Fully automated method for simultaneous determination of total cysteine,cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection

A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355 nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine, glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5–103.0%, 98.3–102.5%, 96.3–99.5% and 97.1–99.1%, respectively. The lower limit of quantification for all thiols was 0.5 μM. The whole unattended instrument acquisition time amounts 13 min.     

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.     

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.     

Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

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 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 determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, 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 artesunic acid and dihydroartemisinin in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A selective reproducible high-performance liquid chromatographic assay for the simultaneous quantitative determination of the antimalarial compound artesunic acid (ARS), dihydroartemisinin (DQHS) and artemisinin (QHS), as internal standard, is described. After extraction from plasma, ARS and DQHS were analysed using an Econosil C₈ column and a mobile phase of acetonitrile–0.05 M acetic acid (42:58, v/v) adjusted to pH 5.0 and electrochemical detection in the reductive mode. The mean recovery of ARS and DQHS over a concentration range of 50–200 ng/ml was 75.5% and 93.5%, respectively. The within-day coefficients of variation were 4.2–7.4% for ARS and 2.6–4.9% for DQHS. The day-to-day coefficients of variation were 1.6–9.6% and 0.5–8.3%, respectively. The minimum detectable concentration for ARS and DQHS in plasma was 4.0 ng/ml for both compounds. The method was found to be suitable for use in clinical pharmacological studies.     

Simultaneous determination of catechins in human saliva by high-performance liquid chromatography

Green tea extracts have been suggested to possess a preventive effect against dental caries. A quantitative method for their anticariogenic substances, catechins, was developed to evaluate their concentrations in human saliva after mouthrinsing with green tea extract. Salivary catechins were extracted to the organic phase after forming a complex with diphenylborate and an ion-pair with tetra-n-butylammonium, and then back-extracted to the acidic aqueous phase. The extract was analyzed by high-performance liquid chromatography using diode array detection at absorption wavelengths ranging from 269 to 278 nm. In reversed-phase chromatography by a gradient elution, eight catechins originating from green tea and an internal standard were separated in 15 min without interfering peaks. All the catechins were simultaneously and selectively determined in the concentration range 0.05–25.0 μg/ml. In replicate spiking experiments with standards, the mean recovery ranged between 86 and 99%, and both intra- and inter-assay C.V.s were within 2.3%. When mouthrinsing with an aqueous solution of green tea extract (5.0 mg/ml) containing eight catechins, the quantitative results revealed that each catechin was retained at μg/ml levels in saliva for up to 60 min.     

Simultaneous determination of procaine,lidocaine, ropivacaine,tetracaine and bupivacaine in human plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated for simultaneous quantification of five local anesthetics in human plasma: procaine, lidocaine, ropivacaine, tetracaine and bupivacaine. In an ice-water bath, 500 μL plasma sample, containing 100 μg/mL neostigmine methylsulfate as anticholinesterase, was spiked with carbamazepine as internal standard and alkalized by sodium hydroxide. Liquid–liquid extraction with ethyl ether was used for plasma sample preparation. The chromatographic separation was achieved on a Kromosil ODS C18 column with a mobile phase consisting of 30 mM potassium dihydrogen phosphate buffer (0.16% triethylamine, pH adjusted to 4.9 with phosphoric acid) and acetonitrile (63/37, v/v). The detection was performed simultaneously at wavelengths of 210 and 290 nm. The chromatographic analysis time was 13 min per sample. The calibration curves of all five analytes were linear between 0.05 and 5.0 μg/mL (r² ≥ 0.998). Precision ranged from 1.4% to 7.9% and accuracy was between 91.7% and 106.5%. The validated method is applicable for simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine for therapeutic drug monitoring and pharmacokinetic study.     

Simultaneous determination of fluvoxamine isomers and quetiapine in human plasma by means of high-performance liquid chromatography

An original HPLC-UV method has been developed for the simultaneous determination of the atypical antipsychotic quetiapine and the geometric isomers of the second-generation antidepressant fluvoxamine. The analytes were separated on a reversed-phase C8 column (150 mm x 4.6mm i.d., 5 microm) using a mobile phase composed of acetonitrile (30%) and a 10.5mM, pH 3.5 phosphate buffer containing 0.12% triethylamine (70%). The flow rate was 1.2 mL min(-1) and the detection wavelength was 245 nm. Sample pretreatment was carried out by an original solid-phase extraction procedure using mixed-mode cation exchange (DSC-MCAX) cartridges; only 300 microL of plasma were needed for one analysis. Citalopram was used as the internal standard. The method was validated in terms of linearity, extraction yield, precision and accuracy. Good linearity was obtained in plasma over the 5.0-160.0 ng mL(-1) concentration range for each fluvoxamine isomer and over the 2.5-400.0 ng mL(-1) concentration range for quetiapine. Extraction yield values were always higher than 93%, with precision (expressed as relative standard deviation values) better than 4.0%. The method was successfully applied to human plasma samples drawn from patients undergoing polypharmacy with the two drugs. Satisfactory accuracy values were obtained, with mean recovery higher than 94%.     

Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.     

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.     

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.     

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 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.