Quantitative, Multicomponent Analysis of Fatty Acids from Cholesteryl Esters by Chemical Ionization Reconstructed Mass Chromatography F. PETTY, Charles B. Stout Laboratory for Neuroscience, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163, J.B. RAGLAND, L.B. KUlKEN, S.M. SABESlN, Department of Medicine, Division of Gastroenterology, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163, and J.D. WANDER, Charles B. Stout Laboratory for Neuroscience, and Department of Biochemistry, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 ABSTRACT

m e t h y l esters ( F A M E s ) , has b e e n used for q u a n t i t a t i v e analysis for a n u m b e r o f years (3-5), sensitivity a n d a c c u r a c y of GC p r o c e d u r e s m a y be c o m p r o m i s e d b y i n t e r f e r e n c e f r o m c o l u m n b l e e d a n d c o e l u t i o n o f peaks, whereas, specificity of d e t e c t i o n d e p e n d s u p o n c o m p l e t e n e s s of s e p a r a t i o n b y t h e c o l u m n a n d m u s t be verified at regular intervals b y t h e use of a u t h e n t i c m i x t u r e s . Because o f t h e e x q u i s i t e selectivity of d e t e c t i o n b y mass s p e c t r o m e t r y (MS), t h e t e c h n i q u e o f c h e m i c a l i o n i z a t i o n (CI) r e c o n s t r u c t e d mass c h r o m a t o g r a p h y (RMC) suffers f r o m n o n e of t h e s e p r o b l e m s , even for peaks h a v i n g e x t r e m e l y low GC signal/noise ratio. In a d d i t i o n , t h e mass s p e c t r u m can be c o n s u l t e d if u n a m b i g u o u s p e a k i d e n t i f i c a t i o n is desired. By this t e c h n i q u e , q u a n t i t a t i o n o f i n d i v i d u a l f a t t y acids a n d t o t a l f a t t y acids in a c o m p l e t e lipid f r a c t i o n is a c c o m p l i s h e d in a single step. Cholesteryl esters (CE) are c h o s e n as i l l u s t r a t i o n in this r e p o r t . MATERIALS AND METHODS

R e c o n s t r u c t e d mass c h r o m a t o g r a p h y using m e t h a n e as a carrier a n d r e a g e n t gas for c h e m i c a l i o n i z a t i o n gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y o f t h e derived m e t h y l esters allows rapid, q u a n t i t a t i v e m i c r o d e t e r m i n a t i o n s of c o m p l e t e cholest e r y l ester f a t t y acid profiles. T h e sensitivity of t h i s m e t h o d is c o n s i s t e n t w i t h completely specific, m u l t i c o m p o n e n t assay at t h e p i c o m o l e level. I n t r o d u c t i o n of t w o h o m o l o g u e s as i n t e r n a l s t a n d a r d s , o n e i n t o t h e i n t a c t biological s p e c i m e n a n d t h e o t h e r a f t e r d e r i v a t i z a t i o n , provides a m e a s u r e o f t h e n e t efficiency of t h e processes o f e x t r a c t i o n a n d derivatization. This p r o c e d u r e m a y b e e x t e n d e d readily t o t h e d e t e r m i n a t i o n o f f a t t y acid profiles in m o s t biological fluids. INTRODUCTION

M a n y t e c h n i q u e s have b e e n d e v e l o p e d for q u a n t i t a t i v e e s t i m a t i o n o f f a t t y acids (1,2); h o w e v e r , m a n y o f t h e s e are n o n s p e c i f i c , insensitive, or t e d i o u s . A l t h o u g h gas c h r o m a t o g r a p h y ( G C ) of derivatized f a t t y acids, usually as t h e i r

Sample Preparation for GC-MS Analysis

A m i x t u r e o f 1 ml o f p o o l e d , n o r m a l h u m a n plasma a n d 6 0 0 pg (901 n m o l e s ) of c h o l e s t e r y l n o n a d e c a n o a t e was e x t r a c t e d w i t h t w o 4-ml

TABLE I Parameters a for Converting b Measured RMC Peak Areas into Molar Concentrations of Individual Fatty Acid Methyl Esters FAME 14:0 16:0 16:1 18:0 18:1 18:2 18 : 3 19:0

20:4

m 0.871 0.826 0.835 0.853 0.832 0.863 0. 836 0.918 0.868

b

r

0.262 0.426 1.308 0.497 0.403 1.747 0.946 0.649 1.259

.99999 .99986 .99983 .99982 .99995 .99996 .99998 .99996 .99994

range 0.01-10 0.0t-50 0.01-10 0.01-10 0.01-50 0.1 -10 0.01-10 0.1 -100 0.1 -10

aFAME = Fatty acid methyl ester; m = slope; b = intercept; r = correlation coefficient of linear regression. bAccording to the expression: [FAMEi] = [17:0] exp{m In(Areal/Areal7:0)+ b}. c[ 17:0] added = 370 nmole/ml in this study. 800

CHOLESTERYL ESTERS BY GC-MS TABLE II

100

Amounts of Fatty Acids and Cholesterol from Cholesteryl Ester Fraction of Normal Human Plasma Coefficient of variation (%)

Fatty Acid a

14:0 7.0 16:0 5.5 16:1 5.2 18:0 11.1 18:1 4.0 18:2 3.8 18:3 10.1 20:4 4.6 Total concentration Total plasma cholesterol b Free plasma cholesterol b Cholesterol in CEc (difference)

0.032

0.430 4.68 7.69-+1.86 2.84-+0.12 4.85

Analysis of F A M E s by GC-MS

measured

45

CH3(CH2~SC02MI

0.027 0.540 0.300 0.108 0.865 2.38

portions of m e t h a n o l : c h l o r o f o r m (2:1); insoluble matter was dispersed partially by interm i t t e n t , vibrational agitation, and sedimented by centrifugation prior to decantation. The c o m b i n e d extracts were c o n c e n t r a t e d to dryness in a stream of nitrogen, affording a residue that was redissolved in the m i n i m u m a m o u n t , ca. 100 pl, of spectroquality heptane and quantitatively transferred to a 5 x 80 cm thin layer chromatographic (TLC) plate (Adsorbosil-5, Applied Science Labs, State College, PA). The TLC plate was developed in hexane: ether:acetic acid ( 9 0 : 2 0 : 1 ) , and c o m p o n e n t s were visualized by exposure to iodine vapors. The region containing the cholesteryl esters (CE) was scraped into a test tube, to which was added a 5-ml p o r t i o n of c h l o r o f o r m : m e t h a n o l : e t h e r (1:1 : 1). The m i x t u r e was agitated vigorously and centrifuged, and the solvent was decanted. The residue was similarly extracted with a second 5-ml p o r t i o n of solvent. The c o m b i n e d solutions were c o n c e n t r a t e d in a stream of dry nitrogen, dissolved in a mixture of 1 ml benzene and 2 ml 0.5 N sodium methoxide in m e t h a n o l and heated to 80 C for 20 min. The transesterified m i x t u r e t h e n was cooled and diluted with 3 ml distilled water and 3 ml ether. The organic layer was separated, washed w i t h a 3-ml p o r t i o n of distilled water, and dried by the addition of a n h y d r o u s sodium sulfate. After decantation f r o m the desiccant, the solution was concentrated to dryness in a nitrogen stream and re-dissolved in 100 gl of ethyl acetate containing 100 #g (352 nmole) of m e t h y l h e p t a d e c a n o a t e ( 1 7 : 0 FAME). were

285

Concentration (#mole/ml)

aDetermined as fatty acid methyl ester by chemical ionization reconstructed mass chromatography. bDetermined by gas chromatography. CCE = cholesteryl esters.

FAMEs

801

using a Finnigan

. . . .

200

,.,I

/ rl&,

2~50

i,

.

].

r

,[.I

300 m/e

....

, . . . . . . .

35[;

400

FIG. 1. Chemical ionization mass spectrum of methyl heptadecanoate (17:0 fatty acid methyl ester) using methane as reagent gas. 1015D MS interfaced to a Finnigan 9500 GC and a Finnigan 6000 Interactive Data System (DS) (Finnigan Corp., Sunnvale, CA). The GC was fitted with a 0.5 m x 2 m m internal diameter (ID) glass c o l u m n packed with 3% EGSS-X coated on Gas Chrom Q. The solution to be analyzed (1/11) was injected at a c o l u m n temperature of 130 C; the effluent f r o m the GC was diverted into a v a c u u m bypass for 30 sec, allowing the solvent to elute, w h e r e u p o n the c o l u m n t e m p e r a t u r e was increased at a rate of 6 C/rain to a final t e m p e r a t u r e of 210 C. The flow rate of methane, 17 ml/min, t h r o u g h the GC was selected to afford a pressure of 0.95 tort, which was optimal for CI in the MS source; electron energy was 150 eV, multiplier setting was 1.7 kV, and pre-amplifier sensitivity was 10 -7 a/V. The DS scanned the range 230-330 ainu at a u n i f o r m rate o f 20 m s e c / a m u during the GC run. After the t e r m i n a t i o n of data acquisition, the DS was directed to recall individual records of the intensity of p r o t o n capture (M + 1) ions corresponding to each of the F A M E s present. The DS was used to calculate the area u n d e r the peak, reproducible within 5% for duplicate inj e c t i o n s , in each such r e c o n s t r u c t e d mass c h r o m a t o g r a m (RMC), and to normalize this area relative to that of 17:0 FAME. Values thus obtained were inserted into the appropriate expression f r o m Table I to give the concentration of CE corresponding to that F A M E in the original sample. Isolation efficiency was calculated as the a m o u n t of 19:0 F A M E measured divided by the a m o u n t o f 19:0 CE added. Calibration curves were calculated f r o m quadruplicate determinations using gravimetrically prepared standard m i x t u r e s of FAMEs. The data in Table II are averages o f triplicate LIPIDS, VOL. 10, NO. 12

802

F. PETTY, J.B. RAGLAND, L.B. KUIKEN, S.M. SABESIN, AND J.D. WANDER

measurements on 3 identical preparations of normal human plasma. Determination of Cholesterol by GC

a/

FRME 1R RUN =3 CI-METHRNE TOTRL ION CURRENT

10C

Total cholesterol and f r e e cholesterol were measured using a Barber-Coleman Model 5000 GC fitted with flame ionization detection and a 46 cm x 4 mm ID glass column packed with 3% OV-1 on Gas Chrom Q. The system was operated isothermally at 240 C, using argon as the carrier gas. Peak areas were estimated by triangulation, and samples were determined in triplicate. The preparation was essentially that of Blomhoff (6) except that (a) sitosterol was introduced into the plasma as an internal standard prior to the extraction procedures, and (b) measured values of cholesterol were adjusted to correct for losses in isolation and handling. RESULTS AND DISCUSSION

The characteristic, proton capture (M + 1) ion appears as the most abundant species in the CI mass spectrum (Fig. 1) of each of the FAMEs in this study, accounting for ca. 50% of the total ionization in all examples. Conversely, the electron impact mass spectrum of a FAME exhibits only low intensity ions at the characteristic, high mass region, the most prominent fragments, m/e 74 and 87, being common to the entire series of FAMEs (7). Thus, CI MS offers both enhanced sensitivity and extreme selectivity as a means of quantitatively detecting FAMEs in a GC effluent; this selectivity carries the added advantages that such potential interferences as biological background and column bleed are generally transparent at the mass number being detected, so that baselines normally are close to zero in the RMCs. Figure 2a illustrates the output from the GC detected as the sum of the ion currents from 230 to 330 amu. Therein, methyl myristate (14:0 FAME) presents a weak signal, that of methyl arachidonate (20:4 FAME) is distorted in shape by interfering column bleed, and those of methyl linoleate (18:2 FAME) and 19:0 FAME coelute. In the RMCs (Figure 2b) of m/e 243, 285, 295, 313, and 319 (M + 1 for 14:0, 17:0, 18:2, 19:0 and 20:4 FAME, respectively), each peak is found on a separate trace, where its area may be measured accurately, in the absence of the interferences present in Figure 2a. We found that the calibration curves empirically gave a linear approximation to a log-log expression (Table I and Fig. 3) rather than to a simple cartesian plot. Published calibration curves for the closely related technique of selective ion monitoring are commonly limited to L1PIDS, VOL. 10, NO. 12

"1 .......

|'"

50 bj

.......

I"

.......

100

I''"111"'1

150

........

200

;I .........

250

I'''''''''l

300

350

SELEGTEDRMCs

I

O0

rr.o

18,2 19,0

14,0

~ .

i...,

.....

I""'";'1''"

50

100

.....

| ' " ' ' " " |

150

200

.........

| ' ' ' " " " |

250

300

.........

|"

350

FIG. 2a. Gas chromatogram of fatty acid methyl ester (FAME) mixture detected by summing all of the ion currents measured by the mass spectrometer; 2b. Composite of reconstructed mass chromatograms (RMCs) for M + 1 ions of 14:0, 17:0, 18:2, 19:0 and 20:4 FAMEs. a tenfold dilution range (8). However, Clark and Foltz (9) reported separate calibration curves having different slopes for 3,6-bis(tri-

803

CHOLESTERYL ESTERS BY GC-MS

109

/ / ~ ~[]

o I0 ~

o

J 16,0

I0 "1

for determination. Furthermore, ( a ) C I - R M C requires only inexpensive internal standards, (b) the selectivity of detection minimizes the GC resolution needed for reliable quantitation, and (c) the mass spectrum is available to verify the identity of the component being measured. Multiple selective ion monitoring "mass fragrnentography" would be slightly more sensitive for this analysis, but (a) commercial instruments presently have facilities to monitor no more than 4 ions, and (b) the mass spectrum would not be available for verification.

16=1 18=0

18=1

18,2 16,3 19:0 20=4

10- 2

I0" 0.01

I 0.1 mole

I I rofio of FAME to

I I0

I I00

17:0

FIG. 3. Calibration plot for area response of fatty acid methyl esters (FAME) relative to 17:0 FAME versus mole ratio of FAMEs to 17:0 FAME; best-fit regression expressions and correlation coefficients are recorded in Table II.

methylsilyl)-morphine over two regions of a wider dilution range. The significance of this observation is unclear at present. Table II contains the amounts of each fatty acid present in the original CE fraction as determined by CI-RMC together with a value for the amount of cholesterol in the CE fraction, determined by subtracting free from total cholesterol content. Agreement between the two values is clearly within the reliability limits of either method of determination. Thus, the present communication demonstrates that CI-RMC offers numerous advantages as a technique for simultaneous, multicomponent assay of fatty acids, i.e., simplicity of operation, speed, and broad dynamic range

ACKNOWLEDGMENTS N. Flynn provided superb technical assistance in the development and performance of the GC-MS assay. This research was supported, in part, by Grants No. RR 05423 and AM 17398 from the US Public Health Service, by Grants-in-Aid from the American Heart Association, the Tennessee Heart Association, and the Veterans Administration, and by the Charles B. Stout Memorial Fund. REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9.

Kates, M., "Techniques of Lipidology," Elsevier, New York, NY, 1972, pp. 502-579. Nelson, G.J., in "Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism," Wiley-lnterscience, New York, NY, 1972, pp. 25-73. J a m e s , A.T., Methods Biochem. Anal., 8:1 (1960). Horning, E.C., A. /Carmen, and C.C. Sweeley, Prog. Chem. Fats Other Lipids 7:167 (1964). A c k m a n , R . G . , Methods Enzymol. 14:329 (1969). Blomhoff, J.P., Clin. Chim. Acta, 43:257 (1973). McLafferty, F.W., "Interpretation of Mass Spectra," 2nd edition, W.A. Benjamin, Inc., New York, NY, 1973, p. 132. Holmsted, B., and L. Palme'r, Advan. Biochem. Psychopharm., 7:1 (1973). Clarke, P.A., and R.L. Foltz, Clin. Chem., 20:465 (1974).

[Received July 2, 1975]

LIPIDS, VOL. 10, NO. 12