High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65±2.3 and 8.9±2.4 μmol/l, while post-methionine load gave tHcy levels of 19.9±5.5 and 26.8±5.5 μmol/l, for women and men, respectively (n=40).     

Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.     

Reversed-phase liquid chromatographic determination of plasma levels of adriamycin and adriamycinol

A method is given for the determination of adriamycin and its main metabolite, adriamycinol in plasma from cancer patients after administration of adriamycin as the free drug or as a complex with DNA.Adriamycin and adriamycinol are extracted in a column from 1 ml of plasma (pH 8.6) using a mixture of chloroform—1-heptanol (8:2). After re-extraction into phosphate buffer pH 2.2, the separation is performed as reversed-phase liquid chromatography on a LiChrosorb RP-2 (5 μm) column with a mobile phase of acetonitrile—water, acidified with phosphoric acid.The precision by quantitation with photometric detection was better than 5% within the range 50–300 ng/ml. Plasma levels of adriamycin and adriamycinol in a cancer patient are presented in this paper.     

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19x ? 1.4 for tazobactam and y = 1.7x ? 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.     

New electron-capture gas-liquid chromatographic method for the determination of mexiletine plasma levels in man

A method for the determination of mexiletine in human plasma by gas—liquid chromatography with electron-capture detection is described. Plasma samples are extracted at pH 12 with dichloromethane after addition of the internal standard, the 2,4-methyl analogue of mexiletine. A derivative is obtained using heptafluorobutyric anhydride; according to gas chromatography—mass spectrometry it is a monoheptafluorobutyryl compound. The minimum detectable amount of mexiletine is 5 pg. Accurate determinations of human plasma levels were performed after oral or intravenous treatment.     

Comprehensive plasma thiol redox status determination for metabolomics

Thiol homeostasis plays an important role in human health and aging by regulation of cellular responses to oxidative stress. Due to major constraints that hamper reliable plasma thiol/disulfide redox status assessment in clinical research, we introduce an improved strategy for comprehensive thiol speciation using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that overcomes sensitivity, selectivity and dynamic range constraints of conventional techniques. This method integrates both specific and nonspecific approaches toward sensitivity enhancement for artifact-free quantification of labile plasma thiols without complicated sample handling. A multivariate model was developed to predict increases in ionization efficiency for reduced thiols when conjugated to various maleimide analogs based on their intrinsic physicochemical properties. Optimization of maleimide labeling in conjunction with online sample preconcentration allowed for simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols as their intact symmetric or mixed disulfides. Identification of low-abundance thiols and various other polar metabolites detected in plasma was supported by prediction of their relative migration times using CE as a qualitative tool complementary to ESI-MS. Plasma thiol redox status determination together with untargeted metabolite profiling offers a systemic approach for elucidation of the causal role of dysregulated thiol metabolism in the etiology of human diseases.     

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

High-performance liquid chromatographic determination of ganciclovir in plasma

A rapid, selective and sensitive isocratic reversed-phase high-performance liquid chromatographic method for the determination of ganciclovir in plasma samples was developed. This method, which was applied to the analysis of plasma ganciclovir from heart transplant patients under ganciclovir therapy for cytomegalovirus infections, represents a suitable analytical tool for drug monitoring and pharmacokinetic investigations.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.     

High-performance thin-layer chromatographic determination of ibuprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for quantitation of ibuprofen from plasma is described. The drug was extracted from acidified plasma with hexane-isopropanol (85:15). The mobile phase composition was n-hexane-ethyl acetate-anhydrous acetic acid (75:25:2). Densitometric analysis of ibuprofen was carried out at 222 nm. The calibration curves of ibuprofen in chloroform and in plasma were linear over the range 2–20 μg. The mean values of intercept, slope and correlation coefficient were 0.0422±0.0018, 0.0356±0.0213 and 0.9976±0.0013 for standard curves in chloroform and 0.1044±0.003, 0.8759±0.0213 and 0.9939±0.001 for standard curves in plasma, respectively. The limit of detection of ibuprofen from human plasma (assay sensitivity) was 50 ng and no interference was found from endogenous compounds. The recovery of ibuprofen from human plasma using the described extraction procedure was about 85%. The mean relative standard deviations for within-day and between-day analyses were 2.24 and 2.6% for 5 μg and 3.67 and 3.2% for 15μg ibuprofen concentration, respectively. The method was utilized to monitor the plasma concentration of ibuprofen post administration of sustained release capsules in human patient volunteers.     

Liquid chromatographic determination of amodiaquine in human plasma

A normal-phase high-performance liquid chromatographic method using dichloromethane- methanol-1M perchloric acid (100:10:0.9, v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher Si column with UV (254 nm) detection has been developed for the determination of amodiaquine and its metabolites desethyl amodiaquine and bisdesethyl amodiaquine in plasma. The limit of quantification was 5 ng ml(-1). Mean within-day and day-to-day coefficients of variation (CV) were 4.10 and 6.27% for amodiaquine, 3.43 and 4.80% for desethyl amodiaquine and 3.53 and 5.23% for bisdesethyl amodiaquine, respectively. Mean extraction recovery of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine from plasma were 82.48, 74.50 and 69.65%, respectively. Chloroquine and its metabolite desethyl chloroquine, quinine, sulfadoxine and primaquine do not interfere in the detection of amodiaquine, desethyl amodiaquine and bisdesethyl amodiaquine in plasma.     

High-performance thin-layer chromatographic determination of flurbiprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for the assay of flurbiprofen in plasma is reported. The drug was extracted from acidified plasma with hexane–diethyl ether (80:20). The mobile phase composition was n-hexane–ethyl acetate–glacial acetic acid (60:30:10). Densitometric analysis of flurbiprofen was carried out at 247 nm. The calibration curves of flurbiprofen in methanol and in plasma were linear in the range 40–400 ng. The mean values of correlation coefficient, slope and intercept were 0.995±0.003, 0.075±0.002 and 4.39±0.05 for standard curves in methanol and 0.992±0.002, 0.066±0.007 and 3.40±0.72 for standard curves in plasma, respectively. The limit of quantitation for flurbiprofen in human plasma was 40 ng, and no interference was found from endogenous compounds. The recovery of flurbiprofen from human plasma using the described extraction procedure was about 87%. The coefficient of variation for within-day and between-day analyses was 2.53% and 3.96% for 200 ng and 1.76% and 2.30% for 400 ng flurbiprofen concentration, respectively. The method was utilized to monitor plasma concentration of flurbiprofen post administration of sustained release capsules in human patient volunteers.     

High-performance liquid chromatographic determination of pentamidine in plasma

This report describes the analysis of pentamidine by isocratic reversed-phase high-performance liquid chromatography (HPLC) using a commercially available compound (melphalan) as the external standard. Previously described assays use ion-pairing HPLC, an internal standard (hexamidine) that is not readily available, and require a relatively large sample size. In the present assay, pentamidine was extracted from plasma using solid-phase extraction and was analyzed using a C₁₈ column and a mobile phase containing 18% acetonitrile, 2% methanol, 0.2 M ammonium acetate and 0.5% triethylamine. The identity of the eluting peaks was verified using a diode array detector. The extraction yield of pentamidine was 82%. The limit of detection was 8.6 ng/ml with a sample size of 100 μl. The inter-day and intra-day coefficients of variation ranged between 0.3% and 10% with an average of 5%. This method was applied to study the pharmacokinetics of pentamidine in rodents.     

Gas chromatographic determination of meprobamate in human plasma

A simplified and rapid gas chromatographic method has been developed for the determination of meprobamate in human plasma. The procedure includes a single-step extraction of alkalinized sample with chloroform, and chromatography on a non-polar fused-silica capillary column with flame ionization detection. The method is accurate (97.7 ± 5.7% at 20 mg/l) and precise (maximum coefficient of variation of 9.5%). It provides an alternative to existing methods and is particularly suitable for toxicological studies.     

Liquid chromatographic determination of irbesartan in human plasma

A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm x 4.6 mm i.d., particle size 5 microm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was approximately 13 min. The validated quantitation ranges of this method were 15-4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3-77.1% with coefficients of variation of 3.7-6.3%. The between- and within-batch precision were 0.4-2.2% and 0.9-6.2%, respectively. The between- and within-batch relative errors (bias) were (-5.5) to 0.9% and (-0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at -70 degrees C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.     

Gas-liquid chromatographic determination of total phenylacetic acid in urine

A gas-liquid chromatographic procedure to measure total phenylacetic acid in urine is described. The method is simple, rapid, and reliable. Normal subjects (N = 48) excreted 141.1 ± 10.1 mg/24 h. Untreated depressed patients (N = 42) excreted 102.77 ± 15.9 mg/24 h. The difference in the means is significant and supports the role of phenylacetic acid as a biological marker in certain kinds of mental illnesses.     

Improved gas chromatographic procedure for the determination of clonazepam levels in plasma using a nitrogen-sensitive detector

A gas—liquid chromatographic procedure (GLC) is described for the determination of clonazepam in plasma. The drug is extracted from buffered plasma at pH 9.0 with diethyl ether and then back-extracted into 6 N hydrochloric acid—6 N sulfuric acid (95:5) and hydrolyzed at 100°C to convert the drug into its benzophenone derivative. The benzophenone derivative of flurazepam is added to plasma as an internal reference standard. Drug derivatives are finally extracted from the neutralized aqueous phase and assayed by GLC. The present procedure makes use of a nitrogen-sensitive detector which is more stable and selective than the commonly employed electron-capture procedure. The sensitivity of the detector for clonazepam is 1 ng/ml.