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.
Received: 31 August 2014; Accepted: 23 October 2014; Published: 01 March 2015
An automated biosensor immunoassay, exploiting surface plasmon resonance detection, is described for the quantitation of bovine serum albumin (BSA) in milk products. Antibody selection, assay conditions and potential non-specific binding interferences were defined. Analytical performance includes a working range of 10–1000 ng/mL, a method detection limit of 0.02 mg/g in milk, an instrument intermediate precision relative standard deviation RSD(iR) of 3.7%, an intermediate precision RSD(iR) of 8.9% for whey protein concentrate, and a single flow cell stable for at least 400 cycles. Accuracy was demonstrated by recovery, compliance with a certified reference material and comparison with a high performance liquid chromatographic method. The technique was applied to the estimation of BSA content of bovine milk, colostrum, whey protein fractions and infant formulae. The change in BSA expression during early bovine lactation and across a production season, and the thermal denaturation of BSA were also investigated.
Received: 01 December 2014; Accepted: 26 February 2015; Published: 7 March 2015
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 nucleotidesupplemented products, precision is within the Standard Method Performance Requirements (SMPR) 2011.008 target reproducibility limit of <11%, with the reproducibility, RSD(R) 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.
Received: 23 February 2015; Accepted: 07 April 2015; Published: 01 July 2015
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).
Received: 02 April 2015; Accepted: 09 October 2015; Published: 01 January 2016
A high performance anion-exchange chromatographic method employing pulsed amperometric detection was applied to the determination of endogenous free and total myo-inositol in bovine milk, for which there is limited information. The contents and trend variability of myo-inositol in milk from extensively pasture-fed cows during early lactation and across a production season were therefore evaluated. Free and total myo-inositol in seasonal milk were within the ranges of 2.3–4.5 mg/hg and 5.3–8.7 mg/hg, respectively. This novel information will both improve understanding of the expression of innate myo-inositol in bovine milk, and provide manufacturers with information that can enhance formulation capability related to the production of cow's milk-based products.
Received: 18 November 2015; Accepted: 05 January 2016; Published: 13 January 2016
Method Review by Stakeholder Panel for Infant Formulas and Adult Nutritionals (SPIFAN)
Method Review by Expert Review Panel for Nutrient Methods.
* This method was awarded the 2017 Method of the Year Award by AOAC International at the AOAC Annual Meeting & Conference, Atlanta, GA, USA, 25th September 2017.
Received: 15 May 2016; Accepted: 15 June 2016; Published: 01 January 2017
show abstract
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.
Received: 31 August 2014; Accepted: 23 October 2014; Published: 01 March 2015
Journal Citation Paper Manuscriptshow abstract
An automated biosensor immunoassay, exploiting surface plasmon resonance detection, is described for the quantitation of bovine serum albumin (BSA) in milk products. Antibody selection, assay conditions and potential non-specific binding interferences were defined. Analytical performance includes a working range of 10–1000 ng/mL, a method detection limit of 0.02 mg/g in milk, an instrument intermediate precision relative standard deviation RSD(iR) of 3.7%, an intermediate precision RSD(iR) of 8.9% for whey protein concentrate, and a single flow cell stable for at least 400 cycles. Accuracy was demonstrated by recovery, compliance with a certified reference material and comparison with a high performance liquid chromatographic method. The technique was applied to the estimation of BSA content of bovine milk, colostrum, whey protein fractions and infant formulae. The change in BSA expression during early bovine lactation and across a production season, and the thermal denaturation of BSA were also investigated.
Received: 01 December 2014; Accepted: 26 February 2015; Published: 7 March 2015
Journal Citation Paper Manuscriptshow abstract
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 nucleotidesupplemented products, precision is within the Standard Method Performance Requirements (SMPR) 2011.008 target reproducibility limit of <11%, with the reproducibility, RSD(R) 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.
Received: 23 February 2015; Accepted: 07 April 2015; Published: 01 July 2015
Journal Citation Paper Manuscriptshow abstract
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).
Received: 02 April 2015; Accepted: 09 October 2015; Published: 01 January 2016
Journal Citation Paper Manuscriptshow abstract
A high performance anion-exchange chromatographic method employing pulsed amperometric detection was applied to the determination of endogenous free and total myo-inositol in bovine milk, for which there is limited information. The contents and trend variability of myo-inositol in milk from extensively pasture-fed cows during early lactation and across a production season were therefore evaluated. Free and total myo-inositol in seasonal milk were within the ranges of 2.3–4.5 mg/hg and 5.3–8.7 mg/hg, respectively. This novel information will both improve understanding of the expression of innate myo-inositol in bovine milk, and provide manufacturers with information that can enhance formulation capability related to the production of cow's milk-based products.
Received: 18 November 2015; Accepted: 05 January 2016; Published: 13 January 2016
Journal Citation Paper Manuscriptshow abstract
Method Review by Stakeholder Panel for Infant Formulas and Adult Nutritionals (SPIFAN) Method Review by Expert Review Panel for Nutrient Methods.
* This method was awarded the 2017 Method of the Year Award by AOAC International at the AOAC Annual Meeting & Conference, Atlanta, GA, USA, 25th September 2017.
Received: 15 May 2016; Accepted: 15 June 2016; Published: 01 January 2017
Journal Citation Paper Manuscript