High-performance liquid chromatographic–tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS–MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC–MS–MS. Linearity was given in the tested range of 50–5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50–5000 ng/ml was 1.47–6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37°C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.     

Optimized determination of trace jet fuel volatile organic compounds in human blood using in-field liquid–liquid extraction with subsequent laboratory gas chromatographic–mass spectrometric analysis and on-column large-volume injection

A practical and sensitive method to assess volatile organic compounds (VOCs) from JP-8 jet fuel in human whole blood was developed by modifying previously established liquid–liquid extraction procedures, optimizing extraction times, solvent volume, specific sample processing techniques, and a new on-column large-volume injection method for GC–MS analysis. With the optimized methods, the extraction efficiency was improved by 4.3 to 20.1 times and the detection sensitivity increased up to 660 times over the standard method. Typical detection limits in the parts-per-trillion (ppt) level range were achieved for all monitored JP-8 constituents; this is sufficient for assessing human fuels exposures at trace environmental levels as well as occupational exposure levels. The sample extractions are performed in the field and only solvent extracts need to be shipped to the laboratory. The method is implemented with standard biological laboratory equipment and a modest bench-top GC–MS system.     

Solid-phase microextraction gas chromatographic–mass spectrometric method for the determination of inhalation anesthetics in urine

Solid-phase microextraction (SPME) has been applied to the headspace sampling of inhalation anesthetics (i.e. nitrous oxide, isoflurane and halothane) in human urine. Analysis was carried out by gas chromatography–mass spectrometry using a capillary column with a divinylbenzene porous polymeric stationary phase. A SPME divinylbenzene–Carboxen–polydimethylsiloxane coated fiber, 2 cm long, was used, and its performances were compared with those of a Carboxen–PDMS in terms of sensitivity, extraction efficiency, extraction time, fiber coating–urine distribution coefficient. For both fibers, linearity was established over four orders of magnitude, limits of detection were below 100 ng/l for nitrous oxide and below 30 ng/l for halogenated. Precision calculated as %RSD was within 3–13% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of anesthetics in the urine of occupationally exposed people (operating room personnel).     

Direct plasma injection for high-performance liquid chromatographic–mass spectrometric quantitation of the anxiolytic agent CP-93 393

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC–ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC–ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10–1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 μl) were performed per column with unattended, automated analysis.     

Gas chromatographic–mass spectrometric method for quantitative determination of ketotifen in human plasma after enzyme hydrolysis of conjugated ketotifen

A validated method for determination of total amount of ketotifen (unchanged and conjugated) in human plasma has been presented. An enzyme hydrolysis of conjugated ketotifen was conducted with combination of β-glucuronidase and arylsulfatase. After the enzyme hydrolysis a solid-phase extraction was applied as a cleaning step. The quantitative determination by gas chromatography with mass-spectrometry detection (GC–MS) was performed. Pizotifen has been used as an internal standard. A reliable hydrolysis as well as a satisfactory accuracy, improved precision in the linear region from 0.500 to 10.0 ng/ml plasma, limit of detection of 0.010 ng/ml and prolonged capillary column life have been achieved.     

Oxidation of glycerophospholipids from biological membranes by reactive oxygen species: liquid chromatographic–mass spectrometric analysis of eicosanoid products

Peroxidation of glycerophospholipids present in cellular membranes results in the formation of a complex mixture, with many products derived from the oxidation of esterified arachidonic acid. Techniques of chromatography and mass spectrometry have facilitated the elucidation of the structure of individual components present as intact glycerophospholipids as well as the oxidized fatty acyl groups liberated from the glycerol backbone by saponification. Previously reported studies are summarized in this overview concerning those oxidized products of arachidonic acid derived from the red blood cell membrane, studied by techniques of electrospray tandem mass spectrometry developed to analyze eicosanoid products.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes: Gas chromatographic–mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17α-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17α-methyltestosterone, produced 6β-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography–mass spectrometry (GC–MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6β-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6β-hydroxyl metabolites with testosterone and 17α-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6β-hydroxylation.     

Gas chromatographic–mass spectrometric analysis of perillyl alcohol and metabolites in plasma

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1′-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC–MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC–MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH₂Cl₂ extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC–MS using NH₃ chemical ionization. POH-d₃ was used as the internal standard for POH while ¹³C-PA-d₂ was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1–2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m² p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.     

Liquid chromatographic–mass spectrometric determination of the novel,recently identified thioTEPA metabolite,thioTEPA-mercapturate,in urine

An assay for the quantitative determination of the mercapturic acid conjugate of N,N′,N″-triethylenethiophosphoramide (thioTEPA-mercapturate) in human urine has been developed. ThioTEPA-mercapturate, a recently identified metabolite of the alkylating anticancer agent thioTEPA, was analyzed using LC–MS and with direct sample injection. Sulphadiazine was used as internal standard. Linearity was accomplished in the therapeutic relevant range of 1–25 μg/ml; recovery was 84% and both accuracy and precision were less than 20% for the lower limit of quantification (1.0 μg/ml) and less than 10% for the other concentration levels. The stability of thioTEPA-mercapturate proved to be satisfactory over a period of 2 months, when kept at −80°C. ThioTEPA-mercapturate urine concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.     

Gas chromatographic–mass spectrometric screening for organic acidemias using dried urine filter paper: determination of α-ketoacids

There are several organic acid disorders that require information on α-ketoacids, such as maple syrup urine disease or α-ketoadipic acidemia. The recovery, stability and diagnostic availability of α-ketoacids in dried urine filter paper analyzed by GC–MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of α-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain α-ketoacids, α-ketoadipate and α-ketoglutarate was stable for at least 28 days, although some α-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.     

Gas chromatographic–mass spectrometric analysis of erythrocyte 3-deoxyglucosone in hemodialysis patients

The erythrocyte levels of 3-deoxyglucosone (3-DG) were measured by a selected ion monitoring method of gas chromatography–chemical ionization mass spectrometry using [¹³C₆]-3-DG as an internal standard. Because the erythrocyte levels of 3-DG measured after deproteinization using ethanol were 18 times higher than those using ultrafiltration, we used ethanol deproteinization for measurement of total 3-DG in the erythrocytes. The concentration of 3-DG was significantly elevated in hemodialysis (HD) patients compared with healthy subjects. Although HD treatment could remove the erythrocyte 3-DG efficiently, its post-HD levels were still elevated compared with the healthy subjects.     

High-performance liquid chromatographic–electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening

Within the pharmaceutical industry, significant resources have been applied to the identification of new drug compound leads through the use of high-throughput screening (HTS). To meet the demand for rapid analytical characterization of biologically active samples identified by HTS, the technique of high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) has been utilized, and the application of this technique specifically for the integration of natural product sample mixtures into modern HTS is reviewed. The high resolution provided by reversed-phase HPLC coupled with the gentle and relatively universal ionization facilitated by the electrospray process has had significant impact upon a variety of procedures associated with the HTS of natural products, including extract sample diversity evaluation, dereplication, structure elucidation, preparative isolation, and affinity-based biological activity evaluation.     

Gas chromatographic–tandem mass spectrometric determination of acetylsalicylic acid in human plasma after oral administration of low-dose aspirin and guaimesal

A fully validated gas chromatographic–tandem mass spectrometric (GC–MS–MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[²H₃]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-μl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC–MS–MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0–1 μg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0–100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.     

Improvement in precision of the liquid chromatographic–electrospray ionization tandem mass spectrometric analysis of 3′-C-ethynylcytidine in rat plasma

During the development of the liquid chromatography with electrospray ionization–tandem mass spectrometry for the quantitative determination of 3′-C-ethynylcytidine (I) in rat plasma, ion suppression caused by the matrix components was observed for I and its structural analogue, 3′-C-ethylcytidine (II) as the internal standard. In the method initially designed, I/II peak area ratios varied according to the degree of matrix effect, which led to the poor precision of the assay. From the examination of the ion suppression behavior for I and II, it was assumed that this phenomenon is attributed to the difference in the retention time between I and II. Based on this assumption, therefore, the methanol content in the mobile phase was changed from 5 to 25% so as to make I and II the same retention time. As a result of this modification of the initial method, the precision expressed as relative standard deviation was improved from 5.2–16.2 to 2.7–4.2% in intra-assay and from 6.8–14.9 to 3.5–7.2% in inter-assay validations.     

Gas chromatographic–mass spectrometric analysis of urinary sugar and sugar alcohols during pregnancy

A refined and simplified method has been developed for the simultaneous analysis of urinary sugar and sugar alcohols after urease treatment by using capillary gas chromatography–mass spectrometry (GC–MS). Since carbohydrate metabolism during pregnancy is considered to be diabetogenic, our interest has been concentrated on understanding the mechanism of the metabolic deviation by assessing the glucose excursion and glucose fluxes. The present study suggests that changes of the levels of glucose, sorbitol, fructose, myo-inositol, and 1,5-anhydro-D-glucitol (1,5-AG) may reflect a mild alteration in carbohydrate metabolism that goes undetected by conventional diabetic indicators.     

Gas chromatographic–mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples

A gas chromatographic–mass spectrometric (GC–MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N,S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337±29%/day in rat liver.     

Gas chromatographic–mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance

A method for analysis of veterinary tranquillizers in urine using gas chromatography–mass spectrometry (GC–MS) is described. Detection limits are 5 μg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 μg/l for haloperidol, 20 μg/l for xylazine and 50 μg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC–MS “positive” results is evaluated and discussed.     

Gas chromatographic—mass spectrometric detection of circulating plasticizers in surgical patients

Gas chromatographic and gas chromatographic—mass specrometric analytical techniques were employed to quantitate and confirm levels of circulating organic plasticizers in critically ill surgical patients. Two plasticizers, dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP), have been identified. DEHP can be found in many plastic medical devices. The DEHP levels were significant soon after transfusion or in the presence of renal dysfunction. The source of DBP is not clear at present and requires further study. The prevention of this contamination and the toxicity of these plasticizers should be investigated to ensure the safe use of plastic medical devices.     

Bioanalysis of β2-agonists by hyphenated chromatographic and mass spectrometric techniques

Recent reports on the misuse of β₂-agonists, both as stimulants and as “anabolic agents” in sports, highlight the importance of screening and confirmation methods for these compounds in anti-doping control procedures. Although only a few analytical methods have been developed for this purpose, the large experience gained, both in pharmacokinetic studies and above all in the control of the residues of β₂-agonists in animal fluids and tissues, can be of great help in the anti-doping field. This paper reviews single-residue (SR) and multi-residue (MR) methods developed for the analysis of β₂-agonists in urine, plasma and hair samples, based on hyphenated chromatographic and mass spectrometric techniques, published in the last ten-year period. The evolution from GC-MS analysis after derivatization, with particular attention to the features of different proposed derivatives, to the most recent applications of coupled-column liquid chromatography (LC-LC) combined with tandem mass spectrometric detection (MS-MS) via a thermospray (TSP) interface is illustrated, and future perspectives in the field are outlined.     

Simultaneous supercritical fluid extraction and chemical derivatization for the gas chromatographic–isotope dilution mass spectrometric determination of amphetamine and methamphetamine in urine

An in-situ supercritical fluid extraction (SFE) and chemical derivatization (ChD) procedure followed by gas chromatography–isotope dilution mass spectrometry (GC–MS) for the determination of amphetamines in urine is described and evaluated. While using celite as the SFE wet-support, the one-pot sample pretreatment procedure also employs ammonium water to alkalize the urine matrix that contains protonated amphetamine (AP) and methamphetamine (MA). The mean recoveries achieved by simultaneous SFE–ChD, i.e., 95% (RSD=3.8%) for AP and 89% (RSD=4.0%) for MA, are significantly better than the corresponding overall recoveries obtained upon stepwise SFE–ChD, suggesting the unreacted trifluoroacetic anhydride (TFA) in the former procedure has strengthened the extracting power of CO₂ fluid as has been evidenced by a control test. As to GC–MS analysis, the optimal qualitative ions and quantitative ions of the respective analytes were determined via a rigorous evaluation process. Thus, the regression calibration curves for AP and MA in urine are linear within 100∼50 000 ng/ml, with correlation coefficients typically exceeding 0.999. The limits of detection determined by two methods for AP and MA vary from 19 to 50 ng/ml, and limits of quantitation from 21 to 100 ng/ml. Precisions calculated for the triplicate analyses of AP and MA in a 500-ng/ml spiked control, two real-case samples and two quasi real-case samples, respectively, using regression calibration are typically below 10%. The method is simple and reliable. It may serve as an alternative to the existing confirmatory protocol for forensic urine drug testing.