HPLC-Fluorescence Detection Method for Concurrent Estimation of Domperidone and Naproxen. Validation and Eco-Friendliness Appraisal Studies.

This work demonstrates a simple and reliable HPLC method with fluorimetric detection for simultaneous estimation of domperidone (DOM) and naproxen (NAP). Successful chromatographic separation was accomplished using Inertsil ODS C18 column (5 µm, 4.6 × 150 mm) with gradient elution of the mobile phase consisting of 0.01 M phosphate buffer (pH 5.5) solution and acetonitrile. The gradient elution started with 25% acetonitrile increased linearly to 65% in 5 min, then kept at this percentage till the end of the run. The mobile phase was pumped at a flow rate of 1.0 mL/min. The excitation wavelength at 284 nm was found suitable for both DOM and NAP since it corresponds to a maximum for the minor component DOM and measurable excitation for NAP, while using 316 and 355 nm as emission wavelengths for DOM and NAP, respectively. Peaks eluted with excellent resolution at retention times 4.4 and 6.3 min for DOM and NAP, respectively. Performance of the proposed method was tested according to ICH guidelines in regard to linearity, ranges, precision, accuracy, robustness, detection and quantitation limits. Calibration curves were linear in the ranges of 0.8-3.6 and 1.0-2.5 µg/mL for DOM and NAP respectively with correlation coefficients not less than 0.9996. The validated method was successfully applied to the analysis of DOM and NAP in their laboratory prepared tablets resembling the commercial dosage form, and assay results were favorably compared with a published reference HPLC method. The method's greenness was assessed using the Analytical Eco-Scale and the novel Analytical Greenness metric (AGREE).

LC-MS bioanalysis of targeted nasal galantamine bound chitosan nanoparticles in rats' brain homogenate and plasma.

Validated LC-MS method for the direct quantitative analysis of galantamine (acetylcholinesterase inhibitor) was developed in rat cerebrospinal fluid and brain homogenate besides rat plasma, utilizing structurally close nalbuphine as an internal standard. After a simple protein precipitation step, samples are separated on 2-µm C18 column kept at 40 °C, using isocratic flow of 80% methanol in pH 9.5 ammonium formate buffer, and retention times were about 1.8 and 2.9 min for galantamine and nalbuphine, respectively. Mass detection with electrospray ionization (ESI) and positive polarity was able to detect 0.2 ng mL-1 galantamine using single ion monitoring mode (SIM) at m/z 288 for galantamine and m/z 358 for nalbuphine. The method showed linearity within the range of 0.5 - 300 ng mL-1. The proposed method was validated according to FDA guidelines. Trueness and precision showed acceptable values at all quality control levels, and recoveries were within 85.6 - 114.3% in all matrices at all runs and with relative standard deviations within 0.2 - 12.4%. The method was used to study in vivo brain uptake and pharmacokinetics of galantamine from brain homogenate and plasma samples following the administration of nasal galantamine-bound chitosan nanoparticles compared to oral and nasal galantamine solutions, in scopolamine-induced Alzheimer's disease rat model.

Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate.

OBJECTIVE: Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). METHODS: Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 µm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 µm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. RESULTS: Method I showed good linearity over the range 10-100 µg/mL for both dugs. Method II's linear ranges were 0.001-0.1 and 0.02-1 µg/mL for BIS and AML, respectively. CONCLUSION: The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. HIGHLIGHTS: To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol.

Validated Stability-Indicating HPLC-DAD Method for the Simultaneous Determination of Diclofenac Sodium and Diflunisal in Their Combined Dosage Form.

A simple, rapid, and highly selective HPLC-DAD method was developed for the simultaneous determination of diclofenac sodium (DIC) and diflunisal (DIF) in pure form and in their combined formulation. Effective chromatographic separation was achieved using a Zorbax SB-C8 (4.6×250 mm, 5 µm particle size) column with a mobile phase composed of 0.05 M phosphoric acid, acetonitrile, and methanol in the ratio of 40:48:12 (by volume). The mobile phase was pumped isocratically at a flow rate of 1 mL/min, and quantification of the analytes was based on measuring their peak areas at 228 nm. The retention times for diflunisal and diclofenac were about 7.9 and 9.5 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 5-100 µg/mL for both drugs with correlation coefficients >0.9998. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from four of their related substances and potential impurities as well as from forced-degradation (hydrolysis, oxidation, photolysis, and dry heat) products. The validated HPLC method was successfully applied to the analysis of DIC and DIF in their combined dosage form (suppositories). The proposed method made use of the diode array detector (DAD) as a tool for peak identity and purity confirmation.

Gradient HPLC-diode array detector stability-indicating determination of lidocaine hydrochloride and cetylpyridinium chloride in two combined oral gel dosage forms.

A simple, rapid, and selective HPLC-diode array detector method was developed for the simultaneous determination of lidocaine hydrochloride (LD) and cetylpyridinium chloride (CPC) in two combined pharmaceutical formulations. Effective chromatographic separation was achieved on a Zorbax SB-C8 (4.6 x 250 mm, 5 microm particle size) column with gradient elution using a mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (v/v) acetonitrile, ramped up linearly to 85% in 5 min, and then was constant until the end of the run. The mobile phase was pumped at a flow rate of 1.2 mL/min. The multiple wavelength detector was set at 214 and 258 nm, and quantification of the analytes was based on measuring their peak areas. The retention times for LD and CPC were about 3.4 and 7.3 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. Calibration curves were linear in the range of 5-200 and 10-400 microg/mL for LD and CPC, respectively, with correlation coefficients > 0.999. The proposed method was proven to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) as well as from forced-degradation products. The validated HPLC method was extended to the analysis of LD and CPC in two combined oral gel preparations for which the two analytes were successfully resolved from the pharmaceutical adjuvants and quantified with recoveries not less than 97.9%.

Validated HPLC determination of the two fixed dose combinations (chlordiazepoxide hydrochloride and mebeverine hydrochloride; carvedilol and hydrochlorothiazide) in their tablets.

Simple, rapid, and selective RP-HPLC methods with UV detection were developed for simultaneous determination of chlordiazepoxide hydrochloride and mebeverine hydrochloride (Mixture I) and carvedilol and hydrochlorothiazide (Mixture II). The chromatographic separation in both mixtures was achieved by using an RP-C8 (octylsilyl) analytical column. For Mixture I, a mobile phase composed of acetonitrile-0.05 M disodium hydrogen phosphate-triethylamine (50 + 50 + 0.2, v/v/v), pH 2.5, was used; the detector wavelength was 247 nm. For Mixture II, the mobile phase consisted of acetonitrile-0.05 M disodium hydrogen phosphate (50 + 50, v/v), pH 4.0, and the detector was set at 220 nm. Quantification of the analytes was based on measuring their peak areas. Both mixtures were resolved in less than 6 min. The reliability and analytical performance of the proposed HPLC procedures were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. The linear dynamic ranges were 2.5-150 and 2.5-500 microg/mL for chlordiazepoxide HCI and mebeverine HCI, respectively, and 0.25-200 and 0.25-150 microg/mL for carvedilol and hydrochlorothiazide, respectively. The validated HPLC methods were successfully applied to the analysis of their commercial tablet dosage forms, for which no interfering peaks were encountered from common pharmaceutical adjuvants.

HPLC-DAD stability indicating determination of nitrofurazone and lidocaine hydrochloride in their combined topical dosage form.

In this work, a simple, rapid, and selective high-performance liquid chromatography (HPLC) method with diode array detection was developed for the simultaneous determination of nitrofurazone (NZ) and lidocaine hydrochloride (LD). The chromatographic separation was achieved by using Zorbax Eclipse XDB-C(18) (4.6 x 150 mm, 5 mum p.s.) analytical column and a mobile phase composed of 0.025 M disodium hydrogen phosphate-methanol-triethylamine (70:30:0.1, v/v/v) (pH 4.0) at a flow rate of 1 mL/min. The detector was set at wavelengths 374 and 220 nm for NZ and LD, respectively, and quantification of the analytes was based on measuring their peak areas. The retention times for NZ and LD were approximately 4.5 and 5.7 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, selectivity, robustness, and detection and quantification limits. The linear dynamic ranges were 0.5-25 and 2.5-100 mug/mL for NZ and LD, respectively, with correlation coefficients > 0.999. The stability-indicating aspects of the proposed method were demonstrated by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) as well as from forced-degradation products. The validated HPLC method was successfully extended to the analysis of the combined topical dosage form (soluble dressing) where no interfering peaks were encountered from the dosage form matrix or the inactive ingredients.

Simultaneous spectrofluorimetric determination of amlodipine besylate and valsartan in their combined tablets.

Amlodipine, a dihydropyridine calcium channel blocker, and valsartan, an angiotensin II receptor blocker, are co-formulated in a single-dose combination for the treatment of hypertension. The combination is used by patients whose blood pressure is not adequately controlled on either component monotherapy. This work describes a simple, sensitive, and reliable spectrofluorimetric method for the simultaneous determination of the two antihypertensive drugs; amlodipine besylate (AML) and valsartan (VAL) in their combined tablets. The method involved measurement of the native fluorescence at 455 nm (λ(Ex) 360 nm) and 378 nm (λ(Ex) 245 nm) for AML and VAL, respectively. Analytical performance of the proposed spectrofluorimetric procedure was statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection, and quantification limits. Regression analysis showed good correlation between fluorescence intensity and concentration over the concentration ranges 0.2-3.6 and 0.008-0.080 µg mL⁻¹ for AML and VAL, respectively. The limits of detection were 0.025 and 0.0012 µg mL⁻¹ for AML and VAL, respectively. The proposed method was successfully applied for the assay of the two drugs in their combined pharmaceutical tablets with recoveries not less than 98.85%. No interference was observed from common pharmaceutical additives. The results were favourably compared with those obtained by a reference spectrophotometric method.

Selective and stability-indicating methods for the simultaneous determination of mexiletine hydrochloride and/or its related substance: 2,6-dimethylphenol.

Four simple, rapid, sensitive, and selective analytical procedures were developed for determination of mexiletine hydrochloride (MX) and/or its related substance: 2,6-dimethylphenol (DMP). The latter is a synthetic impurity for which a maximum pharmacopeial limit is defined. The first method depends on derivative-ratio spectrophotometry, for which the first-derivative signals of the ratio spectra at 259 nm (Deltalambda = 3 nm) are selected for the determination of MX. The second method is based on the spectrofluorometric measurement of MX in alkaline solution in the presence of 15 mM sodium dodecyl sulfate micellar medium at 292 nm (lambdaEx 260 nm). The third method is based on liquid chromatographic (LC) separation of MX and DMP on an RP-C8 column with a mobile phase consisting of 50 mM Na2HPO4-acetonitrile (60 + 40, adjusted to pH 2.4), and quantification of the analytes is achieved with UV detection at 212 nm based on peak area. The fourth method uses the coupling reaction of DMP with 2,6-dibromo-quinone-4-chlorimide (DBQC) in borate buffer to form an intensely colored product that was spectrophotometrically measured using first-derivative amplitudes at 670 nm (Deltalambda = 6 nm) for the determination of DMP. Different variables affecting each method were carefully investigated and optimized. The reliability and analytical performance of the proposed methods, including linearity, range, precision, accuracy, and detection and quantitation limits, were statistically validated. The first 3 methods were successfully applied for the stability-indicating determination of MX in laboratory-prepared mixtures with DMP, as well as for the determination of MX in capsules. Also, the LC and the DBQC spectrophotometric methods permitted the selective determination of DMP in the presence of a large excess of the parent drug at or near the pharmacopeial limit (0.1-1%).