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.

     

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.     

Stability-Indicating HPLC and RP-TLC Determination of Cefpirome Sulfate with Kinetic Study

Two sensitive and selective stability-indicating methods were developed for the determination of the antibiotic cefpirome sulfate in bulk powder, pharmaceutical formulation and in presence of its acid, alkaline, photo- and oxidative degradation products. Method A was based on HPLC separation of cefpirome sulfate in the presence of its degradation products on a reversed phase column C18, 250 × 4.6 mm, 5-μm particle size and mobile phase consisting of 0.1 M disodium hydrogen phosphate dihydrate pH 3.9 adjusted with phosphoric acid–acetonitrile (85:15, v/v). Quantitation was achieved with UV detection at 270 nm. The linear calibration curve was in the range 5.0–50.0 μg mL^?1. Method B was based on reversed phase TLC separation of the cited drug in the presence of its degradation products followed by densitometric measurement of the intact drug at 270 nm. The separation was carried out using disodium hydrogen phosphate dihydrate 2.0 g %w/v, at pH 3.5 adjusted with phosphoric acid–acetone (15:10, v/v) as a developing system. The calibration curve was in the range of 1.0–10.0 μg/spot. The HPLC method was used to study the kinetic of cefpirome sulfate acid degradation. The results obtained were statistically analyzed and compared with those obtained by applying the official Japanese method.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Comparison of Ion-Pairing and Reversed Phase Liquid Chromatography in Determination of Sulfamethoxazole and Trimethoprim

Abstract

Two simple, rapid, and sensitive HPLC methods have been developed for the simultaneous determination of sulfamethoxazole and trimethoprim in their pure and dosage forms, one utilizing reversed phase HPLC and the other ion-pair HPLC. In the reversed phase HPLC method (A) the mobile phase consists of 0.05% aqueous solution of formic acid with pH adjusted to 4.5±0.2 with triethylamine : acetonitrile:tetrahydrofuran 50 : 49 : 1 (v/v), and the mobile phase pumped at flow rate of 1.0 ml min^?1. An Appolo LC18 column (5.0 µm), 250 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. In the ion-pair HPLC method (B) the mobile phase consisted of methanol : buffer 35 : 65 (v/v) with the buffer composed of potassium dihydrogen phosphate 0.3 M and sodium heptan sulfonic acid 5.0 mM. To 500 ml of buffer was added 2.0 ml triethylamine, and then the pH was adjusted to 5.0 with phosphoric acid, and the mobile phase was pumped at a flow rate of 1.2 ml min^?1. A Hypersil C₁₈ column (5.0 µm), 150 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. Linearity ranges for sulfamethoxazole and trimethoprim were 1.0–110 and 1.5–98 µg ml^?1, respectively, with method A and 0.5–100 and 1.0–125 µg ml^?1, respectively, with method (B). Minimum detection limits obtained were 0.1969 and 0.3451 µg ml^?1 for sulfamethoxazole and trimethoprim, respectively, with method A, and 0.1377 and 0.2454 µg ml^?1 with method (B). The proposed methods were further applied to the analysis of tablets containing the two drugs, and the results were satisfied.     

The role of TLC in the screening of inherited metabolic diseases

Summary Thin-layer chromatography (TLC) is a rapid, reliable and inexpensive screening technique for diagnosis of inherited metabolic diseases (IMD). Our screening program encompasses five main situations where the use of TLC is considered to be vindicated: (i) analysis of amino acids; (ii) screening for sugar defects; (iii) detection of pathological oligosaccharidurias; (iv) screening for organic acid disorders; and (v) detection of abnormalities in tryptophan metabolism. Examples are presented of chromatograms obtained from pathological samples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Simple and fast analysis of iohexol in human serums using micro‐hydrophilic interaction liquid chromatography with monolithic column

A simple and rapid method based on micro‐liquid chromatography using a synthetic monolithic capillary column was developed for determination of iohexol in human serums, a marker to evaluate the glomerular filtration rate. A hydrophilic methacrylic acid‐ethylene dimethacrylate monolith provided excellent selectivity and efficiency for iohexol with separation time of 3 min using a mobile phase of 40:60 v/v 50 mM phosphate buffer pH 5/methanol. Four serum protein removal, methods using perchloric acid, 50% acetonitrile, 0.1 M zinc sulfate, and centrifuge membrane filter were examined. The method of zinc sulfate was chosen due to its simplicity, compatibility with the mobile phase system, nontoxicity, and low cost. Interday calibration curves were conducted over iohexol concentrations range of 2–500 mg/L (R² = 0.9997 ± 0.0001) with detection limit of 0.44 mg/L. Intra‐ and interday precisions for peak area and retention time were less than 2.8 and 1.4%, respectively. The method was successfully applied to serum samples with percent recoveries from 102 to 104. The method was applied to monitor released iohexol from healthy subject. Compared with the commercially available reversed‐phase high‐performance liquid chromatography method, the presented method provided simpler chromatogram, faster separation with higher separation efficiency and much lower sample and solvent consumption.     

Two validated chromatographic determinations of an antifungal drug,its toxic impurities and degradation product: A comparative study

Tolnaftate, a thionoester anti‐fungal drug, was subjected to alkaline hydrolysis to produce methyl(m‐tolyl)carbamic acid and β ‐naphthol (tolnaftate impurity A). N‐Methyl‐m‐toluidine, tolnaftate impurity D, was synthesized and structurally elucidated along with tolnaftate alkaline degradation products using IR, H¹NMR and MS. Two stability‐indicating HPTLC and RP‐HPLC methods were developed and validated, for the first time, for determination of tolnaftate, its alkaline degradation products and toxic impurities in the presence of methyl paraben, as a preservative in Tinea Cure^® cream. The proposed HPTLC method depended on separation of the studied components on TLC silica gel F₂₅₄ plates using hexane–glacial acetic acid (8:2, v/v) as a developing system and scanning wavelength of 230 nm. The proposed RP‐HPLC method was based on separation of the five components on an Eclipse plus C₁₈ column. The mobile phase used was acetonitrile–water containing 1% ammonium formate (40:60, v/v), with a flow rate of 1 mL/min and detection wavelength of 230 nm. The proposed methods allowed the assay of tolnaftate toxic impurities, β ‐naphthol and N‐methyl‐m‐toluidine, down to 2%, allowing tracing of β ‐naphthol that could be absorbed by the skin causing systemic toxic effects, unlike tolnaftate, indicating the high significance of such determination. International Conference on Harmonization guidelines were followed for validation.     

Simple Liquid Chromatographic Determination of Minocycline in Brain and Plasma

A new high-performance liquid chromatography assay was developed for the determination of minocycline in plasma and brain. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase consisting of acetonitrile:water:perchloric acid (26:74:0.25, v/v/v) adjusted to pH 2.5 with 5 M sodium hydroxide for elution through a RP8 column (250 × 3.0 mm, i.d.) with UV detection set at 350 nm. The method proved to be accurate, precise (RSD < 20%) and linear between 0.15–20 μg mL⁻¹ in plasma and 1–20 μg mg⁻¹ in brain. The method was successfully applied to a blood-brain barrier minocycline transport study.     

Development and Validation of High Performance Liquid Chromatographic and UV-Derivative Spectrophotometric Methods for the Determination of Sotalol Hydrochloride in Tablets

Abstract

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310 nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93 v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1 nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.     

Sensitive Determination of Water Insoluble Dyes Used as Marking of Commercial Petroleum Products Using High‐Performance Liquid Chromatography with Electrochemical Detection

This paper describes an analytical method using high‐performance liquid chromatographic (HPLC) separation coupled with electrochemical detection to detect three dyes, Solvent Blue 14 (SB‐14), Solvent Blue 35 (SB‐35) and Solvent Red 24 (SR‐24). The dyes were eluted and separated using a reversed‐phase column (C‐8) under isocratic elution with the mobile phase containing a mixture of acetonitrile/ammonium acetate (5.0 mmol L^?1) at the ratio of 75 : 25 (v/v). Two sample pretreatment methods were tested and successfully applied to quantify SB14, SB‐35 and SR‐24 dyes in gasoline samples. The proposed method was simple, fast and suitable to detect and quantify marker dyes in gasoline sample at low concentration.     

High-performance liquid chromatographic determination of urocanic acid isomers in cosmetic products

Summary A method has been developed for the determination of trans and cis urocanic acid in oil-in-water cosmetic emulsions. It involves an extraction of the sample in 1:3 methanol-aqueous NaOH (10⁻³ M), by ultrasonication, which leads to quantitative recoveries, and a reversed-phase HPLC isocratic elution for the analysis of the extract. Chromatography is performed on a C18 column using 0.1 M sodium perchlorate (pH 3.0)-acetonitrile 98:2 (v/v), as the mobile phase, and UV detection at 263 nm. The separation of the isomers is good. Linearity and precision of the method have been assessed.     

Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay

Reversed‐phase and size‐exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed‐phase method was carried out on a Jupiter C₄ column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size‐exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25–250 μg/mL (25.75–25 750 IU/mL) (r ² = 0.9997) and 5–80 μg/mL (515–8240 IU/mL) (r ² = 0.9996), respectively, for reversed‐phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.     

Concurrent detection of cabozantinib as an anticancer agent and its major metabolites in human serum using fluorescence-coupled micellar liquid chromatography

A novel, highly sensitive, simple, and rapid strategy was designed and developed for simultaneous determination of cabozantinib (CBZ) as an anticancer agent and its main metabolites including monohydroxy sulfate (EXEL-1646), N-oxide (EXEL-5162(, amide cleavage product (EXEL-5366), and 6-desmethyl amide cleavage product sulfate) EXEL-1644). Measurements were done through a micellar liquid chromatography (MLC) method coupled with fluorescence detection. The high-performance liquid chromatography (HPLC) was performed using a Kinetex C18 100 Å column as well as acetonitrile, cetyltrimethylammonium bromide (CTAB; 0.2 mol.L^?1), and tris buffer (pH 8.5) solutions as the mobile phase at a 40:50:10 (v/v) ratio. The method’s linearity (20 to 700 ng.mL^?1), limit of detection (LOD; 2.11 to 3.69 ng.mL^?1), limit of quantification (LOQ; 20 to 30 ng.mL^?1), intra- and inter-day precisions (RSD < 4.00%), selectivity, recovery, and robustness were fully evaluated. According to the obtained results, the developed method can be used for simple and rapid (~35 min) quantification of CBZ as an anticancer drug and its major metabolites in human serum samples with high sensitivity and low cost.     

High-performance liquid chromatography of 8-hydroxyquinoline chelates of aluminium and cobalt(III)

Summary The 8-hydroxyquinoline chelates of Co(III) and Al(III) may be separated by high-performance liquid chromatography using a silica column and 5% methanol in chloroform as mobile phase. Using detection at 254 nm, the method provided detection limits of 0.9 ng of Co(III) and 17 ng of Al(III) in a 10 mm³ injection.     

High-performance liquid chromatographic determination/identification of oxytetracycline and sulphadimidine in meat and eggs

Summary A rapid method for the simultaneous determination/identification of residual oxytetracycline (OTC) and sulphadimidine (SDD) in meats (beef, pork, chicken) and eggs by high-performance liquid chromatography (HPLC) was developed. The extraction of OTC and SDD was performed using a Sep-Pak^? CN cartridge. The extracts contained OTC/SDD analytes when examined by HPLC using a LiChrospher^? 100 RP-8 end-capped column and a mobile phase of acetonitrile-acetic acid-water (28:4:68, v/v/v) with a photodiode array detector. The average recoveries from spiked samples (0.1 μg g⁻¹ and 1.0 μg g⁻¹) were in excess of 80.2% with coefficients of variation between 1.5 and 5.0%. The limits of detection for OTC and SDD were 0.05 and 0.02 μg g⁻¹, respectively.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Stability‐indicating chromatographic methods for determination of flecainide acetate in the presence of its degradation products; isolation and identification of two of its impurities

In this work, two stability‐indicating chromatographic methods have been developed and validated for determination of flecainide acetate (an antiarrhythmic drug) in the presence of its degradation products (flecainide impurities; B and D). Flecainide acetate was subjected to a stress stability study including acid, alkali, oxidative, photolytic and thermal degradation. The suggested chromatographic methods included the use of thin layer chromatography (TLC‐densitometry) and high‐performance liquid chromatography (HPLC). The TLC method employed aluminum TLC plates precoated with silica gel G.F₂₅₄ as the stationary phase and methanol–ethyl acetate–33% ammonia (3:7:0.3, by volume) as the mobile phase. The chromatograms were scanned at 290 nm and visualized in daylight by the aid of iodine vapor. The developed HPLC method used a RP‐C₁₈ column with isocratic elution. Separation was achieved using a mobile phase composed of phosphate buffer pH 3.3–acetonitrile–triethylamine (53:47:0.03, by volume) at a flow rate of 1.0 mL/min and UV detection at 292 nm. Factors affecting the efficiency of HPLC method have been studied carefully to reach the optimum conditions for separation. The developed methods were validated according to the International Conference on Harmonization guidelines and were applied for bulk powder and dosage form. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC, TLC, and first-derivative spectrophotometry stability-indicating methods for the determination of tropisetron in the presence of its acid degradates

Three stability-indicating assay methods were developed for the determination of tropisetron in a pharmaceutical dosage form in the presence of its degradation products. The proposed techniques are HPLC, TLC, and first-derivative spectrophotometry (1D). Acid degradation was carried out, and the degradation products were separated by TLC and identified by IR, NMR, and MS techniques. The HPLC method was based on determination of tropisetron in the presence of its acid-induced degradation product on an RP Nucleosil C18 column using methanol-water-acetonitrile-trimethylamine (65 + 20 + 15 + 0.2, v/v/v/v) mobile phase and UV detection at 285 nm. The TLC method was based on the separation of tropisetron and its acid-induced degradation products, followed by densitometric measurement of the intact spot at 285 nm. The separation was carried out on silica gel 60 F254 aluminum sheets using methanol-glacial acetic acid (22 + 3, v/v) mobile phase. The 1D method was based on the measurement of first-derivative amplitudes of tropisetron in H2O at the zero-crossing point of its acid-induced degradation product at 271.9 nm. Linearity, accuracy, and precision were found to be acceptable over concentration ranges of 40-240 microg/mL, 1-10 microg/spot, and 6-36 micro/mL for the HPLC, TLC, and 1D methods, respectively. The suggested methods were successfully applied for the determination of the drug in bulk powder, laboratory-prepared mixtures, and a commercial sample.