Determination of miconazole in pharmaceutical creams using internal standard and second derivative spectrophotometry

A simple method is proposed for miconazole determination in pharmaceutical creams, based on extraction and second derivative spectrophotometry. In the presence of sodium lauryl sulfate (0.5%) and sulphuric acid (0.4 mol l(-1)), the miconazole and internal standard (IS) (methylene blue) were extracted to 100 microl of methylene chloride. The organic phase was evaporated in the nitrogen stream and the dry residue was dissolved in methanol (1.5 ml). The analytical signal was obtained as the ratio between second derivative absorbances measured at 236.9 nm (miconazole) and at 663.2 nm (IS). The use of IS in such multi-stage procedure enabled quite good analytical performance in calibration range 50.0 400 mg l(-1): linear correlation coefficient 0.9995, precision (measured as CV for ten replicates) at 50.0 mg l(-1) and at 400 mg l(-1) of miconazole was 1.5 and 0.5% respectively. Four commercial pharmaceutical creams were analyzed and the results obtained were in good agreement with the results obtained by reversed-phase high performance liquid chromatography (HPLC).     

In situ generation of Co(II) by use of a solid-phase reactor in an FIA assembly for the spectrophotometric determination of penicillamine

A flow injection analysis (FIA) manifold for the determination of penicillamine in pharmaceutical preparations is proposed. The manifold includes a solid-phase reactor for the in situ production of the derivatizing reagent, Co(II) ion, which forms a coloured complex with penicillamine in an alkaline medium. The reactor is prepared by natural immobilization of cobalt carbonate on a polymer matrix, which endows it with a high mechanical and microbiological stability. The cobalt released by passage of a 5 x 10(-4) mol l(-1) sulphuric acid stream at a flow-rate of 2.3 ml min(-1) is merged with a volume of 314 microl of sample containing penicillamine in ammonium-ammonia buffer at pH 9.5 to measure the absorbance at 360 nm. Beer's law is obeyed over the penicillamine concentration range 5-60 mg l(-1). The limit of detection (LOD) of the method is 1 mg l(-1) and its throughput 70 samples h(-1).     

Determination of ranitidine hydrochloride in pharmaceutical preparations by titrimetry and visible spectrophotometry using bromate and acid dyes

Four new methods using titrimetry and spectrophotometry are described for the determination of ranitidine hydrochloride (RNH) with potassium bromate as the oxidimetric reagent and acid dyes, methyl orange, indigo carmine and metanil yellow. In direct titrimetry (method A), the drug is titrated directly with bromate in acid medium and in the presence of excess of bromide using methyl orange indicator. In back titrimetry (method B), the drug is treated with a measured excess of bromate in the presence of bromide and acid, and the unreacted bromine is determined iodometrically. Both spectrophotometric methods are based on the oxidation of RNH by a known excess of bromate in acid medium and in the presence of excess of bromide followed by estimation of surplus oxidant by reacting with either indigo carmine (method C) or metanil yellow (method D), and measuring the absorbance at 610 or 530 nm. In methods B, C and D, reacted oxidant corresponds to the drug content. The experimental conditions are optimized. Titrimetric procedures are applicable over the ranges 1-10 mg (A) and 1-17 mg (B), and the reaction stoichiometry is found to be 1:1 (BrO(-)(3): RNH). In spectrophotometric methods, the absorbance is found to increase linearly with increasing concentration of RNH, which is corroborated by the calculated correlation coefficient (r) of 0.9984 (C) and 0.9976 (D). The systems obey Beer's law for 2-12 and 1-7 microg ml(-1), for methods C and D, respectively. Method D with a molar absorptivity of 9.82 x 10(4) l mol(-l) cm(-1) is found to be more sensitive than method C ( epsilon = 2.06 x l0(4) l mol(-1) cm(-1)). The limits of detection and quantification are reported for both the spectrophotometric methods. The proposed methods were applied successfully to the determination of RNH in tablets and injections. The reliability of the assay was established by parallel determination by the official method and by recovery studies.     

Bioavailability of salvianolic acid B in conscious and freely moving rats

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.     

A flow-through optosensing device with fluorimetric transduction for rapid and sensitive determination of dipyridamole in pharmaceuticals and human plasma

A flow-through optosensor with fluorimetric transduction has been prepared for the sensitive and selective determination of dipyridamole in aqueous solutions and biological fluids. The method is based on a monochannel flow-injection analysis system using Sephadex QAE A-25 resin, placed into a Hellma 176-QS fluorimetric flow-through cell, as an active sorbing substrate. The native fluorescence of dipyridamole fixed on the solid sorbent is continuously monitored at wavelengths of 305 and 490 nm for excitation and emission, respectively. After obtaining the maximum fluorescence intensity, the eluent solution (KH(2)PO(4)/NaOH buffer solution, c(T)=0.05 mol l(-1), pH 6.0) is allowed to reach the flow cell, the analyte is removed, and the resin support is regenerated. When an NaOH (10(-4) mol l(-1))/NaCl (0.1 mol l(-1)) solution is used as carrier solution, at a flow-rate of 1.56 ml min(-1), the sensor responds linearly in the measuring range of 10-500 microg l(-1) with a detection limit of 0.94 microg l(-1) and a throughput of 22 samples per hour (300 microl of sample volume). The relative standard deviation for ten independent determinations (200 microg l(-1)) is less than 0.82%. The method was satisfactorily applied to the determination of dipyridamole in pharmaceutical preparations and human plasma.     

Selective determination of pyridoxine in the presence of hydrosoluble vitamins using a continuous-flow solid phase sensing device with UV detection

A very simple, inexpensive and highly selective flow injection UV spectrophotometric method for the determination of vitamin B(6) is presented. The native absorbance of the analyte is continuously monitored at 290 nm when it is transiently retained on Sephadex SP C-25 cation exchanger gel beads placed in the detection area of a flow cell. The preconcentration on the active solid phase provides by itself a high increase in sensitivity compared with the same procedure carried out without a solid support. The analytical response is linear in the concentration ranges 1-10 and 2-20 microg ml(-1) using 600 and 1250 microl of sample, respectively. The R.S.D. (%) are 0.65 (600 microl) and 0.84 (1250 microl) and the detection limits 0.08 and 0.02 microg ml(-1), respectively. The procedure was successfully applied to the determination of vitamin B(6) in pharmaceuticals containing (among other active principles) hydrosoluble vitamins in much higher concentrations than that tolerated by the method if performed in aqueous solution. Nevertheless they were tolerated using the proposed sensor due to the selective retention of the analyte.     

维生素B2片含量及含量均匀度测定方法的研究

目的：对维生素B：片的含量测定方法进行改进,并对其含量均匀度进行考察。方法：以冰醋酸和沸水为溶剂,立即密塞量瓶、强力振摇约2～3min,并超声振荡约5min使维生素B2溶解,暗室密塞放冷,加水稀释,快速滤过,以1％醋酸溶液为空白,用分光光度法测定含量。结果：方法学考察表明,维生素B：在1．69—42．32mg·L-1的线性范围内呈良好的线性关系（r=0．9999）;平均回收率为100．3％（n=9,RSD0．8％）。结论：本法快速、简便、准确、重现性好。可有效控制维生素B2片的质量。     

Determination of cyanocobalamin, betamethasone, and diclofenac sodium in pharmaceutical formulations, by high performance liquid chromatography

The aim of this work was to develop an analytical method for a simultaneous determination of cyanocobalamin (Vitamin B12), betamethasone, and diclofenac, present in pharmaceutical formulations, by high performance liquid chromatography, assuring rapidity, accuracy, precision, and selectivity. The working conditions were as follows: RP18 column of 125 mm x 4 mm ID and a particle size of 5 microm; mobile phase acetonitrile-water (40:60; v/v) (pH* 3.45) adjusted with acetic acidl flow gradient from 0.8 to 1.9 ml/min.; injection volume of 20 microl; temperature 34 degrees C and detection at 240 nm. The method was adequately validated, and linearity, accuracy, as well as the system, method and interday precision, for each active principle, were determined.     

A comparison of matrix resolution method, ratio spectra derivative spectrophotometry and HPLC method for the determination of thiamine HCl and pyridoxine HCl in pharmaceutical preparation

A comparison of two spectrophotometric methods and a HPLC method were described in this work for the analysis of pyridoxine hydrochloride and thiamine hydrochloride in a vitamin combination. In the first method, A₁¹ (1%, 1 cm) values of these two compounds were calculated using absorbances measured at 246.8 and 290.5 nm in zero-order spectra. The matrix was written for A₁¹ (1%, l cm) values and the concentration of both compounds were determined using ‘Matlab’ software. In the second method, the measurements in the derivative of the ratio spectra were made at 297.8 and 309.5 nm for pyridoxine hydrochloride and at 245.6 and 257.7 nm for thiamine hydrochloride. The calibration graphs were established in the range 8–40 μg/ml of both vitamins. In the HPLC method, the separation of these compounds was realized on a Nucleosil 100-5 C₁₈ column with 0.1 M (NH₄)₂C0₃–water–methanol (5:15:80 v/v) as the mobile phase. Results of spectrophotometric and HPLC procedures were compared.     

Protective effect of vitamin B6 in chromium-induced oxidative stress in liver

The aim of this study was to examine the efficacy of vitamin B6 against chromium (Cr)-induced oxidative stress. Adult male albino Wistar rats (100-120 g) were used in this study. Potassium dichromate, a Cr VI compound, was administered at a dose of 127 mg kg(-1) p.o. Vitamin B6 (pyridoxine hydrochloride) was administered at a dose of 100 mg kg(-1) p.o. either alone or 12 h prior to Cr or simultaneously with Cr. Chromium treatment induced oxidative stress in the liver as measured by increased lipid peroxidation (LPO) and decreased vitamin C, vitamin E, glutathione (GSH), glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione reductase (GR). Both pre- and simultaneous treatments countered Cr-induced oxidative stress; pre-treatment was more effective than concurrent administration. The results demonstrate the antioxidant potential of vitamin B6.     

Direct determination of verapamil in rat plasma by coupled column microbore-HPLC method

This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence detection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such as precolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system without sample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mm Capcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation of compounds and two mobile phases are used as mobile phase A (50mM ammonium phosphate, pH 4.5) and B (50mM ammonium phosphate:acetonitrile=70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 microl of rat plasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min (mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaks during a total run time of 25 min. The calibration curve for verapamil showed good linearity (r(2)=0.9997) over the concentration range of 0.01-2.50 microg/ml. The mean RSD (%) values of intra-day (n=5) and inter-day (n=5) variability of verapamil ranged from 1.96 to 9.06 and 0.62 to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 microg/ml, respectively, for verapamil using 10 microl of rat plasma. An automated coupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3mg/kg of verapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.     

Flow injection determination of levodopa in tablets using a solid-phase reactor containing lead(IV) dioxide immobilized

A flow injection spectrophotometric procedure was developed for determining levodopa in tablets. The determination of this drug was carried out by reacting it with lead(IV) dioxide immobilized in polyester resin packed in a solid-phase reactor and the dopachrome yielded was monitored at 520 nm. The analytical curve for levodopa was linear in the concentration range from 1.0x10(-4) to 1.0x10(-3) mol l(-1) with a detection limit of 8.0x10(-5) mol l(-1). The relative standard deviation (R.S.D.) was 0.2% for a solution containing 4.0x10(-4) mol l(-1) levodopa (n=10), and 90 determinations per hour were obtained.     

New HPLC method for separation of blood plasma phospholipids

The aim of the present work was to develop a new HPLC method for separation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) from small-volume samples of blood plasma. Human plasma glycerophospholipids were separated by liquid-liquid extraction method followed by solid phase extraction (SPE) on aminopropyl columns. Reversed-phase Sephasil C8 column (10 cm x 2.1 mm, I.D. 5 microm) and micropreparative chromatograph "SMART" were used for separation of PC, PE, LPC and PI from SPE phospholipids extract. Binary-step gradient of eluent A: acetonitrile-methanol (130:5, v/v) and B (0.01% trifluoroacetic acid) provided good, fast and reproducible resolution of investigated phospholipids classes in 12 min at 30 degrees C. Eluted phospholipids were detected at wavelengths lambda=235 and 254 nm. This method made it possible to determine quantitatively: 5 microg ml(-1) PC, 1 microg ml(-1) LPC, 4 microg ml(-1) PE and 3 microg ml(-1) PI in blood plasma samples.     

Simultaneous micellar LC determination of lidocaine and tolperisone

A micellar liquid chromatography (MLC) procedure was developed for the simultaneous separation and determination of lidocaine hydrochloride (LD HCl) and tolperisone hydrochloride (TP HCl) using a short-column C18 (12.5 mm x 4.6 mm, 5 microm), sodium dodecyl sulfate (SDS) with a small amount of isopropanol, and diode array detector. The optimum conditions for the simultaneous determination of both drugs were 0.075 mol l(-1) SDS-7.5% (v/v) isopropanol with a flow rate of 0.7 ml min(-1) and detection at 210 nm. The LOD (2S/N) of LD HCl was 0.73 ng 20 microl(-1), whereas that of TP HCl was 1.43 ng 20 microl(-1). The calibration curves for LD HCl and TP HCl were linear over the ranges 0.125-500 microg ml(-1) (r(2)=0.9999) and 1.00-500 microg ml(-1) (r(2)=0.9997), respectively. The %recoveries of both drugs were in the range 98-103% and the %RSD values were less than 2. The proposed method has been successfully applied to the simultaneous determination of TP HCl and LD HCl in various pharmaceutical preparations.     

Fully automated LC method for the determination of sotalol in human plasma using restricted access material with cation exchange properties for sample clean-up

A simple and rapid fully automated bio-analytical method for the liquid chromatographic (LC) determination of sotalol in human plasma has been described. The method is based on the use of a new kind of porous silica restricted access material (RAM) with cation exchange properties for sample clean-up. 100 microl of plasma samples were directly injected into the precolumn coupled on-line to a reversed-phase column (RP-Select B) by means of column switching system. The plasma matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate and methanol (97:3; v/v). By rotation of the switching valve, the analytes were then eluted in back-flush mode for 2 min and transferred to the analytical column by the LC mobile phase constituted of a mixture of methanol and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 1-octanesulphonic acid sodium salt (20:80; v/v). The flow-rate was 1.0 ml/min and sotalol was detected using fluorescence detection at 235 and 300 nm as excitation and emission wavelengths, respectively. The method was then validated using a new approach based on accuracy profile over a concentration range from 5 to 500 ng/ml. The limit of quantitation (LOQ) was 5 ng/ml and the total analysis time was 19 min.     

Assay of artemether, methylparaben and propylparaben in a formulated paediatric antimalarial dry suspension

Two HPLC-UV methods are described for the separate determination of artemether (AM) and the combined preservatives, methylparaben and propylparaben in a pharmaceutical dosage form. These analytes are contained in a dry suspension with a high amount of non-soluble excipients, some of which can interfere with the analysis. This makes their separation and analysis of the actives complex. Moreover, due to the wide difference in concentrations, the three analytes could not be quantitated simultaneously. Artemether was analysed using a reversed-phase Nucleosil C(18) column [5 microm, 125 mm x 4 mm (i.d.)] with a mixture of acetonitrile: potassium phosphate buffer pH 5.0 (0.05 M): water [48:32:10 (v/v/v)] as mobile phase. Due to the low solubility of the hydroxy benzoic acid esters in water, their sodium salts were used in the formulation. Complete separation of these preservatives was achieved on the same type of column as artemether using as eluent acetonitrile: potassium phosphate buffer pH 5.0 (0.05 M) (30:70, v/v). Quantitation was achieved with UV detection at 215 nm for artemether and 254 nm for the parabens, respectively. And in both methods, pump flow rate was 1.0 ml/min, sample injection volume 20 microl, ambient temperature maintained and no prior sample extraction methods were necessary throughout the experiments. Calibration curves were linear at concentration ranges of 4-16 microg/ml, 1-4 microg/ml and 1-10 mg/ml for methylparaben, propylparaben and artemether respectively. The excipient powder interference could be eliminated by diluting the sample and the analytes eluted at relatively short times using these systems. Both methods were further validated in terms of specificity, linearity, precision and accuracy. The procedures prescribed here are simple, selective and can be used for routine quality control and stability indicating tests involving the analysed compounds formulated in complex matrices.     

Flow-injection chemiluminescence detection for studying protein binding of terbutaline sulfate with on-line microdialysis sampling

The binding of terbutaline sulfate to bovine serum albumin was studied in vitro using the technique of microdialysis sampling combined with flow-injection chemiluminescence analysis (FIA-CL). In the presence of formaldehyde, terbutaline sulfate can be oxidized by KMnO(4) to produce high chemiluminescence emission in sulfate acid media. The concentration of terbutaline sulfate is proportional with the CL intensity in the range of 1 x 10(-7)-2 x 10(-5) mol l(-1) with a detection limit of 3 x 10(-8) mol l(-1). The drug and protein were mixed in different molar ratios in 0.067 mol l(-1) phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 microl min(-1) and the dialytic efficiency of terbutaline sulfate under the experimental conditions was 26.3%. The data obtained by proposed microdialysis flow-injection chemiluminescence method was analyzed with Scrathard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scrathard analysis were 4.11 x 10(4) l mol(-1) and 1.06, respectively. The proposed system proved that FIA-CL coupled with on-line microdialysis sampling is a simple and reliable technique for the study of drug-protein interaction.     

Effects of folinic acid and Vitamin B12 on ethanol-induced developmental toxicity in mouse

The objective of this study was to assess whether combined supplementation of folinic acid (FA) and Vitamin B(12) (VB(12)) could suppress ethanol-induced developmental toxicity better than FA alone in mouse embryos cultured in vitro. In this study, exposure to 4.0mg/ml ethanol for 48 h yielded growth retardation and various malformations of the embryos. FA (10(-5), 10(-4)mol/l) or VB(12) (10(-6), 10(-5)mol/l) alone supplementation improved the growth parameters moderately, however combined supplementation of the two vitamins (10(-5)mol/l FA plus 10(-6)mol/l VB(12), 10(-5)mol/l FA plus 10(-5)mol/l VB(12), 10(-4)mol/l FA plus 10(-6)mol/l VB(12) and 10(-4)mol/l FA plus 10(-5)mol/l VB(12)) showed better protective effects, including both the growth and development parameters of the embryos, than either vitamin alone at the same dosage. The present investigation indicated that combined supplementation of folic acid and VB(12) might be a better choice than folic acid alone in the prevention of ethanol-induced birth defects.     

A simple spectrophotometric method for determination of sodium diclofenac in pharmaceutical formulations

A new, simple, rapid and accurate spectrophotometric method is proposed for determination of sodium diclofenac (SD) in pharmaceutical preparations based on its reaction with concentrated nitric acid (63% w/v). The reaction product is a yellowish compound with maximum absorbance at 380 nm. The corresponding calibration curve is linear over the range of 1-30 mg l(-1), while the limit of detection is 0.46 mg l(-1).     

Voltammetric determination of pyridoxine (vitamin B6) by use of a chemically-modified glassy carbon electrode

A novel carbon nanotube-modified glassy carbon electrode was described for the direct determination of pyridoxine. The electrochemical behavior of pyridoxine was investigated, and a well-defined oxidation peak with high sensitivity was observed at the modified electrode. Owing to the unique structure and extraordinary properties of multi-wall carbon nanotube (MWNT), the MWNT-modified glassy carbon electrode shows obvious electrocatalytic activity to the oxidation of pyridoxine, since it greatly enhances the oxidation peak current of pyridoxine as well as lowers its oxidation overpotential. Based on this, a very sensitive and simple voltammetric method was developed for the measurement of pyridoxine. A sensitive linear voltammetric response for pyridoxine was obtained in the concentration range of 5 x 10(-7)-1 x 10(-4)mol/L, and the detection limit is 2 x 10(-7)mol/L using differential pulse voltammetry. Compared with other voltammetric methods, this proposed method possesses many advantages such as very low detection limit, fast response, low cost and simplicity. The practical application of this new analytical method was demonstrated with pyridoxine drugs.