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equaled only by the spectroelectrochemical approach of Jan et al. (1, 2). Selectivity is available through control of the microelectrode potential, making it possible to study the fate of different chemical species in the diffusion layer. Previous workers have determined the mechanism of homogeneous reactions by probing the diffusion layer spectroscopically(25, 26), but this appears to be the first example of studying mechanisms by direct observation of concentration profiles. It should be possible with the present system configuration to obtain concentration profiles for chemical species that are so reactive they exist no more than 5-10 km away from the electrode where they were generated, implying first-order rate constants up to approximately 200 s-l.
LITERATURE CITED (1) Jan, C.C.; McCreery, R. L. Anal. Chem. 1986, 58, 2771. (2) Jan, C.-C.; McCreery, R. L.: Gamble, F. T. Anal. Chem. 1985, 5 7 , 1763. (3) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Wlnqulst, S. Anal. Chem. 1986, 5 4 , 844. (4) Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R . M. Anal. Chem. 1980, 82,848. ( 5 ) Ewlng, A. 0.;Dayton, M. A.; Wightman, R. M. Anal. Chem. IS81, 5 3 , 1842
(6) &Duffle, B.; Anderson, L. B.; Reilley, C. N. Anal. Chem. 1986, 38, 883. (7) Anderson, L. B.; Rellley, C. N. J. Hectroanal. Chem. 1985, IO, 295. (8) Hubbard, A. T.; Anson, F. C. Anal. Chem. 1964, 36, 723.
(9) Sanderson, D. E.; Anderson, L. B. Anal. Chem. 1985, 57, 2388.
(IO) Chklsey, C. E.; FeMman, B. J.; Lundgren, C.; Murray, R. W. Anal.
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Chem 1986. 58, 801. (11) Bard, A. J.; Crayston, J. A.; Kittlesen, G. P.; Shea, T. V.; Wrighton, M. S. Anal. Chem. 1986, 58, 23211. (12) Deakin, M. R. PhD. Thesis, Indiana University, 1986. (13) Lui, H.-Y.; Fan, F A . F.; Lin, C. W.; Bard, A. J. J. Am. Chem. SOC. 1986, 108, 3838. (14) Bard, A. J.; Faulkner, L. R. Electroanalytical Methods; Wlley: New York, 1980; p 566. (15) Kelly, R. S.; Wightman, R. M. Anal. Chlm. Acta 1986, 187, 79. (16) Adams, R. N. Electrochemistry at Solid Electrodes; Marcel Dekker: New York, 1970; p 219. (17) Wilson, A. M.; Epple, D. G. Biochemistry 1966, 5 , 3170. (18) Schmakel, C. 0.; Santhanam, K. S. V.; Elving, P. J. J . Am. Chem. SOC. 1975, 97,5083. (19) Hawley, M. D.; Tatawawadi, S. V.; Piekarskl, S.; Adams, R. N. J. Am. Chem. SOC. 1987, 89, 447. (20) Hawley. M. D.; Feklberg, S. W. J. Phys. Chem. IS66, 70, 3459. (21) Alberts, 0. S.; Shain, I. Anal. Chem. 1983, 35, 1859. (22) Fddberg, S. Nectrasnal. Chem. 1969, 3 , 199. (23) Long, D. E. Anal. Chim. Acta 1969, 46, 193. (24) Demlng, S. N.; Morgan, S. L. Anal. Chem. 1973, 45, 278A. (25) Mayausky, J. S.; McCreery, R. L. Anal. Chem. 1983, 55, 308. (26) Winograd, N.; Kuwana, T. J. Am. Chem. SOC. 1971, 93, 4343.
RECEIVED for review January 20, 1987. Accepted April 23, 1987. This work was supported in part by the National Science Foundation, Grant No. CHE-8411000 and CHE8500529, and was presented in part at the National Meeting of the American Chemical Society, New York, 1986.
Radioimmunologic Determination of Human C-Peptide and Proinsulin in Plasma with Anti-C-Peptide Serum after Prepurification by High-Performance Liquid Chromatography Martin Komjati,*JPeter Nowotny, and Werner Waldhausl I. Medizinische Uniuersitatsklinik, Division of Clinical Endocrinology and Diabetology, Wien, Austria
A method Is descrlbed for analytical separatlon and subsequent determination of human GpepUde and proln8uln In plasma. To this end detennlnatlons of C-peptide were performed after Its separatlon from prolnsulln by hlgh-performance Uqukl chromatography (HPLC). Chromatographic p r a pur'flcatlon was peformed wRh methanol and Sep-PAK CIS extractlon of plasma, foilowed by HPLC elutlon wlth acetonitrile In an ammonlum acetate/acetk acld buffer (pH 4.2). Thereby C-peptlde, lnsulln, and prolnsuln were separated skndtaneotrsiy In 35 % acetonitr#ewlthh 10 mln. Thereafter, Gpeptlde contsbllng fractions were pooled and deealted, and Cpeptkle was measwed by radldmnwloaerayuslng antLCpeptlde antibodies as blndlng reagent. Subsequently, prolnsulln concentratlon was calculated as the dmerence between Its apparent molar concentratlon In plasma minus absolute plasma C-peptlde concentratlon, the lower llmlt for the detection of the latter belng 50 pmoVL. Thls approach for the determlnatlon of plasma C-peptlde and prdnsulln Is of value whenever crogs-reactlansbetween the two hormone mdeHes are to be absolutely avolded.
The availability of pure intact human proinsulin through recombinant DNA technology (I) raised interest in studies dealing with its physiological and therapeutical potential (2, Dedicated t o Prof. Werner Waldhausl, h i s 50th birthday.
M.D., on t h e occasion of
3). Since proinsulin is antigenically related to insulin and C-peptide, severe difficulties arise when one of the two latter compounds has to be estimated in plasma enriched by exogenously administered proinsulin. In this case proinsulin can only be removed either by physicochemical methods, e.g. high-performance liquid chromatography (HPLC), or by immunoprecipitation. The latter method requires highly specific antibodies directed against human proinsulin with negligible cross-reactivity vs. insulin and C-peptide. The generation of such antibodies and their application for human proinsulin radioimmunoassay have been described recently (4-6). Furthermore, to avoid the use of specific anti-human proinsulin antisera, separation by anti-insulin antiserum of insulin and human proinsulin has been used prior to estimation of C-peptide immunoreactivity in serum (7, 8). A number of papers have been published in the recent years on successful separation and analysis of pancreatic polypeptide hormones by reversed-phase HPLC (9-13). However, only one of these studies describes a method, in which pancreatic peptides were separated from biological fluids in order to determine the immunological activities of insulin, C-peptide, and glucagon by radioimmunoassay (13). To overcome the uncertainities of immunological crossreactivity, this paper describes a method for separation by HPLC of human proinsulin from endogenous C-peptide with subsequent measurement of chromatographed C-peptide by radioimmunoassay. Such a procedure is of considerable importance whenever analytical separation of proinsulin and C-peptide is required, particularily so during exogenous ad-
0003-2700/87/0359-2010$01.50/0@ 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987
ministration of proinsulin with parallel measurements of endogenous plasma C-peptide concentration (3). Plasma proinsul'ln concentration was calculated in parallel as the difference between its apparent molar plasma concentration measured directly by radioimmunoassay with anti-C-peptide antibodies and absolute plasma C-peptide concentration determined after its prepurification by HPLC. EXPERIMENTAL S E C T I O N
2011
1 000
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I2 800
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Reagents. Acetonitrile (HPLC gradient grade) was purchased from Baker (Deventer, Holland) and ammonium acetate (analytical grade) from Merck (Darmstadt, West Germany). The aqueous solution of 0.01 M ammonium acetate was adjusted to pH 4.2 by addition of acetic acid. HPLC-grade water was drawn from a Millipore Milli-Q plant (Millipore Co., Bedford, MA), and both the buffer and acetonitrile were filtered through a Millex SR (0.5 pm) and Millex HA (0.45 pm) filter (Millipore), respectively, and ultrasound degassed before use. The synthetic human C-peptide standard and lZ6I-C-peptide were purchased from Mallinckrodt (Dietzenbach,West Germany), biosynthetic human insulin and biosynthetic human proinsulin (lot No. CT-5765-2C)were a gift from E. Lilly (Indianapolis, IN). All other chemicals used were of analytical grade and purchased from Merck. Extraction of Plasma. Two milliliters of heparin (250 U)/ Trasylol(2OOOU/5 mL of blood) plasma samples were spiked with 1251-C-peptide(0.07 kBq; specific activity, 12 GBq/mg), to permit individual estimation of recovery, and precipitated consecutively with 8 mL of methanol in siliconized glass tubes. Thereafter, centrifuged supernatants were diluted with water to 25% methanol and desalted by passing through Sep-PAK C18 cartridges (Waters Associates, Milford, MA). First the cartridge was flushed with 5 mL of methanol, followed by 20 mL of water. The diluted aqueous samples were loaded on the Sep-PAK column and eluted with 3 mL of methanol. Sodium sulfate was used to remove water from the eluent. After centrifugation the methanolic extracts were evaporated to dryness at 30 "C, and consecutively reconstituted with 500 pL of acetonitrile (35%) and ammonium acetate buffer (65%). The dissolved samples were filtered through an activated Millex SR 0.5-pm filter unit (Millipore) and the filters were washed with 400 p L of acetonitrileammonium acetate buffer. The entire sample volume of approximately 1400 pL was quantitatively applied to the HPLC column. Apparatus and Chromatography. HPLC was performed by using Waters equipment (Waters Equipment, Wilford, MA) consisting of two Model 510 solvent delivery pumps, an automated switching valve, a Model Lambda-Max 481 LC spectrometer operated at either 218 or 280 nm, a QA-1 data system, and an automated gradient controller. A precolumn (Guard-PAK, precolumn module, Waters Associates) packed with a C18 cartridge was placed ahead of a 3.9-mm X 30-cm pBondapak C18 liquid chromatography column. In a modification of the two-component system described elsewhere (111,a mobile phase consisting of 0.01 M ammonium acetate, adjusted with acetic acid to pH 4.2, and acetonitrile was used at room temperature and at a flow rate of 1mL/min. After a 10 min long isocratic elution with 35% (v/v) acetonitrile, a linear acetonitrile gradient (2% acetonitrile/min) up to a maximum of 60% acetonitrile was applied to elute all plasma polypeptides and proteins. If separation of insulin and proinsulin was not required, a concave gradient was started immediately after sample injection that increased to 60% acetonitrile within 10 min, followed by 5 min of isocratic elution. Fractions of the eluent were collected at 1-minintervals in siliconized plastic tubes. Radioimmunoassay. C-Peptide-Containing HPLC fractions 3-6 were diluted with water and consecutively applied to Sep-PAK C18 cartridges as described above in order to remove the organic solvent and salt. After elution with methanol and removal of water by sodium sulfate, the samples were evaporated to dryness under a stream of nitrogen and reconstituted with 1000 pL of a phosphate buffer appropriate for C-peptide radioimmunoassay as described previously (14). The assay sensitivity (2 standard deviation, n = 20) for C-peptide was 60 pmol/L and the interassay relative standard deviation was
