Determination of remifentanil in human blood by capillary gas chromatography with nitrogen-selective detection

A validated method for the determination of remifentanil in human blood, applicable to all therapeutic concentrations, using capillary GC with nitrogen-specific detection and fentanyl as the internal standard has been developed. Citrated whole blood samples were extracted into 1-chlorobutane following precipitation of proteins with methanol. The drugs were back extracted into 10 mM HCl and re-extracted into methanol-1-chlorobutane. The extracts were reconstituted in methanol and injected onto a 25-m BPX-5 column. The lower limit of quantitation was 0.2 ng/ml with within- and between-day coefficients of variation of less than 15%.     

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 barbiturates and some neutral drugs in serum using quartz glass capillary gas chromatography

Rapid methods for the glass capillary gas chromatographic determination of barbiturates and some neutral drugs are described. The analysis of barbiturates was performed using a nitrogen—phosphorus selective detector (NPD). The barbiturates were recovered from serum using charcoal adsorption followed by extraction with methylene chloride. The drugs were then alkylated by means of the Claisen carbonate method. Neutral drugs were extracted simultaneously with the barbiturates. The neutral drugs were determined underivatized with a flame ionization detector. In the underivatized form the barbiturates were not stable on the quartz column used. The selectivity of derivatization combined with an NPD was used to determine the barbiturates in the presence of neutral drugs with the aid of retention data.     

Determination of methanol in whole blood by capillary gas chromatography with direct on-column injection

A gas chromatographic procedure was developed for the determination of methanol in small-volume whole blood samples. Samples (100–200 μl) were prepared by protein precipitation, with direct injection of the supernatant on a wide-bore capillary column. The recovery of methanol and acetonitrile (the internal standard) was approximately 90% and did not vary with sample volume. The assay was linear from 2 μg/ml (the limit of detection) through 1000 μg/ml and was highly reproducible (intra-day coefficient of variation <2.5%). Assay performance was assessed following exposure of rats to methanol. The results indicate that the present procedure is suitable for studies of methanol disposition in small rodent species.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Determination of triacylglycerols in serum by capillary gas chromatography with trinonadecanoylglycerol as internal standard

An accurate capillary gas-chromatographic method with trinonadecanoylglycerol as internal standard for determining triacylglycerols in human serum and other biological sources is described. After serum extraction, total triacylglycerol and triacylglycerol species (differing in the number of carbon atoms in the acyl radicals) are directly determined without any further sample manipulation. In addition, from the same gas-chromatographic run the data obtained by the integrator record are compared with those of a computer data acquisition system. Evaluation of the triacylglycerol values resulted in a coefficient of variation (CV) of 2.08% (computer evaluation). Simultaneous evaluation of data obtained from tripalmitoylglycerol and tristearoylglycerol standards resulted in CV of 2.04 and 1.99%, respectively (computer evaluation), and 6.63 and 4.84%, respectively (integrator evaluation). Gas chromatography at lower elution temperature resulted in better separations but enhanced CV values up to about 4%. Triacylglycerol values were not influenced by storage of plasma at -20 degrees C up to 4 days prior to extraction.     

Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic—mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 μg/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at − 20°C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 μg/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.     

Validated capillary gas chromatographic-mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure

BACKGROUND: Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. METHODS: The levels of the diastereomers 2-MCA I and II in human serum were determined by GC-MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCA(total), 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7+/-14.4 years). RESULTS: 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCA(total). The internal standard showed a diastereomer ratio of 2-MCA II-d3 to 2-MCA I-d3 of 0.83+/-0.05, its chemical purity had to be corrected to 90.5+/-0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCA(total), recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCA(total) obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCA(total) over a period of 5 months (n=17). The normal range for 2-MCA(total) in human serum was calculated as 81-266 nmol/l confirming previous findings. CONCLUSIONS: The GC-MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.     

Work in progress: Analysis of the prostaglandins E by glass capillary gas chromatography with electron capture detection

Using a wide bore capillary column coated with D.E.G.S. in combination with a micro volume ⁶³Ni electron capture detector a simple and rapid method with high sensitivity was developed for the analysis of E prostaglandins.For electron capture detection the naturally occurring PGE's have to be transformed to the PGB configuration, esterified and silylated. This method with sensitivity in the low picogram range, tested with prostaglandin standards, will be used for the analysis of prostaglandins and prostaglandin metabolites in biological fluids.     

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 uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Determination of lormetazepam and its main metabolite in serum using micellar electrokinetic capillary chromatography with direct injection and ultraviolet absorbance detection

The use of micellar electrokinetic chromatography for the determination of lormetazepam and its metabolite, lorazepam, in serum samples at a concentration range of therapeutic interest was investigated. The separation was carried out at 30 degrees C and 25 kV, using a 15 mM borate-phosphate buffer (pH 8) with 30 mM sodium dodecyl sulfate as the separation electrolyte and 15% methanol as organic modifier. The analyses were carried out in 20 min under these conditions. Detection limits of 0.5 mg l(-1) were achieved for both benzodiazepines in serum. This method was employed for the quantitative resolution of both drugs (at different concentration ratios) in serum with very good recoveries.     

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection

A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid-liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4-256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.