Determination of folate in infant formula and adult/pediatric nutritional formula by ultra-high performance liquid chromatography-tandem mass spectrometry: First Action 2013.13.

A UHPLC-MS/MS method for the determination of folate (vitamin B9) in infant formula and adult/pediatric nutritional formula was assessed for compliance with standard method performance requirements set forth by the AOAC INTERNATIONAL Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN). A single-laboratory validation (SLV) study was conducted as the first step in the process to validate the method. In the study, 12 matrixes, representing the range of infant and adult nutritional products, were evaluated for folate [the sum of supplemental folic acid plus 5-methyl tetrahydrofolic acid (5-Me THF)]. Method response was linear in the range of 1.0-900 ng/mL, corresponding to 0.33-300 microg/l100 g in reconstituted sample. LOD for folic acid and 5-Me THF, expressed in reconstituted product, were 0.10 microg/100 g and 0.05 microg/100 g, respectively, and LOQ were 0.33 microg/100 g and 0.10 microg/100 g, respectively. Repeatability was <5.3% and intermediate precision was <5.5%. Recovery rates of spiking at 50 and 100% of target values in nonfortified products were within 90-110%. Evaluation of trueness was performed on Certified Reference Material (SRM 1849 Infant/Adult Nutritional Formula) and gave 96.4% of theoretical value. Based on the results of the SLV, the method meets the SPIFAN requirements for AOAC First Action status for the determination of folates in infant formula and adult/pediatric nutritional formula.

Vitamins and carotenoids in human milk delivering preterm and term infants: Implications for preterm nutrient requirements and human milk fortification strategies.

Differences in vitamin and carotenoids content of human milk (HM) produced for infants born at term and preterm is poorly understood. In this study, HM was collected weekly for four and two months post-partum for preterm and term groups, respectively. Nutrients of interest, from single full breast expressions were measured by liquid chromatography coupled with mass spectrometry. Microbiological assay was employed for vitamin B12. When compared at equivalent post-partum age, vitamins B1, B2, B6, and B9 were significantly higher in preterm than in term HM, but only during the first two weeks. No significant differences were observed for A, E, B3 and B12 between groups. Lycopene was the only carotenoid exhibiting a significant higher concentration in term than in preterm HM between weeks 1 and 4 post-partum. When compared at equivalent post-menstrual age, preterm milk was significantly higher for vitamins B1, B2, B3, B6 and B9 and lower levels of vitamins A, E, ß-carotene, ß-cryptoxanthin, lutein, zeaxanthin and lycopene compared to their term counterparts. These results suggest that preterm breastfed infants at term equivalent age may receive lower amounts of these micronutrients than breast-fed term neonates, possibly highlighting the need to supplement or fortify their nutritional intake with vitamins and carotenoids. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT #02052245.

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.

Characterization and quantification of proteins in lecithins.

Several methods for extraction and quantification of proteins from lecithins were compared. Extraction with hexane-2-propanol-water followed by amino acid analysis is the most suitable method for isolation and quantification of proteins from lecithins. The detection limit of the method is 15 mg protein/kg lecithin, and the quantification limit is 50 mg protein/kg. The relative repeatability limits for samples containing 0-500 and 500-5000 mg protein/kg sample were 12.6 and 7.5%, respectively. The protein recovery ranged between 101 and 123%. The protein content has been determined in different kinds of lecithins. The results were as follows: standard soy lecithins (between 232 and 1338 mg/kg), deoiled soy lecithin (342 mg/kg), phosphatydylcholine-enriched soy lecithins (not detectable and 163 mg/kg), sunflower lecithins (892 and 414 mg/kg), and egg lecithin (50 mg/kg). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns of the standard soy and sunflower lecithins are very similar to those of soy flour. The protein profile of the egg lecithin shows several bands with a broad range of molecular masses. The molecular masses of the main proteins of soy lecithins and soy flour have been determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ranged from 10.5 to 52.2 kDa. Most of the major proteins from soy and sunflower lecithins identified by MALDI-MS and electrospray tandem MS belong to the 11S globulin fraction, which is one of the main fractions of soy and sunflower seeds. In addition, the seed maturation protein P34 from the 7S globulin fraction of soy proteins has also been identified in soy lecithins. This protein has been reported as the most allergenic protein in soybean.

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.

Simplified enzymatic high-performance anion exchange chromatographic determination of total fructans in food and pet food-limitations and measurement uncertainty.

A simplified method to determine total fructans in food and pet food has been developed and validated. It follows the principle of AOAC method 997.08, i.e., high-performance anion exchange chromatographic (HPAEC) determination of total fructose released from fructans (F(f)) and total glucose released from fructans (G(f)) after enzymatic fructan hydrolysis. Unlike AOAC method 997.08, calculation of total fructans is based on the determination of F(f) alone. This is motivated by the inherent difficulty to accurately determine low amounts of G(f) since many food and pet food products contain other sources of total glucose (e.g., starch and sucrose). In this case, a correction factor g can be used (1.05 by default) to take into account the theoretical contribution of G(f). At levels >5% of total fructans and in commercial fructan ingredients, both F(f) and G(f) can and should be accurately determined; hence, no correction factor g is required. The method is suitable to quantify total fructans in various food and pet food products at concentrations >or=0.2% providing that the product does not contain other significant sources of total fructose such as free fructose or sucrose. Recovery rates in commercial fructan ingredients and in selected food and pet food ranged from 97 to 102%. As part of a measurement uncertainty estimation study, individual contributions to the total uncertainty (u) of the total fructan content were identified and quantified by using the validation data available. As a result, a correlation between the sucrose content and the total uncertainty of the total fructan content was established allowing us to define a limit of quantitation as a function of the sucrose content. One can conclude that this method is limited to food products where the sucrose content does not exceed about three times the total fructan content. Despite this limitation, which is inherent to any total fructan method based on the same approach, this procedure represents an excellent compromise with regard to accuracy, applicability, and convenience.

Determination of proteins in refined and nonrefined oils.

Five methods using aqueous/organic solvents for the separation of proteins from oils were compared. The extraction with acetone-hexane followed by amino acid analysis was found to be the most suitable method for isolation and quantification of proteins from oils. The detection limit of the method was 0.18 mg protein/kg oil, and the quantification limit was 0.6 mg protein/kg. The relative repeatability limit for samples containing 1-5 mg protein/kg sample was 27%. The protein recovery ranged between 68 and 133%. Using this method, the protein content of 14 refined and nonrefined oils was determined. In none of the refined oils were proteins detected, whereas the protein content of the unrefined oils ranged between undetectable in extra virgin olive oil to 11 mg/kg in rapeseed oil. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with silver staining, many protein bands were visible in the unrefined soy, olive, peanut, and rapeseed oil samples. Proteins bands were not obtained from the refined fish oil. In the other refined oil samples, a few proteins bands could be visualized. Two protein bands with apparent molecular molecular masses of 58 and 64 kDa were always observed in these oils.