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(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 Y.; Sauda, K.; Irnasaka, T.; Ishibashi, N., unpublished work.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
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
3
0.5 1.0 Sodium Hydroxide Concentration ( M I
15
5 10 Reaction Coil Length (m)
Effect of reaction coil length on the fluorescence intensity of -streptomycin.
1.5
Figure 3. Effect of alkallne concentration on the fluorescence intensity of streptomycin.
1
\ ,
7
OD5
01
02
0.3
n- Naphthoquinone-4-sMonate I
.
,
10
20
05
Concentration (mM)
Flgure 4. Effect of P-naphthoquinone-4-sulfonate concentration on the fluorescence intensity of streptomycin.
.
30 40 50 60 T e w e r a t w C)
OA
70
80
90
Flgure 2. Effect of reaction temperature on the fluorescence intensity of streptomycin.
derivatization by high-pressure liquid chromatography were examined by injection 50 KLof a standard solution of SM (7.5 mg/L) into the chromatograph, with a mobile phase flow rate of 1.5 mL/min and an alkaline solution flow rate of 0.5 mL/min. The excitation maximum of the fluorophor in the effluent from the detector was at 347 nm and the emission peak had a maximum a t 445 nm. These data agreed well with those reported by Faure and Blanquet (7). Therefore, the excitation and emission detections of the fluorometer used were a 351-nm lamp and 420-nm cutoff filter, respectively. Reaction Coil. Reaction coils (0.5 mm i.d.) of various lengths were studied to give optimal fluorescence intensity for SM. Figure 1 shows the effect of the coils on the fluorescence intensity. The fluorescence intensity began to plateau with a reaction coil of about 5 m. The use of a 15-m reaction coil exhibited much higher background fluorescence. Therefore, the 10-m reaction coil was used for the determination of SM. Reaction Temperature. The effect of temperature on the reaction of SM with NQS in the alkaline solution was studied in the range of 15 "C to 90 "C (Figure 2). The fluorescence intensity increased with increases in temperature up to a maximum around 65 "C followed by a gradual decrease at high temperatures. Therefore, a reaction temperature of 65 "C was used. Alkaline Concentration. Figure 3 shows the fluorescence intensity for SM in the sodium hydroxide solution a t a concentraton of 0.3-1.5 M. The maximum fluorescence intensity occurred at the sodium hydroxide concentration of 0.5 M.
0
5
10 Time ( m i d
Flgure 5. Chromatograms obtained from the standard solution of streptomycin (A), streptomycin-free serum (B), and patient serum (C).
/3-Naphthoquinone-4-sulfonate (NQS) Concentration. Figure 4 shows the effect of NQS concentration on the fluorescence intensity for SM. The fluorescence intensity increased gradually up to the concentration of 0.3 mM and appeared to plateau between 0.3 and 0.5 mM. The optimal final concentration of NQS was chosen as 0.4 mM in the mobile phase. The solution was used within 1 week. Analytical Studies. Chromatograms. Figure 5 shows typical chromatograms obtained from the standard solution of SM, SM-free serum, and patient serum. SM-free serum shows no peaks that would interfere with the determination of SM. The retention time of SM is 8 min. Analytical Recovery. In order to estimate the analytical recovery, an aqueous solution of SM (7.5 mg/L) and a SMadded serum (7.5 mg/L) were analyzed and their peak heights compared. The recovery was excellent (100.0 A 0.3%).
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
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Table I. Within-Run and Day-to-Day Precision streptomycin
concn in serum, mg/L 7.5 15.0 30.0
coefficient of variation, % within-run day to day (n = 10) (n = 7) 3.02 3.00 2.67
r.0.990 40'
n.42
'OI
,:
3.50 3.10 3.01
Calibration Curve. A linear regression analysis of the calibration curve obtained from the standard sera (5-50 mg/L) yielded the equation, Y = 0.329X + 0.072 (r = 0.999). A high linearity was obtained between peak heights (Y) and SM in serum, even though no internal standard concentration (X) was used. The limit of detection is 0.5 mg/L. Precision. Within-run and day-to-day precision were obtained from three serum pools containing SM 7.5 mg/L, 15.0 mg/L, and 30.0 mg/L. As Table I shows, the coefficient of within-run variation was less than 3.02% and that of dayto-day variation was less than 3.50%. Comparison with Fluorescence Polarization Immunoassay. The values determined by the proposed method on patient sera were compared with those by the fluorescence polarization immunoassay (Figure 6). The correlation coefficient was 0.990. DISCUSSION NQS reacts with guanidine compounds in alkaline solution to yield high-intensity fluorescent products. SM containing two guanidino groups yields similar fluorescent products. The manual method has been reported for the determination of SM using NQS, but the method by HPLC using NQS has not yet been reported (7). The proposed method describes the determination of SM in serum by HPLC using NQS. Since NQS is not stable in alkaline solution, two pumps (the NQS and the alkaline solution) are necessitated for the postcolumn fluorescence derivatization. But the new postcolumn fluorescence derivatization method has one pump (the alkaline solution) because NQS is added in the mobile phase. As reported previously, precipitation of serum proteins with perchloric acid was essentially complete (8). After centrifugation, as the precipitated serum proteins adhered tightly to the bottom wall of the tube, the supernatant was directly injected into the chromatograph. The sample pretreatment described above is greatly simplified and reduces the analytical time significantly. The separation of SM from the interference in serum has been performed by reversed-phase, ion-pair chromatography based on the same process for the other aminoglycoside antibiotics. By the use of octanesulfonate, the retention time of SM with two guanidino groups and one amino group was longer than that of the interferences with fewer amino groups. By the addition of 1,2-ethanedisulfonate,the retention time of SM and the interferences could be shortened. The NQS
1 .
0
*:'*
10
20
30
40
50
FPIA ( r r q / L )
Flgure 6. Comparison with fluorescence polarization immunoassay (FPIA) and high-pressure liquid chromatography (HPLC).
concentration of 0.15-0.50 mM in the mobile phase did not influence the retention time of SM, though NQS is a counterion reagent. The mobile phase containing NQS could be used at least for a week. The procedure described above was simplified greatly by sample pretreatment of deproteinization with perchloric acid. Even though no internal standard was used in the method, precise results were obtained since protein in the sample pretreatment was separated in one step. The method described here is sensitive and accurate. Each analysis requires only 50 p L of patient serum. Total analytical time including sample pretreatment and chromatographic separation was less than 15 min. This method can be used for pharmacokinetic studies and for routine therapeutic monitoring of SM in serum. This new postcolumn fluorescence derivatization method also can be used for the determination of other guanidine compounds. Registry No. Streptomycin, 57-92-1. LITERATURE CITED (1) Barza, Michael; Scheife, Richard T. Am. J. Hosp. Pharm. 1977, 3 4 , 723-737. (2) Mitra, Shyarnal, K.; Yoshikawa, Thomas T.; Guze, Lucien 6.; Schotz, Michael C. Clin. Chem. (Winston-Salem, N.C.) 1979, 2 5 , 1361-1367. (3) Niisson-Ehie, 1. J. Liq. Chromatogr. 1983, 6(S-2), 251-293. (4) Schwenzer, Kathryn S.; Anhak, John P. Antlmicrob. Agents Chemother. 1983, 2 3 , 683-687. ( 5 ) Whall, T. J. ,J. Chromatogr. 1981, 219, 89-100. (6) Kurosawa, Nahoko; Kuribayashi, Shoichi; Owada, Eiji; Ito, Keiji; Nioka, Masanori; Arakawa, Masako; Fukuda, Ryohei J. Chromatogr . 1985, 343, 379-385. (7) Faure, F.; Bianquet, P. Clin. Chim. Acta 1964, 9 , 292-300. (8) Kubo, Hiroaki; Kobayashi, Yoshie; Nishikawa, Takashi Antimicrob . Agents Chemother. 1985, 28, 521-523.
RECEIVED for review March 18,1986. Accepted June 18,1986.
