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.

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.

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.

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.

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.

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.

HPLC determination of midazolam and its three hydroxy metabolites in perfusion medium and plasma from rats.

A new, simple, rapid, sensitive, and repeatable isocratic reverse-phase HPLC method was developed and validated for simultaneous determination of midazolam and its main three hydroxylated metabolites, i.e. 1'-hydroxymidazolam, 4-hydroxymidazolam, and 1',4-dihydroxymidazolam in rat liver perfusate and also plasma. Diazepam was used as an internal standard to ensure precision and accuracy of this method. Analytes were extracted from alkalinized samples into diethyl ether using single-step liquid-liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and sodium acetate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 7.9 and 19.6 microg/L for midazolam and its hydroxy metabolites. The overall recovery for the analytes exceeded 92%, for concentrations twice the limits of detection. The intra- and inter-day precision at three different concentrations never exceeded 8 and 11% variation, respectively. This method is applicable for modeling and description of possible pharmacological interactions on rat (CYP3A1/2) or human (CYP3A4/5) cytochrome P450 enzymes.

The detection of sedatives in hair and nail samples using tandem LC-MS-MS.

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination.

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).

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.

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.

The compatibility and stability of midazolam and dexamethasone in infusion solutions.

The delivery of subcutaneous medication by continuous infusion is common in palliative medicine. Many centers combine multiple medications, but the analytical confirmation of the compatibility and stability of these combinations has rarely been performed. This study examined the compatibility and stability of midazolam and dexamethasone using high performance liquid chromatography. Nine different solutions were prepared in polypropylene syringes by combining these two drugs with 0.9% sodium chloride. When these two drugs were combined in a syringe, there was significant loss of midazolam over 48 hours, with only 60-80% of the initial concentration remaining in syringes stored at 35-39 degrees C. This study demonstrates that cloudiness of a solution is not the only predictor of drug loss and that drug loss may occur even in solutions that remain clear at time of preparation. The clinical implications of these results are that dexamethasone and midazolam should not be combined in syringe driver solutions.

Determination of midazolam and its metabolite as a probe for cytochrome P450 3A4 phenotype by liquid chromatography-mass spectrometry.

This study demonstrated the analysis of midazolam and its metabolites by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. The analytical column was a YMC-Pak Pro C18 (50 mm x 2.0 mm i.d.) using 10 mM ammonium acetate (pH 4.8)-methanol (1:1) at a flow rate of 0.2 ml min(-1). The drift voltage was 100 V. The sampling aperture was heated at 110 degrees C and the shield temperature was 230 degrees C. The lower limits for the detection of midazolam and 1'-hydroxymidazolam were 26.3 and 112.76 pg injected, respectively. The calibration curves for midazolam and 1'-hydroxymidazolam were linear in the range of 0.1-5 microg ml(-1). Within-day relative standard deviations was less than 7%. The method was applied to the determination of midazolam in monkey plasma, and the analysis of midazolam and its metabolites in an in vitro study with recombinant cytochrome P450 (CYP) 3A4. This method is sufficiently sensitive and useful to elucidate the kinetics of midazolam metabolite formation. We also investigated the effect of propofol on the metabolism of midazolam using recombinant CYP3A4. Propofol competitively inhibited the metabolism of midazolam to 1'-hydroxymidazolam by CYP3A4.

Determination of droperidol in plasma by liquid chromatography.

A reversed-phase high performance liquid chromatographic method is described for the determination of droperidol concentrations in plasma. Following extraction, separation of droperidol and the internal standard flurazepam was achieved with a Spherisorb Nitrile, 5 microns, S5CN 250 mm x 4.6 mm column at 200 nm. The mobile phase was phosphate buffer (0.05 M, pH 2.4), acetonitrile and ethanol (65:20:15, v/v/v). The assay was rapid, sensitive and linear over the range 2-4000 ng ml-1. Precision of the assay expressed as the intra- and inter-day relative standard deviations (%RSD) did not exceed 10%. Flunitrazepam, midazolam and nitrazepam were also resolved with this technique and did not interfere with droperidol or flurazepam. Resolution of all five compounds was complete in less than 6 min. The assay was used to study the pharmacokinetics of high dose droperidol infusions during and after cardiac surgery.

Stability of parenteral midazolam in an oral formulation.

Midazolam is increasingly being used for oral sedation in pediatric dentistry. Unfortunately, it is available only as a parenteral formulation in Canada and the United States. Preparation of the parenteral solution for oral use is not uniform and leads the clinician to question the stability of this drug when used in conjunction with these vehicles. Therefore, the purpose of this study was to investigate the chemical stability of parenteral midazolam as an oral formulation to determine its expiry date. This was evaluated using a validated stability-indicating liquid chromatographic method. Midazolam was diluted in orange-flavored syrup to yield concentrations of 0.35, 0.64, and 1.03 mg/ml and then stored at room temperature. Samples were drawn on each of 9 study days (0, 1, 2, 6, 7, 9, 13, 21, and 102) and chromatographed. On each study day, solutions were inspected visually for changes in color, clarity, and appearance of particulate matter. Midazolam concentrations were considered within acceptable limits if they were not less than 90% of the initial concentration. Over the 102-day study period, there was no significant change in concentration in any of the solutions. On day 102, the remaining midazolam was within 7% of the day zero concentration. Therefore, these formulations of midazolam are stable at room temperature for a period of 102 days and would be suitable for clinical use.

[A case of suicide by an intravenous injection of pancuronium].

A 25-year-old male anesthesiologist was found dead in his room with an intravenous drip line connected with his leg. There were several empty ampules of pancuronium, midazolam and buprenorphine around him. Pancuronium and midazolam in the blood and urine samples collected at postmortem examination were detected by spectrofluorometry and ECD-gas chromatography, respectively. The concentrations of pancuronium and midazolam were 0.3 micrograms/g and 0.12 micrograms/g in the blood, respectively; 0.9 micrograms/g and 0.05 micrograms/g in the urine, respectively. The cause of death was determined to be asphyxia due to respiratory muscle paralysis caused by pancuronium.