The quantitation of plasma phytanic acid by mass fragmentography

319

Clinica Chimica Acta, 72 (1976) 319-325 0 Elsevier/North-Holland Biomedical Press, Amsterdam -Printed

in The Netherlands

CCA 8044

THE GUANTITATION FRAGMENTOGRAPHY

OF PLASMA PHYTANIC

ACID BY MASS

G. PHILLIPOU a and A. POULOS b a Endocrine Laboratories, Dept. of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, Woodville, South Australia 5011, and b Dept. of Chemical Pathology, Adelaide Children’s Hospital, Nth. Adelaide, South Australia 5006 (Australia) (Received May 6,1976)

Summary Plasma phytanic acid has been quantitated by mass fragmentography after its successive reduction and derivatization to the corresponding t-butyldimethylsilyl ether. The latter was estimated by selective ion monitoring of its characteristic (M-57) ion (M/z 355). The technique has the advantage of being more rapid, specific and sensitive than existing methods, permitting the determination of phytanic acid at levels >5 E.cg/ml plasma.

Introduction The branchedchain acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is normally only a trace component of human tissue [1,2]. In Refsum’s disease, however, its concentration increases by several orders of magnitude; levels in excess of 500 pg/ml plasma not being uncommon [ 1,3]. Plasma phytanic acid has previously been assayed by gas chromatography (g-c.) as its methyl ester [4]. Errors may occur in the g.c. method however, due to interfering fatty acids which may be unresolved from the methyl phytanate under the same analytical conditions [ 51. As the quantitation of plasma phytanic acid is important both in the diagnosis and assessment of the subsequent dietary treatment of Refsum’s disease [4], we decided to devise a more specific and sensitive assay based on mass fragmentography (m.f.). The advantages of t-butyldimethylsilyl (BDMS) fatty esters in m.f. have been recently reported [6]. However, in the present work problems were encountered in efficiently hydrolysing the plasma lipids to their respective free fatty acids. This correlates with the report that the esterification of phytanic acid is retarded due to steric hindrance exhibited by the 3-methyl group towards nucleophilic attack at the carbonyl carbon atom [ 71.

320

As a consequence, the plasma lipids were reduced to their respective longchain alcohols with lithium aluminium hydride [8]. The only exceptions to this mode of reduction are the sphingolipids, whose amide linkage is converted to the corresponding secondary amine [9]. Long-chain alcohols, such as dihydrophytol produce mass spectra with a virtual absence of characteristic high mass ions [lo], which, therefore, render them unsuitable for m.f. To overcome this problem the alcohols were silylated to form their respective t-BDMS ethers. This class of derivatives has recently been applied to the analysis of steroids by m.f. and g.c.-mass spectrometry (g.c.m.s) [ 11-131. Materials

and methods

Phytanic acid and 14% boron trifluoride in methanol (w/v) were from Applied Science Laboratories. Fatty acid methyl ester standards tadecanoic acid were purchased from Sigma Chemical Co. Long-chain were obtained from Nu-Chek Prep. t-Butyldimethylsilyl chloride was ed from Willow Brook Lab. and was redistilled prior to use. All solvents used were analytical reagent grade and were redistilled use.

obtained and hepalcohols purchasprior to

Plasma Blood was collected from fasted and unfasted healthy adult individuals or from patients clinically diagnosed as having Refsum’s disease. The samples were placed into heparinised containers and centrifuged (500 X g) at room temperature for 10 min. The plasma was then removed and stored at -10°C until it was required for extraction.

Lipid extraction 0.5-1.0 ml of plasma, to which was added an internal

standard (220-440 I-18 heptadecanoic acid) and hydrochloric acid (1 mol/l, 50 ~1) was extracted as described by Folch et al. [ 141. The residue so obtained was dissolved in chloroform/methanol (2 ml, 2 : 1, v/v) containing 4% water, treated as described by Poulos et al. [15], and then redissolved in chloroform/methanol (2 ml, 2 : 1, v/v). The efficiency of the extraction procedure, determined (g.c.) by reextracting the residue and estimating the remaining fatty acid content after transesterification, was >95%.

Preparation of methyl esters An aliquot of the total lipid extract

(0.5-1.0 ml) was transesterified with 14% boron trifluoride in methanol (3 ml) at 100°C for 1.5 h; benzene and methanol (3 ml) were then added and reaction was continued for a further 0.5 h [16]. After the addition of water (3 ml), the reaction mixture was extracted with n-hexane (2 X 3 ml). The total extract was then evaporated to dryness using a rotary evaporator (40°C) and the resulting residue redissolved in chloroform/methanol (1.0 ml, 2 : 1, v/v). Further purification of the methyl esters was achieved by preparative thin-layer chromatography (t.1.c.) as previously described [15]. The recovery of the internal standard, assessed by the addition of an external standard (methyl nonadecanoate) immediately prior to gc., was 80-90%.

321

Preparation of long-chain alcohols An aliquot of the total lipid extract (0.5-1.0 ml) was treated with lithium aluminium hydride as described by Thompson [S] . The reaction mixture was then cooled to 0°C and acidified with hydrochloric acid (4 ml, 4 mol/l) to destroy excess hydride (caution). The mixture was then extracted with ether (2 X 3 ml), and the ethereal extract evaporated to dryness under nitrogen. The residue was dissolved in chloroform/methanol (1 ml, 2 : 1, v/v). Aliquots of the extract (50-100 ~1) were examined by t.1.c. in hexane/diethyl ether/ acetic acid (60 : 40 : l,v/v). Preparation of t-BDMS ethers 0.1-0.4 ml aliquots of the total lipid alcohol solutions were evaporated to dryness and silylated as originally described by Corey and Venkateswarlu [ 171. The final products were dissolved in diethyl ether (1 ml), containing a trace of anhydrous magnesium sulphate. T.1.c. analysis of the reaction mixture confirmed the absence of starting materials. Gas chromatography Aliquots of the methyl ester solution (0.1-0.5 ml) were evaporated to dryness and redissolved in n-hexane (25 ~1). 5 ~1 of this solution was chromatographed in a Varian 1440 GC fitted with a stainless steel column (1.52 m, 6 mm i.d.) containing 15% ethylene glycol succinate on 100/120 mesh Chromosorb W (column temp, 180°C; flow rate, 30 ml * min-‘). The linearity of the detector response between methyl phytanate and methyl heptadecanoate (17 : 0) was confirmed by adding varying amounts of methyl phytanate to fasted plasma (0.5 ml) containing a constant amount of internal standard (220 pg). The plasma samples were then extracted, esterified and purified as described earlier and then examined by g.c. Under the analytical conditions the methyl phytanate/l7 : 0 peak area ratios are directly proportional to added methyl phytanate. Phytanate concentration was calculated from the formula : phytanic

acid @g/ml) =

a = represents

detector

(phytanate peak area ‘) x ~~~224 (17 : 0 peak area) 0.89 a X vb response

of methyl

phytanate

to 17 : 0

b = volume of original plasma sample (ml) c = peak areas determined

by trangulation.

Mass fragmentography GCMS was carried out with an AEI MS-30, single beam mass spectrometer equipped with a multi-peak monitor (6 channels) and interfaced to a Pye 104 GC using a single-stage membrane separator. The G.C. was fitted with a glass column (1.0 m, 2 mm i.d.) containing 1% OV-225 on 100/120 mesh support (column temp., 180°C; helium flow, 35 ml * min-‘). The mass spectrometer was operated under the following conditions: resolution, 1000; ionizing current, 100 /IA; electron energy, 24 eV; ion source temp., 200°C; and molecular separator temp., 240°C. M.f. was performed by selective ion monitoring of M/z 355.3396 and

322

314.2960, which are characteristic ions for the t-BDMS ethers of dihydrophytol and heptadecanol respectively; the dwell time on each mass was 250 ms. The linearity of response between the two ions was established by adding varying amounts of methyl phytanate to fasted plasma (0.5 ml) containing methyl heptadecanoate (220 pg) followed by successive extraction, reduction and silylation as described earlier. Results and discussion The hydrolytic method described is chemically mild and rapid, taking 0.5 h as opposed to the 2 h required for the transesterification procedure. Furthermore, lithium aluminium hydride reacts with all of the major plasma lipids, except cholesterol (Fig. 1). The sphingolipids, which are quantitatively minor plasma components [ 181, do not form fatty alcohols. No steric effects are noted for the reduction since both the straight and branched-chain acids form the respective fatty alcohols in quantitative yield. The alcohols are readily converted to the corresponding t-BDMS ethers, which have characteristic electron impact mass spectra (Fig. 2). An examination of the total untreated lipid extract failed to reveal any significant quantities of long-chain alcohols, thus confirm-

-

-----T--T

Fig.

1.

water respond

Reduction = 60

:

35

of plasma

: 8 (v/v)

to unreduced

(b)

lipid

lipids

as described

n-hexaneldiethyl extract.

reduced

in the text; ether/acetic lipids

and

I

------__-_l

solvent acid

reference

systems = 70

:

marker

30

used

:

(a) chloroform/methanol/

1 (v/v):

respectivelv.

zones

1,

2.

and

3 COT-

323

3

157

83 19’

,111

x10

I

LlLLJL I’

;

I

,’

,’

i’m

L._

100

355

‘3co

350

M/Z lb1

Fig. 2.

Electron

impact

mass

spectrum

of the

t-BDMS

ethers

of (a) heptadecanol

(b) dihydrophytol.

PEAK HEIGHT RATIO

METHYL

Fig. 3. Linearity sample.

of response

with increasing

amounts

PHYTANATE

of methyl

ADDED

phytanate

&IJ)

added

to the original

plasma

324

TABLE

I

DETERMLNATION

OF

Patient

m.f.

A.

PHYTANIC

ACID

1

235

227

198

212

3

214

234

4

217

250

2050

C a

PLASMA

(pg/ml)

2120

264

Control

OF

8.C.

2

B

CONTENT

<

247 5b

a Determined for 5 normal fasted patients. b Lmut of detection due to interfering compounds.

ing the fact that the quantitated lipids were derived from esterified and nonesterified fatty acids. Fig. 3 shows the standard curve obtained for phytanic and heptadecanoic acids in plasma. The response is quite linear in the concentration range used (40-1000 pg/ml). Although relatively large volumes of plasma (0.5-1.0 ml) were extracted the m.f. procedure can equally be applied to the analysis of very small plasma samples (
4

Jib---17:o

,J

Fig. (*

4.

Selective

endogenous

(Xi,‘,‘1

ij

1

ion

detection

n-eicosanoic

of acid)

phytanic (b)

patient

and

heptadecanoic

clinicalIy

diagnosed

(17

)

: 0)

acids

as suffering

in (a) Refsum’s

normal

fasted

disease.

subject

325

The data summarised in Table I demonstrate the excellent agreement between the g.c. and m.f. procedures, and substantiates the fact that fatty acid products are released in comparable yields by both methods. The relatively high minimum detectable limit (5 pg/ml) is mainly a consequence of interfering compounds in the crude extracts, coupled with the low intensity of the phytanate (M-57) ion compared to the corresponding ion for heptadecanol t-BDMS ether. For this reason the (M-56) ion (M/z 314) of the internal standard was monitored so that the response correction between the two compounds would not be too large (Fig. 4). Although a t.1.c. step is not necessary for the g.c. assay of phytanic acid, it does serve the useful purpose of removing trace plasma components that may interfere with the peak area measurement of either the internal standard or phytanate. This degree of purification is unnecessary for the m.f. procedure, due to the increased specificity which requires both retention time and a characteristic mass ion to confirm identity. The described methodology can equally be applied to the multicomponent analysis of plasma fatty acids [19]. However, the minimum detectable limit for each acid will be directly dependent on the intensity of the (M-57) ion in the mass spectrum of its corresponding t-BDMS ether. Preliminary observations suggest that picogram amounts (injected) may be readily detected for saturated fatty alcohol t-BDMS ethers. This ultimate sensitivity may have relevance to both the quantitation of trace lipid components and to the analysis of small plasma samples. References 1

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