Simultaneous Determination Of Related Substances Research Articles

Simultaneous Analysis of Ipratropium Bromide and its Related Substances Using HPLC

Background: The main goal of process-related substances investigations is to understand how impurities should be controlled during the synthetic or purification process to produce a high-quality drug substance. The simultaneous determination of related substances is a very important factor in the quality control of API and pharmaceutical dosage forms. Methods: A new HPLC method was developed for the simultaneous analysis of Ipratropium bromide and its seven related substances. The separation was performed on a waters HPLC ZORBAX Eclipse XDB- C8 column (150 × 4.6 mm, 3.5 μm). The gradient method used mobile phases containing methanol, acetonitrile, trifluoroacetic acid (TFA), water: A: (methanol, acetonitrile, TFA, 500:500:0.3 v/v/v) and B (0.3% TFA in water v/v). The gradient elution conditions were as follows: 0-32.0 min, linear from 10% A to 35% A; 32.0-36.0 min isocratic 35% A; 36.0-42.0 min linear from 35% to 10%A. The wavelength by UV detector was 220 nm and the flow rate was 1.2 mL/min. The injection volume of sample was 20 μL, and the column temperature was 30 ºC. The proposed RP-HPLC method was applied for the determination of seven related substances. Results: All standard curves obtained exhibited good linear regression (r > 0.9939) within the tested range. The average recovery rates were in the range of 98.2-102.0% and RSD was less than 2.9% (n = 3). The process-related impurities were not observed in different batches (<0.025% purity). Conclusion: The method was applied to determine 7 process-related substances in ipratropium bromide and proved to be robust, linear, repeatable, sensitive, selective and easy to perform. The results are valuable for quality control in the manufacturing of ipratropium bromide.

A reliable HPLC-DAD method for simultaneous determination of related substances in TBI-166 active pharmaceutical ingredient

A sensitive, stability-indicating reversed-phase high-performance liquid chromatography with diode array detection (HPLC–DAD) method has been developed for the determination of TBI-166 and its 10 kinds of related impurities. Chromatographic separation was achieved on a Kromasil ODS column (250 mm × 4.6 mm, 5 μm), with a gradient elution of the mobile phase system consisting of acetonitrile and 1% ammonium formate solution (with 0.2% formic acid). The flow rate was 1.0 mL/min, and the detection wavelength was set at 251 nm. The method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to selectivity, linearity, limits, accuracy, precision, and robustness. The calibration curves were linear from LOQ to 150% of the specification limit of impurity with correlation coefficients not less than 0.999. The limits of quantitation were between 0.123 and 0.257 μg/mL. Accuracy for the related substances was estimated by the recovery ranged from 94.6% to 111.2%. The method was proved to be reliable for the determination of related substances in TBI-166 bulk drug, which is essential and important in the quality control.

A simultaneous determination of related substances by high performance liquid chromatography in a drug product using quality by design approach

The combination of Abacavir, Lamivudine and Dolutegravir is an anti-retroviral formulation that displays high efficacy and superiority in comparison to other anti-retroviral combinations. Analysis of related substances in this combination drug product was very challenging due to the presence of nearly thirty peaks including the three active pharmaceutical ingredients (APIs), eleven known impurities and other pharmaceutical excipients. Objective of this study was to develop a single, selective, and robust high performance liquid chromatography method for the efficient separation of all peaks. Initially, one-factor-at-a-time (OFAT) approach was adopted to develop the method. But, it could not resolve all the critical peaks in such complex matrix. This led to the advent of two different HPLC methods for the determination of related substances, one for Abacavir and Lamivudine and the other for Dolutegravir. But, since analysis of a single sample using two methods instead of one is time and resource consuming and thus expensive, an attempt was made to develop a single and robust method by adopting quality by design (QbD) principles. Design of Experiments (DoE) was applied as a tool to achieve the optimum conditions through Response surface methodology with three method variables, pH, temperature, and mobile phase composition. As the study progressed, it was discovered that establishment of the design space was not viable due to the completely distant pH requirements of the two responses, i.e. (i) retention time for Lamivudine carboxylic acid and (ii) resolution between Abacavir impurity B and unknown impurity. Eventually, neglecting one of these two responses each time, two distinguished design spaces have been established and verified. Edge of failures at both design spaces indicate high probability of failure. It therefore, becomes very important to identify the most robust zone or normal operating range (NOR) within the design space with low risk of failure and high quality assurance. For NOR establishment, Monte Carlo simulation was performed on the basis of which process capability index (Cpk) was derived. Finally, the selectivity issue problem faced due to the pH dependency and the dissimilar pH needs of the two critical responses was resolved by introducing pH gradient into the program. This new ternary gradient program has provided a single robust method. Thus, two HPLC methods for the analysis of the combination drug product have been replaced with a selective, robust, and cost effective single method.

Simultaneous determination of related substances of telmisartan and hydrochlorothiazide in tablet dosage form by using reversed phase high performance liquid chromatographic method

Objective:Telmisartan is a potent, long-lasting, nonpeptide antagonist of the angiotensin II type-1 (AT1) receptor that is indicated for the treatment of essential hypertension. Hydrochlorothiazide is a widely prescribed diuretic and it is indicated for the treatment of edema, control of essential hypertension and management of diabetes insipidus. In the current article a new, accurate, sensitive, precise, rapid, reversed phase high performance liquid chromatography (RP-HPLC) method was developed for determination of related substances of Telmisartan and Hydrochlorthiazide in tablet dosage form.Materials and Methods:Simultaneous determination of related substances was performed on Kromasil C18 analytical column (250 × 4.6 mm; 5μm pertical size) column at 40°C employing a gradient elution. Mobile phase consisting of solvent A (solution containing 2.0 g of potassium dihydrogen phosphate anhydrous and 1.04 g of Sodium 1- Hexane sulphonic acid monohydrate per liter of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (mixture of Acetonitrile: Methanol in the ratio 80:20 v/v) was used at a flow rate of 1.0 ml min–1. UV detection was performed at 270 nm.Results:During method validation parameter such as precision, linearity, accuracy, specificity, limit of detection and quantification were evaluated, which remained within acceptable limits.Conclusions:HPLC analytical method is linear, accurate, precise, robust and specific, being able to separate the main drug from its degradation products. It may find application for the routine analysis of the related substances of both Telmisartan and Hydrochlorthiazide in this combination tablets.

Development and validation of a stability indicating RP-HPLC method for the simultaneous determination of related substances of albuterol sulfate and ipratropium bromide in nasal solution

A simple, sensitive and specific RP-HPLC method was developed for the quantification of related impurities of albuterol sulfate (AS) and ipratropium bromide (IB) in liquid pharmaceutical dosage form. The chromatographic separation employs gradient elution using an inertsil C8-3, 250 mm × 4.6 mm, 5 μm columns. Mobile phase consisting of solvent A (solution containing 2.5 g of potassium dihydrogen phosphate and 2.87 g of heptane-1-sulfonic acid sodium salt per liter of water, adjusted to pH 4 with orthophosphoric acid) and solvent B (acetonitrile) delivered at a flow rate of 1.0 ml min −1. The analytes were detected and quantified at 210 nm using photodiode array (PDA) detector. The method was validated as per ICH guidelines, demonstrating to be accurate and precise (repeatability and intermediate precision level) within the corresponding linear range of known impurities of AS and IB. The specificity of the method was investigated under different stress conditions including hydrolytic, oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take place under hydrolytic and oxidative conditions. Robustness against small modification in pH, column oven temperature, flow rate and percentage of the mobile phase composition was ascertained. Lower limit of quantification and detection were also determined. The peak purity indices (purity angle < purity threshold) obtained with the aid of PDA detection and satisfactory resolution between related impurities established the specificity of the determination. All these results provide the stability indicating capability of the method.

Development and Validation of a Reversed-Phase HPLC Method for Separation and Simultaneous Determination of Process-Related Substances of Mirtazapine in Bulk Drugs and Formulations

A simple and rapid reversed-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of related substances of mirtazapine in bulk drugs and pharmaceutical formulations. Six impurities, including one degradation product of mirtazapine, have been separated on a BDS Hypersil (4.6 x 250 mm; particle size 5 microm) column with a mobile phase consisting of 0.3% triethylamine (pH 3.0)-acetonitrile (78:22 v/v) eluted in an isocratic mode and monitored with a photo diode array detector at 215 nm. The chromatographic behavior of all the analytes was studied under variable compositions of different solvent systems, temperatures, buffer concentrations, and pH values. The method was validated in terms of accuracy, precision, and linearity. The inter- and intra-day assay precision was found to be < 0.98% [relative standard deviation; (RSD)] and the recoveries were in the range 95.54-102.22% with RSD < 2.21%. The correlation coefficients for calibration curves for mirtazapine as well as impurities were in the range of 0.9941-0.9999. The method was successfully applied to the analysis of commercial formulations and the recoveries of mirtazapine were in the range of 99.38-100.73% with < 0.52% RSD. The method is useful not only for rapid evaluation of the purity of mirtazapine, but also for the simultaneous determination of related substances in bulk drugs and pharmaceutical formulations.

Development and validation of a capillary electrophoresis method for the simultaneous determination of impurities of escitalopram including the R-enantiomer

A stereospecific capillary electrophoresis assay for the simultaneous determination of related substances and the enantiomeric purity of escitalopram was developed by a central composite face-centered factorial design and subsequently validated. Separations were carried out in a 50 μm, 47/40 cm fused-silica capillary. The optimized conditions included 20 mM phosphate buffer, pH 2.5, containing 0.5 mg/ml β-cyclodextrin and 22 mg/ml sulfated β-cyclodextrin as background electrolyte, an applied voltage of −20 kV and a temperature of 28 °C. Salicylic acid was used as internal standard. The assay was validated for the ( R)-enantiomer of citalopram and the enantiomers of the impurity citadiol in the range of 2.5–150 μg/ml and 2.5–50 μg/ml, respectively. The limit of detection was 0.02% for all compounds, the limit of quantitation 0.05%, relative to a concentration of escitalopram of 5 mg/ml. Intraday precision of migration time and peak area ratio were in the range of 0.17–0.44% and 1.64% and 6.25%, respectively. Relative standard deviations of interday precision ranged between 0.84% and 1.85% in the case of migration times and between 5.20% and 9.28% for peak area ratio. The assay was applied to the determination of the purity of escitalopram in bulk drug and tablets. ( R)-Citalopram and ( S)-citadiol were detected as impurities.