Rapid evaluation of 25 key sphingolipids and phosphosphingolipids in human plasma by LC-MS/MS.
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ABSTRACT:
SUBMITTER: Basit A
PROVIDER: S-EPMC4471391 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundOxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.MethodsA method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.ResultsOur method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.ConclusionThe method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.
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Project description:To support a phase III randomized trial of the multi-targeted tyrosine kinase inhibitor cabozantinib in neuroendocrine tumors, we developed a high-performance liquid chromatography mass spectrometry method to quantitate cabozantinib in 50 μL of human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in water over a 5-min run time. Detection was performed on a Quattromicro quadrupole mass spectrometer with electrospray, positive-mode ionization. The assay was linear over the concentration range 50-5000 ng/mL and proved to be accurate (103.4-105.4%) and precise (<5.0%CV). Hemolysis (10% RBC) and use of heparin as anticoagulant did not impact quantitation. Recovery from plasma varied between 103.0-107.7% and matrix effect was -47.5 to -41.3%. Plasma freeze-thaw stability (97.7-104.9%), stability for 3 months at -80°C (103.4-111.4%), and stability for 4 h at room temperature (100.1-104.9%) were all acceptable. Incurred sample reanalysis of (N = 64) passed: 100% samples within 20% difference, -0.7% median difference and 1.1% median absolute difference. External validation showed a bias of less than 1.1%. This assay will help further define the clinical pharmacokinetics of cabozantinib.
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Project description:Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1?×?100 ?mm, 3.5?µm) or on a polyimide-coated, fused-silica capillary self-packed with 17?cm AquaC18 (3?µm, 125?Å). Quantitation was done using the SRM transitions m/z 233???174 and m/z 219???160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165? pg/mL, LC: 1165-116,500 ?pg/mL) and for NAS (nanoflow LC: 11.0-1095 ?pg/mL).
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Project description:Lenvatinib (LENVA) is an oral antineoplastic drug used for the treatment of hepatocellular carcinoma and thyroid carcinoma. LENVA therapeutic drug monitoring (TDM) should be mandatory for a precision medicine to optimize the drug dosage. To this end, the development of a sensitive and robust quantification method to be applied in the clinical setting is essential. The aim of this work was to develop and validate a sensitive, rapid, and cost-effective LC-MS/MS method for the quantification of LENVA in human plasma. On this premise, sample preparation was based on a protein precipitation and the chromatographic separation was achieved on a Synergi Fusion RP C18 column in 4 min. The method was completely and successfully validated according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, with good linearity in the range of 0.50-2000 ng/mL (R≥0.9968). Coefficient of variation (CV) for intra- and inter-day precision was ≤11.3% and accuracy ranged from 96.3 to 109.0%, internal standard normalized matrix effect CV% was ≤2.8% and recovery was ≥95.6%. Successful results were obtained for sensitivity (signal to noise (S/N) ratio >21) and selectivity, dilution integrity (CV% ≤ 4.0% and accuracy 99.9-102%), and analyte stability under various handling and storage conditions both in matrix and solvents. This method was applied to quantify LENVA in patient's plasma samples and covered the concentration range achievable in patients. In conclusion, a sensitive and robust quantification method was developed and validated to be applied in the clinical setting.
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Project description:Benzoylpaeoniflorin (BP) is a potential therapeutic agent against oxidative stress related Alzheimer's disease. In this study, a more rapid, selective, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to determine BP in rat plasma distinguishing with a monoterpene isomer, benzoylalbiflorin (BA). The method showed a linear response from 1 to 1000?ng/mL (r > 0.9950). The precision of the interday and intraday ranged from 2.03 to 12.48% and the accuracy values ranged from -8.00 to 10.33%. Each running of the method could be finished in 4 minutes. The LC-MS/MS method was validated for specificity, linearity, precision, accuracy, recovery, and stability and was found to be acceptable for bioanalytical application. Finally, this fully validated method was successfully applied to a pharmacokinetic study in rats following oral administration.
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