Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: Collaborative Study, Final Action AOAC 2021.07.

BACKGROUND: Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective and antimicrobial factors to the neonate. OBJECTIVE: To evaluate method reproducibility of AOAC 2021.07 Official First Action method for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. METHOD: Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a 4-parameter calibration regression. RESULTS: After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable HorRatR values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin clean-up LC UV), showed negligible difference between results. CONCLUSIONS: The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official Method. HIGHLIGHTS: A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described.

Enhanced Automated Online Immunoaffinity Liquid Chromatography-Fluorescence Method for the Determination of Aflatoxin M1 in Dairy Products.

BACKGROUND: Aflatoxin M1 (AFM1) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B1, which is converted in the cow liver by hydroxylation to AFM1 and is then expressed in milk. AFM1 is regulated in milk and other dairy products because it can cause serious health issues, such as liver and kidney cancers, in humans and is an immunosuppressant. OBJECTIVE: To optimize the chromatographic protocol and to extend the matrix scope to include a wider range of dairy products: whey powder, whey protein concentrate, whey protein isolate, liquid milk, skim milk powder, whole milk powder, adult nutritional products, and yogurt. METHODS: AFM1 is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM1 in the resulting extract is concentrated using an automated RIDA®CREST IMMUNOPREP® online cartridge coupled to quantification by HPLC-fluorescence. RESULTS: The method was shown to be accurate, with acceptable recovery (81.2-97.1%) from spiked samples. Acceptable precision was confirmed, with a relative standard deviation (RSD) for repeatability of 6.6-11.2% and an RSD for intermediate precision of 7.5-16.7%. Method LOD and robustness experiments further demonstrated the suitability of this method for routine compliance testing. Analysis of an international proficiency trial sample generated results that were comparable with the value assigned from alternative independent methods. CONCLUSION: A method with improved chromatography for high-throughput, routine testing of AFM1 in an extended range of dairy products is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit for purpose. HIGHLIGHTS: Single-laboratory validation of an automated online immunoaffinity cleanup fluorescence HPLC method for AFM1 in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM1 with enhanced chromatographic performance. Method applicable to the analysis of AFM1 in an extended range of milk and milk-based products.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: First Action 2021.07.

BACKGROUND: Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of immunoprotective, growth, and antimicrobial factors to the neonate. OBJECTIVE: To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005. METHOD: Following dilution of the sample in buffer, an automated, label-free, real-time optical biosensor immunoassay was used in a direct assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a 4-parameter calibration regression. RESULTS: The analytical range (0-200 mg/hg), method detection limit (0.8 mg/hg), recovery (96.1-109.2%), and repeatability (1.0-5.3%) complied with the requirements given in the lactoferrin SMPR. The method was shown to be specific for native, intact lactoferrin; thermally denatured lactoferrin generated no measurable binding response. CONCLUSION: The method described is suitable for the quantification of intact, undenatured lactoferrin in milk products, infant formulas (bovine milk protein-based, soy protein-based, and amino acid-based), and adult nutritionals and has been demonstrated to meet the performance requirements defined in SMPR 2020.005. HIGHLIGHTS: A single-laboratory validation (SLV) of an automated biosensor immunoassay for the determination of intact, undenatured lactoferrin is described.

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.

Analysis of α-Tocopherol Stereoisomers in Fortified Infant Formula by Chiral Chromatography.

BACKGROUND: Direct measurement of the bioavailable α-tocopherol content presents a significant analytical challenge and requires chiral separation of the α-tocopherol stereoisomers. OBJECTIVE: The objective of the study was to validate an analytical method for the analysis of α-tocopherol stereoisomers in infant formulas and dairy products. METHOD: Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the α-tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection. RESULTS: The method was shown to be accurate, with spike recoveries of 91.9-108.8% for RRR-α-tocopherol and 90.1-104.7% for α-tocopherol, with no statistical bias against NIST 1849a certified reference material (P-value = 0.54) and an HPLC-UV analytical method (P-value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR-α-tocopherol and 4.6% RSDr (HorRat = 0.4) for α-tocopherol. CONCLUSIONS: A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of α-tocopherol is described. In a single analytical run, the method can quantify: (i) the total α-tocopherol content; (ii) the nutritionally important RRR-α-tocopherol and/or 2 R, 4'-ambo, 8'-ambo-α-tocopherol contents; (iii) the amount of all-rac-α-tocopherol, all-rac-α-tocopheryl acetate, or all-rac-α-tocopheryl succinate fortified into the product. HIGHLIGHTS: An accurate and precise chiral chromatographic method for the analysis of isomeric forms of α-tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection.

BACKGROUND: Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. OBJECTIVE: A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. METHOD: AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. RESULTS: The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81-112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4-12% RSD and intermediate precisions of 5-13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. CONCLUSIONS: A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. HIGHLIGHTS: An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.

Differential Thermal Isomerization: Its Role in the Analysis of Vitamin D3 in Foods.

BACKGROUND: For nutritional purposes, the measurement of vitamin D3 (defined as the sum of vitamin D3 and previtamin D3) is required to obtain an accurate and reliable estimate of its content in foods. An often neglected aspect in the development of methods for the analysis of vitamin D3 is accounting for any potential analytical bias in the results associated with differential thermal isomerization between previtamin D and vitamin D. CONCLUSIONS: For LC-UV methods using a vitamin D2 internal standard, cold saponification, or direct lipid extraction techniques should be avoided, unless chromatographic separation of vitamin D2, vitamin D3, and their previtamin forms is achieved so that UV absorbance corrections can be made. For both LC-UV and LC-MS methods using calciferol internal standards, the simplest solution to avoid analytical bias due to the presence of previtamin D is to utilize heating conditions (typically during saponification) such that previtamin D and vitamin D in the sample and the internal standard reach an equivalent equilibrium state prior to instrumental analysis. Only under such circumstances is the integration of previtamin D unnecessary to obtain accurate results for vitamin D3. HIGHLIGHTS: A detailed discussion of the quantitation of vitamin D3 in food with concise recommendations for avoiding measurement bias as a consequence of differential thermal isomerization.

Determination of Sorbic Acid in Cheese by High Performance Liquid Chromatography.

BACKGROUND: Sorbic acid (E, E-2, 4-hexadienoic acid) is added as a preservative to cheese because of its fungistatic and antimicrobial activity. OBJECTIVE: A facile method for the analysis of sorbic acid that is applicable to sliced processed cheese and grated cheese products. METHOD: A cheese sample and dry-ice mixture was blended and sorbic acid was extracted with methanol and analyzed by HPLC-ultraviolet with external standardization. A large sample size was used to overcome sample inhomogeneity due to imprecise sorbic acid addition techniques during production and sorbic acid migration through the fat over time. RESULTS: The method was shown to be accurate for both processed cheese and grated Cheddar cheese, with acceptable spike recovery (93.7, 103.7%, respectively), and no bias (α = 0.05) against an international reference method (p = 0.59, p = 0.13, respectively) was found. Acceptable precision was confirmed for both processed cheese slices and grated Cheddar cheese, with repeatability of 5.3% and 4.3% relative standard deviation, respectively, and intermediate precision Horwitz ratio values of 1.3 and 1.7 for processed cheese slices and grated Cheddar cheese, respectively. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. CONCLUSIONS: A method for high-throughput, routine testing of sorbic acid is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose.

Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry.

BACKGROUND: Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. OBJECTIVE: A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. METHOD: Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography‒tandem mass spectrometry (LC-MS/MS), with quantitation accomplished by internal standard technique. RESULTS: The method was shown to be accurate, with acceptable recovery (thiamine, 99.3-101.1%; pantothenic acid, 99.2-108.6%). A certified reference material (NIST 1849a), showed no statistical bias (α = 0.05) for thiamine (P = 0.64); although a statistically significant bias (P < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC-MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (RSDr) (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. CONCLUSIONS: This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.

Separation of RRR-α-Tocopherol by Chiral Chromatography.

BACKGROUND: α-Tocopherol can exist as eight possible stereoisomers due to the presence of three chiral carbons. Regulations and industry guidelines necessitate that dietary vitamin E intakes be based on the vitamin E activity of RRR-α-tocopherol. Food products fortified with synthetic all-rac-α-tocopherol or all-rac-α-tocopheryl acetate during manufacturing will require chiral separation of the α-tocopherol stereoisomers for accurate estimation of vitamin E activity. OBJECTIVE: The development of an HPLC method utilizing a chiral column for the chromatographic separation of RRR-α-tocopherol from other α-tocopherol stereoisomers. METHOD: Normal phase liquid chromatographic separation using a polysaccharide-based chiral column with fluorescence detection of α-tocopherol stereoisomers. RESULTS: The described chromatographic method achieves baseline resolution of RRR-α-tocopherol from its stereoisomers. Method selectivity, precision, and robustness were evaluated and acceptable performance was achieved. CONCLUSIONS: The chromatographic method was found to be suitable for application where both RRR-α-tocopherol content and total α-tocopherol content are required for routine compliance testing. HIGHLIGHTS: A robust and precise chomatographic method for the baseline resolution of RRR-α-tocopherol from its stereoisomers was acheived.

Comparison of LC-MS/MS and Enzymatic Methods for the Determination of Total Choline and Total Carnitine in Infant Formula and Milk Products.

BACKGROUND: Choline and l-carnitine are classified as pseudo-vitamins because of their conditionally essential status. As they are involved in multiple physiological metabolic pathways in the human body, they are routinely fortified in infant and adult nutritional formulas. OBJECTIVE: The performance of an LC-MS/MS method for the analysis of choline and carnitine, compared with enzymatic methods in routine use for the analysis of total carnitine and total choline, is described. METHOD: Powder samples were reconstituted, with release of carnitine and choline facilitated by both acid and alkaline hydrolysis and the extract analyzed by LC-MS/MS. Quantitation was by internal standard technique using deuterium-labeled carnitine and deuterium-labeled choline. RESULTS: Method range, specificity, sensitivity, precision, recovery, accuracy, and ruggedness were assessed for milk powders, infant formulas, and soy- and milk-based nutritional products. Spike recoveries of 94.0-108.4% were obtained for both total carnitine and choline, and no statistical bias (α = 0.05) between measured results and certified values (choline: P = 0.36; free carnitine: P = 0.67) was found for NIST 1849a certified reference material (NIST1849a). Precision, as repeatability relative standard deviation (RSD), was 2.0% RSDr for total carnitine and 1.7% RSDr for total choline. Equivalent results for total choline and total carnitine were obtained by LC-MS/MS and enzymatic methods (n = 30). CONCLUSIONS: The described LC-MS/MS method is fit for purpose for routine product compliance release testing environments. This validation study has confirmed that alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable. HIGHLIGHTS: An LC-MS/MS method was evaluated and found to be fit-for-purpose for routine product compliance release testing of infant formula. The LC-MS/MS method was compared with enzymatic methods for the analysis of total carnitine and total choline. Alternative enzymatic assays can be used with confidence in laboratories in which LC-MS/MS platforms are unavailable.

A Rapid Method for the Determination of Biotin and Folic Acid in Liquid Milk, Milk Powders, Infant Formula, and Milk-Based Nutritional Products by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Biotin and folate are B-group vitamins that play a critical role in numerous metabolic reactions, and they are supplemented to infant and adult nutritional formulas as free biotin and folic acid. OBJECTIVE: We describe a rapid method for the analysis of biotin and folic acid that is applicable to liquid milk, milk powders, infant formula, and milk-based nutritional products. METHODS: Samples are autoclaved, centrifuged, filtered, and analyzed by HPLC-MS/MS, with quantitation accomplished by the internal standard technique. RESULTS: The method was shown to be accurate, with acceptable spike recovery (biotin: 96.5-108.2%; folic acid: 92.6-104.4%), and no bias (α = 0.05) against either a certified reference material (biotin: P = 0.70; folic acid: P = 0.23) or established analytical method (biotin: P = 0.10; folic acid: P = 0.48) was found. Acceptable precision was confirmed with repeatability relative standard deviation (RSDr) and Horwitz ratio (HorRat) values (biotin: RSDr = 0.5-5.6%, HorRatr = 0.1-0.6; folic acid: RSDr = 2.0-3.1%, HorRatr = 0.3-0.5). Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. CONCLUSIONS: This rapid method is intended for use in high-throughput laboratories as part of the routine product compliance release testing of biotin and folic acid in the manufacturing of infant formulas and adult nutritional products.

Analysis of Vitamin D2 and Vitamin D3 in Infant and Adult Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: A Multilaboratory Testing Study.

A multilaboratory testing study was conducted on AOAC First Action Method 2016.05 "Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry." Nine laboratories participated in the analysis of duplicate samples of 20 nutritional products. The samples were saponified at high temperature with lipid-soluble components extracted into isooctane; an aliquot was washed and vitamin D derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to form a high-molecular mass, easily ionizable adduct, extracted into acetonitrile and analyzed by reversed-phase LC-tandem MS. Stable isotope-labeled internal standards were used for quantitation to correct for losses in extraction and variation in derivatization and ionization efficiencies. Acceptable precision as RSD was demonstrated; repeatability ranged from 1.9 to 5.8% RSDr and reproducibility values ranged from 6.4 to 12.7% RSDR, with samples meeting the precision limits specified in the vitamin D Standard Method Performance Requirements and the guidelines recommended for the Horwitz ratio. Method accuracy was assessed using NIST 1849a Standard Reference Material, with a P-value of 0.32, indicating an absence of bias against the certified value. As expected, placebo samples not fortified with vitamin D returned negligible results.

Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.

A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the 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 tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.

Current Methods for the Analysis of Selected Novel Nutrients in Infant Formulas and Adult Nutritionals.

Infant formula is designed to provide the human infant with a sole source of nutrition and it is intended to imitate breast milk. In recent years, advances in the science of infant nutrition have led to an increasing number of novel ingredients that are supplemented into infant formula. As the list of these nutritionally important nutrients is lengthy, this review summarizes contemporary analytical methods that have been applied to a representative selection (lutein, carnitine, choline, nucleotides, inositol, taurine, sialic acid, gangliosides, triacylglycerides, oligosaccharides, α-lactalbumin, and lactoferrin).

Analysis of Nucleotide 5'-Monophosphates in Infant Formulas by HPLC-UV: Collaborative Study, Final Action 2011.20.

A collaborative study was conducted on AOAC First Action Method 2011.20: 5'-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula. After the successful analysis of National Institute of Standards and Technology (NIST) 1849a Standard Reference Material (SRM) as a practice sample, 12 laboratories participated in the analysis of duplicate samples of six different infant formula products. The samples were dissolved in high-salt solution to inhibit protein and fat interactions, with the nucleotides [uridine 5'-monophosphate (UMP), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), and cytidine 5'-monophosphate (CMP)] separated from the sample matrix by strong-anion exchange SPE, followed by chromatographic analysis using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique using thymidine 5'-monophosphate. For nucleotide-supplemented products, precision is within the Standard Method Performance RequirementsSM (SMPR) 2011.008 target reproducibility limit of ≤11%, with the reproducibility RSD (RSDR) estimated at 7.1-8.7% for CMP, 7.9-9.0% for UMP, 2.8-7.7% for GMP, 5.5-10.3% for IMP, and 2.7-6.2% for AMP, and Horwitz ratio (HorRat) values of 0.9-1.0 for CMP, 0.9-1.0 for UMP, 0.3-0.7 for GMP, 0.6-1.0 for IMP, and 0.3-0.7 for AMP.

A rapid method for the determination of vitamin D3 in milk and infant formula by liquid chromatography/tandem mass spectrometry.

A rapid method for the determination of vitamin D3 applicable to milk and infant formula products is described. Samples are saponified at high temperature, and lipophilic components are extracted into isooctane in a single tube. Vitamin D3 is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to form a Diels-Alder adduct, which is re-extracted into a small volume of acetonitrile and analyzed by UHPLC-MS/MS with quantification accomplished by an internal standard technique utilizing deuterium-labeled vitamin D3. The analysis of vitamin D3 as the PTAD adduct offers a significant increase in sensitivity and selectivity, allowing for rapid sample preparation and short chromatographic run times. The method was shown to be accurate, with spike recoveries of 94.7-104.7% and no statistical bias against both a certified reference material (P = 0.37, α = 0.05) and a reference LC-UV analytical method (P = 0.09, α = 0.05). Acceptable precision was confirmed with a repeatability RSD of 1.4-4.5% and corresponding HorRat values of 0.1-0.2. This high-throughput method is ideal for routine compliance testing, with more than 50 samples/day achievable by a single analyst.

Evaluation Protocol for Review of Method Validation Data by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Expert Review Panel.

Methods under consideration as part of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals process are to be evaluated against a set of Standard Method Performance RequirementsSM (SMPRs) via peer review by an expert review panel (ERP). A validation protocol and a checklist have been developed to assist the ERP to evaluate experimental data and to compare multiple candidate methods for each nutrient. Method performance against validation parameters mandated in the SMPRs as well as additional criteria are to be scored, with the method selected by the ERP proceeding to multilaboratory study prior to Final Action approval. These methods are intended to be used by the infant formula industry for the purposes of dispute resolution.

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography-tandem mass spectrometry.

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1-112.9% and repeatability relative standard deviations of 1.9-7.2%. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.