Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid—liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3–0.8 pg injected) and reproducible (coefficients of variation 1–10%), and shows good accuracy (93–98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

Sensitive method for the assay of sertindole in plasma by high-performance liquid chromatography and fluorimetric detection

A simple and highly sensitive normal-phase HPLC method is described for determining sertindole concentrations in human plasma using fluorimetric detection. A short C₈ column was used to extract sertindole and the internal standard from plasma; the column was rinsed with acetonitrile, and the analytes were recovered by elution with methanol. This uncommon selectivity between the two solvents allowed clean extraction and near- quantitative recovery of the analytes (> 89%). Separation was done on a 5-μm silica-gel column and detection was performed by fluorimetry, with emission at 340 nm and excitation at 260 nm. The detection and lower quantifiable limits were 0.01 and 0.025 ng/ml, respectively, with no interference from plasma or potential metabolites.     

Determination of semicarbazide-sensitive amine oxidase activity in human plasma by high-performance liquid chromatography with fluorimetric detection

We report here a simple and sensitive method for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human plasma. Benzaldehyde, generated during a 1-h incubation of plasma with benzylamine, is derivatized with the specific aldehyde reagent dimedone after prior deproteinization. Quantitation of the derivatization product is done by automated injection onto an isocratic high-performance liquid chromatographic system with fluorimetric detection. The assay shows good linearity and reproducibility (intra-assay C.V. 7%). Detection limit is 25 mU/1 (= pmol/ml/min). In 51 healthy controls (age 49 ± 13 yr, 20 males) the measured SSAO activity was 352 ± 102 mU/1 (mean ± S.D.). A large number of samples (70–80) can easily be processed in one day by one technician.     

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10 000 pg/ml. Sample extraction (efficiencies >86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantited using post-column UV-photochemical cyclization coupled with fluoremetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 μg/ml of succinylcholine. In a pilot study the plasma concentration—time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Effect of ageing on human plasma glutathione concentrations as determined by high-performance liquid chromatography with fluorimetric detection

A convenient method for the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves direct addition of human plasma to methanolic monobrombimane. for simultaneous protein precipitation and thiol derivatization. The assay was validated by addition of authentic GSH and GSSG to plasma samples. Plasma glutathione levels in Chinese male and female volunteers were found to decrease with increasing age (age groups, 20–30, 30–40, 40–50, 50–60, and >60; mean ± S.E.M. 0.97 ± 0.03, 0.77 ± 0.02, 0.67 ± 0.03, 0.51 ± 0.02, 0.48 ± 0.02 μM for male volunteers and 1.11 ± 0.06, 0.76 ± 0.03, 0.61 ± 0.03, 0.53 ± 0.04 and 0.43 ± 0.04 μM for female volunteers). GSSG levels, in both males and females, did not show a correlation with age. There were no significant differences in GSH or GSSG levels among male and female volunteers of the same age group. These results suggest that elderly persons might be more susceptible to oxidative injury due to decreased plasma glutathione levels.     

Determination of d-serine and related neuroactive amino acids in human plasma by high-performance liquid chromatography with fluorimetric detection

A simple and versatile methodology using high-performance liquid chromatography (HPLC) with fluorimetric detection was developed to simultaneously determine d-serine along with other metabolically related neuroactive amino acids in the glutamatergic system: L-serine, L-glutamate, L-glutamine, and glycine. On-column sensitivity was in the lower picomole range. Of two chiral thiol reagents investigated, amino acid derivatives of o-phthaldialdehyde (OPA) in combination with N-isobutyryl-L-cysteine were found to have consistently higher responses than their corresponding N-tert-butyloxycarbonyl-L-cysteine derivatives. This methodology was applied to the quantitative detection of amino acids in human plasma and lays the foundation for further investigations of the role of neuroactive amino acids in the pathophysiology and treatment of neurological and psychiatric disorders.     

Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

Determination of unbound etoposide concentration in ultrafiltered plasma by high-performance liquid chromatography with fluorimetric detection

Etoposide is a highly protein bound drug, and monitoring the concentration of free drug could help individualize dosage in oncological patients. The cost and difficulty of the standard techniques (equilibration dialysis) has hampered the monitoring of free drugs. We describe a simple HPLC method for the measurement of free etoposide concentration in plasma. Sample preparation involves the ultrafiltration of plasma by a Centrifree device for 30 min at 2000 g and extraction with chloroform. The isocratic separation is performed with a μBondapak phenyl analytical column. Fluorimetric detection is used (288–328 nm excitation and emission wavelengths). Linearity of the calibration curve is excellent between 0.05 and 1 μg/ml. Accuracy and precision are reported at the concentrations 0.06 and 0.4 μg/ml: within-run accuracy is 10% and 6.2%, respectively; between-run accuracy is ⩽1%; within-run coefficients of variation (C.V.) are 10.6 and 5.0%; between-run C.V. are 11.6 and 6.8% respectively. The range of the assay is 0.05 to 1 μg/ml. The feasibility of the technique has been tested in 7 patients treated with oral etoposide for hepatocarcinoma (mean protein binding 91%). We found no interference from endogenous substances, co-administered drugs (alizapride, furosemide, ranitidine) and other antineoplastic agents (doxorubicine, idarubicine, vinblastine, vinorelbine).     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Sensitive column-switching high-performance liquid chromatography method for determination of propiverine in human plasma

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2-200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Sensitive determination method of estradiol in plasma using high-performance liquid chromatography with electrochemical detection

The improvement in the sensitive determination method of estradiol using HPLC with electrochemical detection is described. The improvement was due to the optimization of the potential applied to the electrode of the analytical cell and employment of a guard cell. The detection conditions were optimized from the electrochemical properties of estradiol in acidic and alkaline eluents. The employment of the guard cell drastically decreased the background noise without any reduction in the response of estradiol, and contributed to improvement in the sensitivity. The optimized method combined with pretreatment by liquid-liquid extraction was applied to the determination of estradiol in rat plasma. The detection limit of 8 pg for the standard solution and 24 pg for the plasma sample, which was about 6-8-fold more sensitive compared to the previous reports, was attained.     

Alternative method for determination of ceftazidime in plasma by high-performance liquid chromatography

A high-performance liquid chromatography procedure was developed to analyze ceftazidime concentrations in plasma. The procedure consisted of solid phase extraction followed by ion-pairing reverse-phase chromatography. An excellent linear relationship between ceftazidime peak height measurements and concentrations was demonstrated over the concentration range of 1–200 μg ml⁻¹. The advantage of this assay is the elimination of interference at the ceftazidime elution time that has been noted in previous studies and in our experience. Thus, this study describes an alternative, simple methodology that is clinically useful for analyzing ceftazidime in the research setting.     

Free malondialdehyde determination in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% (v/v) 0.03 M Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.