Stability indicating high-performance thin-layer chromatographic determination of nelfinavir mesylate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method of analysis of nelfinavir mesylate both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-methanol-acetone (7:1.5:1.5, v/v/v). This system was found to give compact spots for nelfinavir mesylate (R_f value of 0.45±0.02). Nelfinavir mesylate was subjected to acid and alkali hydrolysis, oxidation, dry heat treatment and photodegradation. Also the peaks of degraded products were well resolved from the pure drug with significantly different R_f values. Densitometric analysis of nelfinavir mesylate was carried out in the absorbance mode at 250 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r²=0.999±0.002 in the concentration range of 1000-6000 ng per spot. The mean value of correlation coefficient, slope and intercept were 0.999±0.002, 0.014±0.001 and 21.73±1.26, respectively. The method was validated for precision, robustness and recovery. The limits of detection and quantitation were 60 and 140 ng per spot, respectively. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Separation and determination of terbinafine and its four impurities of similar structure using simple RP-HPLC method

A novel reversed-phase HPLC method for the simultaneous determination of active component terbinafine, its one impurity 1-methylaminomethylnaphtalene and three degradation products, β-terbinafine, Z-terbinafine and 4-methyl-terbinafine occurring in pharmaceutical formulations after long-term stability tests, was developed and validated using propylparaben as an internal standard.The chromatographic separation was performed on a NUCLEOSIL 100-5-CN column, mobile phase for separation of all compounds consisted of a mixture of tetrahydrofurane, acetonitrile and citrate buffer pH 4.50 (10:20:70, v/v/v). The analysis time was less than 32 min at flow-rate of 0.8 ml min⁻¹. UV detection was performed at 226 nm. The method was validated and system suitability parameters were investigated. Method robustness and short-term standard solution stability were verified. Limits of detection for terbinafine degradation products/impurity were from 0.023 to 0.098 μg ml⁻¹, limits of quantitation were from 0.078 to 0.327 μg ml⁻¹. The method was applicable for routine determination of terbinafine and all its found impurities of similar structure with sufficient selectivity, precision and accuracy.     

Development and validation of a high-throughput high-performance liquid chromatographic assay for the determination of caffeine in food samples using a monolithic column

The present study reports the development and validation of a high-throughput high-performance liquid chromatographic (HPLC) assay for the determination of caffeine in food samples. The analyte was separated rapidly from sample matrix using a short monolithic column (50 mm × 4.6 mm i.d.). The flow rate was 3.0 mL min⁻¹, while the mobile phase consisted of ACN/water (10:90, v/v). Caffeine was detected directly at 274 nm. Under the optimal HPLC conditions, the sampling rate was 60 h⁻¹. The assay was validated for linearity, LOD and LOQ, precision, selectivity and ruggedness. The case of external calibration versus standard addition for the analysis of real samples was also examined. The proposed assay was applied to the analysis of beverages and coffee samples.     

A Validated, Specific, Stability-Indicating RP-LC Method for Analysis of Gatifloxacin in the Presence of Degradation Products and Process-Related Impurities

A validated, specific, stability-indicating reversed-phase liquid chromatographic method has been developed for quantitative analysis of gatifloxacin, its degradation products, and its process-related impurities in bulk samples and in pharmaceutical dosage forms. Forced degradation of gatifloxacin bulk sample was conducted in accordance with ICH guidelines. Acidic, basic, neutral, and oxidative hydrolysis, thermal stress, and photolytic degradation were used to assess the stability-indicating power of the method. Substantial degradation was observed during oxidative hydrolysis. No degradation was observed under the other stress conditions. The method was optimized using samples generated by forced degradation and sample solution spiked with impurities. Good resolution of the analyte peak from peaks corresponding to process-related impurities and degradation products was achieved on a C₁₈ column by use of a simple linear mobile-phase gradient prepared from mixtures of acetonitrile and an aqueous solution of sodium dihydrogen orthophosphate dihydrate and triethylamine adjusted to pH 6.5 with orthophosphoric acid. Detection was performed at 240 nm. Limits of detection and quantification were established for gatifloxacin and its process-related impurities. When the stressed test solutions were assayed by comparison with gatifloxacin working standard the mass balance was always close to 99.3%, indicating the method was stability-indicating. Validation of the method was performed in accordance with ICH requirements. The method was found to be suitable for checking the quality of bulk samples of gatifloxacin at the time of batch release and also during storage.     

Development and validation of a rapid HPLC method for the determination of five banned fat-soluble colorants in spices using a narrow-bore monolithic column

This work reports a fast and simple liquid chromatographic method for the simultaneous determination of five banned fat-soluble synthetic colorants, namely Sudan I-IV and Para-Red, in spice samples. The analytes were successfully separated isocratically in less than 5 min on the new narrow bore monolithic column, FastGradient^® Chromolith (50 mm × 2.0 mm i.d.) using a mobile phase of 0.1% (v/v) HCOOH/acetonitrile (35/65%, v/v) at a flow rate of 1.5 mL min⁻¹. All colorants were detected at 506 nm. The main parameters (mobile phase composition, flow rate, injection volume) affecting the separation were studied. The proposed method was thoroughly validated in terms of linearity, LODs, precision and accuracy. The method was applied to the determination of the studied azo-dyes in various spices (paprika, chilli and mixed spice powders) after ultrasound-assisted extraction. Satisfactory recoveries, ranging from 92% to 109% were obtained.     

Determination of free captopril in human plasma by liquid chromatography with mass spectrometry detection

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of captopril after precolumn derivatization with p-bromo-phenacyl-bromide in human plasma was validated. Plasma samples were analysed on a monolithic column (Cromolith Performance-RP 18e, 100 mm × 4.6 mm I.D., 3 μm) under isocratic conditions using a mobile phase of a 40:60 (v/v) mixture of acetonitrile and 0.1% (v/v) formic acid in water. The flow rate was 1 mL/min at the column temperature of 30 °C. In these chromatographic conditions, the retention time was 4.4 min for captopril derivative. The detection of the analyte was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were 216, 253, 255, 268, 270 m/z derived from 415 m/z for derivatized captopril. The sample preparation was very simple and consisted in plasma protein precipitation from 0.2 mL plasma using 0.3 mL methanol after the derivatization reaction was completed. Calibration curves were generated over the range of 10-3000 ng/mL with values for coefficient of correlation greater than 0.993 and by using a weighted (1/y²) quadratic regression. The values for precision (CV %) and accuracy (relative error %) at quantification limit were less than 9.9% and 3.9%, for within- and between-run, respectively. The mean recovery of the analyte was 99%. Derivatized samples demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. This is the first reported LC-MS/MS method for analysis of captopril in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of the analyte. The validated LC-MS/MS method has been applied to a pharmacokinetic study of 50 mg captopril tablets on healthy volunteers.     

A Validated Stability Indicating RPLC Method for Tazarotene

A simple, isocratic, rapid and accurate reversed phase high performance liquid chromatography method was developed for the quantitative determination of tazarotene. The developed method is also applicable for the related substance determination in bulk drugs. The chromatographic separation was achieved on a Hypersil C18 (250 mm × 4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid:acetonitrile (15:85, v/v) as a mobile phase. The chromatographic resolutions between tazarotene and its potential impurity A and B were found greater than three. The limit of detection and limit of quantification of impurities were found to be 25 and 75 ng mL⁻¹. The percentage recovery of impurities in bulk drug sample was ranged from 96.8 to 103.5.The percentage recovery of tazarotene in bulk drug sample was ranged from 98.4 to 100.9. The developed RPLC method was validated with respect to linearity, accuracy, precision and robustness.     

Development and validation of a HPLC method for the determination of buprenorphine hydrochloride, naloxone hydrochloride and noroxymorphone in a tablet formulation

A simple isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) was developed for the simultaneous determination of buprenorphine hydrochloride, naloxone hydrochloride dihydrate and its major impurity, noroxymorphone, in pharmaceutical tablets. The chromatographic separation was achieved with 10 mmol L⁻¹ potassium phosphate buffer adjusted to pH 6.0 with orthophosphoric acid and acetonitrile (17:83, v/v) as mobile phase, a C-18 column, Perfectsil Target ODS3 (150 mm × 4.6 mm i.d., 5 μm) kept at 35 °C and UV detection at 210 nm. The compounds were eluted isocratically at a flow rate of 1.0 mL min⁻¹. The average retention times for naloxone, noroxymorphone and buprenorphine were 2.4, 3.8 and 8.1 min, respectively. The method was validated according to the ICH guidelines. The validation characteristics included accuracy, precision, linearity, range, specificity, limit of quantitation and robustness. The calibration curves were linear (r > 0.996) over the concentration range 0.22-220 μg mL⁻¹ for buprenorphine hydrochloride and 0.1-100 μg mL⁻¹ for naloxone hydrochloride dihydrate and noroxymorphone. The recoveries for all three compounds were above 96%. No spectral or chromatographic interferences from the tablet excipients were found. This method is rapid and simple, does not require any sample preparation and is suitable for routine quality control analyses.     

Enantiospecific resolution of rabeprazole by liquid chromatography on amylose-derived chiral stationary phase using photo diode array and polarimetric detectors in series

A simple and rapid liquid chromatographic method for enantioselective separation and determination of R-(+) and S-(−) enantiomers of rabeprazole in drugs and pharmaceuticals using photo diode array (PDA) and polarimetric detectors connected in series was developed. Chiralpak AD-H (250 mm × 4.6 mm) 5 μm column packed with amylose tris(3,5-dimethylphenyl carbamate) as a stationary phase and the mobile phase containing n-hexane:ethanol:2-propanol(75:15:10, v/v/v) in an isocratic mode has yielded baseline separation with resolution greater than 3.0 at 40 °C. Effects of ethanol, 2-propanol and temperature on separation were studied for optimum resolution. Lansoprazole sulphone was used as an internal standard (IS) for quantitative determination of individual enantiomers in bulk drugs as well as pharmaceutical formulations. The method was validated in terms of accuracy, precision and linearity according to ICH guidelines. The linearity of the method was studied in the range of 0.5-50 μg/ml and the r² was >0.9997. The inter- and intra-day precision of assay were determined (R.S.D. < 1%) and the recoveries were in the range of 99.63-100.22% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) were 0.02 μg/ml and 0.07 μg/ml for both the enantiomers, respectively.     

A liquid chromatography/positive ion tandem mass spectrometry method for the determination of cilazapril and cilazaprilat in human plasma

A simple, rapid, and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for simultaneous determination of cilazapril levels and its active metabolite, cilazaprilat, in human plasma using enalapril as internal standard. The acquisition was performed in the multiple reaction monitoring mode; monitoring the transitions: m/z 418.4 > 211.1 for cilazapril and m/z 390.3 > 211.1 for cilazaprilat. The method involves a simple single-step liquid-liquid extraction with ethyl acetate. The analyte was chromatographed on an YMC C₈ reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (10:90, v/v; pH 3.2 with formic acid). Numerous compounds did not interfere with specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted cilazapril, cilazaprilat, and enalapril within 2 min. This method was validated over 0.1-500 ng ml⁻¹ of cilazapril and 0.5-50 ng ml⁻¹ of cilazaprilat. Cilazapril and cilazaprilat were stable in standard solution and in plasma samples under typical storage and processing conditions. The assay was successfully applied to a pharmacokinetic study of cilazapril given as a single oral dose (5 mg) to healthy volunteers.     

Separation and determination of synthetic impurities of sildenafil (Viagra) by reversed-phase high-performance liquid chromatography.

A simple and rapid high-performance liquid chromatographic method for the separation and determination of process-related impurities of sildenafil was developed. The separation was achieved on a reversed-phase C18 column using acetonitrile-0.05 M potassium dihydrogen orthophosphate (70:30 v/v) as a mobile solvent at a flow rate of 1.0 ml/min and UV detection at 230 nm. The method was used not only for quality assurance, but also for monitoring the chemical reactions during the synthesis of sildenafil. It was found to be specific, precise and reliable for the determination of all process-related impurities of sildenafil in bulk drugs and formulations.     

Simultaneous determination of rosiglitazone and metformin in plasma by gradient liquid chromatography with UV detection

A novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in human plasma. The analysis was performed on a phenyl column (250 mm × 4.6 mm i.d., 5 μm) using a gradient method starting with mobile phase composed of acetonitrile:5 mM acetate buffer pH 5.5 (75:25, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The total run time was less than 10 min. Sample preparation included a simple protein precipitation step with acetonitrile. Validation experiments were performed to demonstrate stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 100 ng mL⁻¹ for rosiglitazone and 250 ng mL⁻¹ for metformin. The extraction recoveries were 100.02-105.0% for rosiglitazone and 105.64-103.88% for metformin. The method was applied with success to plasma samples obtained from diabetic patients undergoing treatment with rosiglitazone and metformin.     

A Validated, Specific Stability-Indicating RP-LC Method for Moxifloxacin and Its Related Substances

A validated, specific, stability-indicating reversed-phase liquid chromatographic method has been developed for quantitative analysis of moxifloxacin and its related substances in bulk samples and pharmaceutical dosage forms in the presence of degradation products and process-related impurities. Forced degradation studies were performed on bulk samples of moxifloxacin, in accordance with ICH guidelines, using acidic, basic, and oxidizing conditions, and thermal and photolytic stress, to show the stability-indicating power of the method. Significant degradation was caused by oxidative stress and by basic conditions; no degradation was observed under the other stress conditions. The method was optimized by analysis of the samples generated during the forced degradation studies and sample solutions spiked with the impurities. Good resolution between the analyte peak and peaks corresponding to process-related impurities and degradation products was achieved on a C₁₈ column with a simple linear mobile phase gradient prepared from aqueous sodium dihydrogen orthophosphate dihydrate containing triethylamine, pH adjusted to 3.0 with orthophosphoric acid, and methanol. Detection was performed at 240 nm. Limits of detection and quantification were established for moxifloxacin and its process related impurities. When the stressed test solutions were assayed against moxifloxacin working standard solution the mass balance was always between 99.3 and 100.1%, indicating the method was stability-indicating. The method was validated in accordance with ICH guidelines, and found to be suitable for checking the quality of bulk samples of moxifloxacin at the time of release of a batch and during storage (long term and accelerated stability testing was conducted).     

Determination of Tandospirone and its Impurities in Drug Formulations by LC-UV and LC-MS

This work describes the determination of tandospirone in bulk drug substance and formulated products by a reversed-phase liquid chromatographic method with UV detection. Chromatographic separation was performed on a C₁₈ column with a mobile phase of a binary mixture of methanol and water (70:30, v/v) delivered at a flow rate of 0.5 mL min^–1 and detection was performed at 243 nm. The proposed LC method is selective, precise and accurate for the determination of tandospirone in the presence of its manufacturing impurities with a limit of quantitation of 0.54 g mL^–1. A preliminary study for the identification of the major manufacturing impurities was made by liquid chromatography-mass spectrometry with electrospray ionization source operated in a positive ion mode.     

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC–MS method

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stability-indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film-coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ-C18, 250 × 4.6 mm, 5-μm particle) and mobile phase. Mobile phase A contained KH₂PO₄ buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 μL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r² = 0.9995–0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC–MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.     

Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions

High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol–water–DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76–26.28 μg mL⁻¹ for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL⁻¹, respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol–DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of ≥99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.     

Development and validation of a liquid chromatography-electrosprayionization mass spectrometric method for the determination of dexamethasone in sheep plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometric method for the determination of dexamethasone in sheep plasma has been developed and validated. The samples were extracted using solid-phase extraction cartridges with mixed reversed-phase materials (oasis-HLB). The chromatographic separation was performed on a reversed-phase XTerrra MS C₁₈ column ( mm; 5 μm) using a mobile phase consisting of 65% methanol in water containing 0.1% (v/v) formic acid, pumped at a flow rate of 0.30 ml min⁻¹. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The probe heater temperature was set at 260 °C, the capillary voltage was set at 3.5 kV and the source block voltage (AQA_max) was set at 30 V. The method was fully validated. Calibration graphs were linear (r better than 0.998, n=11), in concentration ranges 6-1000 ng ml⁻¹ for dexamethasone. The intra- and inter-day RSD values were less than 24.1% (n=6). The limits of detection and quantitation for dexamethasone were found to be 1 and 6 ng ml⁻¹, respectively. The efficiency of the solid phase extraction procedure was found to be 92.4% for dexamethasone. The method was further applied to a pilot kinetic study in order to assess the main pharmacokinetic parameters of dexamethasone in sheep.     

Stability indicating high-performance thin-layer chromatographic determination of gatifloxacin as bulk drug and from polymeric nanoparticles

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method for determination of gatifloxacin both as a bulk drug and from polymeric nanoparticles was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed thin-layer chromatography (TLC) aluminium plates precoated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-methanol-concentrated ammonia solution (25%) (5:1:0.9, v/v/v). This solvent system was found to give compact spots for gatifloxacin (R_f value of 0.60 ± 0.02). Densitometric analysis of gatifloxacin was carried out in the absorbance mode at 292 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9953 with respect to peak area in the concentration range of 400-1200 ng spot⁻¹. The mean value (±S.D.) of slope and intercept were 9.66 ± 0.05 and 956.33 ± 27.67, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 2.73 and 8.27 ng spot⁻¹, respectively. Gatifloxacin was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. The drug undergoes degradation under acidic and basic conditions and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of gatifloxacin as bulk drug and from polymeric nanoparticles is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one.     

Liquid-chromatographic separation and determination of process-related impurities, including a regio-specific isomer of celecoxib on reversed-phase C18 column dynamically coated with hexamethyldisilazane.

A simple and rapid reversed-phase high-performance liquid-chromatographic method for the separation and determination of process-related impurities of celecoxib (CXB) in bulk drugs and pharmaceuticals was developed. The separation of impurities viz., 4-methylacetophenone (I), 1-(4-methylphenyl)-4,4,4-trifluorobutane-1,3-dione (II), 4-hydrazinobenzene sulfonamide (III) and a regio-specific isomer [3-(4-methylphenyl)-5-trifluoromethyl-1H-pyrazole-1-yl]-benzenesulfonamide (IV), was accomplished on an Inertsil ODS-3 column dynamically coated with 0.1% hexamethyldisilazane (HMDS) in acetonitrile:water (55:45 v/v) as a mobile phase and detection at 242 nm using PDA at ambient temperature. The chromatographic conditions were optimized by studying the effects of HMDS, an organic modifier, time of silanization and column temperature. The method was validated and found to be suitable not only for monitoring the synthetic reactions, but also to evaluate the quality of CXB.     

Separation and determination of process-related impurities of erlotinib using reverse-phase HPLC with a photo-diode array detector

A simple and rapid reverse-phase high-performance liquid chromatographic (HPLC) method for the simultaneous separation and determination of erlotinib and its process-related impurities in bulk drugs has been developed. Five process-related impurities of erlotinib have been separated on an Inerstsil ODS-3V (C18) column and detected at 254 nm using a photo diode array (PDA). This HPLC method was successfully applied to the analysis of erlotinib bulk drug. The recoveries of erlotinib and process-related impurities were in the range of 92.86-106.23%, and found to be specific, precise and reliable for the determination of unreacted raw materials, intermediates in the reaction mixtures and bulk drugs.