Quantitative Analysis of Complex Mixtures of Steroids and Bile Acids by Gas Chromatography DANIEL K. BLOOMFIELD Department of Medicine, Western Reserve University, Cleveland 6, Ohio
b A practical gas chromatography method for the quantitative analysis of complex mixtures of C-27 steroids and C-24 bile acids, using an argon ionization detector, is described, The method is based on a relationship between relative retention times and responses of steroid nucleus compounds under specific detector conditions. An internal standard is used, eliminating prolonged calibration. The method has been applied successfully to fecal extracts and gives fractional as well as total quantitation.
T
paper describes a n approach to quantitative analysis of C-27 steroids, and C-24 bile acids by gas chromatography (GLC). Separation and identification of these compounds by this method have progressed rapidly. Quantitation has been less satisfactory and recently Sweeley and Chang (6) concluded that "to apply the method to the quantitative determination of steroids in a mixture, the argon ionization detector must be calibrated with each of the steroids present in the mixture." Since my objective has been to quantitate complex biological mixtures, such a method would be impossible if only through lack of standards, not to speak of time. A useful method, empirical and accurate bo -lo%, has been found. I t takes advantage of the relationship between the retention times of C-27 steroids and (2-24 bile acids on a specific type of column and their relative responses in a capillary-type argon ionization detector. HIS
EXPERIMENTAL
Apparatus. -4 Barber-Colman Model 10 instrument with a n argon ionization detection system is used. I t is equipped with an . I 4 1 4 5 detector which is modified t o type -4-4148 (Barber-Colman numbers). This type of detector is designed for use either witl: rapillary columns or LTith packed rolumns equipped with a n effluent For quantitat'ion, however, splitt'pr the toh! effiwnt from a packed column IS passea t,hwugti the detector. The cell is equipj)t,o with a 300-megaohm -4 6-foot l/r-inch
tric Co.) on Chromosorb W 120- to 140mesh (Johns-Manville) is used for all experiments ( 3 ) . Column efficiency varies between 130 and 300 theoretical plates per foot. Quantitative results are independent of column efficiency. Uniform sample injection is achieved by using dilute solutions with a Hamilton 701-iY syringe. Sample size varies from 1 to 9 p1. Before a sample is drawn in the syringe, the syringe is filled with acetone or methanol, all bubbles removed, and brought to the 1-pl. mark. The desired sample volume is then drawn into the syringe and the sample plus the wash injected into the column. Because of the narrow bore of tmhesyringe there is minimal mixing of the sample and t,he solvent behind it,. The solvent wash a l l o w for complete injection of the sample into the column. Duplicate injections can be reproduced within 5% limits. Reagents. Except as noted below all compounds were purchased from commercial sources and crystallized to constant melting points n-hich agreed with known values. Each gave a single peak by gas chroniatography. Cholestane-3p,icu-diol and cholestane-3p,ip-diol were the gift of R. B. Clayton of Cambridge, Mass. Chenodeoxycholic acid was the gift of A. F. Hofmann, Lund, Sweden. Deoxycholic acid from commercial sources could not be crystallized satisfactorily and was purified by preparatd. Individual steroids and bile acids in com5.0
I
O'
Yethyl Cholate
0 1 0 2 0 3 04 0 5 06 07 0 8 09
Response Relative to Cholestane
Ib
Figure 5. Relationship between relative responses and retention times for steroid and bile acid test mixtures VOL. 34, NO. 7, JUNE 1962
739
ples mixtures can be quantitated within the limits of the resolving poiver of the column. Application to Biological Problems. T o apply the method to biological problems several assumptions had to be made. These were: T h a t all peaks which appear a t or beyond specified relative retention times are members of the homologous series being quantitated. I n t h e case of sterols, I have used all peaks appearing a t the time of and after coprostanol. I n the case of bile acids this has included all peaks m-ith relative retention times greater than 1.20 which corresponds to the retention time of the earliest appearing bile acid in the series studied by Sjovall, hfeloni, and Turner (6); that each peak which appears represents a compound or mixture of compounds with relative responses, relative retention times, and linear ranges comparable to the known substances listed in Table I ; and that thermal decomposition does not occur to a significant degree. The data collected from the 13 consecutive substances studied in Table I strongly support these assumptions.
Table
II.
Cholestane Cholesterol Cholestane-3P,7a-diol Cholestane-3P,5a,60-triol
0 0 1 4
2 6 0 0
E
Y Figure 6.
’
12
%by at.
3 5 10 4 172 68 9
Bile Acid Mixture Mg / nil. Cholestane Methyl cholanates-: 30-hydroxy 3a, 120-dihydroxy 30,iol,l20-trihydroxy
Fluorimetrically ( 4 ) , mg. Methyl cholate added, mg. Total bile acid by fluorimetric method, mg. Determination of total fecal bile acids per 2 grams of rat feces by GLC method alone, mg. % recovery of added methyl cholate by GLC method (7.08 - 6.27 = 0.81 mg.)
%by
ut. 1 9
0 2
1 0 3 8
9 6
366 51 9
5 4
Other Methods
Bb
Aa
1.27 0.14
1.27d
0 __
1.41
0 . 14d 0.50 __ 1.91
1.30
1.84
. . ,
108
7.40
7 ,40d
7.40
0.74 8.14
6.27
7.08
0 -
~
110
a Values from typical fractions obtained on adult male rat on low fat, cholesterol-free diet. b Values obtained when standards were added to biological material of column A . c Measured gravimetrically. Values taken from column A.
ANALYTICAL CHEMISTRY
3.8*i
3.2”i-O32;”1
Schematic example of quantitative method
Other support comes from the following facts: Chromatography of supernatant material from digitonin precipitation of nonsaponifiable fecal lipides was nearly free of “steroid” peaks except for a coprostanol residue; most of the “bile acid” peaks observed from rat
Comparison of Gas Chromatographic and Lipide Extracts from Rat Feces Xonsaponifiable Fraction Determination of total fecal sterols per 0.8 gram of rat feces: 9 s digitonide, mg.C By GLC in digitonide supernatant, mg. Cholesterol added Total steroid by digitonide-GLC methods, mg. Determination of total fecal sterols per 0.8 gram of rat feces by GLC method alone, mg. 70 recovery of added cholesterol by GLC method (1.84 - 1.30 = 0.54 mg.) Acidic Fraction Determination of total fecal bile acid per 2 grams of rat
740
~
Asterisks indicate values derived from Figure 5, or calculations d e scribed in text
Table 111.
feces:
1 4.0
Composition of Test Mixtures
Steroid Mixture
m/ ml.
0
feces correlated with relative retention times previously described in the literature (6); decomposition of compounds such as auto-oxidation of cholesterol to 7~,3~-cholestane-dioIJ or dehydration of the latter compound to cholest-7-en-3P-01 would not materially affect quantitation of total sterols since each of the products quantitates without difficulty. To quantitate fecal sterols, the nonsaponifiable lipides from the equivalent of 0.25 to 1.0 gram of rat feces are dissolved in benzene so that the total sterol concentration is in the range of 1 to 3 mg. per ml. A preliminary run of exactly 2 pl, is made to observe the character of the peaks in the steroid areas and to ensure that no major peak will interfere with the cholestane standard, Cholestane as a standard solution is then added to the biological mixture and the volume adjusted so that the cholestane concentration is 0.2 to 0.4 mg. per ml. and with a second injection of 1 to 3 pl. a maximum number of peaks may be quantitated. It is difficult to define categorically the accuracy of the method in such a biological system since as of this date there is no completely satisfactory method for the total quantitation of fecal sterols (W), nor are all necessary standards available. To check the accuracy of the method the following procedure was used: An aliquot of the same nonsaponifiable lipides quantitated by gas chromatography was treated with digitonin and the precipitated sterol digitonides determined gravimetrically. The supernatant from this procedure, Le., the unprecipitated sterol digitonides, was evaporated to dryness in vacuo and the sterol therein recovered by the method of Bergman, m-hich gives yields of over 90y0 ( I ) . This method also quantitatively recovered the cholestane which had been added. The recovered sterols were then quantitated by gas
chromatography and the total fecal sterols assumed to be the sum of the digitonide precipitated sterols plus the supernatant sterols as determined by gas chromatography (Table 111). Analysis of fecal bile acids obtained as the ether extract of acidic fecal lipids after the nonsaponifiable materials have been removed has been more difficult. The methodology is identical to that for sterols except a larger proportion, the equivalent of 0.5 to 2.0 grams of rat feces, is required. The bile acids are quantitatively methylated with ewess diazomethane and dissolved in benzene so that bile acid concentration is 5 to 25 mg. per ml. Cholestane standard is added to a concentration of 0.2 to 0.4 mg. per ml. Values obtained are frequently 10 to 20% lon-er than those obtained by the fluorimetric method of Levin, Irvin, and Johnston (4) applied to feces. Nevertheless, when
known amounts of bile acids are added to fecal extracts, they can be accounted for with the same 10% accuracy (Table 111). It seems, therefore, that the method may be more accurate than fluorimetry in this application to feces. There are few compounds which have retention times similar to bile acids so that interference is not a problem. The method, though empirical, may have wider application with biological substances. With C-21 steroids, for example, if one takes t h e data of Sweeley and Chang (6) and converts relative retention times and responses to androstane as a standard, a plot similar to Figure 5 may be drawn that encompasses allopregnane-3p,20p-diol, pregnane-3,20-dione, 4-pregnene-3,20dione, and 5-pregnen-3p-ol-2O-one, and comes reasonably close to other thermostable steroids. The same holds true for the data of Wotiz and Martin (7‘) if estrone acetate is used as the
standard for a homologous series of acetylated estrogens. LITERATURE CITED
(1) Bergman, IT., J . Bid. Chem. 132, 471 (1940). (2]‘Cook, R. P., Rattray, J. E. M. in Cholesterol,” R. P. Cook, ed.? p. 118, Academic Press, Sew York, 1938. (3) Horning, E. C., Moscatelli, E. A,, SReeley, C. C., Chem. & Ind. (London) 1959, 751. ( 4 ) Levin, S. J., Irvin, J. L., Johnston, C. G., ANAL.CHEM.33, 856 (1961). ( 5 ) Sjovall, J., Meloni, C. R., Turner, D. A., J . Lzpzd Research 2,317 (1961). (6) Sweeley, C. C., Chang, T., ANAL. CHEM.33,1860 (1961). ( 7 ) Wotiz, H. H., Martin, H. F., J . Biol. Chem. 236, 1312 (1961). RECEIVED for revierr January 12, 1962. Accepted April 2, 1962. Work completed during the tenure of an Established Investigationship of the American Heart Association. Work supported by grants from the Heart Societies of Portage County and Cleveland, Ohio.
Proportional Counter Assay of Tritium in Gas Chromatographic Streams J. K. LEE, EDWARD K. C. LEE, BURDON MUSGRAVE, YI-NO0 TANG, JOHN W. ROOT, and F. S. ROWLAND Department of Chemistry, University o f Kansas, lawrence, Kan.
b The assay of tritium b y gas proportional counting of the effluent from a chromatographic column has many advantages. Aside from the precautions common to all gas chromatography practice, the critical point for accurate assay i s maintaining constant efficiency of detection for tritium in various molecular forms. The variations in detection efficiency arise especially from coincidence losses a t high count rates and from composition changes in the operating counter gas during the passage o f a macroscopic peak. The behavior o f helium-methane and helium-propane flow mixtures i s discussed extensively.
G
CHROMATOGRAPHIC separation and immediate radioactive assay of the separated gaseous components form a powerful combination tool for the investigation of many radioactive systems (2, 5, 9). The rapid increase in the availability of tritium, and in our case, the interest in the reactions of recoil tritium atoms, has led to the development of this technique for special application to tritium-labeled molecules (9). The primary design limitation for tritium assay arises from the low energy of the beta particle (Eomax = 18 k. e. v.) emitted in its decay to AS
He3. The practical consequence of this low energy is that the tritiumlabeled molecule must be present in the counting gas; hence the gas-counting tube must be made to operate on the effluent gas from the gas chromatographic separation apparatus The chief advantages of operation of an internal gas proportional flow counter in this manner have been discussed previously (9). The purpose of the present study is to examine the most important factors that affect the accuracy and reproducibility of this method for the analysis of tritiated radioactive components in gas chromatographic streams. I n our laboratory, the technique has been most frequently applied to the analysis of mixtures of labeled hydrocarbons, but many other volatile tritiated molecules have also been examined. The general problems are common to this method of internal counting for other weak beta emitters, and for similar radioactivity measurements in general. This counting system has also handled successfully C14, Ar37, Xe133,and other beta activities. W t h strong beta or gamma-ray emitters, however, it is not necessary to run the effluent gases directly through the counter itself, and many of the problems encountered with tritium are thereby avoided ( 1 ) . Evans and Willard ap-
plied a n external counting technique sensitive to gamma radiation and hard betas for the measurement of halogen radioactivities ( 2 ) . SAMPLE INJECTION
Sample injections are made through a sample chamber bounded by two ground glass T-joints and equipped with a by-pass. The sample loop is filled on a vacuum line with the sample in the gaseous state, and the equipment is equilibrated n i t h carrier gas passing through the by-pass. The injections are then made b y simultaneous turning of the stop-cocks to substitute the sample loop for the by-pass. With relatively nonvolatile samples, a sample loop has been used that is sufficiently big to accommodate all the sample in the gas state. Errors of sampling involved in the operation of any gas chromatograph will also affect the radioactivity measurements. SPECIFIC RADIOACTIVITY A N D OBSERVED COUNTS
This technique of radioactive assay relies on measurement of the number of counts observed in a particular gas chromatographic peak as a measure of the specific radioactivity of the corresponding compound. I n relative VOL 34, NO. 7, JUNE 1962
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