Analysis of Nucleosides and Nucleotides in Milk and Infant Formula

Nucleotides have been routinely supplemented to infant formulas due to the important roles they play in metabolism and to replicate the higher concentrations typically found in human milk. A method utilising anion exchange solid-phase extraction clean-up and liquid chromatography was developed for the rapid, routine determination of supplemented cytidine 5′ monophosphate, uridine 5′ monophosphate, inosine 5′ monophosphate, guanosine 5′ monophosphate, and adenosine 5′ monophosphate in bovine milk-based infant formula. 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 of 1.0–2.3%. An extension study demonstrated the expansion in scope to a wider range of different infant formula products including milk protein and hydrolysate-based products, low and high fat products, soy protein-based and elemental products, adult nutritional and infant formulations, in both ready-to-feed and powder forms. The development of a method to measure the total potentially available nucleosides (TPAN) in human milk has made an important contribution to further understanding the distribution of nucleosides and nucleotides. This method was applied in a lactation study of bovine milk with colostrum and milk samples collected from two herds over the course of the first month post-partum, pooled within each herd by stage of lactation and the TPAN concentrations were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate affinity gel extraction, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the nutritionally available nucleoside pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations. The TPAN method was subsequently applied to the analysis of mature bovine, caprine, and ovine milk. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN (374.1 µmol dL-1), with lower concentrations in caprine milk (97.4 µmol dL-1) and bovine milk (7.9 µmol dL-1). 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 simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry was developed. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution, with mass spectrometric detection, and quantitation by stable isotope labelled internal standard technique. A single laboratory validation study was performed with recoveries of 80.1–112.9% and repeatability relative standard deviations of 1.9–7.2%. The method was validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysate-based infant formula

Determination of total potentially available nucleosides in bovine milk

Bovine colostrum and milk samples were collected from two herds over the course of the first month post-partum, pooled for each herd by stage of lactation and total potentially available nucleosides were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate clean-up, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the available nucleosides pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations

Randomised controlled trial to improve health and reduce substance use in established psychosis (IMPaCT): cost-effectiveness of integrated psychosocial health promotion

Background: There is mounting evidence that people with severe mental illness have unhealthy lifestyles, high rates of cardiovascular and metabolic diseases, and greater risk of early mortality. This study aimed to assess the cost-effectiveness of a health promotion intervention seeking to improve physical health and reduce substance use in people with psychosis. Methods: Participants with a psychotic disorder, aged 18-65 years old and registered on an enhanced care approach programme or equivalent were recruited from community mental health teams in six mental health trusts in England. Participants were randomisation to either standard community mental health team care (treatment as usual) or treatment as usual with an integrated health promotion intervention (IMPaCT). Cost-effectiveness and cost-utility analyses from health and social care and societal perspectives were conducted alongside a cluster randomised controlled trial. Total health and social care costs and total societal costs at 12 and 15 months were calculated as well as cost-effectiveness (incremental cost-effectiveness ratios and cost-effectiveness acceptability curves) at 15 months based on quality of life (SF-36 mental and physical health components, primary outcome measures) and quality adjusted life years (QALYs) using two measures, EQ-5D-3 L and SF-36. Data were analysed using bootstrapped regressions with covariates for relevant baseline variables. Results: At 12-15 months 301 participants had full data needed to be included in the economic evaluation. There were no differences in adjusted health and social care costs (£95, 95% CI -£1410 to £1599) or societal costs (£675, 95% CI -£1039 to £2388) between the intervention and control arms. Similarly, there were no differences between the groups in the SF-36 mental component (−0.80, 95% CI -3.66 to 2.06), SF-36 physical component (−0.68, 95% CI -3.01 to 1.65), QALYs estimated from the SF-36 (−0.00, −0.01 to 0.00) or QALYs estimated from the EQ-5D-3 L (0.00, 95% CI -0.01 to 0.02). Cost-effectiveness acceptability curves for all four outcomes and from both cost perspectives indicate that the probability of the health promotion intervention being cost-effective does not exceed 0.4 for willingness to pay thresholds ranging from £0-£50,000. Conclusions: Alongside no evidence of additional quality of life/clinical benefit, there is also no evidence of cost-effectiveness

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

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

Analysis of 5'-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First action 2011.20

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 pane

AOAC SMPR 2014.005 Biotin in infant formula and adult/pediatric nutritional formula

Intended Use: Reference Method for dispute resolution. 1 Applicability determination: of total biotin in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). 2 Analytical technique: any analytical technique that meets the following method performance requirements is acceptable