Simultaneous determination of trimipramine and its major metabolites by high-performance liquid chromatography

Trimipramine is a tricyclic antidepressant drug often assayed by gas chromatographic or gas chromatographic-mass spectrometry techniques. A high-performance liquid chromatographic method with electrochemical detection is described for the assay of trimipramine and its major metabolites, monodesmethyltrimipramine and 2-hydroxytrimipramine, in plasma. The method is sensitive, accurate and robust and thus suitable for routinely assaying samples following single doses of trimipramine to man. The assay was applied to plasma samples obtained following a single 50-mg dose of trimipramine to healthy volunteers.     

Automated capillary gas chromatographic assay using nitrogen-phosphorus ionization detection for the determination of bepridil in human plasma

A highly sensitive and specific capillary gas chromatographic method for the determination of the antianginal drug bepridil in plasma is described. The capillary gas chromatograph and nitrogen-selective detector combination provides excellent sensitivity for clinical samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 1-ml plasma sample is 1 ng/ml. Standard curves are linear over the concentration range 1-60 ng/ml. Accuracy and precision of the assay, expressed as relative deviation from the true value and relative standard deviation (inter-run) are less than 15% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, sixty samples can be analyzed routinely in one day. The present assay has been successfully cross-validated with a published high-performance liquid chromatographic assay.     

Analysis of the metabolites of ethyl loflazepate by gas chromatography with electron-capture detection

A gas chromatographic assay with electron-capture detection (GC--EC) is described for the metabolites of ethyl loflazepate (Victan), a new benzodiazepine with a potent anti-anxiety activity, in biological fluids. Since the parent drug undergoes a first-pass effect, pharmacokinetic data may only be obtained by measuring the total levels of two of the major metabolites. Accurate data can not be obtained for the metabolites separately since one of them (M1) is chemically transformed to the other (M2) during plasma sampling, storage and extraction. A sensitive, specific and accurate GC--EC assay is developed using a synthetic analogue of M2 as an internal standard. The limit of detection in plasma is approximately 2 ng/ml and the precision about 3% (within-run and between-run). The method is applied to plasma samples collected after oral administration of 2 mg and 4 mg of the drug in tablet form to human volunteers. The results obtained are correlated with those from an existing gas chromatographic--mass spectrometric assay. A very good correlation between the results (inter-laboratory comparison) is obtained, validating both techniques.     

Capillary gas chromatographic-electron-capture assay for the aldose reductase inhibitor imirestat in lens and plasma

A sensitive and selective gas chromatographic-electron-capture assay was developed for the determination of the aldose reductase inhibitor imirestat in lens and plasma. The method involves solid-phase extraction of drug and internal standard from the plasma specimen or lens sample homogenate using "Baker"-10 SPE extraction columns followed by derivatization with pentafluorobenzyl bromide and further purification. Derivatives of drug and internal standard were separated on a fused-silica capillary column and analyzed using a 63Ni electron-capture detector. The limit of detection was 2.5 ng per lens or ml of plasma. The method was used to evaluate the pharmacokinetics of imirestat in human subjects and to quantitate imirestat in animal lens tissue following topical ocular administration.     

Determination of nifedipine in plasma by a rapid capillary gas chromatographic method.

A rapid, specific and reliable gas chromatographic assay procedure for Nifedipine in plasma has been developed. With a single-step solvent extraction, and electron capture detection, the method is sensitive to 0.5 ng/mL of plasma and the standard curve is linear from 0.5 to 500 ng/mL. Samples are protected from light to prevent formation of photodecomposition products. The method has been used to monitor drug concentrations in patients receiving therapeutic doses.     

Solid-phase microextraction for the assay of clozapine in human plasma

Solid-phase microextraction (SPME) was investigated as sample preparation for the assay of the neuroleptic drug clozapine in human plasma. A mixture of human plasma, water, loxapine as internal standard and aqueous NaOH was extracted with a 100 μm-polydimethylsiloxane-(PDMS)-fiber for 30 min. The analyte and internal standard were well separated in the gas chromatogram. The calibration was linear and passed the origin. Accuracy and precision as well as the influence of changes of the matrix were investigated. No interfering drug was found. It is concluded that the method can be used in the therapeutic drug monitoring of clozapine. Received: 23 December 1998 / Revised: 27 January 1999 / Accepted: 31 January 1999     

Assay of underivatized intrazepam and clonazepam in plasma by capillary gas chromatography applied to pharmacokinetic and bioavailability studies in humans

The assay procedure of underivatized, intact nitrazepam and clonazepam in human plasma is described, using gas chromatography with a support-coated open tubular column (OV-17), a solid injection system and electron-capture detection. Clonazepam is used as a internal standard in the assay of nitrazepam and vice versa. Linear calibration curves after a single extraction step were obtained in the concentration range 10--100 ng/ml plasma, with standard deviations less than 4.9%. The sensitivity limit of the method is about 1 ng/ml plasma for both drugs. The method was applied to pharmacokinetic and bioavailability studies of nitrazepam in humans. Seven healthy volunteers received two nitrazepam-containing tablet preparations (5 mg) and plasma concentrations were determined regularly from 15 min to 80 h following drug administration. The mean elimination half-life of nitrazepam was 27 h (range 13-34 h). Considerable intra-individual differences in peak level times between the two preparations were observed, whereas the extent of bioavailability was rather similar.     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Development of capillary zone electrophoresis-electrospray ionization-mass spectrometry for the determination of lamotrigine in human plasma

A method of coupling capillary zone electrophoresis (CZE) with electrospray ionization-mass spectrometry (ESI-MS) detection has been developed for monitoring an antiepileptic drug, lamotrigine (LTG) in human plasma. The CZE-MS was developed in three stages: (i) CZE separation and ESI-MS detection of LTG and tyramine (TRM, internal standard) were simultaneously optimized by studying the influence of CZE background electrolyte (BGE) pH, BGE ionic strength, and nebulizer pressure of the MS sprayer; (ii) sheath liquid parameters, such as pH, ionic strength, organic modifier content, and flow rate of the sheath liquid, were systematically varied under optimum CZE-MS conditions developed in the first stage; (iii) MS sprayer chamber parameters (drying gas temperature and drying gas flow rate) were varied for the best MS detection of LTG. The developed assay was finally applied for the determination of LTG in plasma samples. The linear range of LTG in plasma sample assay was between 0.1-5.0 microg/mL with a limit of detection as low as 0.05 microg/mL and run time less than 6 min. Finally, the concentration-time profile of LTG in human plasma sample was found to correlate well when CZE-ESI-MS was compared to a more established method of high-performance liquid chromatography with ultraviolet detection.     

Reliable routine method for the determination of plasma amitriptyline and nortriptyline by gas chromatography

A gas chromatographic method has been developed for the determination of amitriptyline and nortriptyline in plasma. OV-17 is used in a 1 m long packed column, with a flame ionization detector and an electronic integrator. Five internal standards are added. The base-specific extraction procedure and the method of calibrating the chromatograph are described in detail. The accuracy, precision and reliability of the method are demonstrated by the results of nearly 700 determinations of each drug, at concentrations ranging from 5 to 400 ng/ml in the plasma. An interlaboratory comparison with a double radioactive isotope derivative assay for nortriptyline has also shown satisfactory agreement.     

Quantitation of the enantiomers of rimantadine in human plasma and urine by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric procedure has been developed for the quantitation in plasma and urine of the enantiomers of rimantadine, an antiviral drug effective against type A influenza. The assay utilizes derivatization with an optically active reagent, selective ion monitoring, methane negative-ion chemical ionization (NICI) mass spectrometry and stable isotope dilution. The method has been used to measure concentrations of each rimantadine enantiomer over a range of 2.5-250 and 12.5-1250 ng/ml in the plasma and urine, respectively, of four male volunteers administered rimantadine. In plasma and urine, no differences were observed in the disposition of the unconjugated enantiomers. In urine, one enantiomer, but not both, was released following enzymatic hydrolysis.     

Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug–drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA‐anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra‐assay precision between 1.6 and 10.7%, and inter‐assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra‐assay concentrations within 85.6–108.7% of the expected value, and inter‐assay concentrations within 89.4–104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of midazolam in human plasma by solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of midazolam, a short-acting 1,4-imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid-phase microextraction (SPME) of midazolam using an 85-microm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5-300 ng/mL, relative intra- and inter-assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.     

Gas chromatographic assay for oxcarbazepine and its main metabolites in plasma

The new anti-epileptic drug oxcarbazepine is temperature-labile and decomposes under the conditions of gas chromatography, even when injected into a cooled, inert, fused-silica capillary column. In contrast, the trimethylsilyl derivative of oxcarbazepine is stable. The bis-trimethylsilyl derivatives of the enol of oxcarbazepine and of its active metabolite, 10-hydroxycarbazepine, and the tris-trimethylsilyl derivative of carbazepine-10,11-trans-diol can be synthesized easily at room temperature. Using the readily available carbamazepine as internal standard, a simple gas chromatographic assay was developed for the simultaneous routine measurement of these three compounds at therapeutic levels. This assay is ten times more sensitive to oxcarbazepine than the previously described high-performance liquid chromatographic assays. It involves a single-step solvent extraction, uses a fused-silica capillary column and a flame ionization detector. On processing 0.5 ml of plasma, limits of detection of 10 ng/ml were obtained for oxcarbazepine and 10-hydroxycarbazepine and a limit of detection of 25 ng/ml for carbazepine-10,11-trans-diol.     

Analysis of clomesone in plasma by gas chromatography-electrolytic conductivity detection

A sensitive and specific gas chromatographic (GC) method with electrolytic conductivity detection (ELD) for the analysis of clomesone (2-chloroethylmethylsulfonylmethane sulfonate), a new experimental antitumor alkylating agent, in plasma has been developed for the first time. Clomesone in plasma containing suitable internal standard was extracted with methylene chloride. After evaporation, the residue was analyzed by GC-ELD. Either a 15-m wide-bore DB-17 or a DB-1 column with the corresponding internal standards of propachlor or butachlor, respectively, was used. For the DB-1 column with butachlor as the internal standard, the routine assay limit was 20 ng/ml with linearity from 10 to 2000 ng/ml monitored. The within-run coefficient of variation of eight replicates at 50 ng/ml was 8.0% and the between-run coefficient of variation was 11% at 120 ng/ml. Using this assay procedure, the stability in several aqueous media and protein binding of clomesone were evaluated. In fresh mouse plasma, the half-life of clomesone was less than 1 h, although in aged pooled human plasma the drug was more stable. The mean protein binding value in mouse and human plasma was about 81-85%.     

Determination of the angiotensin converting enzyme inhibitor benazeprilat in plasma and urine by an enzymic method

An enzyme inhibition assay for the angiotensin-converting enzyme (ACE) inhibitor benazeprilat is described. Plasma and urine samples were diluted and endogenous ACE was inactivated by heating. After incubation of the plasma samples with hippuryl-histidyl-leucine as substrate and blank plasma as the source of ACE, released hippuric acid was measured by high-performance liquid chromatography. Urine samples were incubated with [3H] hippuryl-glycyl-glycine and with rabbit lung extract as the source of ACE. Released [3H] hippuric acid was quantified by liquid scintillation counting. Drug standards for the standard curve were prepared in the biological matrix. A cross-check with a gas chromatographic-mass spectrometric method showed good agreement, demonstrating that this enzymic method is suitable for assessing drug bioavailability and pharmacokinetics.     

Determination of the GABAergic antidepressant drug fengabine and some of its metabolites in plasma by capillary gas chromatography with electron-capture detection

A sensitive capillary gas chromatographic method was developed for the determination of fengabine (a GABAergic antidepressant drug) and some of its metabolites in plasma samples. The method involves a single and rapid liquid-liquid extraction of the parent drug and metabolites from plasma buffered at pH 5, evaporation of the organic phase under nitrogen, derivatization to tert.-butyldimethylsilyl ethers and esters and automatic gas chromatography on a fused-silica, silicone-bonded capillary column coupled to an electron-capture detector. The detection limit for fengabine and other compounds is lower than 1 ng/ml in plasma; the method was successfully applied to pharmacokinetic and drug monitoring clinical studies and tested on more than 2000 biological samples and was found not to suffer from endogenous or exogenous interferences.     

Gas chromatographic-mass fragmentographic determination of propiverine and its metabolites in plasma and urine

1-Methyl-4-piperidyl diphenylpropoxyacetate hydrochloride has been developed clinically for the therapy of urinary bladder dysfunction. A gas chromatographic-mass fragmentographic method was developed for the determination of this drug and its seven metabolites in plasma and urine. The sample was first treated with a Sep-Pak C18 cartridge, the methanol eluate was evaporated to dryness, and the resulting residue was redissolved in distilled water. This solution was then extracted with chloroform and adjusted to pH 9.0 with 0.1 M sodium borate solution. The acidified aqueous layers were extracted with ethyl acetate. The chloroform layer, which contained non-polar metabolites, was concentrated to dryness, then subjected to trifluoroacetylation, decomposition and methylation. The extract from the plasma sample was trimethylsilylated. The dried residue of the ethyl acetate layer, which contained polar metabolites, was subjected to methylation, trifluoroacetylation and decomposition. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer and analysed by the selected-ion monitoring method using an internal standard. Detection was limited to 1-2 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of 1-methyl-4-piperidyl diphenylpropoxyacetate hydrochloride and its metabolites in plasma and urine was thus established, and it should prove useful in basic and clinical pharmacological studies.     

Gas chromatographic determination of guanadrel in plasma and urine

To evaluate the pharmacokinetics and drug availability from various dosage formulations, a method for the determination of guanadrel, (1,4-dioxaspiro[4,5]dec-2-ylmethyl)guanidine, in plasma and urine was required. A gas chromatographic procedure, based on formation of a hexafluoroacetylacetone derivative in a two-phase system of water and toluene, was developed. The limit of determination of the method is 5 ng/ml guanadrel in plasma and 15 ng/ml guanadrel in urine. Statistical analyses indicate average recoveries of 98.1 +/- 18.0 and 104.4 +/- 15.6% from plasma and urine, respectively. Mass spectrometric analyses, in conjunction with gas chromatography, confirmed the specificity of the method for intact drug. The procedure was applied successfully to drug absorption studies in humans.     

Development of enantioselective gas chromatographic quantitation assay for dl-threo-methylphenidate in biological fluids

An enantioselective gas chromatographic quantitation assay was developed for the enantiomers of dl-threo-methylphenidate in plasma and urine. dl-threo-Methylphenidate and the internal standard were acylated with N-heptafluorobutyryl-1-prolylchloride under Schotten-Baumann conditions prior to gas chromatographic separation on achiral mixed stationary phases. The derivatives were detected by means of a nitrogen-phosphorus detector. Linear and reproducible calibration curves were obtained over the concentration ranges 0.43-43.25 and 2.16-216.25 ng/ml enantiomer in plasma or urine, respectively. This enantioselective gas chromatographic quantitation assay was applied in a single oral dose disposition study of dl-threo-methylphenidate in a healthy adult volunteer. Stereoselective differences were observed in the plasma concentration-time profiles and cumulative urinary excretion profiles following oral doses of 20 and 40 mg of dl-threo-methylphenidate hydrochloride. Only d-threo-methylphenidate was detectable in plasma after 4 h.