UPLC-MS/MS analysis of the Michaelis-Menten kinetics of CYP3A-mediated midazolam 1'- and 4-hydroxylation in rat brain microsomes.

Midazolam (MDZ) is a short-acting benzodiazepine with rapid onset of action, which is metabolized by CYP3A isoenzymes to two hydroxylated metabolites, 1'-hydroxymidazolam and 4-hydroxymidazolam. The drug is also commonly used as a marker of CYP3A activity in the liver microsomes. However, the kinetics of CYP3A-mediated hydroxylation of MDZ in the brain, which contains much lower CYP content than the liver, have not been reported. In this study, UPLC-MS/MS and metabolic incubation methods were developed and validated for simultaneous measurement of low concentrations of both hydroxylated metabolites of MDZ in brain microsomes. Different concentrations of MDZ (1-500 µM) were incubated with rat brain microsomes (6.25 µg) and NADPH over a period of 10 min. After precipitation of the microsomal proteins with acetonitrile, which contained individual isotope-labeled internal standards for each metabolite, the analytes were separated on a C18 UPLC column and detected by a tandem mass spectrometer. Accurate quantitation of MDZ metabolism in the brain microsomes presented several challenges unique to this tissue, which were resolved. The optimized method showed validation results in accordance with the FDA acceptance criteria, with a linearity ranging from 1 to 100 nM and a lower limit of quantitation of 0.4 pg on the column for each of the two metabolites. The method was successfully used to determine the Michaelis-Menten (MM) kinetics of MDZ 1'- and 4-hydroxylase activities in rat brain microsomes (n = 5) for the first time. The 4-hydroxylated metabolite had 2.4 fold higher maximum velocity (p < 0.01) and 1.9 fold higher (p < 0.05) MM constant values than the 1'-hydroxylated metabolite. However, intrinsic clearance values of the two metabolites were similar. The optimized analytical and metabolic incubation methods reported here may be used to study the effects of various pathophysiological and pharmacological factors on the CYP3A-mediated metabolism of MDZ in the brain.

Drug detection in decomposed cadavers confirms testimonial evidence in a case of serial homicides.

Toxicology investigation on human's buried dead bodies is a rare and challenging task in the forensic field. As requested by the Judicial Authority, this work aimed to verify testimonial evidence that emerged during a criminal investigation involving multiple murder cases. The statements indicated an improper medical administration of one or more alleged drugs (propofol, morphine, diazepam, and midazolam) which presumably caused the deaths. Since the supposed crimes took place several years before, the task of the present work was to obtain results to support the charges. The analyses involved 18 biological samples taken from four exhumed bodies, three of which were female and one male, each buried in a different date and mode. Each sample was treated with specific purification and extraction techniques (LLE - SPE) after the addition of the deuterated analogs of the searched analytes (propofol-d17, morphine-d3, diazepam-d5, midazolam-d4) as internal standards. Afterwards, the extracts were subjected to qualitative analysis by gas chromatography-mass spectrometry-Electron Impact (GC/MS - EI), both in full scan and SIM mode. Propofol, morphine, and diazepam were identified in the corpses. It supports testimonials that were administered just before the deaths occurred.

Near-fatal Intoxication with the "New" Synthetic Opioid U-47700: The First Reported Case in the Czech Republic.

Recreational use of the potent synthetic opioid 3,4- dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide (U-47700) is rising, accompanied by increasingly frequent cases of serious intoxication. This article reports a case of near-fatal U-47700 intoxication. A man was found unconscious (with drug powder residues). After 40 h in hospital (including 12 h of supported ventilation), he recovered and was discharged. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or gas chromatography/mass spectrometry (GC/MS) were used to detect and quantify substances in powders, serum and urine. Powders contained U-47700 and two synthetic cannabinoids. Serum and urine were positive for U-47700 (351.0 ng/mL), citalopram (

LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 and P-glycoprotein probe substrates and their metabolites using subtherapeutic doses.

Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC-MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01-5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/*3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D6*4/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.

Development of a simultaneous LC-MS/MS method to predict in vivo drug-drug interaction in mice.

Cocktail substrates are useful in investigating drug-drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC-MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0-240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUCratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC-MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.

Avaliação da dexmedetomidina e do tramadol, associados ao midazolam, em gatas anestesiadas com isoflurano e submetidas à ovário-histerectomia / Evaluation of dexmedetomidine and tramadol, associated with midazolam, in cats anesthetized with isoflurane, undergoing ovariohysterectomy

Objetivou-se comparar as alterações cardiorrespiratórias e a analgesia pós-operatória promovidas pela dexmedetomidina e pelo tramadol, quando associados ao midazolam, em felinas. Para tal, foram selecionadas 18 gatas hígidas, divididas em dois grupos randomizados: GDM, tratadas com dexmedetomidina (10µg/kg) e GTM, tratadas com tramadol (2mg/kg), ambos associados a midazolam (0,2mg/kg,) IM. Após 15 minutos, procedeu-se à indução anestésica com propofol (1,46±0,79mL), mantendo-se a anestesia com isoflurano. As felinas foram submetidas à ovário-histerectomia, registrando-se as variáveis cardiorrespiratórias 15 minutos após a MPA (M0), 15 minutos após a indução (M15) e sequencialmente a cada cinco minutos, até o término do procedimento cirúrgico (M20, M25, M30, M35 e M40). A avaliação da dor iniciou-se 30 minutos após o término do procedimento cirúrgico (MP30) e sequencialmente em intervalos de 30 minutos (MP60, MP90, MP120). A partir do MP120, as avaliações foram registradas a cada hora (MP180, MP240 e MP360). A associação dexmedetomidina-midazolam infere diminuição inicial de frequência cardíaca (FC) sem significado clínico e está relacionada à sedação mais pronunciada, à analgesia menor e menos duradoura e a episódios de êmese, quando comparada à associação tramadol-midazolam. Ambos os protocolos denotaram estabilidade cardiorrespiratória e podem ser considerados seguros em felinas submetidas à ovário-histectomia.(AU)

The aim of this study was to compare cardiorespiratory changes and post-operative analgesia provided by dexmedetomidine or tramadol, associated with midazolam, in female cats. For that purpose, 18 healthy cats were assigned to two randomized groups: GDM, which received dexmedetomidine (10 µg/kg) and GTM, which received tramadol (2 mg/kg), both associated with midazolam (0.2 mg/kg) IM. After 15 minutes, anesthesia was induced with propofol (1.46±0.79 mL) and maintained with isofluorane. Ovariohysterectomy was performed and cardiorespiratory variables were registered 15 minutes after pre-anesthetic medication (M0), 15 minutes after anesthetic induction (M15), and every five minutes until the end of the surgical procedure (M20, M25, M30, M35 e M40). Pain evaluation started 30 minutes after the surgery (MP30) and sequentially at thirty-minute intervals (MP60, MP90, MP120). After MP120, each evaluation was registered at every hour (MP180, MP240 e MP360). Dexmedetomidine-midazolam association results in decreases on initial heart rate (HR) without clinical relevance and it is related to pronounced sedation, poor and less durable antinociception and vomiting events, when compared to tramadol-midazolam association. Both protocols indicate cardiorespiratoy stability and safety in cats undergoing ovariohysterectomy.(AU)

Characterization of degradation products of midazolam maleate by UHPLC-HR-IT-MSn and NMR.

Forced degradation studies on midazolam maleate were carried out according to ICH guidelines. Midazolam maleate was subjected to acidic and basic hydrolysis, oxidation, photolysis, high humidity and thermal stress conditions, and the resulting degradation products were investigated by HPLC. Significant degradation of the drug was observed under acidic/basic hydrolysis and thermal stress conditions. The thermal degradation product (Impurity I) was isolated using column chromatography and its structure was elucidated by UHPLC-HRIT-MSn and extensive NMR studies, which was not reported in previous literatures. The acidic/basic hydrolytic degradation product (Impurity II) was characterized by UHPLC-HR-IT-MSn technique and previous literature. The fragmentation pathways of these two degradation products are also described in the paper.

Identification of Drugs in Parenteral Pharmaceutical Preparations from a Quality Assurance and a Diversion Program by Direct Analysis in Real-Time AccuTOFTM-Mass Spectrometry (DART-MS).

In healthcare settings drug diversion and impairment of physicians are major concerns requiring a rapid and efficient method for surveillance and detection. A Direct Analysis in Real Time ion source coupled to a JEOL AccuTOFTM time-of-flight mass spectrometer (DART-MS) method was developed to screen parenteral pharmaceutical formulations for potential drug diversion. Parenteral pharmaceutical formulations are also known as injectable formulations and are used with intravenous, subcutaneous, intramuscular and intra-articular administration. A library was created using the mass spectra data collected by a DART-MS operated in switching mode at 20, 60 and 90 V settings. This library contained 17 commonly encountered drugs in parenteral pharmaceutical formulations that included the surgical analgesic: fentanyl, hydromorphone and morphine; anesthetic: baclofen, bupivacaine, ketamine, midazolam, ropivacaine and succinylcholine; and a mixture of other drug classes: caffeine, clonidine, dexamethasone, ephedrine, heparin, methadone, oxytocin and phenylephrine. Randomly selected 200 de-identified parenteral pharmaceutical formulations containing one or more drugs were submitted for analysis to the FIRM Toxicology Laboratory at Virginia Commonwealth University Health and were screened using the DART-MS. The drug contents of the de-identified formulations were previously confirmed by a published high performance liquid chromatography (HPLC) method. The drugs in the formulations were rapidly and successfully identified using the generated library. The DART-MS and HPLC results were in complete agreement for all 200 parenteral pharmaceutical formulations.

Avaliação dos valores de troponina I, eletrocardiograma e ecocardiograma em felinos sedados com cetamina e midazolam, suplementados ou não com oxigênio / Troponin I, electrocardiography and echocardiography values in felines sedated with ketamine and midazolam, supplemented or not with oxygen

Lesões no miocárdio, causadas por baixa perfusão e oxigenação cardíaca, podem ser ocasionadas por fármacos anestésicos, como a cetamina. Essas lesões podem ser identificadas por meio de biomarcadores específicos e, dentre estes, destaca-se a troponina I. O objetivo deste estudo foi avaliar as alterações cardiovasculares com base nos valores de troponina I (TnI), eletrocardiograma (ECG) e ecocardiograma em gatos sedados com cetamina e midazolam, suplementados ou não com oxigênio. Utilizaram-se 12 gatos machos, hígidos, nos quais se avaliaram os valores de troponina I, eletro e ecocardiografia, frequência cardíaca (FC) e pressão arterial sistólica (PAS) no momento basal (M0). Na sequência, os animais foram sedados com a associação de 10mg.kg-1 de cetamina e 0,5mg.kg-1 de midazolam pela via intramuscular. Decorridos aproximadamente 10 minutos, os animais foram alocados aleatoriamente em dois grupos: com e sem suplementação de oxigênio via máscara facial (GCO e GSO, respectivamente), sendo submetidos novamente aos exames citados. Foram coletadas amostras sanguíneas, para dosagem de TnI em seis, 12 e 24 horas após a administração dos agentes anestésicos. Não foram observadas alterações significativas na FC, na PAS e no ECG após a administração dos tratamentos em ambos os grupos. Os valores médios de TnI elevaram-se significativamente em T6 quando comparados ao basal em ambos os grupos, com médias de 0,507±0,335ng/mL no GSO e 0,777±0,505ng/mL no GCO. Na ecocardiografia, o débito cardíaco (DC) reduziu em M1 em ambos os grupos, quando comparados aos valores basais, sendo M0 0,472±0,115 e M1 0,234±0,08 no GSO e M0 0,356±0,095 e M1 0,222±0,09 no GCO, expressos em L/min. Conclui-se que a administração de cetamina e midazolam em gatos hígidos não promove alterações eletrocardiográficas, aumenta os valores de troponina I, com pico de seis horas após a administração, reduz o débito cardíaco, e que a suplementação de oxigênio 100% via máscara facial não atenua tais alterações.

Myocardium injuries caused by low myocardial oxygenation and perfusion might be induced by anesthetics agents like ketamine. These injuries can be detected by specific biomarkers and, among them, troponin I. The aim of this study was to evaluate the cardiovascular changes based on troponin I (TnI) values, electrocardiography (ECG) and echocardiography in cats sedated with ketamine and midazolam, supplemented or not with oxygen. Blood samples were collected from 12 intact male healthy cats for troponin I (T0) and they were then submitted to electrocardiographic and echocardiographic evaluation, as well as measurements of heart rate (HR) and systolic blood pressure (SBP) (M0). Subsequently, they were ketamine-midazolam (10mg.kg-1 and 0,5 mg.kg-1 respectively) anesthetized by intramuscular route. After about 10 minutes, the animals were randomly allocated into two groups with or without oxygen supplementation (GCO or GSO, respectively), again being subjected to the tests mentioned. Blood samples for troponin I were collected at 6, 12 and 24 hours after sedation. HR, SBP and ECG did not change among groups. The TnI values rise significantly in T6 comparing to baselines in both groups (0,507±0,335 ng/mL in GSO and 0,777±0,505 ng/mL in GCO). In echocardiography, the cardiac output decreased at M1, in both groups compared to baseline (M0 0,472±0,115 and M1 0,234±0,08 in GSO and M0 0,356±0,095 and 0,222±0,09 in GCO, L/min). We concluded that ketamine and midazolam sedation in healthy cats did not cause changes electrocardiography, increase troponin I values, with an 6 hours peak after administration, reduces cardiac output and oxygen supplementation, via facial mask, did not attenuated these alterations.

Applying 'Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra' (SWATH) for systematic toxicological analysis with liquid chromatography-high-resolution tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an indispensable analytical technique in clinical and forensic toxicology for detection and identification of potentially toxic or harmful compounds. Particularly, non-target LC-MS/MS assays enable extensive and universal screening requested in systematic toxicological analysis. An integral part of the identification process is the generation of information-rich product ion spectra which can be searched against libraries of reference mass spectra. Usually, 'data-dependent acquisition' (DDA) strategies are applied for automated data acquisition. In this study, the 'data-independent acquisition' (DIA) method 'Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra' (SWATH) was combined with LC-MS/MS on a quadrupole-quadrupole-time-of-flight (QqTOF) instrument for acquiring informative high-resolution tandem mass spectra. SWATH performs data-independent fragmentation of all precursor ions entering the mass spectrometer in 21m/z isolation windows. The whole m/z range of interest is covered by continuous stepping of the isolation window. This allows numerous repeat analyses of each window during the elution of a single chromatographic peak and results in a complete fragment ion map of the sample. Compounds and samples typically encountered in forensic casework were used to assess performance characteristics of LC-MS/MS with SWATH. Our experiments clearly revealed that SWATH is a sensitive and specific identification technique. SWATH is capable of identifying more compounds at lower concentration levels than DDA does. The dynamic range of SWATH was estimated to be three orders of magnitude. Furthermore, the >600,000 SWATH spectra matched led to only 408 incorrect calls (false positive rate = 0.06 %). Deconvolution of generated ion maps was found to be essential for unravelling the full identification power of LC-MS/MS with SWATH. With the available software, however, only semi-automated deconvolution was enabled, which rendered data interpretation a laborious and time-consuming process.

Comparison of information-dependent acquisition, SWATH, and MS(All) techniques in metabolite identification study employing ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS(2) (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MS(All) (also called MS(E)) techniques. We compared these three techniques in their capabilities to produce comprehensive MS(2) data by assessing both metabolite MS(2) acquisition hit rate and the quality of MS(2) spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MS(All)) using an ultrahigh-performance liquid chromatography-qudrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS(2) acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS(2) acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS(2) acquisition with either the SWATH or MS(All) method. However, the quality of the acquired MS(2) spectra decreased in the order of IDA, SWATH, and MS(All) methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS(2) spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MS(All) methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS(2) spectra but with a lower MS(2) acquisition hit rate than the other two methods. SWATH outperformed the MS(All) method given its better quality of MS(2) spectra with an identical MS(2) acquisition hit rate.

Simultaneous determination of probe drugs, metabolites, inhibitors and inducer in human plasma by liquid chromatography/tandem mass spectrometry and its application to pharmacokinetic study.

Cytochrome P450 3A4 (CYP3A4) and UDP-glucuronosyltransferase 1A1 (UGT1A1) are important enzymes responsible for the metabolism of many xenobiotics. To investigate their induction and inhibition properties, administering probe drugs and monitoring their concentration in plasma under the effects of inducers/inhibitors is the gold standard method. A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of midazolam, raltegravir (probe drugs for CYP3A4 and UGT1A1), their major metabolites, 1'-hydroxymidazolam, 1'-hydroxymidazolam glucuronide and raltegravir glucuronide, rifampicin (inducer), ritonavir and ketoconazole (inhibitors). Analytes were extracted from 100µl of plasma using solid-phase extraction followed by chromatographic separation on a reversed-phase C18 column (50mm×2.1mm, particle size 1.8µm). The mass spectrometer was operated under positive ionization mode. Excellent linearity (r(2)≥0.995) was achieved for all. The method was validated and found to be accurate (88-111%), precise (CV%<13) and selective. Matrix effect was acceptable (88-118%) and analytes recovery was reproducible (60-95%). Analytes in plasma were also found to be stable in the autosampler (6°C for 48h) and after two freeze-thaw cycles. We have developed a robust analytical method to simultaneously quantify probes, inducer and inhibitor of important drug metabolism enzymes. The method was successfully applied in a clinical study to investigate the degree of induction and inhibition of CYP3A4 and UGT1A1 among ethnic groups in Singapore.

Synthesis of the impurities during the manufacture of bulk drug midazolam and separation of these impurities by HPLC.

During the manufacture of bulk drug midazolam various impurities arised that can be the related products or degradation products. Structures of eight impurities that can arise during the manufacture of bulk drug midazolam were proposed. In the present work, synthesis of these impurities and their characterization by different spectroscopic techniques have been done. HPLC method was developed for the separation of impurities from the bulk drug. The developed method separates midazolam from its eight impurities/degradation products within a run time of 45 min.

Development and validation of a LC-MS/MS method for the in vitro analysis of 1-hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures.

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1-hydroxymidazolam in plasma are often determined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). However, validated LC-MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1-hydroxymidazolam. Therefore, the aim was to validate and optimize an LC-MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid-liquid extraction with tert-butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed-phase chromatography consisting of a Zorbax Extend-C18 column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC-MS/MS method was validated over linear range of 1.0-500 nm for 1-hydroxymidazolam. The results revealed good inter-assay accuracy (≥85% and ≤115%) and within-day and between-day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (ß-carotene, green tea, milk thistle and St. John's wort) was determined.

Suicide by self-administration of a drug mixture (propofol, midazolam, and zolpidem) in an anesthesiologist: the first case report in Italy.

The authors report an unusual case of suicide of an anesthesiologist, in which the suicide manner and means depend upon the victim's occupation. This is the first case report published in Italy of a death involving propofol and other drugs. The anesthesiologist was found dead with an empty drip still inserted in the hand and another one near his body. Forensic and toxicological findings suggested that the cause of death was a respiratory depression due to a self-administration of a rapidly infused lethal drug mixture. Analytical drug quantification was performed by gas chromatography-mass spectrometry. Blood analysis revealed: zolpidem (0.86 µg/mL), propofol (0.30 µg/mL), midazolam (0.08 µg/mL), thiopental (0.03 µg/mL), and amitriptyline (0.07 µg/mL). Adipose tissue and hair analysis suggested a previous and repeated use of these drugs verifying the fact that in Italy recreational abuse of anesthetic and sedative agents in health care practitioners is becoming an increasing problem.

Determination of illegally abused sedative-hypnotics in hair samples from drug offenders.

As several sedative-hypnotics are distributed illegally and are available domestically through media like the internet, their abuse is becoming a serious social problem. In the present study, four legal cases involving abuse of diazepam, midazolam, and/or zolpidem were proved by hair analysis using a simultaneous quantification method for the determination of diazepam (and its metabolites), lorazepam, midazolam, and zolpidem, which are often illegally abused in Korea, in hair that was developed and validated. Drugs and metabolites in hair were extracted using methanol followed by solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed using gas chromatography-mass spectrometry in selected ion monitoring mode. The validation parameters of the method, including selectivity, linearity, limits of detection and quantification (LOQ), recovery, intra- and interassay precision and accuracy, and processed sample stability, were satisfactory. Moreover, the developed method was successfully applied to actual cases. In case 1, which involved a pop singer who was detained for suspected drug abuse, the concentrations of diazepam and nordiazepam were 5.7 and 2.0 ng/mg in nonpigmented hair and 6.6 and 1.8 ng/mg in pigmented hair, respectively. In case 2, 0.4 ng/mg zolpidem was detected in hair from a drug abuser who purchased illegally through the internet, and 0.2 ng/mg midazolam was detected in hair from an illegal drug seller in case 3. In case 4, diazepam (lower than the LOQ), nordiazepam (0.7 ng/mg), and zolpidem (0.7 ng/mg) were detected in hair from a medical doctor who abused drugs using forged prescriptions.

Distribution of metoprolol, tramadol, and midazolam in human autopsy material.

In this study it was possible to measure the distribution of metoprolol, tramadol, and midazolam in human directly in several compartments. In the legal medicine autopsy material is normally investigated to find out the cause of death. But the results of corresponding toxicology measurements often involve more information. With screening methods drugs were detected without connection to the cause of death. The deceased had either a continual therapeutic treatment, a treatment during an operation, or an unsuccessful urgent therapy. A liquid/liquid extraction and a LC/MS/MS method were developed for the determination of the drug concentrations. Different autopsy materials of about 120 cases were investigated. Most frequently the drugs metoprolol, tramadol, and midazolam could be proved and determined simultaneously. Metoprolol was found in seven cases, tramadol in seven cases and midazolam in thirteen cases. The dosage of the drugs was unknown. Therefore and because of the low number of cases statistic calculations were not meaningful and an individual case study was necessary. In all cases with oral metoprolol application the patients probably took a normal customary continuous dosage. The concentrations of tramadol in blood were in the toxic range in three cases. The distribution of tramadol in the compartments was independent of the dosage. The time between oral intake of metoprolol or tramadol and death was unknown. With the distribution pattern of metoprolol in the compartments it was possible to estimate the duration between drug intake and death. In most cases midazolam was given intravenously during an operation or an unsuccessful urgent therapy. Sometimes the time between dosage and death was documented. The duration between application of the drug and death played the crucial role for the distribution of midazolam in the compartments. Measurements of drug concentrations in human autopsy material deepen the knowledge of the respective drugs' pharmacokinetics.

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1'-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry.

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 microL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 microL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm x 100 mm, 3.5 microm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100-250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.

Validation of a new HPLC method for determination of midazolam and its metabolites: application to determine its pharmacokinetics in human and measure hepatic CYP3A activity in rabbits.

Midazolam (MDZ) is a commonly used benzodiazepine in clinical practice. In addition, its metabolic oxidation is used as a surrogate marker for Cytochrome P450 (CYP) 3A enzyme activity as well. Thus, a new simpler method to measure MDZ and its metabolites is welcomed. Herein we report a new and simple HPLC method with ultraviolet detection for the simultaneous determination of midazolam and its hydroxyl metabolites using lorazepam as an internal standard. A liquid-liquid extraction was used to extract the compounds from rabbit hepatic microsomes and human plasma. The separation was performed on a Zorbax Eclipse XDB C18 column using a mobile phase composed of 0.05 M Na2PO4 (pH 4.5) and acetonitrile mixture (67:33) pumped at 1.2 mL/min. The calibration curves showed good linearity with correlation coefficient higher than 0.999 for all analytes in the range 10-500 ng/mL. Accuracy in the measurement of quality control (QC) samples was in the range 95-106% of the nominal values. The intra-day and inter-day precisions in the measurement of QC samples were less than 11% coefficient of variation. Although less sensitive than GC-MS, the proposed method was adequately sensitive to measure midazolam hydroxylase activity as a marker for CYP3A activity, and was applied to measure midazolam pharmacokinetics in human plasma.

Cyclodextrin-based potentiometric sensors for midazolam and diazepam.

In this work the implementation of benzodiazepine ion-selective electrodes for pharmaceutical formulations control is described. The solid-contact electrodes for midazolam and diazepam are based on polymeric membranes incorporating respectively beta-cyclodextrin and (2-hydroxiproyl)-gamma-cyclodextrin as ionophores, 2-fluorophenyl 2-nitrophenyl ether as plasticizer and potassium tetrakis (p-chlorophenyl) borate as ionic additive. For conventionally shaped midazolam electrode a slope of 61.9+/-1.3 mVdec(-1), a LLLR of 5.7+/-2.7 x 10(-4)gL(-1) and pH range of 2.6-5.4 was obtained, while the corresponding values for diazepam electrodes were of 67.6+/-3.0 mVdec(-1), 4.9+/-1.5 x 10(-2)gL(-1) and 1.9-2.7 pH units, respectively. Membrane optimization was based on the molar ratio between the ionophore and additive for midazolam and on inclusion cavity of cyclodextrin for diazepam. The miniaturization of the above-described electrodes gave rise to potentiometric detectors for sequential-injection lab-on-valve system with similar characteristics albeit the useful lifetime shortened from 1 year to approximately 15 days under continuous operation. The optimized flow conditions were achieved for sample injection volumes of 20 microL propelled towards the detection cell at the flow rate of 16 microLs(-1) during 80s. Real sample analysis revealed statistical accuracy and between-days precision comparable to the general used chromatographic-based procedure.