High performance liquid chromatography in pharmaceutical analyses

Abstract

In testing the pre-sale procedure the marketing of drugs and their control in the last ten years, high performance liquid chromatography replaced numerous spectroscopic methods and gas chromatography in the quantitative and qualitative analysis. In the first period of HPLC application it was thought that it would become a complementary method of gas chromatography, however, today it has nearly completely replaced gas chromatography in pharmaceutical analysis. The application of the liquid mobile phase with the possibility of transformation of mobilized polarity during chromatography and all other modifications of mobile phase depending upon the characteristics of substance which are being tested, is a great advantage in the process of separation in comparison to other methods. The greater choice of stationary phase is the next factor which enables realization of good separation. The separation line is connected to specific and sensitive detector systems, spectrafluorimeter, diode detector, electrochemical detector as other hyphernated systems HPLC-MS and HPLC-NMR, are the basic elements on which is based such wide and effective application of the HPLC method. The purpose high performance liquid chromatography (HPLC) analysis of any drugs is to confirm the identity of a drug and provide quantitative results and also to monitor the progress of the therapy of a disease.1) Measuring presented on the Fig. 1. is chromatogram obtained for the plasma of depressed patients 12 h before oral administration of dexamethasone. It may also be used to further our understanding of the normal and disease process in the human body trough biomedical and therapeutically research during investigation before of the drugs registration. The analyses of drugs and metabolites in biological fluids, particularly plasma, serum or urine is one of the most demanding but one of the most common uses of high performance of liquid chromatography. Blood, plasma or serum contains numerous endogenous compounds often present in concentrations much greater than those of analyte. Analiyte concentrations are often low, and in the case of drugs, the endogenous compounds are sometimes structurally very similar to the drug to be measured. The binding of drugs to the plasma protein also may occur which decreases the amount of free compound that is measured. To undertake the analyses of drugs and metabolites in body fluids the analyst is facet with several problems. The first problem is due to the complex nature of the body fluid, the drugs must be isolated by an extraction technique, which ideally should provide a relatively clean extract, and the separation system must be capable of resolving the drugs of interest from co extractives. All mentioned when we are using high performance liquid chromatography require good selections of detectors, good stationary phase, eluents and adequate program during separation. UV/VIS detector is the most versatile detector used in high performance liquid chromatography it is not always ideal since it is lack of specificity ...

Analytical aspects of biosimilarity issues of protein drugs

“…Method Comment/goal Amino acid composition Amino acid analysis [88] Amino acid composition of the protein is determined Molecular weight (MW) and size 1D SDS PAGE/CE [89] or mass spectrometry, AUC [90] MW of protein is confirmed Amino acid sequence • N/C-terminal sequence Mass spectrometry [24,25] and Edman degradation [26] Amino acid sequence of protein is compared with theoretical sequence/nature and homogeneity of termini is identified to find truncated forms Extinction coefficient (EC) UV absorption [37,91] or calculation after determination of amino acid composition EC of known substance is confirmed Peptide mapping HPLC and/or mass spectrometry [92] Primary structure/Identity of protein is confirmed after digestion S-S bridges HPLC and/or mass spectrometry [9,93] Higher order of structure/location and/or connectivity of S-groups is confirmed Electrophoretic patterns 1D SDS PAGE [89], CGE [94,95], IEF [96] or 2D PAGE [97,98] or CE [99] Identity, homogeneity, purity, aggregates and truncates of substance is established Liquid chromatographic patterns HPLC/RPHPLC [100], IEX [101], SEC [102], AC, HILIC [103,104], HIC [105,106] Identity, homogeneity and purity of substance is established Spectroscopic profiles CD [107][108][109], NMR [22],IR [14,110] Fluorescence [111][112][113] Higher-order structure is determined …”

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