Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Gas chromatography–electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 μm film thickness, β=320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162°C, for 14.2 min; ramp by 30°C/min to 190°C, for 3.5 min; ramp by 30°C/min to 250°C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300°C. The sample (1 μl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5–20 μmol/l range; the limit of detection was <0.25 μmol/l; day-to-day precision, at 2 μmol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 μmol/m³). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 μmol/l vs. 8.1 μmol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 μmol/l vs. 6.9 μmol/l, p<0.001), and evidenced a relationship between MHAs (y, μmol/mmol creatinine) and airborne xylene (x, μmol/m³) (y=0.085+0.34x; r=0.82, p<0.001, n=56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma

An isocratic high-performance liquid chromatographic method for the determination of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. The method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C₁₈ cartridges. Separation was achieved with an ODS column using acetonitrile and phosphate buffer supplemented with octanesulfonic acid (an ion-pairing agent). The pH of the mobile phase (2.5) was optimal with respect to the mode of detection (fluorescence). The method was validated in the range of 5-MTHF concentrations from 0.0625 μmol/l to 4.0 μmol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exceed 8.7%. The method is specific, accurate and sensitive enough to be used in pharmacokinetic studies for the assessment of the systemic availability of 5-MTHF after leucovorin administration to patients as a rescue after high-dose therapy with methotrexate. The limit of detection was 0.17 pmol which corresponds to a plasma concentration of 1.7 nmol/l. Thus, the assay could potentially be used for the measurement of 5-MTHF in the range of physiological concentrations in plasma (5–20 nmol/l).     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

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.     

Simple and rapid method for the measurement of nitrite and nitrate in human plasma and cerebrospinal fluid by capillary electrophoresis

Nitrite and nitrate levels in physiological fluids are commonly used as an index of nitric oxide production. We developed simple and rapid method for the determination of these anions by capillary zone electrophoresis employing borate buffer (pH 10, 100 mmol/l) as running electrolyte. The anions were analyzed in plasma and cerebrospinal fluid (CSF) without deproteinization of the samples. Electrophoresis was carried out in a capillary (36.5 cm×75 μm) at a potential of 15 kV, with on-column UV detection at 214 nm. Mean retention times for nitrite and nitrates were 4.631 and 5.152 min, respectively. The method was linear (r=0.999) within a 1–500 μmol/l concentration range. Physiological levels of nitrate in plasma (40.2 μmol/l) and CSF (15.3 μmol/l) could be determined with good precision (coefficients of variation <6%) and accuracy (recoveries of added nitrate to plasma and CSF were 97.4 and 104.5%, respectively). Measurements of the physiological levels of nitrite in plasma (6.1 μmol/l) and CSF (0.9 μmol/l) were less precise and accurate.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Determination of estramustine phosphate and its metabolites estromustine, estramustine, estrone and estradiol in human plasma by liquid chromatography with fluorescence detection and gas chromatography with nitrogen-phosphorus and mass spectrometric detection

Bioanalytical methods for the determination of estramustine phosphate by liquid chromatography and its four main metabolites estromustine, estramustine, estrone and estradiol by gas chromatography are described. For the estramustine phosphate assay the plasma was purified by protein precipitation followed by a C₁₈ solid-phase extraction. For the metabolite assay the plasma samples were purified by a C₁₈ solid-phase and liquid–liquid extraction procedure and derivatised by silanization. Thereafter, estramustine and estromustine were quantified by gas chromatography with nitrogen-phosphorus detection and estradiol and estrone were quantified by gas chromatography with selected ion monitoring. The methods were validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 2.3 μmol/l for estramustine phosphate, 30 nmol/l for estromustine and estramustine, 12 nmol/l for estrone and 8 nmol/l for estradiol. The results showed good precision and accuracy for estramustine phosphate and the four metabolites. The intermediate precision was 6.2–13.5% (C.V.) and the accuracy was 91.8–103.9%.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

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.     

Rapid and specific high-performance liquid chromatographic method for the determination of iodide in urine

A rapid and specific method for the determination of iodide in urine by high-performance liquid chromatography on an anion-exchange column with electrochemical detection is described. The assay is reproducible as judged by the coefficient of variation of less than 4% at all concentrations used. The limit of detection was 0.1 μ mol, and the calibration graph was linear for concentrations between 0.1 and 200 μmol. Using this method, healthy volunteers were found to excrete 69±39 μmol of iodide per mole of creatinine.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

Measurement and pharmacokinetic study of tetramethylpyrazine in rat blood and its regional brain tissue by high-performance liquid chromatography

We used a rapid, sensitive and reliable high-performance liquid chromatographic method for the determination of tetramethylpyrazine in rat brain tissue and plasma. The lower limit of quantification in plasma and brain tissue was 0.1 μg/ml and 0.1 μg/g, respectively, and only a small amount of plasma (100 μl) or brain tissue (100 μg) was required for analysis. The decline in the concentration of tetramethylpyrazine in plasma was generally two-exponential at a dose of 2, 5, or 10 mg/kg administered intravenously. Concentrations of tetramethylpyrazine in various regions of the brain (cerebral cortex, brainstem, striatum, hippocampus, cerebellum and midbrain) were not significantly different at 15 min following drug administration (10 mg/kg, i.v.). In additional analysis, mean concentration of the tetramethylpyrazine in rat plasma was approximately five-times greater than the drug in brain tissue.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

The role of prostaglandins in the excitatory innervation of the rat urinary bladder

The possible role of PGs in hyoscine-resistant nerve mediated responses of the rat urinary bladder was investigated. Responses to electrical stimulation were inhibited by cinchocaine (30 μmol/l) but were only partially inhibited by a high concentration of hyoscine (25 μmol/l) or by the choline uptake inhibitors, hemicholinium-3 (500 μmol/l) and troxypyrrolidinium (500 μmol/l). Indomethacin (50 μmol/l) produced partial blockade (30%) of responses to electrical stimulation without markedly affecting responses to acetylcholine and the degree of blockade was of a similar order in the presence of hyoscine or troxypyrrolidinium. PGE₂ (0.028 – 2.8 μmol/l) or F_2α (0.029 – 2.9 μmol/l) produced a slowly developing increase in tone and spontaneous activity. Responses to electrical stimulation were at most only slightly increased in the presence of either PG. However, the PGs always increased the responses to electrical stimulation after indomethacin, indomethacin plus hyoscine or indomethacin plus troxypyrrolidinium. Responses to acetylcholine in the presence of indomethacin were not increased by PGE₂. It is concluded that PGE₂ and F_2α do not function as transmitters responsible for resistance to anti-muscarinic drugs in the bladder but may exert a modulating effect on nervous transmission.     

Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl–glycine, homocysteine and glutathione in plasma and cell extracts

A sensitive high-performance liquid chromatographic method for quantification of sulphydryl and disulfide amino acids in human plasma using ultra violet spectrophotometric detection was developed. Precolumn derivatization with 5,5′-dithio-bis-nitrobenzoic acid (DTNB) and an optional pre-derivatization reaction with dithiothreitol allowed both quantitative reduction of disulfides for measurement of total amino acid levels and the measurement of the reduced forms. A dynamic range of 500 nmol/l–750 μmol/l allowed the major analytes of interest to be quantified in plasma without sample dilution. The assay is a sensitive and precise method for the determination of sulphydryl and disulfide amino acids in plasma and cell extracts.