Multiple inert gas elimination technique by micropore membrane inlet mass spectrometry--a comparison with reference gas chromatography.

The mismatching of alveolar ventilation and perfusion (VA/Q) is the major determinant of impaired gas exchange. The gold standard for measuring VA/Q distributions is based on measurements of the elimination and retention of infused inert gases. Conventional multiple inert gas elimination technique (MIGET) uses gas chromatography (GC) to measure the inert gas partial pressures, which requires tonometry of blood samples with a gas that can then be injected into the chromatograph. The method is laborious and requires meticulous care. A new technique based on micropore membrane inlet mass spectrometry (MMIMS) facilitates the handling of blood and gas samples and provides nearly real-time analysis. In this study we compared MIGET by GC and MMIMS in 10 piglets: 1) 3 with healthy lungs; 2) 4 with oleic acid injury; and 3) 3 with isolated left lower lobe ventilation. The different protocols ensured a large range of normal and abnormal VA/Q distributions. Eight inert gases (SF6, krypton, ethane, cyclopropane, desflurane, enflurane, diethyl ether, and acetone) were infused; six of these gases were measured with MMIMS, and six were measured with GC. We found close agreement of retention and excretion of the gases and the constructed VA/Q distributions between GC and MMIMS, and predicted PaO2 from both methods compared well with measured PaO2. VA/Q by GC produced more widely dispersed modes than MMIMS, explained in part by differences in the algorithms used to calculate VA/Q distributions. In conclusion, MMIMS enables faster measurement of VA/Q, is less demanding than GC, and produces comparable results.

Enantioselective determination of ondansetron and 8-hydroxyondansetron in human plasma from recovered surgery patients by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization for quantification of ondansetron and its main metabolite 8-hydroxyondansetron in human plasma was presented. The enantiomeric separation was achieved on a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate). The validation data were within the required limits. The assay was successfully applied to authentic plasma samples. Quantitative results from postoperative patients receiving ondansetron demonstrated a great interindividual variability in postoperative plasma drug concentrations, the metabolites were not detected in their unconjugated form. A wide variation in the S-(+)-/R-(-)-ondansetron concentration ratio between 0.14 and 7.18 is indicative for a stereoselective disposition or metabolism. In further studies CYP2D6 and CYP3A4 genotype dependent metabolism of ondansetron enantiomers as well as of co-administered drugs and clinical efficacy of the medication should be tested.

Analysis of llicit and illicit drugs in waste, surface and lake water samples using large volume direct injection high performance liquid chromatography – Electrospray tandem mass spectrometry (HPLC–MS/MS)

http://www.sciencedirect.com/science/article/pii/S0045653510008891

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples

A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

On the interference of Kr during carbon isotope analysis of methane using continuous-flow combustion–isotope ratio mass spectrometry

Stable carbon isotope analysis of methane (delta C-13 of CH4) on atmospheric samples is one key method to constrain the current and past atmospheric CH4 budget. A frequently applied measurement technique is gas chromatography (GC) isotope ratio mass spectrometry (IRMS) coupled to a combustion-preconcentration unit. This report shows that the atmospheric trace gas krypton (Kr) can severely interfere during the mass spectrometric measurement, leading to significant biases in delta C-13 of CH4, if krypton is not sufficiently separated during the analysis. According to our experiments, the krypton interference is likely composed of two individual effects, with the lateral tailing of the doubly charged Kr-86 peak affecting the neighbouring m/z 44 and partially the m/z 45 Faraday cups. Additionally, a broad signal affecting m/z 45 and especially m/z 46 is assumed to result from scattered ions of singly charged krypton. The introduced bias in the measured isotope ratios is dependent on the chromatographic separation, the krypton-to-CH4 mixing ratio in the sample, the focusing of the mass spectrometer as well as the detector configuration and can amount to up to several per mil in delta C-13. Apart from technical solutions to avoid this interference, we present correction routines to a posteriori remove the bias.

Development of the gas chromatograph – mass spectrometer to investigate volatile species in the lunar soil for the Luna-Resurs mission

Since the analysis of the lunar rocks and soil samples, brought to Earth by the Apollo missions, it is believed that the Moon has a waterless nature and also other volatile species are strongly depleted. Advancement in analysis techniques helped to identify water and other volatile species in lunar volcanic glasses. Additionally, recent lunar space missions detected water and volatile organic compounds in the region of the lunar poles where permanently shadowed craters are existing. All known lunar soil samples available on Earth come from the lunar near side, close to the equator. To verify the most recent measurement results and to enhance the knowledge of the geological history of the Moon it is of high interest to perform in situ measurements on the lunar poles. For this reason the Russian space agency, Roskosmos, developed aprogram for the scientific exploration of the lunar poles. The Gas Analysis Package (GAP) is part of the selected scientific payload aboard the Luna-Resurs Lander. This instrument uses pyrolytic cells and will apply laser spectroscopy, gas chromatography and mass spectrometry to detect and analyze volatile components trapped in the lunar soil. An existing ion optical design of a compact reflectron type time-of-flight mass spectrometer, originally built for the MEAP/P-BACE balloon mission, was chosen as a part of the GAP instrument. The scope of this thesis is the development of the interface between gas chromatography (GC) and this Neutral Gas Mass Spectrometer (NGMS) to perform coupled GC-MS measurements. In the first part of this thesis the interfacing concept was developed and verified by coupling the NGMS prototype to gas chromatography. The second part of this thesis is devoted to the development of the NGMS flight version.

Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.