Optimization of dispersive liquid-liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC-UV.

A simple, rapid, and sensitive method based on dispersive liquid-liquid microextraction combined with HPLC-UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 µL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min(-1), neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C18 column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min(-1) The linear response (r(2) > 0.998) was achieved in the range of 0.005-10 µg mL(-1) with detection limit 0.0005 µg mL(-1) The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3-102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid-liquid microextraction combined with HPLC-UV is a fast and simple method for the determination of chlordiazepoxide in real samples.

Application of Ultrasound-Assisted Surfactant-Enhanced Emulsification Microextraction Based on Solidification of Floating Organic Droplets and High Performance Liquid Chromatography for Preconcentration and Determination of Alprazolam and Chlordiazepoxide in Human Serum Samples.

An improved microextraction method is proposed on the basis of ultrasound-assisted surfactant-enhanced emulsification and solidification of a floating organic droplet procedure combined with high performance liquid chromatography for the preconcentration and quantification of alprazolam (ALP) and chlordiazepoxide (CHL) present in a number of human serum samples. Several parameters affecting the extraction efficiency were investigated by the Plackett -Burman factorial design as the screening design. Then the response surface methodology based on the Box-Behnken design was used to optimize the effective parameters in the proposed procedure. The limits of detection for the proposed method were found to be 3.0 and 3.1 ng mL-1 for CHL and ALP, respectively. The calibration curves obtained for the method were linear in the ranges of 10.0-3,500.0 and 10.0-3,000.0 ng mL-1 for CHL and ALP, respectively, with a good determination coefficient. The recoveries of the drugs in the spiked human serum samples were above 93.0%. The developed method was successfully applied to the analysis of these studied drugs in human serum samples. The pre-treatment of the serum samples was performed using acetonitrile to remove the proteins. The proposed procedure was an accurate and reliable one for the determination and preconcentration of these drugs in blood samples.

Preconcentration and determination of chlordiazepoxide and diazepam drugs using dispersive nanomaterial-ultrasound assisted microextraction method followed by high performance liquid chromatography.

Benzodiazepines (BDs) are used widely in clinical practice, due to their multiple pharmacological functions. In this study a dispersive nanomaterial-ultrasound assisted- microextraction (DNUM) method followed by high performance liquid chromatography (HPLC) was used for the preconcentration and determination of chlordiazepoxide and diazepam drugs from urine and plasma samples. Various parameters such as amount of adsorbent (mg: ZnS-AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), and desorption volume (mL) were investigated by fractional factorial design (FFD) and central composite design (CCD). Regression models and desirability functions (DF) were applied to find the best experimental conditions for providing the maximum extraction recovery (ER). Under the optimal conditions a linear calibration curve were obtained in the range of 0.005-10µgmL(-1) and 0.006-10µgmL(-1) for chlordiazepoxide and diazepam, respectively. To demonstrate the analytical performance, figures of merits of the proposed method in urine and plasma spiked with chlordiazepoxide and diazepam were investigated. The limits of detection of chlordiazepoxide and diazepam in urine and plasma were ranged from 0.0012 to 0.0015µgmL(-1), respectively.

Plasma and cerebrospinal fluid concentrations of chlordiazepoxide and its metabolites in surgical patients.

Thirty otherwise healthy patients received a 100-mg oral dose of chlordiazepoxide HCl just prior to surgical procedures using spinal anesthesia. Fourteen of these patients had also received 100 mg on the night before surgery. Simultaneous samples of venous blood and cerebrospinal fluid (CSF) were taken immediately prior to injection of spinal anesthesia and were assayed for concentrations of chlordiazepoxide (CDX) and its major metabolite, desmethylchlordiazepoxide. Plasma concentrations of CDX ranged from 2.32 to 13.34 mug/ml. Simultaneous CSF concentrations were considerably lower, ranging from 0.04 to 0.34 mug/ml. Equilibration of CDX between plasma and the lumbar sampling site appeared to be complete within 2 hr of the most recent dose. After attainment of distribution equilibrium, simultaneous plasma and CSF concentrations of CDX were hightly correlated (r = 0.76), with a mean CSF-plasma concentrations ratio of only 0.043 (range; 0.02 to 0.06). The limited passage of CDX into human CSF is probably due to extensive binding to plasma protein. Assuming that transfer of CDX from plasma to CSF is governed by passive diffusion, the extent of plasma protein binding of CDX in healthy individuals averages about 96%.

Absorption of oral intramuscular chlordiazepoxide by alcoholics.

The effect of chronic alcoholism on oral and intramuscular plasma levels of chlordiazepoxide (CDX) was assessed. A 50-mg oral dose of CDX resulted in significantly higher plasma levels in the 2 hr following CDX than a 50-mg intramuscular dose administered to acute withdrawing alcoholic subjects. The same CDX dose was administered 7 days later and the same differences were observed between the mean oral and intramuscular plasma levels during the first 2 hr after administration of CDX. Peak concentration occurred significantly sooner after the oral than intramuscular dose of CDX in both the initial dose and the dose given a week later. It was also observed that the areas under the curve for CDX were significantly greater initially than 1 wk later. It is suggested this effect may be at least partially the result of the longer CDX half-lives initially than a week later. The active metabolite, N-desmethylchlordiazepoxide, peaked significantly earlier with the oral dose than with the intramuscular dose after the patient was alcohol free for a week.

Influence of magnesium and aluminum hydroxide mixture on chlordiazepoxide absorption.

Ten healthy male subjects ingested 25 mg of chlordiazepoxide hydrochloride (Librium) with 100 ml of water or with 100 ml of magnesium and aluminum hydroxide (Maalox) in a single-dose crossover study. Multiple venous blood samples drawn during the first 24 hr after each dose were assayed for concentrations of chlordiazepoxide and its major metabolite, desmethylchlordiazepoxide. The antacid prolonged the mean chlordiazepoxide absorption half-time from 11 to 24 min, and in 6 of 10 subjects delayed achievement of the peak blood concentration by from 0.5 to 3.0 hrs. The formation of desmethylclordiazepoxide was also slowed. The areas under the 24 hr blood concentration curve for chlordiazepoxide and for its metabolite were not influenced by the antacid. The apparent elimination half-life of chlordiazepoxide (8.4 and 8.2 hr) was not significantly affected. Administration of chlordiazepoxide with antacid reduces the rate of its absorption but does not alter the completeness of absorption or the apparent rate of elimination.

Effects of caffeine on plasma free fatty acids, urinary catecholamines, and drug binding.

The effects of caffeine (250 mg orally) on plasma free fatty acids (FFA), urinary catecholamines, and drug binding were studied in 16 normal subjects (six men, five women on oral contraceptives, and five women not on oral contraceptives). FFA doubled 1 hr after caffeine, and remained elevated for at least 4 hr. with elevation of each FFA. Urinary excretion of epinephrine and dopamine increased (p<0.05) in the first 2 hr. returning to baseline in the next 2 hr. Plasma binding of chlordiazepoxide, diaxepam, and propranolol was estimated in each of the hourly plasma samples after caffeine; there was no change in percent unbound drug in any of the samples. In vitro addition of oleic acid to plasma samples of four subjects caused a step-wise increase in percent unbound fraction of all three drugs whereas in vitro addition of caffeine did not further alter drug binding. In our study circulating plasma FFA and urinary catecholamine levels were elevated after caffeine ingestion. In spite of a rise in FFA, there was, however, no change in plasma binding of chlordiazepoxide, diazepam, or propranolol.

Disposition of chlordiazepoxide: sex differences and effects of oral contraceptives.

There is considerable interspecies and interdrug variability in the effect of sex differences and oral contraceptive (OC) steroids on hepatic drug elimination. Their influence on the disposition of chlordiazepoxide has been studied in 11 healthy young men (29 +/- 5 yr), 11 healthy young women (28 +/- 5 yr), and 7 healthy women receiving OC steroids (27 +/- 2 yr) for more than 6 months. The elimination half-life (t1/2(beta)) was longer (from 14.8 +/- 5.9 hr to 8.9 +/- 2.5 hr) and protein binding less (95.5 +/- 1.4% and 97.0 +/- 1.2%) in women than in men. Weight-normalized plasma clearances of total drug did not differ, but the clearance of unbound drug was significantly less in women (8.7 +/- 5.0 ml/min/kg) than in men (15.6 +/- 5.3 ml/min/kg). Women on OC steroids had a lower plasma binding (from 93.6 +/- 1.5% to 95.5 +/- 1.4%) and a higher volume of distribution (from 0.62 +-/ 0.23 l/kg to 0.40 +/- 0.14 l/kg) than women not on OC steroids. The elimination t1/2 was longer (from 24.3 +/- 12 hr to 14.8 +/- 5.9 hr) and the clearance of unbound drug lower (from 5.7 +/- 3.0 ml/min/kg to 8.7 +/- 5.0 ml/min/kg) in women on OC steroids than in those not using them, but these differences were not statistically significant.

PIP: The effect of oral contraceptives (OCs) on hepatic drug elimination, in this case chlordiazepoxide, was studied in 11 healthy young men, 11 healthy young women, and 7 healthy young women receiving OCs for more than 6 months. Elimination half-life was longer (14.8-8.9 hours) and the protein binding less (95.5 and 97%) in women than in men. When weights were normalized, plasma clearances of total drug did not differ, but clearance of unbound drug was significantly less in women (8.7 ml/min/kg) than in men (15.6 ml/min/kg). Women taking OCs had a lower plasma binding (from 93.6-95.5%) and a higher volume of distribution (from .62-4 1/kg) than women not taking OCs. Elimination half-life was longer (from 23.4-14.8 hours) and clearance of unbound drug lower (from 5.7-8.7 ml/min/kg) in women on OCs than in those not using them, but these differences were not statistically significant.

Effect of ketoconazole on hepatic oxidative drug metabolism.

Several clinical reports have suggested (but not demonstrated) that ketoconazole, a broad-spectrum antifungal drug, may inhibit hepatic oxidative drug metabolism in man. We recently demonstrated that ketoconazole inhibits caffeine and aminopyrine oxidation in the rat; we now study the influence of ketoconazole on theophylline and chlordiazepoxide kinetics in man. These studies were performed before and after varying doses of ketoconazole within the currently accepted therapeutic range. Ketoconazole had no effect on theophylline clearance, whereas the drug impaired chlordiazepoxide clearance from plasma. After a single dose of ketoconazole, there was a 20% decrease in clearance and a 26% decrease in volume of distribution without evidence of inhibition of drug metabolism. These changes apparently were not related to ketoconazole dose. After repetitive dosing with ketoconazole, chlordiazepoxide clearance decreased by 38% and was associated with reduced concentrations of its first oxidative metabolite, N-desmethylchlordiazepoxide. We conclude that ketoconazole inhibits at least one subset of the hepatic mixed-function oxidase system, but is not as general an inhibitor of hepatic oxidative drug metabolism as cimetidine appears to be. For some coadministered drugs, ketoconazole may also have an effect on other kinetic parameters such as volume of distribution. Therefore, we caution that clinically important drug interactions may occur with the concurrent use of ketoconazole.

Effects of influenza virus vaccine on hepatic drug metabolism.

Experimental and more limited clinical studies have suggested that influenza vaccination may depress the oxidative hepatic metabolism of various drugs and lead to drug toxicity. The alleged mechanism is the formation of interferon and the resulting decrease in cytochrome P-450 available for drug oxidation. Because of the clinical and basic science implications of these reports, we undertook to study the effects of influenza vaccine on the metabolism of three commonly used drugs: chlordiazepoxide, theophylline, and lorazepam. Our healthy male subjects were studied just before and 1 and 7 days after vaccination. As expected, lorazepam metabolism, which proceeds by glucuronidation and not oxidation, was not altered by vaccination. Surprisingly, however, the oxidation of chlordiazepoxide was also not depressed by the vaccine. Theophylline oxidation, which proceeds primarily by microsomal oxidation (demethylation), was significantly decreased 1 day, but not 7 days, after vaccination. Serum alpha-interferon levels rose after vaccination for only about 8 hours, and levels of gamma-interferon rose to about 500 IU/ml at 24 hours, peaked at 72 hours, and returned to normal by 100 hours after dosing. It appeared that the higher the theophylline clearance before vaccination, the greater the degree of clearance depression after vaccination. Thus the inhibition of drug oxidation after influenza vaccination is selective and each drug should be studied individually. The degree of depression of theophylline clearance is small and transient and appears to be greater in subjects with higher prevaccination clearance.

Relationship of plasma levels of chlordiazepoxide and metabolites to clinical response.

The authors report on the relationship between the antianxiety effects of chronically administered chlordiazepoxide (CDX) and plasma levels of CDX and two of its metabolites, desmethylchlordiazepoxide (DMCDX) and demoxepam (DMX). Fifteen subjects with moderate to severe anxiety were studied in a double-blind, placebo-controlled, crossover design. Significant correlations were found between anxiety reduction and DMCDX and DMX plasma levels. No such correlation was observed between anxiety reduction and CDX levels. These data suggest that in chronically treated subjects, DMCDX and DMX have significant antianxiety properties which surpass those of CDX itself.

Absorption rate, blood concentrations, and early response to oral chlordiazepoxide.

Ten healthy male volunteers received chlordiazepoxide (CDX) hydrochloride or matching placebo with Maalox or water in a four-way single-dose crossover trial. Coadministration of CDX with Maalox did not change the completeness of CDX absorption but significantly slowed its rate of absorption and the rate of desmethylchlordiazepoxide (DMCDX) appearance. Self-rating of feeling "spacey" at 1.0 and 2.5 hours were significantly increased over baseline for CDX taken with water but not with Maalox. Increases in "spacey" feelings at 1.0 hours were highly correlated with 0.5-hour but not with 1.0-hour blood levels. Similar findings were observed for self-ratings of "thinking" slowed down. Thus certain subjective effects of antianxiety agents after oral dosage may depend on the rate of drug absorption and may be attenuated or eliminated if the absorption rate is reduced.

Factors influencing blood concentrations of chlordiazepoxide: a use of multiple regression analysis.

Three groups of male and female subjects aged 24-74 years received 25, 100, or 200 mg of chlordiazepoxide hydrochloride by mouth as a single dose or as two divided doses. The relation of plasma or whole blood concentrations for chlordiazepoxide (CDX) and its metabolite, desmethylchlordiazepoxide (DMCDX), to time since the last dose, weight, age, and sex were determined by simple and multiple regression analyses. Both CDX and DMCDX levels were negatively correlated with weight. Concentrations of CDX decreased, while those of DMCDX increased, with the time since the last dose. Lower levels of both drugs were associated with female sex, and lower levels of DMCDX were noted with increasing age. In the largest sample group, age and weight were more important variables than sex in accounting for CDX and DMCDX. Sex was of significance, and more important than time or age in explaining the variance of CDX in one series of observations. Multiple regression analysis is a useful approach to assessing interrelated factors influencing blood levels of drugs, especially when combined with a consideration of the interactive components of variance. Age and sex, in addition to weight and time, may be important factors that deserve further attention.

Trazodone: a simple, clean extraction and rapid quantification by high pressure liquid chromatography.

We have developed an HPLC technique which is precise, rapid and reliable. Sample preparation involves simple alkaline extraction into an organic solvent mixture. Trazodone elutes at 3.2 min in this reverse phase system. There is no requirement for elevated column temperature. The method is linear to 3000 ng/ml. The availability of a technique which is transferrable to the clinical laboratory may help to define the proper role of therapeutic monitoring of trazodone.

Development and retention of tolerance to the sedative effects of chlordiazepoxide: role of apparatus cues.

Groups of rats were pretreated for 5 days with chlordiazepoxide (5 to 50 mg/kg) or with control water injections. On the sixth day the rats were given a test dose of chlordiazepoxide (10 mg/kg), or water. The rats that had received 5 days of pretreatment with chlordiazepoxide were significantly less sedated by the test dose than were those given chlordiazepoxide for the first time, i.e. they had developed tolerance. There were no significant differences between the two pretreatment groups in the extent of tolerance. A second experiment examined the effects of associating drug injections with apparatus cues. This had no effect on the development of tolerance, but had a significant effect on its retention: rats pretreated and replaced in their home cages showed complete recovery from tolerance after two drug-free days, whereas those placed in the apparatus after each day's injection retained some tolerance even after two drug-free weeks.

Simultaneous assay of diazepam, chlordiazepoxide, N-desmethyldiazepam, N-desmethylchloridazepoxide, and demoxepam in serum by high performance, liquid chromatography.

A method is presented for simultaneously determining diazepam and chlordiazepoxide along with their respective major active serum metabolites N-desmethyldiazepam, and N-desmethylchlordiazepoxide and demoxepam. The drugs are extracted from one ml of buffered serum using chloroform containing 5-(p-methylphenyl)-5-phenylhydantoin as an internal standard. The elution is accomplished using a reversed-phase column with a mobile phase consisting of an acetonitrile/methanol/acetate buffer pH 5.0 (200/225/500) at a flow rate of 2.0 ml/min. Absorbance is monitored at 240 nm using a variable wavelength detector. Each chromatographic separation requires approximately 15 minutes at ambient temperature. Of more than twenty drugs tested for possible interference with this procedure, only methaqualone interferes with the internal standard, and phenytoin with demoxepam.

Continuous-infusion flumazenil in the management of chlordiazepoxide toxicity.

Flumazenil is indicated for reversal of sedation from benzodiazepines administered during therapeutic or diagnostic procedures and during induction or maintenance of general anesthesia, as well as for benzodiazepine overdose. Bolus doses of flumazenil are usually adequate to achieve reversal; however, when medical conditions may lead to a prolonged half-life of the benzodiazepine involved, continuous infusion may be warranted. A 67-year-old man with chlordiazepoxide toxicity required a 9-day infusion of flumazenil to prevent resedation and respiratory insufficiency; he initially was admitted to the hospital for alcohol detoxification. Concomitant medical conditions and the metabolism characteristics of each benzodiazepine must dictate the agent of choice. When measures are required to ensure adequate recovery of a patient's respiratory function and mental awareness, such as in patients with benzodiazepine toxicity, consideration of continuous-infusion flumazenil is warranted.

Quantitative determination of chlordiazepoxide and its metabolites in serum by fluorescence TLC--densitometry.

A sensitive fluorescence TLC--densitometric procedure was developed for the specific determination of chlordiazepoxide (I) and its two metabolites, demoxepam (II) and desmethylchlordiazepoxide (III), in serum. After extraction from serum with ether, I, II, and II were separated by TLC and converted with a sulfuric acid spray to pale blue (Rf 0.63), green (Rf 0.54), and blue (Rf 0.45) fluorescence spots, respectively. Quantitation was accomplished by scanning the plate with a densitometer at 390 (I), 430 (II), and 390 (III) nm. The sensitivities were 0.05 (I), 0.01 (II), and 0.01 (III) microgram/ml of serum. The procedure was successfully applied to measurement of I-III in human serum after oral administration of 20 mg of chlordiazepoxide hydrochloride.

Rapid GLC determination of chlordiazepoxide and metabolite in serum using on-column methylation.

A rapid GLC method was developed for the assay of chlordiazepoxide in serum. After chlordiazepoxide was extracted with ether, it was methylated with trimethylanilinium hydroxide in the injection port and detected by electron capture. The assay is simple and sensitive and can be automated for large-scale clinical analysis.

High-pressure liquid chromatographic determination of chloridazepoxide and its major metabolites in biological fluids.

A simple, isocratic, reversed-phase, high-pressure liquid chromatographic procedure was developed for the determination of chlordiazepoxide and its major metabolites in plasma and urine. The within-run coefficient of variation was 3.4-8.0%, and the day-to-day variation was 4.0-8.0%. Recoveries of 80-91% with sensitivity limits of 50 ng/ml were obtained for the parent drug and its metabolites. Plasma and urine samples collected after single intravenous and single oral doses were analyzed using this procedure.