- Email: [email protected]
A general method for quantitative separation of prostaglandins by paper chromatography
ANALYTICAL
BIOCHEMISTRY
68, 6.54-657
A General Method Prostaglandins
(
1975)
for Quantitative Separation by Paper Chromatography
of
Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers ( I ,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Unger’s technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud ( 11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners. This communication describes the quantitative chromatographic separation of PGF,,, PGE,, PGA,, and arachidonic acid on commercially available Whatman SG-8 1 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents. METHODS
Whatman SG-8 1 chromatography paper was cut into 1-in-wide strips about 11 in. long and arranged for descending development. As the solvent front reached a predesignated origin, the sample was quickly introduced onto the moist paper (with the strip remaining in the solvent trough) and the tank resealed. At least 20 ~1 can be applied along the origin; wider strips could be used for larger samples. The spotting solution contained [ l-14C] arachidonic acid, [ l-14C] PG&, [ 5,6-3H] PGF,,, and [5,6-3H] PGA, in methanol. The chromatogram was developed for about 20 cm then scanned for radioactivity using a Packard 720 1 radiochroma654 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved
SHORT
COMMUNICATIONS
655
togram scanner with a Packard 385 recording ratemeter and Disc peak integrator. When necessary, radioactivity was determined independently in 15 ml of toluene/ethanol/Liquifluor (New England Nuclear), 70/30/4.2, using a Packard Model 3320 liquid scintillation spectrometer. The counting efficiency for 14C was about SO%, with the paper causing less than 10% quenching. RESULTS
AND
DISCUSSION
Control experiments showed that count recovery was greater than 90%, that negligible smearing occurred between peaks and that peak width was not appreciably increased by spotting onto the moist paper. The radiometer displayed linear correlation between integrator deflection and radioactivity, between chromatogram and recorder distances and among attenuator settings. The limit of detection for [l”C]arachidonic acid at 58 mCi/mmole was about 2 pmoles. Other means of detection could be applied for nonradioactive prostaglandins, concomitant with altered sensitivity. By applying the sample onto premoistened paper (wet spotting), chromatograms with negligible radioactivity at the origin were obtained using the solvent systems listed in Table 1. Conversely, up to 90% of PGF, was retained at the origin when the sample was allowed to dry on the paper prior to developing (dry spotting). Furthermore, when dry spotted, polar prostaglandins were more completely adsorbed, rendering quantitative separation impossible. To determine the resolving power of the system, the radioactive mixture was chromatogrammed using the following developing solvents and the wet spotting technique. 1. Ethyl acetate/isooctane/acetic
acid (60/40/0.5) acid (9011 O/ 1) I II. Ethyl ether/hexane/acetic acid (85/I S/O. 1) IV. Ethyl ether/methanol/acetic acid (90/2/0.1) V. Chloroform/methanol/acetic acid/water (90/S/ l/0.8) VI. Ethyl ether/methanol/acetic acid (90/l/2) VII. Benzene/chloroform/acetone/methanol/acetic acid (20/20/5/5/l). 11. Ethyl acetate/acetone/acetic
Listed in Table I are the 17~values obtained with these systems along with those obtained with silica-gel tic. Even with dry spotting, the developing solvent of Stamford and Unger (VII) left only negligible quantities of prostaglandins remaining at the origin; however, nonpolar prostaglandins and fatty acids were not resolved. On the other hand, solvents I-VI required wet spotting for quantitative separation. With silica-gel tic, PGD migrates between PGE and PGA, while the
656
SHORT
COMMUNICATIONS
TABLE
1
R, VALUES FOR PROSTAGLANDINS ON PAPER (PC) AND THIN-LAYER CHROMATOGRAPHY (TLC)
PGFl Solvent I II IIIC IV V VI VII
Reference” 4 4s 6 6 7 7 3
PGE,
PGA,
AAb
PC
t1c
PC
tic
PC
t1c
PC
tic
0.05 0.34 0.04 0.19 0.47 0.42 0.54
0.08 0.24 0.02 0.07 0.18 0.17 0.36
0.10 0.46 0.08 0.29 0.62 0.46 0.66
0.08 0.39 0.07 0.16 0.35 0.27 0.50
0.46 0.66 0.41 0.60 0.74 0.66 0.76
0.40 0.79 0.28 0.60 0.60 0.71 NDd
0.70 0.73 0.81 0.75 0.80 0.74 0.82
0.74 0.91 0.90 0.84 0.80 0.94 ND
n R, values for tic from these references. b AA, arachidonic acid. c Hexane substituted for petroleum ether. d ND, not determined.
peroxide intermediates of prostaglandin biosynthesis migrate between PGA and arachidonic acid (6,7). Based on these observations and the results in Table 1, solvent system IV is best suited for general separations using the paper. Solvent systems II and VII adequately separate PGF and PGE, whereas the peroxide intermediates should be best resolved by system III. Analysis and purification of these peroxide intermediates should be simplified by wet spotting, since they are known to rearrange into prostaglandins on contact with dry silica gel (8,9). In addition, 15-keto[ 5,6-3H] PGA,, formed by enzymatic dehydrogenation of [ 5 ,6-3H] PGA,, has been separated from the parent [ 5 ,6-3H] PGA, by solvent system I (10). Irreversible adsorption and consequent retention of nonpolar compounds at the origin of silica-impregnated cellulose paper occurs for separation problems other than those described in this communication. However, in the case of prostaglandins it is sufficiently bothersome that, despite its inherent advantages, paper chromatography is rarely used to separate these mixtures. The wet spotting procedure described herein overcomes this problem and should also be applicable to other separations. REFERENCES 1. 2. 3. 4. 5.
Singh, E. J., and Zuspan, F. P. (1974) Chromafographia 7, 200-202. Singh, E. J., Celic, L., and Swartwout, J. R. (1971) J. Chromatogr. 63, 321-327. Stamford, I F., and Unger, W. G. (1972) J. Physiol. (London) 225, 4P-5P. Humes, J., unpublished results. Andersen, N. H. (1969) J. Lipid Res. 10, 3 16-3 19.
657
SHORT COMMUNICATIONS
6. Miyamoto. T., Yamamoto, S.. and Hayaishi, 0. (1974) Proc. Nar. Acad. Sci. USA 71, 3645-3648. 7. Nugteren, D. H. and Hazelhof, E. (1973) Biochim. Biophys. Acra 326, 448-461. 8. Hamberg, M. and Samuelsson, B. (1973) Proc. Nar. Acad. Sci. USA 70, 899-903. 9. Willis, A. L., Vane, F. M., Kuhn, D. C., Scott, C. G., and Petrin, M. (1974) Prostaglandins
8, 453-507.
10. Oien, H. G., personal communication. 11. Tobias, L. and Paulsrud, J. R., (1975) Prostaglnndins
9, 57-60.
ROBERT Merck Institute for Therapeutic Rahway, New Jersey 07065 Received February 20, 1975;
Research accepted
Muy
IS, 1975
W. EGAN
BIOCHEMISTRY
68, 6.54-657
A General Method Prostaglandins
(
1975)
for Quantitative Separation by Paper Chromatography
of
Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers ( I ,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Unger’s technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud ( 11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners. This communication describes the quantitative chromatographic separation of PGF,,, PGE,, PGA,, and arachidonic acid on commercially available Whatman SG-8 1 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents. METHODS
Whatman SG-8 1 chromatography paper was cut into 1-in-wide strips about 11 in. long and arranged for descending development. As the solvent front reached a predesignated origin, the sample was quickly introduced onto the moist paper (with the strip remaining in the solvent trough) and the tank resealed. At least 20 ~1 can be applied along the origin; wider strips could be used for larger samples. The spotting solution contained [ l-14C] arachidonic acid, [ l-14C] PG&, [ 5,6-3H] PGF,,, and [5,6-3H] PGA, in methanol. The chromatogram was developed for about 20 cm then scanned for radioactivity using a Packard 720 1 radiochroma654 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved
SHORT
COMMUNICATIONS
655
togram scanner with a Packard 385 recording ratemeter and Disc peak integrator. When necessary, radioactivity was determined independently in 15 ml of toluene/ethanol/Liquifluor (New England Nuclear), 70/30/4.2, using a Packard Model 3320 liquid scintillation spectrometer. The counting efficiency for 14C was about SO%, with the paper causing less than 10% quenching. RESULTS
AND
DISCUSSION
Control experiments showed that count recovery was greater than 90%, that negligible smearing occurred between peaks and that peak width was not appreciably increased by spotting onto the moist paper. The radiometer displayed linear correlation between integrator deflection and radioactivity, between chromatogram and recorder distances and among attenuator settings. The limit of detection for [l”C]arachidonic acid at 58 mCi/mmole was about 2 pmoles. Other means of detection could be applied for nonradioactive prostaglandins, concomitant with altered sensitivity. By applying the sample onto premoistened paper (wet spotting), chromatograms with negligible radioactivity at the origin were obtained using the solvent systems listed in Table 1. Conversely, up to 90% of PGF, was retained at the origin when the sample was allowed to dry on the paper prior to developing (dry spotting). Furthermore, when dry spotted, polar prostaglandins were more completely adsorbed, rendering quantitative separation impossible. To determine the resolving power of the system, the radioactive mixture was chromatogrammed using the following developing solvents and the wet spotting technique. 1. Ethyl acetate/isooctane/acetic
acid (60/40/0.5) acid (9011 O/ 1) I II. Ethyl ether/hexane/acetic acid (85/I S/O. 1) IV. Ethyl ether/methanol/acetic acid (90/2/0.1) V. Chloroform/methanol/acetic acid/water (90/S/ l/0.8) VI. Ethyl ether/methanol/acetic acid (90/l/2) VII. Benzene/chloroform/acetone/methanol/acetic acid (20/20/5/5/l). 11. Ethyl acetate/acetone/acetic
Listed in Table I are the 17~values obtained with these systems along with those obtained with silica-gel tic. Even with dry spotting, the developing solvent of Stamford and Unger (VII) left only negligible quantities of prostaglandins remaining at the origin; however, nonpolar prostaglandins and fatty acids were not resolved. On the other hand, solvents I-VI required wet spotting for quantitative separation. With silica-gel tic, PGD migrates between PGE and PGA, while the
656
SHORT
COMMUNICATIONS
TABLE
1
R, VALUES FOR PROSTAGLANDINS ON PAPER (PC) AND THIN-LAYER CHROMATOGRAPHY (TLC)
PGFl Solvent I II IIIC IV V VI VII
Reference” 4 4s 6 6 7 7 3
PGE,
PGA,
AAb
PC
t1c
PC
tic
PC
t1c
PC
tic
0.05 0.34 0.04 0.19 0.47 0.42 0.54
0.08 0.24 0.02 0.07 0.18 0.17 0.36
0.10 0.46 0.08 0.29 0.62 0.46 0.66
0.08 0.39 0.07 0.16 0.35 0.27 0.50
0.46 0.66 0.41 0.60 0.74 0.66 0.76
0.40 0.79 0.28 0.60 0.60 0.71 NDd
0.70 0.73 0.81 0.75 0.80 0.74 0.82
0.74 0.91 0.90 0.84 0.80 0.94 ND
n R, values for tic from these references. b AA, arachidonic acid. c Hexane substituted for petroleum ether. d ND, not determined.
peroxide intermediates of prostaglandin biosynthesis migrate between PGA and arachidonic acid (6,7). Based on these observations and the results in Table 1, solvent system IV is best suited for general separations using the paper. Solvent systems II and VII adequately separate PGF and PGE, whereas the peroxide intermediates should be best resolved by system III. Analysis and purification of these peroxide intermediates should be simplified by wet spotting, since they are known to rearrange into prostaglandins on contact with dry silica gel (8,9). In addition, 15-keto[ 5,6-3H] PGA,, formed by enzymatic dehydrogenation of [ 5 ,6-3H] PGA,, has been separated from the parent [ 5 ,6-3H] PGA, by solvent system I (10). Irreversible adsorption and consequent retention of nonpolar compounds at the origin of silica-impregnated cellulose paper occurs for separation problems other than those described in this communication. However, in the case of prostaglandins it is sufficiently bothersome that, despite its inherent advantages, paper chromatography is rarely used to separate these mixtures. The wet spotting procedure described herein overcomes this problem and should also be applicable to other separations. REFERENCES 1. 2. 3. 4. 5.
Singh, E. J., and Zuspan, F. P. (1974) Chromafographia 7, 200-202. Singh, E. J., Celic, L., and Swartwout, J. R. (1971) J. Chromatogr. 63, 321-327. Stamford, I F., and Unger, W. G. (1972) J. Physiol. (London) 225, 4P-5P. Humes, J., unpublished results. Andersen, N. H. (1969) J. Lipid Res. 10, 3 16-3 19.
657
SHORT COMMUNICATIONS
6. Miyamoto. T., Yamamoto, S.. and Hayaishi, 0. (1974) Proc. Nar. Acad. Sci. USA 71, 3645-3648. 7. Nugteren, D. H. and Hazelhof, E. (1973) Biochim. Biophys. Acra 326, 448-461. 8. Hamberg, M. and Samuelsson, B. (1973) Proc. Nar. Acad. Sci. USA 70, 899-903. 9. Willis, A. L., Vane, F. M., Kuhn, D. C., Scott, C. G., and Petrin, M. (1974) Prostaglandins
8, 453-507.
10. Oien, H. G., personal communication. 11. Tobias, L. and Paulsrud, J. R., (1975) Prostaglnndins
9, 57-60.
ROBERT Merck Institute for Therapeutic Rahway, New Jersey 07065 Received February 20, 1975;
Research accepted
Muy
IS, 1975
W. EGAN