Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

The assay of fentanyl and its metabolites in plasma of patients using gas chromatography with alkali flame ionisation detection and gas chromatography—mass spectrometry

Fentanyl was determined using gas chromatography (GC) and alkali flame ionisation detection (AFID), in the plasma of patients who had received a high single dose (up to 60 μg/kg body weight). The relative standard deviation is 6% for 11 ng/ml while the calculated detection limit is 3.3 ng of fentanyl per 1 ml of plasma. The concentration of fentanyl in patients ranged from 40 to 3 ng/ml of plasma in the first hour after administration. In the plasma of patients treated with fentanyl two metabolites could be detected and identified using GC—AFID and GC—MS.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Analysis of cyclosporine A and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of cyclosporine A (CyA) and the identification of its metabolites in rat urine and feces. The analytes were extracted from waste samples via liquid–liquid extraction. A Turboionspray source was used as a detector. It was operated in a positive ion mode with transitions of m/z 1225 → m/z 1112 for CyA and in a selected multiple reactions monitoring (MRM) mode with transitions of m/z 1239 → m/z 1099 for the internal standard (cyclosporine D, CyD). Linear calibration curves were obtained for CyA concentration ranges of 12.5–250 ng mL^?1 in urine and 2.5–375 ng mg^?1 in feces. The intra- and inter-day precision values (relative standard deviation) obtained were less than 8%, and the accuracy was within ±15% for each of the analytes. Extraction recoveries of CyA and CyD were both over 80%. The identification of the metabolites and elucidation of their structure were performed on the basis of their retention times and mass spectrometry fragmentation behaviors. A total of seven metabolites in rat feces were identified as dimethyl CyA, hydroxy CyA, and dihydroxy CyA after the oral administration of cyclosporine A-Eudragit^® S100 nanoparticles (CyA-NP). Six of these metabolites were also detected in rat urine. A possible metabolic pathway was also proposed. The newly developed method was proven to be sensitive, simple, reproducible, and suitable for the rapid determination of CyA. It was successfully employed to study the excretion of CyA in rats and could be used to better understand the in vivo metabolism of CyA-NP, a potentially effective nanoparticle system.     

Evaluation of plasma enzyme activities using gas chromatography–mass spectrometry based steroid signatures

The simultaneous quantification of 65 plasma steroids, including 22 androgens, 15 estrogens, 15 corticoids and 13 progestins, was developed using gas chromatography-mass spectrometry (GC–MS). The extraction efficiency of the catechol estrogens was improved by the addition of l-ascorbic acid in several steps. All steroids, as their trimethylsilyl derivatives, were well separated with good peak shapes within a 50 min run. The devised method provided good linearity (correlation coefficient, r² > 0.993), while the limit of quantification ranged from 0.2 to 2.0 ng mL^?1. The precision (% CV) and accuracy (% bias) were 2.0–12.4% and 93.5–109.2%, respectively. The metabolic changes were evaluated by applying this method to plasma samples obtained from 26 healthy male subjects grouped according to the pre- and post-administration of dutasteride, which inhibits 5α-reductase isoenzyme types 1 and 2. The levels of three plasma steroids, such as dihydrotestosterone, 5α-androstanedione and allotetrahydrocortisol, were decreased significantly after drug administration, while the levels of testosterone and 5β-androstane-3β,17α-diol were increased. In addition, the ratios of the steroid precursors and their metabolites, which represent the activities of the related enzymes, were z-score transformed for visualization in heat maps generated using supervised hierarchical clustering analysis. These results validated the data transformation because 5α-reductase is an indicator for the biological actions of dutasteride. GC–MS base quantitative visualization might be found in the integration with the mining biomarkers in drug evaluations and hormone-dependent diseases.     

Determination of alkylresorcinol metabolites in human urine by gas chromatography–mass spectrometry

Alkylresorcinols (ARs) are phenolic lipids present at high concentrations in the outer parts of rye and wheat kernels and have been proposed as biomarkers for intake of whole grain and bran products of these cereals. AR are absorbed in the small intestine and after hepatic metabolism two major metabolites, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), are excreted in urine either as such or as conjugates. Urine samples from nine individuals were incubated with different enzymes to assess type and extent of conjugates. In comparison with DHBA, which was mostly found in the free form, the less polar DHPPA was conjugated to a greater extent and the major conjugates were glucuronides. In this method, urine samples were hydrolyzed using β-glucuronidase from Helix pomatia and syringic acid was used as internal standard. Samples, silylated with BSTFA, were analyzed by GC–MS utilizing a BP-5 fused silica capillary column and single ion monitoring of molecular ions (m/z 370 [DHBA], m/z 398 [DHPPA]). Recoveries of DHBA and DHPPA were estimated to be 94% and 93%, respectively. The average intra-assay/inter-assay coefficients of variation were 4.9/5.7% for DHBA and 7.6/9.3% for DHPPA.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry

Urinary metabolic pattern after the therapeutic peroral dose of dihydrocodeine tartrate to six human volunteers has been explored. Using the GC–MS analytical method, we have found that the major part of the dose administered is eliminated via urine within the first 24 h. However, the analytical monitoring of dihydrocodeine and its metabolites in urine was still possible 72 h after the dose was administered. The dihydrocodeine equivalent amounts excreted in urine in 72 h ranged between 32 and 108% of the dose, on average 62% in all individuals. The major metabolite excreted into urine was a 6-conjugate of dihydrocodeine, then in a lesser amount a 6-conjugate of nordihydrocodeine (both conjugated to approximately 65%). The O-demethylated metabolite dihydromorphine was of a minor amount and was 3,6-conjugated in 85%. Traces of nordihydromorphine and hydrocodone were confirmed as other metabolites of dihydrocodeine in our study. This information can be useful in interpretation of toxicological findings in forensic practice.     

Estimation of BTEX in groundwater by using gas chromatography–mass spectrometry

Advanced analytical modern technology such as coupling a gas chromatography to a mass spectrometric technique provides sufficient information to the environmental and analytical chemists to identify the presence of a variety of components of the specific volatile organic product, determine the degree of the product weathering and in some instances estimate the age of the product as well in the testing sample. In this study, we estimated BTEX in groundwater sample by using gas chromatography–mass spectrometry (GC–MS) after standardization of this technique for advancement towards purification check of water samples in the petro-polluted regions of the soil.     

Identification of flavanone metabolites in rat urine by combined gas–liquid chromatography and mass spectrometry

1. The metabolism of flavanone in the rat was studied after oral or intraperitoneal administration of the compound. Flavone and flav-3-ene together with five other unidentified minor metabolites were excreted in the urine. 2. The formation of flavanone metabolites was not suppressed by the administration of high doses of the antibacterial compounds aureomycin and phthaloylsulphathiazole. 3. No aromatic acids that could be attributed to ring cleavage of flavanone were detected. 4. Administration of 100 or 200mg of flavanone daily per rat caused some deaths during the 7-14-day period. 5. The application of combined gas-liquid chromatography/mass spectrometry and proton nuclear-magnetic-resonance spectroscopy to the separation and identification of the flavanone metabolites is described. 6. Measurement of the two major flavanone metabolites was carried out by gas-liquid chromatography.     

Determination of bicyclol in dog plasma using liquid chromatography–tandem mass spectrometry

A sensitive and accurate method for determination of bicyclol in dog plasma was developed. Thermo Scientific TSQ Quantum triple quadrupole system with multiple ion monitoring (MIM) positive scanning mode was applied. Bicyclol and DDB (IS) sodium adduct molecular ions were monitored at m/z 413 and m/z 441 in both Q1 and Q3, respectively. The collision energy in Q2 was set to 15 eV. Precipitation method was employed in the extraction of bicyclol and DDB from the biological matrix. The method was validated over 1–500 ng/mL for bicyclol. The recovery was 96.5–109.5%, and the limit of quantitation (LOQ) detection was 1 ng/mL for bicyclol. The intra- and inter-day precision of the method at three concentrations was 3.3–14.3% with accuracy of 99.9–109.0%. The method was successfully applied to bioequivalence studies of bicyclol controlled-release formulation to obtain the pharmacokinetic parameters.     

Revised method for routine determination of urinary dialkyl phosphates using gas chromatography–mass spectrometry

Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk assessment. In our conventional method, we have employed a procedure including simple liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up (multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main modification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection were approximately 0.15 μg/L for dimethylphosphate (DMP), 0.07 μg/L for diethylphosphate (DEP), and 0.05 μg/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels varying in the range of 0.5–50 μg/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP and 9.6–27.8% for DETP. Between-run precision at 5 μg/L was below 15.7% for all DAPs. The revised method proved to be feasible to routine biological monitoring not only for occupational OP exposure but also for environmental background levels in the general population. Compared to our previous method, the revised method underscores the importance of adding pre-derivatization anhydration for higher sensitivity and precision.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r² > 0.997, n = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n = 5) and inter-day (n = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.     

Liquid chromatography–tandem mass spectrometry analysis of metabolites in rats after administration of prenylflavonoids from Epimediums

The metabolites in rats after administration of icariside II, icariin, epimedin C and extracts of four Epimedium species were investigated. Feces, bile, plasma and urine samples were detected comprehensively using HPLC-ESI-MSⁿ method. The structures of metabolites were identified on the basis of their characteristic fragmentations in MSⁿ experiments. Totally, 54 metabolites were identified in these biosamples. Specific hydrolysis of 7-O glucosides in gut lumen and glucuronic acid conjugation in liver were considered as the main physiologic processes of prenylflavonoids. Icariside II and anhydroicaritin were the major intermediate products in forming of mono- and di-glucuronic acid conjugations in vivo. In general, this study revealed the possible metabolite profiles of prenylflavonoids in rats, and might aid the clinical use of different Epimedium species.     

Determination of urinary valproylcarnitine by gas chromatography—mass spectrometry with selected-ion monitoring

A modified method for the determination of valproylcarnitine in urine samples of patients receiving sodium valproate by gas chromatography—mass spectrometry with selected-ion monitoring is described. The chemically analogous internal standard 2-ethylpentanoylcarnitine was added to the urine samples. Valproic acid and its metabolites were removed by extraction with chloroform at pH 5.0. The samples were then applied onto a C₁₈ Sep-Pak column. Inorganic and water soluble compounds were washed out with water. Valproylcarnitine and internal standard were eluted with methanol and were derivatized to the corresponding acyl-containing lactones by heating at 100°C for 60 min in dimethylformamide. Urinary valproylcarnitine levels of epileptic patients receiving valproate were determined according to the present method. The data obtained might be useful for diagnosis of carnitine deficiency.     

Screening for inborn errors of metabolism using gas chromatography–mass spectrometry

Screening of newborns for inborn errors of metabolism (IEM) in China is both a challenging and undeveloped area for gynecologists and pediatricians. Since 1999, the Capital Institute of Pediatrics has been studied as regards screening for IEM using advanced gas chromatography–mass spectrometry (GC–MS) method in collaboration with the Matsumoto Institute of Life Science (MILS), Japan, and has successfully diagnosed 51 cases of IEM in a total of 393 patients. Galactosemia, phenylketonuria and methylmalonic acidemia were the most frequent disorders among 51 cases of IEM. Treatment by suitable drugs and/or diet therapy was very effective in the most cases.     

Detection and quantification of fatty acid ethyl esters in meconium by headspace-solid-phase microextraction and gas chromatography–mass spectrometry

Meconium fatty acid ethyl esters (FAEEs) are currently used as biomarkers to detect heavy prenatal alcohol exposure. We introduce a novel technique to quantify FAEEs in meconium using headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS). This method improves on previous approaches by decreasing sample preparation time, eliminating the need for organic solvents, and reducing the required sample size. Using 50 mg of meconium, the detection limits of FAEEs ranged from 0.05 to 0.16 nmol/g and had good reproducibility making it ideal for routine analysis of clinical samples.     

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤ r ≤ 0.910) and with 1-OHPYR (0.614 ≤ r ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤ r ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.     

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography–electron-capture mass spectrometry

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC–ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene–divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2–25, 0.4–40, 4–200 and 4–200 pg g⁻¹ plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo-o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93–107%. The repeatability of the method was in the range 4–30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g⁻¹ plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.