Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection

Tryptophan metabolism is disturbed in mental depression, and the induction of indoleamine 2,3-dioxygenase increases kynurenine production. In order to determine this disturbance in patients with chronic hepatitis C and receiving interferon-based immunotherapy, a new and specific HPLC protocol was elaborated. For tryptophan, the assay was linear from 6.25 to 100 micromol L(-1), and the limits of detection (LOD) and quantitation (LOQ) for the method were 0.7 and 8.0 micromol L(-1). For kynurenine, the linearity of calibration was from 0.0625 to 6.25 micromol L(-1), with LOD and LOQ of 2 and 3 nmol L(-1). Reproducibility and repeatability were satisfactory. The method allowed study of human blood serum.     

Determination of ascorbic acid in human plasma with a view to stability using HPLC with UV detection

A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.     

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm × 4.6 mm, 5 μm) at 35 °C under isocratic conditions using 5 mM KH₂PO₄ buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min⁻¹. UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na₂HPO₄ pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 °C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL⁻¹ plasma (r ≥ 0.9927), limits of quantification of 50 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL⁻¹ plasma (r ≥ 0.9900), limits of quantification of 30 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.     

Simultaneous determination of rosiglitazone and metformin in plasma by gradient liquid chromatography with UV detection

A novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in human plasma. The analysis was performed on a phenyl column (250 mm × 4.6 mm i.d., 5 μm) using a gradient method starting with mobile phase composed of acetonitrile:5 mM acetate buffer pH 5.5 (75:25, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The total run time was less than 10 min. Sample preparation included a simple protein precipitation step with acetonitrile. Validation experiments were performed to demonstrate stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 100 ng mL⁻¹ for rosiglitazone and 250 ng mL⁻¹ for metformin. The extraction recoveries were 100.02-105.0% for rosiglitazone and 105.64-103.88% for metformin. The method was applied with success to plasma samples obtained from diabetic patients undergoing treatment with rosiglitazone and metformin.     

Simultaneous determination of cytosine arabinoside and its metabolite,uracil arabinoside,in human plasma using hydrophilic interaction liquid chromatography with UV detection

A practical high‐performance liquid chromatography using a Cosmosil HILIC column and UV detection was developed for determining the concentrations of cytosine arabinoside (Ara‐C) and uracil arabinoside (Ara‐U), which is a major metabolite of Ara‐C, in human plasma. This method was used to determine the plasma concentrations of Ara‐C and Ara‐U in a patient treated with high‐dose Ara‐C therapy for end‐stage renal failure. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Simultaneous analysis of kynurenine and tryptophan in human plasma by capillary electrophoresis with UV detection

Plasma kynurenine (Kyn)/tryptophan ratio has been proposed as a useful marker for the monitoring activation of the cellular immune system. Here, we describe an easy capillary electrophoresis method with UV detection for the separation and detection of Kyn and tryptophan in human plasma using methltryptophan as internal standard. The plasma samples were simply treated with acentonitrile for the elimination of proteins, the supernatant was evaporated, and the dried sample was resuspended with water and directly injected on the capillary without sample derivatization procedures. The use of a run buffer composed by 100 mmol/L Bis-Tris propane at pH 2.15 allowed to baseline resolve the analytes within 9 min. Precision tests indicated a good repeatability of our method both for times (CV< 0.53%) and areas (CV< 2.8%). Moreover, a good reproducibility of intra-assay and interassay tests was obtained (CV < 3.9% and CV < 7.6%, respectively). The obtained limit of detections for Kyn and tryptophane, evaluated at 226 nm, were 0.15 and 0.40 μmol/L, respectively. The method suitability was tested by measuring analyte levels both in healthy volunteers, acute myocardial infarction and chronic kidney disease patients.     

A simple determination of mizoribine in human plasma by liquid chromatography with UV detection

A simple liquid chromatography (LC) method was developed for determination of the therapeutic level of mizoribine in human plasma. After precipitation of plasma proteins with 6% perchloric acid, mizoribine was determined by LC with spectophotometric detection. The peak height for mizoribine was linearly related to its concentrations, which ranged from 0.09 to 3.13 microg/mL. Therefore, the limit of quantitation was considered to be 0.09 microg/mL. The accuracy was 104.96-107.37%. The intra- and interday relative standard deviation values were in the range of 1.10-3.25%. The detection limit was 0.025 microg/mL, defined as a signal-to-noise ratio of 3. The plasma concentrations of mizoribine were not related to the dosage. Because mizoribine was mainly excreted in the urine, the plasma concentrations of mizoribine might be affected by a change in renal function. Therefore, the mizoribine concentration in blood should be monitored and the dosage adjusted, depending on the condition of renal function. It was suggested that the present method may be applied well in the therapeutic drug monitoring for mizoribine.     

Determination of guaifenesin from spiked human plasma using RP-HPLC with UV detection

A rapid, simple, selective and specific high performance liquid chromatography (HPLC) method with UV detection (230 nm) was developed and validated for estimation of guaifenesin from spiked human plasma. The analyte and internal standard (eplerenone) were extracted with dichloromethane. The chromatographic separation was performed on HiQSil C 18HS column (250 × 4.6 mm, 5 μm) with a mobile phase of methanol: water (60: 40%, v/v) at a flow rate of 1 mL/min. Guaifenesin was well resolved from plasma constituents and internal standard. The calibration curve was linear in the range of 100–3200 ng/mL. The heteroscedasticity was minimized by using weighted least square regression with weighing factor of 1/x. The intra- and inter-day % RSD was less than 15. Results of recovery studies prove the extraction efficiency. Stability data indicated that guaifenesin was stable in plasma after three freeze thaw cycles and upon storage at ?20°C for 30 days.     

A highly sensitive method for the determination of adriamycin and adriamycinol in human plasma using HPLC with fluorimetric detection

Summary A simple and very sensitive HPLC method, for the simultaneous determination in human plasma of adriamycin and its metabolite adriamycinol, is described. Plasmas from patients were stored frozen. Thawed samples were extracted by absorption of anthracyclin onto a small C18 column. After evaporation of the eluate and reconstitution of the residue with methanol (100L), 30 to 40L of the mixture were injected into the chromatograph. Separation was obtained using an RP 8 column with a mobile phase of formate buffermethanol-acetonitrile (502327, v/v). A spectrofluorimeter was used as detector. The limit of sensitivity of the assay was 50 pcg/ml of plasma.     

Simple determination of forphenicinol in human plasma and erythrocytes by HPLC with native fluorescence detection

A high performance liquid chromatographic method is described for monitoring forphenicinol, a possible therapeutic drug for cancer and muscular dystrophy, in human plasma and erythrocytes. Forphenicinol in the deproteinized samples was separated from interfering biogenic substances on an aminopropyl-bonded silica (Unicil NH2) column within 10 minutes with isocratic elution, and determined with fluorescence detection. The detection limits for forphenicinol in plasma and erythrocytes are 65 pmol (12.8 ng)/ml and 160 pmol (31.5 ng)/ml, respectively, corresponding to 2 pmol each in a 100 microliters injection volume. The method is very simple, and sensitive enough to permit the quantification of forphenicinol in the blood samples from man dosed with forphenicinol.     

HPLC-MS法同时测定5种抗癫痫药的血药浓度

癫痫是神经系统的一种常见病,不少患者需长期同时服用两种或两种以上抗癫痫药物.本研究采用高效液相色谱-电喷雾离子化质谱法同时测定5种常用抗癫痫药物卡马西平、氯硝西泮、阿普唑仑、艾司唑仑和苯妥英钠的血药浓度,色谱条件为以地西泮作为内标,Zorbax SB-C18(30mm×2.1mm,3.5μm)为色谱柱,甲醇-25mM的乙酸铵水溶液梯度洗脱,样品采用无水乙醚萃取.样品分析以离子阱质谱作为检测器,采用多重反应监测模式(MRM),选择MS2离子提高方法的专属性,5种抗癫痫药物在50～5000ng/mL浓度范围内均具有良好的线性关系,其日内、日间精密度均小于10%,方法回收率均大于90%.研究表明,本方法专属性强,灵敏度高,线性关系良好,适用于这5种抗癫痫药物的血药浓度监测、个体化给药方案的制定、药物滥用监测以及药代动力学研究.关键词高效液相色谱质谱抗癫痫药血药浓度药物分析     

Simultaneous determination of anthracyclines and taxanes in human serum using online sample extraction coupled to high performance liquid chromatography with UV detection

An online SPE‐LC method that can determine both anthracyclines and taxanes simultaneously in human serum samples is reported. The entire method of extraction, separation and UV detection was achieved online by column switching between an SPE column (Biotrap 500 (20×4 mm)) and an analytical column (Zorbax XDB C18, 150×4.6 mm, 5 μm) with a 23 min total cycle time. The method is linear (r²>0.998) over the range of 0.5–25 μg/mL. The analytes of interest are retained on the SPE column with good recovery (84–117%), while proteins and other serum components elute to waste. This online clean‐up is much faster (150 s) and less manual than traditional off‐line extraction methods. Using 0.1 mL spiked serum samples, the LOQ was 0.5 μg/mL. Intra‐ and inter‐day precision were acceptable (≤15% RSD) at and above the LOQ. The method was applied to the analysis of serum samples from patients undergoing chemotherapy with these agents.     

A new HPLC method with fluorescence detection for the determination of memantine in human plasma

A sensitive, selective, simple and fast HPLC method based on the formation of derivative with fluorescamine was developed for the determination of memantine (ME) in human plasma. Separation was achieved on a CN column (200 mm×4.6 mm) using acetonitrile-10 mM orthophosphoric acid containing 1 mL/L triethylamine (45:55, v/v) at a flow rate of 1 mL/min. Emission and excitation wavelengths were 480 and 380 nm, respectively. Amantadine was used as an internal standard. Calibration graphs were rectilinear over the range of 1.0-100.0 ng/mL. Limit of detection and limit of quantification were found to be 0.3 and 1.0 ng/mL, respectively. Intra-day and inter-day relative standard deviation values were found to be <2.03%. Average recovery was also found to be around 94%. Proposed method was applied for the pharmacokinetic study in a healthy volunteer after a single oral administration of 20 mg of ME.     

Determination of a new calcium antagonist in human plasma by liquid extraction enrichment and HPLC with UV detection

Summary An HPLC method has been developed for the determination of SL 85.1016, a new calcium antagonist arylbenzylamide methylthioether derivative. SL 85.1016 and the internal standard, SL 87.0210, are extracted from alkaline human plasma withn-hexane and back extracted into 0.05 M phosphate buffer (pH 2.5; 0.2 ml). Acetonitrile (50 l) is added to the final aqueous extract in order to prevent absorption of the compounds of interest onto the walls of the glass tube; this solution then is partially processed by HPLC on a C₁₈ column with UV detection (254 nm). The determination limit of the method is 2 ng.ml^–1 of SL 85.1016 in human plasma; the response to the drug is linear in the range 2–200 ng.mg^–1.     

Simultaneous determination of first-line anti-tuberculosis drugs by capillary zone electrophoresis using direct UV detection

An alternative methodology for simultaneous analysis of ethambutol, isoniazid, rifampicin and pyrazinamide in pharmaceutical formulations by capillary zone electrophoresis under UV direct detection with an analysis time of 8.0 min is proposed. Background running was based on the effective mobility curve of the analytes and an optimum separation condition was achieved using a 3³ Box-Behnken design, with Brij 35, Cu²⁺ and acetic acid/sodium acetate buffer as factors. An electrolyte consisting of 50.0 mmol L⁻¹ of acetic acid/sodium acetate buffer, 12.5 mmol L⁻¹ of CuSO₄, and standard and sample solutions prepared in 2.00 mmol L⁻¹ of Brij 35 and 12.5 mmol L⁻¹ of CuSO₄ were optimized. After evaluating validation parameters, the method was successfully applied to the analysis of samples in the form of tablets and sachets.