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.

Analysis of Taurine in Infant Formulas and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: First Action 2022.03.

BACKGROUND: Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. OBJECTIVE: To evaluate the analytical performance of a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for compliance with AOAC Standard Method Performance Requirements (SMPR®) for taurine analysis described in SMPR 2014.013. METHOD: Following protein precipitation with Carrez solutions, taurine is extracted and separated by HILIC with detection by triple quadrupole MS using multiple reaction monitoring (MRM). Stable isotope labeled (SIL) taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. RESULTS: The method was shown to meet the requirements specified in the SMPR with a linear range of 0.27-2700 mg/hg RTF (ready-to-feed), a limit of detection of 0.14 mg/hg RTF, acceptable recovery of 97.2-100.1%, and acceptable repeatability of 1.6-6.4% relative standard deviation. Additionally, the method was found to have no statistically significant bias compared with reference values for National Institute of Standards and Technology (NIST) 1849a certified reference material (CRM) (P-value = 0.95) and 1869 CRM (P-value = 0.31), and with results from AOAC 997.05 (P-value = 0.10). CONCLUSIONS: A recent review of the method and validation data by the Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) found that this method met all the criteria for analysis of taurine specified in SMPR 2014.013 and voted to adopt this method as First Action AOAC Official MethodSM2022.03. HIGHLIGHTS: A method for the analysis of taurine in infant formulas and adult nutritionals by HILIC-MS/MS is described. A single-laboratory validation (SLV) study demonstrated the applicability of the method to meet requirements of SMPR 2014.013. In December 2022, the SPIFAN ERP voted to adopt this method as First Action AOAC Official Method 2022.03.

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.

Rapid Analysis of Taurine in Infant Formula and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

BACKGROUND: Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. OBJECTIVE: A rapid compliance method for the analysis of taurine that is applicable to infant formula and milk-based nutritional products is described. METHOD: Following protein precipitation with Carrez solutions, taurine in the sample extract is separated by hydrophilic interaction liquid chromatography (HILIC) with detection by triple quadrupole mass spectrometry using multiple reaction monitoring (MRM). Stable isotope-labeled taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. RESULTS: The method was shown to be accurate, with acceptable recovery of 99.6% (range = 91.1-106.5%). Results for National Institute of Standards and Technology (NIST)-certified reference materials showed no statistical bias for NIST 1849a (P = 0.96) and NIST 1869 (P = 0.88) when compared with reference values. No bias was found when results were compared with those of an international reference method, AOAC Official MethodSM997.05 (P = 0.18). Repeatability was estimated to be 3.1% RSDr (range: 2.4-4.0%, HorRat: 0.3), and intermediate precision was estimated to be 4.9% RSDiR (range: 2.2-7.7%). CONCLUSIONS: Successful single-laboratory validation demonstrates that this rapid method is suitable for use in high-throughput laboratories as part of routine product compliance release testing of taurine in nutritional products. HIGHLIGHTS: A method for the analysis of taurine in infant formula and adult nutritionals by hydrophilic interaction liquid chromatography-mass spectrometry (LC-MS) is described. The method is suitable for use in high-throughput laboratories for routine product compliance testing of taurine. A single-laboratory validation study demonstrated the method to be accurate, precise, and fit for purpose.

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.

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.

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.