BIOMEDICAL CHROMATOGRAPHY, VOL. 5,161-164 (1991)

Medium- and Long-chain 3-Hydroxymonocarboxylic acids: Analysis by Gas Chromatography Combined with Mass Spectrometry L. h r l a n d , D. Ketting, L. Bruinvis and M. Duran University Children's Hospital, "Het Wilhelmina Kinderziekenhuis", Nieuwe Gracht 137, NL-3512 LK Utrecht, T h e Netherlands

Medium- and long-chain 3-hydroxymonocarboxylicacids represent intermediates in the poxidation of fatty acids: they accumulate in the plasma of patients with an inherited deficiency of long-chain 3-hydroxyacylcoenzyme A dehydrogenase. 3-Hydroxy acids with chain lengths varying from 6 to 16 were synthesized by a Reformatzky reaction. Capillary gas chromatograpby of the pertrimethylsilyl derivatives was performed on a CP-SP 19 CB column, coupled to a quadrupole mass spectrometer in the electron impact mode. Calculation of the retention indices showed that the separation of the 3-hydroxy acids from the homologous straight-chainfatty acids may be troublesome, stressing the need for mass spectrometric identification.

INTRODUCTION

The primary diagnosis of inherited defects of fatty acid P-oxidation relies on the analysis of unusual amounts or identities of fatty acids (and their metabolites) in plasma and urine. In this respect deficiencies of carnitine palmitoyltransferase or long-chain acyl-coenzyme A (CoA) dehydrogenase have been associated with the accumulation of long-chain fatty acids in plasma after prolonged fasting (Demaugre et al., 1988; Roe and Coates, 1989). Recently we described a new inborn error of the fatty acid metabolism, viz., long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Duran et al., 1990; Wanders et af., 1989). As in other defects in this area, patients suffer from life-threatening episodes of hypoketotic hypoglycemia. These can be prevented by treatment with a diet enriched with medium-chain triglycerides and carbohydrates. Abnormal amounts of C,-C,, dicarboxylic acids, 3-hydroxydicarboxylic acids, and medium- and long-chain 3-hydroxymonocarboxylic acids were observed in plasma. The major abnormal metabolites in urine were 3-hydroxyadipic acid, adipic acid, suberic acid and its unsaturated analogue. The structure determination of the 3-hydroxymonocarboxylic acids was carried out by gas chromatography combined with mass spectrometry (GUMS). Reference 3-hydroxymonocarboxylic acids were synthesized using the Reformatzky reaction (Furniss et al., 1978). This paper deals with the gas chromatographic analysis of 3-hydroxymonocarboxylic acids and their mass spectrometric behaviour. EXPERIMENTAL Chemicals. 3-Hydroxybutyric acid, butanal, and ethyl abromoacetate were obtained from Fluka. Zinc dust, hexanal, octanal and decanal were purchased from Merck. N,N0269-3879/91/040161-04 $05.00 01991 by John Wiley & Sons, Ltd.

Bis(Trimethylsi1yl)triAuoroacetamide was obtained from Sigma, trimethylchlorosilane from Pierce, iodine from Baker, dodecylaldehyde from Janssen Chimica and tetradecylaldehyde from Aldrich.

Sample preparation, gas chromatographic conditions and mass spectrometry. Organic acids in plasma were analysed by capillary gas-liquid chromatography with a Varian 3700 instrument (Palo Alto, CA, USA) equipped with a flame ionization detector and connected with a Varian DS 604 data system. The WCOT fused silica capillary column (25mx0.25mm i d . ) was coated with CPSil 19 CB, film thickness 0.19 pm (Chrompack Nederland BV, Middelburg, The Netherlands). Nitrogen was used as the carrier gas. Heparinized plasma (0.5 mL) was acidified with one drop of 2mollL HCI and subsequently 50 pL of a 1 mg/mL aqueous internal standard solution of 3-phenylbutyric acid was added. After addition of 0.5 mL of a saturated sodium chloride solution, the plasma was extracted twice with 2 mL ethyl acetate by vigorously shaking for 1 min each time. The two layers were separated by centrifugation at 2000 X g for 10 min. The combined organic phases were dried over anhydrous sodium sulphate. The solvent was then removed by rotary evaporation at 35°C under reduced pressure. The residue was trimethylsilylated with 50pL of a mixture of N,N-bis(trimethylsilyl)trifluoroacetamide, pyridine and trimethylchlorosilane (5:1:0.05 v/v) for 30 min at 60 "C. One pL of the derivatized plasma acids was injected onto the capillary column. The initial oven temperature was kept at 75 "C for 5 min and then programmed (5"/min) to 280 "C. This temperature was maintained for 15 min. G U M S of the compounds was carried out with a Ribermag RlO-IOC (Argenteuil, France) quadrupole mass spectrometer in the electron impact mode. The gas chromatographic conditions of the G U M S combination were identical to those used in the GC procedure, with the exception of helium replacing nitrogen as the carrier gas. The ionizing voltage was set at 70 eV. Receioed 28 June 1990 Accepted (revised) 20 September 1990

162

L. DORLAND E T A L .

a-on-ca 3-0W-C4

3-on-ci2

,.

9-0W-ClO

c1a

C1.3

10

10

-c

(mln)

I I

s-on-ci4 I

I

ao

40

Figure 1. Capillary gas-liquid chromatography of synthetic straight even-numbered 3-hydroxymonocarboxylic acids on a 25 m x 0.25 m m wall-coated CP-SiI 19 CB column. The even-numbered C,,-C, n-alkanes were added as reference compounds together with the internal standard 3-phenylbutyric acid (IS).

Synthesis of reference 3-hydroxy acids. 3-Hydroxycarboxylic acids were prepared by reaction of a linear aldehyde (C, to CI4) with ethyl a-bromoacetate in the presence of zinc dust (Reformatsky reaction) (Furniss et al., 1978). 3-Hydroxyhexanoic acid. Carefully dried zinc dust (30 g, 0.46 mol) and a few crystals of iodine were placed in a 500-mL three-necked flask, equipped with a reflux condenser with a drying tube filled with calcium chloride, a magnet stirrer and a dropping funnel. Dry benzene (150 mL) was added to the zinc dust followed by 30 mL of a mixture of 29 g (0.40 mol) butyroaldehyde and 63 g (0.38 mol) ethyl a-bromoacetate in 75mL benzene. The remainder of this mixture was added through the dropping funnel at such a rate that gentle refluxing was maintained. After an additional two hours stirring and refluxing the reaction mixture was cooled and poured out into 200 g crushed ice with 22.5 mL concentrated sulphuric acid. The aqueous layer was removed with a separatory funnel and discarded. The benzene layer was washed with two 75 mL portions of 2 M sulphuric acid, with 75 mL water, with 75 mL of a 1M sodium hydrogen carbonate solution and finally again with 75 mL water. The benzene layer was dried with anhydrous sodium sulphate and subsequently evaporated to dryness using a rotary evaporator. The residue was dissolved in 270 mL alcohol. Then 450 mL 1M sodium hydroxide solution was added. After mixing, 375 mL water was added and stirring was continued for 20 min. The pH was adjusted to pH 13 with 1M sodium hydroxide solution and subsequently the solution was boiled for one minute. After adding decolorizing charcoal, the solution was filtered, acidified to pH 2 and extracted with ethyl acetate. The combined ethyl acetate extracts were concentrated by rotary evaporation and redissolved in a small volume of a 1M sodium hydrogen carbonate solution (pH 8.5). Silver nitrate was then added, whereupon precipitation started. The filtered crystals were recrystallized from hot water. The higher even homologues of 3-hydroxyhexanoic acid were synthesized in a similar

way using C6, C8,Clo, C12and C14aldehydes. Only the C6 and the C8 acids required the formation of a silver salt for their precipitation; the other acids could be precipitated as such.

RESULTS Figure 1 shows the gas chromatogram of a mixture of synthesized 3-hydroxymonocarboxylicacids with chain lengths varying from C, to CI6and n-alkanes with chain lengths from CI2to C%. The analysis was completed in 60 min. Retention times together with Kovats retention indices (Wehrli and Kovats, 1959) are summarized in Table 1. Based on this gas chromatographic separation we investigated the organic acids in the plasma of two siblings with long-chain 3-hydroxyacyl-CoA deficiency. Both patients revealed increased concentrations for all Table 1. Retention times and Kovats retention indices of 3hydroxymonocarboxylic acids on CP-Sil 19 CB. Oven temperature program: 5 min at 75OC, 75280 "C at 5 "Clmin, 15 min at 280 "C. Nitrogen was used as carrier gas at an inlet pressure of 1.OX lo5Pa. The gas flow rate was 1 mLlmin Acid

Retention time (min)

3-Hydroxybutyric 3-Hydroxyhexanoic 3-Hydroxyoctanoic 3-Phenylbutyric (IS) 3-Hydroxydecanoic 3-Hydroxydodecanoic 3-Hydroxytetradecanoic 3-Hydroxyhexadecanoic aRetention index: methane= 100. etc.

Retention indexa

12.85 1225 17.26 1373 21.78 1541 21.90 1546 26.14 1720 30.24 1904 34.08 2092 37.65 2282 ethane=200, propane=300,

G U M S OF 3-HYDROXYMONOCARBOXYLIC ACIDS

3-hydroxymonocarboxylic acids with chain lengths from C6 to CI6(Duran et af.,1990). Analysis of a urine sample from one of the patients failed to reveal the presence of free 3-hydroxymonocarboxylic acids. However, a urine sample hydrolysed with alkali or by the action of 8-glucuronidase did contain 3-hydroxyoctanoic acid. It is concluded that medium-chain 3hydroxyfatty acids are excreted as glucuronides; longchain 3-hydroxyfatty acids do not appear in the urine. The hydroxy acids were identified by mass spectrometry. Their mass spectra appeared to be very characteristic. Mass spectrometric identification is an absolute condition as lauric acid and oleic acid overlap with 3hydroxydecanoic acid and 3-hydroxyhexadecanoic acid, respectively. Figure 2 depicts the mass spectra of the hydroxy acids from C4to CI6.Explanations for the various fragment ions are given in Table 2. All the spectra contained in a relatively abundant ion with mlz 233, which is a characteristic ion present in the mass spectra of the pertrimethylsilyl derivatives of all 3hydroxy acids, including 3-hydroxydicarboxylic acids. This fragment is the result of the cleavage of the C(3)-C(4) bond with retention of the positive charge at C(3). In no case was a molecular ion observed, but all compounds showed a [M- 151 ion. A fragment at M - 57, representing the loss of a methyl group and of ketene, is characteristic of 3-hydroxy acids. Ketene is formed by the following rearrangement reaction:

J-OTMS-, Th4S%H2

H-

1I

-0TMS

TMSO

163 I

n 248

1'17

3-OH-BUTVRIC A C I D

-I

1

I n 276 3-OH-HEXANOIC ACID

n

304

3-OH-OCTANDIC ACID

n 332 3-OH-OECANOIC ACID

+ CH,=C=O mlz 42 3-OH-DOOECANOIC ACID

-

All the compounds showed losses of 105 mass units, being CH3+TMSOH, and of 131 mass units, being CH,COOTMS. The fragment ions at mlz 191 and mlz 189 both stem from rnlz 233 by losing ketene and carbon dioxide, respectively. This comparison of a homologous series of 3-hydroxymonocarboxylic acids with even-numbered chain lengths shows that these acids can easily be recognized from their characteristic mass spectrometric fragmentation pattern.

83

189

I

163

1

SO3

I

-,255 1

H 388 3-nH-TETRADECWiOIC

373

331 283

I

n

416

3-OH-HEXADECANOIC

DISCUSSION The primary recognition of inherited enzyme defects in the catabolism of amino acids or fatty acids relies on the finding of accumulated characteristic metabolites in body fluids such as plasma and urine. In this respect the gas chromatographic analysis of organic acids has brought to light more than 30 inborn errors of metabolism which are accompanied by an overproduction of organic acids. Originally the defects were almost exclusively situated in the catabolic pathways of amino acids. Only recently were inborn errors of the P-oxidation system of fatty acids added to this collection. No more than a few of the theoretically possible inherited defects of the fatty acid oxidation cascade have been discovered so far. These are mainly the acyl-

ACI

100

300

400

ACID

500

Figure 2. Electron impact mass spectra of the pertrirnethylsilylated C4-CIB 3-hydroxymonocarboxylic acids. All spectra were recorded at an ionisation energy of 70 eV. The other analytical conditions are mentioned in the text.

CoA dehydrogenase deficiencies (Roe and Coates, 1989), which only give rise to the accumulation of straight-chain saturated and unsaturated dicarboxylic acids as well as 0-1 oxidation products and conjugates.

L. DORLAND E T A L .

16.1 ~

Table2. Mass spectral data of trimethylsilylated hydroxymonocarboxylic acids Acid

3-Hydroxybutyric 3-Hydroxyhexanoic 3-Hydroxyoctanoic 3-Hydroxydecanoic 3-Hydroxydodecanoic 3-Hydroxytetradecanoic 3-Hydroxyhexadecanoic

3-

M-15

M-57

M-105

M-131

233 261 289 317 345 373 40 1

191 219 247 275 303 33 1 359

143 171 199 227 255 283 311

117 145 173 201 229 257 285

A defective enoyl-CoA hydratase has not yet been found, and a deficiency of long-chain 3-hydroxy-acylCoA dehydrogenase was only recently established (Wanders et al., 1989). This condition has a very severe prognosis, as several patients have died in early childhood (Wanders et af., 1989; Hagenfeldt et a l . , 1990). Especially in the case of infants dying suddenly and unexpectedly, the only material for metabolic investigation may be plasma. It is reassuring that long-chain 3hydroxy-acyl-CoA dehydrogenase deficiency can be diagnosed by analysing plasma 3-hydroxymonocarboxylic acids (Duran et al., 1990). The plasma 3-hydroxy-fatty acid concentrations are not very impressive: Hagenfeldt et al. (1990) reported values from 1-60 pmollL, figures which are one or two orders of magnitude lower than the free fatty acid concentrations, which are sometimes in the millimolar range (Duran et al., 1988). Analysis of the 3-hydroxyfatty acids by selected ion monitoring has been suggested by Hagenfeldt et al. (1990), which seems a good approach.

Mass spectrometric support for the analysis of 3hydroxy-fatty acids is facilitated by the nature of the mass spectra (Fig. 2). It can easily be seen that all the spectra have an important fragment at mlz 233, representing that part of the trimethylsilylated molecule which remains after cleavage of the C(3)-C(4) bond. Although the relative abundance of this peak has some variation, individual calibration curves can be drawn, enabling the quantitative analysis of all medium- and long-chain 3-hydroxy-fatty acids. The fragment at mlz 233 does not occur in the mass spectra of the trimethylsilylated medium-chain and long-chain fatty acids (Chalmers and Lawson, 1982), hence there is no interference from this group of substances. 3-Hydroxymonocarboxylic acids with chain lengths greater than C, do not usually occur in human urine, the only exception being the patient reported by Kelley and Morton (1988). We have evidence that 3-hydroxyoctanoate may form a glucuronide and is excreted into the urine as such (Duran et al., unpublished results). Apparently the long-chain 3-hydroxymonocarboxylic acids are handled by the kidneys in a similar manner to the free fatty acids, i.e., they are virtually completely retained in the plasma. We have shown that the identification of mediumand long-chain 3-hydroxymonocarboxylic acids is of diagnostic value for the study of inborn errors of fatty acid B-oxidation. Theoretically one should be careful in the interpretation of clinical data, as it may be expected that subjects who are fed with medium-chain triglycerides may accumulate some 3-hydroxyoctanoate and 3hydroxydecanoate. Further investigations in this area are in progress.

REFERENCES Chalrners, R. A. and Lawson, A. M. (1982). OrganicAcidsinMan, p. 447. Chapman and Hall, London-New York. Dernaugre, F., Bonnefont, J. P., Mitchell, G., Nguyen-Hoang, N., Pelet. A., Rimoldi, M.. Donato, S. di and Saudubray, J. M. (1988). Pediatr. Res. 24, 308. Duran, M., Bruinvis, L., Ketting, D., de Klerk, J. B. C. and Wadrnan, S. K. (1988). Clin. Chem. 34, 548. Duran, M., Wanders, R. J. A., De Jager, J. P., Dorland, L., Bruinvis, L., Ketting, D.,IJlst, L. and Van Sprang, F. J. (1991). Eur. J. Pediatr., 150. 190. Furniss, B. S., Hannaford, A. J., Rogers, V., Smith, P. W. G. and Tatchell, A. R. (eds) (1978). Vogel's Textbook of Practical

Organic Chemistry, 4th ed., p. 533. Longrnan, London. Hagenfeldt, L., von Dobeln, U., Holrne, E., Alm, J., Brandberg, G., Enocksson, E. and Lindeberg, L. (1990). J. Pediatr. 116, 387. Kelley, R. I. and Morton, D. H. (1988). Clin. Chim. Acta 175, 19. Roe, C. R. and Coates, P. M. 11989). In The Metabolic Basis of lnherited Disease, ed. by Scriver, C. P., Beaudet, A. L., Sly, W. S. and Valle, D., p. 889. McGraw-Hill, New York. Wanders, R. J. A., Duran, M., IJlst, L., De Jager, J. P., Van Gennip, A. H.. Jakobs, C., Dorland, L. and Van Sprang, F. J. (1989). Lancet i, 52. Wehrli, A., and Kovats, E. (1959). Helv. Chim. Acra 42, 2709.