Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.     

Determination of maprotiline in plasma by high-performance liquid chromatography with chemiluminescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of maprotiline, an antidepressant, in plasma. After a single-step extraction from plasma (100 μl) with n-hexaneisoamylalcohol (19:1, v/v), the drug and desipramine (internal standard) are converted into their chemiluminescent derivatives by reaction with 6-isothiocyanatobenzo[g]phthalazine-1,4(2H,3H)-dione, a new chemiluminescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSKgel ODS-80, using isocratic elution with acetonitrile-100 mM acetate buffer (pH 3.2), and produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline medium. The detection limit for maprotiline added to plasma is 0.36 pmol (0.1 ng)/ml plasma (1.5 fmol on column), at a signal-to-noise ratio of 3.     

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.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Simultaneous determination of aciclovir, ganciclovir, and penciclovir in human plasma by high-performance liquid chromatography with fluorescence detection

A fast, simple and selective HPLC method has been developed for the assay of aciclovir, ganciclovir, and penciclovir in human plasma by coupling HPLC with fluorescence detection. 200 microl plasma, with guanosine 5'-monophosphate as an internal standard, was subjected to protein precipitation with a 7% [v/v] aqueous perchloric acid solution. The 40 microl supernatant was injected into a Diamonsil-5 microm C18 column. Aciclovir, ganciclovir, and penciclovir, with solvents composed of methanol and 0.08% aqueous trifluoroacetic acid solution, were analysed by fluorescence detection at 260 nm (excitation) and 380 nm (emission) using a gradient elution program. The calibration curves of all three analytes were linear between 20 and 2000 ng/ml. The mean absolute recoveries of aciclovir, ganciclovir, and penciclovir were 93.91+/-1.20%, 97.42+/-0.75%, and 99.01+/-3.30%, respectively. The mean inter-day CVs for aciclovir, ganciclovir, and penciclovir, were within 1.29-7.30%, 1.00-5.53%, and 1.19-3.54%, respectively. The intra-day bias for aciclovir, ganciclovir, and penciclovir ranged from -2.01 to 6.33%, 1.81 to 7.37%, and 1.42 to 6.91%, respectively. The method has been validated and applied in pharmacokinetic studies in Chinese adult renal transplant patients.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Simultaneous determination of triamcinolone acetonide and hydrocortisone in human plasma by high-performance liquid chromatography

A validated HPLC method for the simultaneous determination of triamcinolone acetonide and hydrocortisone has been established to monitor the plasma levels of the compounds in healthy volunteers following intramuscular (i.m.) administration of triamcinolone acetonide. Plasma (1.0 ml) was extracted with dichloromethane after addition of the internal standard, fluocortolone. The compounds were separated using a LiChrospher RP 18 column and detected by UV absorbance. Specificity, linearity, as well as the repeatability, intermediate precision and accuracy of the method were established. The limit of quantification was 0.6 ng/ml for triamcinolone acetonide (C.V.=8.7%, R.E.=2.6%, n=6) and 2.0 ng/ml for hydrocortisone (C.V.=8.3%, R.E.=2.8%, n=6). Data on the stability of triamcinolone acetonide in human plasma are presented. Recovery of the compounds and the internal standard have been studied. The results of quality control samples (n=126) determined during routine analysis of volunteer samples are described. Plasma levels of triamcinolone acetonide after i.m. administration of 40 mg of triamcinolone acetonide are presented.     

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.     

Simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine by high-performance liquid chromatography using electrochemical detection

A rapid, selective and sensitive method for the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine has been developed using high-performance liquid chromatography with electrochemical detection.The unchanged drugs and internal standard extracted from plasma and urine were separated by reversed-phase high-performance liquid chromatography. The influence of acetonitrile concentration and of the pH of the mobile phase were investigated. The detection limits were 100 pg for chlorpromazine and for levomepromazine. In comparison with three other detection systems this was found to be the most sensitive method.This method was successfully applied to the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine for pharmacokinetic studies.     

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 microL of human plasma followed by 3 mL of hexane-methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2-10.0 mg/L for voriconazole and 0.05-10.0 mg/L for posaconazole. The biases were comprised between -3 and 5% for voriconazole and -2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) in human plasma. Acidified plasma is deproteinized with acetonitrile which is separated from the aqueous layer by adding sodium chloride. ASA and SA are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: Novapak C₁₈ 4 μm silica,150×4mm I.D.; eluent: 740 ml water, 900 μl 85% orthophosphoric acid, 180 ml acetonitrile) and photometric detection (237 nm). 2-Methylbenzoic acid is used as internal standard. The method allows the determination of ASA and SA in human plasma as low as 100 ng/ml with good precision (better than 10%). The assay was used to determine the pharmacokinetic parameters of ASA and SA following oral administration of 100–500 mg ASA in healthy volunteers.     

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

Simultaneous determination of misonidazole and desmethylmisonidazole in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of misonidazole and desmethylmisonidazole in plasma is described. After plasma is deproteinized with methanol and the diluted supernatant is chromatographed on a C₁₈ reversed-phase column, both compounds are quantitated by means of an internal standard. The coefficients of variation of within-day and day-to-day precision are below 5.0% for misonidazole in the concentration range of 25–250 mg/l and below 6.1% for desmethylmisonidazole in the concentration range of 2.5–25.0 mg/l. Calibration curves are linear and an analytical recovery varying from 97.6 to 99.8% is obtained. The detection limits for misonidazole and desmethylmisonidazole in plasma are 1.4 mg/l and 0.7 mg/l, respectively.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid–liquid and liquid–solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 μm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20–600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20–1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at −20°C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.     

Simultaneous determination of hydroperoxides of phosphatidylcholine, cholesterol esters and triacylglycerols by column-switching high-performance liquid chromatography with a post-column detection system

A method for the simultaneous determination of hydroperoxides of phosphatidylcholines (PC), triacylglycerols (TG) and cholesterol esters (CE) has been developed. A sample was separated into a combined TG and CE hydroperoxides fraction and a PC hydroperoxides fraction on a short silica column. The fractions were introduced into an ODS column and another silica column by a valve-switching device. The PC hydroperoxides were monitored by a post-column detection system with diphenyl-1-pyrenylphosphine, and the TG and CE hydroperoxides were monitored by another switching device. With this system, the hydroperoxides were determined at the picomole level within 32 min. Their detection limits were 2–4 pmol at a signal-to-noise ratio of 3, and the relative standard deviations of the peak areas were 1.6–3.1%. This method was successfully applied to determine lipid hydroperoxides in human plasma.     

Simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) procedure for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma samples is described. A one-step solid-phase extraction (SPE) with C18 cartridges was coupled with a reversed-phase HPLC system. The system requires two mobile phases composed of tetrabutyl ammonium hydrogensulfate (10 mM adjusted to pH 7)-acetonitrile (62:38, v/v) for quinapril, and (25:75, v/v) for quinaprilat elution through a C18 Symmetry column and detection at a wavelength of 215 nm. Calibration curves were linear over the ranges 20 to 1,000 ng/ml for quinaprilat and 10 to 500 for quinapril. The limits of quantification were 20 and 10 ng/ml for quinaprilat and quinapril, respectively. Extraction recoveries were higher than 90% for quinapril and 80% for quinaprilat. This method has been successfully applied to a bioequivalence study of quinapril in healthy subjects.