Identification by Reduction. A second method for the identification of the peroxides was found in the pattern of products obtained by reduction with lithium aluminum hydride followed by liquid-solid chromatography on silicic acid. This is illustrated by reduction of the peroxide from 4-C~4-7-dehydrocholesterol.There were seven distinct radioactive bands eluted as shown in Table I. Band I had A,, 286 mp. This replaced a maximum at 256 mp which was found prior to chromatography and was presumably the A4~6~8(14)-triene-3P-01 derived on the column from dehydration of the A6,*(l"-diene-3P,5oc-diol which is produced as one of three major products in the reduction (5). Band I1 was shown to contain 7-dehydrocholesterol by its ultraviolet spectrum. The A~~7-compound has been shown previously (14, 23) to be one of the three major products in the ergosterol series. From Band IV a compound was obtained to which we assign the structure of 7-cholestene-3P,5a-diol for the following reasons. It had no absorption maximum in the ultraviolet above 220 mp excluding a dienic moiety. End absorption (23) F. Dalton and G. D. Meakins, J. Chem. SOC.,1961, p. 1880.
which it did possess had an intensity which was characteristic of trisubstituted steroidal double bonds (24). These facts are consistent with a A'-structure. The elemental analysis eliminated the presence of three hydroxyl groups and agreed with the expected composition as a hemihydrate of the A7-diol. Finally, the amount of radioactivity indicated the substance was one of three major products of reduction. We have already accounted for two of these in Bands I and 11, and Band IV must then be the remaining one which, as already shown in the ergosterol series (14, 23) is the A7-diol. RECEIVED for review July 7, 1966. Accepted October 7, 1966. A preliminary report of this work was presented a t the annual meeting of the American Society of Biological Chemists in Chicago, April 1964. Support in part by Research Grant No. P-292 of the American Cancer Society and Training Grant No. 5001 and Research Grant No. AM-09100 of the National Institutes of Health is gratefully acknowledged. (24) P. Bladon, H. B. Henbest, and G. W. Wood, Chem. Ind. (London), 1951, p. 866.
Gas Chromatographic Analysis of Cardiac Glycosides and Related Compounds W . E. Wilson, S. A. Johnson, W. H. Perkins, and J. E. Ripley Southern Research Support Center, Veterans Administration Hospital, Little Rock, Ark.
Gas chromatography of several cardiac glycosides has been effected by converting them to their trimethylsilyl (TMS) ether derivatives prior to analysis. The TMS ethers of digoxigenin, digoxigenin monodigitoxoside, digoxigenin bisdigitoxoside, and digoxin have been chromatographed on a single column. The free sterols digoxigenin, digitoxigenin, and gitoxigenin, as well as their TMS ether derivatives, have been chromatographed on packed columns containing a silanized diatomaceous earth coated with 1.0 or 1.6% methyl silicone polymer SE30. The aglycones of digoxin and digitoxin have been quantitatively determined by hydrolyzing the glycosides in dilute hydrochloric acid-dioxane solution, followed by chromatography of the TMS derivatives of the resulting sterols.
THEDEVELOPMENT of gas chramatographic methodology for determination of cardiac glycosides and related compounds is of interest due to its potential applicability to metabolic studies (1) as well as to obvious clinical and pharmaceutical problems. In a previous attempt Jelliffe and Blankenhorn (2) reported the gas chromatography of trimethylsilyl (TMS) ether derivatives of digoxigenin and of digitoxigenin; however, they reported difficulties in the chromatography of the free sterols. They were unable to chromatograph the sterol glycoside TMS ethers. While many varieties of spectrophotometric and fluorometric quantitative analyses have appeared in the literature, (1) J. J. Ashley, B. T. Brown, G. T. Okita, and S . E. Wright, J . Biol. Chem., 232, 315 (1958). (2) R. W. Jelliffe and D. H. Blankenhorn, J . Chromafog.,12, 268 (1963). 40
ANALYTICAL CHEMISTRY
extensive prior purification is usually required if chromophore formation or fluorescence is to be attributed to a particular derivative of a genin. In this connection Jakovljevic ( 3 ) reported the fluorometric determination of digoxin and digitoxin, and presented a comprehensive review of various spectrophotometric assays for cardiac glycosides. In view of the results of Sweeley et al. (4,it was decided to attempt gas chromatographic determination of the TMS derivatives of a variety of cardiac glycosides, aglycones, and related compounds. Quantitative and qualitative determination of the TMS derivatives of digoxigenin and digitoxigen in, as well as of related glycosides, may be achieved by gas chromatography. Structures of the cardiac glycosides and related compounds are indicated in Figure 1. EXPERMENTAL
Chromatographic Homogeneity of Chemicals. Ouabain was purchased from Mann Research Laboratories, 136 Liberty St., New York, N. Y. The remaining cardiac glycosides and related compounds were purchased from the Boehringer Mannheim Corp., 20 Vesey St., New York, N. Y. Cholesterol was obtained from Applied Science Laboratories, Inc., State College, Pa. The homogeneity of the cardiac glycosides and related compounds was ascertained by thin-layer chromatography (Table I). Solvent system A consisted of cyclohexane(3) I. M. Jakovljevic, ANAL.CHEM., 35, 1513 (1963). (4) C . C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J . Am. Chem. Soc., 85, 2497 (1963).
Table I.
R,'s of Cardiac Glycosides and Related Compounds after Thin-Layer Chromatography R j in Solvent System A B
Compound
Figure 1. Structural formulas of cardiac glycosides and relateld compounds Compound Digitoxigenin Digoxigenin Gitoxigenin
Substitution RI - R: = H RI,R3 - Ri = H. Rt
Digitoxin, digoxin, gitoxin Ouabagenin
R1 = 1,3-tridigitoxoside
Ouabain
=
OH
Ri - R3
= H. Rq - Rr = OH R, = rhamnose
acetone-acetic acid (65 :33 :2 volume ratios). Chromatography in solvent system A involved six successive developments to a height of 15 cm above the origin. Solvent system B consisted of chloroform-ethanol (2 :1 volume ratio). Chromatography in solvent system B involved one development to a height of 10 cm above the origin. Thin-layer chromatography was conducted o n plates of Silica Gel G (Merck) 0.25 mm thick which had been prewashed in developing solvent and then activated for 30 minutes a t 110" C. The spots were detected using the Liebermann-Burchard reagent: acetic anhydride-sulfuric acidethanol ( 5 :5 :50 volume ratios). After spraying, the plates were heated to 110" C for 10 minutes for color development. Gitoxigenin monodigitoxoside contained a trace of gitoxigenin. Digitoxigenin bisdigitoxoside contained a trace of digitoxin. Ouabain contained trace amounts of three unidentified compounds. The remaining commercially available sterols and sterol glycosides were homogeneous in the solvent systems used. Preparation of TM8 Derivatives. The TMS derivatives were prepared by adding 1 rnl of silanizing mixture (hexamethyldisilazane-trimethylchlorosilane-pyridine,10 :1 : 10 volume ratios) to the dry alcohol. The mixture, which was contained in a polyethylene-capped, 1-dram glass vial, was shaken intermittently for 30 minutes a t room temperature. The resulting mixture vias transferred to a 2-ml tube capped with a silicon rubber septum, and the suspended particles were sedimented a t 2OClO X g for 30 minutes. The resulting clear solution was injec:ed directly onto the column for analysis. Sample delivery was performed using a Hamilton 10-pl syringe with a Chaney adaptation. In the quantitative analyses, digoxin or digitoxin was hydrolyzed t o yield the corresponding aglycone. Hydrolysis was effected by heating a solution containing 1 to 4 mg of sterol in 1 ml of 0.05N HCl for 15 minutes a t 100" C. (Hydrolysis of 10 mg witl-. 0.05N HCl solution requires heating for 35 minutes, with more extensive dehydration. Dehydration in the larger samples may be minimized by hydrolyzing for a shorter time interval in more concentrated HCl solution.) The 0.05N HCl solution contained 80% dioxane and 20% water. The hydrolyzate was neutralized by adding 0.5 ml of 0.1N ammonium hydroxide, and the resulting solution was evaporated to dryness under a jet of dry nitrogen gas for 2 hl3ur-s.
Digoxin Digoxigenin bisdigitoxoside Digoxigenin monodigitoxoside Digoxigenin Digitoxin Digitoxigenin bisdigitoxoside Digitoxigenin Gitoxigenin Gitoxigenin monodigitoxoside Ouabain Contaminant I in ouabain Contaminant I1 in ouabain Contaminant 111 in ouabain
0.21
0.77
0.29
0.40 0.50 0.52 0.58
0.83
0.74
0.54 0.44 0.00
0.13 0.73 0.82 0.95
Columns and Column Packings. All columns were Ushaped. Columns 1 and 2 were 6 feet long and had internal diameters of 6 mm. Columns 3 and 3' were 1 foot long and had internal diameters of 4 mm. The packing in column 1 consisted of 17; of SE 30 coated on 80- to 100-mesh Gas Chrom Q (Applied Science Labs, Inc.). Columns 2, 3, and 3 ' contained 1 . 6 x SE 30 (Analabs, Inc.) coated on 80- to 100-mesh Gas Chrom Q. The deactivated diatomaceous support was coated by suspending 20 grams of the dry support in 200 ml of 1 SE 30 in toluene for intervals of 20 to 40 minutes. The suspension was filtered on sintered glass and the packing was dried in an oven for 2 hours at 110" prior to filling a column. Packed columns 1 and 2 were conditioned for 24 hours at 300" C, with a nitrogen flow rate of 10 ml per minute, prior to usage. Columns 3 and 3' were conditioned at 340" prior to usage. The number of theoretical plates obtained for the TMS ethers of the sterois varied from 1800 to 2400 when these derivatives were chromatographed on column 1 or 2. The number of plates obtained for the TMS derivatives chromatographed on column 3 varied from 150 to 300. The operating column temperatures are indicated in the legends of the various figures. Injection block temperature was maintained at column temperature. Detector compartment temperature was 320" C for all runs except that shown in Figure 7, when it was 340" C. Chromatograph and Integrator. The instrument used in this study was a Barber-Colman 5000 series chromatograph equipped with hydrogen flame detectors. The detector response was displayed on a chart recorder. The recorder contained a retransmitting slide wire, which was supplied with an external voltage source so that deflection of the recorder pen resulted in production of linearly related dc voltage. This dc voltage was connected to a vuitage-frequency converter whose output of electrical pulses was counted by an electrical counter. The reading of the counter a t any time was proportional to the area under the curve drawn by the pen. RESULTS AND DISCUSSION
Gas Chromatography of Aglycones. Digoxigenin, digitoxigenin, and gitoxigenin were chromatographed on column 2 (Figure 2). The order of retention times is of interest in relation to the structural differences between digoxigenin and gitoxigenin. I t is possible that gitoxigenin undergoes instantaneous dehydration at carbon 14 or carbon 16 under these conditions. Each sterol gave a single peak when chromatographed individually. VOL. 39, NO. 1 , JANUARY 1967
41
W v)
z
4
0
L UI
75
Figure 2.
a 0
+ U W
A
c
A. Gitoxigenin B. Digitoxigenin C. Digoxigenin Nitrogen flow rate 100 ml/min
B
w 0 5 0
Chromatograms on column 2 at 250" C
C
W
2
c U -I W
L
2 5
0 I I
0
5
IO
15 MINUTES
20
25
35
30 I W W
100
F.
w VI
z 0
a
W VI
v)
z
W
0
I
a75 v)
e 7 5
n
K
W
e
0
K
IU
w
0 h
I-
V
: 5 0
i 50 n
W
->
Y
2
I-
I-
4 A
A
W
K 2 5
z 2 5
0
0
0 5
0
10 MINUTES
15
2 0
25
Figure 3. Chromatograms on column 2 at 250" C A. Digitoxigenin TMS ether B. Digoxigenin TMS ether Gitoxigenin TMS ether and digoxigenin TMS ether had identical retention times. Nitrogen flow rate 100 ml/min
5
10
15
20
25
MINUTES
Figure 4. Chromatograms on column 1 at 285" C A. Digoxigenin TRIS ether B. Digoxigenin monodigitoxoside TMS ether C. Ouabain TMS ether Nitrogen flow rate 100 ml/min
100
0
..
7 %
"r
Il i ! ' I I
The TMS ethers of the above-mentioned sterols were chromatographed under otherwise identical conditions and the results (Figure 3) indicated that gitoxigenin T M S ether and digoxigenin TMS ether had identical retention volumes. Chromatography of Sterol Glycosides. The TMS ethers of digoxigenin, digoxigenin monodigitoxoside, and ouabain were separated by chromatography on column 1 (Figure 4). The unpurified ouabain TMS ether gives a slightly fastermoving hump on this column. This hump was not identified, but was presumed to represent contaminants I, 11, and I11 mentioned in Table I.
II
4 Figure 5. Chromatograms on column 3 at 285" C
MINUTES
42
ANALYTICAL CHEMISTRY
A. Digoxigenin monodigitoxoside TMS ether B. Digoxigenin bisdigitoxoside TMS ether Nitrogen flow rate 100 ml/min
100
Table 11. Analysis of Sterol T M S Ether Concentration Mpmoles Average Relative Sterol TMS ether Sample of sample peak standard chromatographed No. injected area" deviation Digoxigenin 1 3.12 46.1 18.04 2 6.66 131.4 14.20 3 9.07 194.9 f6.18 4 12.93 286.2 11.90
C
W
In
z 0 0 In W
75 K
0 IV W
I-
50
Digoxigenin (hydrolyzed)
2
Digoxigenin (from hydrolyzed digoxin)
2
1
1.91 12.35
15.1 271.3
14.74 f2.14
1.93 3.20
13.54 f5.57 16.82 f6.23 15.72 17.68 12.45
W
->
I-
4 J W
K
2 5
Digitoxigenin
3
8.05
4
11.25
21.3 51.9 157.0 244.3
1
2.74 6.36 13.15
32.4 126.2 290.9
1
2 3
0 I r f I
5
0
10
20
15
25
MlNUTES
Figure 6. Chromatograms on column 3 a t 300" C A. Digoxigeniri monodigitoxoside TMS ether B. Digoxigeniri bisdigitoxoside TMS ether C. Digoxin TRlS ether Nitrogen flow rate 125 ml/min
100 W
ln f
0
a.
: u)
75
e 0
u W
Iw 50 n
C
w
-
> I-
4
25
a
0
-5
230
255
IO
I
I
I5 MINUTES
200 305 TEMP ( * C )
20
I
25
330
Figure 7. Chromatograms on column 3 A. Digoxigenini TMS ether B. Digoxigeniri monodigitoxoside TMS ether C. Digoxigeniri bisdigitoxoside TMS ether D. Digoxin TRlS ether Base line compensation in temperature programming interval achieved by bucking output of column 3 against that of column 3' during chromatography. Temperature programming initiated 1 minute after sample injection and proceeded at 6" C per minute over interval from 230" to 330" C. Nitrogen flow at 60 ml/min
I
30
62.0 Digitoxigenin 1 3.96 2 6.94 139.1 (from hydrolyzed 262.2 digitoxin) 3 11.92 a Numbers representing peak areas derived from counter in integrator used with gas chromatograph.
4~8.62 13.01 f3.78 electronic
Figures 5 and 6 represent chromatograms of TMS ethers of the mono-, di-, and trisaccharide derivatives of digoxigenin. The mono- and bisdigitoxoside T M S ethers underwent n o detectable thermal decomposition when injected and chromatographed a t 285" C. However, all of the glycoside T M S ethers underwent between 2 and.10 decomposition when injected directly onto this column at 300" C. In the case of digoxin T M S ether, two additional peaks appeared which had retention times identical t o those of the mono- and bisdigitoxoside T M S ethers of digoxigenin. The T M S ethers of gitoxin and digitoxin had retention times identical to that of digoxin T M S ether when chromatographed o n column 3. Figure 7 represents the results of chromatography of a mixture of the T M S ethers of digoxigenin, digoxigenin monodigitoxoside, digoxigenin bisdigitoxoside, and digoxin o n column 3. Injection of each of these T M S ethers individually, under these conditions, resulted in no detectable thermal decomposition. Quantitative Determination of Cardiac Glycosides. I n order to quantitate the determination of cardiac glycosides, the glycosides were hydrolyzed under carefully controlled conditions to permit minimal dehydration of carbon 14 of the sterol (5). However, approximately 1 dehydration occurred under our optimal conditions, as evidenced by the appearance of peak B in Figure 8 and peak B' in Figure 9. Cholesterol T M S ether was represented in these figures as a reference compound. I n Table I1 are listed the results obtained from chromatographing varying quantities of digoxigenin T M S ether. For each determination ten injections of 1 p1 of sample and ten injections of only the corresponding syringe needle contents were performed. Peak areas were corrected for needle content, which accounted for approximately 6 % of the total uncor rected peak areas. Linearity of detector response and the quantitative recovery of hydrolyzed glycoside aglycone T M S ether are shown ( 5 ) B. Kiliani, Arch. Pharm., 234, 276 (1896). VOL. 39, NO. 1 , JANUARY 1967
43
100.
W
W
v)
VI
z
z
0
0
0
n
;7 5 -
v)
7 5
w
a
a
K 0
K
I-
P V
0
u W
W
I-
w 50 n
500
Y
> -
I-
a
J Y
LL
2 5
0 0
3
6
9
I2
15
MINUTES
Figure 8. Chromatograms on column 1 at 240” C A. Cholesterol TMS ether B. Unknown TMS ether C. Digitoxigenin TMS ether Nitrogen flow rate 100 ml/min
in Figure 10. Quantitation of peak areas for unhydrolyzed digoxigenin TMS ether involved measurement of only one peak, whereas hydrolyzed digoxigenin or hydrolyzed digoxin always yielded two peaks. When more than one peak occurred, the sum of the areas of both peaks was ascertained in computing the points presented in Figure 10. From the four different concentrations of unhydrolyzed digoxigenin TMS ether (Table 11) a standard reference line (best-fit line using the method of least squares) was found to conform to the equation: y = -30.6 2 4 . 6 ~ . The equation for the line which includes all of the points determined for hydrolyzed digoxigenin and hydrolyzed digoxin was y = -29.0 2 4 . 5 ~ . The correlation coefficient for these two sets of data was 0.99, which is indicative of a very high degree of dependability in their direct interrelationship. The equation for a best-fit standard reference line which includes all the points determined for digitoxigenin and hydrolyzed digitoxin was y = -29.7 24.5~. The observation that the intercept of the best-fit straight line in Figure 10 occurs o n the abscissa rather than a t the origin indicates that some irreversible adsorption occurred on the column (6). This, however, does not indicate incomplete conversion of the sterol to the T M S ether, as has recently been reported for estrogens by Lau (7). Digitoxose TMS ether was chromatographed o n a 3 SE 52 liquid phase coated on Chromasorb P (4); the derivative had a retention time that was anticipated from the published results of Sweeley et al. (4). However, it is not possible to determine digitoxose following hydrolysis of digoxin, because the sugar undergoes rearrangement and partial degradation very rapidly in acid solution (8).
300
-
I 0 0
..
w
In
t 0 &
UI
+
+
+
(6) J. M. Richey, H. G. Richey, Jr., andR. Schraer, Anal. Biochem., 9, 272 (1964). (7) H. L. Lau, J . Gas Chromatog., 4, 136 (1966). (8) L. F. Fieser and M. Fieser, “Advanced Organic Chemistry,” p. 138, Reinhold, New York, 1962. 44
0
ANALYTICAL CHEMISTRY
>
/
0
2,Q
6.0 MlLLlMlCROMOLES
14.0
lO.0 OF
SAMPLE
INJECTED
Figure 10. Linearity of detector response to TMS ethers 0 Unhydrolyzed digoxigenin 0 Hydrolyzed digoxigenin
x Hydrolyzed digoxin 0 W
Unhydrolyzed digitoxigenin Hydrolyzed digitoxin
ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance and advice of E. A. Pfeiffer, E. D. Smith, and J. H. Doherty.
RECEIVED for review July 20, 1966. Accepted November 16, 1966.
