Human milk fatty acid composition and its association with maternal blood and adipose tissue fatty acid content in a cohort of women from Europe.

PURPOSE: Human milk (HM) composition is influenced by factors, like maternal diet and body stores, among other factors. For evaluating the influence of maternal fatty acid (FA) status on milk FA composition, the correlation between FA content in HM and in maternal plasma, erythrocytes, and adipose tissue was investigated. METHODS: 223 European women who delivered at term, provided HM samples over first four months of lactation. Venous blood and adipose tissue (only from mothers who consented and underwent a C-section delivery) were sampled at delivery. FAs were assessed in plasma, erythrocytes, adipose tissue, and HM. Evolution of HM FAs over lactation and correlations between FA content in milk and tissues and between mother's blood and cord blood were established. RESULTS: During lactation, arachidonic acid (ARA) and docosahexaenoic acid (DHA) significantly decreased, while linoleic acid (LA), alpha-linolenic acid (ALA), and eicosapentaenoic acid (EPA) remained stable. Positive correlations were observed between HM and adipose tissue for palmitic, stearic, oleic, and polyunsaturated fatty acids (PUFAs). Correlations were found between milk and plasma for oleic, LA, ARA, ALA, DHA, monounsaturated fatty acids (MUFAs), and PUFAs. No correlation was observed between erythrocytes and HM FAs. LA and ALA were more concentrated in maternal blood than in infant blood, contrary to ARA and DHA, supporting that biomagnification of LCPUFAs may have occurred during pregnancy. CONCLUSIONS: These data show that maternal adipose tissue rather than erythrocytes may serve as reservoir of PUFAs and LCPUFAs for human milk. Plasma also supplies PUFAs and LCPUFAs to maternal milk. If both, adipose tissue and plasma PUFAs, are reflection of dietary intake, it is necessary to provide PUFAs and LCPUFAs during pregnancy or even before conception and lactation to ensure availability for mothers and enough supply for the infant via HM.

Quantification of 1,3-olein-2-palmitin (OPO) and Palmitic Acid in sn-2 Position of Triacylglycerols in Human Milk by Liquid Chromatography Coupled with Mass Spectrometry.

This study describes the identification and quantification of fatty acids in the sn-2 position of triacylglycerols (TAG) and of the most abundant TAG regioisomers in human milk by liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS). Over 300 individual TAG species were observed and 1,3-olein-2-palmitin (OPO) was identified as the most abundant TAG regioisomer. Validation of the HPLC-HRMS method showed repeatability and intermediate reproducibility values ranging from 3.1 to 16.6% and 4.0 to 20.7%, respectively, and accuracy ranging from 75 to 97%. Results obtained by the HPLC-HRMS method were comparable to results from the ISO 6800 method for the quantification of palmitic acid in the sn-2 position of TAG (81.4 and 81.8 g 100 g-1 total palmitic acid, respectively). Processing the data obtained with the HPLC-HRMS method is extremely time consuming and, therefore, a targeted method suitable for the quantification of OPO in human milk samples by ultra-performance (UP) LC coupled with triple quadrupole (QQQ) MS was developed and validated. OPO identification and quantification by UPLC-QQQ were based on nominal mass and a fragmentation pattern obtained by multiple reaction monitoring experiments. The method was validated in terms of accuracy and precision by analyzing different aliquots of the same human milk sample over time and comparing the results with values obtained by HPLC-HRMS. Intermediate reproducibility was <15% and trueness comparable to HPLC-HRMS. Quantification of OPO in human milk samples collected at 30, 60 and 120 days postpartum showed that OPO content varies between 333 ± 11.8 and 383 ± 18.0 mg 100mL-1.

Quantification of fatty acids in erythrocytes and plasma by fast gas chromatography.

As a result of the heterogeneous nature of lipid classes in complex biological matrices such as plasma and erythrocytes, it is imperative to have a robust and validated methodology for fatty acid quantification. The effective method presented here combines available methodology of fast gas chromatography and an improvement of the sample preparation methodology before injection into the gas chromatograph. This methodology ensures complete transesterification and quantification of total and individual fatty acids (and not only in relative amounts) by addition of internal standards. We considered sample preparation key, and we established the use of lysis buffer and ethanol for erythrocytes and plasma sample preparation, respectively. Fatty acid profile was determined by acid methylation and fast gas chromatography equipped with a flame ionization detector. The triacylglycerol 13:0, phosphatidylcholine 23:0, and methyl esters 21:0 were used as internal standards. Within the linearity of the calibration, the ratio of the peak area of each fatty acid over the peak area of the internal standard was constant (coefficient of variation ≤ 2.5). Satisfactory repeatability <15% and intermediate reproducibility < 15% were observed. Finally, this validated method was applied to a pre-clinical trial that investigated the impact of dietary fats on accretion of specific fatty acids in plasma and erythrocytes.

Vitamin C in infant formula and adult/pediatric nutritional Formula by ultra-performance liquid chromatography with Ultraviolet detection: first action 2012.22.

During the AOAC Annual Meeting held in Las Vegas, NV from September 30 to October 3, 2012, the Stakeholder Panel on Infant Formula and Adult Nutritionals convened to review single-laboratory validation data submitted for the method, Vitamin C in Adult/Pediatric Formula by Ultra-Performance Liquid Chromatography with Ultraviolet Detection. This method is a modified version of the method "HPLC-UV Determination of Total Vitamin C in a Wide Range of Fortified Food Products" previously published in Food Chem., 94, 626-631 (2006). The SLV data from the modified method were reviewed and compared to the standard method performance requirements (SMPR 2012.012), and it was concluded that the method meets the requirements. The method was approved as AOAC Official First Action. The method is based on the acidic extraction of ascorbic acid in the presence of Tris[2-carboxyethyl] phosphine (TCEP) as a reducing agent. Separation was achieved on a C18 column with a sodium acetate eluent (pH 5.4) combined with TCEP and decylamine as an ion-pairing agent. Accuracy rates were between 90 and 100%. Repeatability RSD (RSD,) ranged from 1.4 to 2.5%, and intermediate reproducibility RSD (RSDiR) ranged from 1.3 to 7.5%.

Choline in infant formula and adult/pediatric nutritional Formula by ultra high-performance liquid chromatography/ Tandem mass spectrometry: AOAC First Action 2012.18.

The method described below is for the determination of choline in infant formula and adult/pediatric nutritional formula by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The single-laboratory validation data were submitted to the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) for review at the AOAC INTERNATIONAL Annual Meeting held September 30 to October 3, 2012 in Las Vegas, NV. The ERP determined that the data reviewed met the standard method performance requirements set by SPIFAN, and the method was approved as AOAC Official First Action. The analytical range was found to be between 0.16 and 3.2 microg/mL. The recovery rates were within 80-120% at 50 and 100% of native levels for all samples. Repeatability precision (RSDr) was < 3%, with intermediate reproducibility (RSDir) no higher than 4%.

Pantothenic acid (vitamin B5) in fortified foods: comparison of a novel ultra-performance liquid chromatography-tandem mass spectrometry method and a microbiological assay (AOAC Official Method 992.07).

A novel method was developed and single-laboratory validated for the determination of free pantothenic acid (vitamin B5) in a wide range of infant and adult fortified food products. The method combines simple sample preparation and chromatographic analysis using ultra-performance LC coupled to tandem MS with positive electrospray ionization. Pantothenic acid was quantified using [13C6, 15N2]-pantothenic acid as an internal standard. Calibration curves were linear between 0.08 and 1.2 microg/mL (r2 = 0.9998), and average recovery varied between 95 and 106%. The method exhibited overall RSD(r) of 1.1% and RSD intermediate reproducibility from 2.5 to 6.0% in infant formulas and cereals. Comparison of results between total and free pantothenic acid showed that the analysis of free pantothenic acid gave a good estimation of total pantothenic acid in the range of products analyzed. The method provides reliable free pantothenic acid results in a wide range of fortified foods (infant and adult nutritionals, cereal products and beverages), and shows good correlation with the microbiological method AOAC Official Method 992.07. It is a more selective, faster, and robust alternative to microbiological determination.

Determination of vitamin B12 in infant formula and adult nutritionals by liquid chromatography/UV detection with immunoaffinity extraction: First Action 2011.08.

At the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," on June 29, 2011, an Expert Review Panel agreed that the method "Determination of Vitamin B12 in Infant Formulas and Adult Nutritionals by Liquid Chromatography/UV Detection with Immunoaffinity Extraction" be adopted AOAC Official First Action status. The method is applicable for the determination of vitamin B12, which includes added cyanocobalamin and natural forms, making it applicable to both fortified and nonfortified products. Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of sodium cyanide (100 degrees C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by LC with UV detection (361 nm). A single-laboratory validation study was conducted on a range of products, including milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method demonstrated linear response over a large range of concentrations, recovery rates of 100.8 +/- 7.5% (average +/- SD), repeatability RSD (RSDr) of 2.1%, and intermediate reproducibility (RSD(iR)) of 4.3%. LOD and LOQ values were 0.10 and 0.30 microg/100 g, respectively, and correlation with the reference microbiological assay was good (R2 = 0.9442). The results of the study were published in J. AOAC Int. 91, 786-793 (2008). The performance characteristics of the method met the standard method performance requirements set forth by the Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action status.

Determination of vitamin B12 in food products by liquid chromatography/UV detection with immunoaffinity extraction: single-laboratory validation.

A fast and simple method to determine vitamin B12 in foods is presented. The method allows, in addition to the determination of added cyanocobalamin, the determination of natural vitamin B12 forms, making it also applicable to nonfortified products, especially those that are milk-based. Vitamin B12 is extracted in sodium acetate buffer in the presence of sodium cyanide (100 degrees C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by liquid chromatography with UV detection (361 nm). The method has been validated in analyses of a large range of products: milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method showed appropriate performance characteristics: linear response over a large range of concentrations, recovery rates of 100.8 +/- 7.5% (average +/- standard deviation), relative standard deviation of repeatability, RSDr, of 2.1%, and intermediate reproducibility, RSDiR, of 4.3%. Limits of detection and quantitation were 0.10 and 0.30 microg/100 g, respectively, and correlation with the reference microbiological assay was good (R2 = 0.9442). The proposed method is suitable for the routine determination of vitamin B12 in fortified foods, as well as in nonfortified dairy products. It can be used as a faster, more selective, and more precise alternative to the classical microbiological determination.

Quantification of glycerophospholipids and sphingomyelin in human milk and infant formula by high performance liquid chromatography coupled with mass spectrometer detector.

Phospholipids and sphingomyelin have a central role in infant nutrition, phospholipid acting as a nutrient carrier of long chain polyunsaturated fatty acids and sphingomyelin having an important role in cognitive function. However, analytical methods to precisely characterize and quantify these compounds in maternal milk are needed. Phospholipids and sphingomyelin were extracted using chloroform and methanol and separated on Polaris 3 Si column 250×2.0mm from Varian and analyzed by high performance liquid chromatography (HPLC) coupled with mass spectrometer detector (MS). The analytical method was validated and repeatability, intermediate reproducibility, and recovery values were calculated. The relative standard deviation of repeatability (CV(r)) and intermediate reproducibility (CV(iR)) values ranged between 2.3 and 7.2% and 9.5 and 17.8%, respectively and the recovery values between 96 and 109%. Finally, the validated method was tested on human milk samples and on infant formula which were analysed also by HPLC coupled with evaporative light scattering detector (ELSD). In human milk, sphingomyelin (9.28mg100mL-1) was the most abundant compound, followed by phosphatidylcholine (5.39mg100mL-1), phosphatidylethanolamine (2.85mg100mL-1) and phosphatidylinositol (1.82mg100mL-1).

Temporal Changes of Human Breast Milk Lipids of Chinese Mothers.

Fatty acids (FA), phospholipids (PL), and gangliosides (GD) play a central role in infant growth, immune and inflammatory responses. The aim of this study was to determine FA, PL, and GD compositional changes in human milk (HM) during lactation in a large group of Chinese lactating mothers (540 volunteers) residing in Beijing, Guangzhou, and Suzhou. HM samples were collected after full expression from one breast and while the baby was fed on the other breast. FA were assessed by direct methylation followed by gas chromatography (GC) analysis. PL and GD were extracted using chloroform and methanol. A methodology employing liquid chromatography coupled with an evaporative light scattering detector (ELSD) and with time of flight (TOF) mass spectrometry was used to quantify PL and GD classes in HM, respectively. Saturated FA (SFA), mono-unsaturated FA (MUFA), and PL content decreased during lactation, while polyunsaturated FA (PUFA) and GD content increased. Among different cities, over the lactation time, HM from Beijing showed the highest SFA content, HM from Guangzhou the highest MUFA content and HM from Suzhou the highest n-3PUFA content. The highest total PL and GD contents were observed in HM from Suzhou. In order to investigate the influence of the diet on maternal milk composition, a careful analyses of dietary habits of these population needs to be performed in the future.

Separation of water-soluble vitamins by reversed-phase high performance liquid chromatography with ultra-violet detection: application to polyvitaminated premixes.

Nine water-soluble vitamins: [thiamine (B1), ascorbic acid (C), nicotinamide (PP), pyridoxine (B6), calcium pantothenate (B5), folic acid (B9), cyanocobalamin (B12), riboflavin (B2) and biotin (B8)] were separated on a YMC-Pack Pro C18 column (250 mm x 4.6 mm, 5 microm particle size) in a single run with a gradient elution of mobile phase consisting of 0.025% trifluoroacetic acid pH 2.6 (solvent A) and acetonitrile (solvent B). The separation was achieved within 17 min with a flow rate of 0.8 ml min(-1) and the detection was performed at two wavelengths (210 and 275 nm). The calibration graphs plotted with six concentrations of each vitamin were linear with a regression coefficient R2 > 0.995. The method was applied for the quantification of vitamins B1, C, PP, B6, B5, B9 B2 and B8 in polyvitaminated premixes (premixes) used for the fortification of infant nutrition products. The sample preparation involves an aqueous extraction of vitamins and two different samples dilution were used prior the LC-analysis. The specificity of the method was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity with the standard of each vitamin. The repeatability of the method was evaluated at different level of concentrations on 12 premixes and the coefficients of variation (CVr) were below 6.5%. The values of the intermediate precision (CV1) were below 9.6% (n = 6). The concentrations of vitamins found in premixes with our method were comparable to the declared values, since no bias was found between the two sets of results at 95% confidence. The simplicity of the procedure should make it highly desirable for quality control of premixes in the food industry.

Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry.

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.