A Novel RP-LC Method for Determination of pK a Values of Some Anticancer Agents and Their Assay

A simple, reliable, and rapid RP-LC method has been developed for the determination of some anticancer drugs (daunorubicin, doxorubicin and vincristine sulfate) in their dosage forms and human urine. These compounds are well separated on a C18 column using the mobile phase consisting of a mixture of acetonitrile (50:50; v/v) at a flow rate of 1.5 mL min⁻¹. The analyte peaks were detected at 235 nm for doxorubicin and daunorubicin, and 220 nm for vincristine. Linearity was obtained in different concentration ranges between 0.10 and 12 μg mL⁻¹ for all compounds. Good sensitivity for all analytes was observed with DAD detection. LOD and LOQ of the method were found satisfying. The proposed method has been extensively validated in accordance with ICH guidelines and obtained results proved that the proposed method was precise, accurate, selective, and sensitive for simultaneous analysis of studied compounds. All analytical procedures including sample preparation, flow rate, and run time were at low levels. Also, pK _a values were determined using the dependence of the retention factor on the pH of the mobile phase. The effect of the mobile phase composition on the ionization constant was studied by measuring the pK _a at different methanol–water mixtures, ranging between 45 and 60 % (v/v).

     

Simultaneous Estimation of Ceftazidime and Ceftizoxime in Pharmaceutical Formulations by HPLC Method

The aim of the present study was to develop a fast, sensitive and reliable method for rapid screening of cephalosporin injectable dosage forms namely ceftazidime and ceftizoxime to the detection of counterfeit and substandard drugs that might be illegally commercialized. Ceftazidime, ceftizoxime and cefixime (IS) were separated in a X-Terra RP-18 column (250 × 4.60 mm ID × 5 ??) and DAD detector set at 290 and 260 nm. The mobile phase consisted of a mixture of methanol:water 20:80 (v/v) at a flow rate of 1.0 mL min^?1. Additionally, in order to find the optimum pH value of separation the pK _a values of studied compounds were determined by using two different methodologies. Aqueous pK _a values of studied compounds have been determined by UV-spectrophotometry and liquid chromatography were used for the determination and direct characterization of the dissociation constants by using the dependence of the capacity factor on the pH of the mobile phase in 20% (v/v) methanol?Cwater binary mixture in which separation was performed. The pH of the mobile phase was adjusted with 25 mM H₃PO₄ to 3.2. The method was shown to be linear, sensible, accurate, and reproducible over the range of analysis and it can be used to pharmaceutical formulations containing a single active ingredient within a short analysis time.     

A Novel RP-LC Method for Determination of pK a Values of Some Anticancer Agents and Their Assay

A simple, reliable, and rapid RP-LC method has been developed for the determination of some anticancer drugs (daunorubicin, doxorubicin and vincristine sulfate) in their dosage forms and human urine. These compounds are well separated on a C18 column using the mobile phase consisting of a mixture of acetonitrile (50:50; v/v) at a flow rate of 1.5 mL min^?1. The analyte peaks were detected at 235 nm for doxorubicin and daunorubicin, and 220 nm for vincristine. Linearity was obtained in different concentration ranges between 0.10 and 12 μg mL^?1 for all compounds. Good sensitivity for all analytes was observed with DAD detection. LOD and LOQ of the method were found satisfying. The proposed method has been extensively validated in accordance with ICH guidelines and obtained results proved that the proposed method was precise, accurate, selective, and sensitive for simultaneous analysis of studied compounds. All analytical procedures including sample preparation, flow rate, and run time were at low levels. Also, pK _a values were determined using the dependence of the retention factor on the pH of the mobile phase. The effect of the mobile phase composition on the ionization constant was studied by measuring the pK _a at different methanol–water mixtures, ranging between 45 and 60 % (v/v).     

A validated RP-LC method for salmeterol and fluticasone in their binary mixtures and their stress degradation behavior under ICH-recommended stress conditions

Simple, accurate, precise and fully validated analytical methods for the simultaneous determination of salmeterol xinafoate and fluticasone propionate in combined dosage forms have been developed. These drugs were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were detected by the proposed method. Additionally, pK _a values of three ionizable drugs (salmeterol xinafoate, fluticasone propionate and thioridazine) were determined using by the dependence of the retention factor on pH of the mobile phase. The effect of the mobile phase composition on the ionization constant was studied by measuring the pK _a in different acetonitrile-water mixtures, ranging between 50 and 65% (v/v) using LC-UV method.     

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C₁₈ column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL⁻¹ for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.

     

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C₁₈ column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL⁻¹ for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.     

Monitoring of the degradation kinetics of diatrizoate sodium to its cytotoxic degradant using a stability‐indicating high‐performance liquid chromatographic method

The X‐ray diagnostic agent sodium diatrizoate (DTA) was studied for chemical degradation. The 3,5‐diamino derivative was found to be the alkaline and acidic degradation product. The 3,5‐diamino degradate is also the synthetic precursor of DTA and it is proved to have cytotoxic and mutagenic effects. A sensitive, selective and precise high‐performance liquid chromatographic stability‐indicating method for the determination of DTA in the presence of its acidic degradation product and in pharmaceutical formulation was developed and validated. Owing to the high toxicity of the degradation product, the kinetics of the acidic degradation process was monitored by the developed RP‐HPLC method. The reaction was found to follow pseudo‐first order kinetics. The kinetic parameters such as rate constant (K ) and half‐life (t _½) were calculated under different temperatures and acid concentrations; activation energy was estimated from the Arrhenius plot. The developed RP‐HPLC method depends on isocratic elution of a mobile phase composed of methanol–water (25:75 v /v; pH adjusted with phosphoric acid), and UV detection at 238 nm. The method showed good linearity over a concentration range of 2–100 μg/mL with mean percentage recovery of 100.04 ± 1.07. The selectivity of the proposed method was tested using laboratory‐prepared mixtures. The proposed method has been successfully applied to the analysis of DTA in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with the official USP method. Validation of the proposed method was performed according to International Conference on Harmonization guidelines.     

Simultaneous densitometric determination of shikonin,acetylshikonin, and β‐acetoxyisovaleryl‐shikonin in ultrasonic‐assisted extracts of four Arnebia species using reversed‐phase thin layer chromatography

A simple, precise, and rapid high‐performance thin‐layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin ( 1 ) together with its derivatives acetylshikonin ( 2 ), and β‐acetoxyisovalerylshikonin ( 3 ) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP‐18 F_254S TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100–600 ng for shikonin and acetylshikonin, and 100–1800 ng for β‐acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1 – 3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.     

High-performance liquid chromatographic determination of neutraceuticals, glucosamine sulphate and chitosan, in raw materials and dosage forms

A simple, rapid, selective and specific high performance liquid chromatographic (HPLC) method was developed to quantitate glucosamine and its (β-1-4)-d-polymeric form chitosan. The chromatographic separation was achieved using an aminophase column and refractive index (RI) detection. The mobile phase consisted of ACN:H₂O:CH₃COOH (50:50:0.02) and pH was adjusted to 4.0. The standard curves for glucosamine sulphate showed linearity (r≥0.99) over the concentration range from 20 to 1000 μg ml⁻¹ for raw materials and dosage forms. The precision of the raw material assay expressed as the relative standard deviation (R.S.D.), was less than 3% at all concentrations.Chitosan, poly-(β-1-4)-d-glucosamine compounds, was hydrolysed with 6 M HCl at 100 °C for 10 h and the released glucosamine was determined by the same HPLC method. The proposed method showed linear relation in concentration ranges of 100-500 μg ml⁻¹.The suggested procedure was applied for the determination of glucosamine sulphate and chitosan in their dosage forms and the validity of the method was further checked by applying the standard addition technique. The method was found to be specific with good linearity, accuracy, precision and is well-suited for quantitation of glucosamine sulphate and chitosan in raw materials and pharmaceutical formulations.     

Determination of chromatographic dissociation constants of some carbapenem group antibiotics and quantification of these compounds in human urine

The dissociation constant values (_s^spK_a) of some carbapenem group drugs (ertapenem, meropenem, doripenem) in different percentages of methanol–water binary mixtures (18, 20 and 22%, v/v) were determined from the mobile phase pH dependence of their retention factor. Evaluation of these data was performed using the NLREG program. From calculated pK_a values, the aqueous pK_a values of these subtances were calculated by different approaches. Moreover, the correlation established between retention factor and the pH of the water–methanol mobile phase was used to determine the optimum separation conditions. In order to validate the optimized conditions, these drugs were studied in human urine. The chromatographic separation was realized using a Gemini NX C₁₈ column (250 × 4.6 mm i.d., 5 µm particles) and UV detector set at 220 and 295 nm. Copyright © 2013 John Wiley & Sons, Ltd.     

TLC Determination of Amitriptyline HCl,Trifluoperazine HCl,Risperidone and Alprazolam in Pharmaceutical Products

A simple, sensitive, and precise thin layer chromatographic (TLC) method for simultaneous analysis of psychopharmacological drugs like amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam in their single dosage forms has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The TLC separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using carbon tetrachloride:acetone:triethylamine (8:2:0.3, v/v/v), as mobile phase. Densitometric measurements of their spots were achieved at 250 nm over the concentration range for amitriptyline HCl (50–1,200 ng spot⁻¹), trifluoperazine HCl (50–1,200 ng spot⁻¹), risperidone (100–2,400 ng spot⁻¹) and alprazolam (25–600 ng spot⁻¹). Limit of detection (LOD) for amitriptyline HCl (20 ng spot⁻¹), trifluoperazine HCl (20 ng spot⁻¹), risperidone (40 ng spot⁻¹) and alprazolam (5 ng spot⁻¹) was obtained. The study showed that TLC was sensitive and selective for determination of amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam using a single mobile phase. This proposed method is able for simultaneous determination of psychopharmacological drugs and also applicable for analysis of pharmaceutical formulations.

     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min⁻¹ and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL⁻¹ (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL⁻¹, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.

     

Determination of pKa values of some antihypertensive drugs by liquid chromatography and simultaneous assay of lercanidipine and enalapril in their binary mixtures

In this study, pK_a values were determined using the dependence of the retention factor on the pH of the mobile phase for three ionizable substances, namely, enalapril, lercanidipine and ramipril (IS). The effect of the mobile phase composition on the ionization constant was studied by measuring the pK_a at different methanol-water mixtures, ranging between 50 and 65% (v/v), using LC-DAD method. Two simple, accurate, precise and fully validated analytical methods for the simultaneous determination of enalapril and lercanidipine in combined dosage forms have been developed. Separation was performed on an X-Terra RP-18 column (250 mm × 4.60 mm ID × 5 μm) at 40 °C with the mobile phase of methanol-water 55:45 (v/v) adjusted to pH 2.7 with 15 mM orthophosphoric acid. Isocratic elution was performed in less than 12 min with a flow rate of 1.2 mL min⁻¹. Good sensitivity for the analytes was observed with DAD detection. The LC method allowed quantitation over the 0.50-20.00 μg mL⁻¹ range for enalapril and lercanidipine. The second method depends on first derivative of the ratio-spectra by measurements of the amplitudes at 219.7 nm for enalapril and 233.0 nm for lercanidipine. Calibration graphs were established for 1-20 μg mL⁻¹ for enalapril and 1-16 μg mL⁻¹ lercanidipine, using first derivative of the ratio spectrophotometric method. Both methods have been extensively validated. These methods allow a number of cost and time saving benefits. The described methods can be readily utilized for analysis of pharmaceutical formulations. The methods have been applied, without any interference from excipients, for the simultaneous determination of these compounds in tablets. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations.     

A Validated Stability Indicating LC Method for Mitotane in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of mitotane, its impurity in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved using a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at a wavelength of 230 nm. The mobile phase consisted of buffer and acetonitrile (25:75, v/v) delivered at a flow rate of 1.0 mL min⁻¹. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 2.5 by orthophosphoric acid. In the developed LC method the resolution (R _s ) between mitotane and its impurity namely Imp-1 was found to be greater than 2.5. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for mitotane and its impurity. This method was capable to detect the impurity of mitotane at a level of 0.003% with respect to test a concentration of 0.2 mg mL⁻¹ for a 10 μL injection volume. The inter- and intra-day precision values for mitotane and its impurity was found to be within 2.0% RSD. The method has shown good and consistent recoveries for mitotane in bulk drugs (99.2–101.5%), pharmaceutical dosage forms (98.2–103.1%) and for its impurity (99.7–102.1%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in basic stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.97%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

     

RP-LC and HPTLC Methods for the Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Combined Tablet Dosage Forms

Two new, rapid, precise, accurate and specific chromatographic methods were described for the simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage forms. The first method was based on reversed phase liquid chromatography using an Eurosphere 100 RP C18 column (250 × 4.6 mm ID, 5 μm). The mobile phase was methanol–0.05% o-phosphoric acid (60:40 v/v) at a flow rate of 1.0 mL min⁻¹. Commercially available tablets and laboratory mixtures containing both drugs were assayed and detected using a UV detector at 270 nm. The second method involved silica gel 60 F₂₅₄ high performance thin layer chromatography and densitometric detection at 254 nm using acetonitrile–ethyl acetate–glacial acid (7:3:0.4 v/v/v) as the mobile phase. Calibration curves ranged between 200–600 and 125–375 ng spot⁻¹ for olmesartan and hydrochlorothiazide, respectively.

     

Simultaneous liquid chromatographic determination of ezetimibe and simvastatin in pharmaceutical products

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm x 4.6 mm id) maintained at 45 degrees C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5-acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5-200 microg/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.     

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL⁻¹ for ciprofloxacin HCl and 0.5–1.5 μg mL⁻¹ for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL⁻¹, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

     

Analysis of Zoledronic Acid and Its Related Substances by Ion-Pair RP-LC

A simple high-performance liquid chromatographic method – ion-pair reversed- phase high performance liquid chromatography (RPIC) has been developed and employed for the analysis of zoledronic acid and its related substances in bulk material and commercial dosage forms. The mobile phase was a mixture of methanol (20%) and 5 mmol L^–1 phosphate buffer (80%) containing 6 mmol L^–1 tetrabutylammonium bromide, adjusted to pH 7.0 with sodium hydroxide. C₈ column was used as the stationary phase. The chromatographic conditions were optimized. The active ingredient – zoledronic acid was successfully separated from its related substances, including remained imidazol-1-yiacetic acid in the synthesis of zoledronic acid and other possible impurities of oxidation and decomposition. The excipients did not interfere with the determination of zoledronic acid in commercial dosage formulations. The method was rapid, simple, accurate and reproducible. It was not only successfully employed for the assay of zoledronic acid in bulk material and pharmaceutical dosage forms but also for the determination of its related substances.     

Determination and Quantification of Pitavastatin Calcium in Tablet Dosage Formulation by HPTLC Method

Abstract

A simple, sensitive, reliable, and rapid HPTLC method has been developed for the determination of pitavastatin calcium in tablet dosage form. Identification and determination were performed on aluminum backed silica gel 60F₂₅₄ washed with methanol. The mobile phase of ethyl acetate‐methanol‐ammonia‐1 drop formic acid (7:2:0.8) calibration plots were established showing the dependence of response (peak area) on the amount chromatographed. The spot were scanned at 245 nm. The method has a linear range of 50–250 ng/spot. The method was validated for selectivity, repeatability, and accuracy. The method was used for determination of the compound in commercial pharmaceutical dosage forms. It is a more effective option than other chromatographic techniques in routine quality control.