Assay of timolol in human plasma using gas chromatography with electron-capture detection

A simple and sensitive gas chromatographic method has been developed for the determination of timolol in plasma using electron-capture detection and propranolol as internal standard. Timolol was extracted using butyl chloride and derivatized using trifluoroacetic anhydride in butyl acetate. The lower detection limit for the assay was found to be 1 ng/ml from 1 ml of plasma. Extracted standards gave within-day precision of 12.55, 9.68 and 3.78% for 1, 20 and 100 ng/ml plasma samples, respectively. A recovery of at least 80% of timolol was found using the extraction method described. The assay was used in a randomized cross-over bioequivalence trial using an oral administration of 20 mg of timolol. Pharmacokinetic parameters compare favourably with other literature values.     

Determination of paroxetine in plasma by high-performance liquid chromatography for bioequivalence studies

A high-performance liquid chromatographic method is described for the determination of paroxetine in human plasma. Dibucaine was used as the internal standard. Paroxetine was isolated by solid phase extraction using a Bond-Elut C₁₈ extraction column. Separation was obtained using a reversed-phase column under isocratic conditions with fluorescence detection. The sample volume was 500 μl of plasma. The intra- and inter-assay accuracy and precision, determined as relative error and relative standard deviation, respectively, were less than 10%. The lower limit of quantitation, based on standards with acceptable relative error and relative standard deviation, was 10 ng ml⁻¹. No endogenous compounds were found to interfere. The linearity was assessed in the range 5–100 ng ml⁻¹. Stability of paroxetine during processing (autosampler) and in plasma was checked. This method proved suitable for bioequivalence studies following multiple doses in healthy volunteers.     

Quantitation of levorphanol in human plasma at subnanogram per milliliter levels using capillary gas chromatography with electron-capture detection

A gas chromatographic method for the determination of levorphanol in human plasma is described. The method utilizes extractive alkylation with tetrabutylammonium cation as the phase-transfer catalyst and pentafluorobenzyl bromide as the alkylating agent, and employs a structural analog, d-3-hydroxy-N-ethylmorphinan, as the internal standard. The pentafluorobenzyl ethers formed are separated by capillary gas chromatography and detected by electron capture. The method has good precision and accuracy for concentrations ranging from 0.25 ng/ml to 100 ng/ml and has been used to measure plasma concentrations as part of a study to evaluate the management of chronic neuropathic pain with levorphanol.     

Determination of acetaldehyde in biological samples by gas chromatography with electron-capture detection

A simple specific assay was developed for the determination of acetaldehyde in biological samples. Acetaldehyde was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with electron-capture detection. The use of this detection method is an important device to which no one drew notice. This procedure was very simple and so sensitive that as little as 500 fmol of acetaldehyde could be measured in aqueous solution. The calibration curve of acetaldehyde was linear at least up to 40 μM. Its recoveries from human plasma and rat liver homogenate were 96.5 and 95.7%, respectively.     

Rapid simple high-performance liquid chromatographic determination of paroxetine in human plasma

A rapid, simple method for the measurement of paroxetine in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described. This method includes only one-step extraction of paroxetine and dibucaine, an internal standard, with chloroform. Their recoveries were around 90%. The mobile phase, 10 mM phosphate buffer–acetonitrile (40:60, v/v) was eluted isocratically. Between- and within-day coefficients of variation were in the range of 1.9–9.4% and 2.3–13.3%, respectively. The detection limit was 0.2 ng/ml. The method we describe can be easily applied to the measurement of plasma paroxetine concentration for pharmacokinetic studies as well as for therapeutic drug monitoring in patients taking paroxetine.     

Determination of plasma, urine, and bovine serum albumin low-molecular-weight carbonyl levels by capillary gas chromatography with electron-capture and mass-selective detection

Peroxidation of lipids produces low-molecular-weight carbonyl compounds, which are reactive with biological nucleophiles. The analysis of these compounds is often difficult. A multicomponent method for the determination of 11 of them in biological samples is reported. The samples are subjected to a pretreatment-derivatization procedure followed by gas chromatographic analysis with either electron-capture detection (ECD) or mass-selective detection (MSD) in the selected-ion monitoring mode. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the derivatization products on a nonpolar gas chromatographic column. The concentration of the derivatization reagent, pH, reaction time, temperature, and presence of extraneous ions were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of low-molecular-weight carbonyl compounds at femtomole levels in several biological materials such as plasma, urine, and bovine serum albumin without interferences. The limits of detection were in the ranges 0.01-0.2 microM for ECD and 0.15-1.5 microM for MSD. The mean procedural recoveries obtained during the method validation were within the range 85-95% and the intra- and interassay standard deviations do not exceed 4.6 and 6.1%, respectively.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

Determination of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection

A method for the determination of trace amounts of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection was established. The column used exhibits excellent thermostability in high-temperature analysis and easy handling and a long lifetime of the column and well shaped peaks on the chromatograms are obtained. With the metal capillary column, it was found to be easier to maintain suitable analytical conditions for the routine assay of triazolam than with a fused-silica column. With this method, 0.5 ng/ml of triazolam in serum can be determined. The method is useful for pharmacokinetic and therapeutic purposes.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Determination of a new blood glucose-lowering agent in biological fluids by capillary gas chromatography using electron-capture detection

A gas chromatographic method for the determination of 5-(4-chlorophenyl)-2-[(4-methylphenyl)sulphonyl]-4-pentynoic acid (I) in plasma (serum) and urine has been developed. After an extraction process, the cleaned-up organic extract was derivatized with diazomethane at ambient temperature. Results are evaluated from peak-height ratios with respect to the appropriate internal standard. The detection limit following extraction of a 1-ml plasma sample is about 20 ng/ml.     

Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection

A novel, highly sensitive method for the determination of pilocarpic acid (PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug presence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopilocarpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in heparinized human plasma during storage. For the determination of PA, the selective extraction of PA from protein-free plasma was accomplished using two different solid-phase extraction (SPE) cartridges in two consecutive SPE steps. After extraction, PA was lactonized with trifluoroacetic acid back to P, and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This procedure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previously described methods. The assay was validated in the concentration range of 1 to 10 ng/ml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the quality control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposure to P, administered by the ocular route, in terms of total plasma PA (P and PA).     

Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography—mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Determination of urinary methylhistamine excretion in male and female rats by gas chromatography with electron-capture detection

A gas-liquid chromatographic method for the determination of methylhistamine in urine is described. Methylhistamine is reacted with 2,6-dinitro-4-trifluoromethylbenzene sulfonic acid and the derivative thus formed is quantitated with electron-capture detection. The twenty-four hour urinary excretion of methylhistamine in the male rat is about five-fold greater than that in the female rat. Amino-guanadine, a diamine oxidase inhibitor, causes a four-to five-fold increase in methylhistamine excretion in both male and female rats. Treatment with pargyline, a monoamine oxidase inhibitor, causes only a small increase in methylhistamine excretion in male and female rats thereby suggesting that oxidative deamination of methylhistamine is a relatively minor pathway.     

Determination of ritodrine in biological fluids of the pregnant sheep by fused-silica capillary gas chromatography using electron-capture detection

A sensitive and selective gas chromatographic assay method employing splitless injection, fused-silica capillary columns and electron-capture detection is reported for the quantitation of the tocolytic drug, ritodrine, in a variety of biological fluids obtained from the pregnant ewe and fetus. This method has improved sensitivity and selectivity over previously published assay procedures. A 25 m × 0.31 mm I.D., cross-linked 5% phenylmethylsilicone, fused-silica capillary column was employed for all analyses. Linearity of response was observed over the range 2.5–75 ng of ritodrine base per 0.05–0.5 ml of biological fluid, representing ≈ 1–75 pg at the detector. The coefficient of variation was less than 10% over the range 2.5–75 ng of added ritodrine. The minimum quantifiable amounts is ≈ 2.5 ng from a 0.5-ml biological fluid sample. Applicability of this method to biological fluids, obtained from ovine subjects, is demonstrated by the analysis of samples obtained during the course of ritodrine placental transfer studies.