Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.     

LC-MS/MS analysis of dextromethorphan metabolism in human saliva and urine to determine CYP2D6 phenotype and individual variability in N-demethylation and glucuronidation

In order to establish a fast screening method for the determination of the CYP2D6 metabolic phenotype a sensitive LC-MS/MS assay to quantify dextromethorphan (DEX) and its O-demethylated metabolite dextrorphan (DOR) in human saliva was developed with limits of quantitation of 1 pmol/ml. Saliva was provided by 170 medical students 2h after oral ingestion of 30 mg (81 micromol) dextromethorphan hydrobromide. Individual ratios of the concentrations DEX/DOR (metabolic ratio, MR(DEX/DOR)) varied more than 25,000-fold (0.03-780). Two groups comprising 156 'Extensive' and 14 'Poor Metabolizers' were clearly distinguished. For the investigation of individual differences in N-demethylation and glucuronidation, four additional metabolites of DEX, 3-methoxymorphinan (MOM), 3-hydroxymorphinan (HOM), and the two O-glucuronides (DORGlu and HOMGlu) were measured by LC-MS/MS analysis of 6-h urine of 24 volunteers. The N-demethylation reactions DEX-to-MOM and DOR-to-HOM defined by the respective MR were significantly correlated. The same holds for the glucuronidation pathways (MR(DOR/DORGlu) versus MR(HOM/HOMGlu)). The three poor CYP2D6 metabolizers excreted relatively high amounts of the parent compound DEX (up to 7 micromol), but only low amounts of glucuronides (DORGlu: 0.4-1.0 micromol; HOMGlu: 0.2-0.7 micromol). For the 21 'Extensive Metabolizers', the two glucuronides were the most abundant, with relatively little interindividual variation (DORGlu: 10-44 micromol; HOMGlu: 5-17 micromol). For the excretion of the glucuronides, two normal distributions provided the best fit, indicating that the determination of the glucuronides alone could allow assignment of the CYP2D6 metabolic phenotype.     

A sensitive LC-MS/MS method for determination of levamisole in human plasma: application to pharmacokinetic study

A sensitive and rapid LC-MS/MS method was developed and validated for the determination of levamisole in human plasma. The assay was based on liquid-liquid extraction of analytes from human plasma with ethyl ether. Chromatographic separation was carried on an Agilent HC-C(8) column (150 mm × 4.6 mm, 5 μm) at 40°C, with a mobile phase consisting of acetonitrile-10 mM ammonium acetate (70:30, v/v), a flow rate of 0.5 mL/min and a total run time of 6 min. Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive electrospray ionization m/z at 205.1→178.2 for levamisole, and m/z 296.1→264.1 for mebendazole (internal standard). The assay was linear over a concentration range of 0.1-30 ng/mL with a lower limit of quantification of 0.1 ng/mL. The coefficient of variation of the assay precision was less than 8.5%. The assay was successfully used to analyze human plasma samples in a pharmacokinetic study where levamisole was administered as a liniment.     

LC-MS/MS法检测人头发中利培酮及其代谢物9-羟利培酮含量的实验研究

目的:建立高效液相色谱-三重四级杆质谱联用(LC-MS/MS)法检测人头发中利培酮(RIP)及其代谢物9-羟利培酮(9-OH-RIP)含量的方法。方法:采用同位素内标氘4-利培酮(RIP-d4)及氘4-9-羟利培酮(9-OH-RIP-d4),流动相A为10 mmol/L醋酸胺溶液(甲酸调p H值为4.0),流动相B为乙腈,A/B=70/30,流速为0.3 m L/min,等度洗脱4.00 min。色谱柱为安捷伦Zorbax SB C18(2.1×50 mm,1.8μm),柱温30℃。准确称取20 mg丙酮清洗过晾干剪碎成粉末的头发样本,加1N氢氧化钠(Na OH)超声2 h,等量酸中和后,加200μL1N氢氧化钠溶液及5.0 m L的甲基叔丁基醚(MTBE)提取涡旋1分钟,3000×g离心5 min后取上清液在40度水浴下氮气吹干后用100μL流动相复溶,进样2μL经LC-MS/MS检测。MRM监测离子对:RIP:m/z 411.2→191.0,9-OH-RIP:m/z 427.2→207.1,RIP-d4:m/z 415.2→195.2,9-OH-RIP-d4:m/z 431.2→211.1。结果:利培酮及9-羟利培酮线性范围分别为0.5-25 ng/mg,0.0025-0.15 ng/mg,提取回收率均70.0%,方法回收率均在85.0%-115.0%之间,线性r均0.999,精密度和重现性RSD均15%。结论:本研究建立了采用LC-MS/MS法检测人头发中利培酮及9-羟利培酮含量的方法,该法快速、简单、准确、重现性好。     

Metabolite profiling of fungi and yeast: from phenotype to metabolome by MS and informatics

Filamentous fungi and yeast from the genera Saccharomyces, Penicillium, Aspergillus, and Fusarium are well known for their impact on our life as pathogens, involved in food spoilage by degradation or toxin contamination, and also for their wide use in biotechnology for the production of beverages, chemicals, pharmaceuticals, and enzymes. The genomes of these eukaryotic micro-organisms range from about 6000 genes in yeasts (S. cerevisiae) to more than 10,000 genes in filamentous fungi (Aspergillus sp.). Yeast and filamentous fungi are expected to share much of their primary metabolism; therefore much understanding of the central metabolism and regulation in less-studied filamentous fungi can be learned from comparative metabolite profiling and metabolomics of yeast and filamentous fungi. Filamentous fungi also have a very active and diverse secondary metabolism in which many of the additional genes present in fungi, compared with yeast, are likely to be involved. Although the 'blueprint' of a given organism is represented by the genome, its behaviour is expressed as its phenotype, i.e. growth characteristics, cell differentiation, response to the environment, the production of secondary metabolites and enzymes. Therefore the profile of (secondary) metabolites--fungal chemodiversity--is important for functional genomics and in the search for new compounds that may serve as biotechnology products. Fungal chemodiversity is, however, equally efficient for identification and classification of fungi, and hence a powerful tool in fungal taxonomy. In this paper, the use of metabolite profiling is discussed for the identification and classification of yeasts and filamentous fungi, functional analysis or discovery by integration of high performance analytical methodology, efficient data handling techniques and core concepts of species, and intelligent screening. One very efficient approach is direct infusion Mass Spectrometry (diMS) integrated with automated data handling, but a full metabolic picture requires the combination of several different analytical techniques.     

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.     

Simultaneous analysis of angiotensin peptides by LC-MS and LC-MS/MS: metabolism by bovine adrenal endothelial cells

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to simultaneously determine the concentrations of angiotensin (Ang) II, Ang 1-7, Ang III, and Ang IV in biological samples. The samples were extracted with C18 solid-phase extraction cartridges and separated by a reverse-phase C18 column using acetonitrile in water with 0.1% formic acid as a mobile phase. Ang peptides were ionized by electrospray and detected by triple quadrupole MS in the positive ion mode. (M+3H)(3+) and (M+2H)(2+) ions were chosen as the detected ions in the single ion recording (SIR) mode for LC-MS. The limits of detection (signal/noise [S/N]=3) using SIR are 1 pg for Ang IV and 5 pg for Ang 1-7, Ang III, and Ang II. Multiple reaction monitoring (MRM) mode was used for LC-MS/MS. The limits of detection (S/N =3) using MRM are 20 pg for Ang IV and 25 pg for Ang 1-7, Ang III, and Ang II. These methods were applied to analyze Ang peptides in bovine adrenal microvascular endothelial cells. The results show that Ang II is metabolized by endothelial cells to Ang 1-7, Ang III, and Ang IV, with Ang 1-7 being the major metabolite.     

Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.     

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC-MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC-MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid-derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC-MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A(4) and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.     

Determination of colistin in human plasma, urine and other biological samples using LC-MS/MS

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to quantify colistin in human plasma and urine, and perfusate and urine from the isolated perfused rat kidney (IPK). Solid phase extraction (SPE) preceded chromatography on a Synergi Fusion-RP column with a mobile phase of acetonitrile, water and acetic acid (80/19/1) at 0.2mL/min. Ions were generated using electrospray ionization and detected in the positive-ion mode. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 0.028microg/mL (human plasma, IPK perfusate and urine)/0.056microg/mL (human urine) to 1.78microg/mL (all four media) for colistin A sulfate; corresponding values for colistin B sulfate were 0.016/0.032 to 1.01microg/mL. Accuracy and precision were within 10%. The LLOQ for colistin A sulfate was 0.028microg/mL in human plasma, IPK perfusate and urine and 0.056microg/mL in human urine; corresponding values for colistin B sulfate were 0.016 and 0.032microg/mL. The low sample volume, short analysis time and low LLOQ are ideal for pre-clinical and human pharmacokinetic studies of colistin.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

Development and validation of Lenalidomide in human plasma by LC-MS/MS

A highly sensitive and ultra-fast high performance liquid chromatography- tandem mass spectrometry (LC–MS/MS) assay is developed and validated for the quantification of Lenalidomide in human plasma. Lenalidomide is extracted from human plasma by Liquid- Liquid Extraction by Ethyl Acetate and analyzed using a reversed phase isocratic elution on a XTerra RP18, (4.6 × 50 mM, 5 µm) column. A 0.1% Formic acid: Methanol (10:90% v/v), is used as mobile phase and detection was performed by Triple quadrupole mass spectrometry LC-MS/MS using electrospray ionization in positive mode. Fluconazole is used as the internal standard. The lower limit of quantification is 9.999 ng/mL for Lenalidomide. The calibration curves are consistently accurate and precise over the concentration range of 9.999 to 1010.011 ng/mL in plasma for Lenalidomide. This novel LC–MS/MS method competes with all the regulatory requirements and shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic and bioequivalence studies in humans.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

Electrospray ionization LC-MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify griseofulvin in human plasma using propranolol hydrochloride as internal standard (IS). Samples were prepared using solid phase extraction and analysed without drying and reconstitution. The analytes were chromatographed on Hypersil, hypurity C18 reverse phase column under isocratic conditions using 0.05% formic acid in water:acetonitrile (30:70, v/v) as the mobile phase. Total chromatographic run time was 3.0 min. Quantitation was done on a triple quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring (MRM) mode to detect parent-->product ion transition for analyte and IS. The method was validated for sensitivity, matrix effect, accuracy and precision, linearity, recovery and stability studies. Linearity in plasma was observed over the concentration range 20-3000 ng/mL for griseofulvin. Lower limit of quantification (LLOQ) achieved was 20 ng/mL with precision (CV) less than 10% using 5 microL injection volume. The absolute recovery of analyte (87.36%) and IS (98.91%) from spiked plasma samples was consistent and reproducible. Inter-batch and intra-batch coefficients of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 7.5%. The accuracy determined at these levels was within +/-4.2% in terms of relative error. The method was applied to a pilot bioequivalence study of 500 mg griseofulvin tablet in six healthy human subjects under fed condition.     

Characterization of the human tear metabolome by LC-MS/MS

The tear film overlying the epithelial cells of the eye's surface is vital to visual function, and its composition is reflective of ocular surface health. The ultrasmall volume of tears poses challenges in its analysis, contributing to the limited number of reports on the tear metabolome. In addition, using a standard clinical method of tear collection posed some confounding factors in metabonomic analysis. We sought to establish an analytical platform for the global characterization of human tear metabolites. Following information dependent acquisition (IDA) directed liquid chromatography-tandem mass spectrometry (LC-MS/MS), isotope pattern matched peak mining was performed using Extracted Ion Chromatogram (XIC) manager within the PeakView software. Sixty metabolites representing diverse compound classes were identified in human tears, most of which have not been previously reported. Selected metabolites were verified using pure standards. Unsupervised chemometric analysis showed good separation between tear samples and blanks (PC1 = 87%, R(2) = 0.91, Q(2) = 0.87). The results demonstrated the potential of our platform for untargeted metabonomic studies of eye diseases.     

An evaporation-free solid-phase extraction method for rapid and accurate analysis of sumatriptan in human plasma by LC-MS/MS

An evaporation-free solid-phase extraction (SPE) method was developed and validated for sumatriptan. High organic washing (50% methanol) and low organic elution (20% methanol) were used and the recovery was greater than 92%. The eluate was injected into a C18 column without evaporation and reconstitution. Sumatriptan was monitored in positive ion mode with mass transition of m/z 296.4-58.1 amu. The calibration curve was 1-100 ng/mL (r>or=0.9923). The inter-day and intra-day precisions ranged from 4.53 to 9.12% and 1.72 to 6.93%, respectively. This method features reduced cost and pollution, clean extract, high speed, and most importantly overall method reliability.     

Development and validation of a selective and sensitive LC-MS/MS method for determination of cycloserine in human plasma: application to bioequivalence study

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20-30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.