Determination of imidazobenzodiazepine-3-car☐amide, a new anxiolytic agent, in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring imidazobenzodiazepine-3-car☐yamide, a new anxiolytic agent, in human plasma. A tetradeuterated analogue of the analyte is used as the internal standard. The drug and its internal standard are (1) extracted from plasma at pH 9 with benzene containing 20% 1, 2-dichloroethane, (2) derivatized with pentafluoropropionic anhydride in the presence of triethylamine and (3) the nitrile derivative of the analyte and internal standard are analyzed by gas chromatography (GC)—negative chemical-ionization mass spectrometry (CIMS) using methane as both GC carrier gas and CI reagent gas. The mass spectrometer is set to monitor the intense (M-HCl)⁻- ions of imidazobenzodiazepine-3-nitrile and its tetradeuterated analogue atm/z 316 andm/z 320, respectively. Quantitation of an experimental plasma sample is based on the comparison of them/z 316 tom/z 320 ion ratio in each sample to that obtained from the analyses of control plasma spiked with various amounts of the drug and a fixed amount of internal standard. The limit of quantitation of the method is approximately 100 pg ml⁻¹ of plasma and the precision (relative standard deviation) at a plasma concentration of 1 ng ml⁻¹ is 4%.     

Validation of a simple gas chromatographic-mass spectrometric method for the determination of gamma-butyrolactone in human plasma

A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

The determination of terbutaline in human plasma by selected ion monitoring of the t-butyldimethylsilyl ether

A method is described for the quantitative determination of terbutaline in 2 ml human plasma. The drug is extracted from plasma as the terbutaline tetraphenylboron ion pair and determined by gas chromatography mass spectrometry of its t-butyldimethylsily ether. Salbutamol is used as internal standard. Quantification is achieved by selected ion monitoring of the ion m/z 482 derived from t-butyldimethylsilyl terbutaline and m/z 495 from t-butyldimethylsilyl salbutamol. The detection limit was estimated to be 250 pg terbutaline ml-1 plasma. The coefficient of variation at the level of 1 ng terbutaline ml-1 was 4.1% (n = 5).     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

Determination of CGS 16617 and stable isotope-labeled CGS 16617, an angiotensin-converting enzyme inhibitor, in human plasma by gas chromatography/mass spectrometry

An analytical method has been developed for the simultaneous determination of a novel orally active angiotensin-converting enzyme inhibitor (CGS 16617) and a stable isotope-labeled analog. Both compounds are isolated from human plasma using an ion-exchange column, derivatized with pentafluoropropionic anhydride and pentafluoropropanol, and analyzed by gas chromatography/mass spectrometry. After splitless injection on a methyl-silicon column, the compound is detected using negative ion chemical ionization with nitrous oxide as a reagent gas. CGS 16617 labeled with four deuteriums and two 13C is used as an internal standard. The accuracy and precision of the method, expressed as the overall mean +/- SD recovery obtained from two sets of 36 quality-control samples used during a clinical study (concentration range 0.2-100 ng ml-1 plasma), was 96.1 +/- 16.2% for unlabeled drug and 97.6 +/- 14.4% for the D4-labeled drug (concentration range 0.2-100 ng ml-1 plasma). The limit of quantification using 1 ml plasma is 0.2 ng ml-1 for both labeled and unlabeled drug.     

Sensitive determination of pethidine in body fluids by gas chromatography-tandem mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography-tandem mass spectrometry (GC-MS/MS). Pethidine and 4'-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C(18) columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25-40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.     

Quantitative determination of nitroglycerin in human plasma by capillary gas chromatography negative ion chemical ionization mass spectrometry

A quantitative method for determination of nitroglycerin in human plasma was developed. Nitroglycerin and the internal standard (butane-1,2,4-triyl trinitrate) were extracted from plasma with pentane. The extracts were analysed by gas chromatography mass spectrometry using fused silica capillary columns and electron capture negative ion chemical ionization. The quantitation limit of the method was about 50 pg ml-1. Linear calibration curves were obtained in the range of 50-1600 pg ml-1. Precision at the level of 100 pg ml-1 was 4%.     

Determination of methimazole in plasma using gas chromatography—mass spectrometry after extractive alkylation

A gas chromatography—mass spectrometry method for quantitation of the thyreostatic agent methimazole in plasma is described. The drug was transferred from the plasma sample and derivatized in one step by extractive alkylation. Either of two alkylating agents benzylchloride or pentafluorobenzyl bromide were used. Deuterium-labelled methimazole was used as internal standard. The precision of the method at the level of 5 ng methimazole per ml plasma was 6%.     

Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen–phosphorus detection (GC–NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179°C at 4°C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10 000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within ±15%.     

Simultaneous determination of tramadol and its major active metabolite O-demethyltramadol by high-performance liquid chromatography with electrochemical detection

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O-demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10–1000 ng/ml for ODMT and 10–10 000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 μl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.     

Microdose clinical trial: quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry

A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm x 2.1 mm i.d., particle size 3.5 microm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10-1000 pg/ml using 200 microl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1-500 ng/ml using 20 microl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 microg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.     

Simultaneous determination of benazepril hydrochloride and benazeprilat in plasma by high-performance liquid chromatography/electrospray-mass spectrometry

An analytical method for simultaneous determination of benazepril and its active metabolite, benazeprilat, in human plasma by high-performance liquid chromatography/electrospray-mass spectrometry was developed and validated. Rutaecarpine was selected as the internal standard. The separation was achieved on a C(18) column with acetonitrile and aqueous solution (0.1% formic acid) as mobile phase with a gradient mode. The quantification of target compounds was using a selective ionization recording at m/z 425.5 for benazepril, m/z 397.5 for benzeprilat and m/z 288.3 for rutaecarpine. The correlation coefficients of the calibration curves were better than 0.992 (n = 6), in the range of 6.67-666.67 ng/ml for benazepril and benazeprilat. The inter- and intra-day accuracy, precision, linear range had been investigated in detail. The method can be used to assess the bioavailability and pharmacokinetics of the drug.     

Determination of rifalazil, a potent antibacterial agent, in human plasma by liquid-liquid extraction and LC-MS/MS

A sensitive assay for determination of rifalazil (also known as ABI-1648 and KRM-1648) in human plasma is described. The analytical method utilizes liquid-liquid extraction of plasma with methyl tert-butyl ether, followed by reversed-phase liquid chromatography with a C18 column and a mobile phase gradient utilizing 0.1% formic acid in water and acetonitrile, respectively. Electrospray mass spectrometry in the positive ion mode with selected reaction monitoring of rifalazil and an isotope labeled internal standard, 13C4-rifalazil (ABI-9901) was used for selective and sensitive detection. The calibration range was 0.050-50 ng/mL plasma using 200 microL plasma sample volume. The absolute extraction recovery of rifalazil from K2-EDTA plasma, evaluated at three concentration levels, was 88.6-97.3%, and the recovery for the internal standard was 96.8%. A study of plasma matrix effects showed a peak area response at 90-99% compared to neat solutions for both rifalazil and the internal standard. Stability evaluation of rifalazil in plasma, whole blood and methanol showed that the analyte stability was adequate when stored under study conditions. The precision, as evaluated in three validation batches, was consistent for fortified plasma quality control (QC) samples at four concentration levels, with < or =6% R.S.D. except for at the lowest quality control level where it was 10.7% R.S.D. The accuracy for QC samples (difference between found and nominal concentration) ranged from -2.3% to 5.1%. Similar precision and accuracy values were obtained over 6 months of routine application of this method. It was concluded that the performance improved markedly during routine operation by replacing a closely related structural analog internal standard with the stable isotope internal standard.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C₄ reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

Synthesis of [(18)O(2)]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography-electron ionization mass spectrometry

A specific method for the quantitative determination of valproic acid in human plasma is presented. Valproate was extracted from acidified plasma by hexane extraction and converted to its trimethylsilyl derivative without sample concentration. The derivatives were analyzed without any further purification. Using gas chromatography-electron ionization mass spectrometry, diagnostic useful fragment ions at m/z 201 and 205 were obtained for valproic acid and [(18)O(2)]valproic acid internal standard, respectively. [(18)O(2)]Valproic acid was synthesized from unlabeled valproate by acid-catalyzed exchange reaction in H(2)(18)O. The method was validated in the expected concentration range of a pharmacokinetic study. Thus, calibration graphs were linear within a range of 0.47-120 microgram/ml plasma. Intra-day precision was 2.29% (0.47 microgram/ml), 2.93% (4 microgram/ml), 3.22% (20 microgram/ml) and 4.40% (80 microgram/ml), inter-day variability was found to be 1.49% (0.47 microgram/ml), 3.79% (20 microgram/ml), 2.74% (40 microgram/ml) and 3.03% (80 microgram/ml). Inter-day accuracy showed deviations of 1.94% (0.47 microgram/ml), 0.53% (4 microgram/ml), -0.32% (20 microgram/ml) and 0.06% (80 microgram/ml). The method is rugged and robust and has been applied to the batch analysis of valproate during pharmacokinetic profiling of the drug.     

Quantitative determination of carbamazepine and carbamazepine-10,11-epoxide in rat brain areas by multiple ion detection mass fragmentography.

A specific and sensitive method is described for the simultaneous determination of carbamazepineand carbamazepine-10,11-epoxide in biological specimens by combined gas chromatography-mass spectrometry. Cytenamide is used as the internal standard for quantitation. Kinetics of the distribution of the drug and its metabolite in plasma and in different areas of rat brain are reported. Determinations are possible at the nanogram level.     

Simultaneous determination of clarithromycin,rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to ?10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.     

Determination of clofilium, a new antifibrillatory agent, in plasma by gas chromatography—mass spectrometry

A sensitive and selective method for the assay of the new quaternary amine antifibrillatory agent clofilium is described. Plasma samples were extracted with dichloromethane (98.5 ± 0.2% recovery) and analyzed by gas chromatography—mass spectrometry operating in the electron-impact mode. The method involves a Hofmann elimination of an N-alkyl radical from clofilium and the internal standard in the presence of a strong nucleophile in the injector of the gas chromatograph. The resulting tertiary amines are chromatographed and detected by selective ion monitoring. The ratio of the clofilium base peak (m/z 224) to the internal standard peak (m/z 210) was linear relative to the plasma clofilium concentration over the range of 25–1000 ng/ml plasma.     

Gas chromatography-negative ion chemical ionisation mass spectrometry using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl derivatives for the quantitative determination of methylphenidate in human plasma

A novel electrophoric derivatisation procedure using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride for the quantitative determination of methylphenidate in human plasma is described. The drug can be quantitatively measured down to 0.006 pg/mL plasma due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent (n-hexane) and reagent solution for 15 min, which, after concentration was measured by GC-NICI-MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope labelled internal standard was used. Validation data are given to demonstrate the usefulness of the assay, including selectivity, linearity, accuracy and precision, autosampler stability, aliquot analysis, robustness, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral administration.