Acid-induced Degradation Products Research Articles

A solvent-free HPLC method for the simultaneous determination of Favipiravir and its hydrolytic degradation product

During COVID-19 pandemic, Favipiravir (FPV) showed a great efficacy against COVID-19 virus, it produced noticeable improvements in recovery of the patients. The aim of this study was to develop a new, green and simple method for the simultaneous determination of FPV and its acid-induced degradation product (ADP) in its pure and pharmaceutical dosage forms. This method will be key for the inevitable development of FPV solution and inhaler formulations. A green micellar RP-HPLC method was developed using an RP-VDSPHERE PUR 100 column (5 µm, 250 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.02 M Brij-35, 0.1 M SDS and 0.01 M potassium dihydrogen orthophosphate anhydrous and adjusted to pH 3.0 with 1.0 mL min−1 flow rate. The detection was performed at 280 nm with a run time of less than six min. Under the optimized chromatographic conditions, linear relationship has been established between peak area and concentration of FPV and its ADP in the range of 5–100 and 10–100 µg mL−1 with elution time of 3.8 and 5.7 min, respectively. The developed method was validated according to the ICH guidelines and applied successfully for determination of FPV in its pharmaceutical dosage form.

Spectrophotometric determination of favipiravir in presence of its acid hydrolysis product

Favipiravir (FAV) has been approved as an antiviral drug used in pandemic corona virus to treat covid-19. It has an amide moiety susceptible to hydrolysis and degradation in acid medium. Therefore, four simple, sensitive, and accurate stability indicating spectrophotometric methods have been developed for the determination of FAV in presence of its acid induced degradation product. The first method describes direct determination of FAV at 323 nm. Dual wavelength method was the second developed one for FAV quantitation by recording the absorbance difference at 322.7 and 270 nm. The third method involves using first derivative peak to peak amplitude at 338.0 and 308.0 nm, while difference spectrophotometry was the fourth suggested method, and it was based on recording the spectral changes at 361.3 nm as pH changes. The obtained calibration curves were linear over 4.0–22.0 µg/mL. Accuracy of the suggested procedures ranged from 99.11 to100.06, while precision results were from 0.80 to1.68. The developed methods were used to determine FAV in pure powdered form, laboratory-prepared mixtures with their degradation product, and pharmaceutical formulation without interference from its acidic degradation product.The greenness was assessed based on GAPI and ACREE metric and was found to be compatible and in reconciliation with green analytical chemistry concepts.Graphical

Validated green high performance liquid chromatographic and capillary electrophoretic methods for estimation of dinitolmide in presence of its acid-degradation product

This study describes two new, green, fast, specific and highly sensitive stability-indicating techniques for determination of Dinitolmide (DM) in presence of its acid induced degradation product without prior separation; Acid degradation was carried out by hydrolysis with 0.1 N HCl; and the acidic degradation pathway was established by IR, and MS techniques. The first is a HPLC-Diode Array detector was performed by isocratic mode on BDS Hypersil C18 column (150 mm × 4.6 mm × 5 μm particle size) at ambient temperature with a mobile phase consisting of 5 mM sodium acetate buffer (pH 5.5): acetonitrile (40:60, v/v), flow rate of 1 mL/min and detection at 210 nm. DM and its acid degradant were eluted at 4.3 and 2.8 min, respectively offering quick analysis. Linearity of the calibration curves confirm good correlation in range of 1–15 μg/mL with recovery % of 100.02 ± 0.460. The LOD and LOQ were 0.243 and 0.737, respectively. The second method is CE -Diode Array detector was carried out at 25 °C using borate buffer (30 mM, pH 9) as background electrolyte solution and detection at 225 nm. The migration times were 2.26 and 2.97 min for DM and its acid degradant, respectively. The linearity range was 5–50 μg/mL with recovery % of 99.55 ± 0.910, LOD and LOQ were 1.010 and 3.050, respectively. The small LOD and LOQ values for both methods confirm high sensitivity of the methods The developed methods were validated according to the ICH guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by a reported method in terms of accuracy, precision, and no significant difference was found. Both methods were successfully applied for estimating DM and its acid degradant in pure and pharmaceutical dosage forms. In addition, the methods were assessed for their greenness profile and CE was greener owing to using safer solvents and less energy consumption.

Comparative study of extension area based methods for spectrophotometric determination of desmopressin acetate in the presence of its acid-induced degradation products

Desmopressin acetate (DPA) is a synthetic analogue of vasopressin used in the treatment of diabetes insipidus, bedwetting, hemophilia A, and elevated levels of urea in the blood. Sensitive and selective stability-indicating methods are needed to be developed and validated for its assay pure and pharmaceutical dosage forms in the presence of its degradation products as no method has been reported for its determination in the presence of its degradants. This work describes a comparative study of five simple stability-indicating spectrophotometric techniques for determination of DPA in presence of its acid-degradation products (acid-degradants) without prior separation. The proposed spectrophotometric techniques (First derivative, Derivative ratio, Ratio difference, Mean centering and Dual wavelength) were developed and validated according to ICH guidelines. Acid degradation was carried out with 0.1 N HCl; the acid-degradants were separated on TLC plates and the acidic degradation pathway was established by IR, H-NMR and MS techniques. The TLC method was based on the separation of DPA and its acid-induced degradation products on silica gel plates using methanol: water (80:20, v/v) as a developing system and UV detection at 254 nm. All assay suggested methods were successfully applied for quantitation of DPA in pure and tablet forms. They are specific, sensitive, precise and accurate. They showed good linearity in the concentration range of 1–14 µg/mL with good correlation coefficients, and limit of detection (LOD) of 0.304, 0.274, 0.167, 0.248 and 0.199 and limit of quantitation (LOQ) of 0.920, 0.829, 0.506, 0.751 and 0.604) for each method, respectively. These methods were successfully applied for the simultaneous determination of DPA in its pure and tablet dosage form in the presence of its acid-degradants. The results obtained were statistically comparable with those of reported HPLC assay method; no significant differences were observed with relevance to accuracy and precision. All the methods are sensitive, selective and can be used for the routine analysis of DPA in its pure and dosage forms.

Comparative Study of the Selectivity Power of Colorimetric Method Over Chromatographic Methods for the Analysis of Valaciclovir Hydrochloride.

Valaciclovir hydrochloride (VAL) is an essential antiviral prodrug used to cure various types of herpes. Analysis of VAL by different analytical techniques demonstrates a persuasive aspect that is favorable in quality control application. This study describes a comparison between colorimetric and chromatographic (RP-high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)-densitometric) methods concerning selectivity and specificity for the determination of VAL in all possible degradation products (alkali- and acid-induced degradation products, namely aciclovir [ACI] and guanine [GUA], respectively) in their synthetic mixture and pharmaceutical formulations. The colorimetric method was accomplished by forming a highly colored complex with ferric hydroxamate reagent measured at 493 nm in the concentration range (0.20-1.60 mg/mL). Both chromatographic methods were successfully applied using ultraviolet (UV) detection at 256 nm in the concentration range (2.00-5.00 µg/mL) for the RP-HPLC method and (10.00-900.00 ng/band) for the TLC-densitometric method. The linearity studies, regression equations, assay parameters, and validation sheet of the proposed colorimetric and chromatographic methods to determine VAL were obtained with highly acceptable values. The International Council for Harmonization (ICH) guidelines were followed to validate the described methods and the statistical comparison regarding both accuracy and precision, and satisfactory results were accomplished. In this study, we configure a full comparative study between different analytical methods for the analysis of challengeable mixture containing the drug of interest, VAL, along with its degradation products, ACI and GUA.

Greenness Assessment of Two Validated Stability-Indicating Chromatographic Methods for Estimating Modafinil Using the Analytical Eco-Scale.

Modafinil (MDF) is one of the drugs used for the treatment of narcolepsy. This manuscript describes the development and validation of two chromatographic stability-indicating methods for MDF in the presence of its acid-induced degradation product. MDF was degraded under different stress conditions and identification of the degradation product was performed using IR and mass spectroscopy. The first method involved TLC, in which plates precoated with silica gel G60 F254 were used and the developing system was dichloromethane- methanol (9:1, v/v). The second method was an eco-friendly (HPLC) method that utilized a C-18 column and ethanol-H2O (30:70, v/v) as a mobile phase with a flow rate of 1 mL/min and UV detection at 220 nm. Good linear relationships were obtained within the ranges 1-10 µg/band and 2-10 µg/mL for TLC-densitometry and HPLC, respectively. The obtained results were statistically compared with those of the official HPLC method and showed no significant difference with respect to their accuracy and precision at P = 0.05. Greenness scores represent excellent green analysis results in comparison with the reported studies. The proposed methods were validated according to ICH guidelines and were applied on bulk powder and pharmaceutical dosage forms using eco-friendly mobile phases in line with worldwide trends. Chromatographic methods have been validated for the estimation of MDF in the presence of its degradation product. Clarification of the degradation pathway and elucidation of the structure were stated for the first time. This is the first published method using greenness assessment metrics for the analysis of MDF.

Second derivative synchronous fluorescence determination of avanafil in the presence of its acid-induced degradation product aided by powerful Lean Six Sigma tools augmented with D-optimal design.

In this work, the quantitative determination of an erectile dysfunctional drug avanafil in the presence of its acid-induced degradation product was achieved via the application of a pre-optimized novel spectrofluorimetric method. The fluorescence emission wavelength was recorded at 370 and 407 nm, after being excited at 268 and 271 nm for avanafil and its acid-induced degradation product, respectively. Direct determination of avanafil based on its native fluorescence is restricted because the emission spectra of both components are heavily overlapped. Therefore, to overcome this constraint, a novel second derivative synchronous fluorescence method was evolved to eliminate this overlapping. The ideal determination wavelength was found to be 377 nm. Augmentation of lean six sigma (LSS) with response surface methodology (RSM) play a significant role in the development of robust specifications to ensure quality at the six sigma level with a high level of statistical confidence and targeted performance. All of the experimental conditions were optimized using D-optimal design as a RSM to select the optimal parameters. In addition, this work includes a graphical representation of the relationships between various variables that can greatly affect the results and the intensity of the synchronous fluorescence.

Validated Chromatographic and Spectrofluorimetric Methods for Analysis of Silodosin: A Comparative Study with Application of RP-HPLC in the Kinetic Investigation of Silodosin Degradation.

Stability indicating determination of pharmaceuticals is crucial, especially for drugs which have few published official analytical methods. Silodosin (SLD) is an FDA approved α1A-adrenoceptor blocker. Efficient analytical methods were suggested, based on different instrumental techniques for quantification of SLD, besides conducting kinetic investigation of its degradation. The first method is based on Reversed Phase High Performance Liquid Chromatography with Photodiode Array Detector (RP-HPLC-PDAD). Detection is done at wavelength 225 nm. The second method is focused on using High Performance Thin Layer Chromatography (HPTLC) and eluting the drug by solvent mixture followed by scanning at wavelength 270 nm. The third method depends on the First Derivative Synchronous Fluorescence Spectroscopy (1DSFS) for analysis of solutions of SLD and its acid and oxidative induced degradation products at Δλ = 90 nm, then determining the first derivative of the spectra and measuring peak amplitudes at 360 nm. Acceptable linearities were found in the concentration range of 0.50-90 μg/mL, 0.10-3.0 μg/band, and 0.05-0.50 µg/mL, for RP-HPLC-PDAD, HPTLC, and spectrofluorimetric methods, respectively. Statistical analysis showed no significant difference between the suggested and the reported method. In monitoring the kinetics of SLD degradation, the order of reactions was determined and effects of degrading agent concentration and temperature on reaction rate were studied. Three analytical methods were developed for the determination of SLD based on RP-HPLC-PDAD, HPTLC, and 1DSFS in bulk and capsule dosage form. In addition, kinetic investigation of SLD degradation was performed using the developed RP-HPLC-PDAD method.

PCCA algorithm as a fingerprint resolution technique for the analysis of ciprofloxacin in the presence of its acid induced degradation product

Pure component contribution algorithm (PCCA) is an accurate analytical method was developed for extracting the contribution of each component from a mixture where the components of this mixture are completely overlapped. It is based on elimination the signal of interfering components using mean centering as a processing tool finally, the pure contribution of each component is extracted form complex signals and that worked as a finger-print resolution for the component. (PCCA) has been successfully applied to UV data of ciprofloxacin hydrochloride (CIP) in the presence of its acidic degradation product (DCIP). The spectrophotometric technique (PCCA) was successfully applied to the laboratory prepared mixtures and the pharmaceutical dosage form where the purity of the extracted signals were tested by calculating the spectral contrast angle (6) where the results were compared to show the capability to recover pure spectral profiles and detect the presence of impurities. The proposed method proved that spectrophotometric techniques can be used for identification and separation of signals, similar to chromatographic techniques. The method validated according to the ICH guidelines and the accuracy, precision and repeatability found to be within the acceptable limits. The selectivity of the proposed method was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can be used for the routine analysis of ciprofloxacin in quality-control laboratories.

Determination of some antiemetic drugs through its native fluorescence or fluorescence quenching of cerrous ammonium sulphate.

The manuscript describes two fluorimetric methods for the determination of some antiemetic drugs namely granisetron HCl, ondansetron HCl and tropisetron HCl, used in the management of nausea and vomiting induced by cytotoxic chemotherapy and radiotherapy. Granisetron HCl solution exhibits a native fluorescence, which can be applied for its determination at 365nm upon excitation at 305nm. The method was applied for the determination of granisetron HCl in drug substance, drug product as well as in presence of its acid induced degradation products. The quantum yield was calculated. The second proposed method is based on measuring the quenching effect induced by ondansetron HCl or tropisetron HCl on the fluorescence intensity of cerrous ammonium sulphate at λem 348nm upon excitation at 250nm in acidic medium. The analysis of quenching data showed that quenching of cerrous ammonium sulphate induced by ondansetron HCl or tropisetron HCl is mainly through dynamic quenching. Various variables affecting fluorescence response were studied and optimized. The obtained results were found to be statistically agreed with those obtained from the official or reported ones. Moreover, the validity of the methods was assessed according to ICH guidelines.

Monitoring of Clotrimazole Degradation Pathway in Presence of its Co-formulated Drug.

Two stability-indicating chromatographic methods for the determination of clotrimazole and its two acid induced degradation products, with dexamethasone acetate without prior separation. First method depends on RP-HPLC utilizing ODS-3 Inertsil C18 column. Mobile phase consists of acetonitrile:phosphate buffer (pH 6.0) in ratio (65:35, v/v) with flow rate 1.5 mL/min and UV-detection at 220 nm. Linearity range 1.0-75.0 μg/mL for clotrimazole and 2.0-75.0 μg/mL for dexamethasone with mean percentage recovery of 99.49 ± 1.10 for CLT and 99.60 ± 1.06 for DA. Second method depends on HP-TLC. Developing system is composed of chloroform:ethyl acetate in the ratio of (5:3.5, v/v), scanned at 220 nm. Linearity range 1.0-12.0 μg/band for clotrimazole and 1.0-20.0 μg/band for dexamethasone with mean R% of 99.33 ± 0.76 for clotrimazole and 99.77 ± 0.99 for dexamethasone. Conditions and parameters affecting the separation of the cited components without interference of the degradation products are tested and optimized. Suitability of the methods for quantization of the drugs concentrations is proven by validation as instructed from the ICH. Validation results and statistical treatment of the data demonstrate reliability of these methods. Kinetics of acid degradation process of clotrimazole are investigated by the proposed HPLC method and the order rate constant, half life and shelf life are computed.

Potentiometric Sensing of Valaciclovir Hydrochloride in the Presence of Its Acid Induced Degradation Product with Real Time Acquisition of the Dissolution Profile from Its Pharmaceutical Formulations

The possibility of obtaining continuous analytical signal excluding any pre-treatment of sample is the most environmental friendly method of analysis. In the current manuscript, a solid contact sensor was fabricated for dual purpose; firstly, determination of valaciclovir hydrochloride (VAL) in existence of its acid induced degradation product, Guanine (GUA) and secondly, real time acquisition of the dissolution profile from its pharmaceutical formulations (Valtrex) and (Valysernex). The sensing membrane was fabricated using a polyvinyl chloride (PVC), sodium tetraphenyl borate as a cation exchanger with bis (2-ethylhexyl) sebacate as a plasticizer. The proposed sensor displayed fast, stable Nernstian responses across VAL concentration range (1 × 10−5 M to 1 × 10−2 M). The opportunities of the proposed sensor with respect to other off-line classical techniques originates from being in-line, time saving, cost-effective, green and simple. ICH guidelines were followed to ensure validation of the proposed method concerning accuracy and precision.

Validated stability-indicating spectrophotometric methods for the determination of Silodosin in the presence of its degradation products

Five simple, rapid, accurate, and precise spectrophotometric methods are developed for the determination of Silodosin (SLD) in the presence of its acid induced and oxidative induced degradation products. Method A is based on dual wavelength (DW) method; two wavelengths are selected at which the absorbance of the oxidative induced degradation product is the same, so wavelengths 352 and 377 nm are used to determine SLD in the presence of its oxidative induced degradation product. Method B depends on induced dual wavelength theory (IDW), which is based on selecting two wavelengths on the zero-order spectrum of SLD where the difference in absorbance between them for the spectrum of acid induced degradation products is not equal to zero so through multiplying by the equality factor, the absorption difference is made to be zero for the acid induced degradation product while it is still significant for SLD. Method C is first derivative (1D) spectrophotometry of SLD and its degradation products. Peak amplitudes are measured at 317 and 357 nm. Method D is ratio difference spectrophotometry (RD) where the drug is determined by the difference in amplitude between two selected wavelengths, at 350 and 277 nm for the ratio spectrum of SLD and its acid induced degradation products while for the ratio spectrum of SLD and its oxidative induced degradation products the difference in amplitude is measured at 345 and 292 nm. Method E depends on measuring peak amplitudes of the first derivative of the ratio (1DD) where peak amplitudes are measured at 330 nm in the presence of the acid induced degradation product and measured by peak to peak technique at 326 and 369 nm in the presence of the oxidative induced degradation product. The proposed methods are validated according to ICH recommendations. The calibration curves for all the proposed methods are linear over a concentration range of 5–70 μg/mL. The selectivity of the proposed methods was tested using different laboratory prepared mixtures of SLD with either its acid induced or oxidative induced degradation products showing specificity of SLD with accepted recovery values. The proposed methods have been successfully applied to the analysis of SLD in pharmaceutical dosage forms without interference from additives.

Simultaneous equation and area under the curve spectrophotometric methods for estimation of cefaclor in presence of its acid induced degradation product; A comparative study

Two simple spectrophotometric methods were developed and validated for determination of cefaclor in presence of its acid induced degradation product; namely simultaneous equation method using two wavelengths 265 and 235 nm Method (A) and Area under the curve method using two wavelength ranges (230–240) nm and (260–270) nm Method (B). The accuracy, precision and linearity ranges of the proposed methods were determined. The methods were validated and the specificity was assessed by analyzing synthetic mixtures containing the drug and its degradate. The two methods were applied for the determination of the cited drug in its pharmaceutical preparation and the obtained results were statistically compared with those of a reported method. The comparison showed that there is no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

Chromatographic Study of Azintamide in Bulk Powder and in Pharmaceutical Formulation in the Presence of Its Degradation Form.

Two specific, sensitive, and precise stability-indicating chromatographic methods were developed, optimized, and validated for the determination of Azintamide (AZ) in the presence of its degradation product. The first method was TLC combined with the densitometric determination of the separated bands. Separation was achieved using silica gel 60 F254 TLC plates and chloroform-acetone-glacial acetic acid (7.5 + 2.1 + 0.4, v/v/v) as the developing system. Good correlations were obtained between the integrated peak area of the studied drug and its corresponding concentrations in the linearity range. The second method used HPLC with UV diode-array detection, in which the proposed method was applied for the quantitative determination of AZ in the presence of its acidic degradation product and the quantitative determination of the acid-induced degradation product of AZ (AZ Deg) using pentoxifylline as the internal standard. The proposed components were separated on a reversed-phase C18 analytical column using acetonitrile-water (50 + 50, v/v). The flow rate was maintained at 0.55 mL/min and the detection wavelength was 260 nm. Linear regressions were obtained in the range of 1-30 and 0.3-16 μg/mL for AZ and AZ Deg, respectively. Different parameters affecting the suggested methods were optimized for maximum separation of the cited components. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and successfully applied for the determination of AZ in its pure powder form and in its pharmaceutical formulation. Both methods were also statistically compared with the reported method with no significant difference in performance observed.

Different Mathematical Processing of the Ratio Spectra for Quantification of Cefaclor in Presence of its Acid Induced Degradation Product

Three simple mathematical spectrophotometric methods based on processing the ratio spectra were developed and validated for determination of cefaclor in presence of its acid induced degradation product; namely First derivative of ratio spectra (1DD) Method (A), Ratio subtraction method (RS) Method (B) and Mean centering of ratio spectra (MCR) Method (C). The accuracy, precision and linearity ranges of the proposed methods were determined. The methods were validated and the specificity was assessed by analyzing synthetic mixtures containing the drug and its degradate. The three methods were applied for the determination of the cited drug in its pharmaceutical preparation and the obtained results were statistically compared with those of a reported method. The comparison showed that there is no significant difference between the proposed methods and the reported method regarding both accuracy and precision.

Stability-indicating methods for the analysis of ciprofloxacin in the presence of its acid induced degradation product: A comparative study

Four rapid, simple, accurate and precise spectrophotometric methods were used for the determination of ciprofloxacin in the presence of its acidic degradation product. The methods under study are ratio derivative, ratio difference, mean centering and dual wavelength. All the methods were validated according to the ICH guidelines and the obtained accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can be used for the routine analysis of ciprofloxacin in quality-control laboratories.

Stability Indicating Spectrophotometric and Chemometric Methods for Determination of Buflomedil in Presence of its Acid Induced Degradation Products

Simple, accurate, sensitive, selective and validated UV spectrophotometric univariate and multivariate methods were developed for determination of Buflomedil (BFM) in presence of BFM degradation products (BFM Deg). The univariate methods includes mean centering of ratio spectra (MCR), ratio difference spectrophotometric method (RDSM) and H-point standard addition method (HPSAM); the first one used the amplitude at 235.7 nm, the second utilized the difference in amplitudes at 250 nm and 282 nm; while, the third method utilized the absorbance values at 247 and 282.4 nm. Linearity was obtained for the proposed methods in the ranges of 10–100 µgmL-1. The percentage recoveries were 100.06±0.963, 100.17±0.962 and 100.90±0.667 for the three methods in a respective order. Multivariate methods involves the application of three chemometric techniques, and classical least square (CLS) principal component regression (PCR) and partial least-squares regression (PLS), which were used for the simultaneous determination of the BFM and its degradation products. The results obtained from the proposed methods, together with the reported one, were statistically compared using one-way analysis of variance (ANOVA). These methods are suitable as stability indicating methods for the determination of BFM in presence of its degradation products either in bulk powder or in pharmaceutical formulations.

Towards an ideal method for analysis of lisinopril in pharmaceutical formulations using a tris(2,2′-bipyridyl)-ruthenium(ii)-peroxydisulfate chemiluminescence system in a two chip device

A novel, rapid, selective and sensitive method has been developed for analysis of lisinopril (LIS) in pharmaceutical formulations using a tris(2,2′-bipyridyl)-ruthenium(II) (Ru(bipy)32+) peroxydisulfate chemiluminescence (CL) system in a two chip device. The parameters that influence the CL signal intensity were carefully optimized. These include pH, flow rates and concentration of reagents used. Under optimum conditions, a linear calibration curve between 0.25 and 50.0 μg mL−1 was obtained. About 180 samples were analyzed per hour while the detection limit was found to be 0.067 μg mL−1. The method was applied for analysis of LIS in pharmaceutical products and was found to be free from interferences of acid-induced degradation (AID) products and other ingredients usually present in these preparations.

Stability‐indicating methods for the determination of mosapride citrate in the presence of its degradation products according to ICH guidelines

In the present work, different spectrophotometric methods and one spectrofluorimetric method have been developed and validated for the determination of mosapride citrate in the presence of its acid-induced degradation products. The drug was subjected to stress stability study including acid, alkali, oxidative, photolytic, and thermal stress degradation. The developed spectrophotometric methods included the use of first order derivative ((1)D), derivative of ratio spectra ((1)DD), mean centring of ratio spectra (MC) and H-point standard additions (HPSAM) spectrophotometric methods. For (1)D method, the peaks amplitudes at 282.8 and 319.6 nm were measured, while for (1)DD method those at 308 nm and 323 nm were measured. Mean centring of ratio spectra method used the values at 317 nm for calibration, while for HPSAM the absorbance at 273 and 288.6 nm were used. These methods were successfully applied for determination of mosapride in the concentration range of 5-70 µg.ml(-1). The spectrofluorimetric method was based on measuring the native fluorescence of mosapride in 0.1 M NaOH using λ(excitation) 276 nm and λ(emission) 344 nm and 684 nm with linearity ranges of 50-3000 ng.ml(-1) and 50-9000 ng.ml(-1), respectively. All the developed methods were validated according to the International Conference on Harmonization (ICH) guidelines and were applied for bulk powder and dosage form. The results obtained were statistically compared to each other using one-way ANOVA testing.