High-pressure liquid chromatographic assay of dextromethorphan hydrobromide, guaifenesin, and sodium benzoate in an expectorant syrup

An ion-pair reversed-phase high-pressure liquid chromatographic assay is developed that allows simultaneous quantitation of guaifenesin, dextromethorphan hydrobromide, and sodium benzoate in an expectorant syrup. The method is rapid and accurate. Average recoveries of 99.6, 99.8, and 99.7% with relative standard deviations of 0.5, 0.9, and 0.2% are obtained for guaifenesin, dextromethorphan hydrobromide, and sodium benzoate, respectively, from laboratory prepared samples. Chromatographic conditions are selected to afford a pH that provides adequate separation of guaifenesin, dextromethorphan hydrobromide, sodium benzoate, and sodium saccharin and a detection wavelength that effectively compensates for the great disparity in quantity between guaifenesin and dextromethorphan hydrobromide present in syrups. The relationships between the retention volume of dextromethorphan hydrobromide and the alkyl chain length as well as the concentration of the counterion are studied. The retention profiles for sodium saccharin, guaifenesin, sodium benzoate, and dextromethorphan hydrobromide in the apparent pH range of 2.5 to 6.6 are established.     

Simultaneous determination of phenylephrine hydrochloride, guaifenesin, and chlorpheniramine maleate in cough syrup by gradient liquid chromatography

A simple and reliable high-performance liquid chromatographic method was developed for the simultaneous determination of mixture of phenylephrine hydrochloride (PHENYL), guaifenesin (GUAIF), and chlorpheniramine maleate (CHLO) either in pure form or in the presence of methylparaben and propylparaben in a commercial cough syrup dosage form. Separation was achieved on a C8 column using 0.005 M heptane sulfonic acid sodium salt (pH 3.4 +/- 0.1) and acetonitrile as a mobile phase by gradient elution at different flow rates, and detection was done spectrophotometrically at 210 nm. A linear relationship in the range of 30-180, 120-1800, and 10-60 microg/mL was obtained for PHENYL, GUAIF, and CHLO, respectively. The results were statistically analyzed and compared with those obtained by applying the British Pharmacopoeia (2002) method and showed that the proposed method is precise, accurate, and can be easily applied for the determination of the drugs under investigation in pure form and in cough syrup formulations.     

A new UPLC approach for the quantitation of ephedrine and guaifenesin in a syrup formulation using multivariate optimization strategy

A new ultraperformance liquid chromatography (UPLC) method with photodiode array detection was developed for the quantitative analysis of a commercial syrup formulation containing ephedrine (EPH) and guaifenesin (GUA). In the development of UPLC method, experimental chromatographic conditions, flow rate, column temperature, and percentage of 0.1?M H₃PO₄ in mobile phase, were optimized using chemometric multivariate strategy. From the application of a 3³ full factorial design, the optimal chromatographic conditions were obtained as the flow rate of 0.29?mL/min, column temperature of 36.4°C, and 56.9% of 0.1?M H₃PO₄ in the mobile phase. The optimal conditions gave us a good chromatographic separation of the analyzed drugs with short analysis runtime within 3?min. Calibration curves for EPH and GUA in the linear working range of 4–64 and 6–96?µg/mL, respectively, were obtained using peak areas detected at 215?nm. Performance and validity of the optimized UPLC method were estimated by analyzing independent binary mixtures, inter-day and intra-day samples, and standard addition solutions containing EPH and GUA substances. It was concluded that the proposed method was a promising approach for the quantitative determination and routine analysis of a commercial syrup formulation of the titled substances.     

Improved skin delivery and validation of novel stability-indicating HPLC method for ketoprofen nanoemulsion

Ketoprofen is a non-steroidal anti-inflammatory drug (NSAID) widely used to treat rheumatoid arthritis and other inflammatory diseases. Normally used by oral route, this drug presents numerous side effects related to this administration route, such as nausea, dyspepsia, diarrhea, constipation and even renal complications. To avoid that, topical administration of ketoprofen represents a good alternative, since this drug has both partition coefficient and aqueous solubility suitable for skin application, compared to other NSAIDs. In this study, we describe the production of a nanoemulsion containing ketoprofen, its skin permeation and in vitro release study and a novel validation method to analyze this drug in the permeation samples and a forced degradation study using skin and nanoemulsion samples. The new HPLC method was validated, with all specifications in accordance with validation parameters and with an easy chromatographic condition. Forced degradation study revealed that ketoprofen is sensitive to acid and basic hydrolysis, developing degradation peaks after exposure to these factors. Concerning in vitro release from the nanoemulsion, release curves presented first order profile and were not similar to each other. After 8 h, 85% of ketoprofen was release from the nanoemulsion matrix while 49% was release from control group. In skin permeation study, nanoemulsion enabled ketoprofen to pass through the skin and enhanced retention in the epidermis and stratum corneum, layer on which the formulation presented statistically different values compared to the control group.     

High-performance liquid chromatographic assay for prostaglandin E1 in various ointment vehicles. Separation and stability testing

A high-performance liquid chromatographic method for the determination of prostaglandin E1 (PGE1) incorporated in white petrolatum, white ointment, hydrophilic petrolatum and Plastibase was investigated. The prostaglandin was separated from the oleaginous vehicles with n-hexane-aqueous acetonitrile. Most of white petrolatum, which is a principal component in the vehicles, remained in the n-hexane layer, and the recovery of the drug from any vehicle attained 100%. The method was applied to stability studies of PGE1 in white petrolatum and macrogol ointment. In which pure PGE1 and PGE1-alpha-cyclodextrin complex (PGE1-CD) were incorporated. The drug remained intact for up to 6 months when stored at 5 degrees C. At 25 and 40 degrees C, pure PGE1 was more stable than PGE1-CD and both PGE1 species were more stable in white petrolatum than in macrogol ointment.     

High-performance liquid chromatographic assay of antiinflammatory drugs incorporated in gel ointments. Separation and stability testing

High-performance liquid chromatographic determinations of gabexate mesilate (FOY), prostaglandin E1 (PGE1), PGE1-alpha-cyclodextrin (PGE1-CD), prednisolone (PD) and butyl flufenamate (BF) incorporated in gel ointment were investigated. The gel ointment is composed of a carboxy vinyl polymer (1.3%, w/w) and a large amount of an aqueous organic solvent. A methanol extraction system offered simultaneous advantages of the removal of the polymer and the recovery of active ingredients from the gel phase. The recoveries of the drugs were 100%. The stabilities of PGE1, PGE1-CD, FOY, PD and BF incorporated in gel ointment, stored at 5, 25 and 40 degrees C for up to 90 days, were investigated.     

Development and validation of a stability-indicating liquid chromatographic method for determination of emtricitabine and related impurities in drug substance

A novel stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for assay and determination of impurities of emtricitabine in drug substance. Emtricitabine was found to be degraded under acidic, alkaline, and oxidative stress conditions and to be more labile under oxidative conditions. The drug proved to be stable to dry heat and photolytic degradation. Resolution of major and minor degradation impurities was achieved on an Intersil ODS-3V column utilizing 10 mM sodium phosphate buffer and methanol (85:15) as mobile phase. Detection was at 280 nm. Validation studies were performed as per ICH recommended conditions. The developed method was found to be linear, accurate, specific, selective, precise, and robust.     

High-performance liquid chromatographic analysis of azlocillin in serum

Summary A rapid and sensitive high performance liquid chromatographic method is described for the determination of azlocillin in serum. This method involves a short manual protein precipitation of the sample followed by an injection into a PR 18 column for separation and quantitation. The mobile phase was a 22% (V/V) solution acetonitrile in phosphate buffer pH 4.8 at a flow rate of 2,5 ml/min. The spectrophotometer detector was set at 220 nm with a sensitivity of 0.08 AUFS.     

High-performance liquid chromatographic analysis of doxacurium, a new long-acting neuromuscular blocker

A rapid and sensitive high-performance liquid chromatographic procedure was developed for the analysis of the new, long-acting neuromuscular blocker doxacurium in the plasma and urine of dog and man and in the bile of dog. Samples were prepared on solid-phase extraction cartridges containing a methyl (C1) bonded phase and were chromatographed on a 15 cm reversed-phase column (C1) using a mobile phase of 0.05 M monobasic potassium phosphate-acetonitrile (30:70, v/v). The compound was detected at 210 nm with a lower limit of quantitation of 10 ng/ml. An inter-assay accuracy of 90-92% was obtained for the analysis of the drug from biological fluids. The method was applied to studies of doxacurium after intravenous administration to dog and man.     

High-performance liquid chromatographic analysis of amiloride in plasma and urine

A high-performance liquid chromatographic method has been developed for amiloride in rabbit plasma and urine which uses a reversed-phase C18 column, a mobile phase (flow-rate 2 ml/min) consisting of 32% acetonitrile in 0.15 M perchloric acid, pH 2.2, and spectrofluorometric detection via excitation at 286 nm. A simple extraction step with ethyl acetate eliminates interfering peaks. Short retention times of about 2.3 and 3.8 min are observed for amiloride and the internal standard, triamterene, respectively. The method can measure 4 ng/ml amiloride in plasma. This assay has been used to explore the pharmacokinetics of amiloride in rabbits. The plasma disposition profile is biexponential after a 50-mg intravenous bolus dose and there is no evidence for saturable elimination at zero-order infusion rates of 1.8, 3.6 and 7.2 mg/h.     

An improved liquid chromatographic method for the analysis of tylosin and its impurities

A selective reversed-phase (RP) liquid chromatographic (LC) method coupled with UV for the determination of tylosin and its related substances is described. The gradient method uses a Capcell pak C18 ACR column (25 cm×4.6 mm id, 5 μm) maintained at a temperature of 60°C. The mobile phases consist of acetonitrile, phosphate buffer pH 5.5 and water: (A; 27.5:10:62.5 v/v/v) and (B; 50:10:40 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. It allows the separation of all known and 22 other unknown related substances (≥0.02%) from the main compound and from one another. The method shows good precision, sensitivity, linearity (between 0.2 μg/mL and 1.25 mg/mL) and robustness. The limit of quantification is 0.2 μg/mL, corresponding to 0.020%. Seven bulk tylosin samples containing a large number of impurities were examined using this method.     

New validated liquid chromatographic and chemometrics-assisted UV spectroscopic methods for the determination of two multicomponent cough mixtures in syrup

Multivariate spectrophotometric calibration and liquid chromatographic (LC) methods were applied to the determination of 2 multicomponent mixtures containing diprophylline, guaiphenesin, methylparaben, and propylparaben (Mixture 1), or clobutinol, orciprenaline, saccharin sodium, and sodium benzoate (Mixture 2). For the multivariate spectrophotometric calibration methods, principal component regression (PCR) and partial least-squares regression (PLS-1), a calibration set of the mixtures consisting of the components of each mixture was prepared in 0.1 M HCl. Analytical figures of merit such as sensitivity, selectivity, limit of quantitation, and limit of detection were determined for both PLS-1 and PCR. The LC separation was achieved on a reversed-phase C18 analytical column by using isocratic elution with 20 mM potassium dihydrogen phosphate, pH 3.3-acetonitrile (55 + 45, v/v) as the mobile phase and UV detection at 260 and 220 nm for Mixture 1 and Mixture 2, respectively. The proposed methods were validated and successfully applied to the analysis of pharmaceutical formulations and laboratory-prepared mixtures containing the 2 multicomponent combinations.     

High-performance liquid chromatographic analysis of isoxazolylpenicillins in plasma and urine samples

Summary A simple and fast liquid chromatographic method is described, applicable to the routine analysis of isoxazolylpenicillins (cloxacillin, dicloxacillin, flucloxacillin) in biological fluids (plasma, urine). The method is based on a simple dilution step employed to destroy the protein binding, which is over 95%, and allows the detection of concentrations down to 10μg/ml. In order to analyze concentrations of less than 10μg/ml, a liquid-liquid extraction with dichloromethane must be executed prior to the reversed-phase analysis with absorbance detection at 206nm. The minimum detectable amounts of the isoxazolylpenicillins with this procedure are between 2.5 and 5.1 ng in 100μl plasma samples. The stability of the penicillin samples in aqueous solutions (stock solutions, eluents) was investigated and no significant degradation was observed during the storage and analysis of the samples. Furthermore, the degree of protein binding was established by using a suitable ultrafiltration technique, and the usefulness of the developed procedures in pharmacokinetic studies was demonstrated.     

High-performance liquid chromatographic analysis of trilostane and ketotrilostane in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for assaying trilostane, a synthetic steroid, and one of its metabolites, ketotrilostane, in small volumes of rat plasma has been developed. A single liquid-liquid extraction was used to isolate the two compounds from acidified plasma prior to the quantitative analysis. The HPLC conditions involved the use of a Spherisorb ODS column (250 mm x 4.6 mm I.D.) and a mobile phase of 1,4-dioxan-Sorenson's buffer at pH 5.0 (52:48, v/v). Ethisterone was used as an internal standard. Trilostane and ketotrilostane were detected by their ultraviolet absorbance at 255 nm. Recoveries greater than 80% and detection limits of 50 ng/ml were obtained for both compounds. Inter-day coefficients of variation were less than 10%.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.     

Development of a New Analytical Method for Determination of Related Components in Nateglinide

An isocratic reverse phase liquid chromatographic (RP-LC) assay method has been developed for the quantitative determination of nateglinide and its related components namely imp-1 and imp-2 in bulk drug and in pharmaceutical dosage form, used for the treatment of type II diabetes mellitus. The developed method is stability indicating and also can be used for stability testing. The chromatographic separation was achieved on C-8, 150 × 4.6 mm, 3.5 μm stationary phase. The LC method employs solution A as mobile phase. Solution A contains a mixture of phosphate buffer pH 3.0: acetonitrile (50:50 v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. In the developed LC method the resolution between nateglinide and its potential impurities namely imp-1 and imp-2 was found to be greater than 5.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid medium, alkaline medium and oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.2%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.