tines (carnitinyl esters) in biological sample matrices
A method for the quantitative determination of carcan be indicative of unusual metabolic conditions, innitine, acetylcarnitine, and total carnitine in tissue cluding disorders of organic acid metabolism (1). Diagwas developed for application to clinical research and nosis and management of these metabolic diseases rediagnosis. Human skeletal muscle and heart speciquires knowledge of carnitine, total carnitine (the sum mens (10-20 mg) were homogenized in 1 ml of water. of carnitine and all acylcarnitines), and individual acyl-Aliquots of the resulting homogenates (50 ml) were excarnitine concentrations. Among the reported procetracted with 1.0 ml of acetonitrile:methanol (3:1) and dures to determine carnitine and/or acylcarnitines in the carnitine-related compounds were isolated using biological matrices are those which use radioisotope columns containing 300 mg of silica gel. Samples were exchange-high-performance liquid chromatography then derivatized with 4-bromophenacyl trifluoro-(HPLC) 1 (2), fast atom bombardment ionization coumethanesulfonate for spectrophotometric detection or pled with mass spectroscopy (FAB/MS) (3), on-column 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate N-demethylation gas chromatography followed by for fluorescence detection and quantified by high-permass spectroscopy (GC/MS) (4), pre-column acylcarniformance liquid chromatography. Fluorometric detectine deamination-GC/MS (5), and pre-column chemition of 2-(2,3-naphthalimino)ethyl ester derivatives afcal derivatization-HPLC (6). These methods do not forded a 500-fold increase in sensitivity when provide an optimal combination of sensitivity, specificcompared to derivatization with 4-bromophenacyl ity, ease of analysis, and minimal capital equipment trifluoromethanesulfonate. This methodology permitcosts that would facilitate wide application in research ted detection of acetylcarnitine in dilute human musand clinical laboratories. To address these issues, we cle homogenates at quantities of 790 fmol of acetylcarpreviously developed pre-column chemical derivatizanitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from tion-HPLC methods for the quantitative determinasubjects performing exercise at high work loads. The tion of carnitine, specific acylcarnitines, and total carmethod permitted quantification of carnitine, acetylnitine in urine and plasma (7,8).
carnitine, and total carnitine (sum of carnitine and all
The purposes of this article are: (1) To describe the acylcarnitines) and demonstrated the specific redistriapplication of the HPLC procedure to tissue homogebution of the carnitine pool from carnitine to acetylnates, (2) to compare the use of 4-bromophenacyl carnitine with exercise above the lactate threshold. trifluoromethanesulfonate and 2-(2,3-naphthalimi-This HPLC method is facile, and provides a sensitive no)ethyl trifluoromethanesulfonate as derivatization and specific approach for use in human biopsy specireagents, and (3) to report the results of exercise studmens.