Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma.

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 x 3.0 mm i.d., particle size 5 microm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml(-1) for all compounds analyzed. The limit of quantitation was 5 ng ml(-1) for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml(-1)) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of < or = 9.1%, an inter-assay precision of < or = 6.0% and an overall accuracy (relative error) of < 4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies.

Plasma concentrations and clinical effects after single oral doses of prazepam, clorazepate, and diazepam.

In a double-blind parallel-group pharmacokinetic and pharmacodynamic study, 31 healthy volunteers received single oral doses of prazepam (10 mg), clorazepate (7.5 mg), or diazepam (5 mg). Appearance in plasma of diazepam and of desmethyldiazepam was rapid after administration of diazepam and clorazepate, respectively, with peak plasma concentrations reached within an average of 1 hour. After oral prazepam, however, desmethyldiazepam appeared in blood slowly, with the highest mean concentration at 6 hours postdosage. Clinical self-ratings of fatigue and of "feeling spacey" were significantly different among groups, with changes over baseline being more marked with clorazepate and diazepam than with prazepam. Thus, differences in absorption rate of orally administered benzodiazepines can lead to differences in the intensity of single-dose effects, despite administration of doses that are equivalent in terms of long-term anxiolytic efficacy.

Duration of benzodiazepine clinical activity: lack of direct relationship with plasma half-life. A comparison of single vs divided dosage schedules of prazepam.

The anxiolytic activity and tolerance of two dosage schedules of prazepam, a long plasma half-life benzodiazepine, were compared under double-blind conditions in two groups of 10 inpatients each who met Research Diagnostic Criteria for Generalized Anxiety Disorder and presented chronic and severe symptomatology. Patients received prazepam 40 mg per day on one of two dosage schedules: divided dosage (DD) - 10 mg in the morning and at noon and 20 mg in the evening; or single dosage (SD) - 40 mg in the evening. The 3 weeks of therapy were preceded and followed by 1 week of wash-out for baseline and follow-up assessments, which were performed weekly with the Hamilton Anxiety Scale, Clinical Global Impression, rating of morning drowsiness and evening worsening of symptoms, and patient self-rating of anxiety by means of a visual analogue scale performed both in the morning and in the afternoon. The results showed a clear superiority of the DD over the SD schedule: better anxiolytic efficacy on the Hamilton Anxiety Scale (P less than 0.0005) and on both morning and afternoon visual analogue scales (P less than 0.01 and P less than 0.0002); less morning drowsiness (P less than 0.0001); and steadier anxiolytic effect during the daytime, as globally rated by the investigator (P less than 0.0001) or measured by morning-afternoon differences on the visual analogue scale (P less than 0.005). These results suggest that plasma pharmacokinetics alone may not be sufficient to predict the duration of benzodiazepine anxiolytic activity.

A salt-free isocratic high-pressure liquid chromatographic method for the quantitation of benzodiazepines in serum.

A simple, rapid high-pressure liquid chromatography (HPLC) method was developed for detecting and quantifying benzodiazepines in serum. Seven major benzodiazepines were extracted from spiked serum samples using solid-phase extraction with prazepam as the internal standard. The eluted drugs were then resolved isocratically by HPLC within 11 min using a reversed-phase C8 column with a mobile phase consisting of acetonitrile, methanol, water, and perchloric acid. All drugs gave responses that varied linearly with concentration over the ranges studied. Within-day imprecision (CV) varied from 3.9 to 14.9%, day-to-day CV from 4.8 to 17.0%, absolute recoveries from 67% to 114%, and detection limits from 10 to 110 ng/mL. Tricyclic antidepressants did not interfere, and clinical results were in good agreement with those obtained by a gas chromatographic method. The advantage of this method is that it uses a salt-free isocratic mobile phase that can be easily manipulated to effect difficult benzodiazepine separations.

Pharmacokinetic profiles of prazepam and 14C-prazepam in rat.

Pharmacokinetics of Prazepam and 14C-Prazepam were studied in rat. Prazepam was measured in blood and plasma by a gas-liquid chromatography assay with an electron capture detector. Its major metabolite, Desmethyldiazepam, was also determined in blood in the same way. Total radioactivity was measured in plasma by scintillation spectrometry. Pharmacokinetic analysis were carried out by two ways; according to compartmental pharmacokinetic models and by statistic moments.

Concentrations of N-descyclopropylmethylprazepam in whole-blood, plasma, and milk after administration of prazepam to humans.

After oral doses of 30 mg of prazepam to humans, N-descyclopropylmethylprazepam (desalkylprazepam, N-desmethyldiazepam) is the only major drug-related compound in plasma. Neither the parent drug, nor its major urinary metabolites were detected in plasma. The overall concentration-time profile of desalkylprazepam in the plasma of females was lower than, and significantly different (p less than 0.001) from that in the plasma of males. However, the mean peak desalkylprazepam concentrations in the plasma of females (265 ng ml-1 +/- 60 S.D.) were not significantly different (p greater than 0.05) from those in males (342 ng ml-1 +/- 60 S.D.). Concentrations declined in the plasma of either sex with similar half-lives (mean 60 h, range 37-93 h). Apparent plasma desalkylprazepam clearances were also similar (mean 60 h, range 37-93 h). Apparent plasma desalkylprazepam clearances were also similar (mean 1.09 l h-1), range 0.74-1.84 l h-1). At 12 h after the last of multiple doses of prazepam (60 mg d-1 for 3 days) to lactating women, mean plasma concentrations of desalkylprazepam were 823 ng ml-1 +/- 200 S.D. and declined with a mean half-life of about 60 h over the time-course studied. There was only slight uptake of desalkylprazepam into blood cells; plasma; whole blood concentration ratios were constant at about 1.6. Concentrations of desalkylprazepam in milk were low at about 10 per cent of the corresponding plasma levels (e.g. 86 ng ml-1 +/- 37 S.D. at 12 h). The data suggest that, expressed on a mg kg-1 basis, exposed neonates could receive about 4 per cent of the maternal dose of prazepam as desalkylprazepam.

Study of lot-to-lot reproducibilities of Bond Elut Certify and Clean Screen DAU mixed-mode solid-phase extraction columns in the extraction of drugs from whole blood.

The lot-to-lot reproducibilities of Bond Elut Certify and Clean Screen DAU columns are described. The recoveries of five test drugs obtained from twelve lots of Bond Elut Certify columns ranged from 84 to 104% with standard deviations of less than 9%. The recoveries of five test drugs obtained from six lots of Clean Screen DAU columns ranged from 81 to 103% with standard deviations of less than 7%. The 95% confidence intervals of the means as obtained by ANOVA demonstrate that there are no significant differences between the tested lots of Bond Elut Certify and Clean Screen DAU columns. Comparison of the two brands shows that both Bond Elut Certify and Clean Screen DAU columns are well acceptable for routine drug screening in systematic toxicological analysis, with a slightly higher overall recovery for the former.

Simultaneous high-performance liquid chromatographic analysis of flunitrazepam and four metabolites in serum.

A high-performance liquid chromatographic method for the simultaneous determination of flunitrazepam and four metabolites, desmethylflunitrazepam (DMF), 7-aminodesmethylflunitrazepam (7-NH2DMF), 7-aminoflunitrazepam (7-NH2F) and 3-hydroxyflunitrazepam (3-OHF), in serum is described. The method involves a simple extraction from alkalinized plasma (pH 9.5) into diethyl ether-chloroform (80:20, v/v). Prazepam was used as an internal standard for the quantification of the five compounds. Separation was achieved with a 10 microns RSil CN column (300 x 3.9 mn I.D.). The detection wavelength was set at 242 nm. The limits of detection ranged from 2.5 to 5 micrograms/l with a limit of quantification of 10 micrograms/l for all analytes.