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Anal. Chem. 1981, 53, 471-474
metabolites. Typical HPLC elution patterns of warfarin and its metabolites in human plasma and urine are shown in Figure 4a,b, respectively. There are trace amounts of endogenous fluorescent contaminants which eluted before warfarin and after all of the metabolites in the human urine sample. As a result, little interference was found for this analytical procedure when urine samples were analyzed. The fluorescent contaminants observed in the urine assay were not found in the human plasma analysis. The concentration of warfarin and its metabolites in the human urine were approximately 20 times higher than in plasma as expected. Analysis of the 24-h urine sample clearly indicated the presence of warfarin, 6- and 7-OH-warfarin, and the warfarin alcohols. A low intensity peak was also observed to elute a t the retention time of 4-OH-warfarin. If the peak is in fact due to 4-OH-warfarin, this represents the first time this metabolite has been observed in man. Within the limits of detection no 8-OH-warfarin was observed. Neither 8- or 4‘OH-warfarin nor any of the warfarin alcohols (R,R;S,S) or (R,S; S a ) were detected in plasma. Thus, it can be concluded that the HPLC separation reported herein coupled with the postcolumn fluorescence reaction system provides a very useful analytical method for the quantitation of warfarin and its known metabolites in human plasma and urine. Since this method does not involve a buffered aqueous system, it is more readily adaptable to the mass spectral analysis of pseudoracemic mixtures. Moreover, its inherent sensitivity permits the use of much smaller plasma samples than are required by other reported methods. This suggests that this technique has the potential to allow pharmacokinetic studies of the metabolites in small animals where blood supply is limited.
ACKNOWLEDGMENT The authors are grateful to Whatman, Inc., for a gift of the
chromatographic columns used in this work. We are also grateful to R. A. O’Reilly, Chief of Medicine, Santa Clara Valley Medical Center, San Jose, CA, for gifts of plasma and urine extracts.
LITERATURE CITED (1) ORellly, R. A.; Aggeier, P. M.; h a g , M. S.; Leong, L. Thromb. Dleth. Haemorrh. 1062, 8, 82. (2) O’Reilly, R. A.; Aggeler, P. M.; Hoag, M. S.; Leong, L. J. Clln. Invest. 1063, 42, 1542. (3) Corn, M.; Berberich, R. Clln. Med. 1067, 13, 126. (4) Nagashima, R.; Levy, 0.J. pherm. Scl. 1060, 58, 845. (5) Lewis, R. J.; Ilinicki, L. P. Clln. Res. 1060, 17, 845. (6) Lewis, R. J.; Ilinldti, L. P.; Carlstrorn, M. Eiochem. Med. 1070, 4 , 378. (7) Lan-Carn, C. A.; Chu-Fong, I. J . Pharm. Scl. 1072, 61, 1303. (8) Kaiser, D. G.; Martin, R. S. J. pherm. Scl. 1074, 63, 1579. (9) Midha, K. K.; McClilveroy, 1. J.; Cooper. J. K. J . pherm. Sci. 1074, 63, 1725. (10) Loomis, C. W.; Racz, W. J. Anal. Chlm. Acta, 1070, 106, 155. (11) Vesseli, E. S.; Shhrely. C. A. Sclence, 1074, 184, 466. (12) Wong, L. T.; Solomonraj, G.; Thohas, B. H. J . Chromtcgr. 1077, 135, 149. (13) Vanhaekn-Fastre. R.; Vanhaelen, M. J . Chromatogr. 1976, 729397. (14) Bjornsson, T. D.; Blaschke, T. E.; Meffin, P. J. J. pherm. Scl. 1077, 66. 142. (15) Fasco, M. J.; Piper, L. J.; Kaminsky, L. S. J. Chromatcgr. 1077, 131, 365. (16) Fasco, M. J.; Cashirn. M. J.; Kaminsky, L. S. J . Liq. Chromatcgr. 1070, 2, 565. (17) Johnson, D. W.; Caills. J. 6.; Christian, 0.D. ACS Symp. Ser. 1070, No. 102, 97. (18) Hermodson, M. A.; Barker, W. M.; Link, K. P. J. Med. Chem. 1071, 14, 167. (19) Trager. W. F.; Lewis, R. J.; Garland, W. A. J . Med. Chern. 1070, 13, 1196. (20) Nakarnura, Hiroshi; Tamura, Zenzo Anal. Chem. 1070, 51, 1679.
RECEIVED for review September 11,1980. Accepted December 1, 1980. This research was supported in part by Research Grants GM 22860 and GM 25136 from the Institute of General Medicine (RAO and WFT) and by Research Starter Grant 8114 from the Society for Analytical Chemists of Pittsburgh.
Laser-Induced Photoacoustic Detector for High-Performance Liquid Chromatography Shohei Oda and Tsuguo Sawada‘ Department of Industrial Chemistty, Faculty of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
Laserinduced photoacoustic spectroscopy (LIPAS) was applied to the monitoring of liquid chromatographic effluents. A piezoelectric transducer ( P n ) constitutes a part of a flow cell as a photoacoustic detector. This specially designed cell is approximately 20 pL In volume. Using three kinds of chloro4 4 dimethyiamino)arobenrene (CI-DAAB) Isomers as test compounds, stabllity, reproduciblllty, linearity, and detection limit for the LIPAS detector were tested and compared with those for the UV detector. The llnearlty was extended over the range of 4 orders of magnitude. The detection Iimlt, calculated by considering the dilution in the detectlon cell with respect to an injection concentratlon, corresponds to 7.9 X I O d In absorpttvlty for LIPAS detection. This vahre represents an approximate 25-foid increase in sensitlvity over UV detection.
High-performance liquid chromatography (HPLC) has several advantages over gas chromatography (GC). For ex0003-2700/81/0353-0471$01.00/0
ample, HPLC enables thermally unstable and nonvolatile compounds to be analyzed. However, a detector for HPLC has not fully been advanced. Recently, mass spectrometry (1)and Fourier transform infrared spectrometry (2,3),coupled with HPLC, have been used for qualitative analysis, whereas UV absorption spectrometry has been widely used for trace detection in liquid chromatographic effluents. Recently, the use of laser excitation in fluorescence analysis has opened up new possibilities for ultrasensitive detection ( 4 , 5 ) . However, the rejection of scattered excitation light is still a tedious problem. In the present paper, laser-induced photoacoustic spectroscopy (LIPAS) is applied to the monitoring of HPLC effluents. The authors have revealed that LIPAS is very suitable for ultratrace analysis (6-8). Its sensitivity is about 2 orders of magnitude greater than that of ordinary absorption spectrometry. It is our goal to combine LIPAS with HPLC to achieve hghly sensitive detection of HPLC effluents. We have designed a flow cell for coupling LIPAS with HPLC and have tested stability, reproducibility, linearity, and detection limit 0 1981 American Chernlcai Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
P Recorder
Generotor
Figure 2. Schematic diagram of the experimental instrumentation arrangement.
Effluent exit tube
I
-
0.2
Eftlent entry tu&
Figure 1. Schematic diagram of LIPAS flow cell and detector assembly.
for the LIPAS detector. Comparison is made with the UV detector. Three kinds of chloro-4(dimethylamino)azobenzene (Cl-DAAB) isomers were used as test compounds. These dyes were very suitable as test compounds because they have absorptions in both the UV and visible region. These dyes have been identified as carcinogenic compounds, and thus their trace analysis has become very important. However, no attempt was made to optimize the chromatographic conditions, since our major interest was to demonstrate the feasibility of LIPAS as a HPLC detector.
EXPERIMENTAL SECTION Flow Cell and Detector Construction. Figure 1 shows the LIPAS flow cell, the body of which is made of brass. The column effluent enters the optical region of the flow cell through an entry tube (0.5 mm id., 10 cm long stainless steel pipe) and effuses through an exit tube (the same size and length as the entry tube). The optical region consists of a channel 1.5 mm i.d. and 11mm long. Optically polished quartz windows (10 mm diameter, 4 mm thickness) were attached at both ends of the channel. A laser beam enters from the center of one of the quartz windows and goes out the center of the other one. The volume of the flow cell is about 20 pL. Photoacoustic signal (PA signal) was detected by a piezoelectric transducer (PZT) disk (7 mm diameter, 1mm thickness, NPM, N-6, supplied by Tohoku Kinzoku Co. Ltd.). A narrow slit (0.3 mm width), cut along the channel, was covered with a fully polished platinum foil (0.1 mm thickness). A PZT disk was attached just behind the platinum foil. One of the output terminals of the PZT disk in contact with a platinum foil was grounded through both the platinum foil and the body of the cell (a brass block). The other terminal was connected to a BNC connector, which was built in the body of the cell. Each quartz window was held against a Teflon seal by a brass ring fastened to the brass block (a body) with four screws. Instrumentation Arrangement for the LIPAS Detector. A schematic diagram of the LIPAS system for the detection of chromatographic effluents is shown in Figure 2. An argon ion laser (Spectra Physics Model 164-031,operating in a single line mode, was modulated rectangularly at a given frequency ranging from a few hertz to a few tens of a kilohertz with an acoustooptic light modulator (Intra-Action Corp., Model AOM-40). A laser beam was focused to a few micrometers with a collecting lens (f = 15 cm). A PA signal from the flow cell was amplified with a pre/lock-in amplifier (NF Co. Ltd., Model LI-574) and recorded with a dual-pen recorder (Rikadenki Kogyo Co. Ltd., Model R-202). The laser beam was monitored with a photocell placed behind the flow cell.
3
2
2
0.1
I
300
I
I
400 Wawlength
’
500 ( nm )
Figure 3. Absor tion spectrum of P‘CIDAAB In methanol: concentration, 1 X 10- M; 10 mm pathlength.
9
Chromatography. A pump (Japan Spectroscopic Co. Ltd., Model TRI ROTAR) was operated at a pressure of 45 kg/cm2 and delivered 1.0 mL/min. This pump had a pulsating flow of approximately 0.5 Hz. A bore column (4.6 mm i.d., 250 mm long) was packed with 10 pm of ODS packing (Toyo Soda Co. Ltd., LS-410). An injection valve with a loop of 20 pL (Rheodyne) was used. Chromatographic effluents emerging from a separation column entered the UV detector (Japan Spectroscopic Co. Ltd., Model WIDEC-100-II) and were then introduced into the LIPAS flow cell. The specificationsof the W detector used in the present experiment are as follows: cell volume, 8 pL; path length, 10 mm; detection limit (S/N = 2), 2 X (absorptivity); spectral resolution, 10 nm; light source, deuterium lamp. Output signals from both a W detector and LIPAS were simultaneously recorded with a dud-pen recorder. A back-pressure was applied to the LIPAS flow cell by lifting a drain bottle up to 1m in height in order to eliminate bubble formation. All of the connections and tubings used in the HPLC system were with stainless steel tubes of 0.25 mm i.d. to minimize dead volume. Reagents. Methanol (spectral grade, Kanto Kagaku Co. Ltd.) was used as the eluting solvent without further purification but was partially degassed before use by stirring under a vacuum for 5 min. Three kinds of chloro-4-(dimethylamino)azobenzene (Cl-DAAB) isomers, that is, 2’Cl-DAAB, 3’Cl-DAAB, and 4’ClDAAB (Tokyo Kasei Co. Ltd.), were used as received. The dyes were dissolved in methanol to make 1 X M stock solutions. The stock solutions were diluted with methanol just before measurement. The absorption spectra of these dye solutions were measured with an absorption spectrophotometer (Shimazu Model MPS5000).
RESULTS AND DISCUSSION The UV visible spectrum of 2’Cl-DUB is shown in Figure 3. The spectra of the other isomers of C1-DAAB were similar to that of 2’Cl-DAAB. The wavelengths used for the detection of these dyes in column effluents were 254 nm for the UV
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
473
~
Table I. Experimental Conditions LIPAS wavelength: 488 nm laser power: 500 mW modulation frequency: 4035 Hz time constant of measurement: 3 s
uv wavelength: 254 nm time constant of measurement: 3 s HPLC
'._ _._.,.._--..".. ._ , 1K
-. .-.
10
. 100
_c..
Modulation Frequency
" . 0 '
,
,\?-&
flow rate: 1.0 mL/min sample injection volume: 20 pL eluting solvent: methanol
10K (Hz )
Figure 4. Dependence of modulation frequency on PA signal, noise, and S/N: test sample, 1 X M P'CI-DAAB solution; injection volume, 20 pL (52 ng); HPLC flow rate, 1 mL/min; laser wavelength, 488 nm; laser power, 500 mW; time constant, 3 s.
detection and 488 nm and 514.5 nm for LIPAS. The molar absorption coefficients of 2'Cl-DAAB were calculated to be 9600 L mol-' cm-' for 254 nm, 7700 L mol-' cm-' for 488 nm, and 1900 L mol-' cm-' for 514.5 nm. Pressure fluctuation, caused by the pulsating flow of a HPLC pump, greatly influenced the PA signal measurement. However, the effect of the pressure fluctuation in the column effluent could be avoided by choosing an appropriate modulation frequency of excitation laser beam. Figure 4 shows the dependencies of modulation frequency upon PA signal, noise bandwidth, and signal to noise ratio. By use of 20 pL (50 ng) of a 2'Cl-DAAB (1 X M) solution as a test sample, PA signals of the column effluents were plotted as a function of the modulation frequency. The wavelength and the power of the laser used in the experiment were 488 nm and 500 mW, respectively. PA signal plotted represents a peak height of the chromatogram, and noise bandwidth corresponds to PA signal fluctuations due to a pulsating flow of the column effluent. The PA signal amplitude exhibited a maximum a t approximately 300 Hz and a second maximum a t around 4 kHz. The PA signal dependency on the modulation frequency obtained in the present experiment differed greatly from the one obtained in our previous papers (6, 7). The difference between the present and the former cells is that the present cell is not closed but open and is about l/mth smaller in volume than the former one. The cell volume was considered to influence the PA signal vs. modulation frequency relationship. This frequency dependency is being studied in detail. An enormously large noise level observed at lower modulation frequencies decreased with increasing modulation frequency and finally attained an almost constant value (-4 nV) above 2 kHz. As was already mentioned, such a large noise level a t the lower frequency was caused by pressure fluctuation due to a pulsating flow in the effluents. As clearly shown in Figure 4, the modulation frequency which gave the best signal to noise ratio (S/N) was a t around 4 kHz. Therefore, subsequent experiments were carried out at 4 kHz. These experimental conditions are summarized in Table I. Figure 5 shows chromatograms of three Cl-DAAB isomer mixtures. The respective concentrations of the isomers were the same (1 X lod MI. The chromatogram obtained by LIPAS showed almost the same resolution as that obtained by UV, although the retention time was different between the two methods. In spite of the fact that the volume of the LIPAS flow cell (20 pL) was about 2.5 times larger than that of the UV flow cell (8 p L ) , the bandwidth of each chromatogram obtained with the LIPAS detector agreed with that obtained by the UV detector within experimental errors. The result indicated that the broadening of bandwidth, caused by dif-
0
2
4 Retention
6 8 Time (min )
Figure 5. Chromatograms of CCDAAB isomers mixture: ( 1 2'CC DAAB, (2) 3'CCDAAB, (3) 4'CCDAAB; concentratlon, 1 X 10-?L M for each isomer. Other experlmental conditions are gtven in Table I.
fusion, was negligible. In the case of LIPAS detection, two comparatively high powered laser wavelengths, 488 and 514.5 nm, were used as the excitation light. The peak height of each chromatogram a t 514.5 nm was about one-fourth lower than that at 488 nm, and the peak height ratio just coincided with the ratio of molar absorption coefficients. The reproducibility of the responses of both the LIPAS and UV detectors was investigated by using 1 X M (52 ng) and 1 X lo4 M (5.2 ng) 2'Cl-DAAB solutions. For the case of LIPAS, the variation coefficients were 3.32% for 1 X lo4 M 2'Cl-DAAB solution, and 2.70% for 1 X M solution for a series of six injections, whereas, for UV detection, coefficients of 3.46% for 1 X lo4 M solution and 2.62% for 1X M solution were found. These results show that the LIPAS detector is fully satisfactory compared with the UV detector. By use of LIPAS and UV detectors, the chromatograms were measured over a wide concentration range of 2'Cl-DAAB solutions, that is, from 6 X M (0.31 ng) to 1 X M (5.2 pg) under the experimental conditions summarized in Table I. In the case of LIPAS, PA signal and concentration exhibited a linear relationship to each other over 4 orders of magnitude, from 6 X lo-* M (0.31 ng) to 5 X lo4 M (2.6 pg). In the case of UV a similar relationship between absorbance and concentration held over the concentration of 1 X lo4 M (5.2 ng). The elution peak could not be detected below 6 X lo-' M (3.1 ng). The slopes of the calibration curves were calculated to be 0.985 f 0.004 for LIPAS and 0.980 f 0.008 for UV detection by linear least-squares fit. The same correlation coefficients
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
4
6 Retention
a Time
10
(min )
Flgure 6. Chromatograms of ultra low concentration of CI-DAAB isomers mixtures: (1) 2'CI-DAAB, (2) 3'CI-DAAB, (3) 4'CI-DAAB;. concentration,3 X lo-' M (1.56 ng). Experimental conditions are given in Table I. were obtained, that is, 0,999, for both methods. Detection limits (S/N = 2) were determined to be 4 x M (0.21 ng) for LIPAS, and 8 X M (4.16 ng) for UV, represented by injection concentration of 2 ' C l - D M . Molar absorption coefficients of 2'Cl-DAAB in the UV and visible regions are somewhat different 7700 L mol-' cm-' for 488 nm and 9600 L mol-' cm-' for 254 nm. The detection limits, calculated by considering the dilution in the detection cell with respect to an injection concentration, correspond to 2.0 x in absorptivity for UV and to 7.9 x lo* for LIPAS. The results show that is 25fold more sensitive than UV. However, this value for LIPAS was determined by a noise level inherent in a lock-in amplifier used in the present experiment. The noise amplitude of the PZT detector of our cell in the kilohertz frequency region was estimated to be 1nV or less, and so the detection limit for LIPAS could be improved by appropriate choice of a measuring system. Figure 6 shows chromatograms of three kinds of C 1 - D M isomers mixtures at concentrations of 3 X M (1.56 ng) with UV and LIPAS detection. As the above concentration range wqs lower than the detection limit of UV detector, three elution peaks in the chromatogram could not clearly be discriminated by UV detection. As already described, stability, reproducibility, linearity, and detection limit of LIPAS detection, required for a HPLC detector, were fully satisfactory in comparison with UV detection. LIPAS is thus expected to be very suitable for chromatographic analysis of ultratrace componentsj. One more advantage of the LIPAS detector is that it is well suited for
a micro HPLC detector because of the ease of construction of a micro volume flow cell without a loss in sensitivity. In the case of the UV detector, the sensitivity tends to decrease with reducing cell volume because of shortening of the light pathlength, whereas the sensitivity of LIPAS is scarcely dependent on light pathlength (9). Further, in the case of micro HPLC, the concentration of the solute in the column with respect to eluting solvent is not as dilute in comparison with an ordinary HPLC (10, 11). Therefore, LIPAS detection would be preferable for combination with a micro HPLC. As PAS gives information complementary with fluorescence, simultaneous chromatographic detection with PAS and fluorescence would be much more useful. Though the LIPAS detectors are available with a wide range electromagnetic radiation, the use of lasers limits its application at the present stage. It is particularly difficult to obtain tunable high-powered IR lasers. However, a comparatively stable UV laser is now available, that is, a second harmonic generation of a pulsed dye laser. We are planning to adopt pulse-laser-induced PAS as a HPLC detector. This would greatly improve detector sensitivity. We have demonstrated the usefulness of LIPAS as a detector for chromatographic analysis. LIPAS will become a very attractive detector, particularly in the fields of biochemical and medical analysis.
ACKNOWLEDGMENT We are grateful to Norio Teramae and Shigeyuki Tanaka for use of their HPLC instruments and their helpful advice during our research. We also thank Kageyasu Kuroki of the chemistry department machine shop for construction of the LIPAS flow cell and Sadao Maruyama for his skillful packing of the separation column.
LITERATURE CITED Tauge, T.; Hirata, Y.; Takeuchi, T. Anal. Chem. 1979, 51, 166-169. Vidrine, D. W.; Mattson, D. R. Appl. Spectrosc. 1978, 32, 502-507. Teramae, N.; Tanaka, S. Spectrosc. Lett. 1980, 13, 117-125. Diebold, G. J.; Zare, R. N. Science 1977, 19, 1439-1441. Hershberger, L. W.; Callis. J. 6.; Christian, G. D. Anal. Chem. 1979, 51, 1444-1446. Oda, S.; Sawada, T.; Kamada, H. Anal. Chem. 1978, 50, 865-867. Oda, S.; Sawada, T.; Nomura, M.; Kamada, H. Anal. Chem. 1979, 57, 686-688. Oda, S.; Sawada, T.; Moriguchi, T.; Kamada, H. Anal. Chem. 1980, 52, 650-653. Burt, J. A. J . Acoust. Soc. Am. 1979, 65, 1164-1174. Ishii, D.: Asai, K.; Hiba, K.; Jondtuchi, T.; Nagaya, M. J. Chromatogr. 1977, 14, 157-168. Tsuda, T.; Novotny, M. Anal. Chem. 1978, 50, 271-275.
RECEIVED for review September 10,1980. Accepted November 17, 1980.
