Esther Campos-Giménez

Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry

This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans.

Application of supercritical fluid chromatography coupled to mass spectrometry to the determination of fat-soluble vitamins in selected food products

In the present manuscript, we describe a fully optimized and validated method suitable to analyse nine compounds (retinyl acetate, retinyl palmitate, retinol, α-tocopherol, α-tocopheryl acetate, cholecalciferol, ergocalciferol, phylloquinone, menaquinone-4) representing the major contributors to the fat-soluble vitamin activity of selected food products (infant formulas, adult nutritionals, infant cereals and mixed meals). Sample preparation involves direct solvent extraction using enzyme-assisted matrix disintegration and methanolic protein precipitation. Direct injection of the extract allows quantification of vitamins A, E and K in only 7 min, while vitamin D is determined after fast derivatization of the extract. Separation is achieved by supercritical fluid chromatography and detection performed by tandem mass spectrometry in positive Atmospheric Pressure Chemical Ionization mode. Results on a Standard Reference Material (SRM 1849a Infant/Adult Nutritional) were not statistically different from reference values. Full validation of the method showed excellent overall performance. Average recovery rate was between 90 and 110% for all vitamins and matrixes. The methodology shows enhanced safety and reduced cost as compared with previously published methods, together with potential for application to more complex matrixes. The full procedure can be easily applied in control laboratories dramatically increasing sample throughput and reducing solvent consumption.

Concentrations of Carotenoids and Tocopherols in Breast Milk from Urban Chinese Mothers and their Associations with Maternal Characteristics：A Cross-Sectional Study

Milk composition remains the best estimate of infant requirements. The aims of this study were to quantify carotenoids and tocopherols in human milk from healthy Chinese mothers, and to explore their associations with lactation stage, region, socio-economic and obstetric characteristics, and dietary intake. Human milk was obtained from 509 healthy mothers, and concentrations of carotenoids and tocopherols were analyzed by Ultra High Performance Liquid Chromatography. The mothers’ socio-economic and obstetric characteristics and dietary intake through a single 24-h dietary recall were evaluated. The median concentrations (μg/100 mL) of each component of 0–4 days, 5–11 days, 12–30 days, 31–60 days, 61–120 days, and 121–240 days postpartum were respectively as follows: β-carotene 8.0, 2.8, 2.1, 1.7, 1.9, 1.8; β-cryptoxanthin 6.2, 3.4, 2.4, 1.7, 1.8, 2.1; lutein 5.7, 7.0, 2.2, 2.9, 2.8, 3.7; lycopene 6.3, 2.5, 1.8, 1.4, 1.4, 1.5; zeaxanthin 1.0, 1.4, 0.8, 0.8, 1.0, 1.1; α-tocopherol 645, 382, 239, 206, 212, 211; γ-tocopherol 68, 63, 70, 73, 68, 88. The levels of those components varied significantly among different lactation stages and presented regional differences. Associations of carotenoid contents with maternal education, delivery mode, and present body mass index were found in multivariate analyses. These results suggested that lactation stage, region, and socio-economic and obstetric factors were associated with human milk concentrations of carotenoids and tocopherols in healthy Chinese mothers.

A Novel Methodology for the Quantification of B-Vitamers in Breast Milk

With this report we present development, validation and application of an analytical methodology for the quantification of 18 water soluble vitamers and secreted or biological forms in breast milk. On a relatively low amount of breast milk (200 µL), we applied isotope dilution-based sample preparation based on a combination of enzymatic treatment and protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Compounds separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. To perform the quantification of 18 water soluble vitamers, procured pooled breast milk was used to build matrixmatched calibration curves, as labelled internal standards were not available for each vitamer. The analytical approach has been validated according to the EMA guidelines. The overall performance of the method was considered adequate, with 0.3- 28.3% and 0.9-32.6% intra and inter-day precision respectively and averaged accuracy reaching 92.2-107.5%. In addition, performed freeze/thaw stability studies showed the potential degradation of some vitamers. We therefore recommend particular attention in sample collection with rather having dedicated aliquots with small volumes. The feasibility of this analytical approach has been evaluated by quantifying various breast milk samples that were procured from an external supplier. The main forms found in breast milk were thiamine monophosphate for B1, flavin adenine nucleotide for B2, nicotinamide for B3, pyridoxal for B6 and 5-methyl tetrahydrofolic acid for B9. In addition, we newly reported nudifloramide as B3 form present in breast milk. With this analytical approach, it will give more confidence to provide a comprehensive assessment of the presence of water soluble vitamins in breast milk. This will enable the accurate evaluation of the nutritional requirements of infants.

The contribution of minor folates to the total vitamin B9 content of Infant formula and clinical nutrition products

In the present study an optimization of trienzyme treatment combining α-amylase, protease and γ-carboxy peptidase allowing complete sample preparation within a working day for the analysis of vitamin B9 (folate) in infant formula and adult/pediatric nutritional products is presented. The optimized sample preparation was applied to a set of samples representing most of the products in the marketplace. Results on Standard Reference Material 1849a were well in agreement with certified values. The main contributor to total folate was folic acid, 5-methyl-tetrahydrofolate was the only minor contributor in milk-based products. Soy-based formulas contained polyglutamates of 5-formyl-tetrahydrofolate. The relative contribution of polyglutamates to the total folate content remained low in the types of product included in this study. The results suggest that a simple di-enzyme treatment could be enough for these products, nevertheless, this should be carefully evaluated prior to making a decision on the use of tri- or di-enzyme treatment.

Pantothenic Acid (Vitamin B5) in Infant Formula and Adult/ Pediatric Nutritional Formula by Ultra-High Pressure Liquid Chromatography/Tandem Mass Spectrometry Method: Collaborative Study, Final Action 2012.16.

In order to determine repeatability and reproducibility of AOAC First Action Method 2012.16 [Pantothenic Acid (Vitamin B5) in Infant Formula and Adult/Pediatric Nutritional Formula by Ultra-High Pressure Liquid Chromatography/Tandem Mass Spectrometry], a collaborative study was organized. The study was divided in two parts: method setup and qualification of participants (part 1) and collaborative study participation (part 2). For part 1, each participating laboratory was asked to analyze two practice samples using the aforementioned method. Laboratories that provided results within a range of expected levels were qualified for part 2, during which each laboratory received 10 samples in blind duplicates. Results have been compared to the Standard Method Performance Requirement (SMPR®) 2012.009 established for pantothenic acid. Precision results (repeatability and reproducibility) were within the limits stated in the SMPR. Repeatability ranged from 1.3 to 3.3%, and reproducibility ranged from 4.1 to 7.0%. Horwitz ratio (HorRat) values were all <1, ranging from 0.33 to 0.69. The AOAC Expert Review Panel on Stakeholder Panel on Infant Formula and Adult Nutritionals Nutrient Methods determined that the data presented met the SMPR and recommended the method for Final Action status, which was then granted by the AOAC Official Methods Board.