Current Pharmaceutical Analysis - Volume 7, Issue 3, 2011

Hypoglycemic drugs are popular for the treatment of Type-2 diabetes. In general, they are administered in oral doses daily and are widely biotransformed in the body. A large number of analytical methods based on chromatographic and electrophoretic techniques for the quantification of hypoglycemic drugs and their metabolites in biological matrices have been described in literature and have been used to support preclinical and clinical studies. In the present review, papers published between 2006 and late 2010 were reviewed with the focus placed on sample preparation procedures and on the separation techniques used. In addition only papers describing validated methods were included. Considering that the achievement of the desired outcome in the control of Type 2-diabetes involves a combination of treatments, bioanalytical methods that simultaneously cover several drugs were also included in this review.

Hepcidin is a small protein involved in iron metabolism that also exhibits antibacterial and antifungal activities. Since its discovery in 2000, hepcidin has been extensively studied in many research centers. Usage of natural and synthetic hepcidin inhibitors can be applied as a powerful therapeutic tool that targets multiple types of anemia. In addition, the protein itself has a potential to placate increased absorption of iron in patients with β-thalassemia. Previous studies have characterized mechanisms of hepcidin synthesis as well as its biological activity, however methods enabling estimation of its concentration in biological materials remain obscure. The physiological role of hepcidin in iron metabolism as well as methods of its determination in serum and urine samples are described in this paper. Furthermore, the possible role of hepcidin and its molecular antagonists in health improvement is considered.

High performance liquid chromatographic and 1st derivative spectrophotometric methods for simultaneous determination of chlorhexidine gluconate (CG) and benzydamine hydrochloride (BH) in gargle and spray preparations used for the treatment of dental illnesses, mouth and faucet infections and providing oral hygiene were developed and validated. CG and BH responses were respectively measured at 271 nm and 324 nm in 1st derivative spectrophotometry. In HPLC; the analysis of CG and BH together with internal standard hydrochlorothiazide (HCT) were performed using an analytical column of Nucleosil 100-5 C18 (5 μm, 250 &times 4,6 mm i.d.). Substances were eluted by a mobile phase consisting of 40 mM triethylamine containing phosphate buffer (10 mM, pH 3.0) and acetonitrile (65:35, v/v) at a flow rate of 1 mL min-1. UV detector was set to 230 nm. Under these chromatographic conditions, retention times of HCT, CG and BH were 3.44, 5.42 and 8.58 min, respectively. Developed methods were validated for the parameters of stability, linearity, sensitivity, accuracy, precision, specificity, ruggedness and robustness given in current ICH Guideline. Linearity ranges for two substances were 1-60 μg mL-1 in HPLC and 1-50 μg mL-1 in 1st derivative spectrophotometry. Developed methods were accurate, precise, specific, sensitive, rugged and robust due to the outcome from validation study results. These methods were applied to the binary mixture preparations of CG and BH (gargle and spray) and their reliability for the analysis of these preparations are presented.

A stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of orbifloxacin in tablet dosage forms. Reversed-phase chromatography was performed on a Symmetry Shield RP-18 Waters®, 250mm×4.6mm i.d., 5 μm particle size column with 5% acetic acid:methanol (80:20, v/v) as mobile phase at a flow rate of 0.7 ml/min. PDA detection was performed at 290 nm. A well resolved peak was obtained for orbifloxacin at a retention time of approximately 7.9 min. For stress studies the drug was subjected to oxidation, hydrolysis, photolysis and thermal degradation. The developed method was validated as per ICH guidelines for the parameters such as system suitability, linearity, limit of quantification (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness. Results obtained were found to be satisfactory. No interference from any excipients in the pharmaceutical dosage form or degradation products was observed.

In this present research work, a new stability indicating assay method was developed for the estimation of trandolapril and its degraded products by isocratic reversed phase chromatographic technique using ultra performance liquid chromatography. The Study involves a comprehensive stress testing of trandolapril which was carried out according to ICH guideline Q1A (R2). The drug was subjected to acid (0.1M HCl), neutral (water) and alkaline (0.1M NaOH) hydrolysis at 80°C, as well as the drug was kept at room temperature with H2O2 for oxidative decomposition. Photolysis was carried out by exposing this drug into sunlight (60,000-70,000 lux) for 2 days. Additionally, the solid drug was subjected to 50°C for 60 days in a hot air oven for thermal degradation. The results reveal that the degradation products of this drug were found in alkaline medium, acidic conditions and also in neutral hydrolysis. Separation of this drug and its degradation products (from various stress conditions) was successfully achieved on a BEH (bridged ethylene hybrid) C18 column utilizing water-acetonitrile in the ratio of 20:80. The flow rate and the detection wavelength for the analysis were 0.2 mL/min and 215 nm, respectively. The method was validated and the response was found to be linear in this drug concentration range of 0.431-2.155 μM/mL (10-50 μg/mL). The mean values (± %RSD) of slope, intercept and correlation coefficient were 2674262 (±0.9), 14924 (±1.02) and 0.9999 (±0.08), respectively. The ±RSD values for intra- and inter-day precision studies were <1% and <2%, respectively. The recovery of this drug ranged between 98.93-100.18% from a mixture of degradation products. The obtained results reveal that the developed method is specific to this drug and selective to the degradation products.

Ocular pharmacokinetic analysis of drugs is useful for evaluation of efficacy of novel ophthalmic formulations. A simple and sensitive ultra performance liquid chromatographic (UPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of dorzolamide (DZA) in rabbit aqueous humor. The aqueous humor samples were deproteinized with acetonitrile prior to UPLC analysis. A chromatographic separation was achieved on a Waters Acquity BEH C18 (100 mm&times2.1 mm, 1.7 μm) column with a mobile phase consisting of 0.1% triethylamine (TEA) in water and acetonitrile (50:50, v/v) at a flow rate of 0.25 ml min-1. Detection of DZA was carried out at 254 nm. The method developed was linear (r2 = 0.9996) over the concentration range of 5 - 800 ng mL-1. Accuracy ranged from 98.23 to 99.53% and precision was between 0.12% and 0.36%. Limit of detection and quantitation of DZA in aqueous humor were 5 and 10 ng mL-1, respectively. The method was applied to determine pharmacokinetics of DZA in aqueous humor, following topical instillation of DZA-solution (DZA-SOL) and DZA nanoparticles (DZA-NPs) to rabbit eyes. The pharmacokinetic results showed that DZA-NPs enhances the ocular bioavailability of DZA by 6-fold compared to DZA-SOL.

This study reports a simple and easy colorimetric method for measuring glucose consumption in tissues. Some technical parameters obtained from previous single factor tests and orthogonal designs were validated by single factor test in this study, and the kinetics of glucose consumption reaction was analyzed. Our results show that the kinetics of glucose consumption fits Michaelis-Menten equation with μmax and Km at 0.0942 g/Lh and 14.83 g/L respectively. Further comparative analysis of glucose consumption in diabetic and normal mice using the reported colorimetric method demonstrates that the rate of glucose consumption in liver, muscle, kidney, and brain from diabetic mice is significantly lower than those from normal control, respectively. The analysis of enzymatic activity and intermediate metabolite in the liver's reaction solution indicates the existence of significant differences between normal and diabetic mice. The significance of the method is more close to the glucose metabolism in vivo and without the need for expensive equipment and specialized technical expertise. With analyses of some enzymes' activities and intermediates involved in glucose metabolism, the present method can be applied in the diagnosis of diabetic patients.

Epipodophyllotoxin and its derivatives are important derivatives of podophyllotoxin which are used in cancer chemotherapy. The conditions for the separation and identification of a mixture of seven diastereoisomer pairs at the C-2 position of epipodophyllotoxin and its esters were optimized using both high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS). The separation was carried out on a BDS Hypersil C18 column with methanol-acetonitrilewater as the mobile phase in a gradient elution program. In the HPLC-ESI/MS spectra, the [M+Na]+ ions of all seven pairs were observed. The ion ratios of the relatively abundant ions of [M-ROH+H]+ (ion 397 or 383) to [M+Na]+, [A+H]+ (ion 313) to [M-ROH+H]+, and [A+H-OCH3]+ (ion 282) to [M-ROH+H]+ of each diastereoisomer pair of the lignans specifically exhibited a stereochemical effect. Thus, using identical chromatographic conditions, the combination of DAD and MS permits the separation and identification of seven diastereoisomers of epipodophyllotoxin at the C-2 position. The results of this work may serve as an effective tool for the separation and detection of other epipodophyllotoxin derivatives and their analogues.