QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

The present studies describe the systematic quality by design (QbD)‐oriented development and validation of a simple, rapid, sensitive and cost‐effective reversed‐phase HPLC bioanalytical method for nevirapine in rat plasma. Chromatographic separation was carried out on a C₁₈ column using isocratic 68:9:23% v/v elution of methanol, acetonitrile and water (pH 3, adjusted by orthophosphoric acid) at a flow rate of 1.0 mL/min using UV detection at 230 nm. A Box–Behnken design was applied for chromatographic method optimization taking mobile phase ratio, pH and flow rate as the critical method parameters (CMPs) from screening studies. Peak area, retention time, theoretical plates and peak tailing were measured as the critical analytical attributes (CAAs). Further, the bioanalytical liquid–liquid extraction process was optimized using an optimal design by selecting extraction time, centrifugation speed and temperature as the CMPs for percentage recovery of nevirapine as the CAA. The search for an optimum chromatographic solution was conducted through numerical desirability function. Validation studies performed as per the US Food and Drug Administration requirements revealed results within the acceptance limit. In a nutshell, the studies successfully demonstrate the utility of analytical QbD approach for the rational development of a bioanalytical method with enhanced chromatographic separation and recovery of nevirapine in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Applying green analytical chemistry for development and validation of RP-HPLC stability indicating assay method for estimation of fenoverine in bulk and dosage form using quality by design approach

A green and robust reverse-phase liquid chromatographic method has been developed for the determination of fenoverine (FEN), by applying combined principles of green analytical chemistry and quality by design approaches on a Spherisorb C18 column (150?×?4.6?mm, 3?µm) with UV detection at 262?nm. A two level fractional factorial design (2^^7-3) Res IV was used for screening of influential chromatographic factors. The critical method parameters actively affecting critical quality attributes (CQAs) were identified and further optimized using Box–Behnken design. The predicted optimum assay conditions comprised of methanol and ammonium acetate buffer 20?mM, in an extent of 81:19% v/v individually having a flow rate of 1.0?mL/min with a column oven temperature of 33°C. The drug was stressed in hydrolytic, oxidative, reductive, thermal, and photolytic conditions. The developed method was validated successfully. The detector response was linear in the concentration of 0.5–160?µg/mL with a limit of detection (LOD) and limit of quantitation (LOQ) as 0.1 and 0.3?µg/mL, respectively. The % recovery was found to be 99.7%. The analytical method volume intensity value for developed method was 45?mL and the environment assessment tool (EAT) score was 41.07. The method is simple, environmentally benign, rapid, and robust for the determination of FEN in bulk and in its dosage form.     

QbD-driven development and validation of an efficient bioanalytical UPLC method for estimation of olmesartan medoxomil

The present studies describe quality by design-based development of bioanalytical ultra performance liquid chromatography method of olmesartan medoxomil. Initially, method objectives were defined and critical analytical attributes (CAAs) earmarked. Method optimization was conducted using a central composite design for optimizing mobile phase ratio and injection volume as the critical method parameters (CMPs) identified from risk assessment and factor screening studies, and evaluated for their influence on peak area, theoretical plates, and asymmetry factor as CAAs. Chromatographic separation was achieved using acetonitrile:water solvent system containing 0.1% orthophosphoric acid (54:46, v/v) as the mobile phase with UV detection at 243 nm. Further optimization of bioanalytical extraction process was accomplished using a Box–Behnken design selecting extraction time, centrifugation speed, and centrifugation time as the CMPs identified from failure mode and effect analysis, and evaluated for percent recovery, peak asymmetry, and theoretical plate count as the CAAs. Establishment of calibration curve indicated linearity between concentration range of 100 and 800 ng mL^?1, excellent accuracy and precision with limit of detection and limit of quantification as 6.2 and 19.0 ng mL^?1, respectively. Drug stability studies indicated mean percent recovery ranging between 92.4 and 97.3% under various stress conditions.     

Development and Validation of RP-UHPLC Method for Quantification of Gliclazide in Bulk and Pharmaceutical Dosage Form Using Quality-by-Design (QbD) Approach: A Shifting Paradigm

The current research endeavours quality-by-design (QbD)-aided chromatographic techniques for the quantification of gliclazide (GLZ) in bulk and pharmaceutical dosage forms. Analytical QbD was initiated by assigning both an analytical target profile (ATP) and critical analytical attributes (CAAs). Furthermore, risk evaluation studies, along with factor screening studies, helped identify critical method parameters (CMPs). Optimisation was carried out using a 3² full factorial design by utilising the identified CMP, that is, flow rate (X1) and pH of buffer (X2) at three different levels along with evaluation of the selected CAA, that is, the retention time (Y1) and the peak area (Y2). In addition, the influence of sole and interactive CMPs on CAAs was checked using the data obtained statistically and with response surface plots. The confirmation of significance (P?<?0.05) of the method parameters was determined using analysis of variance (ANOVA). Chromatographic separation was achieved using a stainless-steel C8 column (25 cm?×?4 mm) in isocratic elution mode using phosphate buffer (pH 3.4) and HPLC-grade acetonitrile (50:50 v/v) as the eluent. The flow rate was adjusted to 1 mL min^?1 and the eluent was detected at 230 nm. The validated method, alongside subsequent stress degradation studies conducted according to the ICH guidelines, further favours it as a highly efficient method for the analysis of regular drugs as well as their degraded products. The method proposed above provided a successful demonstration of the QbD-based approach in developing an extremely sensitive and dependable technique for estimating the GLZ for routine analysis and pre-clinical applications.

     

Integrated quality by design (QbD) and design of experiments (DoE) approach for UFLC determination of telaprevir in rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of telaprevir in Wistar albino rat serum. Principles of quality by design (QbD) were implemented for enhancing the bioanalytical liquid–liquid extraction of telaprevir from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the critical analytical attribute, i.e., %recovery of telaprevir. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:borate buffer of pH 9 (90:10 v/v) flowing at 1.2?mL/min. Photodiode array detection was performed at 270?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 25–10,000?ng/mL. Limit of detection for the developed method was 10?ng/mL. Further, design of experiments (DoE) used for inter-day accuracy and precision study suggested superior method reliability. This integrated QbD- and DoE-based approach ensured the development of a validated and reliable analytical method for optimum bioanalysis of telaprevir in biological matrix.     

Quality-by-design-based development and validation of a stability-indicating UPLC method for quantification of teriflunomide in the presence of degradation products and its application to in-vitro dissolution

A systematic design-of-experiments was performed by applying quality-by-design concepts to determine design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography (UPLC) method in the presence of degradation products. Response surface and central composite quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The response variables such as resolution, retention time, and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D, and design space were studied. The method was developed through UPLC BEH C18 2.1?×?100?mm, 1.7-µ column, mobile phase comprised of buffer (5?mM K₂HPO₄ containing 0.1% triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5?mL?min^?1 and UV detection at 250?nm. The method was developed with a short run time of 1?min. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28–84?µg?mL^?1, 2.8–22.7?µg?mL^?1 with a correlation coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in the range of 100.1–101.7%. The method was validated according to ICH guidelines.     

Development and validation of a generic high‐performance liquid chromatography for the simultaneous separation and determination of six cough ingredients: Robustness study on core–shell particles

A generally applicable high‐performance liquid chromatographic method for the qualitative and quantitative determination of pharmaceutical preparations containing phenylephrine hydrochloride, paracetamol, ephedrine hydrochloride, guaifenesin, doxylamine succinate, and dextromethorphan hydrobromide is developed. Optimization of chromatographic conditions was performed for the gradient elution using different buffer pH values, flow rates and two C₁₈ stationary phases. The method was developed using a Kinetex® C₁₈ column as a core–shell stationary phase with a gradient profile using buffer pH 5.0 and acetonitrile at 2.0 mL/min flow rate. Detection was carried out at 220 nm and linear calibrations were obtained for all components within the studied ranges. The method was fully validated in agreement with ICH guidelines. The proposed method is specific, accurate and precise (RSD% < 3%). Limits of detection are lower than 2.0 μg/mL. Qualitative and quantitative responses were evaluated using experimental design to assist the method robustness. The method was proved to be highly robust against 10% change in buffer pH and flow rate (RSD% < 10%), however, the flow rate may significantly influence the quantitative responses of phenylephrine, paracetamol, and doxylamine (RSD% > 10%). Satisfactory results were obtained for commercial combinations analyses. Statistical comparison between the proposed chromatographic and official methods revealed no significant difference.     

Analytical Quality by Design (AQbD)-oriented RP-UFLC method for quantification of lansoprazole with superior method robustness

A reversed-phase ultrafast liquid chromatography method was developed for quantification of lansoprazole in pharmaceutical dosage form using analytical quality by design approach. Systematic planning and experimentation using design of experiment approach were used for method development and optimization studies. A central composite design was used for optimizing the chromatography, by choosing organic phase proportion and flow rate as the critical method variables for evaluating their effect on critical analytical attributes like resolution, plate number, and tailing factor. The optimal chromatography was accomplished on a C-18 column (250?×?4.6?mm, 5?µm) using methanol:water (70:30, v/v) as mobile phase at a flow rate of 1.0?mL/min. Photo diode array (PDA) detection was performed at 284?nm. Caffeine was used as the internal standard. Method validation studies revealed that the calibration curve was linear over 1.0–300?µg/mL. The method was found accurate with average recovery between 98.99 and 102.87%. The percent relative standard deviation values obtained for precision were as per ICH guideline and within the acceptance limits (<2%). Results of system suitability indicated superior method robustness. In a nutshell, the method was found to be highly suitable for its applicability in the determination of lansoprazole in bulk and tablet dosage form.     

Multiobjective Optimization Approach in Evaluation of Chromatographic Behaviour of Zolpidem Tartrate and Its Degradation Products

Multiresponse optimization methodology in combination with experimental design was employed as a powerful technique for simultaneous optimization of input variables significant for evaluation of chromatographic behaviour of zolpidem tartrate, zolpacid, oxozolpidem, zolpyridine and zolpaldehyde towards various responses. In the first stage of the investigation fractional factorial design was used to decrease the number of variables that should be studied in detail. Among examined variables, pH of the mobile phase, percentage of organic modifier and buffer concentration showed to be statistically important and were consequently optimized with central composite design and Derringer??s desirability function. Four responses were considered, the retention factors of zolpacid and zolpaldehyde (the first and last peak) and the resolutions between critical peaks. Optimal conditions included Luna C₁₈(2) analytical column (250?mm?x?4.6?mm, 5???m particle size), mobile phase consisted of methanol?C10?mM ammonium acetate (68.4:31.6, v/v, pH 5.4) and column temperature of 35???C. The flow rate of the mobile phase was 1?mL?min^?1 and the detection was performed at 254?nm. At the end, the method was successfully validated in accordance with ICH guideline and subsequently applied to the analysis of commercially available zolpidem tartrate tablets.     

Quality by Design enabled enhanced bioanalytical extraction and UFLC determination of vilazodone from rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of vilazodone in Wistar rat serum. Principles of quality by design were implemented for enhancing the bioanalytical liquid–liquid extraction of vilazodone from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the analytical attribute, i.e., %recovery of vilazodone. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:phosphate buffer of pH 7 (85:15 v/v) flowing at 1.5?mL/min. Photodiode array detection was performed at 242?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 100–2,000?ng/mL with acceptable accuracy and precision. Limits of detection and quantitation for the developed method were 50 and 100?ng/mL, respectively. This QbD-based approach was found suitable for routine bioanalysis of vilazodone in the biological matrix.     

Development and validation of a high performance liquid chromatographic method for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets

A simple, sensitive and rapid high performance liquid chromatographic method was developed and validated for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets. Chromatographic separation and detection was carried out on a C-18 column using 0.05 M potassium dihydrogen phosphate buffer (pH 5.0) and acetonitrile in the ratio of 94: 06 (v/v) as mobile phase at wavelength of 225 nm. The method was linear in the concentration range of 3.75–22.5 μg/mL for potassium clavulanate and 15–90 μg/mL for cefadroxil. The flow rate was 1.0 mL/min and the total analysis time was less than 10 min. The mean recoveries was found to be greater than 99% with RSD less than 1.0%. The proposed method was validated by performing linearity, recovery, specificity, robustness, LOD/LOQ and within-day and between-day precision. The chromatographic results obtained from the synthetically prepared tablets show that the method is highly precise and accurate for the simultaneous quantitation of clavulanate potassium and cefadroxil.     

A new UPLC approach for the quantitation of ephedrine and guaifenesin in a syrup formulation using multivariate optimization strategy

A new ultraperformance liquid chromatography (UPLC) method with photodiode array detection was developed for the quantitative analysis of a commercial syrup formulation containing ephedrine (EPH) and guaifenesin (GUA). In the development of UPLC method, experimental chromatographic conditions, flow rate, column temperature, and percentage of 0.1?M H₃PO₄ in mobile phase, were optimized using chemometric multivariate strategy. From the application of a 3³ full factorial design, the optimal chromatographic conditions were obtained as the flow rate of 0.29?mL/min, column temperature of 36.4°C, and 56.9% of 0.1?M H₃PO₄ in the mobile phase. The optimal conditions gave us a good chromatographic separation of the analyzed drugs with short analysis runtime within 3?min. Calibration curves for EPH and GUA in the linear working range of 4–64 and 6–96?µg/mL, respectively, were obtained using peak areas detected at 215?nm. Performance and validity of the optimized UPLC method were estimated by analyzing independent binary mixtures, inter-day and intra-day samples, and standard addition solutions containing EPH and GUA substances. It was concluded that the proposed method was a promising approach for the quantitative determination and routine analysis of a commercial syrup formulation of the titled substances.     

Simultaneous determination of six herbal components in intestinal perfusate by high‐performance liquid chromatography

An effective, accurate and reliable HPLC with UV detection method was developed and validated for quantitation of six components: baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein in intestinal perfusate using rotundin as an internal standard. The chromatographic separation was performed on a Welchrom‐C₁₈ column (250 × 4.6 mm i.d. with 5.0 µm particle size) with a mobile phase consisting of acetonitrile, water, phosphoric acid and triethylamine (30:70:0.2:0.1,v/v) at a flow rate of 1.0 mL/min and a UV detection at 270 nm. The method had a chromatographic run time of 30 min and excellent linear behavior over the investigated concentration ranges observed with the values of r higher than 0.99 for all the analytes. The lower limit of quantification of the analytical method was 0.09 µg/mL for berberine hydrochloride, quercetin, kaempferol and baicalein and 0.18 µg/mL for baicalin and isorhamnetin. The intra‐ and inter‐day precisions measured at three concentration levels were all less than 10% for all analytes. The bias ranged from ?6.91 to 4.33%. The validated method has been successfully applied to investigate the rat intestine absorption profiles of baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein. Copyright © 2009 John Wiley & Sons, Ltd.     

Applications of Monte-Carlo simulation and chemometric techniques for development of bioanalytical liquid chromatography method for estimation of rosuvastatin calcium

In the present work, we investigated the development of a bioanalytical HPLC method of rosuvastatin (RSV) calcium as per the Quality by Design (QbD)-based systematic chemometric tools. At first, the method objectives were framed and critical analytical attributes (CAAs) were chosen. Risk assessment and factor screening was performed using Hybrid Risk Matrix and Plackett–Burman design for identifying vital factors influencing the critical method parameters (CMPs). Monte-Carlo simulation analysis was conducted which confirmed excellent process robustness (Ppk >1.33) for the studied ranges of CMPs. Furthermore, systematic method development was carried out using custom experimental design, where mobile phase ratio, pH, and injection volume were taken as CMPs at three levels. The obtained trials were evaluated for peak area, retention time, theoretical plates, and peak tailing as CAAs. Mathematical response surface modeling was carried out and optimal chromatographic solution was identified using response optimizer plots. Method transfer was made to bioanalytical scale for estimation of the analyte in rat plasma samples. Extensive method validation was performed as per the ICH Q2 guideline, which indicated validation parameters within the acceptable limits. Overall, the studies construed successful development of QbD compliant HPLC method of rosuvastatin with potential utility bioanalytical testing.     

Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of catharanthus roseus by high-performance liquid chromatography

A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus..     

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.     

Validation of an LC-MS/MS method for the quantitative determination of mavoglurant (AFQ056) in human plasma

A simple, sensitive, and selective liquid chromatography/tandem mass spectrometry method was validated for the identification and quantification of mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150?×?4.6 mm, 5 μm) column at 40?±?0.5 °C with a mobile phase consisting of acetic acid in water (0.1 %, v/v)/methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometry, operating with electrospray ionization in positive ion mode and applying multiple reaction monitoring. The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant, and the anticoagulant effect was tested by lithium heparin and K₃EDTA. The method was successfully cross-validated between two bioanalytical sites. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area at the retention time of mavoglurant in zero samples was less than 20 % compared with the mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 and 2,500 ng/mL.     

MultiSimplex and experimental design as chemometric tools to optimize a SPE-HPLC-UV method for the determination of eprosartan in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug eprosartan from human plasma samples. MultiSimplex program was used to optimize the HPLC-UV method due to the number of experimental and response variables to be studied. The measured responses were the corrected area, the separation of eprosartan chromatographic peak from plasma interferences peaks and the retention time of the analyte.The use of an Atlantis dC18, 100 mm × 3.9 mm i.d. chromatographic column with a 0.026% trifluoroacetic acid (TFA) in the organic phase and 0.031% TFA in the aqueous phase, an initial composition of 80% aqueous phase in the mobile phase, a stepness of acetonitrile of 3% during the gradient elution mode with a flow rate of 1.25 mL/min and a column temperature of 35 ± 0.2 °C allowed the separation of eprosartan and irbesartan used as internal standard from plasma endogenous compounds. In the solid phase extraction procedure, experimental design was used in order to achieve a maximum recovery percentage. Firstly, the significant variables were chosen by way of fractional factorial design; then, a central composite design was run to obtain the more adequate values of the significant variables. Thus, the extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer pH 2 as conditioning agent, a drying step of 10 min, a washing step with methanol-phosphate buffer (20:80, v/v) and methanol as eluent liquid. The SPE-HPLC-UV developed method allowed the separation and quantitation of eprosartan from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Determination of key flavonoid aglycones by means of nano‐LC for the analysis of dietary supplements and food matrices

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano‐LC technique. In order to find optimal conditions, capillary columns (75 μm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core–shell (2.6 μm particle size), Hydride‐based RP‐C18 (sub‐2 μm particle size), and LiChrospher® 100 RP‐18 endcapped (5 μm particle size) were evaluated. The method was validated using Hydride‐based RP‐C18 stationary phase, with sub‐2 μm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63–4.68% for peak area), column‐to‐column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 μg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.     

Simultaneous assay of olanzapine and fluoxetine in tablets by column high-performance liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance liquid chromatographic (LC) and high-performance thin-layer chromatographic (TLC) methods for the simultaneous estimation of olanzapine and fluoxetine in pure powder and tablet formulations. The LC separation was achieved on a Lichrospher 100 RP-180, C18 column (250 mm, 4.0 mm id, 5 microm) using 0.05 M potassium dihydrogen phosphate buffer (pH 5.6 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1 mL/min and ambient temperature. The TLC separation was achieved on aluminum sheets coated with silica gel 60F254 using methanol-toluene (40 + 20, v/v) as the mobile phase. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 10-70 and 40-280 microg/mL with mean recovery of 99.54 +/- 0.89 and 99.73 +/- 0.58% for olanzapine and fluoxetine, respectively, by the LC method. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 100-800 and 400-3200 ng/spot with mean recovery of 101.53 +/- 0.06 and 101.45 +/- 0.35% for olanzapine and fluoxetine, respectively, by the TLC method with densitometry. These methods are simple, precise, and sensitive, and they are applicable for simultaneous determination of olanzapine and fluoxetine in tablet formulations.