Construction of Time-Resolved Fluorescence Detector for Amino

pers, and spectroscopic filters. Amino compounds de- rivatized by isothiocyanobenzyl EDTA with Eu3+ chelate are mixed with an enhancer solution postco...
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Anal. Chem. 1997, 69, 1861-1865

Construction of Time-Resolved Fluorescence Detector for Amino Compounds after High-Performance Liquid Chromatography Using Europium Chelate Tetsuo Iwata,* Masaaki Senda, and Yasuyuki Kurosu

JASCO Technical Research Laboratories Corporation, 2097-2, Ishikawa-cho, Hachioji-shi, Tokyo, 192 Japan Akio Tsuji and Masako Maeda

School of Pharmaceutical Sciences, Showa University, 1-5-8, Hatanodai, Sinagawa-ku, Tokyo, 142 Japan

We have constructed a simple, low-cost, time-resolved fluorescence detector (TRFD) for analysis of amino compounds with high-performance liquid chromatography using europium (Eu3+) chelates, which consists of a dcoperated xenon lamp, two synchronized mechanical choppers, and spectroscopic filters. Amino compounds derivatized by isothiocyanobenzyl EDTA with Eu3+ chelate are mixed with an enhancer solution postcolumn and detected by the TRFD. Taking advantage of the long fluorescence lifetime of the Eu3+ chelates, high selectivity against background fluorecein is achieved. To demonstrate the usefulness of the TRFD, fundamental performance test comparison with the conventional fluorescence detector was carried out. Details of the system are also described. Chelates of lanthanide ions are characterized by a delayed fluorescence signal of high intensity. Among them, europium (Eu3+) chelates have unique fluorescence properties, such as long Stokes shift, narrow bandwidth of emission, high quantum yields, and long fluorescence lifetimes ranging from tens of microseconds to submilliseconds.1-7 Because of these properties, Eu3+ chelates have been used as labels for immunological assays instead of radioisotopes for safety consideration. The properties of Eu3+ chelates are suitable for time-resolved fluorescence (TRF) detection.8 When we use a conventional dye as the label, e.g., fluorescein, a high signal-to-noise ratio (SNR) cannot be obtained, as expected, even when using a high-intensity excitation source. This is because of the strong interference due to the background fluorescence and the light scattering of the excitation beam that obscures a fluorescence signal emitted from the analyte. The fluorescence signal of analyte, however, can be detected with high selectivity and sometimes with high sensitivity (1) Okabayashi, Y.; Kitagawa, T. Anal. Chem. 1994, 66, 1448-1453. (2) Schreurs, M.; Somsen, G. W.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1989, 482, 351-359. (3) Wenzel, T. J.; Collette, L. M.; Dahlen, D. T.; Hendrickson, S. M.; Yarmaloff, L. W. J. Chromatogr. 1988, 433, 149-158. (4) Ci, Y.-X.; Li, Y.-Z.; Liu, X.-J. Anal. Chem. 1995, 67, 1785-1788. (5) Takeich, T. Talanta 1982, 29, 397-400. (6) Aihara, M.; Arai, M.; Taketatsu, T. Analyst 1986, 111, 641-643. (7) Schreurs, M.; Hellendoorn, L.; Gooijer, C.; Velthorst, N. H. J. Chromatogr. 1991, 552, 625-634. (8) Soini, E.; Hemmila, I. Clin. Chem. 1979, 25, 353-361. S0003-2700(96)01224-3 CCC: $14.00

© 1997 American Chemical Society

when we use Eu3+ chelates as labels in a pulsed excitation mode. This is because fluorescence lifetimes of many background materials are short (10-4 s), which means that the two decay signals can be easily discriminated from each other by using a time gate after the pulsed excitation. Some previous attempts have been reported in the field of highperformance liquid chromatography (HPLC) or capillary electrophoresis to separate overlapping peaks and to eliminate the background signal.9-12 Most of them, however, were carried out on a nanosecond or a picosecond time scale. An expensive, highspeed pulsed laser source and a sophisticated detection system were required. If we take advantage of the fluorescence properties of Eu3+ chelates, we can easily build a simple and inexpensive TRF detector that is useful for HPLC analysis of various amino compounds. In this article, details of the system and its relative performance in comparison to the conventional fluorescence detector (FD) are described. EXPERIMENTAL SECTION Time-Resolved Fluorescence Detector (TRFD). Figure 1 shows a schematic diagram of the proposed system. A combination of a mechanical chopper (CH1; Model 300, Palo Alto Research, Inc.) and a dc-operated xenon lamp (S; L2194-75W, Hamamatu Photonics Co.) was used for the purpose of timeresolved measurements. In comparison with the use of a xenon flash lamp, the combination has advantages in that a high repetitive excitation frequency of a sample can be achieved, along with high stability and long lifetime of the lamp. A relatively longer pulse duration and a weaker radiation intensity over the flash lamp might be tolerable because of the fluorescence properties of the Eu3+ chelates, i.e., high intensity and long decay times. A sampling of the fluorescence signal emitted from the sample was carried out optically by the second mechanical chopper (CH2) inserted at the emission side. The rotation of CH2 was synchronized with that of CH1 by a phase-locked loop (PLL) (9) Furuta, N.; Otuki, A. Anal. Chem. 1983, 55, 2407-2413. (10) Latva, M.; Ala-Kleme, T.; Bjennes, H.; Kankare, J.; Haapakka, K. Analyst 1995, 120, 367-372. (11) Soper, S. D.; Legendre, B. L., Jr.; Williams, D. C. Anal. Chem. 1995, 67, 4358-4365. (12) Millrer, K. J.; Lytle, F. E. J. Chromatogr. 1993, 648, 245-250.

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Figure 1. Schematic diagram of the entire system: L1-L4, quartz lens (f ) 100 mm, 50φ); L5, L6, BK7 lens (f ) 100 mm, 50φ); L7, BK7 lens (f ) 50 mm, 50φ); AP, aperture; SL, slit; F1, UV band-pass filter; F2, low-pass filter; CH1, CH2, optical chopper; PMT, photomultiplier tube.

circuit. By varying the phase angle of the PLL, we can move the sampling gate to an optimal time position of the fluorescence decay signal. The fluorescence light passing through CH2 is detected by the photomultiplier tube (PMT; R372, Hamamatu Photonics Co.) as a voltage signal. The optical sampling method has advantages over the conventional detector output sampling method in SNR because of the ability to prevent saturation of the detector and to narrow the frequency bandwidth of electronics connected to the detector. On the other hand, a dc bias signal from the PMT may be introduced due to stray light passing through the chopper, which can deteriorate SNR. To avoid this problem, we inserted apertures (AP) and slits (SL) on both sides of the sample cell and choppers. In addition, we used a gatedtype integrator (G, effective time constant 0.4 s) that was triggered by a timing signal from CH2. The gate width and position were optimized to give a maximum SNR. To carry out spectroscopic measurements, a UV band-pass filter (F1; UTVAF-50S-34U, Sigma Koki Co.) was inserted in the path of the excitation beam. The transmission wavelength of the filter was between 280 and 370 nm (at -3 dB points) with a maximum at 340 nm. On the emission side, a low-pass filter (F2; SCF-50S-58O, Sigma Koki Co.) was inserted (-3 dB points, 1862

Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

wavelength of 580 nm). The wavelength characteristics of the two filters are suitable for measuring Eu3+ chelate fluorescence. In comparison with the use of grating monochromators, the filter spectroscopic method has advantages in SNR because of high optical throughput and the effective use of the fluorescence signal along the two wavelength directions. Figure 2 shows a timing diagram of the TRF measurements. A timing diagram for CH1 is shown in Figure 2a; the repetitive excitation frequency of the sample was fixed at 1 kHz, with a 75 µs pulse duration. A dashed line in Figure 2a shows a hypothetical waveform (full width at half-maximum (fwhm) 75 µs) of the excitation light, which is determined by a convolution integral between waveforms due to the aperture width (1 mm) of CH1 and to a spatial distance (∼1 mm) of emission of the xenon lamp. The fluorescence signal from the Eu3+ chelates is shown in Figure 2b. Figure 2c shows a timing diagram of CH2, with a duty ratio of 50%. A dashed line in Figure 2c also shows a hypothetical waveform of the sampling signal (fwhm 500 µs), which is again determined by a convolution between waveforms due to the aperture width (2 mm) of CH2 and to a lateral width (1 mm) of the sample cell. An output signal from the PMT is shown in Figure 2d, which is obtained by multiplying (b) and (c). A timing

Figure 2. Timing diagrams for the TRFD; (a) timing diagram for CH1; (b) fluorescence from the sample; (c) timing diagram for CH2; (d) output signal from PMT; (e) timing diagram for a gated-type integrator. Dashed lines represent actual excitation and emission waveforms.

of the electrical gate for the gated-type integrator is shown in Figure 2e, the time position and duration optimized experimentally were 220 and 190 µs, respectively. An output dc signal that is proportional with the concentration of the Eu3+ chelates is fed into a chart recorder. HPLC System. A flow diagram of the HPLC system is shown in Figure 1. It consisted of plural components: pumps 1 and 2 (plunger type PU-980, JASCO Co.), a six-way sample injector (Rheodyne 7175; 20 µL loop), a mixing coil, the TRFD, and a commercial FD (820-FP, JASCO Co.). Pump 1 is used for delivery of the eluent and pump 2 for delivering postcolumn reagent (enhancer solution). The mixing coil was immersed in a temperature-controlled water bath. The volume in the flow cell used for TRFD and FD was 16 µL. The mobile phase was 20 mM NaPO4 (pH 7.0) containing 20% acetonitrile at a flow rate of 0.1 mL/min. The postcolumn reagent used was TTA solution at a flow rate of 0.02 mL/min. A 50 mm × 0.15 mm i.d. stainless steel column packed with Crest C18T-5 was used. The injection volume of the sample was 10 µL. Chemicals. For HPLC, a sample solution derivatized by isothiocyanobenzyl EDTA with Eu3+ chelate is mixed with an enhancer solution postcolumn. For FIA, on the other hand, a sample solution of europium(III) acetate tetrahydrate (Eu(CH3COO)3‚4H2O, 99.9%) was mixed with the enhancer solution. The enhancer solution consists of a β-diketone, tri-n-octylphosphine oxide (TOPO), and a surfactant (TritonX-100). The β-diketone used was either pivaloyltrifluoroacetone (PTA), benzoyltrifluoroacetone (BFA), or thenoyltrifluoroacetone (TTA). TritonX-100 and europium acetate were purchased from Wako Pure Chemical Industries (Osaka, Japan), and PTA, BFA, TTA, TOPO, and ITCBEDTA were from Dojin (Kumamoto, Japan). Dansyl amino acids (DNS-AA) were manually derivatized from free amino acids in our laboratory. All other reagents were of analytical grade. Preparation of the Enhancer Solution. Stock solutions of a mixed ligand (β-diketone and TOPO) were prepared by dissolving the reagents in sodium acetate buffer containing TritonX-100.

Figure 3. FIA peaks obtained for seven injections of 0.1 µM Eu3+ chelate of PTA in the presence of fluorescein: detection, (b) timeresolved fluorescence, (a) fluorescence (λex 290 ) nm, λem ) 615 nm); injection volume, 20 µL; carrier, 20 mM NaPO4 buffer (pH 7.0) containing 8% acetonitrile; flow rate, pump 1, 0.2 mL/min, pump 2, 0.2 mL/min. Peaks depicted are (1) 0 M fluorescein + 0.1 µM Eu3+ chelate, (2) 0.01 µM fluorescein + 0.1 µM Eu3+ chelate, (3) 0.1 µM fluorescein + 0.1 µM Eu3+ chelate, (4) 1 µM fluorescein + 0.1 µM Eu3+ chelate, (5) 10 µM fluorescein + 0.1 µM Eu3+ chelate, (6) 50 µM fluorescein + 0.1 µM Eu3+ chelate, (7) 90 µM fluorescein + 0.1 µM Eu3+ chelate.

Derivatization of Amino Compounds. To 1 µM-1 mM amine solution (10 µL) were added 5-500 µM Eu3+ chelate of ITCB-EDTA (ITCB-EDTA-Eu, 45 µL) and 100 mM sodium carbonate (pH 9.8, 45 µL). The mixture was incubated for 8 h at 40 °C, and 5-20 µL of the reaction mixture was introduced into the HPLC column. The ITCB-EDTA-Eu solution was prepared by addition of ITCB-EDTA (1 mg) to 5 mL of 0.5 mM europium acetate solution. RESULTS AND DISCUSSION Rejection of Background. To demonstrate the background rejection capability of the TRFD, we operated it in a flow injection analysis (FIA) mode (that is, HPLC system without a separation column) in the presence of a strong interfering fluorescent background. Sample solutions used were 0.1 µM Eu3+ chelates of PTA mixed with various concentrations (0-900 µM) of fluorescein, whose lifetime is less than 5 ns. The injection volume of the sample was 10 µL, and the mobile phase was 20 mM NaPO4 (pH 7.0) containing 8% acetonitrile at a flow rate of 0.2 mL/min (pump 1). The flow rate of the enhancer solution (8 µM PTA, Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

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Table 1. Standard Deviation of Noise for Various Concentrations of Fluorescein in PTA Enhancer Solution Normalized to That of 0 M Fluoresceina concn (M)

TRFL

FL

0 10-7 10-6 10-5 10-4

1.00 1.01 0.99 1.07 1.94

1.00 1.24 1.81 5.00 21.94

a Flow rate, 0.2 mL/min; fluorescence detection, λ ) 290 nm, λ ex em ) 615 nm.

Figure 4. Relationship between TRF intensity and concentration of Eu3+ chelate of PTA by the stopped-flow analysis mode. Concentrations ranging from 10-12 to 10-6 M europium acetate were used in the enhancer solution (8 µM PTA, pH 6.5). Detection limit was 3.3 fmol (S/N ) 3).

pH 6.5) was also 0.2 mL/min (pump 2). For comparison, a conventional FD (820FP, JASCO Co.) was connected in series to the TRFD; the FD excitation and emission wavelengths were 290 and 615 nm, respectively. Figure 3 shows signals obtained from (a) the FD and (b) the TRFD. It can be seen that the signals obtained from the TRFD are constant due to the constant concentration of Eu3+ chelates, whereas those from the FD can be influenced by the concentration-dependent background fluorescence signal of fluorescein. Dynamic Range and Detection Limit. Figure 4 shows the dynamic range of the TRFD. The TRFD was operated in a stopped-flow analysis mode. The sample solution used was europium acetate dissolved in the enhancer solution (8 µM PTA, pH 6.5), which was introduced into the flow cell by pump 1. The linear range, as shown in Figure 4, covers over 4 orders of magnitude, and the detection limit was found to be 2.1 × 10-10 M (3.3 × 10-15 mol). Here, we define the detection limit as the sample concentration that gives SNR ) 3. When we operated the TRFD in the FIA mode (injection volume of sample, 10 µL, flow rates of eluent (20 mM NaPO4) and enhancer, 0.1 mL/min), the detection limit was about 9.9 × 10-9 M (1.6 × 10-13 mol). The TRFD can achieve better sensitivity than that possible with the conventional FD, especially in the presence of high background. This is because shot noise superimposed on the signal is proportional to the square root of the signal intensity. To verify it, we measured two signals from the TRFD and the FD for mixtures at various concentrations (10-7-10-4 M) of fluorescein and the PTA enhancer solution. The standard deviations (SD) of noise normalized to 0 M fluorescein obtained from the TRFD and the FD are listed in Table 1. In comparison with that of the conventional FD, the normalized SD of the TRFD was an order of magnitude smaller, thus improving SNR by a factor of 10. From our experiments, we have found that the detection limit in the FIA mode can be as low as 1.9 × 10-9 M (3.0 × 10-14 mol) with the use of BTA enhancer solution and 1.4 × 10-9 M (2.3 × 10-14 mol) with TTA enhancer solution. In the course of our experiments, we have found that metallic components, such as pipes, tees, reaction coils, injectors, pump units, and microsyringes, will interact with Eu3+ and thus introduce large signal tailing and deterioration in signal reproducibility. To avoid such 1864 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997

Figure 5. Chromatogram of 3-phenyl-1-propylamine-ITCB-EDTAEu by fluorescence detection (a) and TRF detection (b) in the presence of dansyl amino acids. Chromatographic conditions: mobile phase, 80/20 (v/v) 20 mM sodium phosphate (pH 7.0)/acetonitrile; postcolumn reagent, TTA enhancer (80 µM, pH 6.5); flow rate, pump 1, 0.1 mL/min, pump 2, 0.02 mL/min; temperature of mixing coil, 40 °C; column, Crest C18T-5 (0.15 mm i.d. × 50 mm long); sample, 66.7 µM 3-phenyl-1-propylamine-ITCB-EDTA-Eu (10 µL); peak assignment, (A) DNS-Gln, (B) DNS-Ala, (C) 3-phenyl-1-propylamineITCB-EDTA-Eu, (*) byproducts of ITCB-EDTA reaction, (**) byproducts of dansylation.

difficulties, we eliminate the use of metallic components in our system as much as possible. Optimization of Experimental Conditions. We have determined the optimal conditions for HPLC experiments. Parameters to be determined are pH of the buffer, total concentration of the enhancer solution, concentration of β-diketone, that of acetonitrile for HPLC, temperature of mixing coil, etc. The conditions experimentally obtained are as follows: enhancer solution, 80 µM β-diketones, 500 µM TOPO, 1.0% TritonX-100, 200 mM sodium acetate buffer of pH 6.5; mixing coil, length 4 m, 0.25 mm i.d., tefzel, temperature 40 °C; eluent, 20 mM NaPO4 including less than 20% acetonitrile, flow rate 0.1-0.2 mL/min); enhancer solution, flow rate 0.02-0.2 mL/min. The enhancer solution mentioned above was prepared as follows: To 200 mL

of 5% TritonX-100 were added 100 mL of 2.0 M sodium acetate buffer (pH 6.5), 10 mL of 50 mM TOPO, 32 mL of 2.5 mM β-ketone (PTA, BFA, or TTA), and enough distilled water to make 1 L. The enhancing ability was not decreased even after dilution 20 times with water. Although the enhancing ability of the TTA solution was about 7 times superior to that of the PTA solution, the ability was lowered to about one-tenth in several days. On the other hand, that of the PTA solution was stable. That for the BFA solution was strongly influenced by the concentration of acetonitrile. Amino Compound Analyses with the TRFD. To demonstrate the overall performance of the TRFD, we have analyzed 66.7 µM 3-phenyl-1-propylamine-ITCB-EDTA-Eu in the presence of dansyl (DNS)-Gln (1.1 mM) and DNS-Ala (1.9 mM); the fluorescence lifetime of the coexisting material lies in the nanosecond region. A chromatogram obtained from the FD is shown in Figure 5a, and that from the TRFD is shown in Figure 5b. Chromatographic peaks marked A, B, and C are due to DNSGln, DNS-Ala, and 3-phenyl-1-propylamine-ITCB-EDTA-Eu, respectively, and the asterisks represent byproducts. The TRFD gave only the sample and byproduct peaks, while the FD detected extraneous peaks. Furthermore, the insufficiently resolved chro-

matographic peaks from the FD were clearly separated by the TRFD, which demonstrates the effectiveness of the TRFD. CONCLUSIONS Taking advantage of the long fluorescence lifetime of Eu3+ chelates, we have constructed a simple, low-cost TRFD for HPLC analysis of amino compounds. The amino compounds derivatized by isothiocyanobenzyl EDTA with Eu3+ chelate are mixed with an enhancer solution postcolumn. In comparison with the conventional FD, we have found high selectivity, and sometimes high sensitivity can be achieved by the TRFD even in the presence of high background material. ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas “New Development of Rare Earth Complexes”, No. 06241107, from The Ministry of Education, Science and Culture of Japan. Received for review December 3, 1996. February 21, 1997.X

Accepted

AC961224W X

Abstract published in Advance ACS Abstracts, April 1, 1997.

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