Radiation metabolomics. 3. Biomarker discovery in the urine of gamma-irradiated rats using a simplified metabolomics protocol of gas chromatography-mass spectrometry combined with random forests machine learning algorithm

Abstract Radiation metabolomics employing mass spectral technologies represents a plausible means of high-throughput minimally invasive radiation biodosimetry. A simplified metabolomics protocol is described that employs ubiquitous gas chromatography-mass spectrometry and open source software including random forests machine learning algorithm to uncover latent biomarkers of 3 Gy gamma radiation in rats. Urine was collected from six male Wistar rats and six sham-irradiated controls for 7 days, 4 prior to irradiation and 3 after irradiation. Water and food consumption, urine volume, body weight, and sodium, potassium, calcium, chloride, phosphate and urea excretion showed major effects from exposure to gamma radiation. The metabolomics protocol uncovered several urinary metabolites that were significantly up-regulated (glyoxylate, threonate, thymine, uracil, p-cresol) and down-regulated (citrate, 2-oxoglutarate, adipate, pimelate, suberate, azelaate) as a result of radiation exposure. Thymine and uracil were shown to derive largely from thymidine and 2'-deoxyuridine, which are known radiation biomarkers in the mouse. The radiation metabolomic phenotype in rats appeared to derive from oxidative stress and effects on kidney function. Gas chromatography-mass spectrometry is a promising platform on which to develop the field of radiation metabolomics further and to assist in the design of instrumentation for use in detecting biological consequences of environmental radiation release.

Determination of menthol in plasma and urine of rats and humans by headspace solid phase microextraction and gas chromatography--mass spectrometry

A method for the determination of menthol and menthol glucuronide (M-G) after enzymatic hydrolysis in plasma and urine of rats and humans was developed using headspace solid phase microextraction and gas chromatography-mass spectrometry in the selected ion monitoring mode (HS-SPME/GC-MS). The assay linearity for plasma ranged from 5 to 1000 ng/ml. The limit of quantification (LOQ) in plasma was 5 ng/ml. The intra- and inter-day precision for menthol and M-G were < or = 18.1% R.S.D. at the LOQ and < or = 4.0% at higher concentrations. Menthol and M-G were determined in rat and human plasma and urine after administration of menthol.

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.

Analytic validation of a gas chromatography-mass spectrometry method for quantification of six amino acids in canine serum samples.

OBJECTIVE To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS 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.

Informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry (GCxGC-HRMS)

This paper describes informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution mass spectrometry (HRMS). GCxGC-HRMS analysis produces large data sets that are rich with information, but highly complex. The size of the data and volume of information requires automated processing for comprehensive cross-sample analysis, but the complexity poses a challenge for developing robust methods. The approach developed here analyzes GCxGC-HRMS data from multiple samples to extract a feature template that comprehensively captures the pattern of peaks detected in the retention-times plane. Then, for each sample chromatogram, the template is geometrically transformed to align with the detected peak pattern and generate a set of feature measurements for cross-sample analyses such as sample classification and biomarker discovery. The approach avoids the intractable problem of comprehensive peak matching by using a few reliable peaks for alignment and peak-based retention-plane windows to define comprehensive features that can be reliably matched for cross-sample analysis. The informatics are demonstrated with a set of 18 samples from breast-cancer tumors, each from different individuals, six each for Grades 1-3. The features allow classification that matches grading by a cancer pathologist with 78% success in leave-one-out cross-validation experiments. The HRMS signatures of the features of interest can be examined for determining elemental compositions and identifying compounds.

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.