Simple and rapid HPLC method for simultaneous determination of atenolol and chlorthalidone in spiked human plasma

A simple, sensitive and rapid chromatographic method was developed and validated for the simultaneous quantification of atenolol and chlorthalidone in human plasma using hydrochlorothiazide as internal standard (IS). The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyl column with isocratic elution with 10 mM KH₂PO₄ (pH 6.0) – methanol (70:30, v/v) at ambient temperature with flow rate of 1 mL min⁻¹ and UV detection at 225 nm. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range of 0.1–10 μg mL⁻¹. The method was validated in terms of accuracy, precision, absolute recovery, freeze–thaw stability, bench-top stability and re-injection reproducibility. The within- and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze–thaw cycles (deviation <15%). The proposed method was specific for the simultaneous determination of atenolol and chlorthalidone in human plasma where there was no interference from endogenous biological substances.     

Transmission FTIR derivative spectroscopy for estimation of furosemide in raw material and tablet dosage form

A Fourier transform infrared derivative spectroscopy (FTIR-DS) method has been developed for determining furosemide (FUR) in pharmaceutical solid dosage form. The method involves the extraction of FUR from tablets with N,N-dimethylformamide by sonication and direct measurement in liquid phase mode using a reduced path length cell. In general, the spectra were measured in transmission mode and the equipment was configured to collect a spectrum at 4 cm⁻¹ resolution and a 13 s collection time (10 scans co-added). The spectra were collected between 1400 cm⁻¹ and 450 cm⁻¹. Derivative spectroscopy was used for data processing and quantitative measurement using the peak area of the second order spectrum of the major spectral band found at 1165 cm⁻¹ (SO₂ stretching of FUR) with baseline correction. The method fulfilled most validation requirements in the 2 mg/mL and 20 mg/mL range, with a 0.9998 coefficient of determination obtained by simple calibration model, and a general coefficient of variation <2%. The mean recovery for the proposed assay method resulted within the (100±3)% over the 80%–120% range of the target concentration. The results agree with a pharmacopoeial method and, therefore, could be considered interchangeable.KEY WORDS: FTIR, Derivative spectroscopy, Furosemide, Frusemide, Pharmaceutical analysisAbbreviations: API, active pharmaceutical ingredient; DMF, N,N-dimethylformamide; DS, derivative spectroscopy; FTIR, Fourier transform infrared; FUR, furosemide or frusemide; HPLC, high performance liquid chromatographic; MIR, mid infrared     

Determination of mirtazapine in spiked human plasma and tablets by first derivative spectrofluorimetric method

A sensitive first derivative spectrofluorimetric method (¹D-spectrofluorimetry) was developed for the determination of mirtazapine. Calibration graph for mirtazapine determination was established using the first derivative amplitudes of the mirtazapine emission spectrum (λ_ex = 314 nm) in 0.1 M sulphuric acid measured at 375–435 nm from peak to peak, as the analytical signals. Moreover, the ratio of ¹D-spectrophotometric peak amplitudes at these wavelengths was calculated and used for the detection of the presence of interferences. Linearity range was found to be between 1 and 40 ng ml⁻¹ with correlation coefficient (r) = 0.9999. The limit of quantitation (LOQ) was 1.0 ng ml⁻¹ and the limit of detection (LOD) was 0.2 ng ml⁻¹. The proposed method was validated according to ICH; and it has been applied for the drug determination in human plasma without prior extraction and in tablets. The proposed method’s accuracy, reproducibility, selectivity and simplicity suggest its application in quality control analysis of the drug.     

Determination of lamivudine and stavudine in pharmaceutical preparations using chemometrics-assisted spectrophotometry

A simple chemometrics-assisted spectrophotometric method for the simultaneous determination of lamivudine and stavudine in pharmaceutical tablets is described. The UV absorption spectra of the studied drugs, in the range of 200–310 nm, showed a considerable degree of spectral overlapping ([D_i]^0.5 = 94.9%). Resolution of the mixture has been accomplished by using classical least-squares regression analysis (CLS) and principle components regression analysis methods (PCR). Beer’s law was obeyed for both drugs in the general concentration ranges of 2–12 and 3–15 μg ml⁻¹ for lamivudine and stavudine, respectively. The proposed methods were successfully applied for the determination of the two drugs in laboratory prepared mixtures. The overall recoveries percent were found 98.58 ± 1.53–101.30 ± 1.35 (CLS) and 98.62 ± 1.65–101.13 ± 1.04 (PCR) for lamivudine and 98.43 ± 1.62–99.42 ± 1.55 (CLS) and 98.23 ± 1.97–101.20 ± 1.79 (PCR) for stavudine, respectively. The commercial tablets percentage content was found 98.10 ± 2.5–102.47 ± 2.94 (CLS) and 99.12 ± 1.71–100.92 ± 1.54 (PCR) for lamivudine and 96.00 ± 2.94–98.17 ± 1.72 (CLS) and 97.40 ± 1.55–97.80 ± 1.92 (PCR) for stavudine, respectively. Good percentage recoveries and proper statistical data obtained with both the laboratory prepared mixtures and the commercial tablets proved the suitability and efficiency of the proposed procedures for routine analysis and quality control purposes with quite satisfactory precision. A comparison of the obtained results from CLS and PCR were also performed with those obtained from reported method. The obtained F- and t-values obtained indicating no significant differences between the results of the proposed and reported methods.     

Optimization of HPLC method for determination of cefixime using 2-thiophenecarboxaldehyde as derivatizing reagent: A new approach

The determination of cefixime 1 has clinical and analytical importance due to its broad spectrum antimicrobial activity and stability. Cefixime is a significant member of orally active third generation cephalosporin and has excellent activity against many pathogens. It is for first time that we have developed a new HPLC–DAD method for analysis of imine derivative 3 of cefixime by using reflux method at 100 °C for 50 min without any buffer solution. 2 Thiophenecarboxaldehyde (2TCA) 2 was used first time as a derivatizing reagent for cefixime drug. Furthermore, separation of three components, i.e. drug (cefixime), reagent (2TCA) and derivative was carried out using kromasil 100 C-18 (15 mm × 0.46 mm, 5 μm) column with isocratic elution of methanol: 0.1% aqueous formic acid (70:30 v/v) with flow rate of 1 ml min⁻¹ at retention time of 1.8, 2.4 and 3.3 min, respectively; while, total run time was 5 min. The developed method was repeatable with a relative standard deviation (RSD) of 0.81–1.88% for imine derivative. The limit of detection and quantification of imine derivative 3 were obtained within the range of 0.132–0.401 μg ml⁻¹ and compared with cefixime drug as 0.30–0.90 μg ml⁻¹, respectively. However, the formation of imine derivative 3 was confirmed by comparing peak height, retention time and spectral changes. The method is rapid, simple, very stable and accurate for the separation and determination of imine derivative 3 of cefixime 1.     

Determination of Cephalexin Monohydrate in Pharmaceutical Dosage Form by Stability-Indicating RP-UFLC and UV Spectroscopic Methods

An ultra-fast liquid chromatographic method and two UV spectroscopic methods were developed for the determination of cephalexin monohydrate in pharmaceutical dosage forms. Isocratic separation was performed on an Enable C₁₈G column (250 mm × 4.6 mm i.d., 5 μm) using methanol:0.01 M TBAHS (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min. The PDA detection wavelength was set at 254 nm. The UV spectroscopic method was performed at 261 nm and at 256–266 nm for the AUC method using a phosphate buffer (pH=5.5). The linearity was observed over a concentration range of 1.0–120 μg/ml for UFLC and both of the UV spectroscopic methods (correlation coefficient=0.999). The developed methods were validated according to ICH guidelines. The relative standard deviation values for the intraday and interday precision studies were < 2%, and the accuracy was > 99% for all of the three methods. The developed methods were used successfully for the determination of cephalexin in dry syrup formulation.     

Development and validation of a stability indicating UPLC method for determination of ticlopidine hydrochloride in its tablet formulation

The objective of the current study was the development of a simple, precise and accurate isocratic reversed-phase stability indicating Ultra Performance Liquid Chromatography [UPLC] assay method and validated for determination of ticlopidine hydrochloride in solid pharmaceutical dosage forms. Isocratic separation was achieved on a Zorbax SB-C18 (50 mm × 4.6 mm, 1.8 μm) column using mobile phase of methanol–0.01 M ammonium acetate buffer, pH 5.0 (80:20, v/v) at a flow rate of 0.8 ml min⁻¹, the injection volume was 4.0 μl and the detection was carried out at 235 nm by using photo-diode array detector. The drug was subjected to oxidation, hydrolysis, photolysis and heat to apply stress condition. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was linear in the drug concentration range of 62.5–375 μg ml⁻¹ with a correlation coefficient of 0.9999. The precision (relative standard deviation – RSD) of six samples was 1.31% for repeatability and the intermediate precision [RSD] among six-sample preparation was 0.77%. The accuracy (recovery) was between 98.80% and 101.50%. Degradation products produced as a result of stress studies did not interfere with detection of ticlopidine hydrochloride and the assay can thus be considered stability indicating.     

Development and Validation of a Stability-Indicating Capillary Electrophoresis Method for the Determination of Zolpidem Tartrate in Tablet Dosage Form with Positive Confirmation using 2D- and 3D-DAD Fingerprints

The aim of the current study was to develop a simple, precise, and accurate capillary zone electrophoresis method for the determination of zolpidem tartrate in tablet dosage form. Separation was conducted in normal polarity mode at 25°C, 22 kV, using hydrodynamic injection for 10 s. Separation was achieved using a background electrolyte of 20 mM disodium hydrogen phosphate adjusted with phosphoric acid (85%), pH at 5.50, and detection at 254 nm. Using the above optimized conditions, complete determination took place in less than 3 min using amiloride HCl as the internal standard. The method was linear over the range of 3–1000 μg mL⁻¹ with a correlation coefficient of 0.9999. Forced degradation studies were conducted by introducing a sample of zolpidem tartrate standard and pharmaceutical sample solutions to different forced degradation conditions, being neutral (water), basic (0.1 M NaOH), acidic (0.1 M HCl), oxidative (10% H₂O₂), temperature (60°C in oven for 3 days), and photolytic (exposure to UV light at 254 nm for 2 h). Degradation products resulting from the stress studies did not interfere with the detection of zolpidem tartrate and the assay can be considered stability-indicating.     

Stability-Indicating RP-HPLC Method for the Determination of Ambrisentan and Tadalafil in Pharmaceutical Dosage Form

A simple, rapid, and highly selective RP-HPLC method was developed for the simultaneous determination of Ambrisentan (AMB) and Tadalafil (TADA) drug substances in the fixed dosage strength of 10 mg and 40 mg, respectively. Effective chromatographic separation was achieved using a Hypersil GOLD C18 column (150 mm × 4.6 mm internal diameter, 5 μm particle size) with a mobile phase composed of methanol, water, and acetonitrile in the ratio of 40:40:20 (by volume). The mobile phase was pumped using a gradient HPLC system at a flow rate of 0.5 mL/min, and quantification of the analytes was based on measuring their peak areas at 260 nm. The retention times for Ambrisentan and Tadalafil were about 2.80 and 7.10 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 1–20 μg/mL for Ambrisentan and 4–80 μg/mL for Tadalafil with correlation coefficients >0.990. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from the forced degradation (hydrolysis, oxidation, and photolysis) products. The validated HPLC method was successfully applied to the analysis of AMB and TADA in pharmaceutical dosage form.     

Development of a Rapid LC-MS/MS Method for the Determination of Emerging Fusarium mycotoxins Enniatins and Beauvericin in Human Biological Fluids

A novel method for the simultaneous determination of enniatins A, A1, B and B1 and beauvericin, both in human urine and plasma samples, was developed and validated. The method consisted of a simple and easy pretreatment, specific for each matrix, followed by solid phase extraction (SPE) and detection by high performance liquid chromatography-tandem mass spectrometry with an electrospray ion source. The optimized SPE method was performed on graphitized carbon black cartridges after suitable dilution of the extracts, which allowed high mycotoxin absolute recoveries (76%–103%) and the removal of the major interferences from the matrix. The method was extensively evaluated for plasma and urine samples separately, providing satisfactory results in terms of linearity (R² of 0.991–0.999), process efficiency (>81%), trueness (recoveries between 85% and 120%), intra-day precision (relative standard deviation, RSD < 18%), inter-day precision (RSD < 21%) and method quantification limits (ranging between 20 ng·L⁻¹ and 40 ng·L⁻¹ in plasma and between 5 ng·L⁻¹ and 20 ng·L⁻¹ in urine). Finally, the highly sensitive validated method was applied to some urine and plasma samples from different donors.     

HPTLC-densitometric method for simultaneous determination of salmeterol xinafoate and fluticasone propionate in dry powder inhalers

A high performance thin layer chromatography (HPTLC) method was developed and validated for determination of two anti-asthmatic drugs, salmeterol xinafoate and fluticasone propionate in co-formulations. Study was performed on pre-coated silica gel HPTLC plates using n-hexane:ethyl acetate:acetic acid (5:10:0.2) as a mobile phase. A TLC scanner set at 250 nm was used for direct evaluation of the chromatograms in reflectance/absorbance mode. Method was validated according to ICH guidelines. Determination coefficients of calibration curves were found 0.9977 and 0.9936 in the ranges 100–1000 and 200–2000 ng band⁻¹ for salmeterol and fluticasone, respectively. Method had an accuracy of 99.5% for salmeterol and 102.01% for fluticasone. Method had the potential to determine these drugs simultaneously from dosage forms without any interference.     

Simultaneous determination of 3-chlorotyrosine and 3-nitrotyrosine in human plasma by direct analysis in real time–tandem mass spectrometry

A novel method for the simultaneous determination of 3-nitrotyrosine (NT) and 3-chlorotyrosine (CT) in human plasma has been developed based on direct analysis in real time–tandem mass spectrometry (DART–MS/MS). Analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) of the ion transitions at m/z 216.2/170.1 for CT, m/z 227.2/181.1 for NT and m/z 230.2/184.2 for the internal standard, d³-NT. The assay was linear in the ranges 0.5–100 μg/mL for CT and 4–100 μg/mL for NT with corresponding limits of detection of 0.2 and 2 μg/mL. Intra- and inter-day precisions and accuracies were respectively <15% and ±15%. Matrix effects were also evaluated. The method is potentially useful for high throughput analysis although sensitivity needs to be improved before it can be applied in clinical research.KEY WORDS: 3-Nitrotyrosine, 3-Chlorotyrosine, Determintion, DART–MS/MS, Human plasma     

Second derivative ultraviolet spectrophotometry and HPTLC for the simultaneous determination of vitamin C and dipyrone

Two accurate, precise and sensitive thin layer chromatographic (TLC) and second derivative UV-spectrophotometric procedures are described for the simultaneous determination of ascorbic acid and dipyrone in pure form and in pharmaceutical dosage forms. The TLC method involved direct application of methanolic solutions of tested samples on silica gel TLC plates using water:methanol (95:5 v/v) as developing system. The developed plates were then directly scanned at 260 nm using a TLC scanner. The second method depends on second derivative UV-spectrophotometry with zero crossing technique of measurement. Second derivative amplitudes at 280 and 272 nm were selected for the determination of ascorbic acid and dipyrone, respectively. Both methods show good linearity, precision and reproducibility. They are simple and do not require manipulation prior to analysis. The proposed methods have been successfully applied to the determination of the drugs in various pharmaceutical dosage forms such as tablets and ampoules.     

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method

Background

A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride.

Methods

For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 μm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K₂HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min ⁻¹. Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification.

Results

The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products.Regression analysis showed r² > 0.999 for cetirizine dihydrochloride in the concentration range of 650 μg mL ⁻¹ to 350 μg mL⁻¹ for drug substance assay and a r² > 0.999 in the concentration range of 0.25 μg mL⁻¹ to 5 μg mL⁻¹ for degradation products. The method presents a limit of detection of 0.056 μg mL ⁻¹ and a limit of quantification of 0.25 μg mL⁻¹. The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method.

Conclusions

The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.     

Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Phenoxyethanol,Methylparaben, Propylparaben,Mometasone Furoate,and Tazarotene in Topical Pharmaceutical Dosage Formulation

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of phenoxyethanol (PE), methylparaben (MP), propylparaben (PP), mometasone furoate (MF), and tazarotene (TA) in topical pharmaceutical dosage formulation. The desired chromatographic separation was achieved on the Waters X-Bridge™ C18 (50×4.6mm, 3.5μ) column using gradient elution at 256 nm detection wavelength. The optimized mobile phase consisted of 0.1%v/v orthophosphoric acid in water as solvent-A and acetonitrile as solvent-B. The method showed linearity over the range of 5.88–61.76 μg/mL, 0.18–62.36 μg/mL, 0.17–6.26 μg/mL, 0.47–31.22 μg/mL, and 0.44–30.45 μg/mL for PE, MP, PP, MF, and TA, respectively. The recovery for all of the components was in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples, in which the spectral purity of PE, MP, PP, MF, and TA along with the separation of degradation products from the analyte peaks was achieved. The proposed method was successfully applied for the quantitative determination of PE, MP, PP, MF, and TA in a cream sample.     

Assessment by HPLC of the degradation behavior of acitretin under hydrolytic,oxidative, photolytic and thermal stress conditions

Acitretin is a photosensitive oral retinoid with very limited data available on its degradation. The official HPLC method for acitretin determination was insufficient to resolve the degradation products generated during stability studies. Therefore, an isocratic RP-HPLC–UV method was developed for the determination of acitretin in the presence of its related impurities and degradation products. Efficient chromatographic separation was achieved on a Thermo beta-basic column C18 (100 mm×4.6 mm, 5 μm) with mobile phase containing 0.3% (v/v) glacial acetic acid with acetonitrile (ACN) and isopropyl alcohol (IPA) in an isocratic ratio of 70:30 at a flow rate of 1.0 mL/min with the eluent monitored at 360 nm. The method was validated for specificity, linearity, precision, accuracy and robustness. The calibration plot was linear over the concentration range of 50–150 μg/mL with a correlation coefficient (r²) of 0.999. The proposed method was used to investigate the degradation kinetics of acitretin under the different degradative conditions. The degradation rate constant (K), half-life (t_1/2), and t₉₀ were calculated. Degradation of acitretin followed pseudo-first-order kinetics. The drug was found to be less stable under acidic and photolytic degradation conditions: the photolytic degradation constants for acitretin in sunlight and UV light were 0.002698% and 0.0008402% min⁻¹, respectively. The LOD for acitretin and the known impurities were at a level below 0.02%. The method shows consistent recoveries for ACTR (99.8%–101.2%) and also for its known impurities (97.2–101.3%). The method was found to be accurate, precise, linear, specific, sensitive, rugged, robust, and useful for characterizing the stability of this chemical.KEY WORDS: Acitretin, Degradation kinetics, Photolytic degradation, Validation     

Quality by Design-Based Development of a Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Methylparaben,Propylparaben, Diethylamino Hydroxybenzoyl Hexyl Benzoate,and Octinoxate in Topical Pharmaceutical Formulation

A stability-indicating RP-HPLC method has been developed and validated for the simultaneous determination of methylparaben (MP), propylparaben (PP), diethylamino hydroxybenzoyl hexyl benzoate (DAHHB), and octinoxate (OCT) in topical pharmaceutical formulation. The desired chromatographic separation was achieved on the Kinetex^TM C18 (250 × 4.6 mm, 5 μm) column using gradient elution at 257 nm detection wavelength. The optimized mobile phase consisted of a buffer : acetonitrile : tetrahydrofuran (60 : 30 : 10, v/v/v) as solvent A and acetonitrile : tetrahydrofuran (70 : 30, v/v) as solvent B. The method showed linearity over the range of 0.19–148.4 μg/mL, 0.23–15.3 μg/mL, 1.97–600.5 μg/mL, and 1.85–451.5 μg/mL for MP, PP, DAHHB, and OCT, respectively. Recovery for all the components was found to be in the range of 98–102%. The stability-indicating capability of the developed method was established by analysing the forced degradation samples in which the spectral purity of MP, PP, DAHHB, and OCT, along with the separation of the degradation products from the analyte peaks, was achieved. The proposed method was successfully applied for the quantitative determination of MP, PP, DAHHB, and OCT in the lotion sample. The design expert with ANOVA software with the linear model was applied and a 2⁴ full factorial design was employed to estimate the model coefficients and also to check the robustness of the method. Results of the two-level full factorial design, 2⁴ with 20 runs including four centrepoint analysis based on the variance analysis (ANOVA), demonstrated that all four factors, as well as the interactions of resolution between DAHHB and OCT are statistically significant.     

A Stability-Indicating HPLC Method for the Determination of Memantine Hydrochloride in Dosage Forms through Derivatization with 1-Fluoro-2,4-dinitrobenzene

Memantine is chemically a tricyclic amine and is used for Parkinson’s disease and movement disorders. Although several HPLC methods with different derivatization reagents have been developed for the determination of memantine in biological fluids, there are some complications which limit the use of these methods in routine analysis of memantine in in vitro tests. We established a simple, sensitive, precise, and accurate HPLC method for the quantification of memantine in dosage forms. Pre-column derivatization of memantine was performed with 1-fluoro-2,4-dinitrobenzene and the reaction product was separated on a Nova-Pak C18 column. A mixture of acetonitrile and sodium dihydrogenphosphate (pH 2.5; 0.05 M) (70: 30, v/v) was used as the mobile phase. UV detection was performed at 360 nm. Forced degradation studies were performed on a powdered tablet sample of memantine hydro-chloride using acidic (0.1 M hydrochloric acid), basic (0.1 M sodium hydroxide), oxidative (10% hydrogen peroxide), thermal (105°C), photolytic, and humidity conditions. Good linearity (r²=0.999) was obtained over the range of 1–12 μg mL⁻¹ of memantine hydrochloride with acceptable within-day and between-day precision values in the range of 0.05–0.95%. The proposed method was used for the assay determination and dissolution rate study of memantine dosage forms with excellent specificity.     

Validated Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Azelnidipine and Olmesartan in Their Combined Dosage Form

A simple, rapid, and highly selective RP-HPLC method was developed for the simultaneous determination of Azelnidipine (AZL) and Olmesartan (OLM) drug substances in the fixed dosage strength of 16 mg and 20 mg, respectively. Effective chromatographic separation was achieved using a Hypersil GOLD C18 column (150 mm × 4.6 mm internal diameter, 5 µm particle size) with a mobile phase composed of methanol, acetonitrile, and water in the ratio of 40:40:20 (by volume). The mobile phase was pumped using a gradient HPLC system at a flow rate of 0.5 mL/min, and quantification of the analytes was based on measuring their peak areas at 260 nm. The retention times for Azelnidipine and Olmesartan were about 8.56 and 3.04 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 2–48 μg/mL for Azelnidipine and 2.5–60 μg/mL for Olmesartan with correlation coefficients >0.990. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from the forced degradation (hydrolysis, oxidation, and photolysis) products. The validated HPLC method was successfully applied to the analysis of AZL and OLM in their combined dosage form.     

Development and validation of stability-indicating high performance liquid chromatography method to analyze gatifloxacin in bulk drug and pharmaceutical preparations

Quantitative determination of gatifloxacin in tablets, solid lipid nanoparticles (SLNs) and eye-drops using a very simple and rapid chromatographic technique was validated and developed. Formulations were analyzed using a reverse phase SUPELCO® 516 C-18-DB, 50306-U, HPLC column (250 mm × 4.6 mm, 5 μm) and a mobile phase consisting of disodium hydrogen phosphate buffer:acetonitrile (75:25, v/v) and with orthophosphoric acid pH was adjusted to 3.3 The flow rate was 1.0 mL/min and analyte concentrations were measured using a UV-detector at 293 nm. The analyses were performed at room temperature (25 ± 2 °C). Gatifloxacin was separated in all the formulations within 2.767 min. There were linear calibration curves over a concentration range of 4.0–40 μg.mL⁻¹ and correlation coefficients of 0.9998 with an average recovery above 99.91%. Detection of analyte from different dosage forms at the same R_t indicates the specificity and stability of the developed method.