ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
2143
CORRESPONDENCE High Performance Liquid Chromatographic Separation of Insulin, Glucagon, and Somatostatin __
Sir: High performance liquid chromatography (HPLC) has
Table I. Recovery of Hormones fromHPLC Column Using Radioimmunoassay
received wide application in the separation of small molecular weight organic molecules but relatively few reports exist concerning the separation of proteins by this technique ( I ) . T h e major problem seems to be the development of chromatographic supports that possess the necessary hydrophyllic character in addition t o stability a t high pressures. Controlled-porosity glass bonded with glycerylpropylsilane have been developed as supports for exclusion chromatography employing HPLC (I). This support, termed "glycophase G", has been used with some success to separate serum proteins
hormone insulin
amount injected, P ga 117.6 30.1 2.7
120.8 30.8 2.5
103 102 93
54.4 12.1 5.8
45.9 11.4 5.2
84 94 90
glucagon
(2).
Research in this laboratory required rapid separation of the pancreatic hormones insulin, glucagon, and somatostatin. These hormones are proteins having molecular weights of approximately 6000 (insulin), 3500 (glucagon), and 2000 (somatostatin). Since insulin has been separated from other proteins by exclusion chromatography on Sephadex G-50 using acetic acid as an eluant (3),we investigated the possibility that similar solvent systems could be used with HPLC on glycophase-coated controlled-porosity glass. We report here the first H P L C method which produces excellent and rapid separation of these polypeptide hormones.
amount in HPLC eluant, recovery, P gb o/c
somatostatin
9.1 9.1 100 4.4 4.2 97 a Assay was performed on the solutions that were injected into the chromatograph. Assay was performed on the eluant representing the hormone peak detected by a UV monitor. method the pH was increased at a constant molarity of acetate. Figure 1A shows the separation achieved when a 20-min linear gradient, starting at the time of injection, progressed from 0.05 M acetate buffer, pH 3.0, to a 0.5 M acetate buffer of the same p H value. As shovm in Figure lB, separation was also achieved using a p H gradient and 0.05 M acetate buffer. T h e p H progressed linearly from 3.0 to 4.0 over a 20-min period. The peak shape was sharp and symmetrical, and resolution of the proteins appears to approach that which might be expected using gel electrophoresis. Initial attempts to separate the hormones employed relatively long columns (up to 1 m). Sharper peaks and better resolution were produced when much shorter columns were employed. T h e chromatograms shown were obtained using columns of 12.5 and 6 cm, and these short columns require less pressure to maintain flow rates of 1-2 niL/min. We investigated the recovery of various amounts of the hormones subjected to the HPLC procedure. T h e eluant representing each hormone peak was collected and assayed for hormone content by radioimmunoassay. T h e results in Table I show that each hormone injected into the chromatograph could be adequately recovered in the eluate. This was not unexpected since it is known that these hormones are stable under acidic conditions (6). Glycophase-bonded controlled-porosity glass has been reported to produce separation by size exclusion ( 1 ) . It is obvious from the data presented here that the hormones were not separated by size exclusion. Since the smaller molecular weight proteins eluted first, it would appear that separation was produced by another process, possibly adsorption. Only if the proteins were present in the column as aggregates with the somatostatin aggregate being largest. could separation have been achieved by size exclusion. This seems unlikely since Somatostatin aggregates have not been reported and glucagon and insulin form aggregates only a t very high concentrations (i.e.. >1 mg/mLi ( 7 . 8). \Ye cannot be certain that glycophase-bonded porous glass will separate proteins solely by a mechanism other than size
EXPERIMENTAL The chromatograph used consisted of two Model 6000A pumps, a Model 660 solvent programmer, and a U6K injector, all from Waters Associates, Milford, Mass. A Model UA-5 absorbance monitor (Isco, Lincoln, Neb.) operated at 280 nm was employed as the detector. The columns used were stainless steel, 1-mm i.d. and 6 cm or 12.5 cm in length. The chromatographic support, Corning Glycophase-G/CPG-100, particle size 5-10 pm (Lot No. D-07-32-C) was purchased from Pierce Chemical Co., Rockford, Ill. The columns were packed using a slurry in 2-propanol at a pressure of approximately 6000 Ib/in'. Porcine insulin was purchased from Schwarz-Mann, Orangeburg, N.Y., glucagon was donated by Eli Lilly, Indianapolis, Ind., and somatostatin was purchased from Calbiochem, San Diego. Calif. Solutions of these hormones for injection of 20 pL into the chromatograph were prepared in 1 N acetic acid. Recovery of each hormone after injection into the chromatograph was determined using radioimmunoassay. Various amounts of the hormones were injected and the eluant representing the peak for each hormone (detected by LV absorption) was collected and assayed for hormone content. An amount of hormone identical to that injected was diluted appropriately with 0.1 M acetate buffer, pH 3.0. and also subjected to radioimmunoassay. Insulin assay was according to the method of hlakulu et al. (41, glucagon by the procedure of Unger et al. (5): and somatostatin by the method of Pate1 and Reichin (6).
RESULTS AND DISCUSSION Various approaches were used in attempts to separate insulin, glucagon, and somatostatin. We report here the methods which produced the best separation of the hormones. Gradient elution HPLC was required since isocratic conditions produced separation of some but not all of the proteins. Two different types of gradients were utilized and they produced similar results. One procedure involved increasing the molarity of an acetate buffer a t constant p H while in the second c
1978 Aqerican Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
radiolabeled hormones produced in biochemical studies and for purposes such as hormone purification.
ACKNOWLEDGMENT We thank Y. Patel for the analysis of somatostatin.
LITERATURE CITED
0
10
20 m
n
Figure 1. (A) Chromatogram resulting f r o m injection of 7.5 p g somatostatin (s), 7.5 p g o f glucagon (g), and 10 pg of insulin (i) onto a 1 2 . 5 - c m glycophase-G/CPG column. A 20-min linear gradient progressed from 0.05 M acetate buffer, pH 3.0, to 0.5 M acetate buffer p H 3.0 a t a flow rate of 1.0 mL/min. (B) Chromatogram resulting from injection o f 2.5 pg of somatostatin (s), 4 p g of glucagon (g), and 15 p g of insulin (i) onto a 6 cm glycophase-G/CPG column. A 20-min linear gradient progressed f r o m 0.05 M acetate buffer, p H 3.0, to 0.05 M a c e t a t e buffer, p H 4.0, a t a flow rate of 1.0 m L / m i n
exclusion. Porous polystyrene gels, for example, separate by size exclusion and/or adsorption depending on the eluting solvent (9). Further experiments are necessary with glycophase-bonded porous glass supports to determine their mechanism of separation under various conditions. T h e UV absorption detection employed here does not provide sensitivity sufficient to measure physiological concentrations of these hormones. T h e HPLC method should prove to be extremely useful, however, for separation of
(1) F. E. Regnier, K . M. Gooding, and S. Chang, "High-speed liquid chromatography of proteins", in "Contemporary Topics in Analytical Clinical Chemistry", G. M. Hieftje, L. R. Snyder, and M. A. Evenson, Ed., Vol. 1, Plenum Press, New York, 1977, p 1-48. (2) F. E. Regnier and R . Noel, J . Chromafogr. Sci.. 14, 316 (1976). (3) K . J. O'Connor and N. Lazurus, Eiochem. J . , 156, 265 (1975). (4) D. R. Makulu, D. Vichick, P. H. Wright, K. E. Sussman, and P. Yu, Diabetes, 18, 660 (1969). (5) R . H. Unger, E. Aguilar-Parada, W. A. Muller, and A . Eisentraut, J . Clin. Invest., 49, 837 (1970). (6) Y. C. Patel and S. Reichin, "Radioimmunoassay of Somatostatin". in "Methods of Hwmone Radioimmunoassay", B. M. Jaffe and H. R. Behrman, Ed., Academic Press, New York, 1978, in press. (7) D. C. Hodgkin and D. Mercola, "The secondary and tertiary structure of insulin", in "Handbook of Physiology", Sec. 7 Endocrinology, Vol. 1, D. F. Seiner and N. Freinkel, Ed., American Physiol. SOC.,Washington, D.C., 1972, p 139. (8) W. W. Bromer "Chemical and physical properties of glucagon", in ' Glucagon", P. I . Lefebvre and R. H. Unger, Ed., Pergamon Press, New York, 1972, p 27. (9) S. Mori, Anal. Chem., 50, 745 (1978).
L. J. Fischer* R. L. Thies D. Charkowski Department of Pharmacology T h e University of Iowa Iowa City, Iowa 52242 RECEIVED for review August 18, 1978. Accepted September 25, 1978. Research supported in part by USPHS grant GM-12675 and by the Kroc Foundation.
