Quantification of the neuromuscular blocking agent rocuronium and its putative metabolite 17-desacetylrocuronium in heparinized plasma by capillary gas chromatography using a nitrogen sensitive detector

We have developed a sensitive and specific capillary GC (cGC) assay for the quantification of the quarternary aminosteroidal compound rocuronium (roc), a neuromuscular blocking agent, and its putative metabolite 17-desacetylrocuronium (17OH-roc), using 3-desacetylvecuronium (3OH-vec) as an internal standard (I.S.). This novel method has been applied to a pharmacokinetic study with roc, monitoring sixty patients who were classified according to four different body mass index (BMI) groups. The isolation of these drugs from plasma was carried out using a dichloromethane liquid-liquid extraction after ion-pairing of the positively charged ammonium compounds with iodide. To achieve thermal stability, tert.-butyldimethylsilyl-ethers were formed at the 3OH- and 17OH-steroidal positions by reaction with N-methyl-N-(tert.-butyldimethylsilyl)-trifluoroacetamide at 70°C overnight. An automated cGC system fitted with a nitrogen sensitive detector with a specially prepared glass phase bead and a computer controlled data handling system was used to analyze and quantify the compounds, which were separated on a DB1 capillary column with helium as the carrier gas and a temperature program ranging from 120 to 300°C. The method is linear for 50-6400 ng/ml for roc and 80-6400 ng/ml for 17OH-roc. The detection limits were 10 ng/ml for roc and 50 ng/ml for 17OH-roc. The lower limit of quantification was 50 ng/ml for roc and 80 ng/ml for 17OH-roc. Intra-assay coefficients of variation (C.V.s) were 10% and 15% and the inter-assay C.V.s 8-18% and 16-21% for roc and 17OH-roc, respectively.     

Sensitive and specific liquid chromatographic–tandem mass spectrometric assay for barnidipine in human plasma

A sensitive and specific LC–MS–MS assay has been developed and validated for barnidipine (1-benzyl-3-pyrrolidinyl)methyl-2,6-dimethyl-4(m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate). The assay involves a simple and rapid solid-phase extraction procedure. Sample analysis was on a Spherisorb S3ODS2 100 mm×2 mm I.D. column, with a Finnigan TSQ 7000 mass spectrometer, using an electrospray interface and selective reaction monitoring (SRM). The intra- and inter-day precision and accuracy, determined as the coefficient of variation and relative error, respectively, were 11.8% or less. The limit of quantitation was 0.03 ng/ml, and the calibration was linear between 0.03 and 3.0 ng/ml. The method has been used successfully for the measurement of over two thousand human plasma samples from pharmacokinetic clinical trials.     

Liquid chromatographic–mass spectrometric determination of itraconazole and its major metabolite, hydroxyitraconazole, in dog plasma

A fast, reliable and sensitive liquid chromatography–mass spectrometry (LC–MS) assay for the determination of itraconazole and hydroxyitraconazole in dog plasma has been developed. The analysis involves a simple liquid–liquid extraction followed by LC–MS analysis using electrospray ionization in the positive mode. Total separation of itraconazole, hydroxyitraconazole and the internal standard, miconazole, was achieved on a C₁₈ column in 3.5 min using an isocratic mixture of acetonitrile and 10 mM ammonium acetate. The response was linear over four-orders of magnitude, allowing reliable quantification of each species. This paper describes the development of the method and its validation.     

Modified gas chromatographic–mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma

A modified method for the determination of gacyclidine enantiomers in human plasma by GC–MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d₃-gacyclidine) as internal standard was developed. Following a single-step liquid–liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d₃-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (−)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (−)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) <14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r²>0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were <9% at 5, 10 and 20 ng/ml, and <16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d₃-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.     

Gas chromatographic—mass spectrometric determination of ethambutol in human plasma

A quantitative gas chromatographic—mass spectrometric assay has been developed for the determination of ethambutol (EMB) in human plasma. Plasma samples were taken from a patient after oral administration of EMB (with proven tuberculosis infection). Deuterated EMB and a non-deuterated analogue of EMB were synthesized and used as internal standards in this procedure; both gave excellent agreement in the analysis. The derivatizing agent used was trifluoroacetic anhydride (TFAA) and quantitative derivatization was complete in one hour, forming EMB-(TFA). Selective ion monitoring was utilized to monitor the gas chromatographic effluent. Ions were generated by electron impact at 70 eV. The limit of detection was 36 ng EMB per ml plasma. This method is compared with the electron-capture gas chromatographic procedure of Lee and Benet.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Gas chromatographic—mass spectrometric assay for low levels of retinoic acid in human blood

A rapid and sensitive method of analysis for retinoic acid in human blood has been developed using gas chromatography—mass spectrometry for separation and detection. The retinoic acid is isolated by solvent extraction into petroleum ether, and converted to methyl retinoate by reacting with dimethylformamide dimethylacetal. The method has been applied to the study of retinoic acid in human blood after subtotal inunction, total inunction and intravenous injection of retinoic acid. The sensitivity limit of 1 ng/ml blood is realized with a 10-ml blood sample.     

Gas chromatographic–mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30°C, DCBs were absorbed for 15 min by a 100-μm polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography–mass spectrometry (GC–MS). By setting the initial column oven temperature at 20°C, the three isomers were resolved at the baseline level. p-Xylene-d₁₀ was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2–10.7% were found at blood concentrations of 1.0 and 10 μg/ml. Each compound was detectable at a level of at least 0.02 μg per 1 g of whole blood (by full mass scanning). HS-SPME–GC–MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.     

Gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum

A validated gas chromatographic–mass spectrometric method for quantitation of phenylalanine and tyrosine in serum is described. Quantitation of phenylalanine and tyrosine with a non-labelled non-endogenous internal standard, -2-chlorophenylalanine, compared favourably with isotope dilution mass spectrometric quantitation. The 95% reference ranges for phenylalanine, tyrosine and the phenylalanine–tyrosine molar ratio in neonate cord blood serum were determined by isotope dilution mass spectrometry and were found to be 77.1–144.7, 33.3–109.3 μmol/l and 1.1–3.0, respectively.     

Gas chromatographic—mass spectrometric assay to measure urinary N-methylhistamine excretion in man

An assay has been developed for N^τ-methylhistamine, a major metabolite of the autocoid histamine, based on gas chromatography—electron-capture negative-ion chemical ionisation mass spectrometry. N^τ-Methylhistamine was extracted from urine by cation-exchange chromatography and converted to its di-(3,5-bistrifluoromethylbenzoyl) derivative. The latter has good chromatographic properties and gives a negative-ion mass spectrum with the molecular ion (M, m/z 605) as base peak. A commercially available trideuterated analogue of N^τ-methylhistamine was used as internal standard. Basal urinary excretion of N^τ-methylhistamine in five normal subjects was found to be 0.21 ± 0.05 μmol/h (289 ± 74 μmol/mol of creatinine). This value was not significantly altered in these subjects following the infusion of a sub-pharmacological dose of histamine. In eight atopic volunteers, basal urinary excretion of N^τ-methyl-histamine was also not significantly changed following challenge with inhaled allergen.     

Validated gas chromatographic–mass spectrometric assay for determination of the antifreezes ethylene glycol and diethylene glycol in human plasma after microwave-assisted pivalylation

A gas chromatographic–mass spectrometric assay is described for identification and quantification of the antifreezes ethylene glycol (EG) and diethylene glycol (DEG) in plasma for early diagnosis of a glycol intoxication. After addition of 1,3-propanediol as internal standard, the plasma sample was deproteinized by acetone and an aliquot of the supernatant was evaporated followed by microwave-assisted pivalylation. After gas chromatographic separation, the glycols were first identified by comparison of the full mass spectra with reference spectra and then quantified. The quantification has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for EG and DEG were linear from 0.1 g/l to 1.0 g/l. The limit of detection for EG and DEG was 0.01 g/l and the limit of quantification for both was 0.1 g/l. The absolute recoveries were 50 and 65% for the low quality control samples and 51 and 73% for the high quality control samples of EG and DEG, respectively. Intra- and inter-day accuracy and precision were inside the required limits. The glycols in frozen plasma samples were stable for more than 6 months. The method was successfully applied to several authentic plasma samples from patients intoxicated with glycols. It has also been suitable for analysis of EG and DEG in urine.     

Gas chromatographic–mass spectrometric determination of total homocysteine in human plasma by stable isotope dilution: method and clinical applications

The detection and quantitation of slight increases of plasma homocysteine levels is of growing interest. This has prompted us to develop a highly sensitive and accurate capillary gas chromatography–mass spectrometry (GC–MS) method. The method proved to be highly sensitive (DL=0.17 μmol/l) with between- and within-run precision less than 6% and 7%, respectively. Reference values of plasma total homocysteine have been determined for men (n=39) and women (n=36), showing a significant difference (P=0.003) between gender. Preliminary results in cerebrovascular accidents and in venous thrombosis are presented.     

Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for dihydroergotamine and its major metabolite in human plasma

A sensitive and specific procedure for the simultaneous determination of dihydroergotamine (DHE) and its 8'-hydroxylated metabolite (8'-OH-DHE) in human plasma was developed and validated. The analytes were extracted from plasma samples by liquid-liquid extraction, separated through a Zorbax C18 column (50x2.1 mm I.D.) and detected by tandem mass spectrometry with an electrospray ionization interface. Caroverine was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 and 11.0 pg/ml for DHE and 8'-OH-DHE, respectively. The intra- and inter-run precision was measured to be below 9.1% for both DHE and 8'-OH-DHE. The inter-run accuracy was within 4% for the analytes. The overall extraction recoveries of DHE and 8'-OH-DHE were determined to be about 58 and 52% on average, respectively. The chromatographic run time was approximately 2.5 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of DHE and 8'-OH-DHE in a pharmacokinetic study of volunteers who received DHE orally.     

Gas chromatographic—mass spectrometric procedure for the quantitation of chlorpromazine in plasma and its comparison with a new high-performance liquid chromatographic assay with electrochemical detection

A gas chromatographic—mass spectrometric assay using selected ion monitoring is compared with a high-performance liquid chromatographic assay using an electrochemical detector for single-dose studies of the psychotherapeutic phenothiazine drug chlorpromazine. Measurements were made after extraction of chlorpromazine and the internal standard, prochlorperazine, from basified plasma with an isopropanol—pentane solvent mixture. Following evaporation of the organic solvents the residue was reconstituted in a small volume of methanol and subjected to gas chromatographic—mass spectrometric selected ion detection. The residual sample was then evaporated and made up in a larger volume of acetonitrile and analyzed by high-performance liquid chromatography using an electrochemical detector. These specific methods display excellent correlation for plasma concentration determinations in the range of 0.25–10 ng ml⁻¹ and will allow for the study of the pharmacokinetics of chlorpromazine following single low doses of the drug.     

Gas chromatographic–tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative

Gas chromatography–mass spectrometry (GC–MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC–tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC–MS.     

Liquid chromatographic–electrospray tandem mass spectrometric determination of novel antifungal agents in plasma

A rapid, selective, sensitive and reproducible HPLC–electrospray tandem mass spectrometric method has been developed for the analysis of novel triazole antifungal agents, SYN-2869 and its derivatives (SYN-2836, SYN-2903 and SYN-2921), in rat plasma using SYN-2506 as an internal standard. Isolation of these compounds from plasma and sample desalting were performed by a simple extraction procedure involving protein precipitation, vacuum-drying and reconstitution with acetonitrile. For all the agents, linearity was observed over the range of 10–10 000 ng/ml (r≥0.996) and the limit of quantitation was 10 ng/ml using a 100-μl plasma volume. A measurement rate of 400–500 samples/day/instrument could be achieved using this method.     

Rapid solid-phase extraction method for automated gas chromatographic–mass spectrometric determination of nicotine in plasma

The aim of this study was to develop a simple, rapid and sensitive assay of nicotine in plasma for automated gas chromatographic–mass spectrometric analysis. Biological samples were extracted using pre-packed Extrelut-1 columns with 5 ml of ethyl acetate. Quantitative analysis was done using deuterium-labelled nicotine as internal standard. The limit of quantitation was 0.5 ng in 1-ml plasma samples. Precision was ranging from 13.3% to 1.64% (R.S.D.) depending on the concentration, while the deviation was 4.16%. This method has been used for determination of nicotine bioavailability from new, low-dosage, nicotine chewing gum strips.     

Development and validation of a liquid chromatography-tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.     

Gas chromatographic–mass spectrometric confirmation of atractyloside in a patient poisoned with Callilepis laureola

The South African traditional remedy Impila (Callilepis laureola) contains the mitochondrial toxin atractyloside. The plant is sold widely and continues to lead to fatalities in patients. We describe, for the first time, a simple GC–MS procedure for the identification of atractyloside, which we have applied to the gastric washing from a poisoned patient and to extracts of Impila tuber.     

Gas chromatographic—mass spectrometric screening procedure for the identification of formaldehyde-derived tetrahydroisoquinolines in human urine

A gas chromatographic—mass spectrometric method has been developed for the identification of 1,2,3,4-tetrahydroisoquinoline and six metabolites extracted from urine in the picogram range. The derivatization procedure for the substances, formed by reaction of formaldehyde with biogenic amines, employs propionic anhydride and can take place in aqueous medium. In this way artificial formation of these compounds via condensation of biogenic amines with aldehydes or α-keto acids during the work-up procedure is eliminated. The procedure results in hydrophobic compounds, which are quantitatively extractable by liquid—liquid extraction with organic solvents. Further clean-up was performed by solid-phase extraction on C₁₈ sample preparation columns.