Current Pharmaceutical Analysis - Volume 9, Issue 1, 2013

Simvastatin, a lipid-lowering agent of the statins class, is widely used in the treatment of hypercholesterolemia and in the prevention of cardiovascular diseases. Currently, simvastatin is available in the global market in both tablet and capsule forms. No official method has been reported for the assaying or dissolution testing of simvastatin capsules. Dissolution specifications were established to evaluate in vitro release studies for simvastatin capsules in this work. Adequate dissolution test conditions were obtained with 0.5% sodium dodecyl sulfate in 0.01 M monobasic sodium phosphate buffer at pH 7.0±0.05 (900 mL), and paddle agitation at 75 rpm for 45 min was used to investigate the immediate release (IR) of simvastatin capsules. Simvastatin quantified in the dissolution medium for capsules using the developed HPLC method, showed R2>0.98, selectivity (no placebo interference), precision (%RSD<5), recovery (95.0 to 105.0%) and robustness for deliberate changes in the buffer molarity and pH of the media and for different C8 columns (p value>0.05) according to the United States Pharmacopeia (2011) criteria. This work highlights the need to develop suitable dissolution conditions to establish criteria for the analysis of IR solid oral dosage form. The developed HPLC simvastatin quantitation method does not require the previous treatment of aliquots with manganese oxide, as described in the US Pharmacopeia and in the British Pharmacopoeia for ultraviolet spectroscopic methods.

The Medicinal Plant of Corrientes, Argentina, elaborates, distributes and provides free 500 mg cephalexin tablets in hospitals and health care centers. HPLC-UV is the reference method to quantify this antibiotic. The spectrophotometric ninhydrin method was applied for cephalexin quantification and analytical parameters for its validation were determined. The linearity of this method was in the range 0.04-0.20 mg/mL, responding to y = 2.2338x + 0.0007, with R2 = 0.9995. The RSD% found was 0.41, indicating a good repeatability of the analytical procedure. Exactitude in recovery experience was 98.50-101.33%. Comparison using t-tests and F-test indicates that there are no significant differences between results using both methods, with a confidence level of 95%. Specificity and intermediate precision assays were satisfactory. These parameters complete the validation of ninhydrin method, according to requirements of both United States and Argentinean Pharmacopoeia, for its application on cephalexin quality control in generic tablets.

Amikacin (AMK) is a semi-synthetic aminoglycoside antibiotic used under the sulfate salt form and manufactured under parenteral formulations. The determination of the sulfate concentration is important to confirm the completeness of salt formation. Continuing the approach followed by some authors in FTIR spectroscopy, the principle of using analyses of aqueous samples in the mid-IR region, by transmission, was adopted as a tool for the development of the proposed approach. Spectroscopic discrimination between the sulfate counter ion and the carbohydrate moiety was achieved by using derivative spectroscopy. The selected measurement criterion was the amplitude from the minimum of the negative wavenumber band (1054 cm-1) of the 1st order derivative spectrum to a cross-over point coincident with the maximum negative slope of the conventional absorbance spectrum. Linear response (r > 0.999) was observed for sulfate concentrations in the range 2.5 to 25 (mg mL-1), with a detection limit of 0.4 mg mL-1. The sampling frequency was estimated as 13 measurements per hour in a continuous flow mode sample-to-sample with a coefficient of variation < 0.5 % (n = 10). The found equivalent sulfate content in AMK sulfate formulations showed in most cases a good agreement with the values declared on the label of each analyzed pharmaceutical product. The proposed analytical strategy allows the analysis of the sample either directly or just requiring only an aqueous dilution which can be understood as a green analytical approach alternative to methods employing either titrimetric approaches or chromatographic and electrophoretic separation methods.

S-nitrosoglutathione (GSNO) is one of low molecular weight S-nitrosothiols occuring in humans. Nowadays, it is widely used as a nitric oxide donor for in vitro, ex vivo and in vivo experiments related to the investigation of its pathophysiological role as well as in clinical trials, aimed at its potential therapeutic use. Despite numerous reports on this physiological molecule, its quality control does not match the criteria required by competent pharmaceutical authorities. Hereby, an extensive physicochemical characterisation of synthesised and purified GSNO is provided for the first time. Indeed, structural identification including spectrometric, thermal and elemental analyses was consistent with the GSNO structure. An ion-pairing reversed phase HPLC system was developed to assess (i) GSNO content with UV detection at 334 nm, and (ii) fingerprint of its impurities coming from synthesis process and/or storage conditions, at 220 nm. The assynthesised product showed a content of 102.5 %, with respect to a commercially available standard. The identified impurities, i.e. chloride, nitrite, nitrate, reduced glutathione and glutathione disulfide, were also quantified basing on pharmaceutical requirements. Main products released during various storage conditions (pH, temperature, dioxygen, …) were disulfide glutathione and nitrite ion. Recommendations are given for the safe use of GSNO in biological and pharmacological experiments.

This work determines the total protein concentration present in microsome fractions obtained by two distinct extraction procedures: ultracentrifugation and calcium aggregation. Additionally, the batch to batch variability within the extraction procedures used and the microsome stability and activity at -80°C during 90 days were determined using 7- ethoxyresorufin as a marker substract. An HPLC method to determine the activity of the cytochrome CYP450 1A1/2 was developed and fully validated by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity by in vitro microsomal mixed-function biotransformation. The biotransformation product, resorufin, was obtained by incubation of 500 μg of rat liver microsomal proteins. The incubation time was 5 min and the assay was monitored by using an HPLC coupled to a fluorescence detector (λexc 530 nm and λem 582 nm). The total analysis time was 20 min and the sample preparation was a fast and simple protein precipitation. The results show that both extraction procedures are useful tools for the extraction of subcellular fractions allowing the recovery of appropriate protein concentration for further in vitro metabolism studies by cytochrome P450, specifically CYP1A1 and CYP1A2.

A novel 96-microwell-based spectrophotometric assay has been developed and validated for the determination of rosuvastatin calcium (ROS-Ca) in tablets. The formation of a colored charge-transfer (CT) complex between rosuvastatin calcium (ROS-Ca) as n-electron donor and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for the determination of ROS-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9987) was found between the absorbance and the concentration of ROS-Ca in the range of 10-100 μg/well. The limits of detection and quantitation were 2.6 and 7.85 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ROS-Ca in its combined formulations. The assay was successfully applied to the analysis of ROS-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ROS-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposure of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50- fold. Although the proposed assay was validated for ROS-Ca, however, the same methodology could be used for any electron- donating analyte for which a CT reaction can be performed.

An accurate, precise, simple and selective stability-indicating gradient reverse phase ultra performance liquid chromatographic method has been developed and validated for the quantitative determination of rupatadine and montelukast in pharmaceutical formulation in presence of degradation products. The chromatographic separation was performed on Acquity BEH C8 column (100 mm x 2.1 mm I.D., 1.7 μm) by using mobile phase containing a gradient mixture of solvent A (0.02 M KH2PO4, pH 3.0) and B (90:10 v/v mixture of acetonitrile and water) at flow rate of 0.5 mL/min. The detection was carried out at a wavelength of 240 nm. To establish stability indicating capability of the method, drug product was subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal and photolytic degradation. The degradation products were well resolved from rupatadine and montelukast. The developed method was validated as per ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.

This present review submitted high-performance liquid chromatography (HPLC) methods for determining the presence of meloxicam used in clinical treatment within various fields of application, including biological samples and pharmaceutical formulations, between 1990 and 2011. Meloxicam is a cyclooxygenase-2 selective nonsteroidal anti-inflammatory drug (NSAIDs), which is the group most often used in human health care. We compared the methods from the standpoint of sensitivity. HPLC methods with MS/MS detection have become the method of choice in bioanalytical applications.

The aim of this work is to develop and validate a microbiological assay for ceftriaxone with a view to the employment of this method to assess photo-stability in commercial products. The experimental conditions included: (1) ceftriaxone standard solution in concentrations of 2.6 μg/mL, 3.2 μg/mL, 4.0 μg/mL, 5.0 μg/mL and 6.3 μg/mL, using phosphate buffer pH 6.0 as diluent, (2) Petri dishes prepared by adding 21-mL of antibiotic medium as base layer and 4-mL of the same medium, inoculated with Bacillus subtilis (ATCC 6633) in a proportion of 0.5% and (3) incubation at 37ºC for 18 hours. The method showed specificity, good linearity in the range from 2.6 to 6.3 μg/mL, as well as good precision (RSD repeatability of 1.7% and RSD intermediate precision of 2.2%) and accuracy (recovery of 101.1%). Commercial samples of ceftriaxone were exposed to light in their primary and secondary packing, being afterwards directly exposed to light. The potencies of commercial samples were then determined by the developed and validated microbiological assay. The results indicate that primary and secondary packing provide satisfactory protection of ceftriaxone from light.

The quality of clopidogrel (CLP) and acetylsalicylic acid (ASA) combination dosage forms was evaluated for their content, impurities and drug release. An enantiospecific (chiral) method for the quantification of the R-enantiomer of CLP and an achiral method for the determination of the content of CLP and ASA and their impurities were used. The following stationary phases were used: a CHIRALCEL® OJ column (250 mm x 4.6 mm, 10 μm) for the quantification of the R-enantiomer and a Luna C18 column (150 mm x 4.6 mm, 3 μm) for the determination of the content of CLP, ASA and their impurities and for the follow up of the release. Ten commercial batches corresponding to 10 different marketed drugs containing CLP and ASA combination dosage forms (tablets and capsules) were analysed. Although no monograph is available for combined dosage forms of CLP and ASA, most of the samples comply with the United States Pharmacopeia (USP) specification for content and related substances of the individual monographs. However, there are notable differences between samples examined in terms of in vitro drug release.

A simple, rapid and sensitive RP-HPLC method was developed and validated for the quantification of bromocriptine mesylate in bulk drug and tablet formulation. The separation was achieved on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.). The mobile phase consisted of methanol and 20 mM sodium acetate, pH 5 (70:30, v/v) at a flow rate of 1.5 mL min-1 and detection was performed at 300 nm using photodiode array (PDA) detector. The drug was subjected to various ICH prescribed stress conditions including hydrolysis (neutral, acid and alkaline), oxidation, photolysis and thermal degradation. The drug in solution was found to degrade significantly in alkaline hydrolysis and when exposed to sunlight. The proposed method was validated with respect to specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), stability, and robustness as per ICH guideline. The peak purity achieved from PDA detector and satisfactory resolution between drug and its degradants established the specificity of the method. The developed method was found to be successively applied for the quality control of bromocriptine mesylate in bulk drug and tablets as well as the stability indicating studies.

Ketoconazole (1-[4-(4-{[(2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]-methoxy} phenyl)piperazin-1-yl]ethan-1-one) in substantia was irradiated to doses between 25 kGy (standard sterilizing dose) and 800 kGy, using high energy electrons from an electron accelerator. The irradiated and non-irradiated samples were then examined using a variety of spectroscopic (UV, MS, NMR, EPR), chromatographic (TLC, HPLC) and hyphenated (HPLC-MS) methods. With the help of an EPR method the presence of free radicals (1.40 × 1015 spin/g for dose 100 kGy) was detected. The loss of ketoconazole was ca 6%, as determined by the HPLC, for the dose of 800 kGy. The radiolytic yield of the process was 4.52 × 10-7 mol/J for the 25 kGy and it decreased to 1.29 × 10-7 mol/J for the 800 kGy dose. On the basis of the HPLC-MS measurements the structures for five main radiolysis products were proposed. It was found that the main radiodegradation processes are: dehalogenation (loss of Cl atom), dehydrogenation of the piperazine ring, Noxidation, hydrolysis and rapture of the C18-O19 bond. Our experiments indicate that ketoconazole is stable in the range of doses between 25-50 kGy and therefore could probably be sterilized using the standard sterilizing dose of 25 kGy.

A flow-based system with solenoid micro-pumps is proposed for fast simvastatin (SMT) determination in pharmaceutical preparations. The method is based on the inhibition of methyl orange bromination in the presence of the analyte with spectrophotometric detection. A linear response was observed between 5.0 and 35 mg L-1 SMT with a detection limit of 1.2 mg L-1 (99.7% confidence level). The coefficient of variation (n = 20) and sampling rate were estimated as 3.3% and 80 determinations per hour, respectively. Reagents consumption was estimated as 3.5 μg of NaBrO3, 9.5 μg of methyl orange and 400 μg of NaBr per determination, generating 4.5 mL of waste. The results for pharmaceutical samples agreed with those obtained by the reference chromatographic procedure at the 95% confidence level. The procedure is a fast, greener and reliable alternative for SMT determination.

Calixarenes (CAs), with their unique three-dimensional surface, are one of the best known host molecules along with cyclodextrins, and crown ethers. However, these compounds have been less studied compared to cyclodextrins. The present work is devoted to the evaluation of the ability of eleven different calixarenes to form host-guest complexes with eleven pharmaceutical relevant substances (chiral active pharmaceutical ingredients/APIs) in different solutions (acetonitrile, methanol and water), including comparisons between water-soluble calixarenes and three pharmaceutically relevant cyclodextrins (α-, β- and γ-cyclodextrins) by means of circular dichroism spectroscopy (CD). The obtained CD spectra provided the absolute configuration of the chiral APIs, as well as of the interactions with host-molecules. An attempt to understand the complexation mechanism of calixarenes was undertaken based on the CD-spectra of the drugs with different host macrocycles. These results indicate that calixarenes could serve as candidate host molecules in the pharmaceutical researches due to their versatility and the ease of adding different moieties to their upper and/or lower rim, which makes it easier to change the affinity of these cyclooligomers towards target molecules and/or increase the solubility of the calixarenes.