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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 First Action Official Method 2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005., Methods: 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 four-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 Horwitz Ratio (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 cleanup LC-UV), showed negligible difference between results., Conclusion: 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 (MLT) study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official MethodSM., Highlights: A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described., (© The Author(s) 2024. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.

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.

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).

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 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.

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.

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.

A method for the routine determination of 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92-101%) and repeatability (1.0-2.3% RSD). The method was approved for Official First Action status by an AOAC expert review panel.

An RP-HPLC method for the routine determination of supplemented 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in pediatric formulas and milk products is described. Following sample dissolution, potential interferences were removed by anion-exchange SPE. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92-101% and repeatability RSDs of 1.0-2.3%. The method was optimized for the rapid, routine analysis of nucleotide-supplemented bovine milk-based infant and follow-on formulas.

The immunoprotective properties of bovine milk immunoglobulin G (IgG) have led to a recent proliferation of nutritional products incorporating this protein. It has therefore become critical that reliable analytical techniques for the measurement of the IgG content in such products are available. This literature review surveys current methods of analysis for IgG, including separation-based or immuno-based concentration analysis. The review also discusses nutraceutical applications, regulatory issues, stability of IgG and the significance of primary reference material in IgG analysis.

Nucleotides and nucleosides play important roles as structural units in nucleic acids, as coenzymes in biochemical pathways, and as sources of chemical energy. Milk contains a complex mixture of nucleotides, nucleosides, and nucleobases, and because of the reported differences in their relative levels in bovine and human milks, pediatric formulas are increasingly supplemented with nucleotides. Liquid chromatography is the dominant analytical technique used for the quantitation of nucleospecies and is commonly applied using either ion-exchange, reversed-phase, or ion-pair reversed-phase modes. Robust methods that incorporate minimal sample preparation and rapid chromatographic separations have been developed for routine product compliance analysis. This review summarizes the analytical techniques used to date in the analysis of nucleospecies in bovine and human milks and infant formulas.

An affinity liquid chromatography (LC) method for the determination of bovine immunoglobulin G (IgG), using protein G coupled to an agarose support, was modified to permit the quantification of IgG in colostrum-based powders. Sample preparation included pH adjustment to 4.6 to precipitate casein and denatured whey protein. The method was applied to a range of colostrum powders and was compared with the alternative independent methods of surface plasmon resonance immunoassay, radial immunodiffusion, and reversed-phase LC. The method was rapid, and performance parameters included a working range of 10-150 microg IgG and precision relative standard deviation values of <10%.

Automated, rapid, sensitive, and label-free biosensor-based immunoassays for immunoglobulin G (IgG), folate binding protein, lactoferrin, and lactoperoxidase in bovine milk using surface plasmon resonance optical detection with direct binding assay format are described. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration are defined and nonspecific binding considerations evaluated. The technique has been applied to the measurement of these proteins in consumer milks, colostrum, milk products, and infant formulas, and their temporal change during early bovine lactation followed.

An optical biosensor inhibition immunoassay was developed using a specific pantothenic acid-binding protein for the quantitation of free pantothenic acid (vitamin B5) in foodstuffs. Samples were prepared by a simple extraction procedure in buffer, and vitamin content was estimated against authentic calibrants in the same buffer. Performance parameters included a working range of 10-5000 ng/mL, a limit of detection of 4.4 ng/mL, precision relative standard deviation of 5.4-7.1% over a range of concentrations, and recoveries > 95% in the matrixes tested. A wide range of foodstuffs, including National Institute of Standards and Technology reference samples, were tested in 3 independent laboratories and the results were compared with microbiological assay and liquid chromatography/mass spectrometry (LC/MS) methods. The results indicate that the biosensor technique is appropriate for the estimation of pantothenic acid in a wide range of foodstuffs.

An automated, rapid, sensitive, and label-free biosensor-based assay for folate-binding protein (FBP) in bovine milk utilizing surface plasmon resonance optical detection is described. The active concentration of FBP is estimated from its specific interaction with a pteroyl-l-glutamic (folic) acid derivative immobilized on the sensor surface in a direct binding assay format. Milk, colostrum, and milk powders are prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration, have been defined, and nonspecific binding considerations were evaluated. Performance parameters include a working range for FBP in buffer of 0-200 ng/mL, a method detection limit of 0.13 microg/mL in fluid milk, overall instrument response RSD(R) of 0.64%, a mean interassay RSD(R) of 7.3% for skim milk powder, and surface stability over ca. 200 samples. The technique was applied to the measurement of active FBP content of consumer milks, the heat classification of skim milk powders manufactured under a wide range of thermal processing protocols, and change during early bovine lactation.

A simultaneous determination of orotic acid, uric acid, and creatinine in milk is described. Following deproteinization, the sample was analyzed by reversed-phase liquid chromatography, using a highly aqueous cationic ion-pair eluent and photodiode array UV detection. In view of their potential dietary significance, the validated method was applied to survey the influence of species, season, and lactation on their contribution to the nonprotein nitrogen pool in milk. Mature bovine milk contained orotic acid, uric acid, and creatinine in the range of 30-70, 9-24, and 6-12 microg/mL, respectively. Although uric acid and creatinine were present in all milks, orotic acid was essentially absent in nonruminant milks. In contrast to urate and creatinine, expression of orotic acid in bovine milk was strongly dependent on stage of early lactation. The co-existence in mammalian milks of related nucleoside and nucleotide components was also determined.

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.

A interlaboratory study was conducted to evaluate a liquid chromatographic (LC) procedure for the determination of total vitamin C in foods at levels of 5-60 mg/100 g. Emphasis was placed on fruit juices, although selected foods were also included in the study. Following dissolution of sample in water, endogenous dehydroascorbic acid was converted to ascorbic acid by precolumn reduction with dithiothreitol at neutral pH. Total ascorbate was determined by C18 reversed-phase LC with a phosphate eluent at pH 2.5, incorporating dithiothreitol to maintain vitamin C in the reduced form, and UV detection at 254 nm. Seven types of fruit juices and foods were tested by 19 collaborators in 7 countries. Three duplicate juices and foods met the criteria for Youden pairs and yielded repeatability relative standard deviation of 5.80-14.66%. Reproducibility relative standard deviation ranged from 6.36 to 35.54% (n = 10) with HORRAT values of 0.82-4.04. The LC method is suitable for routine use in fruit products and foods containing > 5 mg/100 g vitamin C and is recommended for further validation by AOAC INTERNATIONAL and International Fruit Juice Union.

A simplified, simultaneous determination of vitamins B1, B2, B3, and B6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified methanol-water mobile phase. The dioctylsulfosuccinate counter-ion facilitates unique retention of the pyridine-based vitamins (niacinamide and pyridoxine) and allows for concurrent measurement of both the pyridoxal and riboflavin 5'-phosphate endogenous components of milk. Other naturally occurring undetected vitamin congeners have minimal analytical significance. UV detection is used for niacinamide, and programmed fluorescence detection is used for riboflavin and the B6 vitamins. Thiamine is routinely determined sequentially under modified elution conditions.

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.

Biomolecular interaction analysis was evaluated for the automated determination of vitamin B12 in a range of foods. The analytical technique was configured as a biosensor-based, nonlabeled inhibition protein-binding assay using nonintrinsic R-protein. Sample extraction conditions were optimized, and both ligand specificity and nonspecific binding considerations were evaluated. Performance parameters included a quantitation range of 0.08-2.40 ng/mL, recoveries of 89-106%, agreement against assigned reference values for 3 independent certified food reference materials, and a mean between-laboratory reproducibility relative standard deviation of 4.9%. The proposed method was compared with reference microbiological and radioisotope protein-binding methods for a range of food samples. A wide selection of milks, infant formulas, meats, and liver were evaluated for their vitamin B12 content. The influence of season was studied in herd milk, early lactation was followed for a single animal, and the cobalamin content of bovine, caprine, and ovine milks was compared.

Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2-70 ng/mL, recoveries of 86-102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.

A simple procedure for determination of vitamin K1 was developed for routine compliance monitoring of supplemented infant formula and measurement of endogenous levels in milk and milk powders. Samples are digested with lipase and extracted into hexane; and aliquot is evaporated, reconstituted in methanol, and analyzed by reversed-phase LC. Post-column zinc reduction of phylloquinone facilitates detection by fluorescence. The procedure was subjected to an AOAC collaborative study involving 8 materials, each in blind duplicate, across the range of 5-120 micrograms/100 g solids and including NIST 1846 reference material. Thirty-three laboratories returned valid data which were then statistically analyzed for outliers and precision parameters. Mean RSDR (%) was 6.53 (4.33-10.94), with a mean HORRAT value of 0.33 (0.23-0.43) and RSDr:RSDR ratio of 0.74. K1 isomers (cis and trans) were aggregated with conventional C18 columns, but may be selectively estimated with use of the C30 column.

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.

A collaborative study was conducted on a liquid chromatographic (LC) method for determination of taurine in infant formula and milk powders. Twenty laboratories participated in the analysis of 8 blind duplicates over the range of approximately 3-60 mg/100 g sample. The method involved protein removal, conversion to the dansyl-derivative, and isocratic LC separation with UV and/or fluorescence detection. Following outlier treatment, overall mean RSDR has been estimated at 7.00% for supplemented products with a HORRAT value of 1.1. The poorer precision at endogenous levels establishes a lower limit of determination of about 5 mg/100 g. An overall mean RSDr:RSDR value of 0.7 for all products demonstrated acceptable performance.

A method is described for the determination of phylloquinone and menaquinones following enzymatic digestion, extraction and a single-stage HPLC technique utilizing post-column reduction with zinc and fluorescence detection. The technique is applicable to both routine compliance control of phylloquinone supplemented infant formula powders (30-150 micrograms per 100 g) and fundamental studies of the K vitamins at endogenous levels in fluid milks (0-5.0 micrograms per 100 g). Analytical figures of merit include a detection limit of 30 micrograms on-column, recoveries greater than 98% for both K1 and MK4, an RSDR of 2.35% (K1) and 2.32% (MK4) and a regression correlation of 0.9932 for a wide range of infant formulas when compared against an alternative HPLC-UV technique. MK4 and 2',3'-dihydrophylloquinone, both with undefined bioactivity, were detected at measurable levels in a range of infant formulas. Although the higher menaquinones were found to be essentially absent in the milk of several species, the significant presence of MK4 relative to K1 has been confirmed in all milks examined, with both dominant forms correlated during early lactation in the cow. These observations suggest an as yet unrecognized physiological function for MK4 in infant nutrition.

Vitamin K1 in infant formulas and milk products is determined by reversed-phase liquid chromatography (LC) with UV detection. The sample is hydrolyzed enzymatically, and the vitamin is extracted with hexane. Fractionation by normal-phase semi-preparative LC is followed by analytical LC, with quantitation by the internal standard technique. Recovery of the analyte was 97.4 +/- 2.8%. Linearity was established between 0.05 and 4.0 micrograms/mL. The limit of quantitation is 0.5 microgram/100 g for milk powder, which allows the method to quantitate endogenous levels of vitamin K1.

A simple spectrophotometric assay for free carnitine in milk and supplemented infant formulas has been developed. After acid extraction, carnitine is measured enzymatically through its reaction with carnitine acetyltransferase coupled with acetyl coenzyme A and dithiobenzoate (DTNB). The manually performed method is rapid, accurate, and convenient for routine quality-control analysis of infant formulas. The chemistry is also suitable for automation, for greatly enhanced throughput.

A high-performance liquid chromatographic method is described for the determination of supplemental myo-inositol in infant formulas based on cows' milk. The technique incorporates pre-column derivatization with phenylisocyanate followed by reversed-phase gradient chromatography and ultraviolet detection. The protocol was also applied to a survey of free inositol at endogenous levels in the milk of cows, goats and humans. Nutritional preparations containing inositol at pharmacological levels are also amenable to a simplified isocratic analysis.

A method is described for the simultaneous determination of cholesterol, phytosterols and tocopherols in dairy and non-dairy high-performance liquid chromatography. A facile saponification and extraction scheme is completed rapidly within a single reaction tube. Clean-up is not required, with analysis completed by one of two alternative reversed-phase approaches. Sterols are monitored by short-wave ultraviolet spectrophotometry, and tocopherols by means of a series fluorescence detector. Cholesterol is resolved from the principal plant phytosterols, while the tocopherol congeners are well separated, thereby offering complementary information regarding source-lipid composition in foods under regulatory quality-control conditions. Squalene, an essential metabolic sterol precursor, can also be determined concurrently at the same viewing wavelength.