Development and application of a validated stability-indicating HPLC method for simultaneous determination of granisetron hydrochloride, benzyl alcohol and their main degradation products in parenteral dosage forms

A simple, rapid and sensitive reversed phase high performance liquid chromatographic method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, benzyl alcohol, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron) and benzaldehyde (the main degradation product of benzyl alcohol) in granisetron injections. The separation was achieved on Hypersil BDS C8 (250 mm × 4.6 mm i.d., 5 μm particle diameter) column using a mobile phase consisted of acetonitrile:0.05 M KH₂PO₄:triethylamine (22:100:0.15) adjusted to pH 4.8. The column was maintained at 25 °C and 20 μL of solutions was injected. Photodiode array detector was used to test the peak purity and the chromatograms were extracted at 210 nm. Naphazoline hydrochloride was used as internal standard. The method was validated with respect to specificity, linearity, accuracy, precision, limit of quantitation and limit of detection. The validation acceptance criteria were met in all cases. Identification of the pure peaks was carried out using library match programmer and wavelengths of derivative optima of the spectrograms of the peaks. The method was successfully applied to the determination of the investigated drugs and their degradation products in different batches of granisetron injections. The method was proved to be sensitive for the determination down to 0.03 and 0.01% of granisetron degradation product and benzaldehyde, respectively, which are far below the compendia limits for testing these degradation products in their corresponding intact drugs.     

Selective and quantitative analysis of 4-hydroxybenzoate preservatives by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of 4-hydroxybenzoates and their impurities. These materials are commonly known as parabens and are widely used as preservatives in foods, cosmetics and pharmaceuticals. The method was shown to be selective and quantitative for the methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid. An internal standard, 4-hydroxyacetophenone, was employed to improve injection precision and detector linearity. In addition, 4-hydroxybenzoic acid, the major degradent, could also be monitored at the 0.1% (m/m) level. The method was successfully validated for assay and detection of the impurities in 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester samples and for the determination of 4-hydroxybenzoic acid methyl ester in a liquid pharmaceutical formulation. The determination of paraben content by MEEKC in a liquid sample was consistent with HPLC analysis. This work is the first reported validated MEEKC method and shows that the methodology can be successfully implemented into routine quality control testing.     

Utilization of zirconia stationary phase as a tool in drug control

Zirconia-based stationary phases represent an interesting alternative to silica-based materials. Two zirconia-based stationary phases were studied as an option for use in drug analysis. The different properties of zirconia material, distinct from RP silica-columns, were employed for the development of a novel and rapid stability monitoring HPLC method. This method enables simultaneous control of possible degradation processes of active substance (ibuprofen) as well as antimicrobial excipients (methyl-and propylparaben). The separation of ibuprofen, its two main degradation products 2-(4-isobutyrylphenyl)propionic acid and 4-isobutylacetophenone, parabens, and 4-hydroxybenzoic acid as their degradation product was successfully accomplished on a Zr-CarbonC18 column using a mobile phase consisting of acetonitrile-phosphate buffer (pH 4.8)-propan-2-ol (27:56:17, v/v/v). Detection was performed at 258 nm and the analysis was completed within 17 minutes.     

Stability-Indicating LC Determination of a New Antihypertensive,Olmesartan Medoxomil in Tablets

A stability-indicating liquid chromatographic method was developed and validated for quantitative determination of olmesartan medoxomil (OLM) in coated tablets in the presence of degradation products generated under stress conditions. An isocratic LC separation was performed using a Phenomenex RP-18 column using a mobile phase consisting of water:triethylamine:acetonitrile (60:0.3:40 v/v/v, pH adjusted to 6.3 with phosphoric acid). The flow rate was 1.2 mL min⁻¹ and the detection was achieved with a photodiode array detector set at 257 nm. The response was linear over a range of 10.0 to 30.0 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method was verified subjecting the reference substance and drug product to hydrolytic, oxidative, photolytic, and thermal stress conditions. The method showed a good and consistent recovery (100.2%) with low intra- and inter-day relative standard deviation (RSD) (≤1.0%). A considerable degradation occurred in all stress conditions and the degradation product was well resolved from the main peak. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. Thus, the proposed method was found to be stability-indicating and can be used for routine analysis for quantitative determination of OLM in coated tablets without the interference of major degradation products.

     

Stability-Indicating LC Determination of a New Antihypertensive, Olmesartan Medoxomil in Tablets

A stability-indicating liquid chromatographic method was developed and validated for quantitative determination of olmesartan medoxomil (OLM) in coated tablets in the presence of degradation products generated under stress conditions. An isocratic LC separation was performed using a Phenomenex RP-18 column using a mobile phase consisting of water:triethylamine:acetonitrile (60:0.3:40 v/v/v, pH adjusted to 6.3 with phosphoric acid). The flow rate was 1.2 mL min^?1 and the detection was achieved with a photodiode array detector set at 257 nm. The response was linear over a range of 10.0 to 30.0 μg mL^?1 (r = 0.9999). The specificity and stability-indicating capability of the method was verified subjecting the reference substance and drug product to hydrolytic, oxidative, photolytic, and thermal stress conditions. The method showed a good and consistent recovery (100.2%) with low intra- and inter-day relative standard deviation (RSD) (≤1.0%). A considerable degradation occurred in all stress conditions and the degradation product was well resolved from the main peak. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. Thus, the proposed method was found to be stability-indicating and can be used for routine analysis for quantitative determination of OLM in coated tablets without the interference of major degradation products.     

Determination of Parabens and their Degradation Product p-Hydroxy-Benzoic Acid in Pharmaceutical Dosage Forms by Hptlc Densitometry

Abstract

HPTLC densitometry has been developed for the determination of parabens and their degradation product p-hydroxybenzoic acid in pharmaceutical preparations. The method was used for simultaneous determination of parabens in combination:methylparaben - propylparaben, propylparaben - butylparaben and for the determination their stability in dosage forms (suspensions, ointment). The substances were separated on silica gel with fluorescence indicator in system n-pentane - glacial acetic acid (88:12, v/v). Absorbance measurements(detection of reflectance) of separated parabens was carried out “in situ” at 256 nm using single-level calibration (external standard). For the determination of p-hydroxybenzoic acid, a five-level calibration curve (external standard) was used in the concentration range of 20–100 ng/band. The results were evaluated by linear regression analysis. The relative standard deviation values (R.S.D.%) were in range 1.24–1.89 %.     

Stability-indicating HPLC method development and validation for simultaneous estimation of metformin,dapagliflozin, and saxagliptin in bulk drug and pharmaceutical dosage form

A simple, precise, and rapid stability-indicating reversed-phase-HPLC method was developed and validated for the estimation of metformin (MET), dapagliflozin (DAP), and saxagliptin (SAX) combination in bulk and tablet dosage forms. The proposed method uses a Kromasil C18 column (150 × 4.6 mm, 5 μm) with column oven temperature of 30°C and mobile phase containing a mixture of 60% phosphate buffer (pH = 3) and 40% acetonitrile. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. The detection was carried out at 230 nm using a photodiode array detector, and the total run time was 4 min. The proposed method was validated according to International Council for Harmonisation (ICH) guidelines for specificity, linearity, precision, accuracy, robustness, and solution stability. The method is linear over the range of 125–750 μg/mL for MET, 1.25–7.5 μg/mL for DAP, and 0.625–3.75 μg/mL for SAX. The observed correlation coefficients (R²) for MET, DAP, and SAX are >0.999. The proposed method is precise, and the percentage relative standard deviation was found to be between 0.4 and 0.8. The observed percentage recoveries were between 98.51 and 100.80 for all three compounds. The product was subjected to stress conditions of acid, base, oxidative, thermal, and photolytic degradation. The product was found to degrade significantly in oxidative, acid, and base hydrolysis degradation conditions, and the degradation products were well determined from the active peaks, thus proving the stability-indicating power of the method. The developed and validated stability-indicating reversed-phase-HPLC method was appropriate for quantitative determination of these drugs in pharmaceutical preparations and also for quality control in bulk manufacturing.     

Development and validation of reversed-phase column high-performance liquid chromatographic and first-derivative UV spectrophotometric methods for estimation of voriconazole in oral suspension powder

This research paper describes validated reversed-phase high-performance column liquid chromatographic (RP-HPLC) and first-derivative UV spectrophotometric methods for the estimation of voriconazole (VOR) in oral suspension powder. The RP-HPLC separation was achieved on Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using water-acetonitrile (40 + 60, v/v; pH adjusted to 4.5 +/- 0.02 with acetic acid) as the mobile phase at a flow rate of 1.4 mL/min and ambient temperature. Quantification was achieved with photodiode array detection at 255 nm over the concentration range of 0.1-1 microg/mL with mean recovery of 99.49 +/- 0.83% for VOR by the RP-HPLC method. Quantification was achieved with UV detection at 266 nm over the concentration range of 8-20 microg/mL with mean recovery of 99.74 +/- 0.664% for VOR by the first-derivative UV spectrophotometric method. These methods are simple, precise, and sensitive, and they are applicable for the determination of VOR in oral suspension powder.     

Determination of Danshensu, a major active compound of Radix Salvia miltiorrhiza in dog plasma by HPLC with fluorescence detection.

A simple and sensitive high performance liquid chromatography method has been developed for the determination of Danshensu (3, 4-dihydroxyphenyllactic acid) in dog plasma. Plasma samples were extracted with ethyl acetate. Analysis of the extracts was performed on a reversed-phase column with an aqueous phosphate buffer-acetonitrile (93:7, v/v) mobile phase, and 4-hydroxybenzoic acid was used as the internal standard. Fluorescence detection at 285 nm (excitation) and 320 nm (emission) was employed. Standard curves were linear in the range from 0.125 to 11.3 microg/mL (regression coefficient r > 0.993) on three different days. Mean recovery was determined as 96.4% by analysis of plasma standard containing 0.63, 5.65 and 11.3 microg/mL of Danshensu. The inter-day precision (RSD) ranged from 3.4 to 8.6% at concentrations of 0.125, 1.88, 6.28 and 11.3 microg/mL, and the intra-day precision was better than 7.2%. The detection and quantitation limits were 0.063 and 0.125 microg/mL, respectively. This validated assay was applied to the determination of Danshensu concentration in dog plasma after oral administration of Radix Salvia miltiorrhiza extracts.     

Development and Validation of a Stability Indicating HPLC Method for Determination of Granisetron

The aim of this study was to study the stress degradation of granisetron and analysis of the drug in the presence of its degradation products. Forced degradation studies were conducted on bulk sample using acidic, alkaline, oxidative, heat and photolytic conditions. Granisetron was relatively unstable under acidic, alkaline and oxidative conditions. Separation of granisetron and degradation products was achieved using a Nova‐Pak C₈ column and acetonitrile‐KH₂PO₄ 25 mM (75:25, v/v) as mobile phase with UV detection at 305 nm. The method was linear over the range of 0.2‐15 μg/mL granisetron (r² > 0.999). The within‐day and between‐day precision values were also in the range of 0.5‐4%. The proposed method was successfully applied for quantitative determination of granisetron in tablets and in vitro dissolution studies.     

Simultaneous determination, by capillary zone electrophoresis, of multiple components of different industrial products

Summary Four parabens (esters of 4-hydroxybenzoic acid), effective preservatives against the growth of bacteria, yeast, and mold in numerous industrial products, have been used in this work as model compounds to demonstrate the resolving power of capillary electrophoresis (CE). The simultaneous determination of methyl-(MP), ethyl-(EP), propyl-(PP), and butylparaben (BP) was achieved by capillary zone electrophoresis (CZE) with UV diode-array detection at 294 nm. When run voltage, temperature, and electrolyte concentration and pH were optimized the most effective separation was achieved within 7 min by use of 50 cm (effective length) fused silica capillary tubing and operation at 25kV and 20°C. Background electrolyte comprising 35 mM tetraborate buffer adjusted to pH 10.0 gave the best results. The limits of detection of the optimized method ranged from 0.65 μg mL⁻¹ for BP to 0.81 μg mL⁻¹ for MP; the relative standard deviation was between 0.35 and 0.50%. These results showed that the method enables the determination of the four parabens in commerially available cosmetic and pharmaceutical preparations containing some of the parabens and in an unidentified canned berry fruit juice.     

Development and validation of a reversed-phase liquid chromatographic method for separation and simultaneous determination of COX-2 inhibitors in pharmaceuticals and its application to biological fluids

An isocratic reversed-phase high-performance liquid chromatographic method has been developed for separation and simultaneous determination of COX-2 inhibitors, viz., celecoxib, rofecoxib, valdecoxib, nimesulide and nabumetone, using 4-chloro-2-nitroaniline as internal standard. Good chromatographic separation was achieved using a reversed-phase Inertsil C(18) column with mobile phase consisting of methanol and 0.05% aqueous glacial acetic acid (68:32 v/v) using photodiode array (PDA) detector at 230 nm. It was validated with respect to accuracy, precision, linearity, limit of detection and quantification. The linearity range was found to be 1.0--20 microg/mL and the percentage recoveries were between 97.55 and 100.14. The method is suitable not only for the estimation of active ingredients in pharmaceutical dosage forms but also in vitro estimations in human plasma. It is simple, rapid, selective and capable of detecting and determining COX-2 inhibitors with a detection limit of 0.127--1.040 microg/mL simultaneously.     

MSn,LC‐MS‐TOF and LC‐PDA studies for identification of new degradation impurities of bupropion

Three new degradation impurities of bupropion were characterized through high performance liquid chromatography coupled to photodiode array detection and to time‐of‐flight mass spectrometry. Bupropion was subjected to the ICH prescribed stress conditions. It degraded to seven impurities (I–VII) in alkaline hydrolytic conditions which were optimally resolved on an XTerra C₁₈ column (250 × 4.6 mm, 5 µm) with a ternary mobile phase comprising ammonium formate (20 mm , pH 4.0), methanol and acetonitrile (75:10:15, v/v). The degradation impurities (III–V and VII) were characterized on the basis of mass fragmentation pattern of drug, accurate mass spectral and photodiode array data of the drug and degradation impurities. Compound V was found to be a known degradation impurity [1‐hydroxy‐1‐(3‐chlorophenyl)propan‐2‐one], whereas III, IV and VII were characterized as 2‐hydroxy‐2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorpholine, (2,4,4‐trimethyl‐1,3‐oxazolidin‐2‐yl)(3‐chlorophenyl)‐methanone and 2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorphol‐2‐ene, respectively. Compound III was a known metabolite of the drug. This additional information on the degradation impurities can help in the development of a new stability‐indicating assay method to monitor the stability of the drug product during its shelf‐life as well as in development of a drug product with increased shelf‐life. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a stability-indicating LC method for the assay of lodenafil carbonate in tablets

A stability-indicating liquid chromatographic method has been developed for the quantitative determination of lodenafil carbonate in tablets. The method employs a Synergi Fusion C18 column (250 × 4.6 mm, i.d., 4 μm particle size), with mobile phase consisting of a mixture of methanol-acetic acid 0.1% pH 4.0 (65:35, v/v) and UV detection at 290 nm, using a photodiode array detector. A linear response (r = 0.9999) was observed in the range of 10-80 μg/mL. The method showed good recoveries (average 100.3%) and also intra and inter-day precision (RSD < 2.0%). Validation parameters as specificity and robustness were also determined. Specificity analysis showed that no impurities or degradation products were co-eluting with the lodenafil carbonate peak. The method was found to be stability-indicating and due to its simplicity and accuracy can be applied for routine quality control analysis of lodenafil carbonate in tablets.     

Isolation and characterization of degradation products of citalopram and process-related impurities using RP-HPLC

A reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of citalopram hydrobromide and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on an Inertsil ODS 3V (250x4.6 mm; particle size 5 mum) column using 0.3% diethylamine (pH = 4.70) and methanol/acetonitrile (55:45 v/v) as mobile phase in a gradient elution mode. The eluents were monitored by a photodiode array detector set at 225 nm. The chromatographic behavior of all the related substances was examined under variable conditions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. The method could be of use not only for rapid and routine evaluation of the quality of citalopram in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Three unknown impurities were consistently observed during the analysis of different batches of citalopram. Forced degradation of citalopram was carried out under thermal, photo, acidic, alkaline, and peroxide conditions. The degradation products and unknown impurities were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectroscopy.     

LC-PDA and LC-ESI-MS separation and determination of process-related substances arising from stilbene-type fluorescent whitening agents. Application to monitoring of their photodegradation products in industrial effluents and aqueous environmental systems

A simple and rapid gradient elution high-performance liquid chromatographic method using photodiode array and electrospray ionization mass spectrometric detectors was developed for separation and determination of the process-related substances and photodegradation products of stilbenesulfonic acids, viz. 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNSDA), 4-amino-4'-nitrostilbene-2,2'-disulfonic acid (ANSDA), and 4,4'-diaminostilbene-2,2'-disulfonic acid (DASDA) in industrial waste waters. Gradient elution was carried out using ammonium acetate and acetonitrile as mobile phase and an Inertsil-ODS 3V column for separation. The negative-ion electrospray ionization mass spectra containing [M-H]- ions of sulfonic acids allowed molecular mass determination of unknowns and the structures were proposed on the basis of the fragment ions in the MS/MS spectra.     

Optimization of microwave‐assisted extraction followed by RP‐HPLC for the simultaneous determination of oleanolic acid and ursolic acid in the fruits of Chaenomeles sinensis

An optimized microwave‐assisted extraction (MAE) method and an efficient HPLC analysis method were developed for fast extraction and simultaneous determination of oleanolic acid and ursolic acid in the fruit of Chaenomeles sinensis. The open vessel MAE process was optimized by using a central composite experimental design. The optimal conditions identified were microwave power 600 W, temperature 52°C, solvent to material ratio 32 mL/g and extraction time 7 min. The results showed that MAE is a more rapid extraction method with higher yield and lower solvent consumption. The HPLC–photodiode array detection analysis method was validated to have good linearity, precision, reproduction and accuracy. Compared with conventional extraction and analysis methods, MAE–HPLC–photodiode array detection is a faster, convenient and appropriate method for determination of oleanolic acid and ursolic acid in the fruits of C. sinensis.     

HPLC-UV Assay Method for Clindamycin Palmitate Hydrochloride as Drug Substance and Oral Solution

Abstract

A simple isocratic high performance liquid chromatography (HPLC) method was developed for the determination of Clindamycin palmitate hydrochloride in drug substance and oral solutions. The XTerra RP18 250 mm × 4.6 mm × 5 µ column was used as stationary phase, and the mobile phase was a 0.5% solution of Triethylamine in a 1:9 (v/v) water:methanol mixture adjusted to pH 5.0 with orthophosphoric acid. The detector wavelength was selected at 210 nm and flow rate was maintained at 1.50 ml/min. Forced degradation studies were performed for drug substance, 75 mg/5 ml oral solution and placebo, using acid, base, oxidation, temperature, humidity, and photolytic degradation to demonstrate the specificity of the method. The developed method was validated as per ICH method validation guidelines.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.