[41] Microdetermination of stereoisomers of 2-hydroxy and 3-hydroxy fatty acids

326

GI~NERAL ANALYTICAL METHODS

[41]

[41] Microdetermination of Stereoisomers of 2-Hydroxy and 3-Hydroxy Fatty Acids By

SVEN HAMMARSTROM

2-Hydroxy acids and 3-hydroxy acids are intermediates in animal and plant degradation of fatty acids. 1 3-Hydroxy acids are also intermediates in the biosynthesis of fatty acids 1 and 2-hydroxy acids are constituents of brain cerebrosides and of certain waxes. ~ It is possible to determine the enantiomeric composition of microgram amounts of these acids by gas-liquid chromatography of diastereoisomeric derivatives. 3'*

Derivatives of 2-Hydroxy Acids 2-Hydroxy acid methyl esters are analyzed as O(--)-menthyloxycarbonyl derivatives. About 100 t~g of hydroxy acid is methylated with diazomethane~ and subsequently treated with (--)-menthyl chloroformate2 (--)-Menthyl Chloro]ormate2 (--)-Menthol (15.63 g, 0.1 mole; Kebo AB Stockholm, Sweden) and 11.8 ml (0.1 mole) of freshly distilled quinoline (b.p., 118-120°/20 mm) are added to 100 ml of 12.5% (w/v) phosgene in benzene (Matheson, Coleman & Bell) at 0° in a well ventilating fume hood2 a The mixture is stirred overnight (0 °) with the outlet of the reaction vessel connected, via an empty wash bottle, to a wash bottle containing 20% w/v NaOH in water. The quinoline hydrochloride is filtered off in the fume hood, and the excess phosgene is removed by bubbling nitrogen through the filtrate for about 5 hours (the outlet of the reaction vessel should be connected to the trap with 20% NaOH). The reagent solution is transferred to a 100-ml flask with ground glass stopper which contains calcium carbonate. It contains about 1 ~mole (--)-menthyl chloroformate per milliliter and can be kept without noticeable decomposition at + 4 ° for a year. (--)-Menthyloxycarbonyl Derivatives2 To approximately 100 t~g of 1S. J. Wakil, in "Lipid Metabolism" (S. J. Wakil, ed.), pp. 1--48. Academic Press, New York, 1970. 2]). T. Downing, Rev. Pure Appl. Chem. 11, 196-211 (1961). 8 S. HammarstrSm, FEBS Lett. 5, 192-195 (1969). 4 S. H a m m a r s t r S m and M. Hamberg, Anal. Biochem. 52, 169-179 (1973). 5 H. Schlenk and J. L. Gellerman, Anal. Chem. 32, 1412-1414 (1960). 6 j . W. Westley and B. Halpern, J. Org. Chem. 33, 3978-3980 (1968). 6~ Caution : Phosgene is y e w toxic. Symptoms may be delayed up to 24 hours.

[41]

STERIC

ANALYSES

OF ItYDROXY

ACIDS BY GLC

327

2-hydroxy acid methyl ester in 40 ~l of dry benzene is added 60 td of (--)-menthyl chloroformate solution and 12 ~l of dry pyridine. The mixture is left for 30 minutes at room temperature. During this time, the color of the solution gradually changes from pale yellow to deep purple. Two milliliters of benzene is added and the solution is washed with 3 X 2 ml of water in the reaction tube. The benzene phase is evaporated to dryness after addition of absolute ethanol, and the residue, dissolved in CS~, is quantitatively applied as a band (30 X 4 mm) to a thin-layer chromatography plate. The plate should be coated with 0.25 mm of silica gel G and activated at 120 ° for 45 minutes before use. Benzene-dioxane, 97:3 (v/v), is used as the developing solvent. The compounds are made visible in ultraviolet light by spraying the chromatogram with 0.2% (w/v) 2',7'-dichlorofluorescein in ethanol. The diastereoisomeric 2-hydroxy acid derivatives do not separate during the chromatography but move as a single zone (RI 0.85) closely behind a large zone of excess reagent (R/ 0.95-1.0). The 2-hydroxy acid derivatives are recovered by transferring the zone of silica gel to a glass column and eluting it with 3 X l0 ml of diethyl ether. The solvent is removed and the purified derivative is dissolved in 100 ~l of CS2 for gas-liquid chromatography.

Derivatives of 3-Hydroxy Acids 3-Hydroxy acid methyl esters are converted to 2-D-phenylpropionate derivatives. About 100 ~g of hydroxy acid is methylated with diazomethane 5 and then treated with 2-D-phenylpropionyl chloride. 4 2-D-Phenylpropionyl Chloride. 4 2-DL-Phenylpropionic acid (3 g, 20 mmole; K & K Laboratories Inc., N.Y.) in 60 ml of acetone is added to a solution of (+)-l-phenylethylamine in 20 ml of acetone. The mixture is kept at 70 ° for 5 minutes and then cooled to --20 °. This yields a crystalline salt (ca. 2.8 g) which is recrystallized 3 times from acetone at + 4 °. The yield of the (+)-l-phenylethylammonium salt of 2-D-phenylpropionic acid is about 0.3 g. The salt is dissolved in water, treated with 2 N hydrochloric acid and extracted 3 times with diethyl ether to give 2-D-phenylpropionic acid as a pale yellowish syrup in 11% of the theoretical yield ([a]~5 = +88.0°; C 1, benzene; previously reported [a]~0_ +92.5 ° benzene6). Two-hundred milligrams of 2-D-phenylpropionic acid and 0.24 ml of thionyl chloride (newly distilled from beeswax) are mixed at 0 ° and kept at 70 ° for 30 minutes. Dry benzene is added and the mixture is evaporated to dryness. This step is repeated once to remove the last traces of thionyl chloride. The residue is dissolved in 2.40 ml of dry benzene and stored at + 4 ° in a flask with ground glass stopper.

328

GENERAL ANALYTICAL METHODS

[41]

2-D-Phenylpropionate Derivatives. ~ To about 100 t~g of 3-hydroxy acid methyl ester is added 90 t~l of 2-D-phenylpropionyl chloride solution and 20 ~l of dry pyridine. The mixture is left for 2 hours at room temperature in a dessicator. After evaporation of the solvents the residue is dissolved in chloroform and applied to a thin-layer plate (see above). The chromatogram is developed with chloroform (E. Merck AG., pro analysi, containing 0.6-1.0% ethanol as stabilizer), and the compounds are detected with 2',7'-dichlorofluorescein. The diastereoisomeric 3-hydroxy acid derivatives move as a single zone (RI 0.80). They are eluted as described above but using ethyl acetate instead of diethyl ether.

Steric Analyses The derivatives described above can be resolved on packed columns containing OV-210 (Pierce Chemical Co.) or QF-I on 100-120 mesh Gas Chrom Q (Applied Science). W e use U-shaped glass columns (i.d. 2 or 3 mm, length 180 cm) in an F & M model 400 Biomedical Gas chromatoggraph equipped with a hydrogen flame ionization detector or circular glass columns (i.d. 3 mm, length 180 cm) in an LKB model 9000 gas chromatograph-mass spectrometer. Our column packings contain about 1.5% OV-210 or 5% QF-1 and are generally prepared with the Hi-Eft fluidizer (Applied Science) as described beforeJ A gas ehromatogram of (--) -menthyloxycarbonyl derivatives of a mixture of racemic 2-hydroxy acid methyl esters (C14-C~) is shown in Fig. la. The derivatives of optical isomers give rise to pairs of partially separated peaks. Figure lb shows a gas chromatogram of the same mixture after addition of (--)-menthyloxycarbonyl derivatives of 2-L-hydroxypalmitic acid methyl ester, 2-L-hydroxyarachidic acid methyl ester, and 2,L-hydroxylignoceric acid methyl ester. It is clear that the first peaks of the pairs represent the 2-L-hydroxy acids and the second peaks the D-enantiomers. Figure 2 shows a gas-liquid chromatogram of 2-D-phenylpropionate derivatives of five racemic 3-hydroxy acid methyl esters (C1o-C18). Addition of the same derivative of 3-D-hydroxycapric acid methyl ester showed that the first peaks of the pairs represent the 3-Dhydroxy acids and the second peaks of the 3-L-hydroxy acids. (--)-Menthyloxycarbonyl derivatives are not suitable for steric analyses of 3-hydroxystearic, 9-hydroxystearic, 12-hydroxystearic, or 17-hydroxystearie acid methyl ester on the columns described above. Neither are 2-D-phenylpropionate derivatives of 2-hydroxy acid methyl esters suitable for steric analyses on these columns. The latter derivatives, 7E. C. Horning, in "Gas Phase Chromatography of Steroids" (K. B. Eik-Nes and E. C. Horning, eds.), pp. 1-71. Springer-Verlag, Berlin and New York, 1968.

[41]

STERIC

ANALYSES

OF ttYDROXY

ACIDS BY GLC

329

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FIG. 1. (a) Gas-liquid ehromatogram of (--)-menthyloxycarbonyl derivatives of 2-DL-hydroxy 14:0 Me, 2-DL-hydroxy 16:0 Me, 2-DL-hydroxy 18:0 Me, 2-DLhydroxy 20:0 Me, 2-DL-hydroxy22:0 Me, 2-DL-hydroxy24:0 Me, and 2-DL-hydroxy 26:0 Me. Conditions for gas-liquid chromatography (GLC) : Instrument, F&M model 400; stationary phase, 1.4% OV-210on 100-120 mesh Ga~ Chrom Q; column temperature, 250°; carrier gas, helium with a flow rate of 30 ml/minute. (b) The same chromatogram as in (a) after addition of appropriate amounts of (--)-menthyloxycarbonyl derivatives of 2-L-hydroxy 16:0 Me, 2-L-hydroxy 20:0 Me, and 2-L-hydroxy 24:0 Me. (Reproduced from Ref. 3.) however, are useful also for analysis of 15-hydroxystearic, 16-hydroxystcaric, and 17-hydroxystearic acid as well as for 2-alkanols and 3-alkanols. 4 The retention times of the ( - - ) - m e n t h y l o x y c a r b o n y l derivatives of the 2-hydroxy acid methyl esters in Fig. 1 are expressed as triglyceride carbon units in Table I. Triglyceride carbon units are obtained by plot-

330

[41]

GENERAL ANALYTICAL METHODS

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TABLE I RETENTION DATA FOR (--)-MENTHYLOXYCARBONYL DERIVATIVES OF RACEMIC 2-HYDROXY ACID METHYL ESTERS EXPRESSED AS TRIGLYCERIDE CARBON U N I T S a Triglyceride carbon units Hydroxy acid

2-DL-Hydroxy 2-DL-Hydroxy 2-D~-Hydroxy 2,-DL-Hydroxy 2-DL-Hydroxy 2-DL-Hydroxy 2-DL-Hydroxy

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[41]

STERIC ANALYSES OF HYDROXY ACIDS BY GLC

331

TABLE II C VALUES FOR 2D-PHENYLPROPIONATE DERIVATIVES OF 3-HYDROXY ACID METHYL ESTERS °

Hydroxy acid 3-DL-Hydroxy10:0 3-DL-Hydroxy13:0 3-DL-Hydroxy14:0 3-DL-Hydroxy16:0 3-DL-Hydroxy18:0

C values 20.70 and 23.65 and 24.60 and 26.55 and 28.50 and

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RACEMIC

Separation factor (ratio of retention times of diastereoisomers) 1.06 1.08 1.08 1.08 1.08

The stationary phase was 5% QF-1 on 100-120 mesh Gas Chrom Q, the carrier gas was helium (30 ml/minute), and the column temperature was 200°. (Reproduced from Ref. 4.) ting the logarithm of the retention time of synthetic triglycerides (e.g., tricaprylin, tricaprin, and trilaurin) against their total numbers of carbon atoms (27, 33, and 39) and interpolating the logarithm of the retention time of the compound in question. C values are obtained in the same way but using normal fatty acid methyl esters instead and plotting the logarithm of the retention time against the number of carbon atoms in the fatty acids. Table II gives the retention times, expressed as C values, of the 2-D-phenylpropionate derivatives shown in Fig. 2. This table also gives the separation factors for the isomeric compounds. Figures 3 and 4 show partial mass spectra of the compounds in Figs. 1 and 2, respectively. In all cases, the mass spectra of diastereoisomeric compounds are mutually indistinguishable. Figure 5 shows structural formulas of the (--)-menthyloxycarbonyl derivative of 2-D-hydroxystearic acid methyl ester and of the 2-D-phenylpropionate derivative of 3-D-hydroxystearic acid methyl ester. It also shows some of the fragments which are formed on electron impact. The ions M-137, M-169, M-182, M-199, M-231, M-241, and M-286 in the mass spectra of (--)-menthyloxycarbonyl derivatives (Fig. 3) and the ions M, M-76, M-118, M-149, M-181, M-199, and M-223 in the mass spectra of 2-D-phenylpropionate derivatives (Fig. 4) retain the fatty acid part of the molecule whereas the ions at m/e 214, 138, and 123 (Fig. 3) and those at m/e 297, 167, 150, 137, 132, 129, 125, 121, 111, and 105 (Fig. 4) are formed by elimination of this part of the molecule. The methods described can be used for steric analyses of radioactive hydroxy acids in tracer amounts. 8 8A. J. Markovetz, P. K. Stumpf, and S. HammarstrSm, Lipids 7, 159-164 (1972).

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[42]

GENERAL ANALYTICAL METHODS

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[42] Measurement of Protein in Lipid Extracts By LEWIS C. MOKRASCH In the course of studies of organic-solvent-soluble proteins, a variety of classic protein assay methods have been found to be inapplicable or involving excessive effort to reduce interference as a result of the presence of lipids in the mixture. The simplest resource, extracting the lipids away from the protein by the use of organic solvents, fails because of the solubility of the protein in organic solvents. Attempts to extract the protein from the lipid mixture by the use of aqueous systems are also prone to failure because of unfavorable partition characteristics of the protein, to emulsion formation, turbidities and other problems which preclude the possibility of simple procedures. Historically the protein contents of lipid extracts have been estimated