Current Pharmaceutical Analysis - Volume 10, Issue 2, 2014

A protective effect of α -ketobutyrate on hippocampal neuronal cells treated with H2O2 was tested. α - Ketobutyrate concentrations were as follows: 0.5 mM, 1 mM and 5 mM. Tests were performed at the temperature of 37 ºC. Moreover, the protective effect of α-ketobutyrate on the cytotoxicity of 100 and 40 nmol/4·103 cells treated by hydrogen peroxide was observed. Furthermore, the role of α-ketobutyrate as an energy substrate was also proven using the cis- [Cr(C2O4)(pm)(OH2)2]+ complex as a biosensor for the carbon dioxide uptake process. All the experiments were carried out under in-vitro conditions.

Introduction: Gastrografin® is a commercially available contrast agent, primarily utilised in abdominal computed tomography. In order to conduct a randomised controlled clinical trial to determine whether gastrografin® may confer a therapeutic benefit in a postoperative ileus it was necessary to ensure that re-packaging of gastrografin® during blinding had no effect on the stability of its active diatrizoate salts (DTZ). Aim: To develop a validated and rapid reverse phase High Performance Liquid Chromatography (HPLC) analytical method for the assay of DTZ that could then be utilised to determine the stability of gastrografin®. Methods: A published HPLC method for meglumine amidotrizoate injection was adapted and validated for the stability studies. These were carried out in accordance with ICH guidelines. Results: The HPLC method was validated for specificity (no elution peak with placebo), linearity (r2=0.999), accuracy (≤5%) and precision (≤3%). Gastrografin® exposed to 25ºC and 60% relative humidity remained stable for 30 days with no physical change to the solution. Gastrografin® stored under identical conditions and exposed to 1.2 million lux hours of light remained stable for 7 days. Gastrografin® stored at 4ºC underwent crystallisation; this was readily reversed by agitation and had no discernible effect on 30-day drug stability. Conclusion: Gastrografin® remains stable and active for a minimum of 30 days under standard storage conditions (25ºC, 60% humidity, no light) after re-packaging.

A simple, sensitive, specific, robust, precise and accurate reverse-phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for suitable quantitation of fenofibrate in bulk and pharmaceutical dosage forms. The investigation of various validation parameters such as system suitability, linearity, detection limit, quantification limit, precision, accuracy, specificity, robustness and stability was accomplished in accordance with the International Conference on Harmonization (ICH) guidelines. The isocratic elution of fenofibrate was performed using Agilent 1260 Infinity HPLC system. The column was Capcell PAK C18 (4.6 mm x 250 mm, 5 μm). The mobile phase, consisted of acetonitrile and 0.1% (v/v) H3PO4 (75:25, v/v), was eluted at 2 ml/min. The eluent was monitored at 286 nm by the UV detector for fenofibrate concentration measurement. Stability test proved that fenofibrate in sample solutions remained stable at room temperature throughout the analytical process. The limit of detection (LOD) and limit of quantification (LOQ) furnished by this method were better than those of the previously reported HPLC and some UPLC methods of fenofibrate determination. All other validation parameters appeared within the acceptable limits.

Amikacin (AMK) is a semisynthetic aminoglycoside antibiotic derived from kanamycin A that lacks a strong ultraviolet absorbing chromophore or fluorophore. Greatly enhanced resonance Rayleigh scattering (RRS) signals of AMK can be observed with a conventional fluorescence spectrophotometer when AMK interacts with pontamine sky blue (PSB) and forms an ion-association complex in weakly acidic buffer medium. Based on these characteristics, a sensitive assay for detecting AMK was developed. The maximum RRS peak was observed at 362 nm. The enhancement of the RRS signal was directly proportional to the AMK concentration in the range of 0–1.7 μg/ml, and its detection limit (3σ) was 3.0 ng/ml. The method showed a wide linear range and high sensitivity, and almost no interference could be observed from coexistent substances. In addition, this assay was performed in different pharmaceutical formulations of AMK with satisfactory results. Therefore, the proposed method is promising as an effective means for the determination of AMK.

A simple, rapid and cost-effective NP-TLC method combined with densitometry was developed for determination of estradiol hemihydrate in tablet dosage form. TLC-densitometric analysis was conducted on silica gel 60F254 plates. Benzene + methanol in volume composition 9:1 was used as a mobile phase. The detection was carried out at 200 nm. Applied chromatographic conditions enabled complete separation of estradiol hemihydrate (EL) from its related substance, namely estrone (EN) with simple procedure of sample preparation. Linearity range for estradiol hemihydrate was from 0.50 to 1.50 μg/spot. The detection limit (LOD) and quantitative limit (LOQ) values of estradiol hemihydrate were 0.14 μg/spot and 0.41 μg/spot, respectively. The developed method was found to be precise, accurate, and robust. The assay value of estradiol hemihydrate was consistent with the limit recommended by Pharmacopoeia. The elaborated TLC-densitometric method can be used as an alternative method for the accurate assay of estradiol hemihydrate in tablet dosage form because it fulfills the validation criteria of an analytical method designated for quantity control of pharmaceutical preparations.

A comparative investigation has been developed for the stability constants of several amino acid complexes with divalent metal ions, which have been determined by potentiometric pH titration. Depending on the metal ion–binding properties, vital differences in the building complexes were observed. The present study indicates that in some M(L) complexes, metal ions are arranged in carboxyl groups, but in other M(L)complexes, some metal ions are able to build chelate over amine groups. The results mentioned-above demonstrate that for some M(L) complexes, the stability constants are also largely determined by the affinity of metal ions for amine group. This leads to a kind of selectivity of metal ions, and transfers them through building complexes accompanied with some amino acids. For heavy metal ions, this building complex helps the absorption and filtration of blood plasma. This is an important method in micro-dialysis. In this study the different aspects of stabilization of metal ion complexes regarding the Irving-Williams sequence have been investigated.

Stability indicating a reverse phase high- performance liquid chromatography (RP-HPLC) method for the analysis of Ketoprofen (KP) was developed and validated as per the International Conference of Harmonization (ICH) guidelines, Q1A (R2). Chromatographic separation was achieved on a Capacel Pak (Shiseido, Tokyo, Japan) C18 Type MG column (250 mm × 4.6 mm) 5 μm particle size, using isocratic elution of mobile phase containing the mixture of acetonitrile (ACN) and 0.02 M potassium dihydrogen orthophosphate buffer pH 3.0 (40:60) with a flow rate of 1.0 ml minutes-1. Quantification was achieved with UV detection at 254 nm with a linear calibration curve in the concentration range of 0.5–60 μg ml-1 based on peak area. The method was validated for linearity, system suitability, accuracy, precision, sensitivity, selectivity and robustness. Application of a 3-factor, 3-level design was employed using Box-Behnken Experimental Design (BBD) for the validation of robustness using three factors such as the composition of the mobile phase (X1), flow rate (X2) and pH (X3) whereas the peak area (Y1) and retention time (Y2) were used as responses. The limits of detection (LOD) and the limits of quantification (LOQ) were found to be 35.417 ± 0.52 and 107.352 ± 1.37 ng ml-1 respectively. Linearity was found to be in the range between 0.5–60 μg ml-1 with a significantly higher value of correlation coefficient r2 = 0.999. The proposed method was found sensitive, specific and was successfully applied for the estimation of KP in both bulk drug and optimized nanoemulsion gel (NEG) formulation. The developed method also resolved KP from its degradation products indicating high specificity. The results showed that Quality by Design (QbD) using a Design Space (DS) concept could be effectively applied to optimize HPLC chromatography method with the least number of experimental runs.

In the pharmaceutical world, impurity profiling of an APIs or drug substance and products is an important aspect. It has become an important phase as per various regulatory authorities, to be carried out for all the APIs, may be because of direct relation with the efficacy and safety of drugs. For the characterization of impurities, a number of analytical techniques such as chromatographic, spectroscopic and sophisticated hyphenated techniques have been used. Thus, in the present review applications of various analytical tools such as thin layer chromatography, HPTLC, HPLC, UPLC, gas chromatography, flash chromatography, supercritical fluid chromatography, capillary electrophoresis, micellar electrokinetic chromatography, UV-spectroscopy, IR-spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry, liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry-mass spectrometry, liquid chromatography- nuclear magnetic spectroscopy, high performance liquid chromatography-diode array detector- mass spectrometry, high performance liquid chromatography-diode array detector- nuclear magnetic spectroscopy-mass spectrometry, ultra performance liquid chromatography-mass spectrometry, ultra performance liquid chromatography-mass spectrometrymass spectrometry, chemometrics in impurity profiling of APIs or drug products have been briefly discussed.