sweet taste requirements for FUF, and develop a scientific opinion on measuring sweet taste in food and beverage products beyond FUF. Findings showed an abundance of official analytical methods for determining quantities of carbohydrates and other sweet-tasting molecules in food products and beverages, but no analytical methods capable of determining sweet taste. Furthermore, sweet taste can be determined by standard sensory analysis methods. However, it is impossible to define a sensory intensity reference value for sweetness, making them unfit to verify regulatory compliance for the purpose of international food trade. Based on these findings and recommendations, the Codex Committee on Methods of Analysis and Sampling agreed during its 41st session in May 2021 to inform CCNFSDU that there are no known validated methods to measure sweetness of carbohydrate sources; therefore, no way to determine compliance for such a requirement for FUF.

Free and total carnitine in infant and adult/pediatric nutritional formulas by liquid chromatography/tandem mass spectrometry: first action 2012.17.

Data from a previously published study in J. AOAC Int. 91,130-142 (2008) was presented to the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) during the AOAC Annual Meeting held in Las Vegas, NV, from September 30 to October 3, 2012. The panel reviewed the data submitted and concluded that the method satisfied the standard method performance requirements established by SPIFAN. Based on this information, the panel approved the method as AOAC First Action. The determination of free carnitine involves dilution and filtration of the sample, followed by determination of L-carnitine by LC/MS/MS. The determination of total carnitine includes a saponification procedure prior to dilution and filtration.

Single-laboratory validation of a liquid chromatographic/tandem mass spectrometric method for the determination of free and total carnitine in infant formula and raw ingredients.

A reversed-phase liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of free carnitine (FC) and total carnitine (TC) in infant formula and raw ingredients. Preparation of samples for FC determination required only nonvolumetric dilution in water, followed by filtration. Determination of TC in whey-based samples required a common saponification procedure, which hydrolyzed the acylcarnitines to FC. L-Carnitine standards were prepared in water at 3 concentrations (7.5, 30, and 60 ng/mL). L-Carnitine-d3 was incorporated in the sample and standard preparations as a stable isotope internal standard (ISTD), which helped to normalize signal suppression due to fouling of the ionization source, or matrix effects. A short C8 column (50 x 2.1 mm id) was used for liquid chromatography, with mobile phases consisting of 0.1% heptafluorobutyric acid in water and methanol. L-Carnitine and the ISTD eluted at about 2 min, and the injection-to-injection cycle time was about 11 min. Multiple-reaction monitoring was used for quantitation by monitoring the ion transitions m/z 162 > 103 and 162 > 85 for L-carnitine, and m/z 165 > 103 and 165 > 85 for the ISTD. The values for overall method precision for a whey-based infant formula, a protein hydrolyzate infant formula, and a water-soluble vitamin/amino acid premix were relative standard deviations of 1.32, 1.86, and 3.33%, respectively. The overall mean recoveries were between 97.3 and 102.8%. Additionally, the method results showed good correlation with those obtained for a radioenzymatic assay done in-house.

Determination of endogenous extracellular signal-regulated protein kinase by microchip capillary electrophoresis.

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.