Current Pharmaceutical Analysis - Volume 4, Issue 2, 2008

Four different methods were used to measure the “in vitro” antioxidant capacity of three cobalt complexes recently proposed as anticancer active principles: a new biosensor method and a new pulse voltammetric, or pulse polarographic method, together with two other methods - one fluorimetric and the other spectrophotometric. Results obtained using the different methods are discussed and compared with the anti-metastatic effect recently measured “in vivo” by Russian authors. The good correlation found between anti-tumoral and anti-metastatic activity ranking and in vitro antioxidant activity found by the new voltammetric method suggests interesting hypotheses based on the anti-tumoral effect of these complexes.

Advances in recombinant DNA biotechnology have stimulated demand for the development of protein formulations within the pharmaceutical industry. A major challenge in preparation of protein formulations is their often poor stability during formulation processing and storage. Combinations of analytical techniques that probe different aspects of protein structure are often required to ensure that higher order (secondary and tertiary) structure and biological activity are maintained. Vibrational spectroscopy has received increasing attention as it offers effective, reliable, fast, simple and non-destructive measurement of protein structure. This review describes recent applications of near-infrared, infrared and Raman spectroscopy for protein analysis. This paper is intended to serve as guidance for researchers beginning in spectral analysis. It is hoped that the information herein will encourage and facilitate research associated with the use of these spectroscopic techniques in protein pharmaceutics.

Different fractionation steps have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins. The nature of fractionation (performed either on denatured or native proteins), the efficiency of recovery, the capacity of directly identifying proteins or protein panels, the possibility of associating to other laboratory techniques influence the choice of the methods to be used in different experimental and clinical settings. In this review, the main fractionation techniques (such as electrophoresis, SELDI and liquid chromatography) are described for proteomic studies in clinical field, and their advantages and disadvantages are discussed on the basis of our experience. In particular, liquid-liquid fractionation performed by PF2D is discussed in detail because of its partial automation, its ability to improve mass spectrometry analysis of serum proteins and its potential application in clinical studies.

Glycosaminoglycans (GAGs) are non branched polysaccharides which are raising a great interest among the scientists for their biological roles. In fact GAGs play a pivotal role in several biological events, since they participate in and regulate cell adhesion, migration and proliferation. The quantification and analysis of the fine structure of GAGs are increasingly important not only for understanding many biological processes, but also for elucidate many critical aspects in human pathology development. Chondroitin sulfate (CS), heparan sulfate (HS) and keratan sulfate (KS) are commonly described as sulfated GAGs and these molecules are linked to a core protein forming proteoglycans; the sulfation pattern shows a high level of complexity and it is associated with specific function in the tissues. The only GAG without protein core is hyaluronan (HA), which is produced in almost all tissues, often with a molecular weight of 106 Daltons. Several human tissues contain high amount of GAGs and the change of the quantity and the structure of these macromolecules are described in tissue development and it is commonly associated with diseases. Electrophoretic methods based on the gel separation of 2-aminoacridone labelled HA and CS sulfate Δ-disaccharides, derived from GAG digestion with specific eliminases, have been recently proposed. These new techniques represent a suitable method for GAG fast and sensitive analysis. In this review we will describe the recently achieved methods on the GAG analysis based on the electrophoretic approach in comparison with the more standard chromatographic techniques (HPLC).

A novel coated-graphite selective sensor based on verapamil ion pair with phosphomolybdate (PM) for its flow injection potentiometric (FIP) determination has been described. The sensor was prepared by coating the membrane cocktail containing PVC, plasticizer, and carrier on the surface of graphite rode. Influences of the membrane composition, pH, ions and possible interfering anions on the response properties of the electrode were investigated. The sensor membrane containing 3.6 % Ver-PM ion-pair and 62.0 % dioctylphethalate (DOP) in PVC possesses the best response with quasi- Nernstian slope of 62.6 mV/ decade over a wide concentration range of 6x10-6- 1x10-2 M and a lower limit of detection (LDL) of 1.9 x10-6 M. The developed sensor has been applied for FIP determination of verapamil hydrochloride in pure solution, pharmaceutical preparations and urine. The parameters which control the FIP method have been optimized. The determination of verapamil assay in Verpamil tablets after shelf-storage for more than one year using the proposed sensor has been achieved. The percent of verapamil hydrochloride in tablets was found to decrease as the storage time increases and it reached a value of 73.5 % of its nominal value after 14 months storage.

The majority of enantiomeric separations for purity analysis and quality control continue to be performed by liquid chromatography (LC). However, the trend in preparative scale chromatography is rapidly moving the emphasis to supercritical fluid chromatography (SFC) to reduce raw material, processing and waste costs. Fortunately for the analytical chemist, the same stationary phases are used for both normal phase LC and SFC. Analytical laboratories must be ready to continually address the changing nature of molecules in development in the pharmaceutical industry. In our laboratory, preliminary data analysis of chiral screening strategies indicated that the analytical chromatography results from the SFC runs were as superior to the liquid chromatographic results. Using polysaccharide based stationary phases (Diacel AD, OD, AS, and OJ), SFC apparently produced better selectivity than LC screening systems using the same columns and samples. Both were normal phase techniques. And, while SFC mobile phases present several chromatographic advantages, selectivity should not be one of them. A follow up investigation was initiated to look further into this apparent incongruity. The chromatographic results presented illustrate that the difference resulted from the modifiers used rather than from the chromatographic mode used (liquid versus supercritical fluid).