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. Objectives: 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. Conclusions: 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.
Received: 15 November 2022; Accepted: 29 December 2022; Published: 11 January 2023
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. Methods: 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 Method 2022.03.
Received: 17 April 2023; Accepted: 13 June 2022; Published: 30 June 2023
Foods are analysed for their vitamin content to support the verification of regulatory compliance or to generate food composition data. Many international reference methods for the analysis of vitamins in foods originate from the 1990s. Advances in nutrition science and analytical technology and the continuing evolution of statutory regulations necessitate the need of new or supplementary regulatory standards. We have evaluated recent developments in these areas and conclude that most current international reference methods are no longer fit-for-purpose to accurately determine vitamin content in foods and food supplements. We have made recommendations to consider new and/or updated reference methods and regulatory standards for the analysis of vitamins A, D, E, K, B1, B2, B3, B5, B6, B7, B9, B12, C and carotenoids in foods and food supplements. This area of nutrients may benefit from globally harmonised definitions specifying what compounds to include or exclude for analysis, and applicable bioactivity factors.
Received: 26 February 2024; Accepted: 13 April 2024; Published: 16 April 2023
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. Objectives: To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005. Methods: 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. Conclusions: 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.
Received: 06 March 2022; Accepted: 11 May 2022; Published: 06 June 2022
The analysis of vitamin B12 in infant formulas typically requires the use of cyanide during sample preparation to convert the unstable vitamers (hydroxocobalamin, methylcobalamin and adenosylcobalamin) to cyanocobalamin, the most stable form of vitamin B12. To eliminate the risk to laboratory analysts in handling cyanide, alternative strategies are preferred for the analysis of vitamin B12. This research demonstrates the use of cobalamin-derived α-ribazole (a nucleoside moiety of vitamin B12) to determine total vitamin B12 content. Infant formula samples underwent protein denaturation and sugar removal with subsequent acidic hydrolysis and dephosphorylation employed to release α-ribazole, which was isolated by boronate affinity chromatography then analysed by hydrophilic interaction liquid chromatography with fluorescence detection. The method was validated using bovine- and ovine milk-based infant formula samples. The newly developed method was linear over the range of 0.65–6.48 ng mL−1 with repeatability of 3.78–5.47% relative standard deviation (RSDr, n = 10) and an intermediate precision of 3.59–10.0% RSDiR (n = 10). The limits of detection and quantitation (LOD and LOQ) were 0.4 and 1.2 μg 100 g−1 of dry weight, respectively. Accuracy was 68.9–76.4% and 68.7–80.0% at 50 and 150% of typical B12 concentrations in infant formula, respectively. The validated method was applied to eleven infant formulas and no statistical difference (p = 0.45, α = 0.05) was found when comparing with the results obtained using the AOAC Official Method 2014.02 high performance liquid chromatography with ultraviolet detection that requires the use of cyanide. These results indicate that the newly validated method is not only reliable but also offers a safer alternative for routine vitamin B12 determination in infant formula while maintaining high accuracy and precision.
Received: 30 July 2024; Accepted: 27 September 2024; Published: 28 September 2024
Breast milk is recognised as the best source of nutrition for infant growth and development, and is recommended by public health agencies. Under circumstances where breastfeeding may not be possible, unsuitable, or inadequate, infant formula is an effective substitute for infant feeding, as it is specially formulated to be a complete or partial substitute for human milk. For infants that are not breast-fed, infant formula is the sole source of nutrition in their first 6 months of life; as such it needs to meet all of their nutritional requirements. For this reason, infant formula is one of the most highly regulated food products in the world and is manufactured to rigorous specifications to maintain the highest product quality and safety. This paper briefly describes the development of infant formula products and the evolution of the analytical methods required to quantitate their nutrient content.
show abstract
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. Objectives: 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. Conclusions: 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.
Received: 15 November 2022; Accepted: 29 December 2022; Published: 11 January 2023
Journal Citation Paper Manuscriptshow abstract
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. Methods: 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 Method 2022.03.
Received: 17 April 2023; Accepted: 13 June 2022; Published: 30 June 2023
Journal Citation Paper Manuscript Supplementshow abstract
Foods are analysed for their vitamin content to support the verification of regulatory compliance or to generate food composition data. Many international reference methods for the analysis of vitamins in foods originate from the 1990s. Advances in nutrition science and analytical technology and the continuing evolution of statutory regulations necessitate the need of new or supplementary regulatory standards. We have evaluated recent developments in these areas and conclude that most current international reference methods are no longer fit-for-purpose to accurately determine vitamin content in foods and food supplements. We have made recommendations to consider new and/or updated reference methods and regulatory standards for the analysis of vitamins A, D, E, K, B1, B2, B3, B5, B6, B7, B9, B12, C and carotenoids in foods and food supplements. This area of nutrients may benefit from globally harmonised definitions specifying what compounds to include or exclude for analysis, and applicable bioactivity factors.
Received: 26 February 2024; Accepted: 13 April 2024; Published: 16 April 2023
Journal Citation Paper Manuscriptshow abstract
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. Objectives: To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005. Methods: 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. Conclusions: 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.
Received: 06 March 2022; Accepted: 11 May 2022; Published: 06 June 2022
Journal Citation Paper Manuscriptshow abstract
The analysis of vitamin B12 in infant formulas typically requires the use of cyanide during sample preparation to convert the unstable vitamers (hydroxocobalamin, methylcobalamin and adenosylcobalamin) to cyanocobalamin, the most stable form of vitamin B12. To eliminate the risk to laboratory analysts in handling cyanide, alternative strategies are preferred for the analysis of vitamin B12. This research demonstrates the use of cobalamin-derived α-ribazole (a nucleoside moiety of vitamin B12) to determine total vitamin B12 content. Infant formula samples underwent protein denaturation and sugar removal with subsequent acidic hydrolysis and dephosphorylation employed to release α-ribazole, which was isolated by boronate affinity chromatography then analysed by hydrophilic interaction liquid chromatography with fluorescence detection. The method was validated using bovine- and ovine milk-based infant formula samples. The newly developed method was linear over the range of 0.65–6.48 ng mL−1 with repeatability of 3.78–5.47% relative standard deviation (RSDr, n = 10) and an intermediate precision of 3.59–10.0% RSDiR (n = 10). The limits of detection and quantitation (LOD and LOQ) were 0.4 and 1.2 μg 100 g−1 of dry weight, respectively. Accuracy was 68.9–76.4% and 68.7–80.0% at 50 and 150% of typical B12 concentrations in infant formula, respectively. The validated method was applied to eleven infant formulas and no statistical difference (p = 0.45, α = 0.05) was found when comparing with the results obtained using the AOAC Official Method 2014.02 high performance liquid chromatography with ultraviolet detection that requires the use of cyanide. These results indicate that the newly validated method is not only reliable but also offers a safer alternative for routine vitamin B12 determination in infant formula while maintaining high accuracy and precision.
Received: 30 July 2024; Accepted: 27 September 2024; Published: 28 September 2024
Journal Citation Paper Manuscriptshow abstract
Breast milk is recognised as the best source of nutrition for infant growth and development, and is recommended by public health agencies. Under circumstances where breastfeeding may not be possible, unsuitable, or inadequate, infant formula is an effective substitute for infant feeding, as it is specially formulated to be a complete or partial substitute for human milk. For infants that are not breast-fed, infant formula is the sole source of nutrition in their first 6 months of life; as such it needs to meet all of their nutritional requirements. For this reason, infant formula is one of the most highly regulated food products in the world and is manufactured to rigorous specifications to maintain the highest product quality and safety. This paper briefly describes the development of infant formula products and the evolution of the analytical methods required to quantitate their nutrient content.
Published: 10 October 2024
Journal Citation Paper Manuscript