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Radio gas chromatography of steroid metabolites by collection of radioactive fractions on thin layer chromatography plates
ANALYTICAL
BIOCHEMISTRY
72,391-3%
(1976)
Radio Gas Chromatography of Steroid Metabolites by Collection of Radioactive Fractions on Thin Layer Chromatography Plates’ H. J. G. M. DERKS,
F. A. J. MUSKIET,
AND N. M. DRAYER
Department of Pediatrics,
University Hospital, Groningen,
The Netherlands
Received December 9. 1974; accepted January 30. 1976 A simple and inexpensive technique for the monitoring of radioactive compounds in gas chromatographic effluent is described. Use has been made of the adsorption properties of thin layer chromatography plates to trap eluting radioactive peaks. Performance of the technique is demonstrated by analysis of radioactive steroid mixtures. The efficiency and sensitivity of this technique equal that of more complicated techniques.
Although numerous applications have been found for gas chromatography in steroid analysis (1,2) relatively few publications deal with the gas chromatographic tracing of radioactive steroid metabolites (3-5). The reasons for this are probably the high costs of instrumentation in radio gas chromatography and the moderate sensitivity of continuous flow monitoring techniques (6,7). Confronted with these problems in our work on the identification of steroid metabolites by means of tracer studies, we decided to trap the gas chromatographic effluent on a moving tic plate and count the scraped off adsorbent in a liquid scintillation counter. This versatile method has proved to be highly efficient, sensitive, and also inexpensive. In the present paper the characteristics of the system are demonstrated as well as the performance of the technique in tracing low amounts of radioactivity in steroids. MATERIALS
AND METHODS
Radioactive steroids were purchased from the Radiochemical Centre, Amersham, United Kingdom and from New England Nuclear, Dreieichenhain, Germany, and were purified by tic before use to give at least 95% purity. Nonradioactive reference steroids were obtained from Steraloids, Pawling, New Jersey. Silylation and methoximation reagents were purchased from Pierce Chemical Company, Rockford, Illinois, ready made tic plates from both Machery & Nagel, D&en, Germany and Merck, Darmr This work is supported by the Foundation for Fundamental Medical Research (FungoZWO, The Netherlands). 391 Copyright 0 1976 by Academic Press. Inc. All nghts of reproductmn III any form reserved.
392
DERKS.
MUSKIET
AND DRAYER
FIG. 1. The splitter and heated outlet assembly. The splitter and glass outlet are connected by PTFE shrinkable tubing. The splitting ratio is 1:30, the smaller part being fed to the FID. The bottom half of the cylinder can be separated from the upper part in order to install the glass outlet tubing into the cylinder. The right end of the cylinder is covered and insulated by asbestos.
stadt, Germany, and 2rY2%OV- 1 on Chromosorb G-HP from Chrompack, Vlissingen, The Netherlands. A Packard-Becker model 407 gas chromatograph, equipped with a flame ionization detector (FID) was fitted with a detector splitter and a heated all glass outlet as can be seen in Fig. 1. The outlet assembly consisted of 18 cm of glass tubing (i.d. 2 mm) fitted in a brass cylinder, which was suspended to the movable oven cover. The outer end of the glass tubing was bent downward leading through a hole in the wall of the brass cylinder. The brass cylinder was heated by Pilz heating tape of 170 W power, fed by a variable transformer. The bent tip of the outlet tubing was positioned as close as possible to the adsorbent layer of a tic plate. This plate could be moved horizontally by a Camag Diochrom apparatus. .This latter instrument enables both continuous and stepwise movement of the tic plate. The splitting ratio of the detector splitter is determined by pieces of TABLE
1
GAS CHROMATOGRAPHIC
CONDITIONS
Column: 2 m , 2 mm i.d. glass column packed with 2.5% OV-I on Chromosorb G-HP (80- 100 mesh) Carrier gas: nitrogen 20 ml/min Temperatures: injection port FID Oven program Outlet
260°C 260°C 210-28O’C at 4OC/min 260°C
TRACER
STUDIES
OF STEROID TABLE
RECOVERIESOFTHE DETERMINED
Radioactive
INDWIDLJAL IN A MIXTURE
[I .2-3H]-3~-hydroxy-5a-androstene17.one [7-3H]-3P-hydroxy-5-androstene-17-one [1,2,6,7-SH]-17/3-hydroxy-4-androstene-3-one [I ,2,6.7-3H]-4-pregnene-3 .20-dione [I ,2-3H]-17a-hydroxy-4-pregnene-3,2~dione [1.2.6,7-3H]-17a.21-dihydroxy-4-pregnene-3.1 trione [1,2.6.7-3H]-1 I@. 17u.?l-trihydroxy-4-pregnene3.2O-dione [4-‘*Cl-l I@, 17a,21-trihydroxy-4-pregnene-3,20dione
2 RADIOACTIVE STEROIDS OF NINE STEROIDS”
Abbreviation
steroid*
393
METABOLITES
Relative retention
AS
Percentage
Coefficient of vatiatio#
time
recoveryC
A DHEA T P 170HP
0.685 0.795 0.870 I.111 1.222
82.4 74.2 77.8 70.2 70.9
6.9 7.7 5.2 6.9 7.3
E
I.611
64.8
6.7
F W)
1.722
55.6
6.5
F (“C)
1.722
56.0
6.8
l,20-
a Besides the eight listed radioactive steroids the mixturecontained also 301,20a-dihydroxy-Sppregnane (see text). * injected amount was 1 pg (2000 dpm) for each steroid. c Recovery was measured by scraping off the trapped radioactivity, and counting the fractions of the individual steroids. The recovery was calculated as the counted radioactivity divided by the injected amount. The recovery is not corrected for loss due to the amount that is split off to the FID. d Mean of six experiments,
capillary tubing of appropriate length and diameter, positioned in outletand FID-tubing. To prevent the radioactivity from escaping to the direct environment of the apparatus, the tic trapping equipment was placed in a box sucked off to open air. The FID outlet gas was trapped in an adsorbent (Molekularsieb 3, Merck, Germany) by suction. The gas chromatograph was operated under the conditions mentioned in Table 1. All steroid material was methoximated and persilylated prior to gas chromatography. The reaction conditions were: methoximation-2 hr at 60°C in a 2% solution of methoxylamine. HCl in pyridine; silylation-2 hr at 100°C in a mixture of trimethylsilyl-imidazole, bis- (trimethylsilyl) acetamide and trichlorosilane (3/3/2). To establish the characteristics of the system, radioactive steroid MOTMS derivatives and mixtures of them were injected into the gas chromatograph and the eluate trapped on tic plates with different adsorbents and under varying conditions. Advancement speeds of tic plate and recorder stripchart were, respectively, 1.0 and 0.5 cm/min. The tic plates were scanned for radioactivity by a Berthold tic radioactivity scanner or scraped off in zones corresponding to the positions of the peaks in the gas chromatogram. For uv absorbing steroids (A-4,3 keto structure) the latter method could be checked by viewing the tic plate in uv light (254 nm). The
394
DERKS,
MUSKIET
AND
DRAYER
scraped off adsorbent was put in counting vials and scintillation fluid was added. The vials were counted for 10 min in a Nuclear Chicago Mark II liquid scintillation counter. Results were corrected for quenching by the external standard method. Besides the several radioactive steroids listed in Table 2, also one nonradioactive steroid (3a,20a-dihydroxy-5P-pregnane, “P2”) was used in the experiments. All radioactive steroids were diluted with unlabeled reference steroids to give specific activities of 0.91 &i/mg (2,000 dpm/pg). RESULTS
For maximum recovery of the injected radioactivity on the tic plate it was essential to keep the distance between outlet tip and plate as small as possible. No significant influence on the performance of the trapping process could be detected, when linear gas velocity or column temperature was varied, nor was there any measurable difference in recovery for different tic adsorbents such as silica gel, kieselguhr or alumina. Machery & Nagel silica gel plates of 0.25 mm thickness were eventually selected, as they are easily scraped off. Under the conditions used, no significant band broadening effect resulting from the trapping process was observed, as can be seen in Fig. 2, where both a gas chromatogram and a radioactivity scan of the matching tic plate are shown. After some time, columns tended to show increased irreversible adsorption, resulting in a decrease of peak heights in the gas chromatogram and a similar decrease of recovery of injected radioactivity on the tic plate. A reliable criterion for the column quality with respect to irreversible adsorption was the relative peak height of the used radioactive steroids in the gas chromatogram compared to the peak height of 3a,20a-dihydroxy-So-pregnane. The latter steroid suffered least from increasing irreversible adsorption. During our experiments only columns with comparable and constant relative peak heights were used. The recoveries of the individual radioactive steroids as determined in a mixture are shown in Table 2. Standard deviation for the individual values ranged from 58% of the mean. With respect to sensitivity we found that even if 80 dpm of [l ,2,6,7-3H]-17p-hydroxy-4-androstene-3-one (T) was injected, the radioactivity recovered for this steroid on the tic plate was five times higher than background. Background was measured by counting the radioactivity eluted from the column not coincident to the position of T on the tic plate and correcting this value for an area equal to the size of the T spot. DISCUSSION
The recoveries of the investigated radioactive steroids (Table 2) were satisfactory as compared with other methods (58.9). We observed a
TRACER
STUDIES
OF STEROID
METABOLITES
395
CPS
3
220 20 l/-i
10 r;meCmm
\ ”
0 I
IS’
0 L:‘u I
27
78
1
75 time
Imm
72 )
9
t 6
FIG. 2. Gas chromatogram of a mixture of steroid MO-TMS derivatives and a radioactivity scan of the matching tic plate. Gas chromatographic conditions were as mentioned in Table I. For abbreviations see Table 2. The injected amount was 1.5 pg (3000 dpm) for each steroid. The tic plate advancement was started 6 min after injection with a speed of 1 cm/min. Radioactivity on the tic plate was scanned at a speed of 12 mmihr with a time constant of 100 set and a slit width of 1 mm. The high amount of radioactivity in the F peak results from the higher efficiency of the detector in the tic scanner for 14C radiation.
roughly linear decrease in recovery of the used radioactive steroids with increasing relative retention time. This phenomenon cannot be accounted for by differences in trapping efficiency considering the fact that the method is not sensitive to changes in column temperature and carrier gas velocity. In our opinion this decrease in recovery with increasing relative retention time must be attributed to irreversible adsorption on the column packing. This means that the quantitative determination of the radioactivity in a steroid by this method requires the use of the same steroid with a different label or a different steroid with comparable recovery, in order to correct for trapping efficiency. As already mentioned, no significant band broadening of the radioactivity on the tic plate was observed. This is essential for the accurate re-
396
DERKS,
MUSKlET
AND DRAYER
moval of radioactive spots from the tic plate with the gas chromatogram as a guide. Due to the smooth character of the trapping technique employed here, there was no influence on the stability of the baseline in the gas chromatogram. However in trapping techniques using fraction collectors, adjustments have to be made to keep the baseline stable (8). We regard this method as an inexpensive alternative to other gas chromatographic radiotracing techniques. The main advantage of this method compared to gas flow monitoring techniques, apart from less expense, is higher sensitivity (5,9). Other trapping techniques require complicated fraction collectors or a tape system (lo), while their trapping efficiency does not exceed the efficiency of our system (8). In conclusion it can be stated that this method can be used to detect very low amounts of radioactive steroid metabolites in biological material, thus reducing the amount ofradioactivity to be injected in humans. A study concerning the assay of steroid production rate by this method is in progress. ACKNOWLEDGMENTS We wish to thank Dr. A. Groen and Dr. M. G. Woldringh for providing instrument facilities, and Dr. B. G. Wolters and Mr. J. J. Pratt for their advice.
REFERENCES 1. Eik-Nes, K. B., Horning, E. C. (1968) Gas phase chromatography of steroids, Monographs on Endocrinology (Labhart, A., Mann, T., Samuels, L. T., and Zander, J., eds.), Vol. 2. Springer-Verlag. Berlin. 2. Wotiz, H. H.. and Clark, S. J. (1966) Gas Chromatography in the Analysis of Steroids, Plenum Press, New York. 3. Cronholm. T., Eriksson, H., and Gustafsson, J. A. (1971) Eur. J. Biochem. 19,424. 4. Eriksson, H. (1970) Eur. J. Biochem. 16, 261. 5. Swell, L. (1966)Anal. Biochem. 16, 70. 6. Cram, S. P. (1970) Advances in Chromatography, Giddings, J. C., and Keller. R. A., eds.), Vol. 9, p. 243, Marcel Dekker. New York. 7. Karmen, A. (1967)J. Gas Chromatogr. 5, 502. 8. Cooke, B. A. (1969)Anal. Biochem. 32, 198. 9. Karmen, A., McCaffrey, I., and Kliman. B. (1963) Anal. Biochem. 6, 31. 10. Schiff. R. M. (1966). U.S. PAT 3, 290, 501.
BIOCHEMISTRY
72,391-3%
(1976)
Radio Gas Chromatography of Steroid Metabolites by Collection of Radioactive Fractions on Thin Layer Chromatography Plates’ H. J. G. M. DERKS,
F. A. J. MUSKIET,
AND N. M. DRAYER
Department of Pediatrics,
University Hospital, Groningen,
The Netherlands
Received December 9. 1974; accepted January 30. 1976 A simple and inexpensive technique for the monitoring of radioactive compounds in gas chromatographic effluent is described. Use has been made of the adsorption properties of thin layer chromatography plates to trap eluting radioactive peaks. Performance of the technique is demonstrated by analysis of radioactive steroid mixtures. The efficiency and sensitivity of this technique equal that of more complicated techniques.
Although numerous applications have been found for gas chromatography in steroid analysis (1,2) relatively few publications deal with the gas chromatographic tracing of radioactive steroid metabolites (3-5). The reasons for this are probably the high costs of instrumentation in radio gas chromatography and the moderate sensitivity of continuous flow monitoring techniques (6,7). Confronted with these problems in our work on the identification of steroid metabolites by means of tracer studies, we decided to trap the gas chromatographic effluent on a moving tic plate and count the scraped off adsorbent in a liquid scintillation counter. This versatile method has proved to be highly efficient, sensitive, and also inexpensive. In the present paper the characteristics of the system are demonstrated as well as the performance of the technique in tracing low amounts of radioactivity in steroids. MATERIALS
AND METHODS
Radioactive steroids were purchased from the Radiochemical Centre, Amersham, United Kingdom and from New England Nuclear, Dreieichenhain, Germany, and were purified by tic before use to give at least 95% purity. Nonradioactive reference steroids were obtained from Steraloids, Pawling, New Jersey. Silylation and methoximation reagents were purchased from Pierce Chemical Company, Rockford, Illinois, ready made tic plates from both Machery & Nagel, D&en, Germany and Merck, Darmr This work is supported by the Foundation for Fundamental Medical Research (FungoZWO, The Netherlands). 391 Copyright 0 1976 by Academic Press. Inc. All nghts of reproductmn III any form reserved.
392
DERKS.
MUSKIET
AND DRAYER
FIG. 1. The splitter and heated outlet assembly. The splitter and glass outlet are connected by PTFE shrinkable tubing. The splitting ratio is 1:30, the smaller part being fed to the FID. The bottom half of the cylinder can be separated from the upper part in order to install the glass outlet tubing into the cylinder. The right end of the cylinder is covered and insulated by asbestos.
stadt, Germany, and 2rY2%OV- 1 on Chromosorb G-HP from Chrompack, Vlissingen, The Netherlands. A Packard-Becker model 407 gas chromatograph, equipped with a flame ionization detector (FID) was fitted with a detector splitter and a heated all glass outlet as can be seen in Fig. 1. The outlet assembly consisted of 18 cm of glass tubing (i.d. 2 mm) fitted in a brass cylinder, which was suspended to the movable oven cover. The outer end of the glass tubing was bent downward leading through a hole in the wall of the brass cylinder. The brass cylinder was heated by Pilz heating tape of 170 W power, fed by a variable transformer. The bent tip of the outlet tubing was positioned as close as possible to the adsorbent layer of a tic plate. This plate could be moved horizontally by a Camag Diochrom apparatus. .This latter instrument enables both continuous and stepwise movement of the tic plate. The splitting ratio of the detector splitter is determined by pieces of TABLE
1
GAS CHROMATOGRAPHIC
CONDITIONS
Column: 2 m , 2 mm i.d. glass column packed with 2.5% OV-I on Chromosorb G-HP (80- 100 mesh) Carrier gas: nitrogen 20 ml/min Temperatures: injection port FID Oven program Outlet
260°C 260°C 210-28O’C at 4OC/min 260°C
TRACER
STUDIES
OF STEROID TABLE
RECOVERIESOFTHE DETERMINED
Radioactive
INDWIDLJAL IN A MIXTURE
[I .2-3H]-3~-hydroxy-5a-androstene17.one [7-3H]-3P-hydroxy-5-androstene-17-one [1,2,6,7-SH]-17/3-hydroxy-4-androstene-3-one [I ,2,6.7-3H]-4-pregnene-3 .20-dione [I ,2-3H]-17a-hydroxy-4-pregnene-3,2~dione [1.2.6,7-3H]-17a.21-dihydroxy-4-pregnene-3.1 trione [1,2.6.7-3H]-1 I@. 17u.?l-trihydroxy-4-pregnene3.2O-dione [4-‘*Cl-l I@, 17a,21-trihydroxy-4-pregnene-3,20dione
2 RADIOACTIVE STEROIDS OF NINE STEROIDS”
Abbreviation
steroid*
393
METABOLITES
Relative retention
AS
Percentage
Coefficient of vatiatio#
time
recoveryC
A DHEA T P 170HP
0.685 0.795 0.870 I.111 1.222
82.4 74.2 77.8 70.2 70.9
6.9 7.7 5.2 6.9 7.3
E
I.611
64.8
6.7
F W)
1.722
55.6
6.5
F (“C)
1.722
56.0
6.8
l,20-
a Besides the eight listed radioactive steroids the mixturecontained also 301,20a-dihydroxy-Sppregnane (see text). * injected amount was 1 pg (2000 dpm) for each steroid. c Recovery was measured by scraping off the trapped radioactivity, and counting the fractions of the individual steroids. The recovery was calculated as the counted radioactivity divided by the injected amount. The recovery is not corrected for loss due to the amount that is split off to the FID. d Mean of six experiments,
capillary tubing of appropriate length and diameter, positioned in outletand FID-tubing. To prevent the radioactivity from escaping to the direct environment of the apparatus, the tic trapping equipment was placed in a box sucked off to open air. The FID outlet gas was trapped in an adsorbent (Molekularsieb 3, Merck, Germany) by suction. The gas chromatograph was operated under the conditions mentioned in Table 1. All steroid material was methoximated and persilylated prior to gas chromatography. The reaction conditions were: methoximation-2 hr at 60°C in a 2% solution of methoxylamine. HCl in pyridine; silylation-2 hr at 100°C in a mixture of trimethylsilyl-imidazole, bis- (trimethylsilyl) acetamide and trichlorosilane (3/3/2). To establish the characteristics of the system, radioactive steroid MOTMS derivatives and mixtures of them were injected into the gas chromatograph and the eluate trapped on tic plates with different adsorbents and under varying conditions. Advancement speeds of tic plate and recorder stripchart were, respectively, 1.0 and 0.5 cm/min. The tic plates were scanned for radioactivity by a Berthold tic radioactivity scanner or scraped off in zones corresponding to the positions of the peaks in the gas chromatogram. For uv absorbing steroids (A-4,3 keto structure) the latter method could be checked by viewing the tic plate in uv light (254 nm). The
394
DERKS,
MUSKIET
AND
DRAYER
scraped off adsorbent was put in counting vials and scintillation fluid was added. The vials were counted for 10 min in a Nuclear Chicago Mark II liquid scintillation counter. Results were corrected for quenching by the external standard method. Besides the several radioactive steroids listed in Table 2, also one nonradioactive steroid (3a,20a-dihydroxy-5P-pregnane, “P2”) was used in the experiments. All radioactive steroids were diluted with unlabeled reference steroids to give specific activities of 0.91 &i/mg (2,000 dpm/pg). RESULTS
For maximum recovery of the injected radioactivity on the tic plate it was essential to keep the distance between outlet tip and plate as small as possible. No significant influence on the performance of the trapping process could be detected, when linear gas velocity or column temperature was varied, nor was there any measurable difference in recovery for different tic adsorbents such as silica gel, kieselguhr or alumina. Machery & Nagel silica gel plates of 0.25 mm thickness were eventually selected, as they are easily scraped off. Under the conditions used, no significant band broadening effect resulting from the trapping process was observed, as can be seen in Fig. 2, where both a gas chromatogram and a radioactivity scan of the matching tic plate are shown. After some time, columns tended to show increased irreversible adsorption, resulting in a decrease of peak heights in the gas chromatogram and a similar decrease of recovery of injected radioactivity on the tic plate. A reliable criterion for the column quality with respect to irreversible adsorption was the relative peak height of the used radioactive steroids in the gas chromatogram compared to the peak height of 3a,20a-dihydroxy-So-pregnane. The latter steroid suffered least from increasing irreversible adsorption. During our experiments only columns with comparable and constant relative peak heights were used. The recoveries of the individual radioactive steroids as determined in a mixture are shown in Table 2. Standard deviation for the individual values ranged from 58% of the mean. With respect to sensitivity we found that even if 80 dpm of [l ,2,6,7-3H]-17p-hydroxy-4-androstene-3-one (T) was injected, the radioactivity recovered for this steroid on the tic plate was five times higher than background. Background was measured by counting the radioactivity eluted from the column not coincident to the position of T on the tic plate and correcting this value for an area equal to the size of the T spot. DISCUSSION
The recoveries of the investigated radioactive steroids (Table 2) were satisfactory as compared with other methods (58.9). We observed a
TRACER
STUDIES
OF STEROID
METABOLITES
395
CPS
3
220 20 l/-i
10 r;meCmm
\ ”
0 I
IS’
0 L:‘u I
27
78
1
75 time
Imm
72 )
9
t 6
FIG. 2. Gas chromatogram of a mixture of steroid MO-TMS derivatives and a radioactivity scan of the matching tic plate. Gas chromatographic conditions were as mentioned in Table I. For abbreviations see Table 2. The injected amount was 1.5 pg (3000 dpm) for each steroid. The tic plate advancement was started 6 min after injection with a speed of 1 cm/min. Radioactivity on the tic plate was scanned at a speed of 12 mmihr with a time constant of 100 set and a slit width of 1 mm. The high amount of radioactivity in the F peak results from the higher efficiency of the detector in the tic scanner for 14C radiation.
roughly linear decrease in recovery of the used radioactive steroids with increasing relative retention time. This phenomenon cannot be accounted for by differences in trapping efficiency considering the fact that the method is not sensitive to changes in column temperature and carrier gas velocity. In our opinion this decrease in recovery with increasing relative retention time must be attributed to irreversible adsorption on the column packing. This means that the quantitative determination of the radioactivity in a steroid by this method requires the use of the same steroid with a different label or a different steroid with comparable recovery, in order to correct for trapping efficiency. As already mentioned, no significant band broadening of the radioactivity on the tic plate was observed. This is essential for the accurate re-
396
DERKS,
MUSKlET
AND DRAYER
moval of radioactive spots from the tic plate with the gas chromatogram as a guide. Due to the smooth character of the trapping technique employed here, there was no influence on the stability of the baseline in the gas chromatogram. However in trapping techniques using fraction collectors, adjustments have to be made to keep the baseline stable (8). We regard this method as an inexpensive alternative to other gas chromatographic radiotracing techniques. The main advantage of this method compared to gas flow monitoring techniques, apart from less expense, is higher sensitivity (5,9). Other trapping techniques require complicated fraction collectors or a tape system (lo), while their trapping efficiency does not exceed the efficiency of our system (8). In conclusion it can be stated that this method can be used to detect very low amounts of radioactive steroid metabolites in biological material, thus reducing the amount ofradioactivity to be injected in humans. A study concerning the assay of steroid production rate by this method is in progress. ACKNOWLEDGMENTS We wish to thank Dr. A. Groen and Dr. M. G. Woldringh for providing instrument facilities, and Dr. B. G. Wolters and Mr. J. J. Pratt for their advice.
REFERENCES 1. Eik-Nes, K. B., Horning, E. C. (1968) Gas phase chromatography of steroids, Monographs on Endocrinology (Labhart, A., Mann, T., Samuels, L. T., and Zander, J., eds.), Vol. 2. Springer-Verlag. Berlin. 2. Wotiz, H. H.. and Clark, S. J. (1966) Gas Chromatography in the Analysis of Steroids, Plenum Press, New York. 3. Cronholm. T., Eriksson, H., and Gustafsson, J. A. (1971) Eur. J. Biochem. 19,424. 4. Eriksson, H. (1970) Eur. J. Biochem. 16, 261. 5. Swell, L. (1966)Anal. Biochem. 16, 70. 6. Cram, S. P. (1970) Advances in Chromatography, Giddings, J. C., and Keller. R. A., eds.), Vol. 9, p. 243, Marcel Dekker. New York. 7. Karmen, A. (1967)J. Gas Chromatogr. 5, 502. 8. Cooke, B. A. (1969)Anal. Biochem. 32, 198. 9. Karmen, A., McCaffrey, I., and Kliman. B. (1963) Anal. Biochem. 6, 31. 10. Schiff. R. M. (1966). U.S. PAT 3, 290, 501.