The total potentially available nucleosides (TPAN) in bovine, caprine, and ovine milk were analyzed through the sequential application of phosphatase, pyrophosphatase, and nuclease enzyme treatments prior to high performance liquid chromatographic analysis of released nucleosides. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN, i.e., 374.1 μmol/dL, with lower concentrations in caprine milk (97.4 μmol/dL) and bovine milk (7.9 μmol/dL). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest.
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.
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 labeled 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 NIST 1849a 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.
An optical biosensor assay utilising a monoclonal antibody was developed for the quantitation of the biotin content of milk and paediatric formulae. The method provides a reliable estimate of total biotin accomplished by simple aqueous extraction, combined with heat treatment, prior to automated biosensor analysis. The binding assay was configured under inhibition conditions utilising a sensor surface functionalised with biotin and was subjected to single-laboratory validation. Critical assay factors, including calibration parameters, cross-reactivity, non-specific binding and matrix interferences were evaluated systematically. Assay performance parameters including range, detection limits, precision, recovery and bias were estimated. The method was applied to the routine compliance testing of paediatric formulae and the temporal change in the biotin content of early lactation milk and seasonal milk powder. The assay is an expedient alternative to current HPLC, microbiological and proprietary kit-based immunoassay methods for the determination of the biotin content of milk-based foods.
Lutein is a carotenoid that is considered to be important to the integrity of the retina and is therefore increasingly being supplemented into bovine milk-based paediatric formulae to levels equivalent to those found in human milk. A simple analytical method has been developed and intra-laboratory validated to facilitate routine in-process control of lutein addition. The method involves dilution of a carotenoid premix, followed by C30 reversed-phase liquid chromatographic separation of lutein, zeaxanthin and β‑carotene, with detection and quantitation at 450 nm. The method performance parameters include range (0–700 ng/mL), method limit of detection (0.08 μg/g), recovery (95.5–109.5%) and precision (1.2% RSDr). The method has been applied to an evaluation of lutein recovery (95.6–104.2%) through the manufacture of paediatric formulae, which confirms that lutein loss through the entire process is insignificant.
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 Requirements (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.
Received: 25 August 2011; Accepted: 22 November 2011; Published: 31 December 2011
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
The total potentially available nucleosides (TPAN) in bovine, caprine, and ovine milk were analyzed through the sequential application of phosphatase, pyrophosphatase, and nuclease enzyme treatments prior to high performance liquid chromatographic analysis of released nucleosides. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN, i.e., 374.1 μmol/dL, with lower concentrations in caprine milk (97.4 μmol/dL) and bovine milk (7.9 μmol/dL). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest.
Received: 14 February 2012; Accepted: 14 February 2012; Published: 01 May 2012
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
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.
Received: 28 January 2013; Accepted: 20 March 2013; Published: 05 April 2013
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
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 labeled 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 NIST 1849a 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.
Received: 22 July 2013; Accepted: 1 October 2013; Published: 15 October 2013
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
An optical biosensor assay utilising a monoclonal antibody was developed for the quantitation of the biotin content of milk and paediatric formulae. The method provides a reliable estimate of total biotin accomplished by simple aqueous extraction, combined with heat treatment, prior to automated biosensor analysis. The binding assay was configured under inhibition conditions utilising a sensor surface functionalised with biotin and was subjected to single-laboratory validation. Critical assay factors, including calibration parameters, cross-reactivity, non-specific binding and matrix interferences were evaluated systematically. Assay performance parameters including range, detection limits, precision, recovery and bias were estimated. The method was applied to the routine compliance testing of paediatric formulae and the temporal change in the biotin content of early lactation milk and seasonal milk powder. The assay is an expedient alternative to current HPLC, microbiological and proprietary kit-based immunoassay methods for the determination of the biotin content of milk-based foods.
Received: 25 November 2013; Accepted: 25 February 2014; Published: 22 March 2014
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
Lutein is a carotenoid that is considered to be important to the integrity of the retina and is therefore increasingly being supplemented into bovine milk-based paediatric formulae to levels equivalent to those found in human milk. A simple analytical method has been developed and intra-laboratory validated to facilitate routine in-process control of lutein addition. The method involves dilution of a carotenoid premix, followed by C30 reversed-phase liquid chromatographic separation of lutein, zeaxanthin and β‑carotene, with detection and quantitation at 450 nm. The method performance parameters include range (0–700 ng/mL), method limit of detection (0.08 μg/g), recovery (95.5–109.5%) and precision (1.2% RSDr). The method has been applied to an evaluation of lutein recovery (95.6–104.2%) through the manufacture of paediatric formulae, which confirms that lutein loss through the entire process is insignificant.
Received: 31 July 2014; Accepted: 06 October 2014; Published: 01 January 2015
Citation Journal Paper Manuscript Abstract.ris MLA APA Chicago Harvard Vancouver
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 Requirements (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.