Journal of Chromatography B (v.998-999, #C)
Editorial Board (i).
Liquid chromatography–mass spectrometry analysis of five bisphosphonates in equine urine and plasma by April S.Y. Wong; Emmie N.M. Ho; Terence S.M. Wan; Kenneth K.H. Lam; Brian D. Stewart (1-7).
Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography – tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5 mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples.
Keywords: Amino bisphosphonates; Non-amino bisphosphonates; Horse; Plasma; Urine; Liquid chromatography–mass spectrometry; Tiludronic acid; Doping control analysis;
Simultaneous determination of carboprost methylate and its active metabolite carboprost in dog plasma by liquid chromatography–tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application to a pharmacokinetic study by Lei Yin; Xiangjun Meng; Xiaotong Zhou; Tinglan Zhang; Heping Sun; Zhichao Yang; Bo Yang; Ning Xiao; J. Paul Fawcett; Yan Yang; Jingkai Gu (8-14).
A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method using positive/negative electrospray ionization (ESI) switching for the simultaneous quantitation of carboprost methylate and carboprost in dog plasma has been developed and validated. After screening, the esterase inhibitor, dichlorvos was added to the whole blood at a ratio of 1:99 (v/v) to stabilize carboprost methylate during blood collection, sample storage and LLE. Indomethacin was added to plasma to inhibit prostaglandins synthesis after sampling. After liquid–liquid extraction of 500 μL plasma with ethyl ether-dichloromethane (75:25, v/v), analytes and internal standard (IS), alprostadil-d4, were chromatographed on a CAPCELL PAK Phenyl column (150 × 2.0 mm, 5 μm) using acetonitrile-5 mM ammonium acetate as mobile phase. Carboprost methylate was detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 400.5 → 329.3; the carboprost and IS were detected by negative ion electrospray ionization followed by MRM of the transitions at m/z 367.2 → 323.2, and 357.1 → 321.2, respectively. The method was linear for both analytes in the concentration range 0.05–30 ng/mL with intra- and inter-day precisions (as relative standard deviation) of ≤6.75% and accuracy (as relative error) of ≤7.21% and limit of detection (LOD) values were 10 and 20 pg/mL, respectively. The method was successfully applied to a pharmacokinetic study of the analytes in beagle dogs after intravaginal administration of a suppository containing 0.5 mg carboprost methylate.
Keywords: LC–MS/MS; Prodrug; Carboprost methylate; Carboprost; Esterase inhibitors; Pharmacokinetics; Dog;
Organic solvent-free air-assisted liquid–liquid microextraction for optimized extraction of illegal azo-based dyes and their main metabolite from spices, cosmetics and human bio-fluid samples in one step by Behruz Barfi; Alireza Asghari; Maryam Rajabi; Sedigheh Sabzalian (15-25).
Air-assisted liquid–liquid microextraction (AALLME) has unique capabilities to develop as an organic solvent-free and one-step microextraction method, applying ionic–liquids as extraction solvent and avoiding centrifugation step. Herein, a novel and simple eco-friendly method, termed one-step air-assisted liquid–liquid microextraction (OS-AALLME), was developed to extract some illegal azo-based dyes (including Sudan I to IV, and Orange G) from food and cosmetic products. A series of experiments were investigated to achieve the most favorable conditions (including extraction solvent: 77 μL of 1-Hexyl-3-methylimidazolium hexafluorophosphate; sample pH 6.3, without salt addition; and extraction cycles: 25 during 100 s of sonication) using a central composite design strategy. Under these conditions, limits of detection, linear dynamic ranges, enrichment factors and consumptive indices were in the range of 3.9–84.8 ng mL−1, 0.013–3.1 μg mL−1, 33–39, and 0.13–0.15, respectively. The results showed that –as well as its simplicity, fastness, and use of no hazardous disperser and extraction solvents- OS-AALLME is an enough sensitive and efficient method for the extraction of these dyes from complex matrices. After optimization and validation, OS-AALLME was applied to estimate the concentration of 1-amino-2-naphthol in human bio-fluids as a main reductive metabolite of selected dyes. Levels of 1-amino-2-naphthol in plasma and urinary excretion suggested that this compound may be used as a new potential biomarker of these dyes in human body.
Keywords: Sudan dyes; Biomarker; Air-assisted; One-step; Food; Lipstick; Biological fluid;
Determination of galangin in rat plasma by UPLC and pharmacokinetic study by Wei-hua Feng; Hang-hang Zhang; Yun Zhang; Ming Sun; Jun-long Niu (26-30).
In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography (UPLC) has been developed for the determination of galangin in rat plasma using diazepam as internal standard (IS). After sample preparation by a simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and ultraviolet detection set at a wavelength of 360 nm. The method was linear over the concentration range 10–1000 ng/mL with a lower limit of quantification (LLOQ) of 10 ng/mL. Inter- and intra-day precision (RSD %) were all within 9.5% and the accuracy (RE %) was equal or lower than 8.9%. Recoveries of galangin and IS were more than 78.3%. Stability studies showed that galangin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administration of galangin to rats.
Keywords: Galangin; Rat plasma; Pharmacokinetic; UPLC;
Validation of a UPLC–MS/MS method for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human plasma by Yuji Mano; Takuho Ishii; Koichiro Hotta; Kazutomi Kusano (31-39).
E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. As ER-392710 (M11), a hydrolyzed metabolite, is a main metabolite, a simultaneous assay method for quantification of E6005 and M11 in human plasma has been developed and validated using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). E6005, M11, and each deuterium-labeled compound used as internal standard were extracted from 100 μL human plasma by solid phase extraction then chromatographed on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) under gradient elution. The analytes were detected by selected reaction monitoring in the positive ion mode with the mass transition of m/z 473.1/163.0 and m/z 459.1/149.0 for E6005 and M11, respectively. E6005 and M11 were quantifiable ranging from 1 to 200 ng/mL with no carryover. Accuracy and precision in intra- and inter-batch reproducibility assays were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. Various stability assessments including possible conversion of E6005 to M11 were thoroughly performed to demonstrate the stability of E6005 and M11 in human blood and plasma. The method was successfully applied to support clinical trials.
Keywords: Atopic dermatitis; E6005; UPLC–MS/MS; Validation;
Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine by Daniela Remane; Soeren Grunwald; Henrike Hoeke; Andrea Mueller; Stefan Roeder; Martin von Bergen; Dirk K. Wissenbach (40-44).
During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6 mm × 100 mm).No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45 mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63 mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from −120 mg/L to +110 mg/L with a mean of differences of 29.0 mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples.
Keywords: HPLC-DAD; Validation; Creatinine; Uric acid; Hippuric acid; Urine;
Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography – tandem mass spectrometry by Hans F.N. Kvitvang; Per Bruheim (45-49).
Capillary ion chromatography (capIC) is the premium separation technology for low molecular phosphometabolites and nucleotides in biological extracts. Removal of excessive amounts of salt during sample preparation stages is a prerequisite to enable high quality capIC separation in combination with reproducible and sensitive MS detection. Existing sampling protocols for mammalian cells used for GC–MS and LC–MS metabolic profiling can therefore not be directly applied to capIC separations. Here, the development of a fast filtration sampling protocol for mammalian suspension cells tailored for quantitative profiling of the phosphometabolome on capIC–MS/MS is presented. The whole procedure from sampling the culture to transfer of filter to quenching and extraction solution takes less than 10 s. To prevent leakage it is critical that a low vacuum pressure is applied, and satisfactorily reproducibility was only obtained by usage of a vacuum pressure controlling device. A vacuum of 60 mbar was optimal for filtration of multiple myeloma Jjn-3 cell cultures through 5 μm polyvinylidene (PVDF) filters. A quick deionized water (DI-water) rinse step prior to extraction was tested, and significantly higher metabolite yields were obtained during capIC–MS/MS analyses in this extract compared to extracts prepared by saline and reduced saline (25%) washing steps only. In addition, chromatographic performance was dramatically improved. Thus, it was verified that a quick DI-water rinse is tolerated by the cells and can be included as the final stage during filtration. Over 30 metabolites were quantitated in JJN-3 cell extracts by using the optimized sampling protocol with subsequent capIC–MS/MS analysis, and up to 2 million cells can be used in a single filtration step for the chosen filter and vacuum pressure. The technical set-up is also highly advantageous for microbial metabolome filtration protocols after optimization of vacuum pressure and washing solutions, and the reduced salt content of the extract will also improve the quality of LC–MS analysis due to lower salt adduct ion formation.
Keywords: Capillary ion chromatography; Tandem mass spectrometry; Sampling protocol; Fast filtration; Mammalian suspension cells;
Preparation and characterization of an immunoaffinity column for the selective extraction of aflatoxin B1 in 13 kinds of foodstuffs by Jie Xie; Tao Peng; Jian-Li He; Yu Shao; Chun-Lin Fan; Ying Chen; Wen-Xiao Jiang; Min Chen; Qi Wang; Xing-Yao Pei; Shuang-Yang Ding; Hai-Yang Jiang (50-56).
A rapid and reliable immunoaffinity column (IAC) clean-up based ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed for the determination of aflatoxin B1 (AFB1) in cereals, peanuts, vegetable oils and Chinese traditional food products like sufu and lobster sauce. The immunoaffinity column of AFB1 (AFB1-IAC) was prepared by coupling CNBr-activated Sepharose-4B with the anti-AFB1 monoclonal antibody. The column capacity of IAC was over 260 ng/mL gel. Samples were extracted with methanol–water (60:40, v/v) and the extracts were then purified on an AFB1-IAC before UPLC–MS/MS analysis. The average recoveries of AFB1 in spiked samples at levels of 1.0, 5.0 and 10.0 μg/kg ranged from 72% to 98%, with the relative standard deviations of 1.2–9.3% (n = 6). The limits of qualification ranged from 0.07 to 0.23 μg/kg, which were below the MRLs of AFB1 in the matrices evaluated. In this work, the developed method was suitable for the determination of trace AFB1 residues in 13 kinds of foodstuffs.
Keywords: Immunoaffinity column; Aflatoxin B1; Foodsutffs; UPLC–MS/MS;
Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry by A.E. Kip; H. Rosing; M.J.X. Hillebrand; M.M. Castro; M.A. Gomez; J.H.M. Schellens; J.H. Beijnen; T.P.C. Dorlo (57-62).
Phagocytes, the physiological compartment in which Leishmania parasites reside, are the main site of action of the drug miltefosine, but the intracellular pharmacokinetics of miltefosine remain unexplored. We developed a bioanalytical method to quantify miltefosine in human peripheral blood mononuclear cells (PBMCs), expanding from an existing high performance liquid chromatography-tandem mass spectrometry method for the quantification of miltefosine in plasma. The method introduced deuterated miltefosine as an internal standard. Miltefosine was extracted from PBMC pellets by addition of 62.5% methanol. Supernatant was collected, evaporated and reconstituted in plasma. Chromatographic separation was performed on a reversed phase C18 column and detection with a triple-quadrupole mass spectrometer. Miltefosine was quantified using plasma calibration standards ranging from 4 to 1000 ng/mL. This method was validated with respect to its PBMC matrix effect, selectivity, recovery and stability. No matrix effect could be observed from the PBMC content (ranging from 0.17 to 26.3 × 106 PBMCs) reconstituted in plasma, as quality control samples were within 3.0% of the nominal concentration (precision less than 7.7%). At the lower limit of quantitation of 4 ng/mL plasma, corresponding to 0.12 ng/106 PBMCs in a typical clinical sample, measured concentrations were within 8.6% of the nominal value. Recovery showed to be reproducible as adding additional pre-treatment steps did not increase the recovery with more than 9%. This method was successfully applied to measure intracellular miltefosine concentrations in PBMC samples from six cutaneous leishmaniasis patients up to one month post-treatment.
Reassessment of the antioxidative mixture for the challenging electrochemical determination of dopamine, noradrenaline and serotonin in microdialysis samples by Jolien Van Schoors; Charlotte Lens; Katrien Maes; Yvette Michotte; Ilse Smolders; Ann Van Eeckhaut (63-71).
In recent years, the simultaneous monitoring of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in vivo is advancing due to innovations in miniaturized and fast chromatographic techniques. However, the determination of the most hydrophilic compound, noradrenaline, in microdialysis samples by (ultra-)high performance liquid chromatography ((U)HPLC) with electrochemical detection (ECD) is impeded by a broad solvent front, caused by the addition of antioxidative agents. Hence, an elaborate reassessment of currently used antioxidative mixtures is necessary for further analytical improvements. The proposed mixture, containing 100 mM acetic acid, 0.27 mM Na2EDTA and 12.5 μM ascorbic acid (pH 3.2), is less complex than previously described mixtures and shows minimal ECD interference. It stabilizes the three monoamines in standard solutions and in microdialysis samples, considering both autosampler stability at 4 °C for 48 h and long term stability at −20 °C for a duration of six months. An in vivo microdialysis experiment demonstrates the possibility to monitor changes in extracellular levels of the three monoamines simultaneously in the rat hippocampus with UHPLC–ECD using the optimized antioxidative mixture.
Keywords: In vivo microdialysis; Monoamine neurotransmitters; Stability; UHPLC; Electrochemical detection;
A novel method based on MSPD for simultaneous determination of 16 pesticide residues in tea by LC–MS/MS by Yalin Cao; Hua Tang; Dazhou Chen; Lei Li (72-79).
A novel method based on matrix solid phase dispersion (MSPD) coupled with liquid chromatography-tandem mass spectrometry was established for the determination and the quantification of 16 pesticides (5 carbamates, 4 organophosphorus, and 7 pyrethroids) in various tea. Matrix dispersive sorbent and further cleanup sorbent were applied to improve the efficiency of extraction and purification. PVPP, PSA and GCB were introduced as further cleanup sorbents packed at the bottom of the MSPD to remove co-eluting matrix components. Different experiment conditions, such as type of eluting solvent, its volume, matrix dispersive sorbent, sample to matrix dispersive sorbent mass ratio, and the dosage of cleanup sorbents were thoroughly studied and optimized. It was found that polyvinylpolypyrrolidone (PVPP), an inexpensive and excellent absorbent, could effectively remove polyphenols in tea, which was seldom reported before. The method showed satisfactory linearity over the range assayed 0.9986–0.9999 (1–500 ng g−1 for 5 carbamates and 4 organophosphorus, 2–800 ng g−1 for 7 pyrethroids), the limits of detections (LODs) ranged from 0.01 to 1.38 ng g−1, and the limits of quantifications (LOQs) were ranging from 0.03 to 4.74 ng g−1. The recoveries using this method at three spiked concentration levels (10, 100, and 500 ng g−1) range from 87.7 to 99.6%. The relative standard deviation (RSD) was from 0.2 to 9.6% in all case. The proposed analytical method has been successfully applied for the analysis of 16 pesticides in commercial tea.
Keywords: Tea; Pesticides; MSPD; PVPP; LC–MS/MS;
Development and validation of a sensitive LC–MS/MS assay for the quantification of nizatidine in human plasma and urine and its application to pharmacokinetic study by De-Wei Shang; Zhan-Zhang Wang; Xiao-Jia Ni; Ming Zhang; Jin-Qing Hu; Chang Qiu; Yu-Guan Wen (80-87).
We developed and validated a high performance liquid chromatographic method coupled with triple quadrupole mass spectrometry for analysis of nizatidine in human plasma and urine. The biological samples were precipitated with methanol before separation on an Agilent Eclipse Plus C18 column (100 mm × 46 mm, 5 μm) with a mixture of methanol and water (95:5, plus 5 mM ammonium formate) as the mobile phase at 0.5 mL/min. Detection was performed using multiple reaction monitoring modes via electrospray ionization (ESI) at m/z 332.1 → 155.1 (for nizatidine) and m/z 335.1 → 155.1 (for [2H3]-nizatidine, the internal standard). The linear response range was 5–2000 ng/mL and 0.5–80 μg/mL for human plasma and urine, with the lower limits of quantification of 5 ng/mL and 0.5 μg/mL, respectively. The method was validated according to the biological method validation guidelines of the Food and Drug Administration and proved acceptable. This newly developed analytical method was successfully applied in a pharmacokinetic study following single oral administration of a 150 mg nizatidine capsule in to 16 healthy Chinese subjects. Maximum and endpoint concentrations in plasma and urine were quantifiable, suggesting our method is appropriate for routine pharmacokinetic analysis.
Keywords: Nizatidine; LC–MS/MS; Plasma; Urine; Pharmacokinetics;
Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization by Hiroo Yoshida; Kazuhiro Kondo; Hiroyuki Yamamoto; Naoko Kageyama; Shin-ichi Ozawa; Kazutaka Shimbo; Takahiko Muramatsu; Akira Imaizumi; Toshimi Mizukoshi; Junichi Masuda; Daisuke Nakayama; Yoshihiro Hayakawa; Kyoko Watanabe; Kazuo Mukaibatake; Hiroshi Miyano (88-96).
The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8 min, and the interval time between injections was 12 min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.
Keywords: Plasma free amino acid analysis; Automated precolumn derivatization; LC–MS; UF-Amino Station; Validation;
Negative electrospray ionization mode in mass spectrometry: A new perspective via modeling by Christos I. Gioumouxouzis; Maria G. Kouskoura; Catherine K. Markopoulou (97-105).
Electrospray ionization technique is used for production of gas phase ions without fragmentation and is considered as one of the most sensitive analytical methods for structural characterization of molecules. Nonetheless, the determination of some parameters (physicochemical properties or structural features) that may enhance the signal response especially in the negative ion mode has not yet been clarified. The present work is an attempt to correlate the signal response behavior of 110 compounds used as probes, with their characteristics (molecular descriptors, X variables). In order to quantify this phenomenon, Partial Least Squares which is a software capable of performing linear multivariate analysis was applied. The models derived explore the positive or negative effect of 49 X variables on the signal response of each analyte, expressed as Y variable. The process of gas phase ions formation was verified by both flow injection and column analysis. The models derived are proven reliable for the study of such mechanisms, with small number of components and good linearity (R 2 > 83%, Q 2 > 70%). The present study showed that parameters as pK a, ionization percentage of the analyte, PSA, HBA, ―COOH, water solubility and surface tension of a solid are affecting ion formation. At the same time, slight differentiations of the influence of certain parameters were observed on column injection analysis due to the chromatographic delay of some analytes.
Keywords: Negative ionization mode; Molecular descriptors; Modeling; Partial Least Squares;
Accurate measurement of the essential micronutrients methionine, homocysteine, vitamins B6, B12, B9 and their metabolites in plasma, brain and maternal milk of mice using LC/MS ion trap analysis by J. Efraim Oosterink; Eva F.G. Naninck; Aniko Korosi; Paul J. Lucassen; Johannes B. van Goudoever; Henk Schierbeek (106-113).
Methionine, homocysteine, vitamins B6, B12, B9, and their metabolites are crucial co-factors and substrates for many basic biological pathways including one-carbon metabolism, and they are particularly important for brain function and development and epigenetic mechanisms. These are essential nutrients that cannot be synthesized endogenously and thus need to be taken in via diet. A novel method was developed that enables simultaneous assessment of the exact concentrations of these essential micronutrients in various matrices, including maternal milk, plasma, and brain of neonatal mice. The protocol for analysis of these components in the various matrices consists of a cleanup step (i.e. lipid extraction followed by protein precipitation) combined with a liquid chromatography mass spectrometry (LC/MS) ion trap method with high sensitivity and selectivity (SRM mode). This novel method enables the measurement of these essential nutrients with good recoveries (69–117%), and high intra-day (<10%) and high intra-day precision (defined as <15% for compounds with an isotopologue and <20% for compounds without an isotopologue as internal standard) in plasma, maternal milk, and brain of mice at low and high levels. In addition, lower limits of quantitation (LOQ) were determined for the various matrices in the range for methionine (700–2000 nmol/L), homocysteine (280–460–nmol/L), vitamins B6 (5–230 nmol/L), B12 (7–11 nmol/L), B9 (20–30 nmol/L). Degradation of vitamins and oxidation of homocysteine is limited to a minimum, and only small sample volumes (30 μL plasma, 20 mg brain and maternal milk) are needed for simultaneous measurement. This method can help to understand how these nutrients are transferred from mother to offspring via maternal milk, as well as how these nutrients are absorbed by the offspring and eventually taken up in various tissues amongst the brain in preclinical and clinical research settings. Therefore the method can help to explore critical periods in lactating mothers and developing offspring.
Keywords: B vitamins; Methylation; Stable isotopes; Ion trap mass spectrometry;