Mobile Phase Of Acetonitrile Research Articles

Estimation of Lexisinatide in Bulk and Tablets by RP‐UPLC Method Developed by Quality by Design Concept

AbstractThe primary goal of the proposed study is to establish and validate a low‐cost, sensitive, reliable, and trouble‐free RP‐UPLC process with improved performance for the direct estimation of Lixisenatide in pure powder and fixed‐dose tablet form. The proposed method is optimized by implementing quality by design studies. The method is optimized using quadratic model of central composite design (CCD) of randomized response surface study. Successful separation of Lixisenatide is obtained by the use of Phenyl‐XBD (1.7 µm, 2.1 mm × 100 mm) column and a mobile phase of acetonitrile: 0.1% w/v OPA (60:40) with 0.5 mL min−1 flow rate. The separated Lixisenatide and its degradants are explorated with PDA detector which has the ability to provide comprehensive spectral information, rapid data acquisition, quantitative analysis, and peak purity analysis of all compounds simultaneously. The RT of Lixisenatide is found to be 1.34 min. The lowest concentration of Lixisenatide's limit of detection and limit of quantification makes guarantees regarding the method's sensitivity. For the provided range of linear concentrations (1.25–7.5 µg mL−1), the regression coefficient is found to be 0.999. The computed average percentage recoveries of Lixisenatide in spiked solutions ranged from 98.9% to 101.2%. The % degradation of Lixisenatide in the presence of a variety of forced conditions confirms the existing method's stability. Lixisenatide is significantly photo sensitive when compared to other stressful conditions such as acid, base hydrolysis, and oxidation. The proposed UPLC approach separates Lixisenatide with high sensitivity, a short retention time, and an affordable solvent system.

TANDEM MASS SPECTROMETRIC METHOD FOR THE TRACE LEVEL DETERMINATION OF 2-AMINOPYRIDINE: A POTENTIAL GENOTOXIC IMPURITY IN TENOXICAM API

Objective: This study aimed to develop a highly sensitive method for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam, employing hyphenated techniques. Methods: The determination of 2-amino pyridine was carried out using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in Selected Ion Monitoring mode (SIM). A LiChrospher RP-18 (100×4.6 mm) 5.0 µm column was utilized for the separation. A gradient elution technique was employed with acetonitrile (mobile phase A) and 0.01M ammonium acetate buffer (mobile phase B) in varying ratios. The gradient program (T/%B) was set as 0/5, 2.50/15, 5.00/30, 10.00/50, 15.00/95, 20.00/95. The developed method was validated according to the International Conference on Harmonization guidelines. Results: The limits of detection (LOD) and quantification (LOQ) for 2-amino pyridine were found to be 0.09 ppm and 0.3 ppm, respectively. The method demonstrated accuracy within the range of 89.1% to 106.6% for the analyte. The method's linearity was confirmed through a six-point calibration graph spanning 6 ppm to 75 ppm, corresponding to a concentration of 20 mg/ml of Tenoxicam. Conclusion: Developed hyphenated LC-MS/MS method presented in this study offers a highly sensitive and accurate means for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam. With validated LOD and LOQ values, as well as demonstrated accuracy, this method proves to be a robust quality control tool suitable for the quantitation of 2-amino pyridine at very low concentrations in the pharmaceutical compound Tenoxicam.

Diode Array Detector Based RP-UPLC Method for Simultaneous Estimation of Dapagliflozin Propanediol Monohydrate and Metformin

A simple, rapid, accurate, precise, and reproducible diode array detector-based RP-UPLC method for simultaneous estimation of dapagliflozin propanediol monohydrate and metformin hydrochloride in bulk and tablet dosage form has been developed and validated. The chromatographic analysis was carried out using Acquity UPLC HSS T3 C18 (50 × 2.1mm, 1.8μm) column with a mobile phase consisting of orthophosphoric acid (pH 3.00) and acetonitrile (55:45% V/V) in the isocratic mode, at a flow rate of 0.2mL/min, and eluents were the simultaneous measurement of dapagliflozin propanediol monohydrate and metformin hydrochloride in bulk and tablet dosage form using a diode array detector-based RP-UPLC technique has been developed and validated. The chromatographic analysis was carried out using an Acquity UPLC HSS T3 C18 (50 2.1mm, 1.8 m) column and an orthophosphoric acid (pH 3.00) and acetonitrile (55:45% V/V) mobile phase in the isocratic mode.at a flow rate of 0.2mL/min. Eluents were measured at 222nm. Metformin hydrochloride and dapagliflozin propanediol monohydrate were shown to have retention times of 0.845 minutes and 1.889 minutes, respectively. The linearity was established in the range of 5-15μg/mL for dapagliflozin propanediol monohydrate and 250-750μg/mL for metformin hydrochloride, which had a regression coefficient (r2) greater than 0.999. The % Recovery was found to be 98.77% - 99.57% and 99.15% -101.2% for dapagliflozin propanediol monohydrate and metformin hydrochloride respectively. In a short period,a larger number of samplescan be analyzed due to their short retention time and, therefore, will be economical for routine analysis in the pharmaceutical industry.

Quantification of catalpol in root of Rehmannia glutinosa varieties 19 collected in Phu Tho province by high-performance liquid chromatography

The active ingredient catalpol in the Rehmannia glutinosavariety 19 roots is an important chemical component. Catalpol has hypoglycemia, diuretic, and laxative effects; it is used to support the treatment of diabetes, ischemic cerebral, and dementia, and it is regulated by Vietnamese Pharmacopoeia V (2017) as a marker to check the quality of medicinal herbs. This study aimed to develop a procedure for quantifying catalpol in the root of Rehmannia glutinosa varieties 19 by high-performance liquid chromatography (HPLC) to evaluate the quality of medicinal herbs. The research results have selected suitable chromatographic conditions using the Gemini C18 column (250x4.6 mm, 5 μm), detector at 210 nm, mobile phase acetonitrile - phosphoric acid 0.1%, and flow rate at 0.6 ml/min. The peak area and catalpol concentration have a strong linear correlation with r=0.9999, Y = 21887X + 26213. The procedure has good accuracy and repeatability with a relative standard deviation (RSD) <2%. Quantitative results of catalpol in the root of the R. glutinosa varieties 19 reached 1.25%. The HPLC method is suitable for quantifying catalpol in the root of R. glutinosa varieties 19.

A UPLC-MS/MS method for simultaneous determination of arachidonic acid, stearic acid, and related endocannabinoids in human plasma

Endocannabinoids (eCBs) exert considerable influence over energy metabolism, lipid metabolism, and glucose metabolism within the human body. Among the most biologically active cannabinoids identified thus far are 2-arachidonoylglycerol (2-AG), arachidonoyl ethanolamide (AEA), 1-stearoylglycerol (1-SRG), and stearoyl ethanolamide (SEA), which are derived from arachidonic acid (AA) and stearic acid (SA). However, despite the unique in bioactivities exhibited by eCBs, their determination in plasma has been hindered by the lack of sensitive analytical methods. The aim of this study was to develop and validate a highly sensitive and rapid method using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for accurate measurement of AEA, SEA, 2-AG, 1-SRG, AA, and SA levels in human plasma samples. Sample preparation involved a protein precipitation method and a methyl tert-butyl ether liquid-liquid extraction method. Chromatographic separation was accomplished by utilizing an ACQUITY UPLC BEH C8 column with a mobile phase of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid, flowing at a rate of 0.35 mL/min. AA-d8, 2-AG-d5, and AEA-d8 were selected as deuterated internal standards. The analytes were determined with MRM in both positive and negative ion mode. The lower limit of quantification ranged from 0.1 to 400 ng/mL, and the correlation coefficient (R2) was >0.99. Inter-day and intra-day precision exhibited values of 0.55–13.29% and 0.62%–13.90%, respectively. Recovery and matrix effect were within the range of 77.7%–109.7%, and 90.0%–113.5%, respectively. Stability tests confirmed the acceptability of all analytes. To demonstrate the effectiveness of the approach, it was implemented to assess and compare plasma samples from healthy volunteers (n = 49) and individuals with non-alcoholic fatty liver disease (NAFLD) (n = 62). The study revealed significant differences in AEA, SEA, AA, and SA levels between the two groups.

Development and Validation of a New Reversed Phase HPLC Method for the Quantitation of Azithromycin and Rifampicin in a Capsule Formulation.

This research aimed to develop a new method for simultaneously estimating the presence of azithromycin (AZT) and rifampicin (RIF) in a capsule formulation using reverse-phase high-performance liquid chromatography. The developed method utilized a Gemini column with a 60:40% v/v acetonitrile and potassium dihydrogen phosphate mobile phase, a flow rate of 1mL/min, and an injection volume of 20μL. The detection wavelengths of 210 and 254nm for AZT and RIF, respectively, were used. Validation ensures specificity with a peak purity index > 0.99999 for AZT and >0.99995 for RIF, affirming unambiguous analyte detection. The system suitability test, within acceptable limits, validates method reliability. Linearity calibration curves (R2 = 0.998) cover a 25-150% target concentration range. Accuracy studies employing the standard addition method yield recovery values between 96.6 and 103.9% for both drugs, confirming method accuracy. Precision studies reveal % relative standard deviation values consistently below 2%, highlighting reproducibility. Robustness testing supports method reliability under varying conditions. Application to a pharmaceutical capsule formulation demonstrates the method's practicality, accurately quantifying AZT (98.30%) and RIF (99.37%). This study provides a validated analytical approach for simultaneous quantification in commercial pharmaceutical products containing both drugs, enhancing pharmaceutical quality control for critical antibiotics in complex formulations.

HPLC method for the determination of nifedipine in rat plasma: development, validation, and application to pharmacokinetic drug-herb interaction study

A simple and rapid high-performance liquid chromatography (HPLC) was developed to determine the plasma level of nifedipine in rats after its single administration and combination with herbs. Nifedipine was extracted with acetonitrile to precipitate protein from plasma samples. The separation was implemented on a C18 column with a mobile phase of acetonitrile: water (63:37, v/v). The calibration curve displayed good linearity in the 30–1000 ng/mL range. The lower limit of quantification (LLOQ) was 30 ng/mL. The intraday and interday assay accuracy and precision met the criteria of validation and study sample analysis. The recovery was found to be 101.89%. Stability studies showed that nifedipine was stable after 12 h at room temperature and 21 days at -20 °C. No significant difference was examined between the pharmacokinetic parameters of nifedipine with or without Gynura procumbens leaf extract. The proposed method was helpful for the pharmacokinetic interaction study of nifedipine combined with herbal in rats.

Development of a Stability Indicating UPLC Method for the Determination of Tirbanibulin in Bulk and Its Pharmaceutical Dosage Form.

The primary goal of this study was to create and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for estimating tirbanibulin in pure and dosage form. A UPLC technique was developed using a Waters Acquity UPLC Phenyl (100 x 2.1 mm, 1.7 µm) column. The developed technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Tirbanibulin was separated chromatographically with high resolution using the mobile phase acetonitrile: buffer (30:70 v/v) at 0.5 mL/min, 5 µL injection volume, and 220 nm wavelength. The validated technique was found to be linear in the 1-15 µg/mL range. The detection and quantification limits for tirbanibulin were 0.03 and 0.1 µg/mL, respectively. The percentage relative standard deviation was less than 2%, demonstrating the precision of the developed technique. Furthermore, the recovery rate was nearly 100%, confirming the accuracy of the method. Minor modifications to the chromatographic conditions demonstrated the robustness of the method. The developed analytical method was precise, simple, reproducible, and sensitive. Consequently, it can be used to determine tirbanibulin.

Analytical Method Development and Validation of Beclomethasone Dipropionate and Fusidic Acid in pure and Pharmaceutical Dosage Form (Cream) by RP-HPLC

A simple, rapid, specific, cheap, eco-friendly, and accurate RP-HPLC method for simultaneous estimation of Beclomethasone Dipropionate and Fusidic Acid in pure and combination drugs has been developed and validated. A stationary phase of BDS Hypersil C18 ((250mm x 4.6m, 5m) and a mobile phase of Acetonitrile: Methanol: Orthophosphoric acid (60:20:20v/v) were used for the separation. The flow rate was continued at 1.0 ml/min and the effluent was observed at 230nm. The designated situations were appropriate for separating Beclomethasone Dipropionate and Fusidic acid eluted with a mean retention time of 7.2 and 5.1 minutes, respectively. The planned technique was validated as per the International Council for Harmonisation recommendation. Its linearity, specificity, system suitability, accuracy and precision, limit of detection, limit of quantitation, and robustness are some of the parameters that have been validated. The concentration range produced linear calibration curves of 1-6g/ml and 2-12g/ml for Beclomethasone Dipropionate and Fusidic Acid with R2 = 0.9961 and 0.9963. Percent RSD values for both intraday and interday were determined to be less than 2.0%. Demonstrating the accuracy of the approach. The technique was proven to be reliable. A successful application of the developed RP-HPLC method for the quantification of Beclomethasone Dipropionate and Fusidic Acid in marketed semi-solid dosage form (Cream).

Pharmacokinetics of Anti-Rheumatic drugs Methotrexate and Tofacitinib with its metabolite M9 in Rats by UPLC-MS/MS.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

BIOANALYTICAL OF UPLC METHOD DEVELOPMENT AND VALIDATION OF XANTHORRIZOL AND ITS APPLICATION TO PHARMACOKINETIC STUDY

Objective: A simple, straightforward, ultra-performance liquid chromatography (UPLC) with a fluorescence detector method was developed and validated to determine xanthorrhizol in rat plasma. This method was successfully applied to an oral pharmacokinetic study. Methods: Xanthorrhizol was separated using a C18 column in an isocratic mode using a mobile phase of acetonitrile: water (85:15 v/v) at a 0.4 ml/min flow rate. The fluorescence detector was set at 230 nm excitation and 320 nm emission wavelengths. The method was then applied in the pharmacokinetic study involving 12 Sprague-Dawley rats. Results: The developed bioanalytical methods were found to be linear in the range of 0.078–5 mg/ml with a correlation coefficient of r2=0.999. The percentage recovery of xanthorrhizol was more than 95%, and the relative standard deviation was less than 2. These results indicate that the method is accurate and precise. The limit of detection (LOD) and limit of quantification (LOQ) of the technique were 0.123 µg/ml and 0.373 µg/ml, respectively. Furthermore, the stability studies demonstrated that xanthorrhizol is stable under various analytical conditions. The pharmacokinetic study revealed that the area under the curve (AUC) was 27.23±19.65 (µg. h/ml), the half-life (t 1/2) was 7.71±2.89 h, the mean residence time (MRT) was 13.86±4.06 h while the maximum concentration (Cmax) was 1.58±0.62 µg/ml, and the time to reach the maximum concentration (Tmax) was 1.33±0.20 h. Conclusion: The developed bioanalytical method was reliable and successfully met all validation criteria, making it a robust choice for quantifying xanthorrhizol. Therefore, it may be effectively utilized to determine xanthorrhizol in rat plasma following a pharmacokinetic study.

Development of a dispersive liquid-liquid microextraction method for the determination of plastic additives in seawater.

A method using dispersive liquid-liquid microextraction (DLLME) prior to high performance liquid chromatography-diode array detection (HPLC-DAD) was developed to determine seven additives from the plastics industry (butylated hydroxytoluene, diisodecyl phthalate, irgafos 168, lawsone, quercetin, triclosan and vitamin E) in seawater samples. These compounds can reach seawater due to direct discharge from wastewater treatment plants and leaching from plastics and microplastics. The extraction was performed using 25 mL of seawater, 500 μL of 1-octanol (extraction solvent) and a stirring step instead of dispersive solvent. Additive concentrations were determined by LC-DAD on a C18 column with a mobile phase of acetonitrile and phosphoric acid aqueous solution (pH 3.5) by gradient elution. The analytical recoveries ranged from 82 to 93% for all compounds, except for lawsone (60%). Repeatability and intermediate precision were adequate with RSD < calculated values following the Horwitz equation at the concentration levels evaluated (0.06 and 0.24 mg L-1). All additives exhibited linear matrix calibration curves (R2 > 0.99). Detection limits ranged from 0.009 to 0.028 mg L-1 and quantification limits ranged from 0.027 to 0.084 mg L-1. Finally, the application of the method to real samples verified the method as accurate and applicable to seawater.

Implementation of Quality by Design Approaches for Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Assay Method for Determination of Glycyrrhizin in Nanoformulation.

Glycyrrhizin (GL) is the principal constituent of Glycyrrhiza glabra, having antiallergic, anticancer, anti-inflammatory, and antimicrobial action. The reverse-phase high-performance liquid chromatography (RP-HPLC) analytical method was used to quantitatively estimate GL in a nanoformulation and validated as per International Conference on Harmonization Q2 (R1) standards. A stationary phase of the C18-HL reversed-phase column and a mobile phase of acetonitrile and water were used for effective elution. The chromatographic conditions of RP-HPLC were optimized utilizing a quality-by-design approach to accomplish the required chromatographic separation of GL from its nanoformulation with minimal experimental runs. Optimized RP-HPLC conditions for the assay method consist of acetonitrile (41%) and water, pH 1.8, balanced with phosphoric acid (0.1%) as a mobile phase with a flow rate of 1 mL/min. The retention time was found at 7.25 min, and method validation confirmed its sensitivity, preciseness, accuracy, and robustness.

Design of Experiment Enhanced Development and Validation of RP-HPLC Method for Analysis of Ascorbic Acid and Rutin

This work intends to develop a QbD-based RP-HPLC method and validate rutin (RU) and ascorbic acid (ASC) in combined tablet dosage form. The method is optimized by the central composite design. The independent variables chosen are mobile phase ratio and pH. The dependent variables are retention time for RU (R1), retention time for ascorbic acid ASC (R2), and resolution (R3). Analysis of variance revealed that the method parameters were significant (p &lt;5). Waters Alliance-e2695 [C18 (150x 4.6 mm, 3.5)] was used to separate rutin and ascorbic acid using a mobile phase Acetonitrile: Hexane Sulphonic Acid (pH-2.5/OPA) in a 70:30 ratio, the selected wavelength was 215 nm. Method validation and degradation studies were performed ICH guidelines are followed. The approach was determined to be easy, suitable, accurate, precise, and robust for quantitative analysis of ascorbic acid and rutin.

Improved quantitative determination of (R)- and (S)‐rabeprazole sodium and its metabolites in rat plasma by LC-MS/MS and its application to a toxicokinetic study

There exist two enantiomers: (R)‐ and (S)‐rabeprazole. (R)‐rabeprazole offers specific pharmacokinetic advantages and enhanced therapeutic efficacy, warranting further investigation and development. Here, we developed a simple and rapid chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify rabeprazole enantiomers and their metabolites (rabeprazole sulfoxide and desmethyl rabeprazole enantiomers) and a LC-MS to quantify rabeprazole thioether. As for the chiral LC-MS/MS method, Chiral-AGP column (150 × 4 mm, 5 μm) was used and its mobile phase was acetonitrile (mobile phase A) and 10 mmol/L ammonium acetate (mobile phase B) (linear gradient profile: 0 min, 10 % B; 5 min, 15 % B; 9 min, 15 % B; 9.01 min, 10 % B; 13 min, 10 % B). The multiple reactions monitoring transitions of m/z 360.3 → 242.1, 376.2 → 240.1, 346.2 → 228.2 and 368.2 → 190.2 were opted for quantifying rabeprazole enantiomers, rabeprazole sulfoxide, desmethyl rabeprazole enantiomers and internal standard omeprazole. The analyte samples were prepared by a simple liquid–liquid extraction method. As for the LC-MS method, analytes were separated on a Inertsil® ODS-3 column (4.6 × 150 mm, 5 μm). The mobile phase was acetonitrile-5 mmol/L ammonium acetate water solution (65:35, v/v). ESI+ was used and ion peaks with m/z 344.2 (rabeprazole thioether) and 285.1 (internal standard diazepam) were monitored. Both these 2 methods were validated for specificity, linearity, precision, accuracy, matrix effect and extraction recovery, and, particularly, the stability of analytes under various conditions. We successfully applied these methods to a 13-week toxicokinetic study of rabeprazole in rats after intravenous administration of (R)- (80, 20, 5 mg/kg/d) and racemic (80 mg/kg/d) rabeprazole sodium. The results showed that rabeprazole and its metabolites did not accumulate in rats. However, desmethyl rabeprazole and rabeprazole thioether showed higher exposure and lower clearance rate in the last administration than in the first one. (R)-rabeprazole showed a higher exposure and a slower elimination rate than (S)-rabeprazole in rats. These findings offer experimental evidence and a theoretical foundation for further preclinical investigations and clinical applications of (R)-rabeprazole.

Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography–tandem mass spectrometry

Agastache rugosa contains phenolic compounds and flavonoids, and has been extensively used as a traditional herbal medicine. The major components in Agastache rugosa extract (ARE) are rosmarinic acid, tilianin, and acacetin, for which several analytical techniques have been reported. However, these substances have yet to be simultaneously quantified in human plasma. In this study, we aimed to simultaneously determine the three active components of ARE in human plasma by developing a reliable quantitative analytical method using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation of the plasma samples was achieved using an ACQUITY UPLC® BEH C18 column with a gradient mobile phase of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed using a triple quadrupole tandem mass spectrometer in negative electrospray ionization (ESI−) and multiple reaction monitoring (MRM) modes. The developed quantitative method was validated for the three active components. All three analytes exhibited a linear response over the ranges of 0.5–50 ng/mL for rosmarinic acid, 0.1–20 ng/mL for acacetin, and 0.5–20 ng/mL for tilianin with a weighting factor of 1/x (where x is the concentration). At three quality control (QC) concentration levels (low, medium, and high), including the lower limit of quantitation (LLOQ), acceptable accuracy (±15 %) was achieved in the intra- and interday validations. The concentration of rosmarinic acid was highest in plasma. Tilianin and acacetin appeared and were eliminated earlier in the plasma than rosmarinic acid. This study provides a successfully validated method that can be used in further clinical applications of Agastache rugosa extracts.

Bio-analytical Method for the Pre-clinical Estimation of Donepezil Hydrochloride in Rabbit Plasma by RP-HPLC

A simple, reliable and sensitive RP-HPLC bioanalytical method with PDA as detector was developed for the estimation of donepezil HCl in the rabbit plasma and validated. The biological samples were extracted and analysed using loratadine as internal standard. The sample analysed with Inertsil ODS 150mm x 4.6mm, 5.0µm particle size column using 70:30 v/v of 0.1% trifluoroacetic acid and acetonitrile mobile phase at flow rate of 1mL/min. All the parameters validated were within the limits proposed in bioanalytical method validation guidelines. The linearity was observed in the range of 5-200ng/mL with LOD of 0.9ng/mL. The method was observed to have short analysis time of 6 min and also found to be successful in studying the pharmacokinetic parameters of donepezil HCl.

A Fast HPLC/UV Method for Determination of Ketoprofen in Cellular Media.

A simple, sensitive and quick HPLC method was developed for the determination of ketoprofen in cell culture media (EMEM, DMEM, RPMI). Separation was performed using a gradient on the C18 column with a mobile phase of acetonitrile and miliQ water acidified by 0.1 % (v/v) formic acid. The method was validated for parameters including linearity, accuracy, precision, limit of quantitation and limit of detection, as well as robustness. The response was found linear over the range of 3-100 μg/mL as demonstrated by the acquired value of correlation coefficient R2=0.9997. The described method is applicable for determination of various pharmacokinetic aspects of ketoprofen in vitro.

QUALITY RISK ASSESSMENT AND DESIGNED EXPERIMENTS ORIENTED SIMULTANEOUS QUANTIFICATION OF ASPIRIN AND PANTOPRAZOLE SODIUM USING DRIFTS AND UFLC-DAD METHODS

Systematized and reliable analytical methods are always of great advantage for the quality control of new drug products. Two new analytical methods were developed and validated using the multivariate approach to quantify a unique combination of aspirin and pantoprazole sodium. In the first method, emphasis was on non-destructive identification with quantification of aspirin and pantoprazole at their characteristic diffused reflectance-based infrared absorption band at 1747cm-1(-C=O) and 1303cm-1 (-S-O), respectively. The second method relies on liquid chromatographic separation using a mobile phase of acetonitrile: phosphate buffer pH 3.5 (60:40 V/V) at a flow rate of 1.0 mL min-1 using a C-18 column. At 240 nm, the diode array detection was performed. Employing risk assessment revealed the risky method parameters that may influence the preciseness of the present methods. Nevertheless, these techniques were linear, sensitive and reliable for the quick and simultaneous measurement of the analytes in bulk and proposed fixed-dose commercial formulation.

The Effect of Using Different Anticoagulant Types for Determination of Esomeprazole Levels in Human Plasma by High-Performance Liquid Chromatography

Esomeprazole is a Proton Pump Inhibitor (PPI) drug formulated in delayed-release tablets, that are included in the mandatory bioequivalence test. In vitro method validation used human plasma from the Indonesian Red Cross that used citrate as an anticoagulant. In the implementation of in vivo study, usually using human plasma used EDTA or heparin as an anticoagulant. This study aims to evaluate the effect of using anticoagulant types that may affect the analysis of esomeprazole in human plasma. Optimum chromatographic conditions used column C18 SunfireTM (5 μm, 250 mm x 4.6 mm); column temperature 40°C; mobile phase acetonitrile - phosphate buffer (40:60% v/v) pH 7.6; 1.0 mL/min flow rate with lansoprazole as an internal standard and wavelength 300 nm (PDA). The extraction was carried out by liquid-liquid extraction method using 500 μl plasma and 5 ml dichloromethane as extraction solvent. The result showed that the concentration range of calibration curve linearity in 5 – 1500 ng/mL. Recovery and broad peak response data of esomeprazole in plasma have significant differences between heparin-EDTA and citrate-EDTA anticoagulants (p&lt;0.05), but there is no significant difference for the stability test. In conclusion, heparin is better than EDTA as an anticoagulant for esomeprazole bioanalysis