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Anal. Chem. 1009, 65, 759-762
Speciation of Iodo Amino Acids by High-Performance Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometric Detection Kikuo Takatera; and Tadashi Watanabe' Institute of Industrial Science, University of Tokyo, Roppongi, Minato-ku, Tokyo 106, Japan
Hlgh-performance iiquld chromatography (HPLC) has been coupkd wlth on-llne lnductlvely coupled plasma mass spectrometry (ICPIMS) for the speciation of Iodide ion I- and five iodo amino acids (monolodotyrosine, diiodotyrodne, 3,3',5and 3,3',5'-trllodothyronlne, and thyroxine) commonly found in thyroglobulin. The absolute detectionlimltswere in a range from 35to 130 pgas lodlne this Isroughly 1order of magnttude lower than thoso in conventlonaimethods wing stable isotope of iodine. The rdatlve standard deviation of peak-area species. measurements was i e r than 6% for each of the SIX Baud on thew results, the contents of iodine-containing compouMk in an enzymatic digest of bovine thyroglobulin were determined.
INTRODUCTION Iodine is a biologically essential element, and the metabolic importance of iodine depends heavily on its chemical form. Most of the iodine-containing biological molecules are iodo amino acids, which can be found in thyroid hormones. From a clinical point of view, the speciation of these amino acids in plasma and urine is of much importance in diagnosing thyroid diseases including hyperthyroidism. A number of methods have been reported for the analysis of iodoamino acids, namely 3-monoiodo-~-tyrosine(MIT), 3,5-diiodo-~-tyrosine(DIT), 3,5,3'-triiodo-~-thyronine(Td, 3,3',5'-triiodo-~-thyronine(rT3),and thyroxine (T4)contained in thyroglobulin (Tg). Among them the most frequently used procedure is high-performance liquid chromatography (WLC) with UV detection.'"' The method, usually based on reversedphase HPLC,lv2 has high efficiency in separating these compounds in a relatively short time. However, in applying the method for the analysis of the compounds in serum, bile, and Tg, other hydrophobic amino acids tend to interfere with their quantitation because the UV detection is nonspecific.2 To circumvent this problem, the catalytic activity of iodine in the Sandell-Kolthoff reaction is generally utilized by way of a postcolumn reaction system before UV detection of the eluent.3~4However, a large difference in the catalytic activity of each iodide requires tedious and time-consuming calibration by use of each standard iodide prior to the quantitation. Moreover, the detection limits are not sufficient even with this technique, and careful preconcentration is necessary for accurate determination of trace amounts of T3 and rT3 in human serum for example. (1) Hearn, M. T. W.; Hancock, W. S.; Bishop, C. A. J. Chromatogr. 1978,157, 337-344. (2) Ohmori, T.; Tarutani, 0.;Hosoya, T. Biochem. J. 1989,262,209214. (3) Sorimachi, K.; Ui, N. J. Biochem. 1974, 76, 39-45. (4) Nachtmann, F.; Knapp, G.; Spitzy, H. J. Chromatogr. 1978,149, 693-702. 0000-2700/90/0065-0759$04.00/0
High-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC/ ICP/MS) is a useful technique for trace element speciation, especially for biological trace molecules, because it combines the separative power of HPLC with the highly sensitive and element-specific detection of ICP/MS."13 In the present work we examined the feasibility of using ICPIMS as an iodide detector in reversed-phase HPLC to quantitate iodide ion and iodo amino acids of biological interest.
EXPERIMENTAL SECTION Instrumentation. The HPLC system was composed of a Jasco 880-PU dual piston HPLC pump and a Rheodyne 7125 injector. The reversed-phase column employed was Shiseido CIS SG120(35mm X 4.6-mm i.d.), and the column outlet was directly connected to the nebulizer of an ICP/MS apparatus (Seiko Instrument Inc., SPQ-6100). The instrumental operating parameters are summarized in Table I. The nebulizer gas (argon) was mixed with about 6% of oxygen to prevent carbon accumulation on the sampler. In the ICP/MS measurement, the integration time for the peak counting was set at 0.1 s at one mass before going to the next. Within the total measurement time, three data acquisitions were done per peak, one at the central mass and the other two at f0.125 au from the assumed center. Reagents and Procedure. Potassium iodide and DIT were purchased from Wako Pure Chemical Industries (Osaka,Japan); MIT, Ts, and T4 from Aldrich Chemical Co. (Milwaukee, WI); rT3, Tg (from bovine),and Pronase E (fromStreptomycesgriseus) from Sigma Chemical Co. (Saint Louis, MO). These reagenta were used without further purification. Water deionized with a Milli-Q system (Millipore Filter Co. Ltd.) was used throughout. In the ICP/MS measurement, the iodine single isotope at mass 127 was monitored. HPLC/ICP/MS determination of iodides was done via peak-areacalibrationby use of KI standard solutions, as described previously.12 The system can be operated in either HPLC or a flow injection (FI)analysis mode (FI/ICP/MS) by controllingthe switchingvalve placed between the injection valve and the HPLC column as shown in Figure 1. This facilitated quantitation of samples and standards under the same flow conditions as for the HPLC column. After the sample separation and the ICP/MS detection were completed in about 720 s, the ~
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( 5 ) Crews, H. M.; Dean, J. R.; Ebdon, L.; Massey, R. C. Analyst 1989,
114,895-899. (6) Matz, S. G.; Elder, R. C.; Tepperman, K. J. Anal. At. Spectrom. 1989,4,767-771. (7) Mason, A. Z.; Storms, S. D.; Jenkins, K. D. Anal. Biochem. 1990, 186, 187-201. (8) Gercken, B.; Barnes, R. M. Anal. Chem. 1991, 63, 283-287. (9) Heitkemper, D.; Creed, J.; Caruso, J.; Fricke, F. L. J. Anal. A t . Spectrom. 1989,4, 279-284. (10) Sheppard, B. S.; Shen, W. L.; Carueo, J. A,; Heitkemper, D. T.; Fricke, F. L. J. Anal. At. Spectrom. 1990,5,431-435. (11) Takatera, K.; Watanabe, T. Anal. Sci. 1991, 7,696498. (12) Takatera, K.; Watanabe, T. Anal. Sci. 1992,8,469-474. (13) Mason, A. Z.; Storms, S. D.; Jenkins, K. D. Anal. Biochem. 1990, 186, 187-201.
0 1993 Amerlcan Chemlcai Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
Table I. Experimental Operating Conditions chromatographic parameters Shiseido CAPCELL PAC column C18 SG120
mobile phase flow rate sample loop volume temperature ICP/MS parameters instrumental parameters rf generator frequency spray chamber
nebulizer Cu sampler and skimmer cones orifice diameter operational parameters (in 10% methanol HPLC eluent) rf forward power rf reflected power argon gas flow rates plasma gas auxiliary gas nebulizer gas 02 fraction in nebulizer (in 50% methanol HPLC eluent) rf forward power rf reflected power argon gas flow rates plasma gas auxiliary gas nebulizer gas 02 fraction in nebulizer
(35 mm X 4.6-mm id.) 5-pm-diameterparticles 10% or 50% methanoliO.1M (NH4)2HP04/0.1%&PO4 1 mL/min 20 pL 23 O C 27.12 MHz Scott-type double pass water cooled, 15 O C
Meinhard TR-3042 1.1,0.7 mm
1300 W
