Anal. Chem. 1988, 58, 2649-2653
7 40
Figure 2. Effect of pH on the first and second phases of the fluorescamine-secondary amine reaction.
2849
fluorogenic reagent. If, on the other hand, the pH is slightly higher, the fluorescamine is hydrolyzed and therefore unable to react with the amine. The fluorescamine-secondary amine reaction was found to remain stable for over 24 h. The proposed method does not require the identification of each of the nitrosamines present in a sample (as occurs with the chromatographic method), since the N-nitroso compounds are evaluated as a whole, given their reduction to secondary amines. The sensitivity of this method is also considerable, and the minimum detectable level for this procedure is 10 nM of N-nitroso compounds/mL. Registry No. Ni-AI alloy, 11114-68-4;NDMA, 62-75-9; NEMA, 10595-95-6;NDEA, 55-18-5; NDPA, 621-64-7; NEBA, 4549-44-4; NPBA, 25413-64-3;NPYR, 930-55-2;fluorescamine,38183-12-9; L-leucine-L-alanine,7298-84-2.
LITERATURE CITED its innate fluorescence. In the second phase, the lactonic ring closes up when the previous product reacts with a primary amine (L-Leu-L-Ala). The pH plays a vital role in these two phases of reaction. If the pH is low, the amines could be charged by the protons, making a reaction with fluorescamine impossible. Moreover, an increased acidity could provoke the precipitation of the
(1) Magee, P.; Barnes, J. 8 . J. Cancer 1958, 70, 114-122. (2) Cutaia, A. J. J. Assoc. Off. Anal. Chem. 1982, 65(3), 584-587. (3) Sen, N. P.; Seaman, S.; Karpinsky, K. Assoc. Off. Anal. Chem. 1984, 67(2), 232-235. (4) Samueisson, R. Anal. Chim. Acta 1979, 708, 213-219. (5) Nakamura, H.; Tamura, 2. Anal. Chem. 1980, 52, 2087-2092.
RECEIVED for review May 2, 1986. Accepted June 27, 1986.
Determination of Protein in Human Serum by High-Performance Liquid Chromatography with Semiconductor Laser Fluorometric Detection Kouji Sauda, Totaro Imasaka, and Nobuhiko Ishibashi* Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
Indocyanlne green (ICG) was found to become fluorescent when bound with proteln. Then, protein in human serum was labeled with ICG,and the complex was separated by a gel flitration column. The eluted sample was detected by a fluorometrlc system uslng a semiconductor laser (780 nm, 15 mW) as an exciting source. a,-Lipoprotein and yglobulln were preferentlally comblned wlth I C 0 and gave large peaks In the chromatogram, though the albumin content was more than 10 tlmes larger than those compounds. The detectlon limit was 1.3 pmoi for albumln, which was -1-2 orders of magnitude better than the value obtained by conventlonal spectrophotometric and fluorometric detectors.
Laser fluorometry has been used as a sensitive detector for high-performance liquid chromatography (HPLC) because of its good beam coherence and large photon flux. Various lasers such as a continuous wave (CW) argon ion laser and a pulsed nitrogen laser pumped dye laser are used as a light source for the fluorometric detector in HPLC. Diebold and Zare constructed the windowless flow cell, which was designed to reject background light scatter from the cell windows, and they demonstrated ultratrace analysis of aflatoxins by using a helium-cadmium laser (325 nm) (I). Other researchers have also shown advantages of the laser fluorometric detector and have demonstrated many analytical applications (2-9), but, the lasers have large dimensions and are expensive. Moreover, the lasers have some difficulties in their operation and 0003-2700/86/0358-2649$01.50/0
maintenance. As such, the laser fluorometric HPLC detector has not been practical for use in the commercial instrument. Recently, a semiconductor laser has been developed for the application to a videodisc system, since it is very small apd less expensive. The continuous wave semiconductor laser is currently used as a light source for photoacoustic spectrometry (lo), conventional absorption spectrometry (11-13), fluorometry (14), thermal lens spectrometry (151, and heterodyne spectrometry (16). On the other hand, a picosecond and highly repetitive pulsed semiconductor laser is advantageous for measurements of the fluorescence lifetime by a time-correlated photon counting system (17). Among these spectrometric methods, semiconductor laser fluorometry is most sensitive, allowing sample detection at M levels when a 3-mW laser is used. It is emphasized that blank fluorescence from a solvent is completely negligible in near-infrared fluorometry. However, the wavelength of the semiconductor laser commercially available is limited to 750-1300 nm, and therefore, it is useful only in a few analytical applications (14). Some polymethine dyes are known to have absorption bands at around 600-900 nm. They have large molar absorptivities (>l00OOO) and are strongly fluorescent, and as such, they have been used as laser dyes. Therefore, they have been detected at ultratrace levels by semiconductor laser fluorometry (14, 17,18). However, most samples such as biochemical substances have neither an absorption band nor fluorescence emission in this wavelength region. For determination of such substances, the sample molecule should be labeled with a 0 1986 American Chemical Society
2650
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986 Monochromator
1
1
Lens
Color Filter
I
j
ii----J/ Photomultiplier
(CH2)4 I
SOg
I inlector
N ~
[ L o c k - i n Ampliller
I
I
S03Na
Flgure 2. Chemical structure of indocyanine green (ICG).
Reference Semiconductor
Laser {Current d o n t r o i l e r j Mirror
(CH2)4
1-
Lens
Figure 1. Block diagram of experimental apparatus.
near-infrared fluorescent tag. Unfortunately, no such labeling reagent has, at present, been reported. In this study, we first constructed a HPLC system with a semiconductor laser fluorometric detector and used a polymethine dye of indocyanine green (ICG) as a labeling reagent for protein. We demonstrated trace analysis of protein in human serum after sample separation by HPLC and compared the performance of the semiconductor laser fluorometric detector with that of conventional spectrometric detectors.
and other chemicals such as organic solvents were obtained from Wako Pure Chemical Industries. It is noted that control serum is a standard of human serum prepared to contain proteins at specified concentrations for calibration of the instrument in clinical assay. Water was used as a solvent after redistillation and deionization, but other reagents were used without further purification. ICG (25 mg in a bottle) was dissolved in specifically prepared distilled water (Daiichi Seiyaku Co., 10 mL) and diluted stepwise to the specified concentration. Freeze-dried human serum was dissolved in 5 mL of water and treated by ultrasonic agitation (Yamato, Bransonic 12) for 3 min. The pH of the eluent containing 0.1 M sodium sulfate was adjusted to 6.8 with a buffer solution of 0.2 M sodium dihydrogen phosphate and 0.2 M potassium dihydrogen phosphate.
RESULTS AND DISCUSSION Spectrometric Property of ICG. Many polymethine dyes EXPERIMENTAL SECTION are known to be fluorescent in the near-infrared region, but Apparatus. A block diagram of the experimental apparatus no polymethine dye has been used as a fluorescence labeling is shown in Figure 1. The light source of the semiconductor laser reagent. In our previous study a polymethine dye of 3,3’(Sharp, LT024MD) is modulated to square waves at 100 Hz by diethyl-2,2’-(4,5:4’,5’-dibenzo)thiatricarbocyanine iodide a pulse generator (Hewlett-Packard, 8013B). The output power (NK427) with a positive charge was used for ion-pair solvent and the exciting wavelength of the semiconductor lasers are 15 mW and 780 nm, respectively. The stability of the output power extraction of surfactants with a negative charge into the orwas -1%; it could be reduced to 0.003% by feedback control with ganic solvent (14). As a trial, we added NK427 to the aqueous an integrated circuit (Sharp, IR3C02N) as specified by the solution of albumin for evaluation of its potential capability manufacturer. The laser beam is focused into a flow cell by an of being used as a fluorescence labeling reagent, but no change objective lens (Olympus, LWDMSPlan, 50X). Fluorescence from was observed in the fluorescence spectrum. Protein has a lot the sample passes through a spatial filter to reduce scattering of of positive charges in the molecule, so it is considered that exciting light from the flow cell and is collimated by a lens (focal it does not interact with NK427, which has a positive charge. length 31 mm; diameter 27 mm). It goes through an interference Since most polymethine dyes have a positive charge, we filter (Ditric 15-20785,diameter 19 mm, transmission maximum concluded that a group of these compounds were inadequate 830 nm, transmittance 45 %) and a color filter (Toshiba V-R63, 630-nm cutoff). Fluorescence is focused by a lens (focal length for labeling protein. 60 cm, diameter 28 mm) onto a slit of a monochromator (Jasco, Recently, we noticed that a polymethine dye of ICG was CT-IO, dispersion 8 nm/mm) equipped with a red-sensitive used to diagnose liver activity. It is known that a healthy liver photomultiplier (Hamamatsu, R943-02, 160-930 nm), which is rapidly decomposes ICG in the blood. Thus, liver activity can installed in a cooling system (Hamamatsu, C659-A). The applied be examined by measuring the time-dependent ICG concenvoltage was typically -1500 V. The output signal is fed to a lock-in tration in serum after injection of concentrated ICG into the amplifier (NF Circuit Design Block, LI-570) and is measured by blood stream. The chemical structure of ICG, which has a a chart recorder (Rikadenki, R-50). negative charge, is presented in Figure 2. We are also inThe 100-pL sample was injected into a gel filtration column terested in the fact that the absorption peak shifts to longer (Toyo Soda, TSK-gel G-3000SW) by means of a saple injector wavelengths by complex formation with protein (19). This (Rheodyne, Model 7125). The eluent is delivered by a computer-controlleddual pump system (Toyo Soda, CCPD). The typical implies that ICG is bound to protein and that its spectral flow rate was 1 mL/min. The flow cell is made of nonfluorescent properties are changed by complex formation. Barker has quartz glass, which has a path length of 1 cm and a cell volume already reported that ICG is preferentially bound to globulin of 18 pL. fractions (80%)in serum protein (20). As such, this compound The eluted sample was also detected by a commercial fluoromight be used as a labeling reagent for protein. However, no metric detector (Kyowa, KLF-3080) and by a commercial specfluorescence characteristics have been reported for this comtrophotometric detector (Kyowa, KLC-2290). For fluorescence pound. measurements the photomultiplier was replaced by red-sensitive Figure 3 shows the excitation and emission spectra for free R928 produced by Hamamatsu Photonics, whose spectral response ICG in aqueous solution as well as for ICG combined with extends to 930 nm. The excitation and fluorescence spectra were bovine serum albumin (BSA). The ICG molecule was found measured by a conventional fluorescence spectrometer (Hitachi, MPF-4) and the absorption spectrum by a conventional specto have a large molar absorptivity of 180000 at 780 nm by trophotometer (Shimadzu, UV2OOS). absorption measurement. This value is close to that of porThe detection limit was determined from the ratio of the signal phine, which is known to have the largest molar absorptivity intensity and the base-line drift in the chromatogram. as an organic compound. The fluorescence intensity of ICG Reagents and Procedure. The fluorescent reagent of indoincreases when it is bound with protein, and the emission cyanine green was purchased from Daiichi Seiyaku Co. (Diagmaximum is shifted to 820 nm. Fluorescence of ICG is nogreen) or from Nippon Kanko-Shikiso Kenkyusho (anhydro3,3,3’,3’-tetramethyl-l,l’-bis(4-sulfopropyl)-(4,5:4’,5’-dibenzo)- strongly quenched by dimer formation in aqueous solution. The fluorescence intensity increases by complex formation indotricarbocyaninehydroxidesodium salt (NK2611)). Human with protein since it dissociates the ICG dimer. serum (control serum I), bovine serum albumin (M,= 690001,
ANALYTICAL CHEMISTRY, VOL.
58,NO. 13, NOVEMBER 1986
2651
8 d, -lipoprotein
‘z‘1
EXCITATION
EMISSION
%
c
5
ICG bound on albumin
700
750
800
1
850
W a v e l e n g t h /nm
Figure 3.
Excitation and emission spectra for ICG: excitation wavelength, 765 nm; emission wavelength, 820 nm. The concentrations of ICG and BSA are adjusted to 3.2 X lo-’ M.
The stability of the ICGBSA complex was discussed by measuring the fluorescence spectrum under different ICG and BSA concentrations. First, the fluorescence intensity of ICG (1.6 X lo4 M) was measured by changing the BSA concenM, and tration. The intensity was proportional to 1 X thereafter it deviated from linearity. Second, the fluorescence intensity was measured at the specified concentration of albumin (2 X lo4 M) by changing the ICG concentration. It was linear below 1 X lo4 M, giving a maximum value at 1 X M. A t higher concentrations, the signal decreased probably due to self-absorption of ICG. Third, the ICGBSA M) was injected into a highcomplex (6.4 X 10” M:2 X performance liquid chromatograph, and the eluted sample was collected at the albumin peak for the measurements of absorption and fluorescence spectra. It was found that only 3 molecules of ICG were bound to 100 molecules of BSA. Residual ICG might be adsorbed to the packing material of the gel filtration column. These results show that the ICG molecule is rather poorly bound to protein at present concentration levels. It is noted that ICG in aqueous solution gradually decomposed and the fluorescence intensity became 50% in 2 h, with a further decrease to 25% in 24 h. On the other hand, the fluorescence intensity of ICG bound to BSA decreased to 72% in 2 h and was almost identical in 24 h. Thus, ICG bound to BSA was found to be more stable than ICG in aqueous solution. These results show that ICG is useful as an labeling reagent and can be used in chromatographic determination of protein. Chromatography of Human Serum. The real sample, such as human serum, contains various kinds of protein, whose determination has currently been carried out by HPLC with spectrometric and electrochemical detectors. Gel filtration (21,22) and ion exchange column (23)have been used for the separation of proteins. In our preliminary study ICG was added in the eluent, and the sample of human serum was injected into the HPLC system. Such a procedure was necessary in the determination of protein in human serum by ultraviolet laser fluorometry with a fluorescence labeling reagent of 1-anilinc-%naphthalene sulfate (ANS) (24). Though this method gave background noise from free ANS, dissociation of ANS from protein could be reduced, and therefore protein was determined even at trace levels. Unfortunately, this procedure was not successful for the determination of protein with ICG, since the gel filtration column was damaged by adsorption of free ICG. Next, protein in human serum (concentration for albumin, 1.2 X M)was labeled with 2 X 10” M aqueous ICG and it was injected into the HPLC system. The obtained chromatogram is shown in Figure 4. This procedure was found to give sharp and intense signal peaks originating from ICG-
Retention Time lrnin
Flgure 4. Chromatogram for human serum labeled with ICG. Semiconductor laser fluorometry is used for sample detection. wavelength of the monochromator was adjusted to 840 nm.
The
1
I
’.* t
0 0
10
20
30
R e t e n t i o n Tirne/rnin
Flgure 5. Chromatogram for human serum. Conventional spectrophotometer is used for sample detection: (A) absorption of native protein is measured at 280 nm, and (B) absorption of ICG bound to protein is measured at 740 nm.
labeled protein. Dissociation of the complex may take place during sample separation in the column. However, free ICG seems to be adsorbed to the packing material and gives no ICG peak. From the retention time, peaks denoted as a, b, and c are assigned to al-lipoprotein, a group of y-globulin, and albumin, respectively. The chromatogram was measured at different ICG concentrations to investigate the stability of the M) was complex. When more concentrated ICG (6.5 X used, the major peak originated from albumin. In this case, the column was seriously damaged in a short period by adsorption of free ICG. A t a lower ICG concentration (2 x lo4 M), the major peaks appeared from al-lipoprotein and yglobulin. It is noted that the concentration of albumin in human serum is about 10 times larger than that of globulin and fibrinogen (25). Thus, the binding constants of ICG with al-lipoprotein and y-globulins are much larger than that for albumin. In order to acertain this fact, the chromatograms of human serum were measured with a conventional spectrophotometric detector with and without using ICG. The results are shown in Figure 5. It is apparent that ICG is more preferentially bound to a,-lipoprotein and y-globulin. This tendency is opposite to the case for a hydrophobic probe of ANS, which is more preferentially bound to albumin, which implies that the binding mechanisms might be different for ICG and ANS. Detection Limit. In our previous study we could detect 10 fg of NK427 by injecting the 60-nL sample into the stream and by detecting in a 60-nL micro flow cell without using a
2652
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
T a b l e 1. D e t e c t i o n Limit for BSA L a b e l e d with
ICG
method
detection limit, pmol
semiconductor laser fluorometera conventional fluorometer conventional spectrophotometer
1.3 150
b
230 270
C
The photomultiplier is cooled down to -20 "C. Absorption of ICG bound to protein is measured at 740 nm. cAbsorption of native Drotein is measured at 280 nm. separation column (17). This result implies that the semiconductor laser fluorometric detector is useful for ultratrace analysis of protein. The detection limits for BSA labeled with ICG were investigated by the constructed HPLC system with various spectrometric detectors. The results are summarized in Table I. The detection limits for the conventional absorption detectors are 200-300 pmol, which corresponds to 14-21 pg of BSA. By using a fluorometric detector, the detection limit could be slightly improved to 150 pmol. The detection limit was 8 pmol for the semiconductor laser fluorometric detector, which could be improved to 1.3 pmol(90 ng) when a photomultiplier is cooled to -20 "C. Thus, the detection limits for semiconductor laser fluorometry are 1-2 orders of magnitude better than the values obtained by conventional spectrometric detectors. The improved detection limit is, of course, due to the large photon flux of the semiconductor laser. However, it should be emphasized that good beam coherence and monochromaticity are also necessary for reduction of scattered emission by the spatial and spectral filters. It is noteworthy that background emission from impurities in the eluent is negligible in near-infrared fluorometry. Raman scattering is also negligible, since it appears at around 890 nm (small) and 1053 nm (large), which can be completely rejected by a fluorescence monochromator. Thus, the detection limit is, a t present, determined by the dark current noise of the photomultiplier. As such, the photomultiplier cooling system is useful in improving the detection limit, as demonstrated. The sensitivity of the present HPLC system can readily be improved by increasing the output power of the semiconductor laser. It is noted that a 500-mW laser (780-850 nm) is now commercially available (Spectra Diode Labs, SDL-4450 series) and may be useful for the present purpose. Since the excitation maximum of the BSA-ICG complex is located at around 750 nm, the semiconductor laser that oscillates at 750 nm (Sharp,LT030MD) excites the sample more efficiently, though it has a lower output power at present (3 mW). Application. In this study we demonstrate trace analysis of protein in human serum by stepwise dilution of the standard solution. As a matter of fact, such an application might be very few in practical works. It might be possible to detect a very small amount of protein in the pure aqueous solution by using the present HPLC system, but it may be difficult to detect specified trace protein such as insulin in human serum since so many proteins are included in biological fluid. What is necessary in the next step is specificity of the reagent, since ICG is nonspecifically bound to protein. Fluorescence immunoassay may provide us additional selectivity in trace analysis of protein in biological fluid. It has great selectivity, but it still has difficulty in rejection of the background noise since the real sample contains many fluorescent species. Therefore, greater selectivity is necessary in practical fluorescence immunoassay (26). It is noteworthy that radioimmunoassay is very useful since no radioisotope exists in the real sample. In this sense it is recommended to use a red fluorescence tag for labeling protein since the fluorescence intensity of native protein is weak in this
-
wavelength region. We would like to emphasize that no impurity fluorescence could be observed in near-infrared fluorometry, since no molecule is, as far as we know, fluorescent in the near-infrared region except for a polymethine dye. Because of great selectivity given by immunoassay and near-infrared fluorometry, the present approach might be successful in many analytical applications. For fluorescence immunoassay, a labeling reagent, which can be covalently bound to protein and is fluorescent in the near-infrared region, is necessary. Such a compound has, however, not been developed. We except that the development of a near-infrared labeling reagent may open a new field in practical fluorescence immunoassay of protein. Recently, electronic engineers concentrated on their work in developing a semiconductor laser that oscillates at shorter wavelengths. This allows for tight focus of the laser beam into a small area and increases the recording density of information in the optical disk system. A pulsed semiconductor laser that oscillates a t 579 nm is already reported, though the device should be maintained at a liquid nitrogen temperature (27). We expect that room-temperature operation will be achieved in the near future. If such a laser was available, many fluorescence labeling reagents such as rhodamine dyes could be used. For example, rhodamine isothiocyanate (RITC) is well-known for reacting with an amino group of protein and for undergoing fluorescence in this wavelength region. Thus, the use of RITC readily allows for demonstration of fluorescence immunoassay of protein using semiconductor laser fluorometry. Very recently, the research group of Matsushita Electric Co. reported a new technology for very effieicnet frequency conversion from near-infrared emission (800 nm, 30 mW) to blue-violet emission (1 mW) by using a wave guide of a nonlinear crystal (LiNbOJ (28). Such a visible laser may be widely used for many spectrometric applications, though selectivity may be partially lost since impurity included in biological fluid is weakly fluorescent in the visible region.
ACKNOWLEDGMENT We thank Hideo Watanabe of Toyo Soda for his helpful suggestion that ICG is currently used as a reagent to examine liver activity in the clinical field. We also thank him for providing us with a HPLC system manufactured by Toyo Soda. R e g i s t r y No. ICG, 3599-32-4. LITERATURE CITED Dieboid, G. J.; Zare, R. N. Science (Washington, D.C.)1977, 796, 1439. Sepaniak, M. J.; Yeung, E. S.J . Chromatogr. 1980, 190, 377. Folestad, S.;Johnson, L.; Josefsson, E.; Gab, 6.Anal. Chem. 1982, 5 4 , 925. Todoriki, H.; Hirakawa, A. Y. Chem. Pharm. Bull. 1984, 32, 193. Zare, R. N. Science (Washington, D . C . ) 1984, 226, 298. Richardson, J. H.; Larson, K. M.; Haugen, G. R.; Johnson, D. C.; Ciarkson, J. E. Anal. Chim. Acta 1980, 776. 407. Imasaka, T.; Ishibashi, K.; Ishibashi. N. Anal. Chim. Acta 1982, 742. 1.
Furuta, N.;Otsuki, A. Anal. Chem. 1983, 55. 2407. Ishibashi, K.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1985, 173, 165. Kawabata. Y.: Kamikubo. T.: Imasaka, T.: Ishibashi, N. Anal. Chem. 1983, 55, 1419. Imasaka, T.; Kamikubo, T.; Kawabata, Y.; Ishibashi, N. Anal. Chim. Acta 1983, 753,261. Ohtsu, M.; Kotani, H.; Tagawa, H. Jpn. J . Appl. Phys. 1983, 22, 1553. Chan, K.; Ito, H.; Inaba, H. Appl. Opt. 1983, 22, 3802. Imasaka, T.; Yoshitake, A.; Ishibashi, N. Anal. Chem. 1984, 56, 1077. Nakanishi, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1985, 57, 1219. Lenth, W.; Gehrtz, M. Appl. Phys. Lett. 1985, 4 7 , 1263. Kawabata, Y.; Imasaka. T.; Ishibashi, N. Talanta 1986, 33, 281 Sauda. K.; Imasaka. T.; Ishibashi, N. Anal. Chem., in press. Fox, I. J.; Wood, E. H. Staff Meeting Mayo Clinic 1960, 35, 732. Barker, K. J. Proc. SOC. E x p . Biol. Med. 1988, 122,957. Okazaki, M.; Ohno, Y.; Hara, I . J . Chromatogr. 1980, 221, 257.
Anal. Chem. 1986, 58, 2653-2655
2653
(27) Ikeda, M.; Honda, M.; Mori, Y.; Kaneko, K.; Watanabe, N. Appl. f h y s . Lett. 1904, 4 5 , 964. (28) Nishi-Nlppon Newspaper, June 19, 1986.
(22) Okazaki, M.; Hara, I.BunsekiKagaku 1984, 33,356. (23) Kadoya, T.; Arnano, Y.; Isobe, T.; Kato, Y.; Nakamura, K.; Okuyama, T. Bunseki Kagaku 1984, 33 €287. (24) Kawabata. Y.; Sauda, K.; Irnasaka, T.; Ishibashi, N., unpublished work. (25) Tomono, T.; Toshida, S.; Tokunaga, E. J . W m . Sci. polym. Lett. Ed. 1979, 1 7 , 335. (26) Lidofsky, S. D.; ~masaka,T.; Zare, R. N. Ana/. them, 1979, 5 1 , 1602. I
RECEIVED for review April 25, 1986. Accepted July 1, 1986. This research is supported by Grant-in-Aid for Scientific Research from the Ministry of Education of Japan.
Fluorescence Determination of Streptomycin in Serum by Reversed-Phase Ion-Pairing Liquid Chromatography Hiroaki Kubo,* Yoshie Kobayashi, and Toshio Kinoshita School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan
A new postcolumn fluorescence derivatiration method for the determinatbn of streptomycin in serum by high-pressure liquid chromatography has been developed. The method Is sensitive to 0.5 mg/L using only 50 pL of serum. After the serum proteins are precipttated with percMork acid, the supernatant Is injected into the chromatograph. Streptomycin is separated by reversed-phase ion-pairing chromatography using a mobile phase containing octanesuifonate, 1,2athanedisuIfonate, and P-naphthoquinone-4-suifonateand detected by fluorescence using continuous-flow, postcolumn derivatization with alkaline medium and P-naphthoquinone-4-sulfonate in the mobile phase. Comparison with a fluorescence polarization immunoassay gave a correlation coefficient of 0.990.
Streptomycin (SM) was the first aminoglycoside antibiotic discovered that exhibited a high potency and a broad-spectrum bactericidal action against both gram-negative and gram-positive bacteria, particularly Mycobactrium tuberculosis. Today, it is used primarily in combination with other antimicrobial agents to treat serious enterococcal infections and tuberculosis. Like other aminoglycoside antibiotics, SM has a narrow therapeutic range and exerts nephrotoxicity and ototoxicity (1). Therefore, monitoring SM levels in serum is necessary to achieve the best therapy. Various methods for the determination of aminoglycoside antibiotics except SM were reviewed (2, 3). For SM, only bioassay has been widely used in clinical laboratories. The disadvantages of this method are lack of speed, specificity, simplicity, sensitivity, and precision. Recently, analytical methods for S M by fluorescence polarization immunoassay ( 4 ) and high-performance liquid chromatography (HPLC) using UV detection (5, 6) were reported. Fluorescence polarization immunoassay is rapid and sensitive, but the apparatus and reagent kit for fluorescence polarization immunoassay are expensive. One method of HPLC does not apply to the determination of SM in biological materials. The other method does apply to the determination of SM in serum, but time-consuming sample pretreatment and a large volume (400 KL)of serum are required. This paper describes a new postcolumn fluorescence derivatization method using P-naphthoquione-4-sulfonate, which forms fluorescent products with guanidino groups in SM (7). The method is rapid, accurate, sensitive, and specific for the determination of SM in serum. The values determined by 0003-2700/86/0358-2653$01 SO10
the proposed method were compared with those by fluorescence polarization immunoassay.
EXPERIMENTAL SECTION Apparatus. The chromatographic system was constructed from a Model 6000A solvent delivery pump, a Model U6K injector, and a radial compression separation unit that consisted of a Radial-PAK CIS(10 wm, 10 cm x 8 mm id.) cartridge and a Model RCM-100 module for compressing the cartridge, all from Nihon Waters, Ltd. (Tokyo, Japan). The column effluent was introduced into a Waters M-105 reaction system equipped with a mixing tee, a reciprocating pump, a pulse-dampening device, and a reaction coil consisting of a stainless steel tube (10 m X 0.5 mm id.) in a heating bath, from Nihon Waters, Ltd. As a detector, a Model S-FL-330 fluorometer (Soma Optics Co., Ltd., Tokyo, Japan) equipped with a L-1549-04 lamp (energy maximum at 351 nm, excitation), a 420-nm cutoff filter (emission),and a 25-rL quartz flow cell was used. The detection signal was recorded with a Model VP6621A national pen recorder (Matsushita Communication Industrial, Osaka, Japan). Reagents. Streptomycin sulfate (manifested potency 725 pg/mg) was obtained from Meiji Seika Co. (Tokyo, Japan). Sodium ~-naphthoquinone-4-sulfonate (NQS) was obtained from Wako Pure Chemicals (Osaka, Japan). Sodium octanesulfonate was obtained from Aldrich Chemical Co. (Milwaukee, WI) and disodium 1,2-ethanedisulfonatewas obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Water and acetonitrile used were of liquid chromatographic grade. All other chemicals were of reagent grade. The mobile phase was prepared to contain 20 mM disodium 1,2-ethanedisulfonate,5 mM sodium octanesulfonate, and 0.4 mM NQS in a water-acetonitrile mixture (8020, v/v), adjusted to about pH 3.3 with acetic acid. Serum samples were obtained from the National Sanatorium Nishiniigata Hospital. Procedure. A 50-pL serum sample in a 1.5-mL tapered polypropylene centrifuge tube was vortex-mixed with 50 y L of 3.5% perchloric acid solution for a few seconds. The mixture was centrifuged at lOOOOg for 1 min. A 50-pL aliquot of the supernatant was injected into the chromatograph. Standard sera supplemented with various known amounts of SM (potency, 5-50 mg/L) were prepared and analyzed. Peak height measurements were performed to construct the calibration curve. Every serum was analyzed in duplicate and the results were averaged. Fluorescence polarization immunoassay was performed by using commercially available kits (Abbot-TDX-Streptomycin,Dainabot, Tokyo, Japan). RESULTS Optimization of the Analytical Chromatographic System. The optimum reaction conditions for the postcolumn 0 1986 American Chemical Society
