AQbD-Oriented Development of a New LC Method for Simultaneous Determination of Telmisartan and Its Impurities

Analytical quality by design (AQbD)-oriented liquid chromatographic method development for determination of telmisartan and its impurities A, C, and 1 is determination is presented. Step-by-step process was conducted in order to define reliable design space. At the beginning, critical process parameters with the highest influence on method performance were defined: acetonitrile content in the first (ACN 1) and second (ACN 2) gradient step and time (t ₂) the second gradient step. These factors were varied according to Box–Behnken plan of experiments and their influence on retention times of impurities A and C, S value between telmisartan and impurity 1 and peak capacity were followed. In this way, the relationship between the critical process parameters and critical quality attributes was established. The obtained mathematical models and Monte Carlo simulations were used to identify the design space. Fractional factorial design was applied for experimental robustness testing, and the method was validated to verify the adequacy of selected optimal conditions. Finally, all validation parameters were tested, and adequacy of the method was confirmed. Applicability as a routine method was confirmed by analysis of commercially available tablets.     

A Stability-Indicating LC Method for the Simultaneous Determination of Telmisartan and Ramipril in Dosage Form

A simple, rapid, and precise method is developed for the quantitative simultaneous estimation of telmisartan and ramipril in combined pharmaceutical dosage form. A chromatographic separation of the two drugs was achieved with an ACE 5 C₁₈, 25-cm analytical column using buffer–acetonitrile (55:45 v/v). The buffer used in mobile phase contains 0.1 M sodium perchlorate monohydrate in double distilled water pH adjusted 3.0 with trifluoroacetic acid. The instrumental settings are flow rate of 1.5 mL min⁻¹, column temperature at 30 °C, and detector wavelength of 215 nm using a photodiode array detector. The resolution between ramipril and telmisartan were found to be more than 5. Theoretical plates for ramipril and telmisartan were 13,022 and 6,629. Tailing factor for ramipril and telmisartan was 0.94 and 0.98. Telmisartan, ramipril and their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analysed by the proposed method. Peak homogeneity data of telmisartan and ramipril is obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The described method shows excellent linearity over a range of 20–400 μg mL⁻¹ for telmisartan and 2.5–50 μg mL⁻¹ for ramipril. The correlation coefficient for telmisartan and ramipril are 1. The relative standard deviation for six measurements in two sets of each drug in tablets was always less than 2%. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of telmisartan and ramipril in pharmaceutical preparations.     

Determination of Carbamazepine in Pharmaceutical Dosage Form Using GC-FID

The present work describes the methodology and validation of gas chromatography with flame ionization detection (GC-FID) for the determination of carbamazepine with internal standard (diazepam) in pharmaceutical preparations. The method was linear from 2–30 μg mL⁻¹. The RSD values for precision was less than 9%, accuracy (relative error) was better than 11% (n = 6). The developed method was successfully applied for the assay of pharmaceutical dosage forms which do not require any preliminary separation or treatment of the samples. The RSD values for Tegretol^® tablets (200 mg) and Karberol^® tablets (200 mg) was found to be 4.03 and 3.25%, respectively. The results obtained from this method were compared with the reference method (LC) reported in literature and no significant difference was found statistically.

     

Optimization and Validation of an LC Method for the Determination of Cefdinir in Dosage Form and Human Urine

A simple and reliable liquid chromatographic method has been developed and validated for the determination of cefdinir in human urine and capsule samples. A chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of potassium dihydrogen phosphate (10 mM, pH 4.5)–acetonitrile (90:10, v/v). Quantitation was achieved with UV detection at 285 nm, based on peak area with linear calibration curve at a concentration range of 0.7–39 µg mL^?1. This method was successfully applied for the establishment of an urinary excretion pattern after oral dose.     

Optimization and Validation of an LC Method for the Determination of Cefdinir in Dosage Form and Human Urine

A simple and reliable liquid chromatographic method has been developed and validated for the determination of cefdinir in human urine and capsule samples. A chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of potassium dihydrogen phosphate (10 mM, pH 4.5)–acetonitrile (90:10, v/v). Quantitation was achieved with UV detection at 285 nm, based on peak area with linear calibration curve at a concentration range of 0.7–39 µg mL⁻¹. This method was successfully applied for the establishment of an urinary excretion pattern after oral dose.

     

Simultaneous Determination of Diclofenac Sodium and Rabeprazole Sodium in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid, precise and accurate reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of diclofenac sodium and rabeprazole sodium from pharmaceutical formulations. The method was developed using a HiQ SiL C18 (250 mm × 4.6 mm i.d.) column with a mobile phase consisting of methanol:water, (80:20 v/v), at a flow rate of 1.25 mL min⁻¹. Detection was carried out at 284 nm. Indapamide was used as an internal standard. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the estimation of these drugs in combined dosage forms.

     

Simultaneous Determination of Diclofenac Sodium and Rabeprazole Sodium in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid, precise and accurate reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of diclofenac sodium and rabeprazole sodium from pharmaceutical formulations. The method was developed using a HiQ SiL C18 (250 mm × 4.6 mm i.d.) column with a mobile phase consisting of methanol:water, (80:20 v/v), at a flow rate of 1.25 mL min^?1. Detection was carried out at 284 nm. Indapamide was used as an internal standard. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

Development and Validation of a Stability-Indicating LC Method for Determination of Bexarotene in Softgel Dosage Formulation

This study focuses on a novel liquid chromatographic approach that has been developed and approved for the quantitative determination of bexarotene (BXT), its potential impurities in drug substances and drug products. Chromatographic separation was developed on a Symmetry C₈ (150 × 4.6) mm 5-µm column with a mobile phase containing an isocratic mixture of acetonitrile:DI water:glacial acetic acid (650:350:7.5) v/v/v at a flow rate of 1.2 mL min^?1, and quantitation was carried out using ultraviolet detection at 262 nm for BXT and 290 nm for BHA with a column temperature of 35 °C. The resolution among butylated hydroxyanisole (BHA), BXT and its process-related impurity-A was found to be greater than 5. Regression analysis confers an R value (correlation coefficient) higher than 0.998 for BHA, BXT and impurity-A. The detection level for BXT impurities was found at a level below 0.03% (0.18 µg mL^?1). The inter- and intra-day precisions for BHA, BXT and impurities were evaluated and found to have a %RSD of less than 3.0.     

Development and Validation of LC Method for Simultaneous Determination of Two Binary Mixtures Containing Indapamide

A new sensitive, simple, rapid, and precise RP LC method with hydrochlorothiazide as internal standard has been developed for resolving two binary mixtures, perindopril with indapamide and captopril with indapamide, in pharmaceutical formulations. The drugs were separated at room temperature on a 250 mm × 4.6 mm, 5-μm particle, cyanopropyl column with 10 mm KH₂PO₄, pH 6.0-methanol 55:45 (v/v) as mobile phase at a flow rate of 1 mL min^?1. Detection was at 210 nm. Factors affecting the separation process were studied and optimized. The linearity, accuracy, and precision of the method were good, and the method was successfully applied to the determination of the two binary combinations in synthetic mixtures and commercial pharmaceutical products.     

Stability Indicating HPTLC and LC Determination of Dasatinib in Pharmaceutical Dosage Form

Two sensitive and reproducible methods are described for the quantitative determination of dasatinib in the presence of its degradation products. The first method was based on high performance thin layer chromatography (HPTLC) followed by densitometric measurements of their spots at 280 nm. The separation was on HPTLC aluminium sheets of silica gel 60 F₂₅₄ using toluene:chloroform (7.0:3.0, v/v). This system was found to give compact spots for dasatinib after development (R _F value of 0.23 ± 0.02). The second method was based on high performance liquid chromatography (HPLC) of the drug from its degradation products on reversed phase, PerfectSil column [C₁₈ (5 μm, 25 cm × 4.6 mm, i.d.)] at ambient temperature using mobile phase consisting of methanol:20 mM ammonium acetate with acetic acid (45:55, v/v) pH 3.0 and retention time (t _R = 8.23 ± 0.02 min). Both separation methods were validated as per the ICH guidelines. No chromatographic interference from the tablet excipients was found. Dasatinib was subjected to acid–alkali hydrolysis, oxidation, dry heat, wet heat and photo-degradation. The drug was susceptible to acid–alkali hydrolysis and oxidation. The drug was found to be stable in neutral, wet heat, dry heat and photo-degradation conditions. As the proposed analytical methods could effectively separate the drug from its degradation products, they can be employed as stability indicating.     

Stability-Indicating LC Determination of Nitazoxanide in Bulk Drug and in Pharmaceutical Dosage Form

A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for quantitative determination of nitazoxanide in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced decomposition studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5- C18 (250 mm × 4.6 mm, 5 μm) column, and 50 mM ammonium acetate (pH 5.5 by acetic acid) and acetonitrile (55:45 v/v) as a mobile phase. The detection was carried out at a wavelength of 240 nm. The nitazoxanide was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for nitazoxanide in base, acid and in 30% H₂O₂ conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of nitazoxanide was from (100.55 to 101.25%) in the pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, specificity and robustness. The forced degradation studies prove the stability indicating power of the method.     

Development and Validation of an Improved LC Method for the Simultaneous Determination of Pirfenidone and Its Carboxylic Acid Metabolite in Human Plasma

A sensitive and specific assay based on liquid chromatography with ultraviolet detection was developed for the simultaneous determination of pirfenidone (PFD), a novel antifibrotic agent, and its carboxylic acid metabolite in human plasma. The carboxylic acid metabolite was further identified by mass spectrometric analysis. PFD, its carboxylic acid metabolite and the internal standard methyl-p-aminobenzoate were extracted from plasma by a simple one-step liquid-liquid extraction with ethyl acetate and subsequently separated on a Zorbax SB-C18 column with a mobile phase of trifluoroacetic acid–triethylamine–acetonitrile–water (0.1:0.15:28:71.75, v/v/v/v) and monitored at 314 nm. Extraction recovery was over 70% in plasma. The calibration curves were linear over the concentration range of 0.05–25 μg mL^?1. The limit of detection (LOD) and lower limit of quantitation (LLOQ) in human plasma were 10 and 50 ng mL^?1, respectively. Intra- and inter-assay precision of the method were within 8.6%. The accuracy as expressed by the bias ranged between ?4.5 and 4.0%. The method was successfully applied to determine pharmacokinetic parameters of PFD and its carboxylic acid metabolite after a single oral dose of 200 mg of PFD in healthy volunteers.     

Development and Validation of Hptlc Method for Simultaneous Estimation of Propranolol Hydrochloride and Flunarizine Dihydrochloride in Combined Tablet Dosage Form

JPC – Journal of Planar Chromatography – Modern TLC - A simple, precise, and accurate high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of...     

Stability-Indicating LC Method for the Determination of Olmesartan in Bulk Drug and in Pharmaceutical Dosage Form

A novel stability-indicating LC assay method was developed and validated for quantitative determination of olmesartan in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced degradation studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5-C18 (250 mm × 4.6 mm, 5 μm) column, and 50 mM ammonium acetate (pH-5.5 by acetic acid) and acetonitrile (70:30 v/v) as a mobile phase. The detection was carried out at the wavelength of 235 nm. The olmesartan was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for olmesartan in acid, base and in 30% H₂O₂ conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of olmesartan ranged from (99.89 to 100.95%) in pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, specificity and robustness. The forced degradation studies prove the stability-indicating power of the method.

     

Simultaneous Determination of Etodolac and Acetaminophen in Tablet Dosage Form by RP-LC

A simple, specific, precise and accurate reverse phase liquid chromatographic (RP-LC) method has been developed for the simultaneous determination of etodolac and acetaminophen in tablet dosage form. The chromatographic separation was achieved on a BDS Hypersil C18, 100 mm × 4.6 mm, 5 μm column at a detector wavelength of 274 nm using an isocratic mobile phase consisting of a mixture of 0.05% aqueous orthophosphoric acid and acetonitrile in the ratio of 50:50 (v/v) at a flow rate of 1.0 mL min^?1. The retention times for etodolac and acetaminophen were found to be 1.32 and 4.24 min, respectively. The method was validated for the parameters like specificity, linearity, precision, accuracy and robustness. The method was found to be specific and stability indicating as no interfering peaks of impurities, degradent and excipients were observed. The square of correlation coefficients (R ²) for etodolac and acetaminophen were 0.9996 and 0.9998 while percentage recoveries were 101.32 and 100.94%, respectively. Intra- and inter-day relative standard deviations for both the components were <2.0%. The proposed RP-LC method can be applied for the routine analysis of commercially available formulations of these drugs either as such or in combination.     

Development and Validation of a Novel Gradient LC Method for Simultaneous Determination of Isoniazid and Acetylisoniazid in Human Plasma

The goal of this study was to develop and validate a new gradient high-performance liquid chromatography method for the simultaneous determination of isoniazid (INH) and acetylisoniazid (Ac-INH) in human plasma samples. A C18 reversed-phase column was employed for separation followed by UV detection at 266 nm. The calibration involved the use of five concentration levels ranging from 1 to 20 μg mL^?1 for both analytes. The developed method was validated using ICH guidelines. The calibration curve was found to be linear with correlation coefficient values (r ²) above 0.9991 and the highest RSD% values for intra-day assays were found to be 6.34 and 2.57% for INH and Ac-INH, respectively. The highest RSD% values for inter-day assays were 9.31 and 10.17% for INH and Ac-INH, respectively. LOD was calculated to be 0.1 and 0.15 μg mL^?1 for INH and Ac-INH, respectively. LOQ was calculated to be 0.33 and 0.5 μg mL^?1 for INH and Ac-INH, respectively.     

Development and Validation of a Stability-Indicating UPLC Method for the Determination of Hexoprenaline in Injectable Dosage Form Using AQbD Principles

A novel and efficient stability-indicating, reverse phase ultra-performance liquid chromatographic (UPLC^®) analytical method was developed and validated for the determination of hexoprenaline in an injectable dosage form. The development of the method was performed using analytical quality by design (AQbD) principles, which are aligned with the future requirements from the regulatory agencies using AQbD principles. The method was developed by assessing the impact of ion pairing, the chromatographic column, pH and gradient elution. The development was achieved with a Waters Acquity HSS T3 (50 × 2.1 mm i.d., 1.8 µm) column at ambient temperature, using sodium dihydrogen phosphate 5 mM + octane-1-sulphonic acid sodium salt 10 mM buffer pH 3.0 (Solution A) and acetonitrile (Solution B) as mobile phases in gradient elution (t = 0 min, 5% B; t = 1 min, 5% B; t = 5 min, 50% B; t = 7 min, 5% B; t = 10 min, 5% B) at a flow rate of 0.5 mL/min and UV detection of 280 nm. The linearity was proven for hexoprenaline over a concentration range of 3.50–6.50 µg/mL (R² = 0.9998). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability and injectable dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with enhanced knowledge, which can eventually lead to a reduced regulatory risk, high quality data and lower operational costs.     

Stability-Indicating LC Method for Analysis of Lornoxicam in the Dosage Form

A simple, rapid, and precise method has been developed for quantitative analysis of lornoxicam (Lxm) in pharmaceutical dosage forms. Chromatographic separation of Lxm and its degradation products was achieved on a C₁₈ analytical column with 0.05% (v/v) aqueous trifluoroacetic acid–acetonitrile, 70:30 (v/v), as mobile phase. The flow rate was 1.0 mL min^?1, the column temperature 30 °C, and detection was by absorption at 295 nm using a photodiode-array detector. The number of theoretical plates and tailing factor for Lxm were 6,577 and 1.03, respectively. Lxm was exposed to thermal, photolytic, hydrolytic, and oxidative stress, and the stressed samples were analysed by use of the proposed method. Peak homogeneity data for Lxm in the chromatograms from the stressed samples, obtained by use of the photodiode-array detector, demonstrated the specificity of the method for analysis of Lxm in the presence of the degradation products. The linearity of the method was excellent over the range 10–200 μg mL^?1 Lxm. The correlation coefficient was 0.9999. Relative standard deviations of peak areas from six measurements were always less than 2%. The proposed method was found to be suitable and accurate for quantitative analysis of Lxm and study of its stability.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min⁻¹. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL⁻¹ for isomer I and 0.40–2.40 µg mL⁻¹ for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL⁻¹ for isomer I and 0.0356 and 0.1078 µg mL⁻¹ for isomer II, respectively.

     

RP-HPLC Method for Simultaneous Determination of Sofosbuvir and Ledipasvir in Tablet Dosage Form and Its Application to In Vitro Dissolution Studies

A reversed-phase high-performance liquid chromatographic method was developed for the simultaneous determination of sofosbuvir and ledipasvir in tablet dosage form. The analysis was performed on Luna analytical column 250 × 4.6 mm, 5 µm, octyl silica packing (Si–[CH₂]₇–CH₃) C8, using ammonium acetate buffer solution pH 7.0 and acetonitrile 35:65 % v/v as mobile phase at flow rate of 0.7 mL min⁻¹ for isocratic elution. Detection of sofosbuvir and ledipasvir was performed on a UV detector at 245 nm. The retention times of sofosbuvir and ledipasvir were 4.468 ± 0.013 min and 8.242 ± 0.012 min, respectively, and the total run time was 20 min. The method was validated according to the requirements of the United States Pharmacopeia (category I). The overall recovery of both analytes was 100 ± 1 %; the relative standard deviation for precision and intraday precision was less than 2.0 %. The method was linear with correlation coefficient (r) >0.9999, limits of detection 0.485 and 0.175 µg mL⁻¹, and limits of quantification was 1.619 and 0.586 µg mL⁻¹ for sofosbuvir and ledipasvir, respectively. The method was successfully applied to the assay and in vitro dissolution studies of sofosbuvir and ledipasvir in tablet dosage form.