Determination of 5-~~~ns-Prostaglandin E2 in Prostaglandin E2 igh Performance Liquid Chromatography of Their pNitrophenacyl Esters on a Silver Ion-Loaded Cation Exchange Resin Margaret V. Merritt* and George
E. Bronson
Physical and Analyfical Chemistry Research, The Upjohn Company, Ka/amazoo, Mich. 4900 1
A high performance liquid chromatographic assay of trace amounts of 5-frans-PGE2 in PGE2 has been developed. This assay, capable of assaying as little as 0.20 YO 5-trans-PGE2 with a precision of & I O % , is based on the chromatographic separation of the p-nitrophenacyl esters of these compounds on a silver ion-loaded cation exchange resin. This chromatographic column resolves the PGE2-p-nitrophenacylesters from those of PGA2, PGB2, PGF,,, and PGF2,.
Most prostaglandins isolated from natural sources have the cis configuration at the 5,6 double bond. Prostaglandin A2 (PGA2) extracted from a soft coral, Plexaurea homomalla, however, was found to contain significant amounts of 5trans-PGA2 (1).Prostaglandin Ez (PGE2) derived from this source (2) is expected to be contaminated with 5-trans-PGE2. Although methods for the chemical synthesis of PGE2 are highly stereospecific (3-5), the possibility of the synthetic material containing trace amounts of the trans isomer remains. This possibility required the development of a procedure capable of measuring small amounts of 5-trans-PGE2 in PGE2
0
PG E 2
0
5-TRAN S-PGE 2 Chromatographic separation of a large number of closely related prostaglandins of the E-series as their p -nitrophenacyl esters on microparticulate silica gels has been recently reported (6). Formation of the uv-absorbing ester derivatives was necessary for detection of the prostaglandins in liquid chromatography with the commonly used 254-nm uv detector. Unfortunately, only partial resolution of 5-trans-PGEz and PGE2 was attained with these highly efficient silica gel columns. Weber has reported the separation of the methyl esters of PGAz and 5-trans-PGA2 by liquid chromatography on a reversed-phase column via the use of a mobile phase containing silver perchlorate (7). Because of the greater stability
constant of the 5-cis-PGAz-silver complex, this compound was eluted from the column prior to the trans isomer. Incorporation of silver ion into the solid chromatographic support was expected to reverse the elution order of olefinic isomers and, hence, enhance the sensitivity of the desired assay for 5-trans-PGEz. We report here the assay of this compound in PGE2 by liquid chromatography of the p-nitrophenacyl esters on a silver ion-loaded cation exchange resin as illustrative of the utility of this type solid support in high performance liquid chromatography.
EXPERIMENTAL All chromatography was done on a Varian 8500 dual pump liquid chromatograph with a Model 635 Variscan uv detector. Detection was a t 254 nm. The chromatographic columns were thermostated using a Haake Model F J circulating, constant temperature bath and homemade stainless steel column jackets. A 10-pl Precision Sampling Pressure-Lok syringe was used for sample injection. Chemicals. 2-Bromo-4'-nitroacetophenone (Eastman) and N,N-diisopropylethylamine (Aldrich) were used as received. Prostaglandin samples were obtained from the research labs of The Upjohn Company. The acetonitrile was Burdick-Jackson spectral grade. All other solvents used were Burdick-Jackson distilled in glass. Column Preparation. Six grams of Vydac strong cation exchange resin (The Separations Group) solid core, 30-44 micron particle size were equilibrated overnight a t room temperature in a 0.6 M aqueous solution of silver nitrate (Mallinckrodt). The solution and solids were transferred to a medium porosity Buchner funnel and washed with 800 ml of water; the final wash contained no detectable silver ion. The packing was then washed with 120 ml each of the following sequence of solvents: absolute ethanol, acetone, ethyl acetate, 1,lJ-trichloroethane (dried), and hexane. After drying the packing at 35-40 "C under vacuum for 4 h, a 1m X 2.1 mm i.d. stainless steel column was then packed with mechanical vibration a t reduced pressure. In Situ Preparation of p-Nitrophenacyl Esters. The p-nitrophenacyl esters of the prostaglandins were prepared for chromatography on the silver ion-loaded Vydac column by a modification of the procedure of Morozowich and Douglas (6). A 10-mg portion of the prostaglandin sample was weighed accurately into a 2-ml volumetric flask; 1 mi of a stock acetonitrile solution containing 15 mg/ml of 2bromo-4'-nitroacetophenone was added, followed by 5 pl of N,Ndiisopropylethylamine. The sample was sealed, mixed, and allowed to stand at least 2 h at room temperature. After removal of the acetonitrile with nitrogen, the residue was dissolved in 1ml of chloroform; 0.5 ml of aqueous silver nitrate (200 mg/ml) was added with thorough mixing and the sample was then centrifuged. The chloroform solution was filtered through a Millipore (0.2-pm pore size) filter just prior to chromatography. Precisely 4 pl of this chloroform solution was injected on the column for PGE2 assays. Assay of 5- trancs-PGEz in PGEZ. To construct a calibration curve, a series of samples of PGEz spiked with 5-trans-PGE2 covering the range from 0.20 t o 3.00% was prepared. These samples were derivatized and chromatographed on the silver ion-loaded Vydac column, thermostated at 26 "C, with a mobile phase consisting of a 55/45 ratio solvents A/B. A was a 2/1 (v/v) mixture of chloroform/hexane. B consisted of a 425/75 (v/v) mixture of A/acetonitrile. The flow rate was 30 ml h-l. Samples of PGEz to be assayed for 5-trans-PGE2 content were treated in an identical fashion. Under the above conditions, the p nitrophenacyl esters of prostaglandins A2 (PGAZ) and B2 (PGBz), other possible impurities in PGE2, were eluted with the solvent front.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
e
1851
n
Table I. Chromatographic k’Values of p-Nitrophenacyl Esters of Selected Prostaglandins on Ag(1)-Loaded Vydac Strong Cation Exchange Column Prostaglandin k’ Mobile Phase l o
Mobile Phase 2
PGA2 PGB2 5-trans-PGE2 PGE2 11-epi-PGEz 8-iso-PGE2 PGFi, PGF2, PGAz PGB2 PGEz
0 0
4.5 5.9 4.4 3.5 14.1 43.0 1.1 1.7 12.2
0 Mobile Phase I consisted of a 55/45 ratio of solvents A/B. A was a 2/1 (v/v) mixture of chloroform/hexane,B consisted of a 425/75 (v/v) mixture of A/acetonitrile. Mobile Phase I1 was a programmed mixture of A and B. Pump I contained Solvent A, Pump I1 contained Solvent B. Pump I ran 90%for 10 min followed by stepping Pump I1 at 4%/min to 50%.
Table 11.5- trans-PGEz Content of PGEz Samples Determined by HPLC of their p-Nitrophenacyl Esters on Ag(1)-Loaded Vydac Strong Cation Exchange Resin PGE2 sample
% 5-trans-PGEza
1 2 3
0.81 f 0.05 1.36 f 0.03 0.54 f 0.01 0.67 f 0.03 0.40 f 0.04
4 5 a
Precision is expressed as the standard deviation.
The mobile phase composition was programmed to examine PGEz for the presence of these materials: Pump I contained Solvent A; Pump I1 contained Solvent B. Pump I ran 90% for 10 min followed by stepping Pump I1 at 4%/minto 50%. RESULTS The chromatographic k’ values of the p -nitrophenacyl esters of selected prostaglandins are reported in Table I. A chromatogram of the derivatized PGE2 containing 1.40% of the 5-trans isomer is shown in Figure 1. The chromatographic peak height ratios of the p-nitrophenacyl ester of the 5-trans-PGE2 relative to that of PGE2 from the standard samples were determined. These data as a function of percent of 5-trans-PGEz were fitted to a straight line by least squares analysis; a correlation of 0.9997 and a relative standard deviation of the slope of 1.0% were obtained. The nonzero intercept of these curves was used to compute the 5-trans-PGEz content of the PGE2 used in preparing the standards. TLC had been previously used to show the presence of 5-trans-PGE2 in this sample. The trans content of several lots of PGE2 was calculated from the calibration curve; results of duplicate assays of several lots of PGE2 are reported in Table 11. Three separate batches of the silver ion-loaded Vydac were prepared and used to pack three 1-m columns during the course of this study. Although slightly different mobile phases were used with each column, the reproducibility of column preparation was satisfactory in that approximately the same number of theoretical plates and resolution of the p-nitrophenacyl esters of PGE2 and 5-trans-PGE2 were obtained for each column on initial use. The number of theoretical plates for the 5-trans-PGE2-p-nitrophenacyl ester was ca. 270; 1852
0
4
8 MINUTES
12
Figure 1. Chromatogram of PGE2 containing 1.40% 5-frans-PGE2 as the p-nitrophenacyl esters on Ag(l)-loaded Vydac with CHCI3/hexane/CH&N mobile phase
resolution of 1.2 was obtained for the esters of a (97/3) mixture of PGE2/5-trans-PGEz. A portion of the silver ion-treated Vydac packing was washed with concentrated ammonia. Acidification of these washings with hydrochloric acid yielded a white precipitate. Consequently, we concluded that the Vydac packing had been loaded with silver ion. One column was used continuously for six weeks. Column deterioration was evident in that, a t the end of this time period, the acetonitrile content of the mobile phase had to be reduced from 5.5 to 4.5% to maintain the retention times seen at the start of the study. Resolution of the two PGE2 isomers was, however, unaltered. DISCUSSION Complex-forming stationary phases have been used to achieve high selectivity in a variety of chromatographic systems (8,9).Several groups have recently reported the utility of silver-loaded silica gels for high performance liquid chromatography (HPLC) separations of geometrical isomers (9-13). Our preliminary efforts to resolve the p-nitrophenacyl esters of 5-cis- and trans-PGE2 via HPLC on silver-impregnated silica gel were, however, unsuccessful. Houx observed improved liquid chromatographic resolution of cis and trans alkene isomers on an ion exchange resin loaded with silver ion relative to that attained on silver-impregnated silica gel (14). A report of the preparative separation of 5-cis- and 5-transPGAz on a silver-loaded Amberlyst-15 further suggested that the desired resolution might be attained on a silver-loaded cation exchange packing ( 1 ) . The chromatogram shown in Figure 1illustrates the utility of the Vydac-silver ion column in achieving the separation of the PGE2 olefinic isomers. In addition to resolving the p nitrophenacyl esters of 5-cis- and trans-PGEz from one another, this column separated these materials from the corresponding esters of PGAz, PGB2, PGFl,, and PGF2,. Resolution was not adequate to separate completely the ester of 5-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
trans-PGE2 from those of 11-epi-PGEZ, PGE1, and 8-isoPGE2; other work had previously established the absence of these latter compounds in the PGEz samples examined. Morozowich and Douglas have demonstrated that the base-catalyzed esterification conditions used in our procedure yield quantitative conversion of the prostaglandins to p-nitrophenacyl esters (6). Our modification of that procedure with the addition of silver nitrate was necessary to remove bromide generated in the reaction and excess reagent. In the absence of this clean-up procedure, the silver ion-loaded Vydac column rapidly deteriorated with use, presumably through formation of silver bromide. It was found that the prostaglandin esters were stable to hydrolysis for several days when stored in chloroform following treatment with silver nitrate. Hydrolysis of these esters was significant in one day following silver nitrate treatment with either acetonitrile or methylene chloride as a solvent. Examination of samples under HPLC conditions in which the p-nitrophenacyl esters of PGA2 and PGB2 were resolved from that of PGE2 indicated that the cornbined esterification-silver nitrate treatment did not promote the conversion of PGEz to PGAz and PGB2. The role of the mobile phase in achieving the separation of the p-nitrophenacyl esters of PGEz and 5-trans-PGE2 should be emphasized. Poor resolution of these isomers was obtained with mobile phases of comparable polarity relative to that of those containing acetonitrile. The acetonitrile probably competes with the prostaglandins for Ag(1) binding sites. The role of this particular solvent has been noted in liquid chromatography on other silver-loaded columns (10). The retention volume of the p-nitrophenacyl ester of PGE2 was extremely sensitive to the acetonitrile content; a 1%increase in the acetonitrile content of the mobile phase yielded a 50% reduction in retention volumes. The performance of the Vydac-silver ion column was also dependent on the loading of the column; injection of amounts of the p-nitrophenacyl ester of PGE2 representing more than 50 gg of PGE2 resulted in reduced retention times and loss in resolution of the cistrans isomers. This maximal loading of 50 pg PGE2 limited the sensitivity of the assay to measuring 0.2% 5-trans-PGEz. The sensitivity of this column, both to small alterations in mobile phase composition and column loading, may be attributed to the low capacity of the Vydac support itself. This material consists of a solid core with a thin surface film of the cation exchanger (15). The capacity of the resin is 0.1 mequiv/g. Approximately 5 g of this material were used to prepare the packing for one column. Assuming each site contains a silver ion, only 10 ions per theoretical plate are available per molecule of PGE2 p -nitrophenacyl ester. (Rough
estimates by x-ray fluorescence suggest that the silver ion loading may be as little as 0.01 mequiv/g.) The use of microparticulate silver-ion loaded cation exchnge resins should permit higher column loading and reduced sensitivity to small alterations in the composition of the mobile phases; these systems are currently under investigation. Eight separate determinations of the 5-trans-PGEz in one PGE2 lot were made (see Table 11): % 5-trans-PGE2 was 0.40 f 0.03 at the 95% confidence level. Best previous estimates indicated that less than 1%(fl%)of this isomer was present in this lot of PGE2. The 10%relative standard deviation for these determinations should be a measure of the minimum precision of the assay, since the precision of the measurements increases with increasing 5-trans-PGE2 content. It should be noted that the use of PGEz itself as an internal standard for this assay is valid only if the sum of all impurities is small. Clearly, for routine use of this assay, an alternative internal standard should be used. Although the Vydac-silver ion column showed evidence of some deterioration through continuous use for six weeks, its performance was still adequate for the desired assay. The ease of preparation of the column packing material, the possibility of introducing metal ions (12)of varying complexing properties and consequent selectivity, and the wide choice of solvents compatible with the column packing should make metal-ion loaded cation exchange resins increasingly useful supports for HPLC.
LITERATURE CITED (1) G. L. Bundy, E. G. Daniels, F. H. Lincoln, and J. E. Pike, J. Am. Chem. Soc., 94, 2124(1972). G. L. Bundy, W. P. Schneider, F. H. Lincoln, and J. E. Pike, J. Am. Chem. Soc., 94, 2123 (1972). R. C. Kelly, V. Van Rheenen, I. Schletter, and M. D. Pillai, J. Am. Chem. SOC.,95, 2746 (1973). E. J. Corey, N. M. Weinshenker, T. K. Sehaaf, and W. Huber, J , Am. Chem. SOC..91. 5675 (1969). E. J.' Corey and G. ?. Kwiatkowski, J. Am. Chem. Soc., 88, 5652
(1966). W. Morozowich and S.L. Douglas, Prostaglandins, 10, 19 (1975). D. J. Weber, J. Pharm. Sci., to be published. 0. K. Gaha and J. Janak, J. Chromatogr. (Rev.)68, 325 (1972). R. Aigner, H. Spitzy, and R. W. Frei, Anal. Chem., 48, 2 (1976). R. Vivilecchia, M. Thiebaud, and R. W. Frei, J. Chromatogr. Sci., 10,411 (1972). R. W. Frei, K. Beall, and R. M. Cassidy, Mikrochim. Acta, 859 (1974). F. Mikes, V. Schurig, and E. Gil-Av, J. Chromatogr., 83, 91 (1973). R. R. Heath, J. H. Tumlinson, R. E. Doolittle, and A. T. Proveaux, J. Cbromatogr. Sci., 13, 380 (1975). N. W. H. Houx, S. Voerma, and W. M.F. Jongen, J. Chromatogr., 98, 25 (1974). Applied Science Laboratories, Inc., Catalog 19, 1976, p 48.
RECEIVEDfor review April 26, 1976. Accepted July 26, 1976.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
1853
