Anal. Chem. 2005, 77, 1448-1457
Strategy for the Isolation, Derivatization, Chromatographic Separation, and Detection of Carnitine and Acylcarnitines Paul E. Minkler,† Stephen T. Ingalls,‡ and Charles L. Hoppel*,†,‡,§
Medical Research Service, Louis Stokes Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, and Case Comprehensive Cancer Center and the Departments of Pharmacology and Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cationexchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/ reversed-phase chromatography using a single non-endcapped 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/reversedphase 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 solidphase 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. Carnitine (3-hydroxy-4-(N,N,N-trimethylammonio)butanoate) is a low molecular weight trimethylammonio carboxylate required for the mitochondrial oxidation of long-chain fatty acids.1 * To whom correspondence should be addressed. Phone: (216) 791-3800, ×5657. Fax: (216) 707-5973. E-mail:
[email protected]. † Louis Stokes Department of Veterans Affairs Medical Center. ‡ Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine. § Departments of Pharmacology and Medicine, Case Western Reserve University School of Medicine.
1448 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
Other roles for carnitine include its mass action effect in establishing the steady-state intramitochondrial acyl coenzyme A/coenzyme A ratio2 and peroxisomal fatty acid oxidation.3 These functions are accomplished through the action of acyltransferases, which produce carnitinyl esters (acylcarnitines). Methods for the detection and quantification of carnitine have used a variety of strategies.4 Originally, Marquis and Fritz5 proposed a method for the accurate quantification of carnitine that exploited its recognition by carnitine acetyltransferase. This approach was further refined by using radioactive acetyl coenzyme A.6 Due to technological advances, other more recent approaches have been developed employing HPLC/UV,7 flow injection analysis,8 tandem MS,9 or HPLC/tandem MS.10 Determination of acylcarnitines is challenging and has been addressed using a variety of chromatographic, derivatization, or detection schemes including radioisotopic exchange/HPLC,11 HPLC/UV,12 HPLC/fluorescence,13 GC/MS,14 CE,15 CE/MS,16 and HPLC/tandem MS.17 However, today the most common method used to analyze acylcarnitines begins with the esterification of acylcarnitines by means of high-temperature acylation followed (1) McGarry, J. D.; Brown, N. F. Eur. J. Biochem. 1997, 244, 1-14. (2) Brass, E. P.; Hoppel, C. L. Biochem. J. 1980, 190, 495-504. (3) Jakobs, B. S.; Wanders, R. J. A. Biochem. Biophys. Res. Commun. 1995, 213, 1035-1041. (4) Marzo, A.; Curti, S. J. Chromatogr. 1997, 702, 1-20. (5) Marquis, N. R.; Fritz, I. B. J. Lipid Res. 1964, 41, 184-187. (6) McGarry, J. D.; Foster, D. W. J. Lipid Res. 1976, 17, 277-281. (7) Minkler, P. E.; Hoppel, C. L. Clin. Chim. Acta 1992, 212, 55-64. (8) Manjon, A.; Obon, J. M.; Iborra, J. L. Anal. Biochem. 2000, 281, 176-181. (9) Kodo, N.; Millington, D. S.; Norwood, D. L.; Roe, C. R. Clin. Chim. Acta 1989, 186, 383-390. (10) Vaz, F. M.; Melegh, B.; Bene, J.; Cuebas, D.; Gage, D. A.; Bootsma, A.; Vreken, P.; van Gennip, A. H.; Bieber, L. L.; Wanders, R. J. A. Clin. Chem. 2002, 48, 826-834. (11) Schmidt-Sommerfeld, E.; Zhang, L.; Bobrowski, P. J.; Penn, D. Anal. Biochem. 1995, 231, 27-33. (12) Poorthuis, B. J. H. M.; Jille-Vlckova, T.; Onkenhout, W. Clin. Chim. Acta 1993, 216, 53-61. (13) Kamimori, H.; Hamashima, Y.; Konishi, M. Anal. Biochem. 1994, 218, 417424. (14) Libert, R.; Van Hoof, F.; Thillaye, M.; Vincent, M-F.; Nassogne, M-C.; Stroobant, V.; de Hoffmann, E.; Schanck, A. Anal. Biochem. 1997, 251, 196-205. (15) Vernez, L.; Thormann, W.; Krahenbuhl, S. J. Chromatogr. 2000, 895, 309316. (16) Heinig, K.; Henion, J. J. Chromatogr. 1999, 735, 171-188. (17) Tallarico, C.; Pace, S.; Longo, A. Rapid Commun. Mass Spectrom. 1998, 12, 403-409. 10.1021/ac0487810 CCC: $30.25
© 2005 American Chemical Society Published on Web 02/03/2005
by detection using tandem MS.18,19 Primarily due to its simplicity and rapidity, this approach has been applied with much success to programs for newborn screening20 and contributed greatly to our understanding of the role of acylcarnitines in metabolic disorders. The development of the benchtop electrospray ionization/ion trap MS and its coupling to HPLC has proven to be a substantial technical innovation.21 Previously, we reported HPLC methods for the quantification of carnitine and acylcarnitines in urine,22 plasma,23 and tissue.24 From this knowledge base, we concluded that combining the ion trap MS with an HPLC approach would allow the sensitive and selective detection of carnitine and acylcarnitines. This combination would have the ability to accommodate complex mixtures and distinguish isomeric acylcarnitines. These capabilities are limited with tandem MS. This report describes and illustrates the four essential components of this system: (1) isolation of carnitine and acylcarnitines by protein precipitation/desalting and silica gel cation-exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with the new reagent pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase chromatography of carnitine and acylcarnitine esters, and (4) MS and MS/MS detection. Examples of the chromatographic behavior, performance of the derivatization reagent, and MS characteristics of carnitine and acylcarnitines using this procedure are shown. EXPERIMENTAL SECTION Materials and Sources. Acetonitrile, methanol, acetic acid, and triethylamine were all HPLC grade and purchased from Fisher Scientific (Cleveland, OH). Diisopropylethylamine, L-carnitine chloride, O-acetyl-L-carnitine chloride, and palmitoyl-DL-carnitine chloride were purchased from Sigma (St. Louis, MO). L-Carnitine (N-methyl-d3, 98%) and acetyl-L-carnitine (N,N-dimethyl-d6, 98%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Methyl[14C]-L-carnitine was synthesized from norcarnitine (4-N,N-(dimethylamino)-3-hydroxybutyrate) by methylation using [14C]-methyl iodide.25 Palmitoyl-[14C]-L-carnitine was synthesized from methyl[14C]-L-carnitine and palmitoyl chloride.26 Other acylcarnitines used were synthesized using established methods.27,28 [1-14C]-Palmitic acid was purchased from New England Nuclear (Boston, MA). Radioactivity was measured using a Beckman Coulter LS6500 scintillation counter (Fullerton, CA). 2,4′-Dibromoacetophenone, N-methyl-N-nitroso-p-toluenesulfonamide, and trifluoromethanesulfonic acid were purchased from Aldrich (Milwaukee, WI). Pentafluorobenzoyl chloride was purchased from (18) Millington, D. S.; Norwood, D. L.; Kodo, N.; Roe C. R.; Inoue, F. Anal. Biochem. 1989, 180, 331-339. (19) Rashed, M. S.; Ozand, P. T.; Bucknall, M. P.; Little, D. Pediatr. Res. 1995, 38, 324-331. (20) Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clin. Chem. 2003, 49, 17971817. (21) Jonscher, K. R.; Yates, J. R. Anal. Biochem. 1997, 244, 1-15. (22) Minkler, P. E.; Hoppel, C. L. J. Chromatogr. 1993, 613, 203-221. (23) Minkler, P. E.; Hoppel, C. L. Anal. Biochem. 1993, 212, 510-518. (24) Minkler, P. E.; Brass, E. P.; Hiatt, W. H.; Ingalls, S. T.; Hoppel, C. L. Anal. Biochem. 1995, 231, 315-322. (25) Ingalls, S. T.; Hoppel, C. L.; Turkaly, J. S. J. Labelled Compd. Radiopharm. 1982, IX, 535-541. (26) Minkler, P. E.; Ingalls, S. T.; Kormos, L. S.; Weir, D. E.; Hoppel, C. L. J Chromatogr. 1984, 336, 271-283. (27) Ziegler, H. J.; Bruckner, P.; Binon, F. J. Org. Chem. 1967, 32, 3989-3995. (28) Brendel, K.; Bressler, R. Biochim. Biophys. Acta 1967, 137, 98-106.
Lancaster Synthesis (Windham, NH). Elemental analysis was performed by Galbraith Laboratories (Nashville TN). NMR spectra were obtained with a Varian (Palo Alto, CA) INOVA 600-MHz instrument equipped with an HX probe tunable to 13C and 19F. All spectra were obtained using d-chloroform solutions (20 mg/mL at 25 °C). 1H spectra were acquired with a 45° pulse after a delay of 1 s, and eight transients were collected per spectrum. 1H chemical shifts were calculated relative to 1H-chloroform, which was set to 7.24 ppm. All 13C spectra were 1H-decoupled, acquired with a 30° pulse after a delay of 1 s, and 256 transients were collected per spectrum. These were processed with 1-Hz line broadening and chemical shifts calculated relative to 13C-chloroform, which was set to 77.0 ppm. All 19F spectra were acquired with a 45° pulse after a delay of 1 s, processed with 0.5-Hz line broadening, and 16 transients were collected per spectrum. 19F chemical shifts were calculated relative to R,R,R-trifluorotoluene, which was set to -62.80 ppm. 4′-Bromophenacyl trifluoromethanesulfonate was synthesized as described.29 Solid-phase extraction columns (50 mg of silica Bond Elute) were purchased from Varian (Walnut Creek, CA) and prepared by washing with 0.5 mL of methanol (gravity flow) just before their use. 4′-Bromophenacyl esters of carnitine, 4-(trimethylammonio)butanoic acid, and 6-(trimethylammonio) hexanoic acid were synthesized as described.26 Bovine albumin fraction V solution 7.5% in phosphate-buffered saline was purchased from GIBCO BRL/Life Technologies (Grand Island, NY). Synthesis of Pentafluoro-2-diazoacetophenone.30,31 Caution! Syntheses involving diazomethane should be approached with great care! Diazomethane was prepared from 60 g (2.8 × 10-1 mol) of N-methyl-N-nitroso-p-toluenesulfonamide in a clear fit distillation apparatus.32 The receiving flask was fitted with a straight receiving tube and an addition funnel. A magnetic stirrer was brought into position beneath the receiving flask. A solution of 25 g (1.06 × 10-2 mol) of pentafluorobenzoyl chloride in 100 mL of diethyl ether was added dropwise. The icebath was removed, and the reaction solution was stirred and allowed to warm to room temperature for 1 h. This solution was evaporated under vacuum to leave a slightly viscous yellow oil, which was dissolved in 200 mL of chloroform, treated with MgSO4 and carbon, and filtered twice. The chloroform filtrate was concentrated to ∼75 mL, and 250 mL of hexane was added slowly. The solution was cooled to room temperature and transferred to a freezer for storage overnight at -60 °C. Crystals were collected by vacuum filtration and allowed to dry in air for 5 min before storage in a vacuum desiccator at room temperature. The yield of pale yellow needles was 18.5 g (7.8 × 10-2 mol, 74%), mp 46-48 °C (Fisher Johns micro hot stage, atmospheric pressure, uncorrected). Elemental analysis: Calcd, C, 40.70%; H, 0.43%; F, 40.23%; N, 11.87%. Found, C, 40.31%; H,
