Spectrophotometric and atomic absorption spectrometric determination of ramipril and perindopril through ternary complex formation with eosin and Cu(II)

Two sensitive, spectrophotometric and atomic absorption spectrometric procedures are developed for the determination of ramipril and perindopril. Both methods are based on the formation of a ternary complex, extractable with chloroform, between copper(II), eosin and the two cited drugs. Spectrophotometrically under the optimum condition, the ternary complexes showed an absorption maximum at 535 nm, with apparent molar absorptivities of 6.55 and 4.00×10³ mol⁻¹cm⁻¹ and Sandell’s sensitivities of 5.80×10⁻² and 1.04×10⁻¹μg cm⁻² for perindopril and ramipril, respectively. The solution of ternary complex obeyed Beer’s law in concentration ranges 10–60 and 20–100 μg ml⁻¹ for perindopril and ramipril, respectively. The proposed method was applied to the determination of the two cited drugs in pharmaceutical tablets. The atomic absorption spectrometric method, directly through the quantitative determination of copper content of the organic extract of the complex, was also investigated for the purpose of enhancing the sensitivity of the determination. The spectrophotometric and atomic absorption spectrometric procedures hold their accuracy and precision well when applied to the determination of ramipril and perindopril dosage forms.     

Utility of certain π-acceptors for the spectrophotometric determination of perindopril

Simple, rapid, accurate and sensitive spectrophotometric methods are described for the determination of perindopril. The methods are based on the reaction of this drug as n-electron donor with 2,3-dichloro-5,6-dicyano-p-benzoquinone(DDQ)-7,7,8,8-tetracyanoquinodimethane (TCNQ), tetracyanoethylene (TCNE), chloranil (CL) and p-chloranilic acid (p-CA) as π-acceptors to give highly coloured complex species. The coloured products are measured spectrophotometrically at 588, 843, 419, 550 and 520 nm for DDQ, TCNQ, TCNE, CL and p-CA, respectively, optimization of different experimental conditions is described. Beer's law is obeyed in the range of 20–200 μg ml⁻¹ and colours were produced in non-aqueous media and were stable for at least 1 h. Application of the suggested methods to perindopril tablets are presented.     

Determination of some aminobenzoic acid derivatives: glafenine and metoclopramide

Simple, sensitive and accurate spectrophotometric methods for the determination of glafenine and metoclopramide hydrochloride are described. The first method is based on the oxidation of glafenine with iodic acid in strong acid medium to give a coloured diphenylbenzidine derivative and subsequent measurement of the coloured product at 509 nm. Beer's law is obeyed over the concentration range 2.5–20 μg ml⁻¹. The second method depends on the interaction of metoclopramide hydrochloride with p-dimethylaminocinnamaldehyde, to give a red coloured schiff's base with an absorbance maximum at 548 nm. Obedience to Beer's law is achieved over the concentration range 5–30 μg ml⁻¹. First-derivative method is used to overcome the slight interference of p-dimethylaminocinnamaldehyde reagent blank at the wavelength of measurement. Linearity between the peak heights at 576 nm versus concentration range 5–25 μg ml⁻¹ metoclopromide hydrochloride is obtained. The proposed methods have been successfully applied to the determination of these drugs in commercial products without interference. The validity of the methods is assessed by applying the standard addition technique, the relative standard deviation is less than 1%. The proposed methods are compared with reference methods with good agreement.     

2,6-Dichloroquinone chlorimide and 7,7,8,8-tetracyanoquinodimethane reagents for the spectrophotometric determination of salbutamol in pure and dosage forms

A simple, rapid and sensitive spectrophotometric method for the determination of sulbutamol in pure form and in different pharmaceutical preparations has been developed. The charge transfer (CT) reaction between salbutamol as electron donor and 2,6-dichloroquinone chlorimide (DCQ) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) as a π-electron acceptor have been spectrophotometrically studied. The optimum experimental conditions for these CT reactions have been studied carefully. Beer's law is obeyed over the concentration range of 1.0–30.0 μg ml⁻¹ and 2.0–20.0 μg ml⁻¹ for salbutamol using DCQ and TCNQ, respectively. For more accurate results, Ringbom optimum concentration range is calculated and found to be 10.0 to 30.0 and 8.0 to 20 μg ml⁻¹ for salbutamol using DCQ and TCNQ, respectively. The Sandell sensitivity is found to be 0.011 and 0.010 g cm⁻² for salbutamol using DCQ and TCNQ, respectively, which indicate the high sensitivity of the proposed methods. Relative standard deviations (R.S.D.) of 0.27 to 0.68% and 0.20 to 1.40% (n=5) were obtained for five replicates of salbutamol using DCQ and TCNQ, respectively. The results obtained by the two reagents are comparable with those obtained by British pharmacopoeia assay for the determination of salbutamol in raw materials and in pharmaceutical preparations.     

Determination of meloxicam in bulk and pharmaceutical formulations

Three sensitive and reproducible methods for quantitative determination of meloxicam (mel) in pure form and in pharmaceutical formulations are presented. The first method is high performance liquid chromatography by which the drug is determined in the presence of its degradation products over concentration range 100–500 μg ml⁻¹ with mean percentage accuracy 100.13±0.53. The second method is based on measuring the absorbance of the formed neutral complex between basic methylene blue and mel in phosphate buffer (pH 8) at λ=653.5 nm over concentration range 1–5 μg ml⁻¹ with mean percentage accuracy 99.12±1.18. The third method is based on reaction between 2,3-dichloro-5,6-dicyano-p-benzoquinone resulting in the formation of an intense orange red coloured product after heating in a boiling water bath for 5 min. The coloured product exhibits an absorption maximum at 455 nm, over concentration range 40–160 μg ml⁻¹ with mean percentage accuracy 100.53±1.04.     

Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

A spectrophotometric method is proposed for the determination of oxytetracycline in pharmaceutical preparations. The method is based on the measurement of the absorbance of the molybdate—oxytetracycline complex at 404 nm (pH 5.50; μ = 0.1 M; 20°C). The composition of the complex (1:1) was determined by the application of the spectrophotometric methods of Job and Bent—French (pH 5.50; λ = 390 nm; μ = 0.1 M). The relative stability constant (K′ = 10^4.6) of the complex was obtained by the methods of Sommer and Nash (pH 5.50; λ = 390 nm; μ = 0.1 M; 20°C). The molar absorptivity of the complex was 9.5 × 10³ l mol⁻¹ cm⁻¹. Beer's law was obeyed over the concentration range 2.48–34.78 μg ml⁻¹. The relative standard deviation RSD (n = 10) was 0.27–0.39%. The method proposed can be applied to the assay of oxytetracycline in capsules. The detection limit of oxytetracycline is 2.5 μg ml⁻¹.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

Spectrophotometric determination of clobetasol propionate, halobetasol propionate, quinagolide hydrochloride, through charge transfer complexation

Two spectrophotometric procedures are described for the determination of clobetasol propionate(I), halobetasol propionate(II) (corticosteroids) and quinagolide hydrochloride(III) (prolactin inhibitor). For corticosteroid drugs, the procedures are based on the formation of phenyl hydrazones of the corticosteroids which are subsequently subjected to charge transfer complexation reaction with either 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) as π-acceptor or with iodine as σ-acceptor. Prolactin inhibitor was reacted directly with the previous reagents. The molar ratios of the reactants were established and the experimental conditions were studied giving maximum absorption at 588 and 290 nm with DDQ and iodine methods, respectively for the three drugs. The concentration ranges were 20–150, 50–300, and 20–80 μg ml⁻¹ in DDQ method for (I), (II), and (III), respectively and 13–20, 15–40, and 8–32 μg ml⁻¹ in iodine method for (I), (II) and (III), respectively.     

Spectrophotometric determination of alendronate in pharmaceutical formulations via complex formation with Fe(III) ions

The formation of the complex between alendronate, non-chromophoric bisphosphonate drug important for the treatment of a variety of bone diseases, and iron(III) chloride in perchloric acid solution was studied. The stoichiometric ratio of alendronate to Fe(III) ions in the chromophoric complex was determined to be 1:1. The conditional stability constant was log K′_ave=4.50 (SD=0.15), indicating that the Fe(III)–alendronate complex is a complex of medium stability. The optimum conditions for this reaction were ascertained and a spectrophotometric method was developed for the determination of alendronate in the concentration range 8.1–162.5 μg ml⁻¹, the detection limit being 2 μg ml⁻¹. The method was validated for the direct determination of alendronate in tablet dosage formulations.     

A capillary GC-MS method for analysis of phenytoin and [C3]-phenytoin from plasma obtained from pulse dose pharmacokinetic studies

Stable isotope analogues of phenytoin are useful for pulse dose pharmacokinetic studies in epilepsy patients. A simultaneous assay was developed to quantitate phenytoin (5,5-diphenylhydantoin) and its stable isotope analogue [¹³C₃]-phenytoin (5,5-diphenyl-2,4,5-¹³C₃-hydantoin) from plasma. Quantitation was achieved by GC-MS analysis of liquid/liquid extracted plasma samples, with [²H₁₀]-phenytoin (5,5-di(pentadeuterophenyl)-hydantoin) as an internal standard. The total coefficients of variance (C.V._t) were <7% for phenytoin (2.5–40 μg ml⁻¹) and <10.3% for [¹³C₃]-phenytoin (0.1–6.0 μg ml⁻¹). The accuracy of the assay varied from 87.8–100.1% (phenytoin, 2.5–40 μg ml⁻¹) and 89.6–116.3% ([¹³C₃]-phenytoin, 0.02–6.0 μg ml⁻¹). The assay was tested under in vivo conditions by administration of a pulse dose of the stable isotope analogue to a single rat dosed to steady-state with fosphenytoin, a phenytoin prodrug. The results of the in vivo experiment demonstrate the usefulness of this assay for future pharmacokinetic studies in special population epilepsy patients.     

LC determination of dinitrosopiperazine in simulated gastric juice

A simple and specific reversed phase HPLC method for the determination of dinitrosopiperazine in simulated gastric juice using UV detection was reported. The chromatographic resolution of the analyte and the internal standard isosorbide dinitrate was performed without extraction from the gastric juice on a reversed phase ODS column. Isocratic elution was carried out with methanol–0.02 M sodium dihydrogen phosphate (60:40 v/v, pH 3.0) at a flow rate of 1.0 ml min⁻¹ with UV detection at 238 nm. The calibration graph was linear over the concentration range 0.072–2.88 μg ml⁻¹ of dinitrosopiperazine with minimum detectability (S/N=2) of 0.01 μg ml⁻¹ (8×10⁻⁸ M). Inter-day and intra-day precisions calculated as% RSD were in the range 0.32–0.38% and 0.19–0.25% respectively. Inter-day and intra-day accuracies calculated as% error were in the range 0.18–0.21 and 0.08–0.11% respectively. The proposed method was successfully applied to the study of the possible in–vivo production of DNPZ under the standard nitrosation conditions recommended by WHO.     

A rapid colorimetric method for the determination of Losartan potassium in bulk and in synthetic mixture for solid dosage form

Two new rapid reproducible and economical spectrophotometric methods are described for the determination of Losartan potassium in bulk and in synthetic mixture for solid dosage forms. Both methods are based on the formation of an orange-red and orange ion-pair complex due to the action of Calmagite (CT) and Orange-II (O-II) on Losartan potassium in acidic medium (pH 1.2). Under optimised conditions, they show an absorption maxima at 491 nm (CT) and 486 nm (O-II), with molar absorptivities of 1.74×10³ and 1.75×10³ l mol⁻¹ cm⁻¹ and Sandell's sensitivities of 0.2649 and 0.2637 per 0.001 absorbance unit for CT and O-II, respectively. The colour is stable for 5 min after extraction. In both cases Beer's law is obeyed between 10 and 100 μg ml⁻¹. The proposed method was successfully extended to synthetic mixture for solid dosage forms.     

Spectrophotometeric Determination of Cefuroxime Axetil from bulk and in its tablet dosage form

A simple rapid spectrophotometric method has been developed for estimation of cefuroxime axetil from bulk drug and tablet dosage form by using 1-nitroso-2-napthol and sodium hydroxide. The method is based on the formation of yellow-orange coloured complex of drug with 1-nitroso-2-napthol having absorbance maxima at 424 nm. The Beer’s law is obeyed in the concentration range of 10-50 μg/ml of the drug but more precisely it obeys in the range of 10- 30 μg/ml. The slope and intercept values are 0.0101 and 0.0838, respectively. Results of analysis of this method were validated statistically and by recovery studies. The method is applied to the marketed tablet formulation. Result of analysis of tablet formulation given as percentage of label claim ±standard deviation is 99.17±1.57. The precision and accuracy was examined by performing recovery studies and was found to be 99.50±1.82. Sandell’s correlation coefficient is calculated as 0.4434. The developed method is simple, sensitive and reproducible and can be used for routine analysis of cefuroxime axetil from bulk and tablet dosage form.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.     

Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column

A simple, rapid and convenient high performance liquid chromatographic method, which permits the simultaneous determination of paracetamol, 4-aminophenol and 4-chloracetanilide in pharmaceutical preparation has been developed. The chromatographic separation was achieved on porous graphitized carbon (PGC) column using an isocratic mixture of 80/20 (v/v) acetonitrile/0.05 M potassium phosphate buffer (pH 5.5) and ultraviolet detection at 244 nm. Correlation coefficient for calibration curves in the ranges 1–50 μg ml⁻¹ for paracetamol and 5–40 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide were >0.99. The sensitivity of detection is 0.1 μg ml⁻¹ for paracetamol and 0.5 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available paracetamol dosage forms with recoveries of 98–103%. It is suggested that the proposed method should be used for routine quality control and dosage form assay of paracetamol in pharmaceutical preparations. The chromatographic behaviour of the three compounds was examined under variable mobile phase compositions and pH, the results revealed that selectivity was dependent on the organic solvent and pH used. The retention selectivity of these compounds on PGC was compared with those of octadecylsilica (ODS) packing materials in reversed phase liquid chromatography. The ODS column gave little separation for the degradation product (4-aminophenol) from paracetamol, whereas PGC column provides better separation in much shorter time.     

Selective spectrophotometric determination of phenolic β-lactam antibiotics

Two simple and selective spectrophotometric methods were developed for the quantitative determination of cefoperazone sodium, cefadroxil monohydrate, cefprozil anhydrous and amoxicillin trihydrate in pure forms as well as in their pharmaceutical formulations. The methods are based on the selective oxidation of these drugs with either Ce (IV) or Fe (III) in acid medium to give an intense yellow coloured product (λ_max=397 nm). The reaction conditions were studied and optimized. Beer's plots were obeyed in a general concentration range of 5–30 μg ml⁻¹ with correlation coefficients not less than 0.9979 for the four drugs with the two reagents. The methods are successfully applied to the analysis of pharmaceutical formulations containing amoxicillin, either alone or in combination with potassium clavulanate, flucloxacillin or dicloxacillin. They were also applied to the analysis of the other three studied drugs in vials, capsules, tablets and suspensions with good recovery; percent ranged from 99.7 (±0.46) to 100.32 (±1.05) in the Ce (IV) method and 99.6 (±0.50) to 100.3 (±1.32) in the Fe (III) method. Interferences from other antibiotics and additives products were investigated.     

Spectrophotometric determination of pefloxacin in pharmaceutical preparations

It has been established that the antibiotic pefloxacin (Abaktal) methane-sulphonate reacts with Fe(III) at pH 1.00–8.00 to form a water-soluble complex with maximum absorbance at 360 nm. The composition of the complex, determined spectrophotometrically by the application of Job's, molar-ratio and Bent—French's methods, was pefloxacin: Fe(III) = 1:1 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The relative stability constant, obtained by the methods of Sommer and Asmus was 10^5.02 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The molar absorptivity of the complex at 360 nm was found to be 4.8 × 10³ l mol⁻¹ cm⁻¹, Beer's law was followed for pefloxacin concentrations of 2.15–85.88 μg ml⁻¹. The lower sensitivity limit of the method was 2.15 μg ml⁻¹. The relative standard deviation (n = 10) was 0.57–1.07%. The method can be applied to the rapid and simple determination of pefloxacin in aqueous solutions and tablets.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Determination of clotrimazole in mice plasma by capillary electrophoresis

In addition to its antifungal activity, clotrimazole attracts interest as an anti-inflammatory drug. In order to correlate this effect with plasma concentrations in mice, a capillary electrophoretic method was developed. Sample preparation was carried out by protein precipitation using methanol. Quantification of clotrimazole was achieved by means of capillary electrophoresis using ketoconazole as an internal standard (IS). The background electrolyte (BGE) composed of a Tris buffer solution (100 mM, pH 3.0, adjusted with acetic acid) and methanol (8:2, v/v). Injection was carried out electrokinetically with 10 kV over a time period of 20 s. A special rinsing procedure utilizing a sequence of a SDS/methanol solution, a sodium hydroxide solution, water and BGE, was applied to enhance the reproducibility. With this procedure, an intermediate precision (day-to-day precision) of the area ratios of clotrimazole and IS of 5.0% for 0.5 μg ml⁻¹ and 2.6% for 10 μg ml⁻¹ was obtained. In summary, with the described capillary zone electrophoresis (CZE) method it is possible to handle small sample volumes of 60 μl, to detect clotrimazole concentrations of 0.3 μg ml⁻¹ (limit of detection), and to quantify clotrimazole down to concentrations of 0.5 μg ml⁻¹ (limit of quantification).     

Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC

First-derivative ultraviolet spectrophotometry and high-performance liquid chromatography (HPLC) were used to determine valsartan and hydrochlorothiazide simultaneously in combined pharmaceutical dosage forms. The derivative procedure was based on the linear relationship between the drug concentration and the first derivative amplitudes at 270.6 and 335 nm for valsartan and hydrochlorothiazide, respectively. The calibration graphs were linear in the range of 12.0–36.1 μg ml⁻¹ for valsartan and 4.0–12.1 μg ml⁻¹ for hydrochlorothiazide. Furthermore, a high- performance liquid chromatographic procedure with ultraviolet detection at 225 nm was developed for a comparison method. For the HPLC procedure, a reversed phase column with a mobile phase of 0.02 M phosphate buffer (pH 3.2)-acetonitrile (55: 45; v/v), was used to separate for valsartan and hydrochlorothiazide. The plot of peak area ratio of each drug to the internal standard versus the respective concentrations of valsartan and hydrochlorothiazide were found to be linear in the range of 0.06–1.8 and 0.07–0.5 μg ml⁻¹, respectively. The proposed methods were successfully applied to the determination of these drugs in laboratory-prepared mixtures and commercial tablets.