Anal. Chem. 1988, 58, 289-292 (13) Iwasakl, S.;Tanaka, H.; Nakazawa, K.; Arima, M. J . Chromafogr. 1985, 341, 182-186. Preston, M. R. J . Chromafogr. 1983, 275, 178-182. Salazar, A. R.; Baines, A. D. Anal. Biochem. 1985, 145, 9-13. , Yamamoto, T.; Shlmizu, H.; Kato, T.; Nagatsu, T. Anal. Biochem. 1984, 142, 395-399. (17) Tamm, C.; Hodes, M. E.; Chargaff, E. J . Blol. Chem. 1952, 195, 49-63. (la) Hurst, R. 0.; Marko, A. M.; Butler, G. C. J . B b l . Chem. 1953, 2 0 4 , 847-855. (19) Mayers, V. L.; Spizizen, J. J . Bioi. Chem. 1954, 2 1 0 , 877-884.
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(20) Hashizume, T.; Sasaki, Y, Anal. Biochem. 1988, 2 4 , 232-242. (21) Darllngton, R. W.; Randall, C. C. Virology 1983, 19, 322-327. (22) Shapiro, H. S. In “CRC Handbook of Biochemistry”; Sorber, H. A., Ed.; The Chemical Rubber Co.: Cleveland, 1970; p H-84. (23) Borenfreund, E.; Fitt, E.; Bendich, A. Nature (London) 1961, 191, 1375-1376.
RECEIVED for review August 6,1985. Accepted September 23, 1985.
Application of Precolumn Reaction to High-Performance Liquid Chromatography of Qinghaosu in Animal Plasma Zhao Shishan* and Zeng Mei-Yi Institute of Chinese Materia Medica, Academy of Traditional Chinese Medicine, Beijing, People’s Republic of China
A precolumn reactlon of llquld chromatography of qinghaosu and a method for the determlnatlon of qlnghaosu In anlmal plasma are descrlbed. Qinghaosu was converted to the UVabsorbing compound 0260 by treatlng It with 0.16% NaOH at 45 O C for 30 mln and thereafter ackllfylng the soiutlon with acetic acld. Optimum conditions of the precoiumn reaction were Investlgated. Plasma samples were extracted with ethyl acetate. After evaporatlon, qlnghaosu In the residue was converted to 0260 by using the precolumn reaction and determlned by hlgh-performance liquid chromatography. Detectlon limit was 3 ng. Relative deviation was less than 6%. Recovery at 5 X lo-* g/mL, 5 X lo-’ g/mL, and 5 X lo-’ g/mL was 107%, 105%, and 94%, respectively.
Precolumn reaction has been successfully used in liquid chromatographic measurements to enhance their sensitivity and selectivity. Qinghaosu is a very effective new antimalarial constituent of the Chinese traditional herbal drug Artemisia annua L. (I). Its medicinal significance has called for intensive chemical, phytochemical, and biological studies (2-4). Determination of qinghaosu in body fluids and in other samples such as plant extracts and medicinal preparations has been one of the objectives in research. In this aspect, a method of high sensitivity is required for the determination of qinghaosu in samples of biological importance as, for instance, in pharmacokinetic study. Qinghaosu does not possess any sensitive and specific spectrometric characteristics. Therefore, there is a need for its modifications or derivation for a favorable spectrometric detection. Zeng (5) and co-workers reported that treating qinghaosu with sodium hydroxide solution gave a resultant, called Q292, having a maximum absorbance of its UV spectrum at 292 nm. Q292 could be further converted into another W-absorbing compound at 260 nm, called Q260, by acidifying its solution (see Scheme I). It might be expected that the modification reaction for Qinghaosu to Q260 could be utilized as a precolumn reaction for liquid chromatographic measurement at high sensitivity. In a previous article (6)we have reported the chemical equilibrium between Q292 and Q260, their UV-absorption characteristics, and the RP-C18 liquid chromatographic behavior of Q260. In this work, optimum conditions of the precolumn reaction have been investigated, 0003-2700/86/0358-0269$01.50/0
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Q292
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while a high-performance liquid chromatographic method for the determination of qinghaosu in animal plasma has been established based on this precolumn reaction.
EXPERIMENTAL SECTION Chemicals. The qinghaosu crystal used was purified in the authors’ laboratory. Q260 was prepared according to ref 6. All other chemicals were of analytical-reagent grade. Apparatus. The W spectrophotometer used was a Shimadzu Model UV-300 (Japan). The HPLC system consisted of a LC4A chromatograph, a SPD-PAS UV detector, a 4 mm i.d. X 25 cm stainless steel column packed with LiChrosorb-RP18 (10 pm) of E. Merck (West Germany), and a CR-2 AX Chromatopac microprocessor, all manufactured by Shimadzu (Japan). Conditions for the Precolumn Reaction. A stock solution of 0.100 mg/mL of qinghaosu was prepared by using ethanol as solvent. To investigate the reaction conditions, 4 mL of NaOH solution at certain concentration was pipetted in a 10-mL flask and, when necessary, prewarmed in a water bath at a given temperature for 5 min. One milliliter of stock solution was then added and the time was counted. After a certain reaction time in the water bath, 4 mL of acetic acid solution of the same molar concentration as the NaOH solution was added. The mixture was brought to room temperature with cooling water and adjusted to 10 mL with ethanol. A 1O-wL portion of the mixture was injected onto the column and peak area was measured. Standard Procedure for the Precolumn Reaction. A 1-mL ethanolic solution of qinghaosu sample was pipetted into a 10-mL flask and mixed with 4 mL of 0.2% NaOH solution. The mixture was warmed in a water bath at 45 O C for 30 min. After being cooled with water, the mixture was neutralized and diluted to 10 mL with 0.1 M acetic acid in 20% ethanol. This solution was taken directly for chromatography. Extraction Behavior of Qinghaosu and Q260. To observe the extraction behavior of qinghaosu and Q260 from water into ethyl acetate, 2 mL of a 0.100 mg/mL ethanolic solution of qinghaosu or Q260 was mixed with 18 mL of distilled water in a separatory funnel. The mixture was adjusted to a certain pH value with dilute hydrochloric acid or sodium hydroxide solution and then extracted with 20 mL of ethyl acetate. Ten milliliters of 0 1986 American Chemical Society
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the organic phase was transferred into a 10-mL flask and evaporated with nitrogen gas. In the case of qinghaosu, the residue was dissolved in 1 mL of ethanol and further treated according to the standard procedure described above. In the case of Q260, the residue was dissolved in 10 mL of a water-ethanol mixture (82, v/v). The extracted percentages of qinghaosu and Q260 were given by the ratios between their peak areas and that of a 0.0100 mg/mL qinghaosu sample prepared according to the standard procedure and a 0.0100 mg/mL standard solution of Q260, respectively. Preparation of Plasma Samples. A known amount of qinghaosu suspended in 1 drop of Tween-80 and 4 mL of distilled water was orally administered to rats or rabbits. Blood samples were drawn before the administration and after administration at given intervals. For rats the 100-pL blood samples were drawn through a heparinized glass capillary in an eyesocket and centrifuged at 2000 rpm for 8 min. The plasma was transferred quantitatively to a test tube containing 2.0 mL of a 0.5% NaCl solution with a microsyringe and stored in a refrigerator until taken for extraction. For rabbits the 1-mL blood samples were drawn through a syringe inserted in an auricle vein and transferred in a heparinized test tube. It was centrifuged at 2000 rpm for 8 min. The plasma was removed to a test tube containing 2.5 mL of a 0.5% NaCl solution with a 500-pL syringe. The test tube was stored in a refrigerator until taken for extraction. A plasma sample was extracted twice with equal volumes of ethyl acetate. After the addition of 0.1 mL of 5 M (NH4)2C03 solution, plasma sample was again extracted with 2 mL of ethyl acetate. The extracts were combined in a test tube of 8 mL and evaporated to dryness with nitrogen gas. The residue was dissolved with 0.500 mL of ethanol and then mixed with 2.00 mL of 0.2% NaOH solution. The mixture was treated in a water bath at 50 "C for 30 min. Immediately after being cooled with water, the mixture was extracted with 2.5 mL of ethyl acetate. The organic phase was discarded. After the addition of 1 drop of 6 N HCl, the sample solution was extracted twice with 2.5 mL of ethyl acetate. The extracts were combined and evaporated in a small test tube. The residue was added to a 0.01 M acetic acid-sodium acetate buffer solution (methanol-water, 2:8, v/v). The final volume was 0.250 mL for plasma sample of rat and 0.500 mL for that of rabbit. The sample solution was then taken for chromatography. The injection volume was 200 MLfor plasma sample of rat and 100 pL for that of rabbit. mg, 1.25 X Recovery. For the study of recovery 1.25 X lo-' mg, and 1.25 X 10+ mg of qinghaosu in ethanol were pipetted into 6-mL test tubes, respectively. The solvent was evaporated with nitrogen gas. The residue in each tube was mixed thoroughly with 0.25 mL of rat plasma and 2.25 mL of 0.5% NaCl solution. The mixtures were treated according to the above preparation procedure of plasma samples. Recovery ratios were obtained by comparing the peak area of each test solution with that of a standard one at the same concentration level. Precision. Plasma samples were divided in four aliquots. They were treated according to the preparation procedure of plasma samples. Curves of Peak Area Response against Wavelength. Five blood samples were drawn within 2 h after administration of qinghaosu to a rat or a rabbit. Their plasma fractions were combindd and treated proportionally in the same way as the preparation of plasma samples. The sample solution was measured simultaneously against a standard solution at different wavelengths. Graphs of peak area vs. wavelength were drawn. Chromatographic Conditions. Chromatographic conditions were as follows: mobile phase, 0.01 M Na2HP04-NaH2P04buffer (water-methanol, 55:45, v/v); flow rate, 1mL/min; wavelength, 260 nm; detector sensitivity (AUFS), 0.01; temperature, ambient temperature; quantitative data processing, two point absolute calibration curve method using peak area. Calibration solutions were prepared according to the standard procedure mentioned above. RESULTS AND DISCUSSION Optimization of the Conditions of the Precolumn Reaction. The conversion from qinghaosu to Q260 completes in two individual steps: first from qinghaosu to Q292, then from Q292 to Q260. In fact, the conversion from Q292 to Q260
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Figure 1. Effect of concentration of sodium hydroxide solution on the conversion from Qinghaosu to Q292: temperature, 45 OC; reaction time, 30 min.
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is a rapid and reversible acid-base equilibrium depending on pH. Therefore, the first step from qinghaosu to Q292 is the determinative step. It was affected by the concentration of sodium hydroxide in reaction solution, the reaction temperature, and time. Figure 1 shows the effect of the concentration of sodium hydroxide solution on the conversion from qinghaosu to Q292 for a given reaction temperature and time. It is obvious that the optimal final concentration of sodium hydroxide solution should be 0.16%. Concentration higher than 0.2% resulted in decrease in peak area. The temperature effect on the conversion is shown in Figure 2. The reaction was speeded up by increasing temperature (see also Figure 3). The proper temperature lay in the range from 25 to 48 OC for the concentration of sodium hydroxide
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
reaction solution. For the optimal sensitivity, precision, and favorable reaction time, the final concentration of sodium hydroxide in reaction solution, the reaction temperature and the reaction time should be 0.16%, 45 "C, and 30 min, respectively, as have been mentioned above in the standard procedure. Unlike Q292, Q260 is very stable. As soon as Q292 was converted into Q260, no change in peak area could be observed even after 1 week in comparison with a freshly prepared standard solution. It can be seen in Figure 3 that a Q292 solution should be converted into Q260 solution by acidifying it within about 1 h after the conversion from qinghaosu to Q292 was completed. UV Spectra. The UV absorption characteristics of qinghaosu and Q260 are shown in Figure 4. The absorption coefficients of qinghaosu and Q260 are 3.7 X lo2 L/(mol.cm) (203 nm) and 1.3 X lo4 L/(mol-cm) (259 nm), respectively. Practical Application to Plasma Samples. The extraction of qinghaosu from water phase with ethyl acetate was quite efficient. It was hardly affected by pH (see Figure 5). Q260 is a carboxylic acid. Changes in pH had profound effect on its extraction from water into ethyl acetate (see Figure 5). This property could be utilized for the separation of Q260 from impurities in plasma sample. As has been described in the preparation procedure of plasma samples, the reaction mixture was extracted with ethyl acetate after the alkaline treatment to remove some impurities. Then the mixture was acidified and Q260 was extracted with ethyl acetate. This process was useful to eliminate possible interferences and to protect the separation column. Before quantitation the identification of the Q260 peaks of plasma samples was accomplished by comparing the retention data and the curves of peak area against wavelength between samples and standard. Figure 6 and Figure 7 show the chromatograms of plasma samples of a rat and a rabbit before and after administration and a standard solution. Figure 8 and Figure 9 illustrate the peak area responses of plasma samples of a rat and a rabbit against wavelength in comparison with that of a standard solution, respectively. Because the concentration of plasma samples was very low, an on-line scanning of the UV spectrum was impossible. According to Beers law the ratio between the peak area response of a sample and that of a standard (Aspl/AsM)should be constant at different detection wavelengths. Both the retention data and the curves of peak area response indicated the identity of the considered peaks of plasma samples to that of the standard. The detection limit (SIN = 10) of the present method was 3 ng. In Figure 8 the original chromatographic peak of 4 ng
Q260
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Wavelength (nm)
Figure 4. UV spectra of qinghaosu and (2260: qinghaosu, 1.O mg/mL in methanol; (2260,0.050 mg/mL in eluent used in the chromatographic procedure. 100-
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0.16% and the reaction time was 30 min. Over this range less than a deviation of 1% in peak area could be caused by a deviation of 1 "C in temperature. Figure 3 depicts the time course with 0.16% sodium hydroxide solution at different temperatures. The curves reached their plateaus in 45 min at 25 "C and in less than 5 min at 50 "C, respectively. They began to lower after a certain stable period. It could be concluded from Figures 1 , 2 , and 3 that Q292 is not very stable. Its stable period depends on both the temperature and the concentration of sodium hydroxide in
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
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Figure 7. HPLC chromatograms of a standard solution and of the plasma samples of a rabbit before and after the administration of qinghaosu (dose, 150 mg/kg): (A) standard, 250 ng in 25 pL solution injected; (Bl) before administration; (B2) 5 min after administration; (C) 20 min after administration; (D)60 min after administration.
of 5 X mg/mL, 5 x lo-' mg/mL, and 5 x lo4 mg/mL. The recovery obtained was 107%, 105%, and 94%, respectively. The relative standard deviation (n = 4)was less than 1 % for the determination of pure qinghaosu, which indicated good precision for the procedure of the precolumn reaction, while that for the analysis of plasma samples was less than 6%. The concentrations of qinghaosu vs. peak area is linear. The linearity over the observed range from 5 ng to 0.5 pg was satisfactory.
CONCLUSION The conversion reaction from qinghaosu to Q260 is a specific quantitative reaction. The present method is suitable for the analysis of qinghaosu in biological samples. The method may be used to analyze other kinds of samples with modification of the separation system.
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ACKNOWLEDGMENT
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The authors wish to thank the collaboraters of the second department of pharmacology at our institute for supplying the plasma samples. R e g i s t r y No. Q260, 88104-60-3;quinghaosu, 63968-64-9. I
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LITERATURE CITED
Wavelength (nml
Figure 8. Oraph of peak area response of a plasma sample of a rat against wavelength in comparison with that of a standard solution: (0) standard, 100 ng; (0) plasma sample.
of qinghaosu standard converted into Q260 injected on column is represented. Its height is equal to about 15a. Because of the on-column preconcentration effect of the chromatographic procedure, injection volume within 1mL has no effect on peak area response (6). According to the concentration levels of qinghaosu in plasma samples, recovery was tested in rat plasma at the levels
(1) Qinghaosu Antimalaria Coordinating Research Group Chin. Med. J , (8eijing. Engl. Ed.) 1979, 92, 811-816. (2) Qinghaosu Antimalaria Coordinating Research Group Yaoxue Tongbao 1979, 74, 49-53.
(3) Liu, Jing-Ming; NI, Mu-Yun; Fan, Ju-Fen; Tu, You-You; Wu, Zhao-Hua; Wu, Yu-Lin; Chou, Wei-Shan Acta Chim. Sin. 1979, 3 7 , 129-141. (4) He, Xi-Chun; Zeng, Mei-Yi; Li, Guo-Feng; Liang, Zheng Acta Bot. Sin. (Engl. Trans/.)1983, 25, 87-90. (5) Zeng, Mei-Yi; Li, Lan-Na; Chen, Shu-Feng; Li, Guang-Yi; Liang, XiaoTian; Chen, Marie; Clardy, Jon Tetrahedron 1983, 3 9 , 2941-2946. (6) Zhao, Shishan; Zeng, Mei-Yi Planta Med. 1985, 304 (3). 233-237.
RECEIVED for review June 25, 1985. Accepted September 13, 1985.
