scholarly journals Human Milk Metabolomics Using Biocrates AbsoluteIDQ® p180 Kit Assay (OR06-04-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Daniela Hampel ◽  
Setareh Shahab-Ferdows ◽  
Muttaquina Hossain ◽  
M Munirul Islam ◽  
Tahmeed Ahmed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Milk ◽  
Biogenic Amines ◽  
Human Plasma ◽  
Analytical Techniques ◽  
Milk Composition ◽  
Funding Sources ◽  
Carry Over ◽  
Gates Foundation ◽  
Student’S T

Abstract Objectives Targeted metabolomics are commercially available for human plasma, but not for human milk. However, metabolite analyses could provide a novel and efficient approach to understanding human milk composition and relationships to maternal and infant status. Methods Pooled human milk was used to evaluate and validate the Biocrates AbsoluteIDQ® p180 kit for human milk metabolomics (40 acylcarnitines, 42 acids/biogenic amines, 91 phospholipids, 15 sphingolipids, sum of hexoses) using an ABSciex 5500QTRAP mass spectrometer in LC-MS/MS and flow injection analysis (FIA) mode. In a feasibility study, milk collected <6 mo lactation from A) Bangladeshi healthy mothers (BMI >18.5, n = 12) and from B) mothers with stunted infants (HAZ-score < −2; n = 13) were analyzed. Results 120 of the detectable 188 assay metabolites were found in the pooled milk, including all of the sphingolipids and amino acids. Additional internal standards (IS) were prepared for lysine and some biogenic amines for higher accuracy. Higher amounts of glutamate, taurine, and putrescine in milk required higher levels of calibrators than for plasma in LC-MS/MS mode. For metabolites of low abundance diluted calibrators (0.25 and 0.5) were added. FIA provided results for 94 of 146 metabolites above LOD without any carry-over. Metabolite recoveries (levels) varied between 64.1 and 127.0%. Intra-assay variations (6 replicates) for all detectable metabolites ranged between 3.4 and 18.4%. Human milk given to healthy compared to malnourished infants was higher in the amino acids citrulline, glutamate, glycine, and phenylalanine, and carnitine, while histamine and dodecanoylcarnitine were lower (Student's t-test, P < 0.05 for all). Conclusions The AbsoluteIDQ® p180 can be used for human milk application and thus allows the application of the same assay for both human plasma and milk, enhancing comparability of results by reducing analytical bias due to different analytical techniques. Funding Sources Bill & Melinda Gates Foundation (OPP1148405 & OPP1164613), USDA/ARS Intramural Project (5306–51,530-019–00).

scholarly journals Validation and Application of Biocrates AbsoluteIDQ® p180 Targeted Metabolomics Kit Using Human Milk

Nutrients ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1733 ◽  
Author(s):  
Hampel ◽  
Shahab-Ferdows ◽  
Hossain ◽  
Islam ◽  
Ahmed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Milk ◽  
Biogenic Amines ◽  
Milk Composition ◽  
Milk Analysis ◽  
Analysis Tool ◽  
Targeted Metabolomics ◽  
Internal Standards ◽  
Liquid Chromatography Tandem Mass ◽  
Height For Age

Human-milk-targeted metabolomics analysis offers novel insights into milk composition and relationships with maternal and infant phenotypes and nutritional status. The Biocrates AbsoluteIDQ® p180 kit, targeting 40 acylcarnitines, 42 amino acids/biogenic amines, 91 phospholipids, 15 sphingolipids, and sum of hexoses, was evaluated for human milk using the AB Sciex 5500 QTRAP mass-spectrometer in liquid chromatography-tandem mass-spectrometry (LC-MS/MS) and flow-injection analysis (FIA) mode. Milk (<6 months lactation) from (A) Bangladeshi apparently healthy mothers (body mass index (BMI) > 18.5; n = 12) and (B) Bangladeshi mothers of stunted infants (height-for-age Z (HAZ)-score <−2; n = 13) was analyzed. Overall, 123 of the possible 188 metabolites were detected in milk. New internal standards and adjusted calibrator levels were used for improved precision and concentration ranges for milk metabolites. Recoveries ranged between 43% and 120% (coefficient of variation (CV): 2.4%–24.1%, 6 replicates). Milk consumed by stunted infants vs. that from mothers with BMI > 18.5 was lower in 6 amino acids/biogenic amines but higher in isovalerylcarnitine, two phospholipids, and one sphingomyelin (p < 0.05 for all). Associations between milk metabolites differed between groups. The AbsoluteIDQ® p180 kit is a rapid analysis tool suitable for human milk analysis and reduces analytical bias by allowing the same technique for different specimens. More research is needed to examine milk metabolite relationships with maternal and infant phenotypes.

scholarly journals High-Throughput Analysis of Water-Soluble Choline and Related Metabolites in Human Milk

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1171-1171
Author(s):  
Daniela Hampel ◽  
Setareh Shahab-Ferdows ◽  
Ngoc Nguyen ◽  
Gilberto Kac ◽  
Allen Lindsay
Keyword(s):  
Sample Preparation ◽  
Human Milk ◽  
Water Soluble ◽  
Process Efficiency ◽  
National Council ◽  
High Throughput Analysis ◽  
Standard Curve ◽  
Funding Sources ◽  
Gates Foundation ◽  
Acquity Uplc

Abstract Objectives Choline and related metabolites play important roles in metabolic processes. Inadequate provision of these nutrients to the exclusively breast-fed infant can negatively impact its healthy growth and development. Methods We developed an UPLC-MS/MS method for analyzing choline (Cho), phospho-choline (PCho), glycerophospho-choline (GPCho), total choline (tCho = Cho + PCho + GPCho), betaine, carnitine, creatinine, dimethyl glycine (DMG), methionine, and trimethylamine N-oxide (TMAO) in human milk. Results Optimized results were obtained using a Phenomenex Luna Silica (2) column, 100 × 2 mm, 3 µm, and a gradient of 0.1% aqueous propionic acid (A) and acetonitrile (B) from 60% to 90% A over 2 min (Waters ACQUITY UPLC I-Class - SCIEX 4500TQ mass spectrometer). Sample preparation required only 5–10µL of milk, diluted 1:80 in methanol/water, 4:1, v/v, prior to analysis. Quantification was done using isotopically labeled internal standards and an external standard curve. Pooled human milk used for method validation showed recovery rates from 108–131% for all analytes, and an overall process efficiency from 54 to 114%. All standard curves revealed good linearity (r &gt; 0.999). Milk from apparently healthy Brazilian mothers (1–120days pp) revealed large concentration ranges within and between analytes (IQR, mg/L): Cho 10.3, 20.3; GPcho 48.7, 101; PCho 134, 221; tCho 120, 166; betaine 0.25, 0.53; carnitine 3.35, 5.06; creatinine 2.92, 3.90; DMG 0.26, 0.54; methionine 0.47, 0.90. 63% of the milk samples reached the tCho value used for the Adequate Intake (1–6 mo). Conclusions Our newly implemented method enabled the simultaneous analysis of water-soluble forms of choline and related metabolites in human milk in minute amounts of sample, and requiring only minimalistic sample preparation. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306–51,530-019–00), and CNPq (Brazilian National Council for Science and Technology).

scholarly journals Human Milk Metabolic Profiling Using Biocrates MxP® Quant 500 Kit

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 874-874
Author(s):  
Daniela Hampel ◽  
Setareh Shahab-Ferdows ◽  
Gilberto Kac ◽  
Lindsay Allen
Keyword(s):  
Fatty Acids ◽  
Human Milk ◽  
Small Molecules ◽  
Metabolic Profiling ◽  
National Council ◽  
Funding Sources ◽  
Upper Limit ◽  
Limit Of Quantitation ◽  
Gates Foundation ◽  
Comparability Of Results

Abstract Objectives Comprehensive metabolic profiling of human milk is a useful tool for examining its composition and relationship to maternal and infant metabolism and status. Methods A pooled milk sample was used to evaluate the Biocrates MxP® Quant 500 kit for human milk metabolomics (107 small molecules and 523 lipids) using an ABSciex 5500QTRAP mass-spectrometer in LC-MS/MS and flow injection analysis (FIA) mode. Additionally, milk from Brazilian mothers (A: 2–8, B: 28–50, C: 88–119d postpartum, ntotal = 25) was analyzed. Results 424 of the 630 assay metabolites were detected in the milkpool above the limit of quantitation (LOQ); 31 metabolites were below the lower limit of quantitation (LLOQ), while 7 were above the upper limit (ULOQ), mostly free fatty acids including arachidonic acid, docohexaenoic acid, and eicosapentaenoic acid. Bile acids were only detected below LLOQ. Concentrations measured in 5 different sample volumes (2–20μL, n = 10) showed satisfactory reproducibility (CV 3.7–20.0%) for 458 metabolites. Acceptable intraday variation (80–120%, 6 replicates) was achieved for 409 metabolites when spiking with 3 levels of QC-standards, but only for 127 metabolites after dilution (1:2 and 1:5). However, 396 metabolites revealed a good intraday variation when only considering the 1:2-dilution. Conclusions The MxP Quant® 500 kit was successfully employed for human milk providing data for over 400 metabolites in 10μL milk. and thus offers ability to use the same assay for both human plasma and milk, enhancing comparability of results by reducing analytical bias and increasing our ability to study milk as a biological system. Funding Sources Bill & Melinda Gates Foundation (OPP1148405), USDA/ARS Intramural Project (5306–51,530-019–00), and CNPq (Brazilian National Council for Science and Technology).

scholarly journals Development and Validation of a Method for Quantification of Favipiravir as COVID-19 Management in Spiked Human Plasma

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3789
Author(s):  
Mohammad Hailat ◽  
Israa Al-Ani ◽  
Mohammed Hamad ◽  
Zainab Zakareia ◽  
Wael Abu Dayyih
Keyword(s):  
Human Plasma ◽  
Internal Standard ◽  
Weighting Factor ◽  
Hplc Method ◽  
Bioanalytical Method Validation ◽  
Spiked Human Plasma ◽  
Fda Guidance ◽  
Significant Difference ◽  
Us Fda ◽  
Carry Over

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1–60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data’s heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday’s % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at −20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.

Human milk composition and the effects of pasteurisation on activity of components

2021 ◽  
Author(s):  
Syaza Y. Binte Abu Bakar ◽  
Malinda Salim ◽  
Andrew J. Clulow ◽  
Kevin Nicholas ◽  
Ben J. Boyd
Keyword(s):  
Human Milk ◽  
Milk Composition

scholarly journals Determination of Branched-Chain Amino Acids in Food Supplements and Human Plasma by a CE-MS/MS Method with Enhanced Resolution

2021 ◽  
Vol 22 (15) ◽  
pp. 8261
Author(s):  
Juraj Piestansky ◽  
Michaela Matuskova ◽  
Ivana Cizmarova ◽  
Dominika Olesova ◽  
Peter Mikus
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Background Electrolyte ◽  
Branched Chain Amino Acids ◽  
Fused Silica ◽  
Suitable Alternative ◽  
Enhanced Resolution ◽  
Validation Parameters ◽  
Branched Chain

In the presented study, a capillary electrophoresis-mass spectrometry method combining high separation efficiency and sensitive detection has been developed and validated, for the first time, to quantify branched chain amino acids (valine, isoleucine, leucine) in commercial food and sport supplement samples and human plasma samples. The separations were performed in a bare fused silica capillary. The background electrolyte was composed of 500 mM formic acid with pH 2.0. The plasma sample pretreatment was realized by simple protein precipitation with acetonitrile. Injection of a short zone of highly basic electrolyte before the sample injection and application of the negative pressure on the separation were accompanied by enhanced resolution of the isobaric amino acids—isoleucine and leucine. The developed method was characterized by favorable validation parameters, such as linearity (r2 > 0.99), accuracy and precision, the limit of detection, lower limit of quantification, or robustness. These parameters were more than sufficient for the quantification of branched chain amino acids in various samples. The determined concentrations of branched chain amino acids in food and sports supplements were in very good agreement with the content declared by the manufacturer. The investigated concentrations of branched chain amino acids were in the range 294.68–359.24 µM for valine, 91.76–95.67 µM for isoleucine, and 196.78–251.24 µM for leucine. These concentrations fall within the physiological limits. The developed CE-MS/MS method represents a suitable alternative to traditional approaches used in branched chain amino acid quality control and bioanalysis.

scholarly journals Free and Total Amino Acids in Human Milk in Relation to Maternal and Infant Characteristics and Infant Health Outcomes: The Ulm SPATZ Health Study

Nutrients ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 2009
Author(s):  
Joris H.J. van Sadelhoff ◽  
Linda P. Siziba ◽  
Lisa Buchenauer ◽  
Marko Mank ◽  
Selma P. Wiertsema ◽  
...  
Keyword(s):  
Amino Acids ◽  
Health Outcomes ◽  
Human Milk ◽  
Infant Development ◽  
Infant Health ◽  
Child Growth ◽  
Health Study ◽  
Maternal Characteristics ◽  
Tract Infections ◽  
Total Amino Acids

Free amino acids (FAAs) are important regulators of key pathways necessary for growth, development, and immunity. Data on FAAs in human milk (HM) and their roles in infant development are limited. We investigated the levels of FAAs and total amino acids (TAA, i.e., the sum of conjugated amino acids and FAAs) in HM in relation to infant and maternal characteristics and immunological conditions. FAA and TAA levels in HM sampled at 6 weeks (n = 671) and 6 months (n = 441) of lactation were determined using high-performance liquid chromatography. Child growth was ascertained at 4–5 weeks and at 6–7 months of age. Child allergy and lower respiratory tract infections were assessed in the first years of life. Associations of amino acid (AA) levels in HM with child growth and health outcomes were determined by Spearman correlation and modified Poisson regression, respectively. Free glutamine, glutamate, and serine in 6-week HM positively correlated with infant weight gain in the first 4–5 weeks of age. Maternal pre-pregnancy weight and body mass index (BMI) were negatively correlated with free glutamine and asparagine in 6-week and 6-month HM and positively correlated with the sum of TAAs in 6-month HM, but significance was lost following confounder adjustment. Free glutamine was lower in 6-month HM of mothers with an allergy (either active or non-active). No consistent associations were found between FAAs in HM and child health outcomes. However, potential negative associations were observed between specific FAAs and the risk of food allergy. These results suggest that specific FAAs play a role in infant growth. Moreover, these findings warrant further investigations into the relation of FAAs in HM with infant health outcomes and maternal allergy.

scholarly journals High-Temperature Short-Time and Holder Pasteurization of Donor Milk: Impact on Milk Composition

Life ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 114
Author(s):  
Diana Escuder-Vieco ◽  
Juan M. Rodríguez ◽  
Irene Espinosa-Martos ◽  
Nieves Corzo ◽  
Antonia Montilla ◽  
...  
Keyword(s):  
High Temperature ◽  
Human Milk ◽  
Milk Composition ◽  
Donor Human Milk ◽  
High Temperature Short Time ◽  
Lactose Concentration ◽  
C 30 ◽  
Milk Banks ◽  
Short Time

Holder pasteurization (HoP; 62.5 °C, 30 min) is commonly used to ensure the microbiological safety of donor human milk (DHM) but diminishes its nutritional properties. A high-temperature short-time (HTST) system was designed as an alternative for human milk banks. The objective of this study was to evaluate the effect of this HTST system on different nutrients and the bile salt stimulated lipase (BSSL) activity of DHM. DHM was processed in the HTST system and by standard HoP. Macronutrients were measured with a mid-infrared analyzer. Lactose, glucose, myo-inositol, vitamins and lipids were assayed using chromatographic techniques. BSSL activity was determined using a kit. The duration of HTST treatment had a greater influence on the nutrient composition of DHM than did the tested temperature. The lactose concentration and the percentage of phospholipids and PUFAs were higher in HTST-treated than in raw DHM, while the fat concentration and the percentage of monoacylglycerides and SFAs were lower. Other nutrients did not change after HTST processing. The retained BSSL activity was higher after short HTST treatment than that following HoP. Overall, HTST treatment resulted in better preservation of the nutritional quality of DHM than HoP because relevant thermosensitive components (phospholipids, PUFAs, and BSSL) were less affected.

Sign in / Sign up

Export Citation Format

Share Document