Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R 2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Dried Blood Spot Testing for Seven Steroids Using Liquid Chromatography-Tandem Mass Spectrometry With Reference Interval Determination in the Korean Population

BACKGROUND: Conventional screening for congenital adrenal hyperplasia (CAH) using immunoassays generates a large number of false-positive results. A more specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been introduced to minimize unnecessary follow-ups. However, because of limited data on its use in the Korean population, LC-MS/MS has not yet been incorporated into newborn screening programs in this region. The present study aims to develop and validate an LC-MS/MS method for the simultaneous determination of seven steroids in dried blood spots (DBS) for CAH screening, and to define age-specific reference intervals in the Korean population. METHODS: We developed and validated an LC-MS/MS method to determine the reference intervals of cortisol, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, corticosterone, and 11-deoxycorticosterone simultaneously in 453 DBS samples. The samples were from Korean subjects stratified by age group (78 full-term neonates, 76 premature neonates, 89 children, and 100 adults). RESULTS: The accuracy, precision, matrix effects, and extraction recovery were satisfactory for all the steroids at three concentrations; values of intra- and inter-day precision coefficients of variance, bias, and recovery were 0.7-7.7%, -1.5-9.8%, and 49.3-97.5%, respectively. The linearity range was 1-100 ng/mL for cortisol and 0.5-50 ng/mL for other steroids (R2>0.99). The reference intervals were in agreement with the previous reports. CONCLUSIONS: This LC-MS/MS method and the reference intervals validated in the Korean population can be successfully applied to analyze seven steroids in DBS for the diagnosis of CAH.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Measurement of Leukocyte Arylsulfatase A Activity Using a Natural Substrate

No abstract available.

Quantification of Human Plasma-Busulfan Concentration by Liquid Chromatography-Tandem Mass Spectrometry

BACKGROUND: Busulfan, an alkylating agent administered prior to hematopoietic stem cell transplantation, has a narrow therapeutic range and wide variability in metabolism. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for rapid and accurate quantification of plasma busulfan. METHODS: Busulfan was separated and detected using an LC system containing a C18 column equipped with MS/MS. The sample was eluted with a mobile phase gradient for a total run time of 10 min. Plasma busulfan concentration was quantified against a 6-point standard curve in a multiple reaction monitoring mode at mass-to-charge (m/z) 264.1 > 151.1. Precision, recovery, matrix effect, linearity, detection capability, carryover, and stability were evaluated. The range of plasma busulfan concentration was obtained by analyzing samples from 9 children receiving busulfan. RESULTS: The coefficients of variation of within-run and within-laboratory precision were all below 5%. Recoveries were all within the range of 100-105%. Linearity was verified from 0 to 5,000 ng/mL. Limit of detection and limit of quantification were 1.56 and 25 ng/mL, respectively. Carryover rate was within allowable limits. Plasma busulfan concentration was stable for 2 weeks at -20degrees C and -80degrees C, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4degrees C. The plasma busulfan concentrations were between 347 ng/mL and 5,076 ng/mL. CONCLUSIONS: Our method using LC-MS/MS enables highly accurate, reproducible, and rapid busulfan monitoring with minimal sample preparation. The method may also enable safe and proper dosage.

Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4degrees C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 micromol/h/L (mean 10.6) with a SD of 5.06 micromol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 micromol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 micromol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.

Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4degrees C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 micromol/h/L (mean 10.6) with a SD of 5.06 micromol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 micromol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 micromol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.