LC-ESI-MS-MS Determination of Rat Plasma Protein Binding of Major Flavonoids of Flos Lonicerae Japonicae by Centrifugal Ultrafiltration

A simple, precise, accurate, selective, and sensitive reversed-phase LC–UV method has been developed for simultaneous analysis of diltiazem and non-steroidal anti-inflammatory drugs (NSAIDs) in the bulk drug, tablet dosage forms, and human serum. Chromatographic separation of the drugs was performed at ambient temperature on a C₁₈ stationary phase with 80:20 (v/v) methanol–water, pH 3.1 ± 0.02, as isocratic mobile phase. The mobile phase flow rate was initially 0.5 mL min^?1 then increased to 1 mL min^?1. All the NSAIDs were well separated from each other and from diltiazem. Total run time was 10 min. The assay was successfully applied to pharmaceutical formulations and serum and there was no chromatographic interference from tablet excipients. The method was linear in the range 1.25–50 μg mL^?1 both for diltiazem and the NSAIDs. The suitability of this HPLC method for quantitative analysis of the drugs was proved by validation in accordance with International Conference on Harmonization (ICH) guidelines. The validation results, and results from statistical analysis of the data, demonstrated the method was reliable.     

Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form

A simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method is described for the simultaneous determination of levofloxacin hemihydrate and ornidazole in tablet dosage form. The method is based on the HPTLC separation of the two drugs followed by densitometric measurements of their spots at 298 nm. The separation is carried out on Merck TLC aluminium sheets of silica gel 60 F254 using n-butanol-methanol-ammonia (5:1:1.5, v/v/v) as mobile phase. The linearity is found to be in the range of 50-250 and 100-500 ng/spot for levofloxacin hemihydrate and ornidazole, respectively. The method is successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients are found. The suitability of this HPTLC method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines.     

Simultaneous densitometric analysis of amlodipine,hydrochlorothiazide, lisinopril,and valsartan by HPTLC in pharmaceutical formulations and human plasma

A simple, accurate, and precise high-performance thin layer chromatographic (HPTLC) method has been developed and validated for the simultaneous quantification of antihypertensive drugs, amlodipine (AML), hydrochlorothiazide (HCTZ), lisinopril (LIS), and valsartan (VAL) in their pharmaceutical formulations and human plasma. Separation of the drugs was performed on aluminum-backed layer of silica gel 60?F₂₅₄ using a mixture of methanol–dichloromethane–glacial acetic acid (9.0:1.0:0.1, v/v/v) as the mobile phase. Densitometric determination of the separated spots was done at 215?nm. The retention factors (R_f) obtained under the optimized conditions were 0.56, 0.75, 0.29, and 0.67 for AML, HCTZ, LIS, and VAL, respectively. Linearity of the method was established in the range of 200–1,500?ng/band for AML, 300–1,500?ng/band for HCTZ, 400–2,000?ng/band for LIS, and 1,000–7,000?ng/band for VAL. The limit of detection/limit of quantitation of the method found were 54.21/164.28, 77.27/234.15, 83.45/252.87, and 156.48/474.19?ng/band for AML, HCTZ, LIS, and VAL, respectively. To determine the drugs in spiked plasma samples, solid phase extraction was performed, which provided highly consistent and quantitative recovery for all four drugs. The method was satisfactorily applied for the analysis of different tablet formulations and proved to be specific and accurate for the quality control of these drugs.     

Development and validation of stability-indicating HPLC method for the simultaneous determination of paracetamol, tizanidine, and diclofenac in pharmaceuticals and human serum

A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.     

Comparison of second derivative-spectrophotometric and reversed-phase HPLC methods for the determination of prednisolone in pharmaceutical formulations.

Second derivative-spectrophotometric and high-performance liquid chromatographic methods for the determination of prednisolone in pharmaceutical formulations have been developed. Determination of prednisolone in tablets was conducted by using a second-order derivative UV spectrophotometric method at 250 nm (n = 5). Standards for the calibration graph ranging from 5.0 to 35.0 microg/ml were prepared from stock solution. The proposed method was accurate, with 98% recovery value, and precise, with a coefficient of variation (CV) of 1.38. These results were compared with those obtained by an exclusively developed isocratic reversed-phase high-performance liquid chromatography (HPLC) method. An isocratic reversed-phase Bondapak C(18) column with acetonitrile-citrophosphate buffer (pH 5; 45:55 v/v) mobile phase was used and UV detector was set to 241 nm using 11 alpha-hydroxyprogesterone as an internal standard. Calibration solutions used in HPLC were in the range from 2 to 300 microg/ml. Results obtained by derivative UV spectrophotometric method were comparable to those obtained by HPLC method, as far as analysis of variance (ANOVA) test, F(calculated), 0.762 and F(theoretical), 3.89, results were concerned.     

Simultaneous Determination of Ceftriaxone Sodium and Non Steroidal Anti‐Inflammatory Drugs in Pharmaceutical Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of ceftriaxone in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher Star, C18 (5 μm, 250 × 4.6 mm) column at room temperature using methanol:water:acetonitrile (80:15:5 v/v/v) as a mobile phase, pH adjusted at 2.8 with ortho‐phosphoric acid and at a flow rate of 1.0 mL/minute, while UV detection was performed at 270 nm. The results obtained showed a good agreement with the declared content. The method shows good linearity in the range of 2.5‐25 μg/mL ceftriaxone serum concentrations with a correlation coefficient 0.999 (inter‐ and intra‐day RSD < 2.0%). The limit of detection and quantification for ceftriaxone and NSAID's in pharmaceutical formulation and serum were in the range 0.51‐1.54 μg/mL. Analytical recovery was >98.1%. The proposed method may be used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs i.e. tiaprofenic acid, naproxen sodium, flurbiprofen, diclofenac acid and mefenamic acid alone or in combination with ceftriaxone from raw materials, dosage formulations and in serum. The established HPLC method is rapid, accurate and selective, because of its sensitivity and reproducibility.     

Validated LC Method, with a Chiral Mobile Phase, for Separation of the Isomers of Fexofenadine Hydrochloride

A rapid, simple and sensitive high-performance liquid chromatographic method (HPLC) has been developed to assay atomoxetine HCl in capsules. The HPLC analysis used a reversed phase C18 (150 × 4.6 mm i.d. 5 μm particle size) analytical column and a mobile phase consisting of monobasic potassium dihydrogen orthophosphate and acetonitrile (95:5 v/v), with UV detection at 269 nm. The validation data showed that the assay is sensitive, specific and reproducible for determination of atomoxetine HCl in this dosage form. Calibration curves were linear from 1 to 10 μg mL⁻¹ (R ² > 0.997). The accuracy of the method ranged from 98.13 to 101.5%. Mean inter- and intra-assay relative standard deviations (RSD) were less than 1.0%. The proposed method provided an accurate and precise analysis of atomoxetine HCl in its pharmaceutical dosage form.     

Stability indicating HPLC method for the simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations

ABSTRACT: BACKGROUND: A simple, specific, and fast stability indicating reverse phase liquid chromatographic method was established for instantaneous determination of moxifloxacin and prednisolone in bulk drugs and pharmaceutical formulations. RESULTS: Optimum chromatographic separations among the moxifloxacin, prednisolone and stressinduced degradation products were achieved within 10 minutes by use of BDS Hypersil C8 column (250 X 4.6 mm, 5 mum) as stationary phase with mobile phase consisted of a mixture of phosphate buffer (18 mM) containing 0.1% (v/v) triethylamine, at pH 2.8 (adjusted with dilute phosphoric acid) and methanol (38:62 v/v) at a flow rate of 1.5 mL min-1. Detection was performed at 254 nm using diode array detector. The method was validated in accordance with ICH guidelines. Response was a linear function of concentrations over the range of 20-80 mug mL-1 for moxifloxacin (r2 [greater than or equal to] 0.998) and 40-160 mug mL-1 for prednisolone (r2 [greater than or equal to] 0.998). The method was resulted in good separation of both the analytes and degradation products with acceptable tailing and resolution. The peak purity index for both the analytes after all types of stress conditions was [greater than or equal to] 0.9999 indicated a complete separation of both the analyte peaks from degradation products. The method can therefore, be regarded as stabilityindicating. CONCLUSIONS: The developed method can be applied successfully for simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations and their stability studies.     

Simple determination of betamethasone and chloramphenicol in a pharmaceutical preparation using a short monolithic column coupled to a sequential injection system

This contribution describes the use of a new separation method based on a reversed-phase sequential injection chromatography (SIC) technique for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops. A short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25 x 4.6 mm column with a 5 mm precolumn (Merck, Germany) and a FIA1ab 3000 system (USA) with a 6-port selection valve and 5 mL syringe were used for sequential injection chromatographic separations in this study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 mL/min; UV detection was at two wavelengths, i.e., 241 and 278 nm (absorption maxima of betamethasone and chloramphenicol, respectively). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) >0.999; repeatability of determination (RSD) in the range 0.8-1.7% at two different concentration levels, and detection limits in the range 0.5-1.0 mg/mL. The chromatographic resolution between compound peaks was greater than 2.1 and the analysis time was less than 8 min under optimal conditions. The developed sequential injection chromatography method was compared with the HPLC method and was found to be applicable for routine analysis of active compounds in pharmaceutical preparations.     

Validated HPLC method for simultaneous estimation of khellol glucoside,khellin and visnagin in Ammi visnaga L. fruits and pharmaceutical preparations

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. Our study focuses on developing simple and rapid method utilising HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously. Their concentrations were determined in A. visnaga L. fruits at different developmental stages and in pharmaceutical formulations together with following up them during shelf life. Separation was accomplished using HPLC. Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of water:methanol:tetrahydrofuran (50:45:5, v/v/v). Peaks were detected at 245 nm. The suggested method for the determination of KG, KH and VS was successful in determining the analytes of interest without any interference of other compounds and matrix. All validation parameters were satisfactory and the procedure was relatively easy and fast as extracts are evaluated without previous steps of purification.     

Use of micellar mobile phases for the chromatographic determination of clorazepate, diazepam, and diltiazem in pharmaceuticals

An ODS-2 column, a micellar mobile phase of high elution strength containing 0.1M sodium dodecyl sulfate and 3% (v/v) butanol, and ultraviolet detection at 230 nm are used for the determination of either of two benzodiazepines (clorazepate and diazepam) and a benzothiazepine (diltiazem) in pharmaceuticals. The procedure is shown to be competitive against conventional chromatography with methanol-water mobile phases, especially for diltiazem. The composition of the micellar mobile phase is selected using a predictive strategy based on an accurate retention model and assisted by computer simulation. Calibration graphs are linear at least in the 2.5 to 20 microg/mL, 4 to 20 microg/mL, and 5 to 40 microg/mL ranges for clorazepate, diazepam, and diltiazem, respectively. The intra- and interday repeatabilities (%) are clorazepate (1.7, 5.2), diazepam (0.43, 3.7), and diltiazem (0.36, 3.1). Limits of detection are well below the concentrations of the drugs found in the commercial pharmaceutical preparations analyzed. The drug contents evaluated with the proposed procedure are compared with the declared contents given by the manufacturers. The achieved percentages of label claim are usually between 95 and 104%.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

High-performance liquid chromatographic determination of amphotericin B in a liposomal pharmaceutical product and validation of the assay

A validated high-performance liquid chromatographic method is presented to quantitate amphotericin B (AB) in a liposomal pharmaceutical formulation. The analysis is based on the chromatographic separation of AB and 1-amino-4-nitronaphthalene (the internal standard) on a C18 muBondapac reversed-phase column with a mobile phase consisting of a mixture of acetonitrile and 0.02 M ethylenediamine tetra-acetic acid disodium salt at pH 5.0 (45:55, v/v). The chromatographic analysis time is less than 10 min, and the validation of the assay shows that it is selective, accurate, and linear for the concentration range of 2.50 to 7.50 microg/mL with a detection limit of 0.00500 microg/mL. The within-day and between-day relative standard deviation values are 1.26% (n = 18) and 1.25% (n = 8), respectively. The method described conforms to the validation of compendial methods used for finished pharmaceutical products in general and offers a reliable, quick, and cost-effective procedure for examining the consistency or quality-control analysis of AB in liposomal products. It can also be applied for the determination of AB in other nonliposomal lipid-based drug delivery systems that are on the market.     

Simultaneous determination of clopidogrel and aspirin by RP-HPLC from bulk material and dosage formulations using multivariate calibration technique

A rapid, simple, and easy method for the simultaneous determination of clopidogrel and aspirin from bulk material and dosage formulations in the presence of meloxicam as internal standard has been developed. Separation was carried out on a Purospher star C(18) (5 μm, 250 × 4.6 mm) column at ambient temperature. The mobile phase consisted of methanol-water (80:20, v/v), the pH of the mobile phase was adjusted to 3.4 with ortho-phosphoric acid and pumped at a flow rate of 1 mL/min using isocratic pump system. Multivariate chromatographic calibration technique was subjected to high-performance liquid chromatography (HPLC) data for simultaneous quantitative analysis of binary mixtures of clopidogrel and aspirin. HPLC data based on the analyte peak areas were obtained at five wavelengths (225, 230, 235, 240, and 245 nm). The mathematical algorithm of multivariate chromatographic calibration technique is based on the use of the linear regression equations. Calibration plots for clopidogrel and aspirin were constructed at each wavelength by using the peak areas corresponding to the concentrations of each active compound. This multivariate chromatographic method was also applied to a commercial pharmaceutical dosage form containing clopidogrel and aspirin.     

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans

A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C(18) column and detected by ultraviolet absorbance at 239 nm. The use of methanol-water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a fl ow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0-300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4-104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 +/- 14.3 ng/mL at 3.1 +/- 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 +/- 83.1 ng.h/mL.     

Simultaneous Quantitation of Captopril and NSAID's in API,Dosage Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of captopril in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher star C18 (5 μm, 25 × 0.46 cm) column at room temperature using methanol:water (80:20 v/v) as a mobile phase, pH adjusted at 2.8 with o‐phosphoric acid and at a flow rate of 1.0 mL min⁻¹, while UV detection was performed at 227 nm. The limit of detection and quantification for captopril were 1 and 0.35 ng mL⁻¹, while that for (NSAID's) i.e. flurbiprofen, ibuprofen, diclofenac sodium and mefenamic acid LOD were 0.2, 1, 2 and 0.4 ng mL⁻¹ respectively and LOQ were 0.9, 2.9, 8 and 1 ng mL⁻¹ Analytical recovery was > 98.1%. The method used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs (NSAID's) i.e. ibuprofen, flurbiprofen, diclofenac sodium and mefenamic acid alone or in combination with captopril from API (active pharmaceutical ingredients), dosage formulations and in human serum. The established method is rapid (RT < 12 min), accurate (recovery > 98.1%), selective (no interference of excepients and other commonly used drugs and food) and sensitive (LOQ 3.5 ng mL^;‐1) and reproducible (SD ± 0.003).     

A Validated Stability Indicating RP-LC Method for Nitazoxanide, a New Antiparasitic Compound

The present paper describes stability indicating reverse phase high-performance liquid chromatography (RP-HPLC) assay method for nitazoxanide in bulk drugs. The developed method is also applicable for the related substances determination in bulk drug. The drug substance was subjected to stress conditions of hydrolysis, photolysis and thermal degradation. The considerable degradation of nitazoxanide was observed under base and peroxide hydrolysis. The drug was found to be stable in other stress conditions attempted. The chromatographic separation of the drug was achieved on reversed-phase C-18 column. Eluents were monitored on photo-diode array detector at a wavelength of 240 nm. The mobile phase was aqueous 0.005 M tetra butyl ammonium hydrogen sulphate and acetonitrile (45:55, v/v). In the developed HPLC method, resolution between nitazoxanide and its potential impurities, namely Imp-A (5-nitro-1,3-thiazol-2-amine), Imp-B (N-(5-nitro-1,3-thiazol-2-yl) acetamide) and Imp-C (2-{[(5-nitro-1,3-thiazol-2-yl) amino] carbonyl} phenyl 2-(acetyloxy) benzoate) was found greater than three. The developed RP-HPLC method was validated with respect to response function, accuracy, precision, specificity, stability of analytical solutions and robustness. Also to determine related substances and assay determination of nitazoxanide that can be used to evaluate the quality of regular production samples. The developed method can also be conveniently used for the assay determination of nitazoxanide in pharmaceutical formulations.     

Isolation and characterization of degradation products of citalopram and process-related impurities using RP-HPLC

A reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of citalopram hydrobromide and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on an Inertsil ODS 3V (250x4.6 mm; particle size 5 mum) column using 0.3% diethylamine (pH = 4.70) and methanol/acetonitrile (55:45 v/v) as mobile phase in a gradient elution mode. The eluents were monitored by a photodiode array detector set at 225 nm. The chromatographic behavior of all the related substances was examined under variable conditions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. The method could be of use not only for rapid and routine evaluation of the quality of citalopram in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Three unknown impurities were consistently observed during the analysis of different batches of citalopram. Forced degradation of citalopram was carried out under thermal, photo, acidic, alkaline, and peroxide conditions. The degradation products and unknown impurities were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectroscopy.     

Development and validation of a novel RP-HPLC method for the analysis of reduced glutathione

The objective of this study was the development, optimization, and validation of a novel reverse-phase high-pressure liquid chromatography (RP-HPLC) method for the quantification of reduced glutathione in pharmaceutical formulations utilizing simple UV detection. The separation utilized a C18 column at room temperature and UV absorption was measured at 215 nm. The mobile phase was an isocratic flow of a 50/50 (v/v) mixture of water (pH 7.0) and acetonitrile flowing at 1.0 mL/min. Validation of the method assessed the methods ability in seven categories: linearity, range, limit of detection, limit of quantification, accuracy, precision, and selectivity. Analysis of the system suitability showed acceptable levels of suitability in all categories. Likewise, the method displayed an acceptable degree of linearity (r(2) = 0.9994) over a concentration range of 2.5-60 μg/mL. The detection limit and quantification limit were 0.6 and 1.8 μg/mL respectively. The percent recovery of the method was 98.80-100.79%. Following validation the method was employed in the determination of glutathione in pharmaceutical formulations in the form of a conjugate and a nanoparticle. The proposed method offers a simple, accurate, and inexpensive way to quantify reduced glutathione.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.