ANALYTICAL

BIOCHEMISTRY

199,%-105

(1991)

Analysis of Acylcarnitines as Their N-Demethylated Ester Derivatives by Gas Chromatography-Chemical Ionization Mass Spectrometry Zhi-Heng

Huang,

Douglas

A. Gage, Loran

MSU-NIH Mass Spectrometry Facility, Department East Lansing, Michigan 48824-1319 -

Received

May

L. Bieber,

Michigan

C. Sweeley’ State University,

24,199l

A novel approach to the analysis of acylcarnitines has been developed. It involves a direct esterification using propyl chloroformate in aqueous propanol followed by ion-pair extraction with potassium iodide into chloroform and subsequent on-column N-demethylation of the resulting acylcarnitine propyl ester iodides. The products, acyl N-demethylcarnitine propyl esters, are volatile and are easily analyzed by gas chromatography-chemical ionization mass spectrometry. For medium-chain-length (C,-C1.J acylcarnitine standards, detection limits are demonstrated to be well below 1 ng starting material using selected ion monitoring. Wellseparated gas chromatographic peaks and structurespecific mass spectra are obtained with samples of synthetic and biological origin. Seven acylcarnitines have been characterized in the urine of a patient suffering from medium-chain acyl-CoA dehydrogenase deficiency. 0 1991 Academic Press, Inc.

Carnitine is essential for the mitochondrial/3-oxidation of long-chain fatty acids. It functions by transporting activated long-chain fatty acids across the inner membrane of mitochondria to become a source of mitochondrial long-chain acyl-CoAs. Carnitine is also involved in the detoxification of specific short- and medium-chain fatty acids that are nonmetabolized or poorly metabolized. In certain metabolic disorders, such fatty acids can be excreted via acylcarnitines, thereby leading to a urinary accumulation of specific acylcarnitines, particularly in organic acidurias (1). For some of these disease states, qualitative and quantitative analyi To whom

and Charles

of Biochemistry,

correspondence

should

be addressed.

sis of urinary free carnitine and acylcarnitines enable the recognition of the specific metabolic defect that may not be easily detected by an organic acid screen (2). To date, a major limitation of such approaches has been the lack of a rapid, sensitive, and specific analytical procedure for detection and quantitation of individual acylcarnitines. The analysis of acylcarnitines has been reviewed extensively (3-6). Current methods involve the enzymatic exchange of radioactive L-carnitine into the acylcarnitine pool, followed by HPLC separation and determination of the radioactivity in individual separated peaks. Although this approach (7,s) allows specific individual acylcarnitines to be identified and quantitated, it is time-consuming and requires that all of the acylcarnitines in the sample be substrates for the carnitine acyltransferase used, a criterion not fulfilled by many samples which have unusual acylcarnitines. Esterification of the carboxyl group of carnitine with a chromophore such as ap-bromophenacyl group has also been used for HPLC analysis of acylcarnitines (9). Although sensitive, contamination of samples by other compounds containing carboxyl groups is a serious limitation of this approach. Two mass spectrometric approaches have also been introduced, one employing fast atom bombardment mass spectrometry (FAB-MS)’ and the other thermo’ Abbreviations used: FAB-MS, fast atom bombardment mass spectrometry; TSP-HPLC-MS, thermospray high-performance liquid chromatography mass spectrometry; CID-MS-MS, collision-induced-dissociation tandem mass spectrometry; CF-FAB-MS, continuous flow fast atom bombardment mass spectrometry; MCAD, medium-chain acyl-CoA dehydrogenase; GC-CI-MS, gas chromatography-chemical ionization mass spectrometry; PCF, n-propyl chloroformate.

98 All

Copyright 0 1991 rights of reproduction

000%2697/91$3.00 by Academic Press, Inc. in any form reserved.

CHROMATOGRAPHIC

ANALYSIS

OF

ACYLCARNITINE

l. :s%,~ M,,N+Afy opr OCOR

o

ii.

1.

aq.KI rt,5

OCOR

99

DERIVATIVES

Fie3Ntw

OPr

0

~-+

0

RCO-0

min

MQN

wopr

+

RCOzMe

0 -MeI

Me2N w

w

Acyl

SCHEME

OPr OCOR

on-column

N-demethylcarnitine Pr ester (ADMCPE)

SCHEME

1

spray liquid chromatography mass spectrometry (TSPHPLC-MS). The former is suitable for examining acylcarnitines in biological fluids, either underivatized or preferably as methyl esters (10-13). Usually, it permits detection in unpurified urine at concentrations below 50 nmol/ml. One major limitation associated with this technique is its restricted ability to distinguish isomers of the same formula weight, if used without an on-line or off-line chromatographic means of separation. Isomeric acylcarnitines can be differentiated by high-energy collision-induced-dissociation tandem mass spectrometry (CID-MS-MS) of the quasi-molecular ions produced by FAB ionization on a four-sector (BE-EB) instrument (11); however, MS-MS analysis using linked scanning at constant B/E ratio on a two-sector instrument or employing low-energy CID is reported to be unable to reveal the structural details of the acyl substituents (10,ll). A quantitative assay has also been reported (14) for determining concentrations of free carnitine and acylcarnitines in physiological fluids. Nevertheless, in view of the severe discrimination in ion intensities of acylcarnitines with different acyl chain lengths (lo), probably due to differences in surface activity, such analyses are only possible when coupled with isotope dilution techniques. Therefore, though FAB-MS or FAB-MS-MS provide unequivocal structural characterization and have been used in a sophisticated method based on continuous-flow FAB-MS (CF-FAB-MS) for handling complex mixtures (X5), the expense and complexity of instrumentation suggest that their potential use will be to provide confirmatory evidence for acylcarnitines determined by other less expensive and simpler procedures. TSP-HPLC-MS of intact acylcarnitines

Me \ 7 Me - N+ Me'> C-10

0

OCOR --

Nu:

l"@N

/\(\I( RCO-0

OMe 0

Acyl N-demethylcarnitine Me ester (ADMCME) SCHEME

2

3

o

represents a further important achievement (12,16,17). It gives reasonable separation as well as distinctive mass spectra for individual acylcarnitines. The sensitivity of this technique at physiological levels of acylcarnitines is still being tested, but, because of its inaccessibility in most clinical laboratories, this approach cannot be considered feasible for routine assays at this time. Recently, an elegant procedure has been reported by Lowes and Rose (18), which involves chemical derivatization whereby the zwitterionic acylcarnitines are cyclized by heating with a nonnucleophilic base, diisopropylethylamine (Hunig’s base) to produce volatile @-acyloxybutyrolactones which are subsequently analyzed by GC-MS. An important advantage is that the resulting derivatives still retain the acyl unit attached to the basic carnitine moiety, unlike the hydrolysis approach (see for example, Ref. (12)) in which the molecular integrity of acyl group with its carnitine parent is lost. By this method, octanoylcarnitine has been successfully detected in the urine of a child suffering from medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Here we report a rapid and sensitive method that has been developed for the analysis of this biologically important family of molecules. In this approach, urinary acylcarnitines are first converted to their n-propyl esters (ACPE) which, after being taken up as corresponding iodides into an organic phase by ion-pair extraction, are then subjected to gas chromatography-chemical ionization mass spectrometry (GC-CI-MS). Nucleophile-assisted thermolytic N-demethylation takes place as an aliquot of the derivative is injected onto the GC column. Consequently, mass spectra of analytes are recorded as their corresponding acyl N-demethylcarnitine propyl esters (ADMCPE) (Scheme 1). In this paper, methodological aspects including sample pretreatment, chemical derivatization, behavior under GC-CI-MS, and application to pediatric urine samples are described. MATERIALS

AND

METHODS

Materials Acylcarnitine hydrochlorides were prepared (see below) or purchased from Sigma Chemical Co., Inc. (St. Louis, MO). Valproylcarnitine was a gift from Dr. L.

100

HUANG

.I

5

1,o

20

ET

AL.

min

TIC

(a)

1oc '

5

80 172 60

40

i 56

20

I 0

. .' 500

FIG. 1.

(a) Total and 12:0 carnitines

ion current derivatized

, 1000

, 1500 Scan

(TIC) trace and mass chromatograms as DMCPE (peaks l-4). Programmed

0 I

5, Ill/Z

of m/z 58 and m/t 172 from a mixture of equal amounts at lO”C/min between 150-300°C; (b) CI mass spectrum

Carter (Medical College of Georgia). DL-Carnitine hydrochloride, ethyl and propyl chloroformates, and dibasic acid anhydrides and chlorides were purchased from Aldrich Chemical Co. (Milwaukee, WI). MCAD deficiency urine samples were obtained from Dr. Eberhard Schmidt-Sommerfeld (Department of Pediatrics, University of Chicago). Synthesis w-Carboxyacylcarnitine hydrochlorides. w-carboxyacylcarnitines-HCl from dibasic acids were prepared by a modification of a procedure described earlier (19). DLCarnitine-HCl (500 mg, 2.54 mmol.), 3-methylglutaric

of 6:0,&O, lO:O, of 12:0 DMCPE.

anhydride (1000 mg, 7.80 mmol), and trichloroacetic acid (1 g) were mixed and warmed to 60°C for 4 h. Then, the cooled reaction mixture was triturated with water (50 pl)-n-PrOH (500 ~1) and diluted with diethyl ether (lo-20 ml) until the solution turned turbid. The mixture was allowed to stand at -1O’C for several hours, and the precipitate formed was recrystallized twice from PrOHdiethyl ether to afford 3-methylglutaryl carnitine-HCl (3-methyl-4-carboxybutyryl-carnitine-HCl) as colorless crystals. Yield: 70%. The product was analyzed by FAB-MS (matrix: m-nitrobenzyl alcohol). Adipylcarnitine-HCl (5-carboxyvaleryl-carnitine-HCl) was similarly prepared from adipoyl chloride in 40% yields. Derivatization Esterification of acylcarnitines was performed by a modification of the procedure reported by Husek (20).

r

Me2N-ofoPr -3 .e,N-(-;;6ol

[M+H]

RCO-0

+OH

RCO-0 min

2

I

i

a

10

12

Me2N+j+~,,

~e2&../,(opr -= m/z 172

6

i------G-l

-RCOPH

-RCO,H

4

2

*OH a

/

d

b

-CO 1

Me2N+=CH2

Me,N

Inks4

-a

ml,? 5s

t

I

M%NyoPr + .CH,CO-0 ndz 231

FIG. 2. pyl esters

Fragmentation (ADMCPE)

+OH

200

Me2NvoPr

CH+HCO-0

+ OH

FIG. 3.

IT& 244 (258, 272.. .) pathways of acyl N-demethylcarnitine under CI-MS.

pro-

400

600

600 Scan

Total ion current (TIC) trace and mass chromatograms of m/z 58 and 172 in a mixture of equal amounts of isomeric acylcarnitines. Peak 1, pivalyl (trimethylacetyl); peak 2, iso-5:O; peak 3, 5:O; peak 4, valproyl (a-propylvaleryl); and peak 5, 8:O DMCPE. Programmed at 2O“C/min from 100 to 300°C.

CHROMATOGRAPHIC

ANALYSIS

OF

ACYLCARNITINE

101

DERIVATIVES

68

(a:

(b)

5.Carbopropoxyvaleryl DMCPE

3-Methyl-4-carbopropoxybutyryl DMCPE

200

360

[MI"]

I

'8

0 300

FIG. 4. CI mass butyryl DMCPEs.

spectra

of adipyl-

and 3-methylglutarylcarnitine

derivatives:

To 25 pg of acylcarnitine-HCl in 100 ~1 of water-nPrOH-pyridine (25O:lOO:lO v/v/v) was added 5 ~1 of npropyl chloroformate (PCF). The mixture was briefly vortexed (with evolution of CO, in some cases) and allowed to stand at room temperature for 5 min. Then 5 ~1 of 30% aqueous solution of potassium iodide was added. The resulting clear solution was extracted (vortex) with 2 x 100 ~1 of chloroform to remove the iodide salt of betaine ester from the aqueous phase. The combined organic layers were evaporated to dryness under nitrogen. The residue (ACPE iodides) was reconstituted in 25 ~1 of ethyl acetate and an aliquot was analyzed by GC or GC-MS (1-2 ~1 for each run). The derivatives thus obtained are quite stable and can be stored in a refrigerator for 8-12 weeks without appreciable decomposition. GC-CI-MS A JEOL AX-505H double focusing mass spectrometer coupled with a Hewlett-Packard 58905 gas chromatograph was used in this study. GC separations employed DB-1 (30-m length X 0.25 mm i.d. fused silica capillary columns with a 0.25-pm film coating of methyl silicone) available from J. & W. Scientific (Ranch0 Cordova, CA). GC conditions were as follows: injection port temperature 26O”C, initial column temperature 1OO’C (or 150°C for higher homologs) for 2 min, program rate 5 or lO”C/min (or 20”C/min for higher homologs) to a final temperature of 300°C. Direct (splitless) injection

MezN m

Opr Pro&

Pro&-CH,;CH2~CH2yX2CO-0 I

,

i

5 :

i j

‘m/z

: -: m/z

-

(a) 5-carbopropoxylvaleryl

and (b) 3-methyl-4-carbopropoxy-

was used. Helium gas flow was approximately 1 ml/min. MS conditions were as follows: interface temperature 28O”C, ion source temperature was not critical and ranged from lOO-2OO”C, the reagent gas was methane at a pressure to maintain the source chamber at ca. 5 X 10e5 Torr, electron energy was 200 eV. The scan rate of the mass spectrometer was 50-500 Da in 1 s/scan. FAB-MS FAB mass spectra were obtained with a JEOL HX110 double focusing mass spectrometer (EB configuration) operating in the positive ion mode. Ions were generated by bombarding the sample with a beam of Xe atoms (6 keV) produced by a JEOL FAB gun. The accelerating voltage was 10 kV, the resolution was set at 1000, and the scan rate was set at 2 min from m/z o-1500. Data were acquired in a single scan. Pretreatment

of Urine Samples

Pretreatment of urine samples was performed by a procedure reported by Millington et al. (13): 500 ~1 of crude urine, after being spiked with 10 pg of valproylcarnitine-HCl, was applied to a 0.7 x 5.0-cm column of anion-exchange resin (AG l-X8, 200-400 mesh, Clform, Bio-Rad Laboratories, Richmond, CA) and eluted with 4 ml of deionized/distilled water. The eluant was passed through a reversed-phase column (C-18,500 mg, Baxter Healthcare Corp., Muskegon, MI; prewashed

oer

[M+Hl+

-

/ (CnH2n)

\

CO,PK Ce O+

-42

-

(CnH2n)

CO,H

/ \

cs

o+

231

m/z 244 n=4,

258

SCHEME

4

m/z

171

SCHEME

n=4,

5

m/z

129

102

HUANG

10

11

12

13

14

15

316

ET

AL. 10

5

m’” TIC

6:0

min

15

6 13

172 5:o

L 274

1,.

2000

,

2200

2400

I

2600

2600

c............, 200

3000

derivatized grammed

SIM of m/z as DMCPE at lO”C/min

172, 274, and 316 for 5:0 and 8:0 carnitines at 1 nglpl EtOAc (sample size: 1 ~1). Profrom 100 to 300°C.

with methanol and deionized/distilled water), which was then eluted with 4 ml of methanol. The eluant was evaporated to dryness under a stream of nitrogen, leaving a residue which was ready for further derivatization. RESULTS

AND

DISCUSSION

Derivatization While studying the reaction of acylcarnitines with a number of nucleophiles (Nu:), we found (Z. H. Huang, unpublished data, 1990) another type of head-to-tail interaction different from that described by Lowes and Rose (18) which may occur as indicated in Scheme 2. In addition, this N-demethylation process was greatly facilitated when a preformed acylcarnitine ester instead of a free acylcarnitine was used in the reaction. Based on these observations, we employed a two-step derivatization procedure involving a preliminary esterification

Matrix: m-Nitrobenzyl alcohol (‘matrix peak)

ml

FIG. 6. derivatized standard:

ml

.m

FAB-MS (matrix: m-nitrobenzyl alcohol) as Pr esters in MCAD deficiency urine valproylcarnitine, 10 pg/500 ~1 urine).

4cm

ml Ill,.?

of acylcarnitines sample (internal

600

600

.

.

.

..I

1000 SCEVl

SCW

FIG. 5.

. 400

FIG. 7.

TIC and mass chromatogram of m/z 172 for MCAD deficiency urine sample (internal standard: valproyl carnitine, 10 ng/500 ~1 urine). Programmed at 5”C/min from 150 to 300°C. Components other than acylcarnitines were identified as (peak no., [M + H], structure): 1,202, valerylglycine Pr ester; 2,313,10:1 FAPE (fatty acid Pr ester); 4,276,0-propyloxycarbonyl carnitine; 5,222, hippuric acid Pr ester; 7,241, 12:l FAPE; 11,278, unidentified; 14,299,16:0 FAPE; 16, 313, di-Pr dodecenedioate; 17,360, unidentified; 18,325,1&l FAPE.

followed by secondary dequaternization to convert the zwitterionic acylcarnitines to their volatile N-demethylated esters (i.e., norcarnitine esters). Initially, a direct esterification is carried out by using HCl in PrOH (12), or, preferably, by using the procedure of Husek (20), in which the sample is esterified by carboxyalkylation with PCF in aqueous propyl alcohol in the presence of pyridine. The reaction proceeds rapidly at room temperature and is completed within 5 min. The product, after being spiked with a nucleophile dopant and extracted into organic solvent, is ready for further treatment. Propyl chloroformate is preferred over other lower homologs because it provides, while retaining enough volatility, more lipophilic propyl esters that are more favorably distributed in the organic phase during extraction and hence affords a more complete conversion and recovery, especially for short-chain acylcarnitines. In addition, as will be discussed later, acyl N-demethylcarnitine propyl esters (ADMCPE) exhibit more abundant diagnostic mass spectral fragment peaks to facilitate definitive identification of the individual acylcarnitines. It is well known in solution chemistry that N-dealkylation of quaternary compounds can be accomplished in a number of ways, including thermal degradation (21), dequaternization induced by nucleophiles (e.g., mercaptides (22,23), thiourea, triphenylphosphine, or sodium azide (24)) and organic bases (e.g., 1,4diazabicyclo[2.2.2]octane (Dabco) (25)). Carnitine itself was converted to the corresponding N-demethyl analog by prolonged heating with PhSNa (26) or PhSH + Me,NCH,CH,OH (27). However, these variations are not suitable for reaction on a microscale. In an effort to

CHROMATOGRAPHIC

ANALYSIS

k+H]

OF

ACYLCARNITINE

103

DERIVATIVES

Peak

(a

6Tlh G---q

13 8:O DMCPE

311

40. 172

20.

&

‘;,,

200

58

212 0.

,

;,

,I

,.;

,I

:

L,

255 231 ;,

1,

300 ,.,,,

I

,I,,

343 .I

I,

T

200

250

300

350

400

50

150

100

200

250

300

50

FIG.

B.

CI mass

spectra

of selected

100

peaks:

15 1O:l

(a) peak

12: 8:l

1

Acylcarnitines Found in MCAD Deficiency Urine by GC-CI (methane)-MS of DMCPE Derivatives Peak

no.

3 6 8 9 10 12 13 15 a Octanoyl

[M+H],

m/z 274 288 316 302 314 314 316 342

N-demethylcarnitine

Structure 5:0 6:0 Valproyl 8:0 8:l 8:l 8:0 1O:l

DMCPE DMCPE DMCPE DMCEE” DMCPE DMCPE DMCPE DMCPE

Et ester.

DMCPE

342

[M+H]

(Cl

150

evaluate the possibility for promoting a clean conversion to a volatile derivative, various reagents including ammonium thiocyanate, potassium fluoride, sodium thiophenoxide, dithiothreitol, N- methylimidazole, and N,N,N’,N’-tetramethylguanidine were tested in our laboratory, and potassium iodide was found to be the most efficient. Importantly, dequaternization is brought about by an in situ reaction occurring on the GC column or in the injection port under conditions specified under Materials and Methods. Similar on-column demethyl-

TABLE

400 IT/Z

IT/Z

Peak

350

Peak

area, 5.7 12.4 100.0 4.0 5.3 t1.0

67.1 4.0

%

isomer;

(b) peak

13: 8:O; and (c) peak

15: 1O:l DMCPE.

ation has been reported in the GC analysis of neostigmine (28) and pancuronium-type neuroblockers (29), where in the latter case the free hydroxylic groups were protected prior to analysis by using t-butyldimethylsilylation (heating for up to 80 h). Attempts to perform the reaction by heating acylcarnitine esters with KI in acetonitrile in a microvial resulted in a mixture of degradation products with significant amounts of free fatty acids or methyl esters. The choice of the reagent is critical to avoid adverse side reactions. For example, acetylcholine was reported to undergo N-demethylation during pyrolysis GC (30); however, in our hands, a retro-Michael type reaction may dominate if the thermal process with AC esters is conducted in the absence of iodide ion, thus leading to the formation of N,N-dimethylaminocrotonic acid and fatty acid esters (Scheme 3). Behavior

under GC-U-MS

Figure 1. shows the reconstructed mass chromatograms obtained from the GC-MS analysis of a mixture of homologous acylcarnitines and the mass spectrum of the laurylcarnitine derivative (12:0 DMCPE). The CI mass spectra of all acylcarnitine derivatives are characterized by abundant [M + l]+ ions, which are

104

HUANG

often accompanied by reagent gas adduct ions [M + 29]+ and [M + 41]+. The [MH-60]+ ion is formed due to loss of PrOH. Two important fragment peaks in the spectra are represented by m/z 58, [Me,N+ = CH,], a fragment derived from the basic terminus, and m/z 172 [often accompanied by m/z 171 and 200 (=171 + 29)], the “aminocrotonate” ion arising from the loss of RCOOH from [M + H]+. Another series of characteristic fragment peaks for straight-chain acylated carnitine derivatives are m/z 231 and 244, 258, 272, etc. A plausible explanation for the formation of these ions is McLafferty rearrangement together with simple cleavages of the acyl group at contiguous positions along the chain. Other common fragments prominent in these mass spectra occur at m/z 84,112,130, and 190. A small, nevertheless easily recognizable acylium ion peak is also displayed in the low mass end of the spectrum for fatty acyl moieties containing more than six carbon atoms, which is useful for indicating the size of the acyl group. All these ions may be accounted for by the fragmentation pathways presented in Fig. 2. Acyl N-demethylcarnitine propyl ester derivatives exhibit very good GC properties, allowing the separation of isomeric acylcarnitines. Figure 3 shows the chromatographic separation of a mixture consisting of three isomerit C,- and two isomeric C,-acylcarnitine derivatives. In all cases, distinctive mass spectra were recorded that permit the chain feature of individual isomers to be recognized. Thus, Valery1 (5:O) carnitine shows m/z 231, 244, 258 (loss of a 3-, 2-, and l-carbon unit, respectively); the isovaleryl (iso-5:0) analog shows m/z 231 and 258 (loss of a 3- and l-carbon unit); while the pivalyl (trimethylacetyl) derivative shows only m/z 258 (loss of l-carbon unit). Subtle, but important characteristics are also seen in the C8 isomeric pair. The 8:0 carnitine derivative exhibits a McLafferty rearrangement peak at mlz 231; while the corresponding rearrangement product occurs at m/z 273 (42 mass shift due to the presence of a propyl substituent branched at a-carbon) in its counterpart, valproyl (a-propylvaleryl) DMCPE. Acylcarnitines derived from dibasic acids behave in a similar manner, although they contain a second propyl ester function on the terminal acyl group carboxyl. Glutaryl and adipyl-carnitine derivatives show typical fragments for straight-chain acyl esters at m/z 231 (McLafferty rearrangement), 244, and 258 (weak) (Fig. 4a). However, this ion series is interrupted when branching occurs in the chain. Consequently, the consecutive abundant peaks at m/z 231 and 258 are a characteristic pattern in the spectrum of 3-methylglutarylcarnitine derivative, i.e., 3-methyl-4-carbopropoxybutyryl N-demethylcarnitine propyl ester (Fig. 4b) (Scheme 4). Moreover, the size of the dibasic acyl group is indicated by acylium ion pairs separated from each other by 42 Da (Scheme 5).

ET

AL.

Analysis

of Clinical

Samples

The present method has proved to be successful for the quantitative analysis of nanogram amounts of valeryl- and octanoyl-carnitines, providing a linear relationship between concentration (weight) and peak area. For example, in the range of lo-850 ng/pl, the correlation coefficients were 0.996 and 0.998, respectively (5 points). Lower detection limits were obtained by selected ion monitoring MS (three channels) for a standard solution of Valery1 and octanoyl carnitine derivatives, allowing 1 ng (1 ng in 1~1 of EtOAc) to be detected without difficulty (Fig. 5). In this study no attempt was made to determine the ultimate detection limits for these derivatives. The present results correspond to 3 nmol/ml urine, a value well below the physiologically normal level (>50 ng/mg creatine), and show that the present method is much more sensitive than most FABMS assays (5,10,14) which report detection limits in the order of ca. 50 nmol/ml urine. To circumvent the possible interference of other matrix constituents of urine, we found that removal of anionic species on an AGl-X8 anion exchange column, followed by sample enrichment using a reversed-phase C-18 minicolumn, as described by Millington et al. (13), provided a suitably purified acylcarnitine fraction. A comparison of the GC peak area ratios produced by a sample of 8:0 carnitine (25 pg) in normal urine, which had been spiked with an equal quantity of valproylcarnitine (internal standard), with that from an identical mixed sample, without being treated prior to derivatization, suggested a 57% recovery efficiency by the described purification procedure. A urine sample (500 ~1) taken during a fasting period from an infant suffering from MCAD deficiency, after being purified, spiked, and derivatized, was subjected to analysis. Fig. 6 shows the FAB spectrum in which four peaks representing acylcarnitine species can be detected; i.e., 5:0 ([M + H]+ = 288), 6:0 ([M + HI+ = 302]), 81 ([M + H]+ = 328), and8:O (octanoylandvalproyl, [M + H]+ = 330). When examined by GC-CI-MS, the sample exhibited a chromatographic profile (Fig. 7) in which seven acylcarnitines could be detected and identified. The results are listed in Table 1. The utility of this method lies in the ease with which the acyl N-demethylcarnitine propyl ester derivatives can be chromatographically separated and in their characteristic mass spectra that are indicative of the structure of individual acylcarnitines (Figs. 8a-c). Even for components that are closely eluting such as the 8:1/ 80 carnitine pair (Figs. 8a and 8b), their spectra enable unambiguous identification. Further application of this approach will be reported in a separate study. In consideration of the fact that the majority of current analytical efforts have been focused on the use

CHROMATOGRAPHIC

ANALYSIS

of FAB-MS or FAB-MS-MS for the analysis of acylcarnitines, our approach based on a novel derivatization strategy and its application to pediatric urine samples is particularly encouraging. The present method, owing to the extreme simplicity and unambiguous characterization it can achieve, may constitute a potentially useful tool in the study of fat metabolism. It should also be possible to integrate this approach into a routine metabolic profiling procedure for use in clinical practice. To summarize, the advantages include: (i) Simple operation: The two-step derivatization can be carried out with great ease and is applicable to carnitine esters derived from mono- as well as dibasic acids. (ii) Good GC separation and distinctive mass spectra of the derivatives permit qualitative and quantitative analysis of complex mixtures. (iii) High sensitivity: The low nanogram detection limits make analysis possible at physiological levels. (iv) High overall efficiency of analysis: Generally, the analysis of a crude urine sample can be completed within 1 h-15 min for urine pretreatment, 15 min for derivatization, and 15-30 min for GC-MS analysis. There are still some problems to be resolved. The first is the partial thermal degradation of acylcarnitine derivatives into fatty acid esters which occurs during analysis of higher acylcarnitines (with the acyl group > C,,). Such an adverse reaction can possibly be minimized by the use of properly chosen esters that are more volatile and more thermostable under the higher temperatures required for the chromatographic separation of these compounds. Secondly, it remains to be demonstrated that mass spectrometric analysis of intact N-demethylcarnitine propyl ester derivatives will allow other features of the acyl substituents (type and position of unsaturation and/or substitution) to be characterized. Further study of this question with the help of CID and MS-MS is currently underway in this laboratory.

OF

ACYLCARNITINE

2. Roe, C. R., Millington, D. S., Maltby, D. A., and Kinnebrew, P. (1986) J. Pediutr. 108,13-18. 3. Marzo, A., Cardace, G., Monti, N., Muck, S., and Martelli, E. A. (1990)

The authors gratefully acknowledge stimulating discussions with Professor J. T. Watson. Sincere thanks are due to Dr. P. HuSek (Institute of Endocrinology, Academy of Sciences of Czechoslovakia, Prague) for providing information on chloroformate esterification procedure. This work was supported, in part, by NIH Grants DK18427 (LLB) and RR00480 (J. T. Watson). The latter provided support for use of instruments at MSU-NIH Mass Spectrometry Facility. Thanks are also expressed to Dr. Eberhard Schmidt-Sommerfeld (Department of Pediatrics, University of Chicago) for useful discussion and provision of clinical samples and to Dr. L. Carter (Medical College of Georgia) for the gift of valproylcarnitine.

M. (1987)

527,

247-258.

S., and Rose, M. E. (1989)

6. Millington,

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