Current Pharmaceutical Analysis - Volume 9, Issue 4, 2013

Analysis of n3 fatty acids in serum samples has clinical applications in supplementation trials, but the analysis can be challenging due to low levels, stability issues and intra-individual variation. This study presents the single laboratory validation of a gas chromatographic-mass spectral (GC-MS) assay for analysis of fatty acid methyl esters (FAME) using sensitive single ion monitoring and provides data on fatty acid stability under different sample handling conditions. Recovery of total fatty acids from serum with Folch extraction was optimized and parallelism tests with spiked samples indicated that the serum matrix did not interfere with mass spectral quantitation. Precision and accuracy of the assay at the lowest limit of quantitation and at low, medium and high levels met with accepted guidelines for single laboratory validation. Several storage conditions that can be encountered with clinical samples also were evaluated for impact on fatty acid levels in serum. Serum from blood that was stored refrigerated for 3 days yielded similar results as serum that was prepared and frozen at -80°C immediately. Serum storage at room temperature for 3-24 hours and serum subjected to one freeze/thaw cycle had minimal effects on fatty acid levels. The intra-individual variability in pregnant women was reasonably small, with significant correlation coefficients ranging from 0.35 to 0.76 for blood drawn between 12-20 weeks versus 34-36 weeks of gestation. These results indicate that GC-MS with single ion monitoring is valid for the analysis of total fatty acids in clinical samples, even when blood processing cannot be performed in a timely manner.

This work aims to develop and validate a simple methodology to quantify the most used antiretroviral, zidovudine (AZT), in rat plasma for preclinical studies. Some assays have been previously reported to quantify AZT in plasma; however, the majority of these methods uses complicated extraction methods. This method uses only 100 μL plasma samples, which were precipitated with 100 μL acidified acetonitrile containing an internal standard (IS). The plasma extracts were injected directly in a chromatographic system consisting of a UV–Vis detector (set at 266 nm) and an RP-C18 column. The mobile phase was an isocratic mixture of acetonitrile, methanol and 0.01% v/v aqueous formic acid (20:20:60 v/v/v) at a flow rate of 0.8 mL/min. Intra- and inter-day assay precision and accuracy variability were lower than 20% for the limit of quantification and 15% for higher concentrations. The applicability of the method was demonstrated by an in vivo study performed in rats. They were treated orally with an AZT-containing syrup and intravenously with an AZT solution. The calculated bioavailability of the syrup (56.7%) was in accordance with the literature. Therefore, this method can be used as an alternative to quantify plasma AZT in preclinical studies.

The preparation of a parenteral solution for oral use leads the clinician to question the stability of drugs when used in conjunction with flavored oral suspending vehicles. Therefore, the purpose of this study was the evaluation of the stability of midazolam, frequently used as an oral premedication in children, in extemporaneously prepared pediatric syrup, using micellar electrokinetic capillary chromatography (MEKC). The optimization of parameters affecting electrophoretic separation provided adequate separation of the analyte of interest, the internal standard (nitrazepam) and the syrup ingredients in a short time of 8 min. As a separation buffer 10 mM sodium tetraborate, 30 mM SDS and 15% methanol were used. During the separation process analytes were detected at 254 nm. Suitable linearity ranged from 1 to 50 μg/ml (r= 0.998), sensitivity (limit of detection 0.12 μg/ml) and recovery (in the range of 100.1% for 50 μg/mL to 119.0% for 1 μg/mL) were achieved. In this experiment syrup samples were stored at different temperatures (4°C, 25°C and 40°C) in amber glass bottles to protect them from access to light. The analysis of properly prepared samples was carried out for 30 days, indicating the concentration of active substances and the pH of the syrup. Studies have confirmed that the most stable formulation was one stored in a refrigerator. The elaborated method can find reasonable application in the determination of midazolam in pharmaceutical formulations, as a stability-indicating assay.

Measurement uncertainty provides indication of the quality of the results and its usefulness for compliance or non-compliance decisions. On the other hand, pharmaceutical equivalence is an important step in confirmation of similarity and interchangeability of pharmaceutical products. The aim of this work was to establish a procedure to estimate the measurement uncertainty for metronidazole quantification by high performance liquid chromatography (HPLC) and apply this uncertainty in the assessment of pharmaceutical equivalence. According to our results, the expanded uncertainty of metronidazole quantification by HPLC was about 3.5%, considering a coverage factor of 2.00 and a 95% confidence level. Uncertainties associated to the accuracy, precision and area of the peaks are the most significant. The measurement uncertainty approach was compared to the two one-sided tests (TOST) for equivalence. Both tests allow us to confirm pharmaceutical equivalence among generic and reference products and similar and reference products of metronidazole injection.

In this article we present an update on the applications of chemiluminescence coupled with flow injection analysis for the determination of pharmaceuticals. Several reported applications of flow and sequential injection chemiluminescence (FI-CL/SI-CL) in pharmaceuticals concern greatly on quality control, investigation of dissolution of particular formulations or process control of production of pharmaceuticals and their presence in physiological fluids. A number of advances in the development of FI-CL methods have been reported in recent years on pharmaceutical analysis. In the present review we have covered six years period from Jan-2007 to Dec-2012 discussing FI-CL/SI-CL in the liquid phase as an analytical tool for the determination of pharmaceuticals in diverse samples. We reviewed the methods as per general CL reaction involved with the help of 321 cited references. Interesting features of the FI-CL methods like the use of molecularly imprinting polymers for selectively and specially designed cells/microfluidic devices or lab-on-a-chip for better detection or sensitivity were also presented.

In this research, we aimed to develop a new triadimefon microemulsion according to the pseudoternary phase diagrams which could greatly decrease the usage of organic solvents while improve stability and antifungal activity of triadimefon preparation. The microemulsion was successfully prepared using toluene as solvent, emulsifier HSH as surfactant, and methanol as co-surfactant. And its optimal formulation was 4/3/3 (w/w) [oil/SAA (mixture of emulsifier and methanol)/water] selected according to the pseudoternary phase diagrams. In this formulation, SAA consists of emulsifier HSH and methanol with a weight ratio of 4/3. Triadimefon microemulsion did not freeze at -18 °C; moreover, the low temperature stability of triadimefon preparations was greatly improved, which was one of the key findings. After observing appearance, centrifuging, and diluting, type of the microemulsion was determined to oil in water. All of the other physicochemical properties including heat storage stability, emulsion stability, and cloud point met the standard of pesticide microemulsion. Its antifungal activities against Rhizoctonia solani AG1-IA including mycelium growth, sclerotia formation and germination were statistically significant higher than that of 20% triadimefon emulsion from the t-test done by IBM SPSS 19.

The aim of this study is optimization of experimental factors in a UV-visible spectrophotometric method for simultaneous determination of dorzolamide hydrochloride and timolol maleate in eye drops. For this purpose, partial least squares-1 method was developed for resolving overlapped UV-visible spectra of these compounds. Principal component number, wavelength range (number of wavelengths) and data preprocessing applications were investigated as experimental factors. The affected experimental response in this method is PRESS (Prediction Error Sum of Squares) values. Response Surface Methodology (RSM) method was used for interpretation of relationships between these factors and PRESS values, and results were shown as equations and contour graphs. Multivariate calibration techniques which were used in the method development have some advantages such as using whole spectrum for the simultaneous analysis of the mixture component that allow to minimize the effects of noise peak, shorter analysis time, low cost and simple instrumentation without any chemical pre-treatment. This optimized method was validated and applied to a pharmaceutical preparation, eye drops, and results were compared with a literature method statistically and no significant difference was observed.