986 LCGC VOLUME 19 NUMBER 9 SEPTEMBER 2001 www.chromatographyonline.com amino acid profiling in routine clinical analysis (1,2). As analysts started using capillary elec- trophoresis (CE) in the 1990s, they were able to successfully use most of the popular reagents from LC analyses in electromigra- tion techniques. The remarkable progress in detection technologies — such as diode- array ultraviolet (UV), laser-induced fluores- cence, evaporative light-scattering, and mass spectrometry (MS) detection with various ionization principles — provided new opportunities in amino acid determination. The advances in evaporative light-scattering and MS techniques restored new interest in native amino acid analyses. Despite the remarkable improvements in separation and detection technology, sample preparation before analysis remains an inte- gral and often the most time-consuming part of the methodology. Because of the matrix complexity, preparation of biological samples such as body fluids usually is diffi- cult and laborious, so that even excellent separation and detection methods may fail to deliver the performance and ruggedness required for routine use. This fact is recog- nized more often in application laboratories but seldom in the academic community. Expedient sample preparation, therefore, is of primary importance in routine applica- tions, and the lack of it is the possible reason that a limited number of robust, user- friendly methods for analyzing amino acids The authors briefly review current strategies of amino acid analysis and present a novel approach to a simplified sample preparation of body fluids. Their procedure involves two consecutive extraction steps: a solid- phase step and a liquid–liquid step, during which the analytes are selectively isolated and rapidly alkylated while being transferred into an organic phase with a high degree of purity. The total time of sample preparation and gas chromatography (GC) analysis is approximately 15 min. The derivatives can be analyzed by GC with flame ionization, nitrogen–phosphorus, or mass spectrometry detection or by liquid chromatography with mass spectrometry detection, all of which give this procedure a versatility lacking in other current techniques. Advances in Amino Acid Analysis Petr Hus ˇek and Petr S ˇ imek* Institute of Endocrinology, Narodni Avenue 8, CZ-116 94 Prague 1, Czech Republic, e-mail phusek@endo.cz * Institute of Entomology, Department of Analytical Biochemistry, Czech Academy of Sciences, Branisovska 31, CZ-370 05 C ˇ eské Bude ˇjovice, Czech Republic, e-mail psimek@entu. cas.cz Address correspondence to P. Hus ˇek. very new advancement in separa- tion techniques subsequently is applied in the bioanalytical field, in which the class of protein amino acids usually is first to be examined. Because of large differences in their chemical structures, which span from nonpolar to highly polar side chains, amino acids have always presented an analytical challenge in terms of sample preparation, separation, and detection. Numerous analytical methods have been developed in the past 50 years, and researchers have made impressive achievements in the fields of derivatization, separation, and detection of this compound class. The ion-exchange-based separation of amino acids, followed by postcolumn nin- hydrin detection, was introduced into com- mon use in the 1950s and has remained important. Starting in the mid-1950s, gas chromatography (GC) analysis methods dominated the field for two decades. Deriva- tization approaches were developed with the purpose of increasing analyte volatility by removing as many active hydrogen atoms as possible from functional groups in amino acid molecules. Thereafter, the significant progress made in reversed-phase liquid chro- matography (LC) stationary-phase develop- ment and in the new field of precolumn derivatization provided improved separa- tions of analyte derivatives. Several methods proved reliable enough to address body fluid E Removable brochure ➞