Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

Chromatographic method for the determination of diazepam,pyridostigmine bromide,and their metabolites in rat plasma and urine

This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The compounds were extracted using C₁₈ Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1±7.7, 83.5±6.4, 83.9±5.9, 71.3±6.0 and 77.7±5.6, and from urine 79.4±7.9, 83.1±6.9, 73.6±7.7, 74.3±7.1 and 77.6±5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin,a novel highly lipophilic camptothecin derivative,in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2±0.5 min and 8.0±0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at −70°C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Radioimmunoassay of plasma androsterone

A method is described and evaluated for the determination of androsterone in peripheral venous plasma (1.0 ml) from men and women. The procedure involves addition of labelled internal standard and extraction with diethyl ether. Aliquots (10 %) are removed for radioimmunoassay. An antiserum to androsterone-17-carboxymethyl oxime-bovine serum albumin and tritiated androsterone complete the system. The practical systematic errors have been determined by replicate analyses. The range of values (mean ± S.D.) in plasma from 40 healthy men are 54 ± 32 ng/100 ml, and the corresponding values for women 46 ± 28.     

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (±)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar☐ylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the elected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02–0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.     

Sensitive gas chromatographic method for the determination of cinnarizine and flunarizine in biological samples

A sensitive method has been developed for the determination of the vasoactive compounds cinnarizine and flunarizine in plasma, urine and milk samples from man and animals. The procedure involves the extraction of the drugs and their internal standard from the biological samples at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol).The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 0.5 ng/ml of biological fluid and extraction recoveries were sufficiently high (87–94%).The method was applied to plasma samples from bioavailability studies of both cinnarizine and flunarizine in healthy volunteers, and to plasma, urine and milk samples from flunarizine-treated dogs.     

Simultaneous gas chromatographic determination of lorcainide hydrochloride and three of its principal metabolites in biological samples

A method is described for the determination of the antiarrhythmic drug lorcainide hydrochloride and its three main metabolites in plasma, urine, faeces and tissues from man and animals. The procedure involves the extraction of the parent drug, its metabolites and the internal standard from the biological materials at different alkaline pH values, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol). After silylation of the different phenolic and the N-dealkylated metabolites, analyses were carried out by automated gas—liquid chromatography with electron-capture detection. The method has a sensitivity limit of 5 ng for lorcainide, and 10–20 ng for the various metabolites, per millilitre of plasma.The method was applied to urine, faeces, plasma and tissue samples from man and animals. It was also suitable for automatic sample analysis.     

Determination of diphenylpyraline in plasma and urine by high-performance liquid chromatography

A rapid, reliable and specific reversed-phase high-performance liquid chromatographic procedure is described for the determination of diphenylpyraline hydrochloride at nanogram concentrations in plasma and urine. After extraction of the drug with n-pentane-2-propanol (50:1) from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with methanol and chromatographed using a 5-μm Asahipak ODP-50 C₁₈ column with UV detection at 254 nm. The elution time for diphenylpyraline was 7.9 min. The overall recovery of diphenylpyraline from spiked plasma and urine samples at concentrations ranging from 53 to 740 ng/ml were 94.65% and 92.29%, respectively. Linearity and precision data for plasma and urine standards after extraction were acceptable. The limit of detection was 15 ng/ml for both plasma and urine samples at 0.002 AUFS.     

Simplified method for determination of the tetracyclic antidepressant mianserin in human plasma using gas chromatography with nitrogen detection

A simplified gas chromatographic method for determination of the antidepressant drug mianserin in human plasma is described. Application of a nitrogen-sensitive detector reduces the assay procedure to extraction, concentration and gas chromatographic determination. The method is suitable to determine mianserin in human plasma at the 1 ng/mol level on a routine basis. At the 20 ng/ml level the deviation of the mean from the true value and the relative standard deviation amount to 1.0% and 6.8%, respectively.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Determination of Ro 19–6327 (Lazabemide) in human plasma and urine by gas chromatography-negative chemical ionization mass spectrometry

A sensitive and specific analytical method was developed for determination of Ro 19–6327 (Lazabemide) in human plasma and urine samples to provide pharmacokinetic data from clinical trials. The new method employs a simple liquid-liquid extraction to isolate the drug from biological samples. The extract is reacted to form the trifluoroacetyl derivative of Ro 19–6327 and then analyzed by gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The lower limit of quantitation of the assay is 0.05 ng/ml for plasma and 5.0 ng/ml for urine, based on 1-ml aliquots. No interferences from anticoagulants, collection devices, or endogenous constituents of plasma and urine were observed. Recovery (64.3%), inter-assay precision (<8% R.S.D.), and accuracy (>85%) of the method were considered acceptable. The assay proved reliable enough to be automated for unattended sample analysis of approximately 50 samples daily. In an additional series of tests, Ro 19–6327 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Determination of (E)-5-(2-bromovinyl)-2′-deoxyuridine in plasma and urine by capillary electrophoresis

(E)-5-(2-Bromovinyl)-2′-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography—negative chemical ionization mass spectrometry using a particle beam interface

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography—negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid—liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml⁻¹. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC—MS method.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.     

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 μl of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 μg/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.     

A high-pressure liquid chromatographic method for plasma phenylacetic acid,a putative marker for depressive disorders

An HPLC procedure for the determination of total phenylacetic acid (PAA) in human plasma is described. After precipitation of plasma proteins with 0.4 n HClO₄, the supernatant was hydrolyzed with 1.5 n HCl at 100°C for 5 h, and PAA was extracted with benzene. From the organic layer PAA was back-extracted into 0.5 ml of 0.1 n NaOH. After neutralization with HCl the sample was directly injected onto the HPLC column (C₁₈). An ultraviolet detector at 210 nm was used to monitor PAA. The plasma PAA values for a control population (536.18 ± 54.99 ng/ml, N = 10) (X ± SE) obtained by the described method are in agreement with values reported using GC/MS methods. Depressed subjects showed significantly lower values (327.64 ± 45.44 ng/ml, N = 10), supporting the view that PAA may be a marker for depressive disorders.     

Determination of levamisole in plasma and animal tissues by gas chromatography with thermionic specific detection

A rapid and sensitive method has been developed for the determination of the anthelmintic levamisole in plasma and tissues from man and animals. The procedure involves the extraction of the drug and its internal standard from the biological material at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol). Several extraction steps can be omitted, however, whenever the gas chromatographic background permits and some operations can be simplified using Clin Elut^TM extraction tubes.The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 5 ng, contained in 1 ml of plasma or in 1 g of the various tissues, and recoveries were sufficiently high (79–86%).The method was applied to human plasma samples in a comprehensive bioavailability study of levamisole in healthy volunteers, and to plasma and tissues in a residue trial in cattle. The effect of the blood collection technique on the plasma levels was also studied and pointed to decreased plasma concentrations when Vacutainer^® tubes were used.