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 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.     

Gas chromatographic—mass spectrometric analysis of tar compounds formed during pyrolysis of rice husks

Pyrolysis of agricultural waste to produce fuel gas involves formation of tars as noxious by-products. In this paper the qualitative analysis of tars formed during pyrolysis of rice husks is presented, based on identification by gas chromatography—mass spectrometry and interpolation of retention times on a polyaromatic hydrocarbon index scale. The influence of some reaction parameters on product formation is briefly discussed.     

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.     

Gas chromatographic—mass spectrometric determination of glutamic acid decarboxylase activity in subregions of rat brain

A quantitative gas chromatographic—mass spectrometric method has been developed for the determination of glutamic acid decarboxylase (GAD) activity in subregions of rat brain. The five subregions analyzed, weighing approximately 2.51 mg each, were globus pallidus, entopeduncular nucleus, ventromedial thalamus, and substantia nigra medial and lateral. The activity of the GAD enzyme has been determined indirectly by measurement of γ-aminobutyric acid (GABA) using γ-[2,2-²H₂]aminobutyric acid as the internal standard. Both compounds were quantitatively converted to trimethylsilyl-GABA and trimethylsilyl-[²H₂]GABA in 90 min with hexamethylchlorosilane, trimethylchlorosilane, pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide silylating agents. Using selective ion monitoring and electron impact ionization at 70 eV, the limit of detection was 15 ng GABA per mg tissue. This method is compared with a fluorimetric procedure.     

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.     

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.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

Artifact formation during gas chromatographic—mass spectrometric analysis of a methylsulfinyl-containing metabolite

During gas chromatographic—mass spectrometric analysis using a heated injector, 1-methylthio-4-methylsulfinyltetrachlorobenzene degraded to form tetrachlorothioanisole. Similar reductive defunctionalizations have been reported during in vivo metabolisms. Caution should be used to distinguish metabolites from artifacts which may be formed during the analysis of methylsulfoxides.     

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic—mass spectrometric assay

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.     

Gas chromatographic—mass spectrometric determination of plasma 5-fluorouracil after administration of 1-hexylcarbamoyl-5-fluorouracil to dogs and humans

A simple, sensitive and specific method for determining 5-fluorouracil (5-FU) in plasma after the administration of 1-hexylcarbamoyl-5-fluorouracil (HCFU) was developed using gas chromatography—mass spectrometry. Thymine was used as the internal standard. After removal of interfering substances with chloroform, diethyl ether and Amberlite XAD-2 resin, 5-FU and thymine were extracted with 16% n-propanol in diethyl ether and methylated with trimethylanilinium hydroxide. Fragment ions at m/e 158 and 154, the molecular ion of the dimethyl derivatives of 5-FU and thymine, respectively, were used to monitor 5-FU and thymine. The sensitivity of the method is 10 ng/ml, which is sufficient to determine the 5-FU levels in plasma after the administration of therapeutic doses of HCFU to patients.     

Gas chromatographic—mass spectrometric methods of analysis for detection of 11-nor-Δ-tetrahydrocannabinol-9-carboxylic acid in biological matrices

Gas chromatographic—mass spectrometric methods of analysis for the detection of 11-nor-Δ⁹-tetrahydrocinnabinol-9-carboxylic acid, a major metabolite of Δ⁹-tetrahydrocannabinol, are reviewed. Emphasis is on analytical methodology including numerous derivatization techniques developed specifically for this analyte. The majority of procedures cited in the literature were developed to detect this metabolite in the blood and urine of man.     

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.     

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.     

A gas chromatographic—mass spectrometric study of profiles of volatile metabolites in hepatic encephalopathy

Volatile organic substances present in blood plasma and cerebrospinal fluids of certain control groups of human subjects and cirrhotic patients some of whom were suffering from hepatic encephalopathy were quantitatively analysed and identified. A rapid, reproducible, direct injection capillary column gas chromatographic method was developed for the concentration and detection of such volatiles at mg/l and lower concentrations. Of at least forty volatiles detected, twenty-one were identified. The mean concentration of one of these, 3-methylbutanal, was found to be significantly elevated (p < 0.01) in chronic encephalopathics (2.37 ± 0.79 mg/l, n = 18), when compared to the controls (0.30 ± 0.08 mg/l, n = 20). Furthermore, the concentration of this component increased with the clinically diagnosed severity of the encephalopathic state. The presence of 3-methylbutanal is related to leucine, a branched-chain amino acid linked with hepatic encephalopathy.     

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–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Automated sample preparation and gas chromatographic–mass spectrometric analysis of urinary androgenic anabolic steroids

This paper presents an automated method for extracting anabolic agents from urine samples for their GC–MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 μl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.