Current Pharmaceutical Analysis - Volume 6, Issue 3, 2010

A biomarker is a measure of the interaction occurring in the biological systems between the organism and the potential risks that may be of chemical, physical, or biological character. The measured response of the body caused by this interaction can be of physiological, functional or biochemical character and can be captured at the cellular or molecular level. Steroids, especially cortisol and cortisone, are valuable biomarkers serving primarily the assessment of the organism's reaction to exposure to chronic stress. The problem of stress and its impact on the development of endocrinological, metabolic, psychiatric and cardiovascular diseases has been the subject of intense research for some years. Studies on cortisol and cortisone in biological materials such as serum, plasma, urine, or saliva were applied to measure stress levels in the body. The advantage of urine-based measurements lies in non-invasive nature of the method of sampling. The serum cortisol and cortisone levels may be increased in certain disease states such as diabetes, obesity, hyperthyroidism, and in pregnancy. Therefore, measurement of free cortisol in urine is the most reliable way to diagnose either stress disorder, or the functioning of the hypothalamic-pituitary-adrenal (HPA) axis. In the review, several validated high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC) assays for determination of steroids in urine samples were compared. Because these methods are automated, simple, rapid, and sensitive, they can be applied easily to the analysis of urine samples. However, HPLC is sometimes limited in its separation efficiency, and the consumption of organic solvents is relatively high. On the other hand, the important drawback of MECC is that sometimes it cannot be sensitive enough to determine low concentrations, e.g., when the sample injection volume is low and the optical path-length short. Nevertheless, it finally seems that MECC offers greater separation efficiency than HPLC and significantly reduces the analysis time and the operating costs compared to HPLC. In summary, those methods carry a great analytical potential, especially because urine, used as biological matrix and treated as a biological fluid, is easy to obtain in non-invasive collection procedures.

During the last decade there has been an increased focus on the application of steroids as biomarkers in biomedical practice. The analysis of steroid hormones in biological samples of plasma or serum is currently routinely used in clinical diagnosis being an essential source of information on not only metabolic pathways, but also disorders of the metabolism. More importantly, the steroid hormones may reduce cancer development on the endocrinal basis, reveal abuse of anabolic substances, or even depression incidences. In the case of biomedical research, quantitative determination of steroids in serum or plasma creates an opportunity to diagnose diseases efficiently in their early stages and to monitor a patient for a possible recurrence of a disease after therapy. Therefore, an accurate measurement of steroids in the plasma or serum has become important for the contemporary medicine, even if troublesome especially in view of their low concentration in biological samples. The most common methods of steroid quantification in clinical practice nowadays include immunoassays such as radioimmunoassay or enzyme immunoassay. However, the main disadvantage of the immunoassay techniques consists in cross reactivity of the antibodies used in the assay with the related hormones. On the other hand, there is a number of applications where the analysis is performed with the use of modern separation techniques. The aim of this review is to present the development and applicability of the various analytical methods, all based on separation techniques and used for determination of steroids in the clinical laboratory, taking into account the recent progress in those areas. The review shows the advantages of high-performance liquid chromatography applied to determine non-volatile and thermally labile steroids and steroid conjugates. It also demonstrates that the currently used mass spectrometry offers practically useful structural information on the composition of steroids. A number of techniques and methods are compared and critically discussed pondering finally their specific analytical requirements like sensitivity, specificity, simplicity, limit of detection, and quantification. Nonetheless, plasma or serum sampling seems to remain one of the most convenient methods for population screening purposes.

Steroid hormones are important in controlling human body functions as a part of the endocrine system together with neuronal systems and the immune system. Application of the assay of the steroid hormones treated as biomarkers was recently illustrated in certain cases, for example in clinical diagnosis of stress, the Cushing syndrome, congenital adrenal hyperplasia, and infertility, as well as in the field of sports medicine. The assessment of the steroid hormones in the body fluids has so far been typically based on serum and urine. However, the use of saliva as the diagnostic medium has recently grown in popularity among the scientists and clinicians because of sample collection, which is quick, uncomplicated, and non-invasive. Moreover, steroid hormones are not bound to protein in saliva. Therefore, salivary determination is an excellent approach for evaluation of free steroid hormones. The present study provides an overview of the analytical methods applied for salivary steroid measurements in the current clinical laboratory practice. It describes and thoroughly discusses the recent achievements associated with optimisation of the analytical conditions for the steroid assay, obtained through application of the modern separation techniques such as liquid chromatography, mass spectrometry, versus non-separation techniques such as the immunological methods. Moreover, the issues associated with optimization of the extraction procedures are presented, since sample pre-treatment is the most limiting and crucial step in analyses of biological fluids. In addition, the study evaluates the consequences of any pre-analytical variation preceding the application of the assay methodologies, stemming from the collection strategy and the subsequent storage conditions. It further provides several examples of application in diverse fields of interest such as psychology, pharmacology, clinical endocrinology, or sports medicine.

Sulfonamides, also known as “sulfa drugs”, derived from sulfanilamide (p-aminobenzenesulfonamide) are used in both veterinary and human medicine. In veterinary medicine they are widely used in farm animal feedstuff and fish cultures for prophylactic and therapeutic purposes. In human medicine they are used to treat systemic infections caused by susceptible organisms. Some members of this class include: sulfathiazole, sulfamethazine (sulfadimidine), sulfamerazine, sulfadiazine, sulfapyridine, sulfabromomethazine, sulfaethoxypyridazine, sulfamethoxypyridazine, sulfadimethoxine and sulfachlorpyridazine. Sulfonamides are effective against Gram-positive and Gram-negative bacteria. Some protozoa, such as coccidians, Toxoplasma species and plasmodia, are generally sensitive. Chlamydia, Nocardia and Actinomyces species are also sensitive. A common disadvantage in all antimicrobial agents is resistance, which is widespread in many animal and human populations. Resistance to sulfonamides in human medicine has severely restricted clinical usefulness. Emergence of drug resistant strains of bacteria, has led to replacement of drug by other semi-synthetic antibiotics to a large extent. However in the third world countries they are of great value. Sulfonamides are well distributed in all body tissues. High concentrations can be found in bile, cerebrospinal fluid, prostatic fluid and sputum. Sulfonamides are metabolized in the liver but are primarily excreted unchanged in the urine. There are exceptions, however. A large proportion of sulfamethoxime is metabolized by the liver and only thirty percent is excreted unchanged by the kidneys. Occasionally severe side effects are observed with sulfonamides and potentiated sulfonamides (e.g. with trimethoprim). The sulfonamides can cause hepatic necrosis, serum sickness like syndrome, acute hemolytic anemia, agranulocytosis and Stevens-Johnson syndrome. Hypersensitivity is also very common. Therefore analytical methods for the determination of sulfonamides in pharmaceuticals and biological samples are of great importance. HPLC methods have been discussed herein. HPLC can provide a valuable tool for generating highly pure preparations for characterizing the antimicrobial activities. In the present review article, column and mobile phase conditions as well as sample preparation issues are taken into consideration. A brief discussion on chemical structure, spectrum of activity and action mechanism of sulfonamides has also been provided. The time frame of papers covered by this review article starts at 1974 and ends at 2009.

Development of new style chromatographic packing materials and improvement of their performance are important in the study of pharmaceutical analysis. Monoliths are separation media in a format that can be compared with a single large 'particle' that does not contain the interparticular voids typical of packed beds. In recent years, monolithic supports as stationary phases in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have gained significant interest due to their ease of preparation, high reproducibility, versatile surface chemistries and fast mass transport. Generally, depending on the nature of the monolithic material, organic polymer-based monolithic columns and silica-based monolithic columns can be identified. The silica monoliths prepared from tetraalkoxysilane by a sol-gel method can provide either micrometer-size through-pores or high specific surface areas and can be well suited for small molecules in HPLC modes. Organic polymer-based monoliths including monolithic molecularly imprinted polymers have been applied not only in separation and analysis of biomacromolecules, but also in treatments of the samples before injected into the chromatograph in biological fluids because it allows the simultaneous removal of matrix compounds and preconcentration of the analytes. The most commonly reported organic polymers are based on polystyrenes, polymethacrylates and polyacrylamides. Capillary electrophoresis with both organic polymer-based and silica-based monolithic columns has also been used for the separation of small molecules. This review summarizes the current achievements and their application of organic polymer-based monolithic columns and silica-based monolithic columns for both HPLC and CE in solid-phase extraction (SPE) and pharmaceutical analysis. The potential of these columns is demonstrated with separations involving various drugs in different chromatographic modes.