Sensitive LC Method Research Articles

Opti-nQL: An Optimized, Versatile and Sensitive Nano-LC Method for MS-Based Lipidomics Analysis.

Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.

Enantioselective separation of eight antihistamines with α1-acid glycoprotein-based chiral stationary phase by HPLC: Development and validation for the enantiomeric quality control

In the present paper, a sensitive and selective chiral LC method has been developed for enantiomeric separation and determination of eight antihistamines in pharmaceutical formulations. Enantiomeric separation was achieved on the α1-acid glycoprotein-based chiral stationary phase known as Chiralpak AGP. The effects of the mobile phase composition, type and proportion of organic modifier and the concentration and pH of the buffer on retention and resolution were fully investigated. All enantiomers of target analytes were successfully separated with the mobile phase consisting of methanol and 10 mM ammonium acetate buffer, at a flow rate of 0.9 mL min−1. Calibration curve of each enantiomer was linear over the concentration range of 5.0–100 μg mL−1, with the correlation coefficient above 0.9990. The limits of detection and quantification were below 0.80 and 3.18 μg mL−1, respectively. The recoveries were in the range of 98.0–102.4% with the relative standard deviation lower than 2.88% for all enantiomers. The validated method was demonstrated to be suitable for the quality control of the enantiomeric composition of antihistamines present in pharmaceuticals. Finally, the proposed chiral LC method was applied to the determination of antihistamines enantiomers in commercially available tablets containing racemic mixtures of compounds. Quantitative results of the commercial tablets demonstrated that the difference between the declared and determined values did not exceed 1.45%, and chiral antihistamines drugs were present in the form of racemates.

Determination of Vitamin D3 Content in High, Low and Zero Fat Food Using High Performance Liquid Chromatography

AbstractBackground: Vitamin D, has a significant role in bone metabolism and helps calcium absorption in the body. There are only few vitamin D assay methods available for zero fat food.Aims of Study: (1) To develop an accurate and sensitive LC method for the quantification of vitamin D3 in food by optimization of each step of the analytical method: Extraction, sample preparation, separation and detection. (2) To validate the developed method. (3) To apply the method to quantify the total vitamin D3 in food from several species.Material and Methods: In this study, a rapid, simple, and economical reversed phase liquid chromatographic method was described for the determination of vitamin D3, in some high, low and zero fat samples (milk products, cereal and chewing gum samples). The isolation of fat soluble vitamins includes a saponification step and an extraction step with petroleum ether and diethyl ether. The vitamin D3 content of samples was determined by reversed phase liquid chromatog-raphy. Ultra violet-Visible (UV-VIS) detector and C18 column were used for this purpose.Results: The linearity of standard curves of vitamin D3 were 10-200 mg/ml expressed of the correlation coefficient r2 = 0.9992). The detection (LOD) and quantification (LOQ) limits were 5.09 and 15.42mg/ml, respectively. The accuracy was 101.37±4.37.Conclusion: The described reversed-phase HPLC method is favorable compared with other published HPLC-UV methods (20 and 21) because of its stability-indicating nature, short run time and wide analytical range with outstanding linearity, accuracy and precision. The proposed method allows the determination of vitamin D3 in a single chromatographic run and is suitable for the analysis of the stability of vitamin D3. The obtained results from the assay of vitamin D3 in commer-cial nutrition supplements confirmed that the method is appropriate for the routine analysis of various food samples.

Development and Validation of a Fast Reversed Phase Liquid Chromatographic Method for the Analysis of Ethionamide in Dosage Forms

Background: Ethionamide (ETA) is widely used as one of the agents for the treatment of multidrug resistant tuberculosis. Although quality control is an important issue, a fast LC-UV method for the assay and impurity determination of ETA was lacking. So, the aim of this study was to develop such a method to evaluate drug products and follow up dissolution tests of ETA tablets. Methods: Chromatographic separation was achieved using a Hypersil BDS C18 (150 mm×4.6 mm, 3 µm) column kept at 30 °C. The mobile phase consisted of 300 mL of acetonitrile, 0.14 g of NH4H2PO4 and 700 mL of water pumped at a flow rate of 1.0 mL/min. UV detection was performed at 287 nm. The developed LC method was validated according to the ICH guidelines. Results: Validation results show that the developed LC method is specific, linear, sensitive, precise, accurate and robust. Forced degradation studies revealed that the generated degradation products did not interfere with ETA and known impurities, thus proving the specificity of the developed LC method for the assay of ETA tablets and quantification of impurities. Conclusion: A robust, selective, sensitive and fast LC method has been developed and validated for analysis of ETA and its main impurities in tablets. Keywords: Ethionamide, impurities, assay, method development, validation, liquid chromatography.

A validated chiral LC method for the enantiomeric separation of AT13148 on polysaccharide-based chiral stationary phase

A sensitive and accurate LC method for the determination of AT13148 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.8 was accomplished within 15 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane: 2-propanol: diethylamine (85:15:0.1, v/v) as mobile phase at a flow rate of 1 mL min−1. Eluted analytes were monitored by UV absorption at 254 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 7 and 500 μg mL−1 (n = 11), and the recoveries between 98.24 and 100.99% were obtained, with relative standard deviation lower than 1.32%. LOD and LOQ for AT13148 were 2.46 and 7.38 μg mL−1 and for its enantiomer were 2.54 and 7.49 μg mL−1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AT13148, especially for the analysis of bulk samples.

Validation of a rapid and sensitive reversed-phase liquid chromatographic method for the quantification of prenylated chalcones and flavanones in plasma and urine

Prenylated chalcones and flavanones from hops (Humulus lupulus), such as 6-prenylnaringenin (6PN), 8-prenylnaringenin (8PN), xanthohumol (XN) and isoxanthohumol (IXN), are increasingly investigated for their potential health benefits, but validated and easy-to-implement methods for their sensitive quantification in biological samples are scarce. We therefore developed and validated a reversed-phase HPLC method with UV and fluorescence detection for the sensitive analysis of 6PN, 8PN, XN and IXN in human plasma and urine. Separation of all analytes and naringenin (a potential internal standard) was achieved within 20min using gradient elution and a pentafluorophenyl column. The method was validated according to the guidelines for bioanalytical method validation of the Center for Drug Evaluation and Research of the U.S. Food and Drug Administration. Recovery, precision and accuracy deviated <20% from the known values, with the exception of XN, which isomerized to IXN during sample preparation. Using the sum of XN+IXN as a measure of XN in the samples improved recovery, precision and accuracy to <25% deviation from the known values. Sensitivity (lower limits of detection and quantification) was comparable to published LC methods with UV-detection or better. The lower limit of quantification for IXN was 1.8ng/mL, which is near the reported sensitivity of tandem MS detection of 1.0ng/mL. In conclusion, we report a validated and sensitive LC method for the quantification of prenylated chalcones and flavanones in human plasma and urine with potential for adaptation to other sample matrices.

Determination of Enantiomeric Purity of GSK962040 Based on Liquid Chromatography Using Chiral Stationary Phase Combined with a Pre-column Derivatization Procedure

A sensitive and accurate LC method was developed and further validated for the determination of enantiomeric purity of GSK962040. Before separation, a pre-column derivatization procedure was performed. Baseline separation with a resolution higher than 1.9 was accomplished within 15 min using a Chiralpak AD-H (250 × 4.6 mm; particle size 5 μm) column, with n-hexane: 2-propanol (85:15 v/v) as mobile phase at a flow rate of 1 mL min−1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within the concentration range between 4 and 200 μg mL−1 (n = 8), and recoveries between 98.15 and 101.48% were obtained, with relative standard deviation (RSD) lower than 1.42%. The LOD and LOQ for the Boc-GSK962040 were 1.23 and 4.15 μg mL−1 and for its enantiomer were 1.38 and 4.76 μg mL−1, respectively. The developed method was also evaluated and validated by analyzing bulk samples with different enantiomeric ratios of GSK962040. It was demonstrated that the method was accurate, robust and sensitive, and also had practical utilities for real analysis.

HPLC Determination of Enantiomeric Purity of Letermovir Based on CHIRALPAK AD.

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of {(4S)-8-Fluoro-2-[4-(3-methoxyphenyl)-1-piperazinyl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydro-4-quinazolinyl} acetic acid (Letermovir). Baseline separation with a resolution >2.8 was accomplished within 10 min using a CHIRALPAK AD (250 mm × 4.6 mm; particle size 5 μm) column, with n-hexane/2-propanol (80:20 v/v) as mobile phase at a flow rate of 1 mL min-1. The eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity as well as resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within the concentration range between 0.003 and 1 mg mL-1 (n = 14), and the recoveries between 98.24 and 101.43% were obtained, with relative standard deviation <1.29%. The limit of detection (LOD) and limit of quantitation (LOQ) for Letermovir were 0.96 and 3.15 μg mL-1; those for its enantiomer were 1.01 and 3.39 μg mL-1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of Letermovir, especially for the analysis of bulk samples.

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane:2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min−1. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL−1 (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL−1; those for its enantiomer were 0.70 and 2.34 μg mL−1, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min−1. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL−1 (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL−1; those for its enantiomer were 1.49 and 4.94 µg mL−1, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

HPLC Determination of Enantiomeric Purity of PF-04136309 Based on a Chiral Stationary Phase

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of (S)-N-[2-(3-{trans-4-hydroxy-4-[5-(pyrimidin-2-yl)pyridin-2-yl] cyclohexylamino} pyrrolidin-1-yl)-2-oxoethyl]-3-(trifluoromethyl) benzamide (PF-04136309). Baseline separation with a resolution higher than 1.8 was accomplished within 40 min using a CHIRALPAK AD (250 × 4.6 mm; particle size 5 μm) column, with n-hexane:2-propanol (70:30 v/v) as mobile phase at a flow rate of 1 mL min−1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as the resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within a concentration range between 0.01 and 1 mg mL−1 (n = 9), and recoveries between 98.17 and 101.28 % were obtained, with relative standard deviation (RSD) lower than 1.44 %. The LOD and LOQ for PF-04136309 were 3.59 and 11.54 μg mL−1 and for its enantiomer were 3.39 and 11.28 μg mL−1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of PF-04136309, especially for the analysis of bulk samples.

Method Development and Validation of Amlodipine, Gliquidone and Pioglitazone: Application in the Analysis of Human Serum

A sensitive LC method has been developed for the simultaneous determination of co-administered drugs amlodipine, gliquidone and pioglitazone in bulk drug, pharmaceutical formulations, and human serums. The assay involved an isocratic elution of analytes on a Purospher® STAR RP-18 end capped (5 μm, 25 x 0.46 cm) column at ambient temperature with a mobile phase consisting of methanol: water: acetonitrile (80:10:10 v/v/v) at a flow rate of 0.7 mLmin-1 at 230 nm. The pH was adjusted to 3.50 with phosphoric acid and glibenclamide was used as an internal standard. The linearity of the method was found to be 5-50 μgmL–1 for all the drugs with correlation coefficients were 0.9991, 0.9992 and 0.9963; the limit of detection was found to be 0.34, 0.06 and 0.33 μgmL–1 for amlodipine, gliquidone and pioglitazone, respectively. All the validated parameters were in the acceptable range. The recovery rates of drugs were 98.2-101.53 %, 100.40-101.60 % and 98.25-101.20 % in human serum for amlodipine, gliquidone and pioglitazone, respectively. This method is applicable for the analysis of drugs in bulk drug, formulations, serum and in clinical samples without interference from excipients or endogenous serum.

A Rapid and Sensitive LC Method for Determination of Diastereomeric Purity of Tenofovir Alafenamide

A rapid and sensitive LC method was developed and validated for the determination of diastereomeric purity of tenofovir alafenamide (GS-7340). Baseline separation with resolution >2.8 was achieved within 17 min on a CHIRALPAK AD-3 (250 × 4.6 mm; particle size 3 μm) column using n-hexane:2-propanol (60:40 v/v) as the mobile phase at a flow rate of 1 mL min−1. The analytes were detected by UV absorbance at 260 nm. The effects of ethanol, 2-propanol, and temperature on diastereomeric selectivity and resolution of diastereomerism were evaluated. The method was extensively validated and proved to be robust. The recoveries were between 98.17 and 102.84 % with <1.93 % relative standard deviation. The limit of detection and limit of quantitation for GS-7339 were 0.77 and 2.56 μg mL−1 and for GS-7340 were 0.61 and 2.04 μg mL−1, respectively. This method was extensively proved to be accurate, stable, rapid, and sensitive for the determination of diastereomeric purity of tenofovir alafenamide (GS-7340) in bulk samples.

Simultaneous Quantification of Glibenclamide, Simvastatin, and Quercetin by Using LC-UV Method and Its Application to Pharmacokinetic Study in Rats

A sensitive, precise, and simple LC method for the simultaneous quantification of glibenclamide, simvastatin, and quercetin in rat plasma has been developed and validated. The chromatographic separation was achieved on a cyano column (250 mm × 4.6 mm, 5 µm) maintained at room temperature, using isocratic elution with methanol : acetonitrile : 10 mM potassium dihydrogen orthophosphate, pH adjusted to 4.5 with o-phosphoric acid (8 : 32 : 60, v/v) and detected using UV-VIS detector. Plasma samples were deproteinated with 0.1% perchloric acid and acetonitrile for extraction of the glibenclamide, simvastatin, and quercetin which resulted in their high recoveries. LC calibration curves based on the extracts from the rat plasma were linear in the range of 50–1000 ng mL−1 for all the three drugs. The limit of quantification was 50 ng mL−1. The described method was successfully applied to study the pharmacokinetics of glibenclamide, simvastatin, and quercetin following oral administration, in combination to Sprague-Dawley rats.

Determination of Moxifloxacin and its Oxidation Products with Kinetic Evaluation Under Potassium Permanganate Treatment in Acidic Solution by Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry

A simple, sensitive, and reproducible ultra-performance LC method for the determination of moxifloxacin (MOXI) oxidation stability under permanganate treatment in acidic conditions (pH 3.0-6.0) was developed. Besides the MOXI peak [retention time (RT) = 2.58], four additional products (RT = 0.86, 0.91, 1.42, and 1.89) were observed in all conditions tested. The oxidation process followed second-order reaction kinetics and depended upon solution acidity. The highest reaction rate constant was observed at pH 3.0, and this value decreased as the pH was increased to 6.0. The oxidation products were characterized, and their fragmentation pathways, derived from MS/MS data, were proposed. Two of these products were identified as hydroxyl derivatives of MOXI and two others as their oxidation product analogs with molecular ions of 418.4 and 416.4 m/z, respectively.

A Novel Validated Ultra-Performance Liquid Chromatography Method for Separation of Eszopiclone Impurities and its Degradants in Drug Products

A selective, specific, and sensitive ultra-performance LC (UPLC) method was developed for determination of eszopiclone and its degradation products. The chromatographic separation was performed with a Waters ACQUITY UPLC system and BEH C18 column using gradient elution with mobile phases A and B. Mobile phase A was 0.01 M phosphate buffer with 0.2% (w/v) 1-octane sulfonic acid sodium salt as an ion pair reagent, adjusted pH 2.2 with orthophosphoric acid-acetonitrile (85 + 15, v/v). Mobile phase B was pH 2.2 buffer-acetonitrile (20 + 80, v/v). UV detection was performed at 303 nm. Eszopiclone and its impurities were chromatographed with a total run time of 13 min. A calibration study showed that the response for each of the impurities A, B, C, and D was linear between concentrations of 0.02 and 7.2 microg/mL (r2 > or = 0.999). The method was validated over this range for precision, intermediate precision, accuracy, linearity, and specificity. For the precision study, RSD of each impurity was <5% (n = 6). The method was found to be precise, accurate, linear, and specific. The proposed method was successfully used for determination of eszopiclone impurities in pharmaceutical preparations.

Liquid chromatographic method for the simultaneous determination of captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulation, and human serum by programming the detector

A highly sensitive LC method with UV detection has been developed for the simultaneous determination of coadministered drugs captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulations, and human serum at the isosbestic point (235 nm) and at individual λmax (220, 255, and 238 nm, respectively) by programming the detector with time to match the individual analyte's chromophore, which enhanced the sensitivity with linear range. The assay involved an isocratic elution of analytes on a Bondapak C18 (10 μm, 25 × 0.46 cm) column at ambient temperature using a mobile phase of methanol/water 80:20 at pH 2.9 and a flow rate of 1.0 mL/min. Linearity was found to be 0.25-25, 0.10-6.0, and 0.20-13.0 μg/mL with correlation coefficient >0.998 and detection limits of 7.39, 3.90, and 9.38 ng/mL, respectively, whereas calibration curves for wavelength-programmed analysis were 0.10-6.0, 0.04-2.56, and 0.10-10.0 μg/mL with correlation coefficient >0.998 and detection limits of 5.79, 2.68, and 3.87 ng/mL, respectively. All the validated parameters were in the acceptable range. The recovery of drugs was 99.32-100.39 and 98.65-101.96% in pharmaceutical formulation and human serum, respectively, at the isosbestic point and at individual λmax . This method is applicable for the analysis of drugs in bulk drug, tablets, serum, and in clinical samples without interference of excipients or endogenous serum components.

Simultaneous Quantification of Aliskiren, Valsartan and Sitagliptin by LC with Fluorescence Detection: Evidence of Pharmacokinetic Interaction in Rats

A sensitive, precise and simple LC method for the simultaneous quantification of aliskiren, valsartan and sitagliptin in rat plasma has been developed and validated. The chromatographic separation was achieved on a C18 column (250 mm × 4.6 mm, 5 μm) maintained at room temperature, using isocratic elution with acetonitrile/20 mM ammonium acetate buffer (35:65, v/v), pH adjusted to 4.85 with glacial acetic acid, and detected using a fluorescence detector. Liquid–liquid extraction of the aliskiren, valsartan and sitagliptin from the rat plasma with t-butyl methyl ether resulted in their high recoveries. LC calibration curves based on the extracts from the rat plasma were linear in the range of 25–2,000 ng mL−1 for aliskiren and sitagliptin and 50–4,000 ng mL−1 for valsartan. The limits of quantification were 25 ng mL−1 for aliskiren and sitagliptin and 50 ng mL−1 for valsartan. The precision and accuracy of the method were well within the generally accepted criteria for biomedical analysis. The described method was successfully applied to study the pharmacokinetics of aliskiren, valsartan and sitagliptin following oral administration, individually as well as in combination in Sprague–Dawley rats. The results of the study implied the occurrence of pharmacokinetic interaction upon the co-administration of these three drugs.

Simultaneous LC determination of rosuvastatin, lisinopril, captopril, and enalapril in API, pharmaceutical dosage formulations, and human serum

A sensitive, economical, and rapid LC method for the quantification of CVD’s rosuvastatin, lisinopril, captopril, and enalapril in bulk drug, pharmaceutical dosage formulations and in human serum has been developed and validated. Chromatographic separation was performed by methanol:water (75:25 v/v) as mobile phase whose pH was adjusted to 3.0 by o-phosphoric acid; flowing at a rate of 1.0 mL min−1 through pre-packed Purospher Star C18 (5 μm, 25 × 0.46 cm) column at ambient temperature. UV detection was performed at isosbestic point 214 nm. The method shows good linearity in the range of 0.5–25 μg mL−1 for rosuvastatin (R 2 = 0.998) and 1–50 μg mL−1 for lisinopril, captopril and enalapril (R 2 = 0.999). All LOD values were ≤130 and LOQ ≤ 370 ng mL−1 for API and human serum. The % recoveries of all drugs were between 98.07 and 102.51% in dosage formulations and human serum. The ICH guidelines were followed for the validation of method. The method can be employed for pharmacokinetic studies, routine QC analysis of dosage formulations, and laboratory analysis in blood samples of high cholesterol and hypertension patients.

Sensitive LC method for the simultaneous determination of diacerein and aceclofenac in tablet dosage form

Background: Diacerein and aceclofenac drug product combination is used for the pain management in osteoarthritis. The main aspects of the present study is to develop stability indicating, sensitive reverse phase high-performance liquid chromatographic method for the simultaneous determination of diacerein and ceclofenac in tablet dosage form. Were, the chromatographic separation was achieved on Kromasil C-18, 150×4.6 mm, 3.5-μm analytical column using mobile phase double distilled water (pH 2.7 with glacial acetic acid)-acetonitrile (45:55v/v). Detector was set at 256 nm. Results: The described method shows excellent linearity over a range of 75–2.5 μg/mL for diacerein and 150–5 μg/mL for aceclofenac. The correlation coefficient for diacerein is 0.9999 and for aceclofenac is 0.9997. Conclusions: The proposed method was found to be suitable for simultaneous determination and stability study of diacerein and aceclofenac in tablet dosage form.