Benzodiazepine stability in postmortem samples stored at different temperatures.

Benzodiazepine (lorazepam, estazolam, chlordiazepoxide, and ketazolam) stability was studied in postmortem blood, bile, and vitreous humor stored at different temperatures over six months. The influence of NaF, in blood and bile samples, was also investigated. A solid-phase extraction technique was used on all the studied samples, and benzodiazepine quantification was performed by high-performance liquid chromatography-diode-array detection. Benzodiazepine concentration remained almost stable in all samples stored at -20°C and -80°C. Estazolam appeared to be a stable benzodiazepine during the six-month study, and ketazolam proved to be the most unstable benzodiazepine. A 100% loss of ketazolam occurred in all samples stored over 1 or 2 weeks at room temperature and over 8 or 12 weeks at 4°C, with the simultaneous detection of diazepam. Chlordiazepoxide suffered complete degradation in all samples, except preserved bile samples, stored at room temperature. Samples stored at 4°C for 6 months had a 29-100% decrease in chlordiazepoxide concentration. The data obtained suggest that results from samples with these benzodiazepines stored long-term should be cautiously interpreted. Bile and vitreous humor proved to be the most advantageous samples in cases where degradation of benzodiazepines by microorganisms may occur.

[Stability of estazolam in dog tissues preserved in formaldehyde solution].

OBJECTIVE: To investigate the stability of estazolam in biological samples preserved in formaldehyde solution. METHODS: The dog was given intragastric administration of estazolam with a dose of 37.6 mg/kg and killed 2 h later. Heart, liver, kidney and brain of the dog were cut up into 1 g and preserved in 4% formaldehyde solution respectively. The content of estazolam in biological samples and formaldehyde solution were analyzed by HPLC at different times. RESULTS: The content of estazolam in heart, liver, kidney and brain or in formaldehyde solution reduced gradually followed with the extention of preservation time. At the 63rd day, estazolam content in four tissues were 0.8%, 1.7%, 1.0% and 2.2% of the original content respectively. CONCLUSION: Estazolam in tissues can diffuse into formaldehyde solution and decomposed quickly, so biological samples contained estazolam should not be preserved in formaldehyde solution.

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics.

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.

Determination of hypnotic benzodiazepines (alprazolam, estazolam, and midazolam) and their metabolites in rat hair and plasma by reversed-phase liquid-chromatography with electrospray ionization mass spectrometry.

Sensitive determination of benzodiazepines i.e., alprazolam (ALP), estazolam (EST), and midazolam (MDZ), and their metabolites, was carried out by reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The chromatography separations were achieved using a semi-micro HPLC column (3 microm particle size; 100 x 2.0 mm, i.d.) with acetonitrile-water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at protonated-molecular ions [M+H](+) of parent drugs and the metabolites. The proposed procedure was applied to the determination in hair shaft of Dark Agouti rats after intraperitoneal (i.p.) administration of benzodiazepines twice a day for 5 days. Various metabolites together with parent drugs were identified in the hair shaft, 1-hydroxyalprazolam (1-HA) and 4-hydroxyalprazolam (4-HA) from ALP administration; 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-one (K-EST) from EST administration; 1-hydroxymidazolam (1-HM) and 4-hydroxymidazolam (4-HM) from MDZ administration. A few unknown metabolites were also detected in the hair samples. These structures were elucidated with acetylation using acetic anhydride and pyridine. The time course studies of parent drugs and the metabolites in both hair root and plasma were also carried out after single i.p. administration of benzodiazepines. The results suggested that the concentrations of parent drugs and the metabolites in the hair samples were mainly dependent upon those in the plasma.

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry.

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.