Measurement of carnitine precursors, epsilon-trimethyllysine and gamma-butyrobetaine in human serum by tandem mass spectrometry.

Methods using tandem mass spectrometry for measurement of epsilon-trimethyllysine and gamma-butyrobetaine in human serum are described. Precursor ion scan analysis of a methylated sample was applied for gamma-butyrobetaine measurement. However, for epsilon-trimethyllysine measurement, homoarginine interfered with the methylated sample during precursor ion scan analysis. To overcome this interference, the sample was propylated and acetylated prior to precursor ion scan analysis. The obtained values resembled those obtained by enzymatic or HPLC measurement. Using tandem mass spectrometry, all members of the carnitine family, free carnitine, acylcarnitines, gamma-butyrobetaine, epsilon-trimethyllysine can be analyzed in 0.1 ml of serum. Thus, the proposed method appears to be suitable for clinical application, especially in the pediatric field.     

Determination of carnitine and acylcarnitines in biological samples by capillary electrophoresis-mass spectrometry

Free carnitine and acylcarnitines (carnitine esters) play an important role in the metabolism of fatty acids. Metabolic disorders can be detected by abnormal levels of these compounds in biological fluids. Capillary electrophoresis-mass spectrometry has the advantage of combining an efficient separation technique with highly selective detection. Therefore, we have developed a method for the determination of carnitine and several of its esters implementing electrospray capillary electrophoresis-mass spectrometry in the positive ion selected reaction monitoring mode. A sheath-flow interface with a mixture of 2-propanol or methanol, water and acetic acid as sheath liquid and nitrogen as nebulizing gas was used. The zwitterionic analytes migrated as cations in the applied electric field using ammonium acetate-acetic acid or formic acid electrolytes. Separations were performed in aqueous, mixed organic-aqueous and non-aqueous media. The influence of the electrolyte composition on the separation efficiency was investigated. The electrospray conditions have been optimized regarding ion current stability and sensitivity. Ammonium acetate (10 mmol/l)-0.8% formic acid in water or 6.4% formic acid in acetonitrile-water (1:1) were used as running buffers for the determination of carnitine and acylcarnitines in human biological samples. Methanol extracts of dried blood spots were analyzed as well as urine and plasma following sample preparation via solid-phase or liquid-liquid extraction. Recoveries approaching 100% were achieved depending on the analytes and sample preparation procedures employed. Endogenous carnitine and acetylcarnitine were determined at concentrations between 2.7 and 108 nmol/ml in normal human urine and plasma. Other acylcarnitines were detected at levels of below the limit of detection to 12 nmol/ml. Good precision (0.8 to 14%) and accuracy (85 to 111%) were obtained; the achieved limits of quantitation (0.1 to 1 nmol/ml) are sufficient to characterize carnitine and acylcarnitine levels occurring as markers for metabolic disorders.     

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry

BackgroundResidual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH₄), the cofactor of PAH.MethodsA robust LC–ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice.ResultsThe PAH assay was linear for phenylalanine and tyrosine (r² ≥ 0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were < 5.3% and < 6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 μmol/L BH₄) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain.ConclusionsThis novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions.     

Quantification of free mycophenolic acid by high-performance liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 microl) using ultrafiltration. The analytes were extracted from the ultrafiltrate (200 microl) using C18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 microg/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 microg/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was <8 and <9%, respectively. Further, analysis of pooled patient plasma produced an intra-day imprecision of 6.6%. The signal-to-noise ratio at the limit of quantification (2.5 microg/l) was approximately 5:1. The mean absolute recovery (n=6) of mycophenolic acid and the internal standard were 76.0+/-13.5% and 86.0+/-9.1%, respectively. The method reported provides an accurate and precise quantification of free mycophenolic acid over a wide analytical range and thus can be used for routine monitoring and pharmacokinetic studies.     

Evaluation of urinary acylglycines by electrospray tandem mass spectrometry in mitochondrial energy metabolism defects and organic acidurias

We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.     

Measurement method for hydroxylated polychlorinated biphenyls in the blood of Yusho patients by liquid chromatography-electrospray tandem mass spectrometry

Hydroxylated polychlorinated biphenyls (OH-PCBs) are formed as major metabolites of PCBs by cytochrome P450 enzyme-mediated oxidation. It has been reported that their total concentration in serum samples of Yusho patients ranged from 390 to 1300 pg/g. We developed a measurement method for OH-PCBs in blood samples by LC/MS/MS. This method is effective at determining the concentrations of PCDDs, PCDFs, Co-PCBs and OH-PCBs from the same sample without special treatment of the sample. The concentration of OH-PCBs in the blood of Yusho patients was examined using this method. The major OH-PCB metabolites were 4-OH-CB187 (54-906 pg/g-wet), 4-OH-CB146 + 3-OH-CB153 (32-527 pg/g-wet), 4-OH-CB109 (ND-229 pg/g-wet) and 4'-OH-CB172 (ND-143 pg/g-wet). The total OH-PCBs ranged from 95 to 1740 pg/g-wet.     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry.

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC-MS-MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1-22 microg/kg. The limits of detection were below 6 microg/kg and the limits of quantification for most benzimidazoles were below 10 microg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Measurement of partial pressure of gases in liquids by mass spectrometry.

The experimental setup of mass spectrometric determination of gas contents in liquids has been modified for continuous and discontinuous measurement of partial pressure of gases in liquids. The inlet system consists of a stainless steel capillary with slits covered by a silicone rubber membrane. Several gases can be measured simultaneously under static conditions and in flowing liquids. The measurement and calibration procedure is described. Results of the analysis of the test criteria, reproducibility, detection limit, response time, and depletion are presented. The difficulties in discontinuous measurement of oxygen in blood are explained by the complex permeation-diffusion process at the membrane and the form of the dissociation curve. With regard to solubility, physically dissolved gases can be determined without problems down to tensions of about 0.01 mm Hg. Continuous measurement of oxygen and carbon dioxide partial pressure in liquids, including blood, is possible with the described system.     

Postanalytical tools improve performance of newborn screening by tandem mass spectrometry

PurposeThe purpose of this study was to compare performance metrics of postanalytical interpretive tools of the Region 4 Stork collaborative project to the actual outcome based on cutoff values for amino acids and acylcarnitines selected by the California newborn screening program.MethodsThis study was a retrospective review of the outcome of 176,186 subjects born in California between 1 January and 30 June 2012. Raw data were uploaded to the Region 4 Stork Web portal as.csv files to calculate tool scores for 48 conditions simultaneously using a previously unpublished functionality, the tool runner. Scores for individual target conditions were deemed informative when equal or greater to the value representing the first percentile rank of known true-positive cases (17,099 cases in total).ResultsIn the study period, the actual false-positive rate and positive predictive value were 0.26 and 10%, respectively. Utilization of the Region 4 Stork tools, simple interpretation rules, and second-tier tests could have achieved a false-positive rate as low as 0.02% and a positive predictive value >50% by replacing the cutoff system with Region 4 Stork tools as the primary method for postanalytical interpretation.ConclusionRegion 4 Stork interpretive tools, second-tier tests, and other evidence-based interpretation rules could have reduced false-positive cases by up to 90% in California.Genet Med16 12, 889–895.     

Simultaneous quantification of isoniazid,rifampicin, ethambutol and pyrazinamide by liquid chromatography/tandem mass spectrometry

A remediable cause of poor treatment response in drug‐susceptible tuberculosis (TB) patients may be low plasma levels of one or more of the first‐line anti‐TB drugs. The aim of this work was to develop an accurate and precise LC‐MS/MS method for simultaneous quantification of all four first‐line anti‐TB drugs in plasma suitable for therapeutic drug monitoring (TDM). To adjust for degradation and losses during sample preparation, isotopically labeled compounds were used as internal standards. Plasma samples spiked with internal standards were extracted using protein precipitation with methanol and acetonitrile. Simultaneous separation of all four drugs was accomplished with a Chromolith Reversed‐Phase column and mobile phases consisting of water, methanol, ammonium acetate and formic acid with subsequent mass spectrometric quantification. The linear range of the calibration curve for isoniazid was 0.5–10 mg/L, for rifampicin 0.75–30 mg/L, for ethambutol 0.25–10 mg/L and for pyrazinamide 4–80 mg/L. The lower limit of quantification was 0.5 mg/L, 0.75 mg/L, 0.25 mg/L and 4.0 mg/L, respectively. Precision estimated by the coefficient of variation was <15% for all four drugs. The LC‐MS/MS method can readily be used for simultaneous quantification of first‐line anti‐TB drugs in plasma and is well suited for TDM.     

Measurement of iron bioavailability by means of stable 54Fe and mass spectrometry.

Stable isotopes represent a useful tool for the assessment of biokinetic parameters in in vivo studies on humans. A procedure is described to evaluate the bioavailability of iron in pharmaceutical preparations by means of 54Fe as a tracer and mass spectrometry for the determination of time dependent changes in the isotope ratio of 54Fe/56Fe in red blood cells. Iron tablets with an increased portion of 54Fe were administered to iron deficient subjects and red cell iron utilization was used as a measure of iron bioavailability. Iron utilization was derived from changes in the 54Fe/56Fe ratio as evaluated by means of fast atom bombardment-mass spectrometry (FAB-MS) on processed blood samples. A good intraindividual reproducibility was observed for blood samples drawn at various times after application of the trial drug. Figures for bioavailability and its interindividual variations were in the range expected from comparable studies on similar iron preparations using radioiron as tracers. The results obtained show that quantitative data of bioavailability from pharmaceutical iron preparations may be obtained without radiation exposure of the subjects investigated.     

Determination of gestrinone in human serum by liquid chromatography--electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography-electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5-177 ng/ml (r2 > or =0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3-13.7 to -4.8-3.0%. The inter-day precision and accuracy ranged from 5.5-14.8 to -6.7-3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Effect of fish skin mucus on the soluble proteome of Vibrio salmonicida analysed by 2-D gel electrophoresis and tandem mass spectrometry

Vibrio salmonicida is the causative agent of cold-water vibriosis in farmed marine fish species. Adherence of pathogenic bacteria to mucosal surfaces is considered to be the first steps in the infective processes, and proteins involved are regarded as virulence factors. The global protein expression profile of V. salmonicida, grown with and without the presence of fish skin mucus in the synthetic media, was compared. Increased levels of proteins involved in motility, oxidative stress responses, and general stress responses were demonstrated as an effect of growth in the presence of mucus compared to non-mucus containing media. Enhanced levels of the flagellar proteins FlaC, FlaD and FlaE indicate increased motility capacity, while enhanced levels of the heat shock protein DnaK and the chaperonin GroEL indicate a general stress response. In addition, we observed that peroxidases, TPx.Grx and AhpC, involved in the oxidative stress responses, were induced by mucus proteins. The addition of mucus to the culture medium did not significantly alter the growth rate of V. salmonicida. An analysis of mucus proteins suggests that the mucus layer harbours a protein species that potentially possesses catalytic activity against DNA, and a protein with iron chelating activity. This study represents the first V. salmonicida proteomic analysis, and provides specific insight into the proteins necessary for the bacteria to challenge the skin mucus barrier of the fish.     

A simple,robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry,microarray and PCR

Carbapenemases should be accurately and rapidly detected, given their possible epidemiological spread and their impact on treatment options. Here, we developed a simple, easy and rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based assay to detect carbapenemases and compared this innovative test with four other diagnostic approaches on 47 clinical isolates. Tandem mass spectrometry (MS-MS) was also used to determine accurately the amount of antibiotic present in the supernatant after 1 h of incubation and both MALDI-TOF and MS-MS approaches exhibited a 100% sensitivity and a 100% specificity. By comparison, molecular genetic techniques (Check-MDR Carba PCR and Check-MDR CT103 microarray) showed a 90.5% sensitivity and a 100% specificity, as two strains of Aeromonas were not detected because their chromosomal carbapenemase is not targeted by probes used in both kits. Altogether, this innovative MALDI-TOF-based approach that uses a stable 10-μg disk of ertapenem was highly efficient in detecting carbapenemase, with a sensitivity higher than that of PCR and microarray.     

Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry

The aim of the project was to develop a fast and reliable method for the quantification of the three tetracyclines: tetracycline, oxytetracycline and chlortetracycline in urine. The method is based on column-switching high-performance liquid chromatography with detection by MS-MS. Buffer is added to the sample before it is injected into the chromatographic system, and the first column which is an internal surface reversed-phase column separates the tetracyclines from the bulk of other compounds in urine. The tetracyclines are collected and concentrated on the analytical column before they are separated and eluted into the mass spectrometer in which the tetracycline are detected. The mass spectrometer is a triple quadrupole instrument and is equipped with an electrospray ion source. The MH+ ions are selected in the first quadrupole and collisionally activated in the collision cell. Upon collision, activation all three tetracyclines form fragment ions which could be assigned as: [M+H-H2O-NH3]+ which are selected in the sond mass filter. The detection limits for all three tetracyclines are about 10 ppb, and the calibration curves are linear from 10 to 1000 ppb.     

Detection and identification of morphine in urine extracts using thin-layer chromatography and tandem mass spectrometry.

The application of tandem mass spectrometry to the analysis and identification of morphine following thin-layer chromatography is described. FAB-mass spectrometry and mass spectrometry-mass spectrometry were performed following chromatography on silica gel high-performance thin-layer chromatography plates. The successful application of this simple methodology to a urine extract suggests that this approach has practical utility for confirming the identity of abused drugs detected by thin-layer chromatography.     

串联质谱技术在脂肪酸氧化代谢病诊断中的应用研究

目的探讨利用串联质谱技术检测干血滤纸片中酰基肉碱水平,诊断脂肪酸氧化代谢病。方法对象为2941例临床遗传性代谢病高危儿童,利用串联质谱技术检测患儿干血滤纸片中酰基肉碱水平,结合临床资料和常规生化结果,进行脂肪酸氧化代谢病诊断。结果诊断了14例脂肪酸氧化代谢病(0.5%),其中肉碱棕榈酰转移酶Ⅰ缺乏症1例,肉碱棕榈酰转移酶Ⅱ缺乏症1例,短链酰基辅酶A脱氢酶缺乏症1例,中链酰基辅酶A脱氢酶缺乏症7例,极长链酰基辅酶A脱氢酶缺乏症2例,多种酰基辅酶A脱氢酶缺乏症2例。结论通过串联质谱技术检测干血滤纸片中酰基肉碱水平,可对部分脂肪酸氧化代谢病进行诊断。