Development and validation of liquid chromatographic and UV derivative spectrophotometric methods for the determination of famciclovir in pharmaceutical dosage forms

A high-performance liquid chromatographic method and a UV derivative spectrophotometric method for the determination of famciclovir, a highly active antiviral agent, in tablets were developed in the present work. The various parameters, such as linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation were studied according to International Conference on Harmonization guidelines. HPLC was carried out by using the reversed-phase technique on an RP-18 column with a mobile phase composed of 50 mM monobasic phosphate buffer and methanol (50 : 50; v/v), adjusted to pH 3.05 with orthophosphoric acid. The mobile phase was pumped at a flow rate of 1 ml/min and detection was made at 242 nm with UV dual absorbance detector. The first derivative UV spectrophotometric method was performed at 226.5 nm. Statistical analysis was done by Student's t-test and F-test, which showed no significant difference between the results obtained by the two methods. The proposed methods are highly sensitive, precise and accurate and therefore can be used for its Intended purpose.     

Validation of an HPLC method for quantitation of MDMA in tablets

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method is developed and validated for the quantitation of 3,4-methylenedioxymethamphetamine (MDMA) in tablets. The chromatographic separation is achieved with potassium phosphate buffer (pH 3.2)-acetonitrile (9:1, v/v) as mobile phase, a Chromspher B column, and UV detection at 210 nm. The calibration curve is linear from 1.4 to 111 microg/mL. The percent relative standard deviation for intra- and interday precision studies is 2.7% each. The measurement uncertainty is estimated to 9%. The method is specific and successfully used for routine quantitation of MDMA in tablets.     

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Comparative Spectrophotometric,Spectrofluorometric, and High‐performance Liquid Chromatographic Study for the Quantitative Determination of the Binary Mixture Felodipine and Ramipril in Pharmaceutical Formulations

Abstract

Two‐component mixtures of felodipine (FLD) and ramipril (RMP) were assayed by derivative UV spectrophotometry, spectrofluorometry, and high performance liquid chromatography (HPLC). The spectrophotometric methods included a zero‐crossing first‐ and second‐order derivative procedure and a derivative compensation technique for the determination of binary mixtures with overlapping spectra. The spectrofluorometric method was based on first‐ and second‐order derivatives of the emission spectra (zero‐crossing point). Results from these methods were compared with those obtained by an exclusively developed isocratic reversed phase HPLC method. A reversed‐phase Adsorbosil DS analytical column, with methanol‐acetonitrile‐water (50∶30∶20, v/v) mobile phase at a flow rate of 1.5 ml/min, was used with a UV detector. The temperature was set at 25±0.2°C. Results obtained by the spectrophotometric and spectrofluorometric methods were comparable to those obtained by the HPLC method, as far as analysis of variance (ANOVA) test results were concerned. It is concluded that the developed methods are equally accurate, sensitive, and precise; with direct and simple application to pharmaceutical formulations of felodipine and ramipril combination, without interference from common pharmaceutical adjuvants.     

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.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Determination of foscarnet (trisodium phosphonoformate) in pharmaceutical preparations by high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic method with UV detection has been developed and validated for the quantitative determination of foscarnet in isoosmotic sodium chloride aqueous solution. The mobile phase consisted of mixture of methanol:water (30:70 v/v), containing 1 mm tetrahexylammonium hydrogen sulphate at pH 5.80. The analyte was separated on a reversed-phase C(18) column packed with 4 microm spherical particles of octadecylsilane. Hydrochlorothiazide was used as internal standard. UV detection at 232 nm allowed a quantification limit of 50 microg/mL. The assay was linear from 50 to 4000 microg/mL. The coefficient of variation was < or =2.52% for intra-assay precision and < or =3.49% for inter-assay precision. The deviation from the nominal value ranged from -0.57 to 0.47% for the same-day accuracy and from -0.75 to 3.06% for day-to-day accuracy.     

Determination of theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by high-performance liquid chromatography

Theobromine, theophylline, and caffeine are determined simultaneously by a rapid and selective reversed-phase high-performance liquid chromatography (HPLC) method with UV detection in by-products of cupuacu and cacao seeds. The determination is carried out in the raw and roasted ground cupuacu seeds and in the corresponding powders obtained after pressure treatment. The by-products of both cupuacu seeds and cacao seeds are obtained under the same technological conditions. The HPLC method uses isocratic elution with a mobile phase of methanol-water-acetic acid (80:19:1) (v/v) at a flow rate of 1 mL/min and UV absorbance detection at 275 nm. Total elution time for these analytes is less than 10 min, and the detection limit for all analytes is 0.1 mg/g. The amounts of theobromine and caffeine found in all the cupuacu samples are one or more orders of magnitude lower than those from cacao. Theophylline is found in all cacao samples except for the roasted ground paste, and it is only found in the roasted ground paste in the cupuacu samples.     

Determination of linezolid in pharmaceutical dosage forms by liquid chromatography and ultraviolet spectroscopy

A liquid chromatographic (LC) method and a UV spectrophotometric method were developed and validated for quantitative determination of linezolid in tablets and injection. The isocratic LC analyses were performed on an RP18 column using a mobile phase composed of methanol-water-acetonitrile (40 + 40 + 20, v/v/v) at a flow rate of 1.0 mL/min. The UV spectrophotometric method was performed at 251 nm. The analytical methods were validated according to International Conference on Harmonization guidelines. The calibration graphs were linear (correlation coefficient > 0.999) in the studied concentration ranges of 0.1 to 10 microg/mL for LC and 2 to 16 microg/mL for UV spectrophotometry. The relative standard deviation values for intraday and interday precision studies were <2%, and the accuracy was >98% for both methods. The specificity of the LC method was proved using forced degradation. Statistical analysis showed no significant difference between the results obtained by the 2 methods. The proposed methods are precise and accurate and can be applied directly to the oral and parenteral pharmaceutical preparations of linezolid.     

Determination of amlodipine in pharmaceutical dosage forms by liquid chromatography and ultraviolet spectrophotometry

A liquid chromatography (LC) method and an ultraviolet (UV) spectrophotometric method were developed and validated for quantitative determination of amlodipine in tablets and compounded capsules. The isocratic LC analyses were performed on an RP18 column using a mobile phase composed of 0.1% (v/v) ortho-phosphoric acid (pH 3.0) -acetonitrile (60 + 40, v/v) at a flow rate of 1.0 mL/min. The UV spectrophotometric method was performed at 238 nm. The analytical methods were validated according to International Conference on Harmonization Guidelines. The calibration graphs were linear [correlation coefficient (r) > 0.999] in the studied concentration range of 10-30 microg/mL for LC and 10-35 microg/mL for UV spectrophotometry. The relative standard deviation values for intraday and interday precision studies were less than 2%, and the accuracy was greater than 98% for both methods. The specificity of the LC method was proved using forced degradation. Statistical analyses showed no significant difference between the results obtained by the 2 methods. The proposed methods are precise and accurate and can be applied directly and easily to the oral pharmaceutical preparations of amlodipine.     

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and isoconazole nitrate in creams

A new RP-LC method and two new spectrophotometric methods, principal component regression (PCR) and first derivative spectrophotometry, are proposed for simultaneous determination of diflucortolone valerate (DIF) and isoconazole nitrate (ISO) in cream formulations. An isocratic system consisting of an ACE C18 column and a mobile phase composed of methanol-water (95 + 5, v/v) was used for the optimal chromatographic separation. In PCR, the concentration data matrix was prepared by using synthetic mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 29 wavelengths in the range of 242-298 nm for DIF and ISO in the zero-order spectra of their combinations. In first derivative spectrophotometry, dA/dlambda values were measured at 247.8 nm for DIF and at 240.2 nm for ISO in first derivative spectra of the solution of DIF and ISO in methanol-water (3 + 1, v/v). The linear ranges were 4.00-48.0 microg/mL for DIF and 50.0-400 microg/mL for ISO in the LC method, and 2.40-40.0 microg/mL for DIF and 60.0-260 microg/mL for ISO in the PCR and first derivative spectrophotometric methods. These methods were validated by analyzing synthetic mixtures. These three methods were successfully applied to two pharmaceutical cream preparations.     

Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism

Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.     

Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography

An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges.     

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

Summary A reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 m reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 l sample volume of a biological fluid.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

Determination of mycophenolic acid in human plasma by high-performance liquid chromatography

The development, validation and evaluation of high-performance liquid chromatography (HPLC) method for quantifying mycophenolic acid in human plasma is described. The method involved protein precipitation using acetonitrile, after addition of terazosin as an internal standard. Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing UV detection at 215 nm. The mobile phase consisted of 0.02 M potassium dihydrogenphosphate solution adjusted to pH 6.9 with 2 M potassium hydroxide solution-acetonitrile (80:20 (v/v)) at a flow rate of 1.5 ml/min. The total run time was 21.0 min. The assay was linear from 0.2 to 25 microg/ml with goodness of fit (r2) greater than 0.99 observed with three precision and accuracy batches during validation. The observed mean recoveries were 89.3 and 98.0% for drug and internal standard, respectively. The applicability of this method to pharmacokinetic studies was established after successful application during a 34-subject bioavailability study. The method was found to be precise, accurate and specific during the study.     

HPLC and chemometrics-assisted UV-spectroscopy methods for the simultaneous determination of ambroxol and doxycycline in capsule

High-performance liquid chromatography (HPLC) and multivariate spectrophotometric methods are described for the simultaneous determination of ambroxol hydrochloride (AM) and doxycycline (DX) in combined pharmaceutical capsules. The chromatographic separation was achieved on reversed-phase C(18) analytical column with a mobile phase consisting of a mixture of 20mM potassium dihydrogen phosphate, pH 6-acetonitrile in ratio of (1:1, v/v) and UV detection at 245 nm. Also, the resolution has been accomplished by using numerical spectrophotometric methods as classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS-1) applied to the UV spectra of the mixture and graphical spectrophotometric method as first derivative of the ratio spectra ((1)DD) method. Analytical figures of merit (FOM), such as sensitivity, selectivity, analytical sensitivity, limit of quantitation and limit of detection were determined for CLS, PLS-1 and PCR methods. The proposed methods were validated and successfully applied for the analysis of pharmaceutical formulation and laboratory-prepared mixtures containing the two component combination.     

Reverse phase high-performance liquid chromatographic determination of primary and secondary aliphatic alcohols as phthalate monoesters by UV detection

Summary Determination of primary and secondary aliphatic alcohols in aqueous and nonaqueous solutions by high-performance liquid chromatography (HPLC) is described. The alcohol is derivatized with phthalic anhydride to yield the corresponding monophthalate. Separation of the derivative is achieved on an ODS-10 reverse phase (RP) column by elution with an appropriate isocratic mobile phase of acetonitrile and aqueous phosphoric acid (0.1%). Detection is accomplished by UV absorption at 230 nm or 280 nm giving linear calibration plots, for alcohols in aqueous solution from 0.05% to 0.50% (w/v) and in pyridine solution from 0.0025% to 0.0250% (w/v).     

加压毛细管电色谱-紫外检测法分析糖皮质激素及其在头发检测中的应用

采用反相加压毛细管电色谱与紫外检测联用技术,建立了一种高效、简便的糖皮质激素分析方法,适用于头发中糖皮质激素的检测。使用C18反相色谱柱,流动相为pH 8.0, 1.5 mmol/L的Tris-乙腈(65:35, v/v),检测波长为245 nm、分离电压为~10 kV、反压为10.5 MPa、泵流速为0.05 mL/min,进行等度洗脱,倍他米松、地塞米松、泼尼松、泼尼松龙、醋酸泼尼松龙、醋酸氢化可的松、醋酸可的松、皮质脂酮等8种激素在20 min内实现快速分离。各组分的质量浓度线性范围达到3个数量级,检出限(S/N=3)在μg/g水平,迁移时间和峰面积的相对标准偏差(RSD)分别小于4.8%和7.4%。将所建立方法应用于头发样品分析,检测前采用蛋白酶水解提取和净化处理样品,不同浓度糖皮质激素的回收率为71%～85%。该研究为糖皮质激素药物暴露监测以及压力检测提供了新手段,有望用于滥用药物的控制和临床诊断。