RESUMO
ACP-105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue-specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP-105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP-105, a selective and reliable LC-MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, accurate mass, and retention time. Under the current experimental condition, a total of 19 metabolites were detected in ACP-105 drug administered equine urine samples. The study results suggest the following: (1) ACP-105 is prone to oxidation, which gives corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (2) along with oxidation, there is a possibility of elimination of water molecule (dehydration) from the third position of the tropine moiety, resulting in the dehydrated analogs of corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (3) from the study on the metabolites using LC-MS/MS, it is clear that the fragmentation pattern is identical and a great number of fragment ions are common in all the metabolites and the parent drug. (4) The ACP-105 and its metabolites were detected for up to 72 h; thus, the result is a valuable tool for evaluating its use and/or misuse in sport.
Assuntos
Androgênios/urina , Compostos Azabicíclicos/urina , Cavalos/urina , Espectrometria de Massas em Tandem/métodos , Administração Oral , Androgênios/administração & dosagem , Androgênios/metabolismo , Animais , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/metabolismo , Cromatografia Líquida/métodos , Dopagem Esportivo , Feminino , Masculino , Detecção do Abuso de Substâncias/métodosRESUMO
AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this study was to investigate the metabolism of AC-262536 in the horse following in vitro incubation and oral administration to two Thoroughbred horses, in order to identify the most appropriate analytical targets for doping control laboratories. Urine, plasma and hair samples were collected and analysed for parent drug and metabolites. Liquid chromatography-high-resolution mass spectrometry was used for in vitro metabolite identification and in urine and plasma samples. Nine phase I metabolites were identified in vitro; four of these were subsequently detected in urine and three in plasma, alongside the parent compound in both matrices. In both urine and plasma samples, the longest detection window was observed for an epimer of the parent compound, which is suggested as the best target for detection of AC-262536 administration. AC-262536 and metabolites were found to be primarily glucuronide conjugates in both urine and plasma. Liquid chromatography-tandem mass spectrometry analysis of post-administration hair samples indicated incorporation of parent AC-262536 into the hair following oral administration. No metabolites were detected in the hair.
Assuntos
Compostos Azabicíclicos/metabolismo , Cavalos/metabolismo , Naftalenos/metabolismo , Substâncias para Melhoria do Desempenho/metabolismo , Administração Oral , Animais , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/urina , Cromatografia Líquida , Cabelo/química , Cavalos/sangue , Cavalos/urina , Naftalenos/administração & dosagem , Naftalenos/sangue , Naftalenos/urina , Substâncias para Melhoria do Desempenho/administração & dosagem , Substâncias para Melhoria do Desempenho/sangue , Substâncias para Melhoria do Desempenho/urina , Receptores Androgênicos/metabolismo , Detecção do Abuso de Substâncias , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Benzodiazepinas/metabolismo , Hipnóticos e Sedativos/metabolismo , Detecção do Abuso de Substâncias/métodos , Alprazolam/análogos & derivados , Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/metabolismo , Compostos Azabicíclicos/urina , Benzodiazepinas/sangue , Benzodiazepinas/urina , Cromatografia Líquida/métodos , Diazepam/análogos & derivados , Toxicologia Forense , Humanos , Hipnóticos e Sedativos/análise , Hipnóticos e Sedativos/sangue , Hipnóticos e Sedativos/urina , Limite de Detecção , Piperazinas/sangue , Piperazinas/metabolismo , Piperazinas/urina , Medicamentos Indutores do Sono/sangue , Medicamentos Indutores do Sono/metabolismo , Medicamentos Indutores do Sono/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Zolpidem/sangue , Zolpidem/metabolismo , Zolpidem/urinaRESUMO
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.
Assuntos
Compostos Azabicíclicos/urina , Modelos Teóricos , Oxazepam/urina , Piperazinas/urina , Saliva/química , Detecção do Abuso de Substâncias/métodos , Adolescente , Adulto , Área Sob a Curva , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/análise , Esquema de Medicação , Feminino , Voluntários Saudáveis , Humanos , Limite de Detecção , Masculino , Oxazepam/administração & dosagem , Oxazepam/análise , Piperazinas/administração & dosagem , Piperazinas/análise , Detecção do Abuso de Substâncias/normas , Fatores de Tempo , Adulto JovemRESUMO
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.
Assuntos
Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/urina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Gasosa-Espectrometria de Massas , Hipnóticos e Sedativos/sangue , Hipnóticos e Sedativos/urina , Piperazinas/sangue , Piperazinas/urina , Espectrometria de Massas em Tandem/métodos , Compostos Azabicíclicos/metabolismo , Humanos , Hipnóticos e Sedativos/metabolismo , Espectrometria de Massas , Piperazinas/metabolismoRESUMO
Date-rape drugs have the potential to be used in drug-facilitated sexual assault, organ theft and property theft. Since they are colorless, tasteless and odorless, victims can drink without noticing, when added to the beverages. These drugs must be detected in time, before they are cleared up from the biofluids. A simultaneous extraction and determination method in urine for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin (an anticonvulsant and antiepileptic drug) with LC-MS/MS was developed for the first time with analytically acceptable recoveries and validated. A 4 steps liquid-liquid extraction was applied, using only 1.000mL urine. A new age commercial C18 poroshell column with high column efficiency was used for LC-MS/MS analysis with a fast isocratic elution as 5.5min. A new MS transition were introduced for barbital. 222.7>179.8 with the effect of acetonitrile. Recoveries (%) were between 80.98-99.27 for all analytes, except for GHB which was 71.46. LOD and LOQ values were found in the ranges of 0.59-49.50 and 9.20-80.80ngmL(-1) for all the analytes (except for GHB:3.44 and 6.00µgmL(-1)). HorRat values calculated (between 0.25-1.21), revealed that the inter-day and interanalist precisions (RSD%≤14.54%) acceptable. The simultaneous extraction and determination of these 8 analytes in urine is challenging because of the difficulty arising from the different chemical properties of some. Since the procedure can extract drugs from a wide range of polarity and pKa, it increases the window of detection. Group representatives from barbiturates, z-drugs, ketamine, phenytoin and polar acidic drugs (GHB) have been successfully analyzed in this study with low detection limits. The method is important from the point of determining the combined or single use of these drugs in crimes and finding out the reasons of deaths related to these drugs.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hipnóticos e Sedativos/urina , Extração Líquido-Líquido/métodos , Compostos Azabicíclicos/química , Compostos Azabicíclicos/isolamento & purificação , Compostos Azabicíclicos/urina , Barbitúricos/química , Barbitúricos/isolamento & purificação , Barbitúricos/urina , Ciências Forenses , Humanos , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/isolamento & purificação , Ketamina/química , Ketamina/isolamento & purificação , Ketamina/urina , Limite de Detecção , Modelos Lineares , Fenitoína/química , Fenitoína/isolamento & purificação , Fenitoína/urina , Piperazinas/química , Piperazinas/isolamento & purificação , Piperazinas/urina , Piridinas/química , Piridinas/isolamento & purificação , Piridinas/urina , Estupro , Reprodutibilidade dos Testes , Oxibato de Sódio/química , Oxibato de Sódio/isolamento & purificação , Oxibato de Sódio/urina , ZolpidemRESUMO
Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics which have been prohibited in sport as anabolic agents according to the regulations of the World Anti-Doping Agency (WADA) since 2008. Within the past three years, numerous adverse analytical findings with SARMs in routine doping control samples have been reported despite missing clinical approval of these substances. Hence, preventive doping research concerning the metabolism and elimination of new therapeutic entities of the class of SARMs are vital for efficient and timely sports drug testing programs as banned compounds are most efficiently screened when viable targets (for example, characteristic metabolites) are identified. In the present study, the metabolism of ACP-105, a novel SARM drug candidate, was studied in vivo in rats. Following oral administration, urine samples were collected over a period of seven days and analyzed for metabolic products by Liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Samples were subjected to enzymatic hydrolysis prior to liquid-liquid extraction and a total of seven major phase-I metabolites were detected, three of which were attributed to monohydroxylated and four to bishydroxylated ACP-105. The hydroxylation sites were assigned by means of diagnostic product ions and respective dissociation pathways of the analytes following positive or negative ionization and collisional activation as well as selective chemical derivatization. The identified metabolites were used as target compounds to investigate their traceability in a rat elimination urine samples study and monohydroxylated and bishydroxylated species were detectable for up to four and six days post-administration, respectively.
Assuntos
Compostos Azabicíclicos/urina , Cromatografia Líquida/métodos , Dopagem Esportivo/prevenção & controle , Receptores Androgênicos/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Anabolizantes , Animais , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/análise , Feminino , Humanos , Hidrólise , Hidroxilação , Masculino , Ratos , Ratos Sprague-Dawley , Detecção do Abuso de Substâncias/métodosRESUMO
A simple liquid chromatography-tandem mass spectrometry method was validated to allow determination of zopiclone (ZOP), N-desmethylzopiclone (NDZOP), zopiclone N-oxide (ZOPNO) and 2-amino-5-chloropyridine (ACP) in urine at concentrations up to 3,000 ng/mL within 3.5 min. This method was used for quantitative analysis of the analytes in authentic urine samples obtained 10 h after oral administration of zopiclone (Imovane(®)) and in aliquots of the same urine samples after different storage conditions. In addition, pH of each studied urine sample was measured over time. The results showed that formation of ACP occurred at elevated pH and/or temperature by degradation of ZOP, NDZOP and ZOPNO. This method was also applied to samples obtained from two female victims of drug-facilitated assault. One sample had been exposed to long-term storage conditions at different temperatures and at pH >8.2, which resulted in high concentrations of ACP. The other sample, which was exposed to pH <6.5, showed no formation of ACP. ACP is formed both from ZOP and from its metabolites NDZOP and ZOPNO depending on the pH of the urine, time of storage and/or the temperature conditions. For correct interpretation in forensic cases, ZOP, its major metabolites and ACP should be analyzed. When ACP is identified in urine, the concentrations of ZOP, NDZOP and ZOPNO should be interpreted with great caution.
Assuntos
Compostos Azabicíclicos/urina , Hipnóticos e Sedativos/urina , Piperazinas/urina , Piridinas/urina , Compostos Azabicíclicos/metabolismo , Cromatografia Líquida de Alta Pressão , Voluntários Saudáveis , Humanos , Concentração de Íons de Hidrogênio , Hipnóticos e Sedativos/metabolismo , Limite de Detecção , Piperazinas/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Avibactam, a novel non-ß-lactam ß-lactamase inhibitor with activity against Ambler class A, class C, and some class D enzymes is being evaluated in combination with various ß-lactam antibiotics to treat serious bacterial infections. The in vivo mass balance recovery and metabolite profile of [(14)C] avibactam (500 mg/1-h infusion) was assessed in six healthy male subjects, and a series of in vitro experiments evaluated the metabolism and drug-drug interaction potential of avibactam. In the mass balance study, measurement of plasma avibactam (using a validated liquid chromatography-tandem mass spectrometry method) and total radioactivity in plasma, whole blood, urine, and feces (using liquid scintillation counting) indicated that most of the avibactam was excreted unchanged in urine within 12 hours, with recovery complete (>97% of the administered dose) within 96 hours. Geometric mean avibactam renal clearance (158 ml/min) was greater than the product of unbound fraction of drug and glomerular filtration rate (109.5 ml/min), suggesting that active tubular secretion accounted for some renal elimination. There was no evidence of metabolism in plasma and urine, with unchanged avibactam the major component in both matrices. Avibactam demonstrated in vitro substrate potential for organic anion transporters 1 and 3 (OAT1 and OAT3) proteins expressed in human embryonic kidney 293 cells (Km > 1000 µM; >10-fold the Cmax of a therapeutic dose), which could account for the active tubular secretion observed in vivo. Avibactam uptake by OAT1 and OAT3 was inhibited by probenecid, a potent OAT1/OAT3 inhibitor. Avibactam did not interact with various other membrane transport proteins or cytochrome P450 enzymes in vitro, suggesting it has limited propensity for drug-drug interactions involving cytochrome P450 enzymes.
Assuntos
Antibacterianos/metabolismo , Compostos Azabicíclicos/metabolismo , Inibidores de beta-Lactamases , Adulto , Animais , Antibacterianos/sangue , Antibacterianos/urina , Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/urina , Transporte Biológico , Radioisótopos de Carbono , Membrana Celular/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Interações Medicamentosas , Fezes/química , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Taxa de Depuração Metabólica , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Microssomos/metabolismo , Pessoa de Meia-Idade , Coelhos , Especificidade por SubstratoRESUMO
Zolpidem and zopiclone (Z-compounds) are non-benzodiazepine hypnotics of new generation that can be used in drug-facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z-compounds and their metabolites by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC-HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy- and three hydroxy- metabolites, that could be also detected by gas chromatography/mass spectrometry (GC-MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N-demethyl and N-oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC-MS/MS method for the determination of Z-compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC-MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA.
Assuntos
Compostos Azabicíclicos/metabolismo , Compostos Azabicíclicos/urina , Hipnóticos e Sedativos/metabolismo , Hipnóticos e Sedativos/urina , Piperazinas/metabolismo , Piperazinas/urina , Piridinas/metabolismo , Piridinas/urina , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Limite de Detecção , ZolpidemRESUMO
This study was conducted to determine the safety, tolerability, and pharmacokinetics of intravenous doses of ceftaroline fosamil administered in combination with the novel non-ß-lactam ß-lactamase inhibitor avibactam in healthy adults. In the single-dose, open-label arm, 12 subjects received single 1-h intravenous infusions of ceftaroline fosamil alone (600 mg), avibactam alone (600 mg), and ceftaroline fosamil in combination with avibactam (600/600 mg) separated by 5-day washout periods. In the multiple-dose, placebo-controlled, double-blind arm, 48 subjects received intravenous infusions of ceftaroline fosamil/avibactam at 600/600 mg every 12 h (q12h), 400/400 mg q8h, 900/900 mg q12h, 600/600 mg q8h, or placebo for 10 days. Ceftaroline and avibactam levels in plasma and urine were measured by liquid chromatography coupled with tandem mass spectrometry. No significant differences in systemic exposure of ceftaroline or avibactam were observed when the drugs were administered alone versus concomitantly, indicating that there was no apparent pharmacokinetic interaction between ceftaroline fosamil and avibactam administered as a single dose. No appreciable accumulation of either drug occurred with multiple intravenous doses of ceftaroline fosamil/avibactam, and pharmacokinetic parameters for ceftaroline and avibactam were similar on days 1 and 10. Infusions of ceftaroline fosamil/avibactam were well tolerated at total daily doses of up to 1,800 mg of each compound, and all adverse events (AEs) were mild to moderate in severity. Infusion-site reactions were the most common AEs reported with multiple dosing. The pharmacokinetic and safety profiles of ceftaroline fosamil/avibactam demonstrate that the 2 drugs can be administered concomitantly to provide an important broad-spectrum antimicrobial treatment option.
Assuntos
Antibacterianos/farmacocinética , Compostos Azabicíclicos/farmacocinética , Cefalosporinas/farmacocinética , Adulto , Antibacterianos/sangue , Antibacterianos/urina , Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/urina , Cefalosporinas/sangue , Cefalosporinas/urina , Cromatografia Líquida , Método Duplo-Cego , Esquema de Medicação , Combinação de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Placebos , Espectrometria de Massas em Tandem , CeftarolinaRESUMO
A simple and reliable HPLC method was developed and validated for the simultaneous determination of the hypnotic drug, zopiclone (ZPC) and its degradation product and main impurity, 2-amino-5-chloropyridine (ACP). The analyses were carried out on BDS Hypersil phenyl column (4.6 mm × 250 mm, 5 µm particle size) using micellar mobile phase consisting of 0.15M SDS, 10% n-propanol, 0.3% triethylamine, and 0.02 M orthophosphoric acid (pH 3.5) with timed programmable fluorescence detection. The proposed method was found to be rectilinear over the concentration ranges of 0.5-10.0 µg/mL for ZPC and 2.5-50 ng/mL for ACP. Moreover, the method was applied for the determination of ZPC in commercial tablets with mean percentage recovery of 99.06±1.49. The results of the proposed method were statistically compared with those obtained by the comparison method revealing no significance differences in the performance of the two methods regarding accuracy and precision. Furthermore, the proposed method was applied for the detection and determination of ACP in human urine as a marker for ZPC intake.
Assuntos
Compostos Azabicíclicos/urina , Cromatografia Líquida de Alta Pressão/métodos , Piperazinas/urina , Piridinas/urina , Espectrometria de Fluorescência/métodos , 1-Propanol/química , Adulto , Compostos Azabicíclicos/química , Contaminação de Medicamentos , Estabilidade de Medicamentos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Modelos Lineares , Micelas , Piperazinas/química , Piridinas/química , Reprodutibilidade dos Testes , Dodecilsulfato de Sódio/química , Comprimidos/química , TemperaturaRESUMO
A fast liquid chromatographic/tandem mass spectrometric method was developed for the simultaneous determination in human urine of seventeen benzodiazepines, four relevant metabolites together plus zolpidem and zopiclone. The sample preparation, optimized to take into account the matrix effect, was based on enzymatic hydrolysis and liquid-liquid extraction. The separation of the twenty-three analytes was achieved in less than eight minutes. The whole methodology was fully validated according to UNI EN ISO/IEC 17025:2005 rules and 2006 SOFT/AAFS guidelines. Selectivity, linearity range, identification (LOD) and quantitation (LOQ) limits, precision, accuracy and recovery were evaluated. For all the species the signal/concentration linearity was satisfactory in the 50-1000 ng/mL concentration range. The limits of detection ranged from 0.5 to 30 ng/mL and LOQs from 1.7 to 100.0 ng/mL. Precisions were in the ranges 5.0-11.8%, 1.5-11.0% and 1.1-4.4% for low (100 ng/mL), medium (300 ng/mL) and high (1000 ng/mL) concentration, respectively. The accuracy, expressed as bias% was within ± 25 % for all the analytes. The recovery values, evaluated at 300 ng/mL concentration, ranged from 56.2% to 98.8%. The present method for the determination of several benzodiazepines, zolpidem and zopiclone in human urine proved to be simple, fast, specific and sensitive. The quantification by LC-MS/MS was successfully applied to 329 forensic cases among driving re-licensing, car accidents and alleged sexual violence cases.
Assuntos
Benzodiazepinas/urina , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Compostos Azabicíclicos/urina , Medicina Legal/métodos , Humanos , Hidrólise , Extração Líquido-Líquido/métodos , Piperazinas/urina , Piridinas/urina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coleta de Urina/métodos , ZolpidemRESUMO
The study of human metabolism of endo-8[bis(2-chlorophenyl)methyl]-3-(2-pyrimidinyl)-8-azabicyclo[3.2.1]octan-3-ol (SCH 486757) after a 200-mg oral dose of the drug to healthy volunteers in the first-in-human study is presented. The structural elucidation of two unique metabolites, which were detected in the process of metabolite characterization in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS), is described. These metabolites (M27 and M34) were initially detected in human plasma at high levels (>35% of the LC-MS response of the parent drug). Additional LC-MS experiments (hydrogen/deuterium exchange and accurate mass measurement) were used to determine structures of metabolites. It was found that both metabolites were formed through a loss of the C-C bridge from the tropane moiety with the conversion into a substituted pyridinium compound. This metabolic process has not been reported previously. Because of the apparent high abundance of metabolites based on the LC-MS response, actual circulating amounts of these metabolites relative to the parent drug were determined semiquantitatively to evaluate their coverage in preclinical species. With the use of reference standards, it was shown that the LC-MS response of M27 and M34 in human plasma was much higher than that of the parent compound. Actual amounts of M27 and M34 metabolites were less than 5% of the level of the parent drug; therefore, additional assessment was not required.
Assuntos
Antitussígenos/metabolismo , Compostos Azabicíclicos/metabolismo , Compostos de Piridínio/metabolismo , Pirimidinas/metabolismo , Receptores Opioides/agonistas , Animais , Antitussígenos/sangue , Antitussígenos/farmacocinética , Antitussígenos/farmacologia , Antitussígenos/urina , Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/farmacocinética , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/urina , Biotransformação , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Conformação Molecular , Compostos de Piridínio/sangue , Compostos de Piridínio/química , Compostos de Piridínio/urina , Pirimidinas/sangue , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/urina , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Receptor de NociceptinaRESUMO
Zopiclone is a nonbenzodiazapine hypnotic used for the treatment of insomnia. Significant side effects include daytime drowsiness, dizziness, lightheadedness, bitter taste, dry mouth, headache, and upset stomach. A single method for confirmation and quantitation of zopiclone was developed for biological specimens and tissues. Zopiclone is extracted from the biological matrix using solid phase extraction technology. The drug is confirmed using gas chromatography mass spectrometry for toxicological and forensic purposes.
Assuntos
Compostos Azabicíclicos/sangue , Compostos Azabicíclicos/urina , Cromatografia Gasosa-Espectrometria de Massas/métodos , Piperazinas/sangue , Piperazinas/urina , Compostos Azabicíclicos/análise , Humanos , Piperazinas/análise , Extração em Fase SólidaRESUMO
Sleep disorders are common conditions that affect about 40 million people in the U.S every year, the most common of which is insomnia, which is characterized by difficulty falling or staying asleep. Zolpidem (Ambien) is a non-benzodiazepine prescription drug that is used to treat insomnia and is often preferred over the commonly used benzodiazepines due to a lesser side effect profile. This is because the non-benzodiazepine binding is more selective to GABA-A receptors versus the non-selective binding of benzodiazepines. With the increasing popularity of non-benzodiazepines, drug abuse and driving-while-impaired cases involving sleep-inducing drugs have risen. Therefore, a highly sensitive and rapid homogeneous immunoassay (EMIT-type assay) has been developed for the detection of zolpidem in urine. The zolpidem antibody is highly specific and does not cross-react with other newer sleep aids such as zopiclone and zaleplon. This assay has a detection limit of 5 ng/mL for zolpidem in urine. Further evaluation of this assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of authentic urine samples demonstrated that the accuracy of the assay is greater than 90%. Because this assay is designed to measure the non-conjugated drug in urine, it resulted in simplification for gas chromatography-MS or LC-MS-MS confirmation methods that do not require urine hydrolysis before solid-phase extraction or liquid-liquid extraction.
Assuntos
Técnica de Imunoensaio Enzimático de Multiplicação , Hipnóticos e Sedativos/urina , Piridinas/urina , Detecção do Abuso de Substâncias/métodos , Acetamidas/imunologia , Acetamidas/urina , Compostos Azabicíclicos/imunologia , Compostos Azabicíclicos/urina , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/imunologia , Piperazinas/imunologia , Piperazinas/urina , Valor Preditivo dos Testes , Piridinas/administração & dosagem , Piridinas/imunologia , Pirimidinas/imunologia , Pirimidinas/urina , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem , ZolpidemRESUMO
Adsorptive stripping voltammetric (AdSV) techniques were proposed for the direct quantitative determination of zopiclone (ZP) in spiked human urine and tablet dosage forms for first time. The electrochemical oxidation and determination of ZP were easily carried out on glassy carbon electrode (CGE) using a variety of voltammetric techniques. Different conditions were investigated to optimize the analytical determination of ZP. The dependence of the intensities of currents and potentials on pH, concentration, scan rate, deposition time, deposition potential, and nature of the buffer were investigated. Oxidation of ZP was found to be adsorptive-controlled and irreversible. The best results for the determination of ZP were obtained by using differential pulse adsorptive stripping (DPAdSV) and osteryoung square wave voltammetric (OSWAdSV) techniques in Britton-Robinson buffer at pH 7.08 after a pre-concentration period of 120 s at 0.60 V. The peak current showed a linear dependence on the ZP concentration in the range of 6 x 10(-7) to 2 x 10(-5) mol L(-1) for both techniques. The achieved limits of detection and quantitation were 2.78 x 10(-7) and 5.28 x 10(-7) mol L(-1) for DPAdSV; 1.70 x 10(-7) and 5.78 x10(-7) mol L(-1) for OSWAdSV, respectively. The proposed techniques were successfully applied to direct determination of ZP in tablet dosage form and spiked human urine samples. Excipients did not interfere with the determination. Precision and accuracy of the developed method were checked by recovery studies in tablet dosage forms and spiked urine samples.