Strategy for the isolation, derivatization, chromatographic separation, and detection of carnitine and acylcarnitines.
A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cation-exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase chromatography using a single non-end-capped C8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversed-phase HPLC results in separation of reagent byproducts from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid-phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by MS and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.
Minkler
Paul E
PE
Medical Research Service, Louis Stokes Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, USA.
Ingalls
Stephen T
ST
Hoppel
Charles L
CL
D000097:Acetonitriles / Q000737:chemistry; D000098:Acetophenones / Q000737:chemistry; D000108:Acetylcarnitine / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods; D002331:Carnitine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D008099:Liver / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry; D000432:Methanol / Q000737:chemistry; D015394:Molecular Structure; D010172:Palmitoylcarnitine / Q000032:analysis / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry; D058428:Silica Gel; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods
D000097:Acetonitriles; D000098:Acetophenones; D012709:Serum Albumin; C116917:acylcarnitine; D010172:Palmitoylcarnitine; D058428:Silica Gel; D000108:Acetylcarnitine; D012822:Silicon Dioxide; C043597:4'-bromophenacyl triflate; D002331:Carnitine; D000432:Methanol; C032159:acetonitrile