RP-HPLC Method for Simultaneous Estimation of Nitazoxanide and Ofloxacin in Tablets

A reverse phase high performance liquid chromatography method was developed for simultaneous estimation of nitazoxanide and ofloxacin in tablet formulation. The separation and quantification was achieved by Hiq Sil C₁₈V Size 4.6 mm Ø ^*250 mm column in isocratic mode, with mobile phase consisting of acetonitrile-methanol-0.4 M citric acid, (60:30:10, v/v/v). Citric acid used to stabilize nitazoxanide and ofloxacin in mobile phase. The mobile phase was pumped at a rate of 0.6 ml/min and the detection was carried out at 304 nm. The retention time of ofloxacin and nitazoxanide was found to be 3.122 and 5.902 min, respectively. The method was validated for linearity, accuracy, and precision. Linearity for ofloxacin and nitazoxanide were in the range 2-36 μg/ml and 5-90 μg/ml, respectively. The developed method was found to be accurate, precise and selective for simultaneous estimation of ofloxacin and nitazoxanide in tablets.     

Simultaneous Determination of Ofloxacin and Ornidazole in Solid Dosage Form by RP-HPLC and HPTLC Techniques

The objective of this work was to develop and validate simple, rapid and accurate chromatographic methods for simultaneous determination of ofloxacin and ornidazole in solid dosage form. The first method was based on reversed phase high performance liquid chromatography, on Intersil C(18) column (250 mm, 4.6 i.d.), using acetonitrile:methanol: 0.025M phosphate buffer, pH 3.0 (30:10:60 % v/v/v) as the mobile phase, at a flow rate of 1 ml/min at ambient temperature. Quantification was achieved with UV detection at 318 nm over a concentration range of 2-40 μg/ml for ofloxacin and 5-100 μg/ml for ornidazole. The mean retention time of ofloxacin and ornidazole was found to be 4.04 min and 5.83 min, 6.77 min (isomers), respectively. The amount of ofloxacin and ornidazole estimated as percentage of label claimed was found to be 100.23 and 99.61%, with mean percent recoveries 100.20 and 100.93%, respectively. The second method was based on TLC separation of these drugs using silica gel 60F(254) aluminium sheets and dichloromethane:methanol:25% ammonia solution (9.5:1:3 drops v/v) as mobile phase. Detection was carried out at 318 nm over the concentration range of 20-100 ng/spot for ofloxacin and 50-250 ng/spot for ornidazole. The mean R(f) value of ofloxacin and ornidazole was found to be 0.16 and 0.56, 0.78 (isomers), respectively. The amount of ofloxacin and ornidazole estimated as percentage of label claimed was found to be 100.23 and 99.61% with mean percent recoveries 100.47 and 99.32%, respectively. Both these methods were found to be simple, precise, accurate, selective and rapid and could be successfully applied for the determination of pure laboratory prepared mixtures and tablets.     

Determination of fluvoxamine and its metabolite fluvoxamino acid by liquid-liquid extraction and column-switching high-performance liquid chromatography

This study describes a new simultaneous determination of fluvoxamine and fluvoxamino acid by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1.5 ml of plasma using chloroform-toluene (15:85, v/v), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C18 analytical column for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), acetonitrile and perchloric acid (60%) (62.4:37.5:0.1, v/v/v) and was delivered at a flow rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.8-153.6 ng/ml for fluvoxamine and 0.6-115.2 ng/ml for fluvoxamino acid, and their good linearity (r > 0.998) were confirmed. Intra-day coefficient variations (CVs) for fluvoxamine and fluvoxamino acid were less than 6.6 and 6.0%, respectively. Inter-day CVs for corresponding compounds were 6.3 and 6.5%, respectively. Relative errors ranged from -18 to 9% and mean recoveries were 96-100%. The limit of quantification was 1.2 and 0.9 ng/ml for fluvoxamine and fluvoxamino acid, respectively. This method shows successful application for pharmacokinetic studies and therapeutic drug monitoring.     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

Stability-indicating HPLC assays for the determination of prilocaine and procaine drug combinations

Stability-indicating, reversed phase high-performance liquid chromatographic (HPLC) methods have been developed for the determination of several procaine hydrochloride and prilocaine hydrochloride combinations. The separation and quantitation of epinephrine-prilocaine and epinephrine-procaine drug combinations were achieved on a phenyl column using a mobile phase of 80:20% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid sodium salt-acetonitrile at a flow rate of 1 ml x min(-1) and UV detection at 254 nm. The method showed linearity for the epinephrine and prilocaine hydrochloride mixture in the 0.25-2.5 and 8-200 micro g ml(-1) ranges, respectively. The intra- and inter-day relative standard deviations (RSDs) ranged from 0.26 to 2.05% and 0.04 to 0.61% for epinephrine and prilocaine hydrochloride, respectively. The epinephrine and procaine hydrochloride mixture yielded linear ranges of 0.25-2.0 and 5-100 micro g ml(-1) and intra- and inter-day RSDs ranged from 0.23 to 1.88% and 0.07 to 0.26% for epinephrine and procaine hydrochloride, respectively. The assays were shown to be suitable for measuring epinephrine-prilocaine and epinephrine-procaine combinations in their respective injection dosage forms. Stability-indicating HPLC assays were also developed for several other procaine drug combinations since their monographs are present in the USP 24; however, quantitation was not investigated since these combinations are not commercially available. A mobile phase consisting of 80:20% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid-acetonitrile was utilized for the levonordefrin-tetracaine-procaine drug combination, while a mobile phase consisting of 70:30% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid sodium salt-acetonitrile was utilized for the separation of levonordefrin-procaine-propoxycaine and norepinephrine-procaine-propoxycaine. All separations were achieved on a phenyl column at a flow rate of 1 ml x min(-1) and UV detection at 254 nm.     

Determination of capsaicinoids in topical cream by liquid-liquid extraction and liquid chromatography

A reversed-phase liquid chromatography (LC) method has been developed, optimised and validated for the separation and quantitation of capsaicin (CP) and dihydrocapsaicin (DHCP) in a topical cream formulation. Sample preparation involves liquid-liquid extraction prior to LC analysis. The method uses a Hypersil C(18) BDS, 5 micrometer, 250x4.6 mm I.D. column maintained at 35 degrees C. The mobile phase comprises methanol, water, acetonitrile (ACN) and acetic acid (47:42:10:1, v/v/v/v) at a flow rate of 1.0 ml/min. Robustness was evaluated by performing a central composite face-centred design (CCF) experiment. The method shows good selectivity, linearity, sensitivity and repeatability. The conditions allow the separation and quantitation of CP and DHCP without interference from the other substances contained in the cream.     

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.     

Systematic Approach for Trace Level Quantification of 2-N-butyl-4-spirocyclopentane-2-imidazole-5-one Genotoxic Impurity in Irbesartan Using LC-MS/MS

2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one has been highlighted as a potential genotoxic impurity in irbesartan. A sensitive LC-MS/MS method was developed and validated for the determination of 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one in irbesartan. Good separation between 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one and irbesartan was achieved with Symmetry C18 (100×4.6 mm, 3.5 μm) column using 65:35 v/v mixture of 0.1% formic acid and acetonitrile as mobile phase with a flow rate of 0.7 ml/min. The proposed method was specific, linear, accurate, and precise. The calibration curve shows good linearity over the concentration range of 0.1-2.0 μg/ml, which matches the range of limit of quantitation-20×limit of quantitation of estimated permitted level (1.0 μg/ml) of 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one. The method was validated as per International Conference on Harmonization guidelines and was able to quantitate 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one impurity at 1.0 μg/ml with respect to 2 mg/ml of irbesartan. 2-N-butyl-4-spirocyclopentane-2-imidazoline-5-one was not present in the three studied pure and formulation batches of irbesartan and the developed method was a good quality control tool for quantitation of 2-N-butyl-4-spirocyclopentane-2-imidazole-5-one at very low levels in irbesartan.     

Development and validation of a new HPLC method for determination of lamotrigine and related compounds in tablet formulations

A simple HPLC method was developed and validated for quantitation of lamotrigine and its related substances which may coexist in solid pharmaceutical dosage forms. The HPLC separation was achieved on a C18 mu-Bondapack column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-monobasic potassium phosphate solution (35:65, v/v) containing orthophosphoric acid to adjust pH to 3.5 at a flow rate of 1.5 ml/min. The UV detector was operated at 210 nm, and column temperature was adjusted at 40 degrees C. The method was validated for specificity, linearity, precision, accuracy, robustness and limit of quantitation. The degree of linearity of the calibration curves, the percent recoveries of lamotrigine and related substances, the limit of detection and quantitation, for the HPLC method were determined. The method was found to be simple, specific, precise, accurate, and reproducible. The method was applied for the quality control of commercial lamotrigine tablets to quantify the drug and its related substances and to check the formulation content uniformity.     

A stability indicating LC method for felodipine

A stability indicating reversed-phase liquid chromatographic (RP-LC) method was developed for the assay of felodipine as a bulk drug and in pharmaceuticals. The chromatography was performed on a C18 column. Eluents were monitored by UV detection at 238 nm using the mobile phase methanol-potassium dihydrogen orthophosphate (pH 3.5; 0.01 M) (75:25, v/v). The method was statistically validated for linearity, accuracy, precision and specificity. The linearity of felodipine peak area responses was demonstrated within the concentration range of 1-7 microg/ml. The limits of detection and quantitation were 150 and 500 ng/ml, respectively. The method was demonstrated to be precise, accurate and specific with no interference from the tablet excipients and separation of the drug peak from the peaks of the degradation products (oxidative degradation, photodegradation, acid and base degradation). The results indicated that the proposed method could be used in a stability assay.     

Simultaneous determination of lisinopril and H2 antagonists in API, formulations and human serum by using two different HPLC systems

An isocratic reversed phase high-performance liquid chromatographic method has been developed for the simultaneous determination of lisinopril and H₂ antagonists (cimetidine, ranitidine, and famotidine) in bulk, dosage formulations, and human serum at 225?nm. Chromatographic separation was achieved using mobile phase, acetonitrile:water (70:25 v/v) adjusted to pH 3.0 via phosphoric acid 85% with flow rate of 1?ml/min at room temperature. Calibration curves were linear over range of 0.7–12.50?μg/ml for H₂ antagonists and 2.5–100?μg/ml for lisinopril with a correlation coefficient?±?0.999. Limit of detection and limit of quantitation were in the ranges of 0.07–10.4?ng/ml. Intra- and inter-day precision and accuracy results were 98.0–102%.     

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine

A method was developed for the separation and quantification of the anti-nerve agent pyridostgmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1 H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C₁₈ Sep-Pak^R cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C₁₈ column, and UV detection at 280 nm. The compounds were separated using gradient of 1–85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9±9.5, 73.7±9.8, 88.6±9.3, 83.9±7.8, and from urine 69.1±8.5, 74.5±8.7, 85.9±9.8, 83.2±9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.     

Validated HPLC method for determination of chlorzoxazone in human serum and its application in a clinical pharmacokinetic study

A high performance liquid chromatographic (HPLC) method for the determination of chloroxazone in human serum using phenacetin as internal standard (IS) is described. Protein precipitation is used for preparation of the sample. A mobile phase consisting of acetonitrile and 0.5% acetic acid in water mixture (40:60 v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/VIS detector set at 287 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 96% over a concentration range of 1 to 100 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 1, 10, 50, and 100 micrograms/ml ranged from 0.9 to 5.1%. The inter-day RSD ranged from 0.6 to 3.0%. The method is simple, sensitive and has been successfully used in pharmacokinetic study conducted in healthy human volunteers.     

Validation of a RP-HPLC method for the assay of formoterol and its related substances in formoterol fumarate dihydrate drug substance

A stability-indicating reversed-phase high performance liquid chromatographic (HPLC) method has been developed and validated for the assay of formoterol fumarate and the related substances, namely, formoterol fumarate desformyl and formoterol fumarate acetamide analogs, in the active pharmaceutical ingredient. The separation was achieved by isocratic elution using an Alltech Alltima C₁₈ (150×4.6 mm) column, a mobile phase consisting of ammonium acetate (50 mM; pH 5.0)–ethanol (65:35, v/v), a flow rate of 1.0 ml/min and UV detection at 242 nm. The detection and quantitation limits were 0.03 and 08 μg/ml, respectively, while the linear range of detection was between 0.03 and 255 μg/ml. Comparative determinations of formoterol fumarate in three lots of bulk drugs using the proposed HPLC method and the standard potentiometric titration method of pharmacopoeia show that both methods are equivalent for pure drug substance assay. However, the HPLC method allowed the separation and quantitation of the impurities not achievable with the official methods in the bulk drugs. This study shows that the proposed method is accurate, linear, and sensitive as stability indicating assay method for formoterol fumarate in the bulk drug.     

LC method for the determination of oxcarbazepine in pharmaceutical preparations

A simple and accurate HPLC method for determination of oxcarbazepine (OXC) in a new tablet formulation is described. Chromatographic separation was achieved on a Diamonsil C18 column using a mobile phase consisting of acetonitrile, potassium phosphate monobasic buffer (pH 6.8) and water (36:8:56, v/v) at a flow rate of 1.0 ml/min. Absorbance was monitored at 255 nm where OXC has maximum absorption. The linear range of detection for OXC was from 9.96 to 99.6 microg/ml. The proposed method was validated for selectivity, precision, accuracy and limits of detection and quantitation, etc.     

Determination of desloratadine in drug substance and pharmaceutical preparations by liquid chromatography

A simple and selective LC method is described for the determination of desloratadine in drug substance and pharmaceutical preparations. Chromatographic separation was achieved on a Diamonsil BDS C18 column using a mobile phase of a mixture of methanol, 0.03 mol/l heptanesulphonic acid sodium and glacial acetic acid (70:30:4, v/v) at a flow rate of 1.0 ml/min with detection at 247 nm. The developed method was validated in terms of selectivity, linearity, limit of quantitation, precision, accuracy and solution stability. The proposed LC method achieved satisfactory resolution between desloratadine and loratadine possibly present in desloratadine drug substance and other impurities in the mother liquor of the synthetic process. It can be used for the synthetic process control and determination of desloratadine in drug substance and pharmaceutical preparations.     

Ultraperformance liquid chromatography tandem mass spectrometric method for direct quantification of salbutamol in urine samples in doping control

A fast and reliable quantitative method for salbutamol using direct analysis of the urine sample by ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) has been developed. Urine samples were spiked with salbutamol-d₆ (internal standard), and, then, they were diluted with ultrapure water (1:1, v/v). Aliquots of 1 μl of the mixture were directly analyzed by UPLC/MS/MS. The chromatographic separation was performed in a UPLC BEH C₁₈ (100 mm × 2.1 mm, 1.7 μm) column with a mobile phase contained 0.01% formic acid in ultrapure water (v/v) and 0.01% formic acid in acetonitrile (v/v), using gradient elution at 0.6 ml/min. The temperature of the column was set to 45 °C. The total run time was 3.2 min. Electrospray ionization in positive ion mode was used under multiple reaction monitoring (MRM) at different collision energies. Nitrogen and argon were used as desolvation and collision gas, respectively. The method was shown to be linear from 200 to 5000 ng/ml (r² > 0.99). The limit of quantitation was estimated in 200 ng/ml. Intra-assay precision and accuracies, evaluated by using quality control samples containing 550 and 1100 ng/ml salbutamol, were always better than 8.4%. The intermediate precision was estimated to be in the range of 5.6–8.9%. The method was shown to be reliable when applying to routine samples, and the short analysis time resulting from a simple sample preparation and a fast instrumental analysis makes it of great interest for antidoping control purposes.     

Development and Validation of a Liquid Chromatographic Method for Estimation of Dicyclomine Hydrochloride,Mefenamic Acid and Paracetamol in Tablets

Liquid chromatographic method was developed for simultaneous quantitative determination of dicyclomine hydrochloride, mefenamic acid and paracetamol in their combined dosage form. The separation was achieved using a C₁₈ column (250×4.6 mm id, 5 μm) using acetonitrile:20 mM potassium dihydrogen phosphate 70:30 (v/v) adjusted to pH 4 using orthophosphoric acid as mobile phase at a flow rate of 1 ml/min and detection at 220 nm. Separation was completed within 12 min. The retention times of dicyclomine hydrochloride, mefenamic acid and paracetamol were 3.8, 9.3 and 2.5 minutes respectively. The proposed method was found to have linearity in concentration range of 10–100 μg/ml for dicyclomine hydrochloride, 0.05-10 μg/ml for mefenamic acid and 0.1−20 μg/ml for paracetamol. The developed method has been statistically validated and was found to be simple, precise, reproducible and accurate. The developed and validated method was successfully used for the quantitative analysis of commercially available dosage form.     

A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.