Tenofovir, emtricitabine, lamivudine and dolutegravir concentrations in plasma and urine following drug intake cessation in a randomized controlled directly observed pharmacokinetic trial to aid point-of-care testing.

BACKGROUND: Poor adherence to ART and pre-exposure prophylaxis (PrEP) can impact patient and public health. Point-of-care testing (POCT) may aid monitoring and adherence interventions. OBJECTIVES: We report the pharmacokinetics of tenofovir [dosed as tenofovir disoproxil (TDF) and tenofovir alafenamide (TAF)], emtricitabine (FTC), lamivudine (3TC) and dolutegravir (DTG) in plasma and urine following drug cessation to evaluate adherence targets in urine for POCT. METHODS: Subjects were randomized (1:1) to receive DTG/FTC/TAF or DTG/3TC/TDF for 15 days. Plasma and spot urine were collected on Day 15 (0-336 h post final dose). Drug concentrations were quantified using LC-MS, and non-linear mixed-effects models applied to determine drug disposition between matrices and relationship with relevant plasma [dolutegravir protein-adjusted 90% inhibitory concentration (PA-IC90 = 64 ng/mL) and minimum effective concentration (MEC = 324 ng/mL)] and urinary thresholds [tenofovir disoproxil fumarate 1500 ng/mL]. RESULTS: Of 30 individuals enrolled, 29 were included (72% female at birth, 90% Caucasian). Median (range) predicted time to plasma dolutegravir PA-IC90 and MEC were 83.5 (41.0-152) and 49.0 h (23.7-78.9), corresponding to geometric mean (90%) urine concentrations of 5.42 (4.37-6.46) and 27.4 ng/mL (22.1-32.7). Tenofovir in urine reached 1500 ng/mL by 101 h (58.6-205) with an equivalent plasma concentration of 6.20 ng/mL (4.21-8.18). CONCLUSIONS: These data support use of a urinary tenofovir threshold of <1500 ng/mL (tenofovir disoproxil fumarate-based regimens) as a marker of three or more missed doses for a POCT platform. However, due to low dolutegravir concentrations in urine, POCT would be limited to a readout of recent dolutegravir intake (one missed dose).

Characterizing Lomerizine metabolites in camel urine: High-resolution mass spectrometry method development and validation for enhanced doping control.

RATIONALE: Lomerizine (LMZ) is an antimigraine drug that works as a calcium channel blocker and has selective effects on the central nervous system. It is metabolized into trimetazidine (TMZ), which is a prohibited substance owing to its performance-enhancing effects in both human and animal sports. Effective doping control measures are imperative to distinguish the source of TMZ in samples to ensure integrity and fairness of the sport, therefore a comprehensive analysis of LMZ metabolites is essential to identify potential biomarkers in camel urine for effective doping control. METHODS: Camel urine samples were collected from four healthy animals following a single oral administration of LMZ at a dosage of 1 mg/kg body weight. In vitro studies were conducted using homogenized camel liver samples. Lomerizine and its metabolites were extracted using solid-phase extraction and analyzed with a Thermo Fisher Orbitrap Exploris liquid chromatography mass spectrometry system. The acquired data was processed with the Compound Discoverer software. RESULTS: The study conducted a comprehensive analysis of LMZ metabolites in camels and identified 10 phase I and one phase II metabolites. The primary pathway for the formation of phase I metabolites was de-alkylation, while phase II metabolite was formed through alkylation of the parent drug. The study provided valuable insights into the unique metabolic pathways of LMZ in camels under specific experimental conditions. CONCLUSION: The developed method enables the detection and characterization of LMZ and its metabolites in camels. The identified metabolites has the potential to act as marker metabolites for the distinctive detection of LMZ in camel urine to ensure efficient analytical strategies for routine doping control applications.

An Innovative Approach to Characterize Clinical ADME and Pharmacokinetics of the Inhaled Drug Nemiralisib Using an Intravenous Microtracer Combined with an Inhaled Dose and an Oral Radiolabel Dose in Healthy Male Subjects.

An innovative open-label, crossover clinical study was used to investigate the excretion balance, pharmacokinetics, and metabolism of nemiralisib-an inhaled phosphoinositide 3-kinase delta inhibitor being developed for respiratory diseases. Six healthy men received a single intravenous microtracer of 10 µg [14C]nemiralisib with a concomitant inhaled nonradiolabeled 1000 µg dose followed by an oral 800 µg dose of [14C]nemiralisib 14 days later. Complementary methods including accelerator mass spectrometry allowed characterization of a range of parameters including oral absorption (Fabs), proportion of nemiralisib escaping gut wall metabolism (Fg), hepatic extraction (Eh), fraction of dose absorbed from inhaled dose (Flung), and renal clearance. Intravenous pharmacokinetics of nemiralisib were characterized by low blood clearance (10.0 l/h), long terminal half-life (55 hours), and high volume of distribution at steady state (728 l). Nemiralisib exhibited moderate inhaled and oral bioavailability (38% and 35%) while Flung was 29%. Absorption and first-pass parameters were corrected for blood renal clearance and compared with values without correction. Any swallowed nemiralisib was relatively well absorbed (Fabs, 0.48) with a high fraction escaping gut wall metabolism and low extraction by the liver (Fg and Eh being 0.83 and 0.10, respectively). There were no major human plasma metabolites requiring further qualification in animal studies. Both unchanged nemiralisib and its oxidative/conjugative metabolites were secreted in bile, with nemiralisib likely subject to further metabolism through enterohepatic recirculation. Direct renal clearance and metabolism followed by renal clearance were lesser routes of elimination. SIGNIFICANCE STATEMENT: A number of innovative features have been combined into one small clinical study enabling a comprehensive description of the human pharmacokinetics and metabolism of an inhaled molecule. Design elements included an intravenous 14C tracer administration concomitant with an inhalation dose that enabled derivation of parameters such as fraction absorbed (Fabs), the proportion of drug escaping first-pass extraction through the gut wall and liver (Fg and Fh) and hepatic extraction (Eh). Entero-test bile sampling enabled characterization of biliary elimination pathways.

Absorption, metabolism, and excretion of [14C]evogliptin tartrate in male rats and dogs.

The objective of this study was to determine the absorption, excretion, and metabolism of a novel, oral antihyperglycemic drug, evogliptin, in male rats and dogs. Plasma, urine, feces, and expired air samples were collected after a single oral dose administration of [14C]evogliptin, samples were analyzed by measuring overall radioactivity levels using high-performance liquid chromatography (HPLC), and radioactivity levels were measured by utilizing LC-tandem mass spectrometry (LC-MS/MS). The total amounts of radioactivity excreted in urine, feces, and expired air up to 168 h after administration of [14C]evogliptin tartrate to rats (30 mg evogliptin/kg) and dogs (10 mg evogliptin/kg) were 96.7% and 96.8% of initial doses administered, respectively. The extent of urinary and fecal excretion in the rat up to 168 h constituted 29.7% and 66.5% of the given dose, respectively; and in dog was 43.3% and 53.5%, respectively. A total of 23 possible metabolites were detected with radiochromatograms of plasma, urinary, and fecal samples, but only the structures of 12 metabolites were identified via LC-MS/MS analysis. Evogliptin was the major component. Regarding the total radiochromatographic peak areas, peaks 9 (evogliptin acid) and 11 (hydroxyevogliptin) were the major metabolites in rats, and peaks 8 [4(S)-hydroxyevogliptin glucuronide], 15 [4(S)-hydroxyevogliptin], and 17 [4(R)-hydroxyevogliptin] were the predominant metabolites in dogs. Data demonstrated that evogliptin was the major component excreted in urine and feces of rats and dogs, but the metabolite profiles varied between species.

Effects of renal impairment on the pharmacokinetics and pharmacodynamics of a novel dipeptidyl peptidase-4 inhibitor, evogliptin (DA-1229).

Evogliptin is a novel potent and selective dipeptidyl peptidase-4 (DPP-4) inhibitor. The aim of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of evogliptin in participants with renal impairment (RI). An open-label, parallel-group clinical study was conducted in participants with mild, moderate and severe RI and in matched participants with normal renal function (NRF). A single oral 5-mg dose of evogliptin was administered and serial blood and urine samples were obtained to assess the PK and PD characteristics of evogliptin. Baseline urine samples were collected to evaluate endogenous CYP3A metabolic markers. The plasma exposure to evogliptin and degree of DPP-4 activity inhibition increased with decreasing renal function. The mean areas under the concentration-time curves from 0 to 120 hours were increased 1.2-, 1.8- and 1.98-fold in the mild, moderate and severe RI groups, respectively, compared with the NRF group. The levels of CYP3A metabolic markers were lower in the RI group than in the NRF group. The increase in the plasma concentration of evogliptin is unlikely to result in changes in its efficacy or safety, considering the results of previous clinical studies.

[Progress on Determination and Analysis of Zopiclone in Biological Samples].

As a new hypnotic, zopiclone is widely used in clinical treatment. There are many methods for determination of zopiclone, including spectrophotometry, chromatography and chromatography mass spectrum, etc. Present paper reviews different kinds of biological samples associated with zopiclone, extraction and purification methods, and determination and analysis methods, which aims to provide references for the relevant research and practice.

Utilization of Stable Isotope Labeling to Facilitate the Identification of Polar Metabolites of KAF156, an Antimalarial Agent.

Identification of polar metabolites of drug candidates during development is often challenging. Several prominent polar metabolites of 2-amino-1-(2-(4-fluorophenyl)-3-((4-fluorophenyl)amino)-8,8-dimethyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone ([(14)C]KAF156), an antimalarial agent, were detected in rat urine from an absorption, distribution, metabolism, and excretion study but could not be characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) because of low ionization efficiency. In such instances, a strategy often chosen by investigators is to use a radiolabeled compound with high specific activity, having an isotopic mass ratio (i.e., [(12)C]/[(14)C]) and mass difference that serve as the basis for a mass filter using accurate mass spectrometry. Unfortunately, [(14)C]KAF156-1 was uniformly labeled (n = 1-6) with the mass ratio of ∼0.1. This ratio was insufficient to be useful as a mass filter despite the high specific activity (120 µCi/mg). At this stage in development, stable isotope labeled [(13)C6]KAF156-1 was available as the internal standard for the quantification of KAF156. We were thus able to design an oral dose as a mixture of [(14)C]KAF156-1 (specific activity 3.65 µCi/mg) and [(13)C6]KAF156-1 with a mass ratio of [(12)C]/[(13)C6] as 0.9 and the mass difference as 6.0202. By using this mass filter strategy, four polar metabolites were successfully identified in rat urine. Subsequently, using a similar dual labeling approach, [(14)C]KAF156-2 and [(13)C2]KAF156-2 were synthesized to allow the detection of any putative polar metabolites that may have lost labeling during biotransformations using the previous [(14)C]KAF156-1. Three polar metabolites were thereby identified and M43, a less polar metabolite, was proposed as the key intermediate metabolite leading to the formation of a total of seven polar metabolites. Overall this dual labeling approach proved practical and valuable for the identification of polar metabolites by LC-MS/MS.

Molecularly imprinted polymeric stir bar: Preparation and application for the determination of naftopidil in plasma and urine samples.

In this study, molecularly imprinting technology and stir bar absorption technology were combined to develop a microextraction approach based on a molecularly imprinted polymeric stir bar. The molecularly imprinted polymer stir bar has a high performance, is specific, economical, and simple to prepare. The obtained naftopidil-imprinted polymer-coated bars could simultaneously agitate and adsorb naftopidil in the sample solution. The ratio of template/monomer/cross-linker and conditions of template removal were optimized to prepare a stir bar with highly efficient adsorption. Fourier transform infrared spectroscopy, scanning electron microscopy, selectivity, and extraction capacity experiments showed that the molecularly imprinted polymer stir bar was prepared successfully. To utilize the molecularly imprinted polymer stir bar for the determination of naftopidil in complex body fluid matrices, the extraction time, stirring speed, eluent, and elution time were optimized. The limits of detection of naftopidil in plasma and urine sample were 7.5 and 4.0 ng/mL, respectively, and the recoveries were in the range of 90-112%. The within-run precision and between-run precision were acceptable (relative standard deviation <7%). These data demonstrated that the molecularly imprinted polymeric stir bar based microextraction with high-performance liquid chromatography was a convenient, rapid, efficient, and specific method for the precise determination of trace naftopidil in clinical analysis.

Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing.

Designer piperazines are emerging novel psychoactive substances (NPS) with few high-throughput screening methods for their identification. We evaluated a biochip array technology (BAT) immunoassay for phenylpiperazines (PNP) and benzylpiperazines (BZP) and analyzed 20,017 randomly collected urine workplace specimens. Immunoassay performance at recommended cutoffs was evaluated for PNPI (5 µg/L), PNPII (7.5 µg/L), and BZP (5 µg/L) antibodies. Eight hundred forty positive and 206 randomly selected presumptive negative specimens were confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Assay limits of detection for PNPI, PNPII, and BZP were 2.9, 6.3, and 2.1 µg/L, respectively. Calibration curves were linear (R (2) > 0.99) with upper limits of 42 µg/L for PNPI/PNII and 100 µg/L for BZP. Quality control samples demonstrated imprecision <19.3 %CV and accuracies 86.0-94.5 % of target. There were no interferences from 106 non-piperazine substances. Seventy-eight of 840 presumptive positive specimens (9.3 %) were LC-HRMS positive, with 72 positive for 1-(3-chlorophenyl)piperazine (mCPP), a designer piperazine and antidepressant trazodone metabolite. Of 206 presumptive negative specimens, one confirmed positive for mCPP (3.3 µg/L) and one for BZP (3.6 µg/L). BAT specificity (21.1 to 91.4 %) and efficiency (27.0 to 91.6 %) increased, and sensitivity slightly decreased (97.5 to 93.8 %) with optimized cutoffs of 25 µg/L PNPI, 42 µg/L PNPI, and 100 µg/L BZP. A high-throughput screening method is needed to identify piperazine NPS. We evaluated performance of the Randox BAT immunoassay to identify urinary piperazines and documented improved performance when antibody cutoffs were raised. In addition, in randomized workplace urine specimens, all but two positive specimens contained mCPP and/or trazodone, most likely from legitimate medical prescriptions. Graphical Abstract Biochip array technology (BAT) immunoassay for designer piperazines detection in urine. In chemiluminescent immunoassay, the labeled-drug (antigen) competes with the drug in the urine. In the absence of drug, the labeled-drug binds to the antibody releasing an enzyme (horseradish peroxidase) to react with the substrate and producing chemiluminescence. The higher the drug concentration in urine, the weaker the chemiluminescent signal is produced. All presumptive positive specimens and randomly selected presumptive negative specimens were analyzed and confirmed by a liquid chromatography high-resolution mass spectrometry with limit of quantification of 2.5 or 5 µg/L.

Development of LC-MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics.

A rapid and highly sensitive liquid chromatography­tandem mass spectrometric (LC-MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse-phase C18 column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1­3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra- and inter-day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats.

[Analysis of primary metabolites of ranolazine in dog urine by LC-MS(n)].

Ranolazine and metabolites in dog urine were identified by LC-MS(n). Dog urine samples were collected after ig 30 mg x kg(-1) ranolazine, then the samples were enriched and purified through solid-phase extraction cartridge. The purified samples were analyzed by LC-MS(n). The possible metabolites were discovered by comparing the full scan and SIM chromatograms of the test samples with the corresponding blanks. Seventeen phase I metabolites and fourteen phase II metabolites were identified in dog urine. Three metabolites were identified by comparing with the control article. The metabolites were formed via the following metabolic pathways: O-demethylation, O-dearylation, hydroxylation, N-dealkylation, amide hydrolysis, glucuronidation and sulfation. The LC-MS(n) method is suitable for the rapid identification of drug and its metabolites in biologic samples.

Simultaneous determination of 10 new psychoactive piperazine derivatives in urine using ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry.

With the rapid development of synthetic drugs, novel piperazine derivatives, as an increasingly important class of new psychoactive substances (NPS), have attracted global attention owing to their increasing demand in the illicit drug market. In this study, ten piperazine derivatives were analyzed in urine samples after pre-treatment with ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) combined with gas chromatography-tandem mass spectrometry (GC-MS/MS). This simple approach involved the use of urine samples (1 mL) adjusted to pH 12, which was added to 100 µL of n-hexane and subjected to ultrasonication for 3 min to completely disperse the sample in the n-hexane solution. The resulting turbid suspension was centrifuged at 10,000 rpm for 3 min, and the supernatant was extracted and analyzed using GC-MS/MS. Under the optimized conditions presented in this study, the linear relationship between the analytes was good within 10-1500 ng/mL, and the correlation coefficient (r) was between .9914 and .9983. The limit of detection (LOD) was 0.3-2 ng/mL (S/N = 3), and the lower limit of quantification (LLOQ) was 10 ng/mL (S/N = 10) with the recovery of the analytes of interest from the spiked samples being 76.3%-93.3%. This method has been used to analyze real-world samples; our study shows that the UA-LDS-DLLME approach can be used for rapid analysis while consuming minimal solvent for the simultaneous determination of a range of analytes. This method has the potential for use in clinical analyses and forensic toxicology.

Metabolism, pharmacokinetics, and excretion of the 5-hydroxytryptamine1b receptor antagonist elzasonan in humans.

The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for ∼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.

A gas chromatography/mass spectrometry method for the determination of sildenafil, vardenafil and tadalafil and their metabolites in human urine.

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis.

Head shop compound abuse amongst attendees of the Drug Treatment Centre Board.

The use of "Head Shop" compounds has received much media attention lately. There is very little research in the current literature with regard to the extent of the usage of these substances amongst the drug using population in Ireland. We conducted a study to examine the extent of the usage of Mephedrone, Methylone and BZP amongst attendees of Methadone maintenance programs at the DTCB. Two hundred and nine samples in total were tested. The results showed significant usage of these compounds amongst this cohort of drug users, with 29 (13.9%) of samples tested being positive for Mephedrone, 7 (3.3%) positive for Methylone and 1 (0.5%) positive for BZP.

Metabolic activation of TM5441 in vitro and in vivo: Formation of reactive metabolites and human enzymes involved.

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5'-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.

Development and Validation of an LC-MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction.

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 µg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified ß-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Disposition and metabolism of radiolabeled casopitant in humans.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)-(GW679769)] is a novel neurokinin-1 receptor antagonist being developed for the prevention of chemotherapy-induced and postoperative nausea and vomiting. The disposition of [(14)C]casopitant was determined in a single-sequence study in six healthy male subjects after single-dose 90-mg i.v. and 150-mg oral administration. Blood, urine, and feces were collected at frequent intervals after dosing. Plasma, urine, and fecal samples were analyzed by high-performance liquid chromatography/mass spectrometry coupled with off-line radiodetection for metabolite profiling. Moreover, urine was also analyzed with (1)H-NMR to further characterize metabolites. Plasma pharmacokinetic parameters for casopitant, a major metabolite (M13, coded as GSK525060), and total radioactivity were determined. Absorption of radioactivity after oral administration appeared to be nearly complete; elimination was principally via the feces both after oral and intravenous administration. Urinary elimination accounted for only <8% of total radioactivity. The main circulating metabolites were a hydroxylated derivative, M13 (coded as GSK525060), and, after oral administration, a deacetylated and oxidized metabolite, M12 (coded as GSK631832). In addition, many other metabolites were identified in plasma and excreta: the principal route of metabolism included multiple oxidations, loss of the N-acetyl group, modifications or loss of the piperazine group, and cleavage of the molecule. Casopitant was extensively metabolized, and only negligible amounts were excreted as unchanged compound. Some phase II metabolites were also observed, particularly in urine.

Detection Time of Oxazepam and Zopiclone in Urine and Oral Fluid after Experimental Oral Dosing.

Data from previous experimental studies on the detection time of oxazepam and zopiclone in biological matrices are limited. The aim of this study was to examine the detection time in urine and oral fluid after single oral doses of oxazepam and zopiclone. Ten healthy volunteers received 25 mg of oxazepam in the evening of Day 1 and 7.5 mg of zopiclone in the evening of Day 3. Urine and oral fluid samples were collected twice daily for 9 days, with an additional sampling the day after ingestion of zopiclone. A total of 19 samples of both urine and oral fluid from each participant were analyzed using fully validated chromatographic methods. The median detection time for oxazepam was 91 h (range 73-108) in urine and 67 h (range 50-98) in oral fluid. The median detection time for zopiclone in urine was 49 h (range 25-98) and 59 h (range 48-146) in oral fluid. The metabolite zopiclone N-oxide showed a detection time of 36 h (range 25-84) in urine. The area under the concentration-time curve (AUCTotal) in urine corrected for creatinine was 150 µmol/L/mmol/L*h (range 105-216) for oxazepam and 1.60 µmol/L/mmol/L*h (range 0.79-4.53) for zopiclone. In oral fluid, the AUCtotal was 673 nmol/L*h (range 339-1,316) for oxazepam and 2,150 nmol/L*h (range 493-4,240) for zopiclone. In conclusion, oxazepam can be detected longer in urine than in oral fluid, while zopiclone can be detected longer in oral fluid than in urine. The high AUCTotal for zopiclone in oral fluid shows that the transfer into oral fluid is significant. In certain individuals the detection time of zopiclone in oral fluid is long. These results can be helpful when interpreting drug testing analyzes.

An available strategy based on accurate mass by ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry technology to characterization of metabolic profile of palbociclib in rat urine, feces and bile.

Palbociclib (named PD 0332991) is a novel highly selective cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitor, which has been approved by the Food and Drug Administration (FDA) for the treatment of hormone-receptor-positive advanced breast cancer. This present study developed a comprehensive strategy to investigate the metabolic profile of palbociclib in rat urine, feces and bile samples based on an ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR MS). A total of 29 metabolites, including 18 phase I metabolites and 11 phase II metabolites, were detected and identified. The metabolic pathways included hydroxylation, oxidation, dehydrogenation, N-dealkylation, carbonylation, oxidative deamination, acetylation, glucuronidation, sulphate conjugation as well as the crossover of multiple metabolic pathways in vivo, and 16 of these metabolites were proposed for the first time. This study showed an insight into the metabolism of palbociclib in vivo, which may provide relevant chemical information for subsequent studies in the future.