Current Pharmaceutical Analysis - Volume 2, Issue 3, 2006

In HIV-infection, therapeutic drug monitoring (TDM) has been proposed to optimize highly active antiretroviral therapy (HAART) response. Pharmacokinetics of protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) efavirenz and nevirapine have been proved suitable for monitoring their plasma drug levels. In contrast, nucleoside reverse transcriptase inhibitors (NRTI) contain lack of a certain correlation between plasma levels and drug efficacy. Currently, TDM under HAART is limited by some uncertainty which pharmacokinetic predictor to prefer and on which drug concentration or range to rely on. In a therapy naive patient trial (ATHENA) on indinavir or nelfinavir based drug regimens, the study arm with use of TDM resulted in prevention of virological failure or treatment discontinuation due to drug toxicity. Current data ascribed increasing importance to TDM in ritonavir boosted PI combinations especially for lopinavir, atazanavir and fosamprenavir. Besides, further applications are proposed in selected patients groups (i.e. children, pregnancy, renal or hepatic dysfunction, weight loss) to confirm adequate drug concentrations and manage drug-drug- interactions. PI double-boosting in salvage regimens in patients with multiple NRTI associated mutations should also require TDM for handling dose adjustments, moreover, treatment experienced patients might benefit from TDM in combination with viral resistance testing. Limitations to the application of TDM are considered as non standardized procedures or techniques permitted to correlate pharmacological and virological data therefore, an approach towards standardization is needed. So far the inhibitory quotient (IQ) (necessitating phenotypical resistance testing) or the genotypic inhibitory quotient (GIQ) (referring to the number of viral mutations using a genotypic resistance test), could become increasingly important in this context. In assessing adherence, TDM allows to confirm the correct medication intake, but only the last few doses taken by a patient will be reflected. Without TDM, numerous interactions by co-administered drugs (antibiotics, antacid drugs, hypnotics, herbals, life-style drugs) turn out to possibly lead to obscure interpretations and individual medical care remains elusive. Currently, the interpretation of PI and NNRTI drug levels is based on increasing experience using TDM. Even though TDM in HAART is not the gold standard of care, yet, it provides security in guiding patients on an individual therapy and might be linked with pharmacogenomics in resource-rich countries.

Molecular imprinted polymers are man-made materials with predetermined selectivity towards one or more analytes. They have been used as selective stationary phases in several analytical separative techniques. This review discusses the possible uses of imprinted polymers, with a particular emphasis on their use as high pressure and electrochromatographic stationary phases for resolution of optical enantiomers, materials for fast screening of libraries and selective solid-phase extraction for sample clean-up and preconcentration.

Allergen-specific immunotherapy (SIT) is an effective form of treatment for IgE-mediated type I allergy and represents the only curative approach. Presently SIT is performed with complex natural extracts that contain allergens (representing active pharmaceuticals ingredients, APIs) together with non-allergenic proteins and other macromolecules. These extracts are difficult to standardize and it is not possible to define each component quantitatively. In order to overcome such difficulties in standardization and batch-to-batch consistency, highly purified recombinant allergens (rallergens) and hypoallergenic variants may be considered as novel candidates for the production of high quality therapeutic preparations offering safer and more effective SIT. The availability of detailed information on the characteristics of r-allergens makes it possible to apply a variety of methods in order to ensure purity, identity, quantity, safety and potency compared to allergen extracts. Besides determination of potential process-related contaminants such as endotoxin and residual host-cell-protein a central consideration focuses on the investigation of the folding status, as pathologic IgE-binding is highly dependent on the conformation of the molecules. The primary structure on the other hand determines the therapeutic potential through the specific T cell epitopes and the ability to induce a curative reorientation of the T-cell response. Suitable methods for evaluation of the relevant properties include immunologic methods such as ELISA and Western blots together with robust chromatographic and electrophoretic techniques. Promising results from early clinical studies suggest that the use of rallergens for SIT will become a reality and therefore it is appropriate to consider how all aspects of the quality of these proteins might be evaluated.

There is increasing in vitro and in vivo evidence indicating that acyl glucuronides of various drugs are chemically reactive and potentially cause organ toxicity. Such conjugates are chemically unstable, undergoing hydrolysis and pH-dependent intramolecular migration to generate isomers and covalent binding with various tissue proteins to result in drug-protein adducts. This review highlights the reactivity of drug acyl glucuronides and commonly used analytical techniques for these metabolites and resultant drug-protein adducts used in preclinical and clinical studies. The stability of acyl glucuronides is dependent on many factors including pH, temperature, nature of the aglycon, and the presence of plasma or albumin. Drug acyl glucuronides may cause toxicity either through changes in the functional properties of the modified proteins, through initiation of antigen-mediated immune responses, or unknown mechanisms. The conjugates and sometimes the drug-protein adducts can achieve appreciable blood concentrations following drug administration. With careful sample handling procedures (e.g. quick cooling and acidification) during preclinical and clinical studies, drug acyl glucuronides in biological matrixes can be reliably identified and quantitated using appropriate analytical methods such as HPLC, LC-MS and NMR techniques. The drug-protein adducts can also be identified and determined using these highly sensitive chromatographic techniques after proper sample processing. In addition, their protein adducts with plasma or cellular proteins can be determined after electrophoretic separation, followed by blotting. ELISA techniques have been used to assess the presence of antibodies against acyl glucuronide-protein adducts in vivo. Further studies are needed to explore the clinical and toxicological implications of acyl glucuronides of various drugs and to establish the relationships between the toxicity of drugs and their acyl glucuronide levels by close monitoring using above analytical techniques.

DHPLC (denaturing high-performance liquid chromatography) has established itself as one of the most powerful tools for DNA variation screening and allele discrimination for the characteristics of sensitivity, accuracy and efficiency that allow a rapid detection of SNPs, insertions and deletions. Moreover, it has the advantages of fullautomation and cost effectiveness. Aside from its well-known application in identifying mutations in human diseases, it has been also implemented in the quantitative measurement of gene expression and the analysis of single nucleotide extension products. In this paper, we aimed at summarizing the general properties and the main applications of DHPLC in the biomedical field as well as comparing this newly developed biotechnology with other conventional pre-screening methods for mutation scanning, such as single strand conformational polymorphisms (SSCP), conformation sensitive gel electrophoresis (CSGE) and two dimensional gene scanning (TDGS).

Fumonisins, first identified in 1988, are naturally-occurring mycotoxins mainly produced by Fusarium verticillioides and F. proliferatum, food-borne fungi widely distributed in crops, and occur as one of the most common contaminants of corn and corn-based foods and feeds. The most abundant fumonisin, fumonisin B1 (FB1), is associated with a range of toxicological effects in animals including equine leukoencephalomalacia, porcine pulmonary edema, and rodent carcinogenicity. In humans, FB1 has been associated with high rates of esophageal cancer and the International Agency for Research on Cancer evaluated the FB1 derived from F. verticililoides as Group 2B, i.e. a possible human carcinogen. Furthermore, FB1 has been found to be a potential cause of human neural tube defects. Fumonisins bear a remarkable structural similarity to sphingosine and their mode of toxic action is in part elucidated in that they may inhibit ceramide synthase, causing accumulation of bioactive intermediates of sphingolipid metabolism. In spite of the need for efficient analysis, the quantification of fumonisins is a difficult task, since they do not bear suitable chromophores nor fluorophores for detection. Practical fluorometric-based high-performance liquid chromatography analysis methods for derivatized fumonisins have been developed since 1990 and a method using o-phthalaldehyde as a fluorescent reagent has been validated. Recently, mass spectrometry analysis has been used to identify fumonisins as liquid chromatography-mass spectrometry interfaces became more widely available. This appears to be the superior method to perform quantitative and qualitative analysis without the need of derivatization procedures. Very recently, the application of tandem mass spectrometry for confirmation and quantification of mycotoxins including fumonisins has been reported. This paper reviews those liquid chromatographic methods developed for this group of mycotoxins.

This review surveys recent publications on the Electrospray Ionisation - Mass Spectrometry (ESI-MS) of drugs of small molecular mass taken from selected drug classes using the Web of Knowledge database. The structural classes are antibiotics/antibacterials, steroids, cannabinols, antidiabetics, immunosuppressants, antitumour drugs, antiretroviral drugs, nonsteroidal antiinflammatories, mucolytic drugs, anticoagulants, cyclooxygenase inhibitors, stimulants, noradrenergic agents, anti-TB drugs and erectile dysfunction agents. Fragmentation information that these drugs exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers is also provided. An appraisal of applications for the period 2004-2005, again taken from the Web of Knowledge database, of the technique liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) to the detection and determination of these drugs, primarily in biomatrices, is then made. Information on such aspects as sample concentration, LC separation conditions, recoveries from biological media and limits of detection (LODs) are provided.

The scope of this study was to investigate the fate and behavior of selected pharmaceuticals and main metabolites under defined conditions in model systems simulating slow sand filtration. For this purpose, analytical procedures were developed for the simultaneous identification and quantification of carbamazepine (CBZ), diclofenac (DCF), ibuprofen (IBU) and sulfamethoxazole (SFM). The following main metabolites were synthesized for method development: N-1-glucuronide-sulfamethoxazole (Glucu-SFM), N-4-acetyl-sulfamethoxazole (Ac-SFM) and 2- hydroxyibuprofen (OH-IBU). The procedures comprised solid phase extraction (SPE) for enrichment using polymeric material (Oasis HLB) and determination of the analytes with both GC/MS and LC/ESI-MS. For GC/MS, an additional derivatization step was necessary. Best results were obtained with the application of diazomethane as derivatizing agent. As a consequence, the determination of DCF, IBU and OH-IBU was preferably carried out by GC/MS, whereas SFM, Ac- SFM and Glucu-SFM were determined by LC/ESI-MS. CBZ could be sufficiently analyzed by both techniques, however, GC/MS is preferred because of much lower interferences by sample matrix. LC/ESI-MS allowed the measurement of the active drugs and their metabolites with quantification limits down to 10 ng/L in the MS/MS mode while GC/MS permitted the quantification in the same range or lower for some analytes, except SFM and its metabolites. The developed method was applied to real surface water samples from the river Ruhr, Germany.