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%.     

Analysis of beta-hydroxy-beta-methyl butyrate in plasma by gas chromatography and mass spectrometry

A method for measuring the branched chain hydroxy acid beta-hydroxy-beta-methyl butyrate (HMB, a product of leucine catabolism) has been described. A [2H6]HMB internal standard was added to plasma and standards, and samples were extracted with diethyl ether, backextracted into neutral phosphate, dried, and derivatized for gas chromatography and mass spectrometry. The natural HMB was monitored at 175 amu and the deuterated HMB was monitored at 181 amu. Standard curves were linear to at least 25 microns and were quantitatively recovered from plasma. Basal concentrations of plasma HMB were from 1 to 2 microM in sheep and increased three- to fourfold when leucine's alpha-ketoacid (alpha-ketoisocaproate, KIC) was fed to lambs. This method can also be adapted to quantitate KIC and other branched chain ketoacids in plasma during the same run.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

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%.     

Determination of plasma propofol levels using gas chromatography—mass spectrometry with selected-ion monitoring

Propofol (2,6-diisopropylphenol, I.C.I. 35 868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic—mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether—pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within −1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

Quantitative determination of tiamenidine hydrochloride in human serum using gas chromatography mass spectrometry

A method has been developed for the blood level determination of the antihypertensive agent tiamenidine hydrochloride. The serum samples are mixed with deuterium labelled tiamenidine hydrochloride as an internal standard and extracted with methylene chloride. The extracts are derivatized with heptafluorobutyric acid anhydride and analysed by means of gas chromatography mass spectrometry using the selected ion monitoring technique to measure the molecular ion intensities of the bis-heptafluorobutyryl derivatives of tiamenidine hydrochloride and of the internal standard. Using 5 ml serum, the limit of detection is 0.2 ng ml-1 with an accuracy of +/- 0.17 ng (Syx of the calibration curve).     

Quantitative analysis of trimethylsilyl derivative of hydroxyurea in plasma by gas chromatography-mass spectrometry

Hydroxyurea is an antitumor drug widely used in the treatment of sickle cell disease. The drug has been analyzed in biological fluids by a number of high-performance liquid chromatography (HPLC) methods. This paper describes a fast and highly reliable capillary gas chromatography-mass spectrometry (GC-MS) procedure that was developed for the detection and quantitation of hydroxyurea in plasma. The compound and its labeled internal standard were liquid extracted from plasma and derivatized with BSTFA before analysis. The detection limit of the assay was 0.078 microg/ml and the limit of quantitation was 0.313 microg/ml with linearity up to 500 microg/ml. Intra-day variation, as coefficient of variation (C.V., %) over the selected concentration range, was 0.3-8.7% and inter-day variation was 0.4-9.6%.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

Gas chromatographic–mass spectrometric determination of serum mexiletine concentration after derivatization with perfluorooctanoyl chloride, a new derivative

Mexiletine is an antiarrhythmic agent used in the treatment of ventricular arrhythmia. The drug has a narrow therapeutic window which necessitates monitoring its serum concentrations. We describe a gas chromatographic–mass spectrometric analysis of mexiletine using selected ion monitoring. Mexiletine was extracted from alkaline serum with dichloromethane and then derivatized with perfluorooctanoyl chloride. The derivatization reaction was completed in 20 min at 80°C. We used N-propylamphetamine as the internal standard. The ions monitored were m/z 122, 454 and 575 for the derivatized mexiletine and m/z 91, 118, 440 and 452 for the derivatized internal standard. The within-run precision at a serum mexiletine concentration of 1 mg/l was 1.9% (mean=0.98, S.D.=0.019 mg/l, n=7) and the between-run precision was 2.5% (mean=0.99, S.D.=0.025 mg/l, n=7). The assay was linear for serum mexiletine concentrations of 0.2 to 4 mg/l. The detection limit was 0.1 mg/l. The average recoveries of mexiletine and the internal standard were 80% and 84%, respectively at a mexiletine concentration of 1 mg/l. There was no carry over problem in our assay. We observed a good correlation between mexiletine concentrations measured by a reference laboratory (GC) and by our new GC–MS assay.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

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%.     

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%.     

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).     

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.     

High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine (VEN) and its three metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in human plasma has been developed and validated. Estazolam was used as the internal standard. The compounds and internal standard were extracted from plasma by a liquid-liquid extraction. The HPLC separation of the analytes was performed on a Thermo BDS HYPERSIL C18 (250 mm x 4.6 mm, 5 microm, USA) column, using a gradient elution program with solvents constituted of water (ammonium acetate: 30 mmol/l, formic acid 2.6 mmol/l and trifluoroacetic acid 0.13 mmol/l) and acetonitrile (60:40, V/V) at a flow-rate of 1.0 ml/min. All of the analytes were eluted within 6 min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. Calibration curves in spiked whole blood were linear from 4.0-700 ng/ml, 2.0-900 ng/ml, 3.0-800 ng/ml and 2.0-700 ng/ml for VEN, ODV, NDV and DDV, respectively, all of them with coefficients of determination above 0.9991. The average extraction recoveries for all the four analytes were above 77%. The methodology recoveries were higher than 91%. The limits of detection were 0.4, 0.2, 0.3, and 0.2 ng/ml for VEN, ODV, NDV and DDV, respectively. The intra- and inter-day variation coefficients were less than 11%. The method is accurate, sensitive and reliable for the pharmacokinetic study of venlafaxine as well as therapeutic drug monitoring (TDM).     

Estimation of carboxylic acid metabolite of clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic study

A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm x 4.6 mm i.d., 5 microm analytical column maintained at 30 degrees C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0-32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at -70 degrees C for 48 days and at ambient temperature for 24h. In the auto-sampler maintained at 15 degrees C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.     

Selective and rapid liquid chromatography-mass spectrometry method for the determination of lercanidipine in human plasma

A specific liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed and validated for the determination of lercanidipine, a dihydropyridine calcium channel blocker, in human plasma. Lercanidipine R-D3 was used as internal standard (IS). The drug was extracted from plasma using liquid-liquid extraction technique utilizing hexane: ethyl acetate as extraction solvent. The samples were analyzed using a prepacked Thermo Hypersil C(8) column and a mobile phase composed of a mixture of aqueous acetic acid and triethylamine in methanol. An ion trap mass spectrometer equipped with electrospray ionization (ESI) source operating in the positive ion mode was used to develop and validate the method. The method was proved to be sensitive and specific by testing six different human plasma batches. Linearity was established for the concentration ranges of 0.1-16 ng/ml with a regression factor of 0.9996. The lower limit of quantitation was identifiable and reproducible at 0.1 ng/ml with a precision of 7.2%.