Spectrophotometric and liquid chromatographic determination of trimebutine maleate in the presence of its degradation products

Three methods are presented for the determination of trimebutine maleate (TM) in the presence of its degradation products. The first method was based on a high performance liquid chromatographic (HPLC) separation of TM from its degradation products using an ODS column at ambient temperature with a mobile phase consisting of acetonitrile-5 mM heptane sulfonic acid disodium salt (45:55, v/v, pH 4) with UV detection at 215 nm. The second method depends on using first derivative spectrophotometry (1D) by measurement of the amplitude at 252.2 nm. The third method depends on using first derivative of the ratio spectrophotometry (1DD) by measurement of the amplitude at 282.4 nm where a normalized spectrum of 3,4,5-trimethoxy benzoic acid is used as divisor. The proposed HPLC and 1D methods were used to investigate the kinetics of acidic and alkaline degradation processes. The pH-rate profile of degradation of TM in Britton-Robinson buffer solutions within the pH range 2-11.9 was studied.     

High performance liquid chromatographic determination of oxeladin citrate and oxybutynin hydrochloride and their degradation products

Two high performance liquid chromatographic (HPLC) methods are presented for the determination of oxeladin citrate (OL) and oxybutynin hydrochloride (OB) and their degradation products. The first method was based on HPLC separation of OL from its degradation product using a Nucleosil C(18) column with a mobile phase consisting of acetonitrile -0.1% phosphoric acid (60:40 v/v). The second method was based on HPLC separation of OB from its degradation product using a VP-ODS C(18) column with a mobile phase consisting of acetonitrile/0.01 M potassium dihydrogen phosphate/diethylamine (60:40:0.2). Quantitation was achieved with UV detection at 220 nm based on peak area. The two HPLC methods were applied for the determination of OL or OB, their degradation products, methylparaben and propylparaben in pharmaceutical preparations. The proposed methods were used to investigate the kinetics of acidic and alkaline degradation processes of OL and OB at different temperatures and the apparent pseudofirst-order rate constant, half-life and activation energy were calculated. The pH-rate profiles of degradation of OL and OB in Britton-Robinson buffer solutions within the pH range 2-12 were studied.     

First derivative spectrophotometric and LC determination of benoxinate hydrochloride and its degradation products

Two methods are presented for the determination of benoxinate HCI and its acid and alkali-induced degradation products using first derivative (1D) spectrophotometry with zero-crossing measurements and liquid chromatography. Benoxinate HCl was determined by measurement of its first derivative amplitude in mcllvaine's-citric acid phosphate buffer pH 7.0 at 268.4 and 272.4 nm in the presence of its alkali- and acid-induced degradation products, respectively. The acid- and alkali-induced, degradation products were determined by measurement of their first derivative amplitude in the same solvent at 307.5 nm. The LC method depends upon using a mu bondapak CN column with a mobile phase consisting of acetonitrile-water triethylamine (60:40:0.01, v/v) and adjusted to apparent pH 7. Quantitation was achieved with UV detection at 310 nm based on peak area. The proposed methods were utilized to investigate the kinetics of the acidic and alkaline degradation processes at different temperatures. The pH-rate profile of degradation of benoxinate HCl in Britton-Robinson buffer solutions was studied.     

First derivative ratio spectrophotometric,HPTLC-densitometric,and HPLC determination of nicergoline in presence of its hydrolysis-induced degradation product

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories.     

High performance liquid chromatographic determination of etofibrate and its hydrolysis products

High performance liquid chromatographic (HPLC) method is presented for the determination of etofibrate (EF) and its hydrolysis products. The method was based on HPLC separation of EF from its hydrolysis products using cyanopropyl column at ambient temperature with mobile phase consisting of acetonitrile-10 mM potassium dihydrogen phosphate, pH was adjusted to 4.1 using phosphoric acid (50:50, v/v). Quantitation was achieved with UV detection at 221 nm based on peak area. The flow rate was 1.5 ml min(-1). The proposed method was used to investigate the kinetics of acidic hydrolysis process of EF at different temperatures and the apparent pseudo first-order rate constant, half-life and activation energy were calculated. The kinetics of alkaline hydrolysis process of EF using 0.01 M sodium hydroxide at different temperatures cannot be studied as the drug is rapidly hydrolyzed in alkaline medium. The pH-rate profile of hydrolysis of EF in Britton-Robinson buffer solutions within the pH range 2-10 were studied.     

Application of HPLC and HPTLC for the simultaneous determination of tizanidine and rofecoxib in pharmaceutical dosage form

Two methods are described for the simultaneous determination of tizanidine and rofecoxib in binary mixture. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 311 nm. The separation was carried out on Merck HPTLC aluminium sheets of silica gel 60 F254 using toluene:methanol:acetone (7.5:2.5:1.0, v/v/v) as mobile phase. The linear regression analysis data was used for the regression line in the range of 10-100 and 100-1500 ng/spot for tizanidine and rofecoxib, respectively. The second method was based on HPLC separation of the two drugs on the reversed phase kromasil column [C18 (5 microm, 25 cm x 4.6 mm, i.d.)] at ambient temperature using a mobile phase consisting of phosphate buffer pH 5.5 and methanol (45:55, v/v). Flow rate was 1.0 ml/min with an average operating pressure of 180 kg/cm2. Quantitation was achieved with UV detection at 235 nm based on peak area with linear calibration curves at concentration ranges 10-200 and 100-2000 microg/ml for tizanidine and rofecoxib, respectively. Both methods have been successively applied to pharmaceutical formulation. No chromatographic interference from the tablet excipients was found. Both methods were validated in terms of precision, robustness, recovery and limits of detection and quantitation. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of tizanidine and rofecoxib determination in dosage form by means of HPTLC and HPLC method.     

First derivative spectrophotometric, TLC-densitometric, and HPLC determination of acebutolol HCL in presence of its acid-induced degradation product

Three methods are presented for the determination of acebutolol HCl in presence of its acid-induced degradation product. The first method was based on measurement of the first derivative amplitude of acebutolol HCl at 266.6 nm. The second method was based on separation of acebutolol HCl from its acid-induced degradation product followed by densitometric measurement of the spots at 230 nm. The separation was carried out on silica gel 60 F254, using ethanol-glacial acetic acid (4:1, v/v) as mobile phase. Second order polynomial equation was used for the regression line. The third method was based on high performance liquid chromatographic (HPLC) separation of acebutolol HCl from its acid-induced degradation product on a reversed phase, ODS column using a mobile phase of methanol-water (55:45, v/v) with UV detection at 240 nm. The first derivative spectrophotometric method was utilized to investigate the kinetics of the acid degradation process at different temperatures.     

Validated stability-indicating HPLC method for the determination of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) and its degradation products

High-performance liquid chromatographic method was developed for the quantitative determination of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) and its degradation products. Forced degradation studies were performed on bulk sample of DDB using acid (1N hydrochloric acid), alkaline (0.1N sodium hydroxide), oxidation (0.33% hydrogen peroxide), heat (70 degrees C) and photolytic degradation. The chromatographic method was fine tuned using the samples generated from forced degradation studies. Good resolution between the peaks corresponds to degradation products and the analyte was achieved on 5 microm ODS column (Luna, Phenomenex, USA). The mobile phase consists of a mixture of acetonitrile and water (60:40, v/v). Quantitation was achieved with UV detection at 235 nm based on peak area. The proposed HPLC method was utilized to investigate the kinetics of acidic, alkaline and oxidative degradation processes of DDB at different temperatures and the apparent pseudo first-order rate constant, half-life and activation energy were calculated. The pH-rate profiles of degradation of DDB in Britton-Robinson buffer solutions within the pH range 2-11 were studied. The developed method was validated with respect to linearity, accuracy, precision, robustness and forced degradation studies prove the stability-indicating power of the method.     

Spectrophotometric and liquid chromatographic determination of fenofibrate and vinpocetine and their hydrolysis products

Several spectrophotometric and HPLC methods are presented for the determination of fenofibrate, vinpocetine and their hydrolysis products. The resolution of either fenofibrate or vinpocetine and their hydrolysis products has been accomplished by using numerical spectrophotometric methods as partial least squares (PLS-1) and principal component regression (PCR) applied to UV spectra; and graphical spectrophotometric methods as first derivative of ratio spectra (1DD) or first (1D) and second (2D) derivative spectrophotometry for vinpocetine and fenofibrate, respectively. In addition HPLC methods were developed using ODS column with mobile phase consisting of acetonitrile-water (80:20, v/v, pH 4) with UV detection at 287 nm for fenofibrate and a mobile phase consisting of acetonitrile-10 mM KH2PO4, containing 0.1% diethylamine (60:40, v/v, pH 4.6) with UV detection at 270 nm for vinpocetine. The proposed methods were successfully applied for the determination of each drug and its hydrolysis product in laboratory-prepared mixture and pharmaceutical preparation. The proposed HPLC and derivative spectrophotometric methods were used to investigate the kinetics of acidic and alkaline hydrolytic processes of each drug. The pH-rate profile of hydrolysis of each drug in Britton-Robinson buffer solutions was studied.     

Voltammetric determination of cilazapril in pharmaceutical formulations

A sensitive adsorptive stripping voltammetric method for the measurement of cilazapril in 0.04 M Britton-Robinson buffer (pH 9.0) solution was described. The method was based on the adsorptive accumulation of the drug at a hanging mercury drop electrode (HMDE), followed by differential pulse voltammetry. The response was evaluated with respect to pre-concentration time, pH effect, accumulation potential, accumulation time and scan rate. The peak potential was -0.60 V (vs. Ag/AgCl). The peak current was directly proportional to the concentration of cilazapril with a detection limit of 17.6 ng ml(-1) at an accumulation time of 10 s. The reduction process was irreversible and the wave showed adsorptive characteristics. The results were compared to those obtained using a HPLC procedure. A reversed-phase C18e column with aqueous phosphate buffer (pH 3.5; 0.125 M)-acetonitrile (67:33, v/v) mobile phase and benazapril as internal standard was used. UV detector was set at 254 nm. Results obtained in HPLC were comparable to those obtained by adsorptive stripping voltammetric method.     

Derivative spectrophotometric, thin layer chromatographic-densitometric and high performance liquid chromatographic determination of trifluoperazine hydrochloride in presence of its hydrogen peroxide induced-degradation product.

Three methods are presented for the determination of trifluoperazine HCl in presence of its hydrogen peroxide induced degradation product. The first method was based on measurement of first (1D) and second (2D) derivative amplitudes of trifluoperazine HCl in 0.1 N hydrochloric acid at the zero crossing point of its sulfoxide derivative, main degradation product, (at 268.4 and 262.5 nm for 1D and 2D, respectively). The second method was based on the separation of trifluoperazine HCl from its sulfoxide derivative followed by densitometric measurement of the intact drug spot at 255 nm. The separation was carried out on Merck aluminum sheet of silica gel 60 F(254), using chloroform-methanol (7:3 v/v) as mobile phase. The third method was based on high performance liquid chromatographic separation of trifluoperazine HCl from its sulfoxide derivative on reversed phase, ODS column, using a mobile phase of acetonitrile-phosphate buffer pH 4.2 (60:40 v/v) at ambient temperature. Quantitation was achieved with UV detection at 255 nm based on peak area. The first derivative spectrophotometric method was utilized to investigate the kinetics of the hydrogen peroxide degradation process at different temperatures. The apparent pseudo first-order rate constant, half life and activation energy were calculated.     

Application of LC and HPTLC-densitometry for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide

Two methods are described for the simultaneous determination of benazepril HCl and hydrochlorothiazide in binary mixture. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 238 and 275 nm for benazepril HCl and hydrochlorothiazide, respectively. The separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F(254,) using ethyl acetate-methanol-chloroform (10:3:2 v/v) as mobile phase. Second order polynomial equation was used for the regression line in the range 2-20 and 2.5-25 microg/spot for benazepril HCl and hydrochlorothiazide, respectively. The second method was based on HPLC separation of the two drugs on reversed phase, ODS column at ambient temperature using a mobile phase consisting of acetonitrile and water (35:65 v/v) and adjusting to pH 3.3 with acetic acid. Quantitation was achieved with UV detection at 240 nm based on peak area with linear calibration curves at concentration ranges 10-60 and 12.5-75 microg ml(-1) for benazepril HCl and hydrochlorothiazide, respectively. The two proposed methods were successfully applied to the determination of both drugs in laboratory prepared mixtures and in commercial tablets. No chromatographic interference from the tablets excipients was found.     

Simultaneous HPLC determination of ketoprofen and its degradation products in the presence of preservatives in pharmaceuticals

A novel and quick high-performance liquid chromatography (HPLC) method with UV spectrophotometric detection was developed and validated for the determination of five compounds in topical gel. The described method is suitable for simultaneous determination of active component ketoprofen, two preservatives methylparaben and propylparaben and two degradation products of ketoprofen--3-acetylbenzophenone and 2-(3-carboxyphenyl) propionic acid--in a topical cream after long-term stability tests using ethylparaben as an internal standard. The chromatographic separation was performed on a 5microm Supelco Discovery C18 column (125mm x 4mm i.d., Sigma-Aldrich); the optimal mobile phase for separation of ketoprofen, methylparaben, propylparaben, degradation products 3-acetylbenzophenone and 2-(3-carboxyphenyl) propionic acid and ethylparaben as internal standard consists of a mixture of acetonitril, water and phosphate buffer pH 3.5 (40:58:2, v/v/v). At a flow rate of 1.0ml min(-1) and detection at 233nm, the total time of analysis was less than 10min. The method was applied for routine analysis (batch analysis and stability tests) of these compounds in topical pharmaceutical product.     

High performance liquid chromatographic and thin layer densitometric methods for the determination of risperidone in the presence of its degradation products in bulk powder and in tablets

Two reproducible stability indicating methods were developed for the determination of risperidone (RISP) in presence of its degradation products in pure form and in tablets. The first method was based on reversed phase high performance liquid chromatography (HPLC), on Lichrosorb RP C 18 column (250 mm i.d., 4 mm, 10 μm), using methanol:0.05 M potassium dihydrogen phosphate pH 7 (65:35 (v/v)) as the mobile phase at a flow rate of 1 ml min⁻¹ at ambient temperature. Quantification was achieved with UV detection at 280 nm over a concentration range of 25–500 μg ml⁻¹ with mean percentage recovery of 99.87 ± 1.049. The method retained its accuracy in the presence of up to 90% of RISP degradation products. The second method was based on TLC separation of RISP from its degradation products followed by densitometric measurement of the intact drug spot at 280 nm. The separation was carried out on aluminum sheet of silica gel 60F₂₅₄ using acetonitrile:methanol:propanol:triethanolamine (8.5:1.2:0.6:0.2 (v/v/v/v)), as the mobile phase, over a concentration range of 2–10 μg per spot and mean percentage recovery of 100.1 ± 1.18. The two methods were simple, precise, sensitive and could be successfully applied for the determination of pure, laboratory prepared mixtures and tablets. The results obtained were compared with the manufacturer's method.     

HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products

A stability-indicating HPLC method was developed to simultaneously determine cisatracurium besylate and propofol in mixtures. The effects of organic modifier, ionic strength and the pH of the mobile phase on resolution and retention were investigated. A baseline separation was achieved on an octadecylsilane column with an isocratic mobile phase of acetonitrile–ammonium formate (pH 5.2; 0.3 M) (50:50, v/v). Cisatracurium and propofol were confirmed by both retention time and mass-to-charge ratio using LC-MS. The degradation products of cisatracurium were identified by ESI positive-ion detection as Hofmann elimination and ester hydrolysis products of cisatracurium. There were no propofol degradation products observed. The quantitation of the two drugs was accomplished using UV detection at 280 nm. This method showed linearity for cisatracurium besylate and propofol in the 8–128 and 37–592 μg ml⁻¹ ranges, respectively. Accuracy and precision were in the 0.4–1.4 and 0.4–2.9% ranges respectively, for both analytes.     

Simultaneous quantification of stavudine, lamivudine and nevirapine by UV spectroscopy, reverse phase HPLC and HPTLC in tablets

In the present study, simultaneous quantification of stavudine (SV), lamivudine (LV) and nevirapine (NV) in tablets by UV spectroscopy, reverse phase HPLC (RP-HPLC) and HPTLC methods were developed. In the UV multi-component spectral method, SV, LV and NV was quantified at 266, 271 and 315 nm, respectively. In the RP-HPLC method, the drugs were resolved using a mobile phase of 20 mM sodium phosphate buffer (containing 8 mM 1-octanesulphonicacid sodium salt):acetonitrile (4:1, v/v) with pH adjusted to 3.5 using phosphoric acid on a C18-ODS-Hypersil (5 microm, 250 mm x 4.6 mm) column in isocratic mode. The retention time of SV, LV and NV was 2.85, 4.33 and 8.39 min, respectively. In the HPTLC method, the chromatograms were developed using a mobile phase of chloroform:methanol (9:1, v/v) on precoated plate of silica gel 60 F254 and quantified by densitometric absorbance mode at 265 nm. The Rf of SV, LV and NV were 0.21-0.27, 0.62-0.72 and 0.82-0.93, respectively. Recovery values of 99.16-101.89%, percentage relative standard deviation of <0.7 and correlation coefficient (linear dynamic range) of 0.9843-0.9999 shows that the developed methods were accurate and precise. These methods can be employed for the routine analysis of tablets containing SV, LV and NV.     

Use of the zirconia-based stationary phase for separation of ibuprofen and its impurities

A new reversed-phase liquid chromatographic method using zirconia-based stationary phase was developed for determination of ibuprofen, its related compounds and its main degradation products. The chromatographic separation was successfully achieved on the Discovery Zr-PS column (150 mm x 4.6 mm i.d., 5 microm), using a mobile phase methanol-phosphate buffer (pH 4.5; 0.05 M)-tetrahydrofurane (21:74:5, v/v/v) and the flow rate 0.5 ml min(-1). The UV detection was performed in dual wavelength mode (219 and 258 nm) to detect all compounds of interest. The column temperature was set on 60 degrees C to shorten the analysis time and improve the peak symmetry. The method is simple, rapid and cuts down the amount of hazardous waste produced in the analysis. The assay is completed within 22 minutes.     

ICH guidance in practice: validated stability-indicating HPLC method for simultaneous determination of ampicillin and cloxacillin in combination drug products

Ampicillin and cloxacillin were degraded together under different stress test conditions prescribed by International Conference on Harmonization. The samples so generated were used to develop a stability-indicating high performance liquid chromatographic (HPLC) method for the two drugs. The drugs were well separated from degradation products using a reversed-phase (C-18) column and a mobile phase comprising of acetonitrile:phosphate buffer (pH 5.0), which was delivered initially in the ratio of 15:85 (v/v) for 1 min, then changed to 30:70 (v/v) for next 14 min, and finally equilibrated back to 15:85 (v/v) from 15 to 20 min. Other HPLC parameters were: flow rate, 1 ml/min; detection wavelength, 225 nm; and injection volume, 5 microl. The method was validated for linearity, precision, accuracy, specificity and selectivity. It was also compared with the assay procedures given in British Pharmacopoeia for individual drugs. Similar results were obtained, indicating that the proposed single method allowed selective analysis of both ampicillin and cloxacillin, in the presence of their degradation products formed under a variety of stress conditions. The developed procedure was also applicable to the determination of instability of the drugs in commercial products.     

Validation of a HPLC method for the quantification and purity determination of SK3530 in drug substance and tablet

SK3530.2HCl, (2-(5-(4-(2-hydroxyethyl)piperazin-1-ylsulfonyl)-2-n-propoxyphenyl)-5-ethyl-7-n-propyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one dihydrochloride), is a novel a new phosphodiesterase type V (PDE V) inhibiting agents. The pharmaceutical development of SK3530 necessitated the availability of an assay for the quantification and purity determination of SK3530 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase high performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with a CapcellPack MG (4.6 mm x 150 mm, 5 microm) column with ammonium acetate buffer (pH 4.0, 20 mM)-acetonitrile (60:40, v/v) as the isocratic mobile phase and UV detection at 250 nm. The method has been shown good chromatographic separation for SK3530 and the other related substances. The method was found to be linear 200-300 microg/ml, precise and accurate. Stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indication capacity. Moreover, the use of LC-MS and on-line diode array detection enabled us to propose structures for degradation products.     

Determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by LC

The purpose of this study was to set up a HPLC method to separate adrenergic amines (dl-octopamine, dl-synephrine and tyramine) and to determine their content in fruits, extracts and herbal products of Citrus aurantium L. var. amara. A rapid method for the quantitative analysis of these amines is described, based on their separation by RP-HPLC technique with UV detection. The analysis were conducted on a Lichrospher RP-18 column at room temperature, using a mobile phase consisting of 0.02 M citric acid–0.02 M NaH₂PO₄ (7:3 v/v) and adjusted to a final pH of 3. The detection was at 220 nm. Since some of these amines are chiral compounds and their enantiomers showed different pharmacological activity, the direct separation of synephrine enantiomers was carried out with HPLC on a β-cyclodextrin stationary phase. The mobile phase consisted of methanol–NaH₂PO₄ 25 mM pH 3.5 (20:80 v/v) and tetrabutylammonium hydrogen sulfate 10 mM in ratio of 30:70 v/v in isocratic condition and the detection was at 220 nm. The two proposed methods were applied to the analysis of fruits, extracts and herbal products of C. aurantium L. var. amara. Taking into account that some authors have reported that l-synephrine may be converted into its d-form by high temperature, this optical isomerization was monitored by the same HPLC method used for the separation of enantiomers.