Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for the determination of doxazosin in human plasma was developed and validated. Prazosin was used as internal standard. After extraction twice with ethyl acetate, chromatographic separation of doxazosin in human plasma was carried out using a reversed-phase Apollo C18 column (250 x 4.6 mm, 5 microm) with mobile phase of methanol-acetonitrile-0.04 m disodium hydrogen orthophosphate (22:22:56, v/v/v) adjusted to pH 4.9 with 0.9 m phosphoric acid and quantified by fluorescence detection operated with an excitation wavelength of 246 nm and an emission wavelength of 389 nm. The lower limit of quantification (LLOQ) of this assay was 1 ng/mL using 500 microL human plasma. Linearity was established over the range 1-25 ng/mL (r2 > 0.9994). The intra- and inter-day accuracy ranged from 90.5 to 104.4% and the coefficient of variation were not more than 8.6% for both intra- and inter-day precision, over the range of the calibration curve. The absolute recoveries of doxazosin and prazosin from human plasma were more than 91%. Doxazosin demonstrated acceptable short-term, long-term and freeze-thaw stability in human plasma. The assay has been successfully applied to plasma sample ana-lysis for pharmacokinetic study.     

Simultaneous quantification of curcuminoids and xanthorrhizol in Curcuma xanthorrhiza by high-performance liquid chromatography

A new method for simultaneous quantification of curcuminoids and xanthorrhizol (XNT) in Curcuma xanthorrhiza was developed and validated using high-performance liquid chromatography with diode-array UV–Vis detector. The chromatographic separation was achieved on a Phenomenex C18 at room temperature with the mobile-phase acetonitrile ?0.001% formic acid in gradient elution system and delivered at a flow rate of 1?mL/min. Detection wavelength 425?nm was used for curcuminoids and 224?nm for XNT. System suitability, linearity, precision, accuracy, limit of detection, limit of quantitation, and stability were evaluated and were found in good agreement with Association of Official Analytical Chemists guidelines for single-laboratory validation. The proposed method was found to be precise, accurate, and reliable and also could be applied for the simultaneous quantitative analysis of curcuminoids and XNT in C. xanthorriza raw material and its herbal medicinal product.     

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as 'estoraque do campo' or 'cuia de brejo', has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts.     

Simultaneous quantification of seven bioactive components in Caulis Lonicerae Japonicae by high performance liquid chromatography

This study presents a new HPLC method for the simultaneous determination of seven major components, namely chlorogenic acid, caffeic acid, loganin, sweroside, secoxyloganin, rutin and luteolin 7-O-glucoside in Caulis Lonicerae Japonicae, a commonly used traditional Chinese medicinal herb derived from the caulis of Lonicera japonica Thunb. These seven compounds, belonging to the chemical types of phenolic acids, iridoids and flavonoids, were separated on a C18 column (250 x 4.6 mm, 5.0 microm) with the column temperature at 30 degrees C. The mobile phase was composed of (A) aqueous acetic acid (0.4%, v/v) and (B) acetonitrile using a gradient elution of 10% B at 0-12 min, 10-17% B at 12-25 min and 17% B at 25-35 min. The flow rate was 1.0 mL/min and detection wavelength was set at 245 nm. The limit of detection (S/N = 3) ranged from 0.10 to 0.23 microg/mL and the limit of quantification (S/N = 10) ranged from 0.69 to 3.56 microg/mL. All calibration curves showed good linear regression (r2 > 0.9990) within the test ranges. The intra- and inter-day precisions as determined from sample solutions were below 1.24 and 2.28%, respectively. The recoveries for seven compounds were found to range from 94.2 to 103.6%. This verified method has been successfully applied to evaluation of commercial samples of Caulis Lonicerae Japonicae from different markets in China.     

Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin,lomefloxacin and ciprofloxacin in human plasma

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC‐FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single‐step precipitation of plasma proteins followed by vortex‐mixing and centrifugation. Chromatographic separation was achieved within 7 min on a reversed‐phase C₁₈ column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)–methanol (82:18, v/v) pumped isocratically at 1.2 mL/min. The detector was set at excitation/emission wavelengths of 278/450 nm. Calibration curves were linear (r² ≥ 0.994) in the range of 0.02–5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC‐FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic‐based studies with those antimicrobial agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.     

Simultaneous qualitation and quantification of four phytoecdysones in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array detection

A high-performance liquid chromatographic method with diode array detection was developed and validated to simultaneously identify and quantify four phytoecdysones,i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-S inokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C(18) column with 6% tetrahydrofuran aqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analytes were determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of the method included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linear regression (r(2) >or= 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng, respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. The obtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analysis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locations were highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration of phytoecdysones.     

Box-Behnken assisted development and validation of high-performance liquid chromatography method for the simultaneous determination of doxorubicin and vorinostat in polymeric nanoparticles

While histone deacetylase inhibitors, such as vorinostat, demonstrate a significant effect against hematological cancers, their application for solid tumor treatment is limited. However, there is strong evidence that combinatorial administration of vorinostat and genotoxic agents (e.g., doxorubicin) enhances the antitumoral action of both drugs against tumors. We developed a high-performance liquid chromatography method for the simultaneous determination of doxorubicin and vorinostat in polymeric nanoparticles designed to provide the parenteral administration of both drugs and increase their safety profile. We performed separation on Nucleodur C-18 Gravity column with a mixture of 10 mM potassium dihydrogen phosphate buffer pH 3.9:ACN (90:10 v/v) as mobile phase at 240 nm. The method was linear within the concentration range of 4.2–52.0 μg/ml for both drugs with limits of detection and quantification of 3.5 and 10.7 μg/ml for doxorubicin and 2.5 and 7.7 μg/ml for vorinostat, respectively. The method was precise and accurate over the concentration range of analysis. Drug loading was 5.4% for doxorubicin and 0.8% for vorinostat. Degradation of doxorubicin after irradiation was less than 5%, while the amount of vorinostat decreased at 88% under the same conditions. Thus, the validated method could be adopted for routine simultaneous analysis of doxorubicin and vorinostat in polymeric nanoparticles.     

Development and validation of a generic RP‐HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines

A novel generic reverse phase high performance liquid chromatography (RP‐HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over‐the‐counter US‐marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min^?1 and injection volume was 10 μL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R² > 0.999), over the concentration ranges 20–120 μg mL^?1 for acetaminophen, 75–450 μg mL^?1 for dextromethorphan, 31.25–187.5 μg mL^?1 for doxylamine, 25–150 μg mL^?1 for phenylephrine, 25–150 μg mL^?1 for aspirin, 6.5–39 μg mL^?1 for caffeine and 12–72 μg mL^?1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP‐HPLC generic method is applicable for routine analysis of cold and cough over‐the‐counter products.     

Quantitation of tolmetin by high-performance liquid chromatography and method validation

A high-performance liquid chromatographic (HPLC) assay method for assessing the degradation of tolmetin (TLM) is developed and validated under acidic, basic, and photoirradiated conditions. The HPLC method includes an Inertsil 5 ODS-3V column (250- x 4.6-mm i.d.), guard column of Inertsil 7 ODS-3V (50- x 4.6-mm i.d.), mobile phase of CH(3)OH-1% HOAc (64:36, v/v), and UV detection at 254 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its degradants. The established assay method exhibits good selectivity and specificity suitable for stability measurements. From the intra- and interday tests of six replicates, the coefficients of variation are between 0.20% and 1.77% for the former, and 0.12% and 3.40% for the latter. Recoveries are found to be 98.7-101.7%. TLM is determined to be more reactive when exposed to light and acidic conditions, yet TLM is stable in a basic medium. A kinetic study of the photodegradation of TLM shows that it follows an apparent first-order reaction in three alcoholic solvents.     

Stability-indicating RP-HPLC method for simultaneous quantitation of tramadol and aceclofenac in presence of their major degradation products: Method development and validation

Primary objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (HPLC) method for simultaneous quantitation of tramadol and aceclofenac in presence of their degradation products. The drugs were subjected to various International Conference on Harmonization recommended stress conditions, such as acid hydrolysis, alkaline hydrolysis, peroxide oxidation, thermolysis, and photolysis. The major degradation products got well resoluted from the analytes in HPLC analysis with a mobile phase composed of a mixture of 0.01?M ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) through a Phenomenex Gemini C18 (250?mm?×?4.6?mm, 5?µm particle size) column. The method was linear over a range of 15–60?µg/mL for tramadol and 40–160?µg/mL for aceclofenac concentration. The analytes were detected at a wavelength of 270?nm. The method was validated and found to be specific, accurate, precise, stable, and robust for its intended use. The method can be recommended for its future use in routine quality control, accelerated and real-time stability analysis of the formulations containing tramadol and aceclofenac combination.     

Development and validation of a liquid chromatography method for analysis of colistin sulphate

Summary The peptide antibiotic Colistin has been successfully separated into more than thirty components on a YMC-Pack Pro, C-18 column with a mobile phase of acetonitrile—sodium sulphate (0.7% m/v)—phosphoric acid (6.8% v/v dilution of 85% m/m H₃PO₄)—water (21.5∶50∶5∶23.5) at 1.0 mL per min and detection at 215 nm. Baseline resolution of the major components, colistin A and B, and many minor components was obtained. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

高效液相色谱法快速测定生物样品中的视黄酸

建立了快速测定生物样品中全反式(all-trans-),9-顺式(9-cis-),13-顺式(13-cis-).视黄酸(RA)的高效液相色谱分析方法.经2次液-液萃取提取生物样品中的视黄酸后.直接应用高效液相色谱法同时测定了3种视黄酸同分异构体的含量.采用Waters C18反相柱(3.9×150mm),V(乙腈):V(0.1%冰醋酸溶液)=86:14为流动相,流速1.0 mL/min,柱温为25℃,检测波长为350 nm.3种视黄酸同分异构体的线性范围均为5-500 ng/mL,r2均大于0.999;检出限均为1 ng/mL;提取回收率为92.7%-101.8%,方法回收率为102,4%-104.4%;日内精密度小于8.3%,日间精密度小于11%.本方法适用于不同生物样品中视黄酸的定量研究.     

Development and validation of UPLC method for simultaneous quantification of carvedilol and ivabradine in the presence of degradation products using DoE concept

A methodical design-of-experiments were performed by applying quality-by-design concepts to establish a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the presence of degradation products. Response-surface, central-composite design, and quadratic model were employed for statistical assessment of experimental data using the Design-Expert software. Response variables such as resolution and retention time were analyzed statistically for chromatographic screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were considered for method optimization. The method was developed using C8 [100?×?2.1?mm, 1.8?µ] UPLC column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50 v/v, the flow rate of 0.4?mL minute^?1 and UV detection at 285?nm for both Carvedilol and Ivabradine. The method was developed with a short run time of two minutes. The method was found to be linear in the range of 25.0–199.9?µg?mL^?1 and 8.9–21.3?µg?mL^?1 for Carvedilol and Ivabradine, respectively with a correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8% and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH Q2 (R1) guidelines.     

Assay of naproxen by high-performance liquid chromatography and identification of its photoproducts by LC-ESI MS

A rapid, accurate and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of naproxen and its photodegradation products in methanol was developed and validated. An Inertsil 5-ODS-3V column (5 microm, C18, 250 x 4.6 mm i.d.) was used with a mobile phase of acetonitrile-methanol-1% HOAc in H2O (40:20:40, v/v/v). UV detection was set at 230 nm. The developed method satisfies system suitability criteria, peak integrity and resolution for the parent drug and its photoproducts. The intraday and interday standard deviations of five replicate determinations for five consecutive days at the working concentrations of 5.0, 10, 25, 50, and 100 microm were 0.23-0.98 with coefficients of variance (CVs) of between 0.96 and 4.56% for the former, and 0.14-1.15 with CVs of between 1.13 and 3.82% for the latter. The percentage recoveries were determined to be 98.34, 99.19, 100.18, 102.97 and 99.81%, respectively, at the five concentrations between 5.0 and 100 microm. The limit of quantitation of naproxen was determined to be 0.29 microg/mL, while the detection limit was 64 ng/mL. Four major photoproducts were observed from the HPLC chromatogram using a Panchum PR-2000 reactor which equipped with 8 W x 16 low-pressure quartz mercury lamps as the light source for irradiation of a naproxen sample in methanol. The structures of the photoproducts were confirmed by LC-ESI MS.     

Development and validation of a fast high pressure liquid chromatography method for the analysis of lignocellulosic biomass hydrolysis and fermentation products

A simple, precise, and accurate 10-min high pressure liquid chromatography (HPLC) method was developed and validated for the analysis of organic acids, alcohols, and furans from processing biomass into renewable fuels. The method uses an H⁺ form cation-exchange resin stationary phase that has a five-fold shorter analysis time versus that in the traditional method. The new method was used for the analysis of acetic acid, ethanol, 5-hydroxymethyl furfural, and furfural. Results were compared with a legacy method that has historically has been used to analyze the same compounds but with a 55 min run time. Linearity was acceptable on the new method with r² > 0.999 for all compounds using refractive index detection. Limits of detection were between 0.003 and 0.03 g/L and limits of quantification were between 0.1 and 0.01 g/L. The relative standard deviations for precision were less than 0.4% and recoveries ranged from 92% to 114% for all compounds.     

Simultaneous determination of cyromazine,melamine and their biodegradation products by ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatography with ultraviolet detection method for the determination of cyromazine, melamine and its biodegradation products (ammeline, ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separate the six analytes with a mobile phase consisting of perchloric acid-ammonia solution and acetonitrile, under gradient elution and variable flow rate. The detection wavelengths were 205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline and ammelide. For analysis of sediment samples, the extraction solution containing acetonitrile, ammonia and water (80:10:10 by volume) was used to extract the analytes from sediment matrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limit was 0.1 μg g^?1 for melamine and cyromazine, 0.2 μg g^?1 for ammeline and ammelide, 1.2 μg g^?1 for cyanuric acid and 1.0 μg g^?1 for biuret in sediment matrix. The recoveries of these compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%. Finally, the proposed method was successfully applied to the analysis of the sediment sample near the wastewater outlet of a melamine-producing factory.     

Development of validated RP HPLC method with fluorescence detection for simultaneous quantification of sacubitril and valsartan from rat plasma

In the present work, a sensitive, accurate and precise RP HPLC method has been established for simultaneous determination of sacubitril and valsartan from rat plasma by using irbesartan as an internal standard. Separation of analytes were carried out on monolithic column using 10?mM phosphate buffer (pH 2.6), methanol and acetonitrile in a proportion of 50:25:25 (v/v). Analytes were monitored using fluorescence detector maintained at an excitation wavelength of 249?nm and an emission wavelength was set at 380?nm till the elution of valsartan as well as internal standard and switched online to 320?nm for sacubitril. Analysis of analytes from rat plasma was carried out by protein precipitation using methanol and acetonitrile. Valsartan and sacubitril showed good linearity in the concentration range of 1–250?ng/ml and 1–200?ng/ml respectively, with good correlation coefficient (r²?≥?0.998). Further, the precision and accuracy of the proposed procedure were suitable as the percent relative standard deviation and percent relative error were well within the acceptable range. The average percent of recovery from the rat plasma was found to be 96.6% and 97.6% for valsartan and sacubitril respectively. The newly proposed method can be used for regular pharmacokinetic studies because of simplicity in sample preparation, short analysis time (<5?min) and good sensitivity.     

Development and validation of high-performance thin-layer chromatographic method for determination of amygdalin

ABSTRACT

A new method for the extraction and quantitative determination of amygdalin has been proposed. Accelerated solvent extraction was applied for the extraction, and reversed-phase high-performance thin-layer chromatography method was developed, validated, and applied for the determination of amygdalin in the extracts of apricot, plum, almond, and peach kernels. The chromatographic system used was RP-18 silica, as stationary phase and acetonitrile/water (50:50, v/v), as mobile phase. Densitometric scanning was performed at 210 nm. The method was validated with respect to specificity, linearity, precision, and accuracy. The results showed that the peak area responses were linear within the concentration range of 2.5–50.0 µg/spot (R² = 0.9984). The limit of quantification was 4.28 µg/spot, and the detection limit 1.28 µg/spot. The intra-day and inter-day reproducibility, in terms of %RSD, were in the range of 0.81–1.15 and 1.32–1.89, respectively. The accuracy data were in the range from 99.98 to 100.56%. The method is linear, quantitative and reproducible, and could be used as an efficient and economical green chromatographic procedure for the determination of amygdalin in the fruit kernel.