Determination of phenelzine in human plasma with gas chromatography—mass spectrometry using an isotope labeled internal standard

A quantitative gas chromatographic—mass spectrometric assay was developed for the determination of phenelzine in human plasma. Phenelzine, in aqueous solution or in plasma reacts at room temperature with pentafluorobenzaldehyde to form quantitatively a hydrazone derivative. The derivative has good gas chromatographic characteristics. The assay utilizes selected ion monitoring in a gas chromatographic effluent, the molecular ion generated by electron impact ionization of phenelzine derivative. Phenelzine-d₇ was synthesized and used as an internal standard. The assay can measure 2 ng/ml of the drug with about 10% precision.The method was used for the determination of steady state levels of phenelzine in the plasma of patients taking a therapeutic dose of the drug.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Quantification of dimethindene in plasma by gas chromatography—mass fragmentography using ammonia chemical ionization

A gas chromatographic—mass fragmentographic method using ammonia chemical ionization for the determination of dimethindene in human plasma is described. The drug was isolated from plasma by liquid—liquid extraction with hexane—2-methylbutanol. Plasma components were separated on a capillary column coated with chemically bonded methyl silicone. For detection of dimethindene, its quasi-molecular ion (M + H⁺) was mass fragmentographically monitored after chemical ionization with ammonia as reagent gas. Dimethindene was quantified using methaqualone as the internal standard: the quantification limit in plasma was 0.2 ng/ml, the within-run precision was 8.0% and the inter-run precision 5.6%. The plasma concentration—time profile was established after a single dose of 4 mg of dimethindene with an average maximum concentration of 5.5 ng/ml, detectable up to 48 h post application.     

Valproic acid analysis in saliva and serum using selected ion monitoring (electron ionization) of the tert.-butyldimethylsilyl derivatives

A highly sensitive ion monitoring method for the determination of valproic acid in saliva and in serum has been developed based on the gas chromatographic—mass spectrometric analysis of the tert.-butyldimethylsilyl derivatives. Extraction methods are simple and the techniques for derivatization are rapid and convenient. Selected ion monitoring was carried out using electron ionization conditions and a common ion m/z 201 (M⁺ − 57) present in valproic acid and the internal standard octanoic acid. The lower limit of sensitivity that has acceptable precision for assay purposes is 0.1 mg/l based on a 200-μl sample size. The ion monitoring method (derivatized) was compared to a gas chromatographic method (underivatized) for serum valproate assays and found to be essentially identical.The assay methodology was used in a kinetic study of valproic acid in two normal subjects. Saliva levels of drug were found to give reasonably good correlations with serum total and with serum free concentrations of drug in both individuals.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Gas chromatographic—mass fragmentographic determination of homopantothenic acid in plasma

A gas chromatographic—mass fragmentographic method was developed for the determination of homopantothenic acid in plasma. Acidified plasma was deproteinized by extraction with chloroform and subsequently the aqueous layer was extracted with ethyl acetate. The organic layer containing homopantothenic acid was reduced to dryness, and the resulting residue was redissolved in N,O-bis(trimethylsilyl)trifluoroacetamide—pyridine solution to allow trimethylsilylation. Aliquots of this solution were injected into the gas chromatograph—mass spectrometer and analyzed by the selected ion monitoring method using l-ascorbic acid as an internal standard. The detection limit for homopantothenic acid was 5 ng/ml of plasma.A precise and sensitive assay for the determination of homopantothenic acid in plasma was established.     

Application of negative-ion chemical ionization isotope dilution gas chromatography—mass spectrometry to single-dose bioavailability studies of mefloquine

An electron-capture negative-ion chemical ionization gas chromatographic—mass spectrometric assay for mefloquine, an antimalarial drug used in the treatment of drug-resistant Plasmodium falciparum malaria, is described. The method, developed in support of bioavailability studies involving the co-administration of different tableted formulations of the drug and an aqueous solution of its ¹³C₃-labeled analog, enables quantification of both dosage forms. Quantitative analysis of extracted plasma samples was performed on the O-tert.-butyldimethylsilyl (t-BDMS) derivative of the drug by selected-ion monitoring, using a VG Trio 2000 quadrupole mass spectrometer and monitoring the [M — t-BDMSOH]^−√ ions of the analytes. The method, incorporating [²H₆]mefloquine as an internal standard, demonstrated good accuracy and precision over the 1–200 ng ml⁻¹ range, with correlation coefficients greater than 0.990 for all standard curves and a detection level of 50 fg on-column. Replicate analysis of plasma samples over a 90-day period exhibited a mean intra-day and inter-day variation of less than 4.5% and 5.5%, respectively. The high stability and sensitivity of the assay, combined with the inherent selectivity of mass spectrometric detection, make the method well-suited for such studies.     

Gas chromatographic—mass spectrometric method for routine monitoring of 5-fluorouracil in plasma of patients receiving low-level protracted infusions

A gas chromatographic—mass spectrometric (GC—MS) method is described which quantitates 5-fluorouracil (5-FU) plasma levels ranging from 0.5 to 50 ng/ml. The analysis uses two internal standards, 1,3-[¹⁵N₂]-5-fluorouracil and 5-chlorouracil. Extraction and derivatization of the pyrimidine bases were accomplished in a single step using acetonitrile. Compounds were analyzed as their 1,3-dipentafluorobenzyl derivatives by electron-impact MS, and the GC—MS analysis was automated with respect to sample injection and data reduction. Stability of the analysis was demonstrated by continuous unattended analysis of 5-FU in human plasma for periods of up to three days with no deterioration of the quantitative results. The method is applicable to quantitating 5-FU plasma levels in patients receiving protracted infusions of the drug for colorectal cancer or other malignancies.     

Automated theophylline assay using gas chromatography and a mass-selective detector

An automated gas chromatographic—mass spectrometric assay for theophylline is described. Theophylline is extracted from plasma or urine (50 μl) and transformed into an N-pentyl derivate. The internal standard used for quantitation is [1,3-¹⁵N, 2-¹³C]theophylline. The detection is performed by monitoring the molecular ions 250 for theophylline and 253 for the internal standard with a quadrupole mass specific detector HP 5790 A. The system has been fully automated: injection, calibration, assay, calculation. The method shows excellent analytical parameters: linearity between 2 and 40 μg/ml; day-to-day reproducibility 1.82% for a concentration of 15 μg/ml; repeatability 0.75% (15 μg/ml) and 0.33% (30 μg/ml). Accuracy is also excellent. Due to the use of an internal standard labelled with stable isotopes, the specificity and high analytical quality of the method make it useful as a reference method to compare with routine theophylline assays.     

Improved selective ion monitoring mass-spectrometric assay for the determination of n,n-dimethyltryptamine in human blood utilizing capillary column gas chromatography

A gas—liquid chromatographic—mass spectrometric procedure is described for the assay of dimethyltryptamine (DMT) in whole blood. The use of a glass capillary column in combination with selective ion monitoring results in an assay with a high degree of specificity and sensitivity. 5-Methoxy-DMT is used as an internal standard and carrier in the isolation procedure. The superior resolving characteristics of the capillary column (as compared to previously employed packed columns) allows monitoring of the intense m/e 58 ion arising from the DMT side-chain. A sensitivity limit of 10 pg/ml blood is realized with a 10-ml blood sample.     

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.     

Rapid mass spectrometric analysis for ascorbate and related organic acids in small volumes of plasma for use in pediatric subjects

A gas chromatographic–mass spectrometric isotope dilution method was developed for analysis of ascorbate on 10 μl samples of plasma. This assay was reproducible (standard deviation of less than 4%) and gave values for plasma ascorbate content within 8% of our previously published gas chromatographic–mass spectrometric method. Non-specific sample preparation allowed other analytes to be determined on the same sample by adjusting data acquisition parameters and adding the appropriate internal standard. Analysis on 28 subjects fell within the expected range for plasma ascorbate 68±29 μm (11.9±5.0 μg/ml) and established a normal range for plasma threonate of 28.1±2.4 μm (3.8±0.4 μg/ml).     

Determination of plasma cocaine and ethylcocaine (cocaethylene) in mice using gas chromatography—mass spectrometry and deuterated internal standards

A gas chromatographic—mass spectrometric method is described for the determination of cocaine and ethylcocaine (cocaethylene) from mouse plasma microsamples (50 μl). [²H₃]Cocaine and [²H₅]ethylcocaine served as internal standards, analytical separations were performed on a (5% phenyl)methylpolysiloxane capillary column, and detection was by selected-ion monitoring of electron-impact generated fragment ions [M --- CO₂Ph]. Pilot study plasma concentrations of ethylcocaine following coadministration of cocaine and ethanol were less than 5% of the parent drug.     

Derivatization and gas chromatographic—mass spectrometric detection of anabolic steroid residues isolated from edible muscle tissues

A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C₁₈ solid-phase extraction columns, the extract was injected onto a C₁₈ reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic—mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate.     

Determination of bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

Determination of Ro 23-7637 in dog plasma by multidimensional ion-exchange—reversed-phase high-performance liquid chromatography with ultraviolet detection

Ro 23-7637 (I) is a new drug under development for the treatment of metabolic diseases. A high-performance liquid chromatographic—ultraviolet detection (HPLC—UV) analytical procedure for its analysis in dog plasma was developed and is reported here. Following C₁₈ solid-phase extraction, the sample is applied to a strong cation-exchange column in the first dimension. The analyte and internal standard, Ro 24-4558 (II), are transferred to a base-deactivated C₁₈ reversed-phase column in the second dimension (orthogonal separation mechanism), with UV detection at 254 nm. The reversed-phase solid-phase extraction provides a gross isolation of the drug, based on hydrophobicity. The first-dimension ion-exchange separation allows neutral species and anions to elute with the column void volume, while separating basic species according to pK_a. The second dimension provides a high-resolution separation dependent upon the hydrophobicity of the sample species. The rationale for using orthogonal multidimensional chromatography was that an exhaustive examination of reversed-phase and normal-phase columns invariably resulted in co-elution of I with endogenous plasma components, which limited the sensitivity of the method. We have utilized C₁₈ solid-phase extraction, followed by multidimensional HPLC—UV, to develop an accurate and precise analytical method whose limit of quantitation, 5 ng/ml using 0.5 ml of plasma, is determined by inherent detector sensitivity. Increased sensitivity can be readily achieved by using more sample or by using microbore HPLC on the second dimension.     

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.     

Routine screening and quantitation of urinary corticosteroids using bench-top gas chromatography—mass-selective detection

A method was developed for the routine screening, confirmation and quantitation of corticosteroids in human urine using bench top capillary gas chromatography (GC)—mass-selective detection. The free and conjugated corticosteroid fractions were isolated by liquid—liquid partition. After evaporation to dryness under vacuum the corticosteroid residues were derivatized to form the methyloxime trimethylsilyl ether derivatives. Both GC retention data and characteristic spectral data based on authentic reference standards were used for the identification and quantitation of cortisol, cortisone, tetrahydrocortisol and tetrahydrocortisone in the ppb (ng/ml) concentration range. The method is simpler and more efficient than the other GC—mass spectrometric (MS) techniques. It is also more sensitive than the liquid chromatographic—MS method.     

High-performance liquid chromatography–tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine

A sensitive, selective and accurate high-performance liquid chromatographic–tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C₁₈) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6–5 nmol/l for the studied compounds in plasma samples.     

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic–mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 μM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-α-ethyl-2-oxo-pyrrolidine acetamide in a dog.