Current Pharmaceutical Analysis - Volume 3, Issue 4, 2007

High temperature liquid chromatography is currently well established in separation sciences. The decrease of mobile phase viscosity with temperature provides fast separations with high efficiency, limiting excessive backpressure. Furthermore, the water polarity decreases with temperature allowing a reduction of the organic modifier content in the mobile phase. These advantages are already observed for temperature below 100°C. Recently, the commercialization of dedicated instrumentation for preheating and cooling the mobile phase, and of new stationary phases stable at high temperature containing carbon, zirconia, titania or polymeric material, allows a routine use of high temperature liquid chromatography (HTLC). The latter stationary phases could however exhibit high retentions and different selectivity in comparison with the traditional silica-based columns. Therefore, embedded, hybrid or new silica- based stationary phases, possessing similar chromatographic behaviour and good thermal resistance at temperatures as high as 100°C, are often selected. Kinetic, stability and chromatographic performance of a silica-based stationary phase (Zorbax Stable Bond) were evaluated up to a temperature of 120°C. This chromatographic support was successfully used for separating numerous drugs at ambient and high temperatures. Improvements of performance due to temperature increase were reported, such as the decrease in analysis time (separations in only few seconds) and the peak shape improvements (higher efficiency and lower asymmetry). Furthermore, several modifications of selectivity and the possibility to use a lower content of organic modifier into the mobile phase were demonstrated.

Protein and peptides production through recombinant techniques has benefited from numerous years of successful research, and is widely accepted as the approach of choice. As a result of these advances, many peptides and proteins are being formulated as biopharmaceuticals; however, to develop these macromolecules into stable formulations still remains a great challenge. Formulation problems in solution arise from the complex native structure of these macromolecules and are often manifested as physical instability, e.g. unfolding, aggregation, and/or precipitation. Understanding these effects is fundamental for research, development, production, and quality control. A variety of different techniques including HPLC, SEC, native gel electrophoresis and other electrophoretic techniques, analytical ultracentrifugation, fluorescence spectroscopy, Fourier transform/IR spectroscopy, mass spectrometry, light scattering, membrane osmometry, and UV spectroscopy have been used to study these processes. The assessment of product quality i.e. identity, content and purity has a major role in the manufacturing process of biopharmaceuticals. The biotechnological production process itself usually shows high variability, which introduces high product diversity. Even from production batches, only limited amounts of material may finally be approved for use and made available, requiring sensitive analytical technology. The purpose of the current review is to describe and discuss the latest advances in analytical methodologies used to secure final product quality.

Sequential-injection analysis (SIA) is a sample handling and manipulation technique developed to address some limitations of traditional on-line analysis approaches, mainly flow-injection analysis (FIA). The heart of a SIA manifold is a multi-position selection valve. The common port of this valve is connected to a so-called “holding coil” (essentially a long piece of coiled tubing) while the selection ports of the valve can be connected to various flow-through modules. The sample to be analyzed is initially aspirated and stored in the holding coil and can be further serially processed by directing it to appropriate modules in which both physical manipulation and/or chemical transformations may take place. SIA has found numerous applications in the field of clinical and bio-analytical chemistry by exploiting its potential as a sample-handling and manipulation technique. This review will cover the most significant applications of SIA in this area including on-line dilution/mixing, liquid and solid-phase extraction and pre-concentration, immunoassays, enzymatic methods, coupling to separation modules, miniaturization and other approaches for the determination of compounds with biological and clinical significance.

Two metformin carbon paste and PVC electrodes are proposed for the determination of metformin. Different methods for electrode fabrication (including formation of the ion pair in situ, soaking of the electrode in the ion pair aqueous solution, in addition to the classical modification of the electrode matrix with the ion pair) are described for both carbon paste and PVC electrodes. Electrodes based on in situ ion pairs of metformin with sodium tetrakis (4-fluorophenyl) borate or tungstophosphoric acid respectively showed the best performance compared with the other methods of preparation. These electrodes exhibit a linear response with a good Nernstian slope over a concentration range 10-5-10-1 M of metformin in a working pH range between 4 and 8. The effect of the electrode matrix composition and plasticizer on the performance of both carbon paste and PVC electrodes are investigated for each fabrication method. The proposed technique was applied successfully for the determination of metformin in pure solutions and in dosage forms with good accuracy and precision. The results were compared statistically with those given by the reported method.

The simultaneous spectrophotometric determination of Atorvastatin calcium (ATV) and Amlodipine besylate (AML) in tablets in the presence of the overlapping spectra was accomplished with the derivative spectrophotometry (DS) and the partial least squares (PLS) approaches without using any chemical pre-treatment. In this study, the concentration model was based on the absorption spectra in the range of 220-400 nm for 25 different mixtures of ATV and AML. The calibration curve was linear over the concentration range of 10-160 and 3-33 μg mL-1 for ATV and AML, respectively. These two methods were tested by analyzing the synthetic mixtures of the above drugs and they were applied to the real samples, containing two commercial pharmaceutical preparations of the subjected drugs. A comparative study was carried out using the experimental results obtained from the two analytical methodologies. The root mean squares differences (RMSD) with PLS and DS were 0.6618 and 1.2555 for ATV and 0.1589 and 0.3490 for AML, respectively. It should also be mentioned that the accuracy of the PLS method was better than that of DS.

A novel economic procedure for stereoselective separation and determination of R(+) - and S(-)- ropivacaine was described using different thin layer chromatographic plates and different cyclodextrins at different temperatures. The spots were detected either with iodine vapors or UV lamp 254nm, followed by densitometric measurements at 262nm. Comparative study was achieved using different cyclodextrins namely, hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (M-β-CD), and Dimethyl-β-Cyclodextrin (DM-β-CD) as chiral selectors. The mobile phase enabling successful resolution of (±) ropivacaine was acetonitrile: water (17:3 v/v) containing 1mM of DM-β-CD at ambient temperature 25±20C. All variables affecting the resolution, such as concentration of different chiral selectors, temperature, and pH were investigated and the conditions were optimized. The procedure provided a linear response over the concentration range of 1.25 - 35 μg/spot for determination of R (+)- and S- (-)enantiomers (r = 0.9998, n = 8), (r = 0.9998, n = 6) with acceptable precision (% RSD <1.5) and accuracy (% RE= -1.18 to 2.00). Limits of detection and quantification were found to be 0.29μg/spot and 0.96 μg/spot for -(R) and 0.26μg/spot and 0.86μg/spot for -(S) respectively. The developed method was validated and proved to be robust. Ropivacaine sample solution was found to be stable for one weak in methanol. The proposed method was found to be selective and accurate for identification and quantitative determination of enantiomeric purity of ropivacaine in bulk powder and pharmaceutical dosage form.