Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 microns) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5-100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Pharmacokinetics and bioequivalence study of rhein as the main metabolite of diacerein

A simple, rapid and fully-validated liquid chromatographic method (RP - HPLC) with fluorescence detection was developed for the analysis of rhein (as the main metabolite of diacerein) in human plasma. The separation was performed using an ODS C₁₈ column with a mobile phase consisted of acetonitrile:methanol:phosphate buffer pH 6.8 and the flow rate was1.0 mL/min. The flourimetric detection was performed at 2 excitation wavelengths ʎ_ex = 440 nm & 338 nm and one emission wavelength at ʎ_em = 520 nm. The developed method was validated according to Food and Drug administration (FDA) guidelines for bioanalytical method validation. The pharmacokinetic parameters of the test and the reference were determined and the analysis of variance (ANOVA) between parameters of the two brands was calculated. The relative bioavailability was found to be 89%. This method was successfully applied for the routine bioequivalence analysis of diacerein in plasma.     

Capillary zone electrophoresis applied to the determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite in pharmaceuticals and urine

A capillary zone electrophoresis method has been developed for the quantitation of antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine. The separation of the compounds was performed in a fused-silica capillary filled with the running electrolyte, which consisted of a 60 mM borate buffer solution at pH 9.5. Under the optimized experimental conditions, the separation took less than 5 min. The analysis of urine samples required a previous solid-phase extraction step using C8 cartridges. The method was successfully applied to the determination of the drug and its metabolite in urine samples obtained from three hypertensive patients (detection limits of 115 ng ml(-1) for cilazaprilat and 125 ng ml(-1) for cilazapril) and to pharmaceutical dosage forms. The method was validated in terms of reproducibility, linearity and accuracy.     

高效液相色谱法测定精浆中过氧化脂质水平及其在男性不育症研究中的应用

采用高效液相色谱法测定精浆中过氧化脂质(lipid peroxidation,LPO)含量,研究了有正常生育能力的男子和不育症患者精浆中LPO含量水平差异及其对男子不育症的影响。精浆样品经酸化后,分解生成的丙二醛(malondialdehyde,MDA)与硫代巴比妥酸(thiobarbituric acid,TBA)缩合反应形成紫红色产物,以Lichrospher C18化学键合硅胶为固定相,0.025 mol/L KH2PO4 (pH 6.2)-甲醇(体积比为58∶42)为流动相进行色谱等度分离     

The bioequivalence study of baclofen and lioresal tablets using capillary electrophoresis

A simple and robust analytical method for rapid separation and sensitive quantification of baclofen in human plasma by capillary electrophoresis technique was developed. Electrophoretic separation was optimized and successfully performed using simple sodium tetraborate aqueous solution. Observed detection limit in biological material was 10 ng. Using UV detection at 200 nm excellent linearity (r = 0.999) was observed over the concentration range from 0.025 to 1.0 microg mL(-1). The described method has been validated and applied to the quantitative determination of baclofen in human plasma. The bioavailability of Baclofen (Polpharma) and Lioresal (Novartis) in 18 healthy volunteers was investigated. The results indicate bioequivalence of the reference and Baclofen preparations.     

A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection

A new, simple, economical and validated high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method has been developed and optimized for different experimental parameters to analyze the most common monothiols and disulfide (cystine, cysteine, homocysteine, methionine, reduced (GSH) and oxidized glutathione (GSSG)) and ascorbic acid present in human plasma and erythrocytes using dopamine as internal standard (IS). Complete separation of all the targets analytes and IS at 35 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved using 0.05% TFA:methanol (97:3, v/v) as a mobile phase pumped at the rate of 0.6 ml min⁻¹ using electrochemical detector in DC mode at the detector potential of 900 mV. The limits of detection (3 S/N) and limits of quantification (10 S/N) of the studied compounds were evaluated using dilution method. The proposed method was validated according to standard guidelines and optimization of various experimental parameters and chromatographic conditions was carried out. The optimized and validated HPLC-ECD method was successfully applied for the determination of the abovementioned compounds in human plasma and erythrocytes. The method will be quite suitable for the determination of plasma and erythrocyte profile of ascorbic acid and aminothiols in oxidative stress and other basic research studies.     

Use of high-performance liquid chromatographic and microbiological analyses for evaluating the presence or absence of active metabolites of the antifungal posaconazole in human plasma

Posaconazole (SCH 56592) is a novel broad spectrum triazole antifungal agent that is currently in phase III clinical trials for the treatment of systemic fungal infections. This study was initiated to determine if orally administered posaconazole to humans would result in the formation of active metabolite(s). Plasma samples from a multiple-rising dose study in healthy volunteers were analyzed by validated HPLC and microbiological methods. The HPLC analysis involved extraction with a mixture of organic solvent (methylene chloride-hexane) followed by separation on a C18 column and quantification by UV absorbance at 262 nm. The microbiological assay was performed utilizing an agar diffusion method using Candida pseudorropicalis ATCC 46764 as the test organism. Potency was determined by comparing the growth inhibition zones produced by the test sample to those produced by standard concentrations prepared in plasma. Individual and mean plasma concentration-time profiles were similar for both HPLC and microbiological assays. The area under the plasma concentration-time curves of the microbiological and HPLC results were similar with a mean (RSD) ratio of 105.5% 15.3%), indicating that there was no relevant biologically active metabolite of posaconazole in human plasma.     

Validation of a solid phase extraction-high performance liquid chromatographic method for the determination of eprosartan in human plasma

In this work, a solid phase extraction-reversed phase high performance liquid chromatographic (SPE-RP-HPLC) method with photometric detection for monitoring the antihypertensive drug eprosartan has been validated in order to assure good quantitation of eprosartan in plasma samples obtained from patients under cardiovascular treatment. This analytical method was developed by using experimental design and quantitation was accomplished with the internal standard method. No interferences were observed from endogenous compounds of plasma and other drugs which are commonly co-administered in elderly patients. The recoveries of eprosartan from plasma samples, measured at three levels of the linear concentration range (150-4000 ng/mL) were found to be between 93.4 and 102.8%. The intraday and interday precision and accuracy (measured by relative standard deviation, RSD, and relative error, RE, respectively) were always lower than 13% (RSD) and 4% (RE). Stability studies showed that eprosartan stock solutions are stable for at least 3 months when stored at 8 degrees C and plasma samples containing the drug were stable at least during the whole analytical method.     

Validated Stability-Indicating HPLC–DAD Determination of the Antihypertensive Binary Mixture of Carvedilol and Hydrochlorothiazide in Tablet Dosage Forms

A simple and selective HPLC with diode array detection stability-indicating method was developed for the simultaneous determination of the antihypertensive drugs carvedilol (CRV) and hydrochlorothiazide (HCT) in their combined formulations. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6 × 250 mm, 5 μm) with gradient elution of the mobile phase composed of 0.025 M phosphoric acid and acetonitrile at a flow rate of 1 mL min^?1. The multiple wavelength detector was set at 242 nm for measurement of CRV and 271 nm for HCT. Quantification was based on measuring the peak areas. The cited drugs were resolved with retention times 4.9 and 6.7 min for HCT and CRV, respectively. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–300 and 5–200 μg mL^?1 for CRV and HCT, respectively, with correlation coefficients >0.9996. Both drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drugs from their forced degradation products. Moreover, specificity of the method was verified by resolution of both drugs from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined tablet dosage forms. The proposed method made use of DAD as a tool for peak identity and purity confirmation.     

A sensitive column-switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies

A simple and sensitive column‐switching HPLC‐UV method was developed for the simultaneous determination of aripiprazole, a novel atypical antipsychotic drug, and its active metabolite, dehydroaripiprazole in human plasma. Aripiprazole, its active metabolite and 7‐[5‐[4‐(3‐chloro‐2‐methylphenyl)‐1‐piperazinyl]pentyloxy]‐3,4‐dihydro‐2(1H)‐quinolinone (OPC‐14558) as an internal standard were extracted from 1 mL of plasma using a mixture of chloroform/n‐heptane (3:7, v/v), and the extract was injected into a column I (TSK BSA‐ODS/S precolumn, 5 μm) for cleanup and column II (C₁₈ STR ODS‐II analytical column, 5 μm) for separation. Peaks were detected with an UV detector set at a wavelength of 254 nm, and the total time for chromatographic separation was ～20 min. Mean absolute recoveries were 74.0 and 74.7% for aripiprazole and dehydroaripiprazole, respectively. Intra‐ and inter‐day CVs were less than 7.5 and 7.1% for aripiprazole concentrations ranging from 2 to 600 ng/mL, and 9.2 and 4.5% for dehydroaripiprazole concentrations ranging from 2 to 160 ng/mL. The validated concentration ranges for this method were 1–500 ng/mL and the limits of detection were 0.5 ng/mL for both aripiprazole and dehydroaripiprazole. This method was applied to pharmacokinetic study in human volunteers and patients taking aripiprazole.     

Development and validation of a reversed-phase HPLC method for monitoring of synthetic reactions during the manufacture of a key intermediate of an anti-hypertensive drug

A reversed-phase high-performance liquid-chromatographic method for monitoring of reactions involved in process development of a key intermediate of antihypertensive drugs, e.g, doxazosin mesylate, prazosin, alfuzosin, terazosin, etc., has been developed and validated. The HPLC profiles of impurities of 4-amino-2-chloro-6,7-dimethoxyquinazoline were used as fingerprints to follow the synthetic procedures in the manufacturing unit. The separation was accomplished on an Inertsil ODS-3 column with isocratic elution using acetonitrile-ammonium acetate (10 mM; pH 4.0; 50:50 v/v) as mobile phase and a photodiode array detector set at 240 nm at ambient temperature. The method was validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The method could detect the impurities at a level of 0.01 to 0.20 microg/mL and it was found to be suitable not only for monitoring of reactions but also for quality assurance of 4-amino-2-chloro-6,7-dimethoxyquinazoline.     

Development and validation of a high‐performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies

A sensitive and selective RP‐HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD‐20A UV–vis) at a λ_max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol–acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100–2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method.     

Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine

A novel capillary zone electrophoresis method is described for the determination of taurine in plasma. The method is rapidly executed and is highly selective for taurine as separation is based on the difference in ionisation of this amino acid from that of other amino acids. Following addition of homotaurine as internal standard, plasma proteins were precipitated with acetonitrile and the supernatant was derivatised with fluorescamine in the presence of a borate buffer. Capillary electrophoresis (CE) separations were carried out in reverse polarity mode at 27.5 kV on a Beckman P/ACE MDQ CE instrument, equipped with a diode array detector (DAD) set at 266 nm. The sample tray was cooled to 5 degrees C and separations were carried out at 20 degrees C. The fused-silica capillary was 50.2 cm in length (40.2 cm to detector) with an internal diameter of 75 microm. A capillary conditioning solution was applied daily in order to suppress the residual electroosmotic flow (EOF). The method, which was validated using feline plasma as the blank matrix, was shown to be linear and reproducible over the concentration range 2.5-100 microg/mL. The coefficients of variation (CVs) of replicate analyses were less than 4.5% at 1 microg/mL taurine in feline plasma and less than 3% for 2.5 microg/mL in human plasma. Recovery was estimated at 99.2% with a CV of 4.85%. It has been demonstrated that quantitation in aqueous solution yields similar results to those obtained by interpolation on a plasma calibration curve provided that subtraction for the taurine peak in unspiked plasma is carried out and that a suitable internal standard is employed.     

Evaluation and differentiation of the Betulaceae birch bark species and their bioactive triterpene content using analytical FT-vibrational spectroscopy and GC-MS

A new simple, rapid and sensitive reversed-phase liquid chromatographic method was developed and validated for the simultaneous determination of sulpiride (SUL) and mebeverine Hydrochloride (MEB) in the presence of their impurities and degradation products. The separation of these compounds was achieved within 6 min on a 250 mm, 4.6 mm i.d., 5 m particle size Waters^?-C18 column using isocractic mobile phase containing a mixture of acetonitrile and 0.01 M dihydrogenphosphate buffer (45:55) at pH = 4.0. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence-detection at excitation 300 nm and emission at 365 nm. The concentration-response relationship was linear over a concentration range of 10- 100 ng/mL for both MEB and SUL with a limit of detection 0.73 ng/mL and 0.85 ng/mL for MEB and SUL respectively. The proposed method was successfully applied for the analysis of both MEB and SUL in bulk with average recoveries of 100.22 ± 0.757% and 99.96 ± 0.625% respectively, and in commercial tablets with average recoveries of 100.04 ± 0.93% and 100.03 ± 0.376% for MEB and SUL respectively. The proposed method was successfully applied to the determination of MEB metabolite (veratic acid) in real plasma simultaneously with SUL. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 100.36 ± 2.92 and 99.06 ± 2.11 for spiked human plasma respectively. For real human plasma, the mean% recoveries (n = 3) were and respectively.     

Multiresidue HPLC analysis of ten quinolones in milk after solid phase extraction: validation according to the European Union Decision 2002/657/EC

A rapid and sensitive analytical method was developed for the residue analysis of ten quinolones (enoxacin (ENO), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), oxolinic acid (OXO), nalidixic acid (NAL), and flumequine (FLU)) in cow's milk. The analytes were extracted from milk by a deproteinization step followed by a simple SPE cleanup procedure using LiChrolut RP-18 Merck cartridges. Recoveries varied between 75 and 92%. HPLC separation was performed at 25 degrees C using an ODS-3 PerfectSil Target (250 x 4 mm(2)) 5 microm analytical column (MZ-Analysentechnik, Germany). The mobile phase consisted of a mixture of TFA 0.1%-CH(3)CN-CH(3)OH, delivered by a gradient program at the flow rate of 1.2 mL/min. Elution of the ten analytes and the internal standard (caffeine, 7.5 ng/microL) was completed within 27 min. Column effluent was monitored using a photodiode array detector, set at 275 and 255 nm. The developed method was validated according to the criteria of Commission Decision 2002/657/EC. The LODs of the specific method of quinolones' determination in milk varied between 1.5 and 6.8 ng/microL.     

HPLC Determination of Imidazoles with Variant Anti-Infective Activity in Their Dosage Forms and Human Plasma

A suitable HPLC method has been selected and validated for rapid simultaneous separation and determination of four imidazole anti-infective drugs, secnidazole, omeprazole, albendazole, and fenbendazole, in their final dosage forms, in addition to human plasma within 5 min. The method suitability was derived from the superiority of using the environmentally benign solvent, methanol over acetonitrile as a mobile phase component in respect of safety issues and migration times. Separation of the four anti-infective drugs was performed on a Thermo Scientific^® BDS Hypersil C₈ column (5 µm, 2.50 × 4.60 mm) using a mobile phase consist of MeOH: 0.025 M KH₂PO₄ (70:30, v/v) adjusted to pH 3.20 with ortho-phosphoric acid at room temperature. The flow rate was 1.00 mL/min and maximum absorption was measured with UV detector set at 300 nm. Limits of detection were reported to be 0.41, 0.13, 0.18, and 0.15 µg/mL for secnidazole, omeprazole, albendazole, and fenbendazole, respectively, showing a high degree of the method sensitivity. The method of analysis was validated according to Food and Drug Administration (FDA)guidelines for the determination of the drugs, either in their dosage forms with highly precise recoveries, or clinically in human plasma, especially regarding pharmacokinetic and bioequivalence studies.     

Validated Stability-Indicating HPLC–DAD Determination of the Antihypertensive Binary Mixture of Carvedilol and Hydrochlorothiazide in Tablet Dosage Forms

A simple and selective HPLC with diode array detection stability-indicating method was developed for the simultaneous determination of the antihypertensive drugs carvedilol (CRV) and hydrochlorothiazide (HCT) in their combined formulations. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6 × 250 mm, 5 μm) with gradient elution of the mobile phase composed of 0.025 M phosphoric acid and acetonitrile at a flow rate of 1 mL min⁻¹. The multiple wavelength detector was set at 242 nm for measurement of CRV and 271 nm for HCT. Quantification was based on measuring the peak areas. The cited drugs were resolved with retention times 4.9 and 6.7 min for HCT and CRV, respectively. Analytical performance of the proposed HPLC procedure was thoroughly validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–300 and 5–200 μg mL⁻¹ for CRV and HCT, respectively, with correlation coefficients >0.9996. Both drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the drugs from their forced degradation products. Moreover, specificity of the method was verified by resolution of both drugs from more than 20 pharmaceutical compounds of various medicinal categories. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined tablet dosage forms. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

     

Capillary column gas chromatographic method using electron-capture detection for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma

A rapid, specific and direct method based on capillary column gas chromatography with electron-capture detection is described for the simultaneous determination of nicardipine, a new calcium antagonist, and its pyridine metabolite II in human plasma. In this method, the nicardipine, its pyridine metabolite II and internal standard are extracted from the plasma and then partially purified by acid-base partitioning prior to the final injection onto the capillary column gas chromatograph for quantification by means of an electron-capture detector. The quantification limit of the method is 1 ng/ml of plasma for both nicardipine and its pyridine metabolite II. The coefficients of variation for nicardipine and the pyridine metabolite II at concentrations of 1-50 ng/ml are less than 7% and less than 9% (n = 4), respectively. The method has been validated against a previously developed high-performance liquid chromatographic method (sensitivity 5 ng/ml).