Development and validation of a reverse-phase liquid chromatographic method for the assay of lidocaine hydrochloride in alginate-Gantrez microspheres

A simple, fast and reliable reverse-phase high-performance liquid chromatographic (HPLC) method was developed for the assay of lidocaine hydrochloride (LH) in Gantrez-alginate microspheres. Separation was achieved in a LiChrospher C18 column, using a mobile phase consisting of acetonitrile:ammonium acetate (0.0257 M) adjusted to pH 4.85 with acetic acid, in the ratio 70:30 (v/v) and a flow rate of 0.6 mL/min. The detection was made with a diode array detector measuring at the maximum for the compound. The validation study demonstrated that the method was precise, accurate and linear over the concentration range of analysis with a limit of detection of 0.001 mg/mL. The limit of quantification was 0.002 mg/mL. Linear regression analysis in the range of 0.8-2.4 mg/mL gave correlation coefficients higher than 0.995. The method developed was applied to the analysis of lidocaine in microsphere samples in order to evaluate in next papers, the encapsulation efficiency of different formulations.     

Development and validation of RP-HPLC method for cetrimonium bromide and lidocaine determination

The simple and rapid RP-HPLC method, for the simultaneous determination of lidocaine and cetrimonium bromide in the presence of pellet color corrigent, was developed. Separations were performed on a Beckman Ultrasphere ODS 4.6 mm x 15 cm, 5 microm particle column at 40 degrees C. The mobile phase consisted of water phase and acetonitrile (72:28 V/V), pH value of the mobile phase was adjusted to 2.0 with 85% ortophosphoric acid. Bisacodil was used as an internal standard. The flow rate was 1 ml/min and UV detection was performed at 208 nm. The proposed RP-HPLC method was validated and all the parameters for the validation of the method are given. According to the obtained results, the developed method was found to be suitable and accurate for the determination of these drugs in commercial formulations.     

A reversed-phase high-performance liquid chromatographic assay for the determination of N-acetylcysteine in aqueous formulations

A stability-indicating assay is described for the determination of N-acetylcysteine in aqueous pharmaceutical formulations. The sample is diluted to an appropriate concentration with dilute aqueous orthophosphoric acid. An aliquot of the solution, containing added l-tyrosine as an internal standard, is chromatographed using a 10-mum C(18) stationary phase with dilute orthophosphoric acid (pH 2.0) containing 0.5% w/v of sodium perchlorate as the mobile phase. The assay, which has a relative standard deviation of about 0.8%, can also be used as a test for related impurities in N-acetylcysteine. It is also suitable for determining the N-acetylcysteine content of the drug substance.     

Improved reversed-phase liquid chromatographic method combined with pulsed electrochemical detection for the analysis of amikacin

A two-step gradient liquid chromatographic method combined with pulsed electrochemical detection is described for the determination of amikacin and its impurities. The mobile phase is composed of an aqueous solution containing 1.8 g/l sodium 1-octanesulphonate, 14 ml/l tetrahydrofuran, 50 ml/l of phosphate buffer pH 3.0 and sodium sulphate, which was 20 g/l in mobile phase A and 28 g/l in mobile phase B. 0.5 M sodium hydroxide was added post-column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18, 5 microm, 250 mm x 4.6 mm i.d.) column was the most suitable. Compared to previously published investigations, the proposed method showed higher sensitivity and efficiency, allowing the separation of the main component amikacin from 16 impurities, 7 of which were of unknown identity. A central composite experimental design was used to assess the robustness. The method showed good repeatability and linearity in the assay range. The method was further applied to analyze some commercial samples.     

Development and validation of a liquid chromatographic method for the determination of branched-chain amino acids in new dosage forms

A reversed-phase liquid chromatographic method (RP-LC) is proposed and validated for the analysis of branched-chain amino acids (l-leucine, l-isoleucine and l-valine) in new pharmaceutical formulations. The pre-column derivatization reaction of these amino acids with 2,4-dinitrofluorobenzene (DNFB) has been investigated considering the matrix effect. The compound reacts at 60 degrees C for 10 min at pH 9 with the amino function, in presence of cetyltrimethylammonium bromide (CTAB), to give adducts that have been separated on a RP amide C16 column and detected at lambda=360 nm. Linear responses were observed for each derivative. The intra-day precision (R.S.D.) was 相似文献   

Improved penetration of aminoglycosides and fluoroquinolones into the aqueous humour of patients by means of Acuvue contact lenses

Objectives: In order to improve the penetration of topically applied drugs in ophthalmology, the suitability of hydrophilic contact lenses (Acuvue, Vistacon, power −1.0 D) as a drug delivery system for antibiotics was tested. A prospective study was undertaken to determine the transcorneal penetration of five topically applied aminoglycosides and fluoroquinolones into the aqueous humour of patients. Methods: Two hundred and sixty-five patients undergoing cataract extraction received 0.3% gentamicin, kanamycin, tobramycin, ciprofloxacin or ofloxacin solution by two different modes of administration: either as eye drops (nine drops every 15 min, starting 2 h prior to surgery) or by means of a drug delivery system (Acuvue contact lenses soaked for 1 h in eye drop solution without preservatives, 1–5 h prior to surgery). At the beginning of cataract extraction, 50–100 μl aqueous fluid was aspirated from the anterior chamber and immediately stored at −80 °C. Antibiotic concentrations were measured using fluorescence polarisation immuno-assays (aminoglycosides) or high-performance liquid chromatography (fluoroquinolones). Results: After soaking for 1 h in 0.3% eye drop solutions, Acuvue contact lenses released about 190–250 μg aminoglycoside and ofloxacin and 1000 μg ciprofloxacin. These amounts are considerably lower or in the same order of magnitude than obtained with application of eye drops (1350 μg). From the aminoglycosides tested, only gentamicin and tobramycin, but not kanamycin, were able to penetrate into the aqueous humour of patients. After the wearing of antibiotic-soaked lenses, mean aqueous humour concentrations were higher than after the use of eye drops. This difference reached significance in tobramycin (1.09 (1.30) μg · ml⁻¹ vs 0.49 (0.79) μg · ml⁻¹), ciprofloxacin (1.23 (0.60) μg · ml⁻¹ vs 0.38 (0.33) μg · ml⁻¹) and ofloxacin (5.55 (2.53) μg · ml⁻¹ vs 0.56 (0.37) μg · ml⁻¹). The percentage of patients with aqueous humour concentration above the MIC₉₀ of Staphylococcus epidermidis, the most common cause of postoperative endophthalmitis, was 92% and 100% after wearing ciprofloxacin- or ofloxacin-soaked lenses, respectively. Conclusion: Gentamicin and tobramycin penetrated into the aqueous humour of patients, whereas kanamycin was not able to overcome the corneal barrier. Acuvue contact lenses soaked in 0.3% eye drop solutions can release sufficient amounts of gentamicin, ciprofloxacin and ofloxacin to produce bacteriostatic concentrations in the humor aquosus. Acuvue contact lenses can be recommended as a drug delivery system for fluoroquinolones. Received: 15 October 1998 / Accepted in revised form: 16 December 1998     

Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10min at ambient temperature) in the dark in sodium acetate buffer (80mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150mmx4.6mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was 相似文献   

Reversed-phase high-performance liquid chromatographic method for the assay of 1,4-dioxane in sulphated polyoxyethylene alcohol surfactants

A rapid high-performance liquid chromatographic method has been developed for the assay of 1,4-dioxane in ethoxylated fatty alcohol sulphates. After solid-phase extraction using Bakerbond C₁₈ cartridges, samples were directly analysed on a LiChrospher CH-8 reversed-phase column with UV detection at 200 nm and an acetonitrile-water eluent. Recovery of 1,4-dioxane from the surfactant matrix was 95.7% in the 40 to 120 μg g⁻¹ range. The minimum quantifiable amount was 18 μg g⁻¹. The procedure is simple, reproducible, specific and suitable for routine analyses of commercial surfactants.     

Development and validation of a high-performance liquid chromatographic method for bioanalytical application with rimonabant

A simple and feasible high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of rimonabant in human plasma. The chromatographic separation was carried out in a Hypersil BDS, C₁₈ column (250 mm × 4.6 mm; 5 μm). The mobile phase was a mixture of 10 mM phosphate buffer and acetonitrile (30:70, v/v) at a flow rate of 1.0 ml/min. The UV detection was set at 220 nm. The extraction recovery of rimonabant in plasma at three quality control (QC) samples was ranged from 84.17% to 92.64%. The calibration curve was linear for the concentration range of 20–400 ng/ml with the correlation coefficient (r²) above 0.9921. The method was specific and sensitive with the limit of quantification of 20 ng/ml. The accuracy and precision values obtained from six different sets of QC samples analyzed in separate occasions ranged from 88.13% to 106.48% and 0.13% to 3.61%, respectively. In stability tests, rimonabant in human plasma was stable during storage and assay procedure. The method is very simple, sensitive and economical and the assay was applied to human plasma samples in a clinical (pharmacokinetic) study of rimonabant.     

Tumour necrosis factor-alpha levels in aqueous humour and serum from patients with uveitis: the involvement of HLA-B27

SUMMARY

Objective: To study the local and systemic levels of the tumour necrosis factor-α in patients with active uveitis and to determine the implication of TNF-α in rheumatological uveitis and to observe if this relationship is more significant in the B27 positive patients.

Patients and methods: Patients were selected on the basis of a diagnosis of uveitis of any aetiology. Data from 23 patients were stratified into two categories according to the presence or absence of systemic rheumatic disease. The first group comprised nine patients with rheumatic disease; the second group contained 14 patients without rheumatic disease. The patients were also sub-classified into those who were HLA-B27 positive (14 patients) and those who were not. TNF-α levels in serum and aqueous humour from a group of 16 patients with uncomplicated cataracts were analysed as a control group.

Results: In the control group (n?=?16) the serum TNF-α concentration was 13.1?±?2.9pg/ml and the aqueous humour concentration of TNF-α was 0.56?±?1.53?pg/ml. In uveitis patients (n?=?23) the serum TNF-α concentration was 35.35?±?26.77?pg/ml and the aqueous humour concentration of TNF-α was 15.1?±?1.70?pg/ml (p?<?0.01). In HLA-B27 positive patients (n?=?9) the serum TNF-α concentration was 45.56?±?34.17?pg/ml and the aqueous humour concentration of TNF-α was 15.89?±?0.93?pg/ml. In HLA-B27 negative patients (n?=?14) the serum TNF-α concentration was 28.79?±?19.38?pg/ml and aqueous humour concentration of TNF-α was 14.57?±?1.91?pg/ml (p?<?0.01).

Conclusions: The concentration of TNF-α in aqueous humour in patients who are HLA-B27 positive is significantly greater than in those who are B27 negative. No significant differences in the concentrations of TNF-α in serum or aqueous humour in patients with or without rheumatic diseases were detected. TNF-α is a cytokine that may participate actively in the pathogenesis of clinical uveitis.     

Simultaneous determination of Ciprofloxacin and the active metabolite of Prulifloxacin in aqueous human humor by high-performance liquid chromatography

A rapid and simple method for determining two fluoroquinolones (FQNs), namely Ciprofloxacin and Ulifloxacin, this being the last active metabolite of Prulifloxacin, in aqueous human humor (AHH) has been developed and validated. The calibration data resulted linearly correlated in the 4-500 ng/mL concentration range with 8 ng/mL lower limit of quantification (LLOQ) for Ciprofloxacin, and 5-600 ng/mL concentration range with 6 ng/mL LOD for Ulifloxacin. The proposed analytic procedure has been validated by testing quality control sample (QCS) of AHH probed with the two FQNs at 10, 50, 500, and 1000 ng/mL concentration values. Validation of the method has been checked by accuracy and precision data of intra-day and long-term experiments. The two FQN concentrations have been measured by HPLC technique with UV detection at 278-nm wavelength for the AHH of patients to whom were supplied oral doses of FQNs (500 mg) twice in a day, within 1-24h before the surgery intervention of cataract. The average concentration of Ciprofloxacin resulted 186 ng/mL and that of Ulifloxacin 78 ng/mL. The nice quality of the proposed analytic procedure means that it may be suitable for in vivo studies of pharmacokinetics regarding these substances in the AHH medium.     

Ultra high-pressure liquid chromatographic assay of moxifloxacin in rabbit aqueous humor after topical instillation of moxifloxacin nanoparticles

The present report describes a rapid and sensitive ultra high-pressure liquid chromatography (UHPLC) method with UV detection to quantify moxifloxacin in rabbit aqueous humor. After deproteinisation with acetonitrile, gradient separation of moxifloxacin was achieved on a Waters Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm) column at 50 °C. The mobile phase consisted of 0.1% trifluoroacetic acid in water and acetonitrile at a flow rate of 0.4 ml/min. Detection of moxifloxacin was done at 296 nm. Method was found to be selective, linear (r = 0.9997), accurate (recovery, 97.30–99.99%) and precise (RSD, ≤1.72%) in the selected concentration range of 10–1000 ng/ml. Detection and quantitation limit of moxifloxacin in aqueous humor were 0.75 and 2.5 ng/ml, respectively. The aqueous humor levels of moxifloxacin after single topical instillation in three formulations, i.e. moxifloxacin solution (Moxi-SOL), anionic nanoparticles (Moxi-ANP) and cationic nanoparticles (Moxi-CNP) were investigated. A fourfold increase in the relative bioavailability was observed with the Moxi-CNP (AUC_0→t, 3.14 μg h/ml) compared with Moxi-SOL (AUC_0→t, 0.79 μg h/ml) and Moxi-ANP (AUC_0→t, 0.72 μg h/ml) formulation. The results indicate that the cationic nanoparticle increases ocular bioavailability of moxifloxacin and prolong its residence time in the eye.     

Development and validation of a reversed-phase liquid chromatographic method for analysis of aspirin and warfarin in a combination tablet formulation

A stability-indicating liquid chromatographic method for the simultaneous analysis of aspirin and warfarin in warfarin sodium/aspirin combination (DuP 647) tablets has been developed and validated. This paper presents linearity, accuracy, precision, robustness, recovery, limits of detection and quantitation, and cross-validation data. The method has been shown to be specific and stability-indicating, and to give results comparable to existing methods for the individual components. Solution stability has been optimized for routine analysis.     

Development and validation of a reversed-phase liquid chromatographic method for analysis of degradation products of estradiol in Vagifem tablets

A stability-indicating liquid chromatographic method for the determination of degradation products and impurities in Vagifem, estradiol vaginal tablets has been developed and validated. Vagifem is a low dose preparation containing only 25microg 17beta-estradiol in a tablet matrix of 80mg (a drug to excipient ratio of 1:3200). This paper presents the rationale for the optimization of the sample preparation in order to minimize placebo interference as well as validation data for linearity, accuracy, precision, ruggedness, specificity and limits of detection and quantification. Data shows that the method is suitable for routine analysis of minute amounts of estradiol impurities.     

Development and validation of a dissolution method for warfarin sodium and aspirin combination tablets

A dissolution method for warfarin sodium-aspirin combination tablets was developed which utilizes USP Apparatus 1 (baskets) at 50 rpm with 900 ml of phosphate buffer (pH 6.8; 0.05 M) medium at 37°C. A reversed-phase liquid chromatographic method was also developed for the simultaneous determination of warfarin sodium, aspirin and salicylic acid on an octadecylsilica column using acetonitrile-tetrahydrofuran-glacial acetic acid-water (23:5:5:67, v/v/v/v) as the mobile phase with UV detection at 282 nm. Validation data were obtained which demonstrate that the dissolution methodology is accurate, precise, linear and rugged for the combination tablets.     

Development and validation of a reversed-phase liquid chromatographic method for analysis of estradiol valerate and medroxyprogesterone acetate in a tablet formulation

A simple and accurate liquid chromatographic method was developed for estimation of estradiol valerate and medroxyprogesterone acetate in pharmaceuticals. Drugs were chromatographed on a reverse phase C18 column, using a mixture (30:70) of ammonium nitrate buffer and acetonitrile and eluants monitored at a wavelength of 280 nm. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for its linearity, accuracy, precision and selectivity. Due to its simplicity and accuracy, the authors believe that the method may be used for routine quality control analysis. It does not require any specific sample preparation except the use of a column guard before the analytical column and suitable prefilter attached to the syringe prior to injection.     

Development of a liquid chromatographic method for the determination of related substances and assay of d-cycloserine

d-cycloserine or d-4-amino-3-isoxazolidinone is an antibiotic produced by Streptomyces garyphalus and Streptomyces orchidaceus. d-Cycloserine is used in the second line treatment of tuberculosis and is often used in developing countries. Therefore, expensive high-tech techniques are not recommended for analysis. Here, a liquid chromatography method with ultraviolet detection (LC-UV) is described using a base deactivated column (Hypersil BDS column; 25 cm x 4.6 mm I.D.) kept at 45 degrees C. The gradient method uses mobile phases containing acetonitrile (ACN), 20mM sodium octane sulphonate (SOS), 0.2M potassium dihydrogen phosphate buffer pH 2.8, water: A: (4:70:10:16v/v/v/v) and B: (17:70:10:3v/v/v/v). The method proved to be robust, linear, repeatable, sensitive, selective and easy to perform. For the related substances test 50 microl of a 0.5 mg/ml d-cycloserine solution is injected. For assay, a concentration of 0.1 mg/ml is proposed to avoid overloading of the detector.     

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 μm Zorbax Dikema C₁₈ column (150 mm × 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine–acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 μg/ml in plasma and 0.007 and 0.14 μg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 μg/ml and nikethamide ranged from 1.25 to 106.8 μg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 μg/ml of lidocaine and 8.0 to 72.4 μg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.     

Development and validation of a stability-indicating high performance liquid chromatographic assay for benoxinate

Benoxinate is a local anaesthetic used for ophthalmic applications. The aim of this study was to develop a rapid and simple stability-indicating method for the determination of benoxinate formulated for ophthalmic use, evaluate its long-term stability and identify its major degradation product. Benoxinate was eluted on a 10 microm Spherisorb phenyl column, 250 x 3.2 mm, with a mobile phase consisting of acetonitrile-buffer (pH 3.5) (35:65, v/v), pumped at 0.8 ml min(-1) flow rate. The buffer was composed of sodium dihydrogen phosphate (50 mM), sodium hydrogen sulfate (2.5 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The analyte was quantified spectrophotometrically at 308 nm. The chromatograms of benoxinate formulations obtained by this method showed benoxinate (t = 4.5 min) well resolved from its degradation product (t = 2.3 min), which was separately identified by means of HPLC-MS as 4-amino-3-butoxybenzoic acid. The assay was demonstrated to have high accuracy, precision and linearity. The method was implemented in investigating the long-term stability of benoxinate 0.4% ophthalmic solutions. The method was found to be simple, quick and selective in determining benoxinate concentrations in fresh and aged preparations.     

Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney

Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from 1 to 25 μg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation <10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) over the concentration range studied (r²>0.99). The assay was used to study the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2–3 months, n=6), adult (6–9 months, n=5) and aged (12–16 months, n=3) male Sprague–Dawley rats at an initial drug concentration of 20 μg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased by only 2-fold in aged (2.2±0.42 ml/min per gram) compared to young (4.6±0.70 ml/min per gram, P<0.05) rats. Furthermore, renal excretion ratio was significantly higher in aged rats (27±8.0 vs. 15±5.0, P<0.05). These preliminary findings challenge the ‘Whole Nephron Hypothesis’ that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.