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Anal. Chem. 1986, 58,285-289
that values of S for typical peptide mixtures are only weakly dependent on peptide t, values. Origin of Equation 13 (figure-of-merit procedure). Equation 13 was derived in the following fashion. When chromatographic conditions are changed for a given sample, changes in relative peak position (for different compounds) often occur. For example, a change in mobile phase composition is often used to alter band spacing, and peak reversals are then common. Chromatographers who are familiar with such examples of peak reversal commonly keep track of individual compounds within the sample by comparing between chromatograms on the basis of their relative size and retention. That is, peaks with similar areas and similar retention times are likely to represent the same compound in the two chromatograms. We constructed several representative examples of such peak reversal, using a range of different areas and relative retentions for two peaks. We then studied different functions of relative peak area and peak retention in an effort to provide as accurate a match as possible between peaks representing the same compound. In this way eq 13 was eventually derived. We subsequently tested eq 13 against a number of actual chromatograms where peak identity was known (steroids, herbicides, etc., changing mobile phase composition), and where peak reversal was rather common. For these examples it was found that eq 13 usually gave the correct identifications; i.e., correctly matched the compounds in chromatogram 1with those in chromatogram 2. Exceptions to this were occasionally noted and found to be related to several effects, one of which is change in peak area with mobile-phase composition. In these cases this difficulty could be overcome by making smaller changes in mobile phase composition between the two chromatograms being compared. The use of eq 13 in the present study is less subject to these errors, because changes in mobile phase composition were less serious than in the latter studies with the steroids and herbicides, and because changes in F can be made small enough to avoid major peak reversals with resulting erroneous peak assignments. Thus we recommend increasing (or decreasing)
flow rate slowly for the second run, then by larger amounts in succeeding runs. This practice was used in the myoglobin study, where flow rates selected were 0.5, 0.61, 0.7, 1.5, and 3.8 mL/min. This allows the more accurate assignment of peak identity to the initial runs, following which eq 10 can be used to confirm assignments in later runs.
LITERATURE CITED Rosenthal, D. Anal. Chem. 1982, 54, 63. Davis, J. M.; Giddlngs, J. C. Anal. Chem. 1983, 55, 418. Glajch, J. L.; Klrkland, J. J. Anal. Chem. 1983, 55,319A. Grego, B.; Lambrou, F.; Hearn, M. T. W. J. Chromatogr. 1983, 266, 89. Vensel, W. H.; Fujita, V. S.;Tarr, G. E.; Margoliash, E.; Kayser, H. J. Chromatogr. 1983, 266, 491. Bennett, H. P. J. J. Chromatogr. 1983, 266, 501. Terabe, S.;Nishi, H.; Ando, T. J. Chromatogr. 1981, 212, 295. Wu, S.;Tseng, M.-J.; Wang, K.-T. J. Chromatogr. 1982, 242, 369. Snyder, L. R. I n "High-Performance Llquid Chromatography"; Horvath, Cs., Ed.; Academic Press: New York, 1981; Vol. 1, p 207. Stadalius, M. A.; Gold, H. S.;Snyder, L. R. J . Chromatogr. 1984, 296, 31. Hearn, M. T. W.; Grego, B. J. Chromatogr. 1983, 266, 75. Quarry, M. A.; Grob, R. L.; Snyder L. R. Anal. Chem., submitted for publication. Quarry, M. A.; Grob, R. L.; Snyder, L. R. J. Chromatogr. 1984, 285, 19. Meek, J. L.; Rosetti, 2. L. J. Chromatogr. 1983, 211, 15. Schoenmakers, P. J.; Billiet, H. A. H.; De Galan, S. A. J. Chromatogr. 1979, 185, 179. Cohen, K. A.; Dolan, J. W.; Grillo, S.A. J. Chromatogr. 1984, 316, 359. Van der Zee, R.; Welllng, G. W. J . Chromatogr. 1982, 244, 134. Kerlavage, A. R.; Hasan, T.; Cooperman, B. S. J. Biol. Chem. 1983, 258,6313. Snyder, L. R.; Stadallus, M. A.; Quarry, M. A. Anal. Chem. 1983, 55, 1412A. Stadalius, M. A.; Quarry, M. A.; Snyder, L. R. J . Chromatogr. 1985, 327, 27. Allen, G. "Laboratory Techniques in Biochemistry and Molecular Biology-Sequencing of Proteins and Peptides"; Elsevler Science Publishers: New York, 1981. Laub, R. J.; Purnell, J. H. J. Chromatogr. 1975, 712, 17. Aguilar, M.-I.; Hodder, A. N.; Hearn, M. T. W. J. Chromatogr. 1985, 327,115.
RECEIVED for review February 4,1985. Resubmitted August 30, 1985. Accepted September 13, 1985.
Quantitation of Nucleic Acids at the Picogram Level Using High-Performance Liquid Chromatography w'ith Electrochemical Detection Johan B. Kafil,' Hung-Yuan Cheng, and Thomas A. Last* Analytical, Physical and Structural Chemistry, S m i t h Kline and French Laboratories, F90, Philadelphia, Pennsylvania 19101 Hlgh-performance liquid chromatography with amperometrlc detedtlon was used to quantitate nuclelc aclds at levels down to 100 pg. The method Is based on hydrolysis and quantltatlon of the purine bases. The detectlon limit for adenine was 0.1 pmol and for guanlne was 0.05 pmol. The method compared well with uitravlolet absorptlon at 258 nm and ethldlum bromlde fluorescence, for quantlatlon of DNA from four dlfferent sources.
The growing interest in recombinant DNA technology has led to the need for a sensitive and precise method of quanPresent address: Quality C o n t r o l Department, H o f f m a n n - L a Roche, Inc., Nutley NJ 07110.
titating nucleic acids. Various methods for quantitating DNA/RNA have been reported; however, each one has some particular drawback with respect to quantition at low levels. The ultraviolet (UV) absorbance measurement at 258 nm is simple to perform, and the detection limit is approximately 100 ng/mL based on an absorptivity of lo4 cm-l mol-l of phosphate ( I ) , but many other compounds absorb UV light in this region. Several colorimetric procedures (2)exist that are based on the reaction between ribose and either diphenylamine or orcinol, following hydrolysis of the nucleic acid. These procedures yield detection limits of 1000 ng/mL and are subject to interferences from other compounds ( 3 , 4 ) . Techniques based on the fluorescence of ethidium bromide (5, 6) or 4',6-diamidino-2-phenylindole(7) yield detection limits in the range of 1-10 ng/mL, but these values are based
0003-2700/86/0358-0285$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
on the final DNA concentration in the dye solution. The original DNA solution would have to be 10-fold more concentrated in order to allow for control of pH and ionic strength. Furthermore, nucleoproteins interfere with all of the intercalating dyes (8). Radioimmunoassay procedures can detect as little as 5 pg of some types of DNA (9);however, the method is not quantitative due to variability of the immunoresponse with respect to the DNA source and sample treatment. Methods based on hybridization (10,11) are very sensitive, with detection limits on the order of 50 pg of DNA, using complementary 32P-labeledRNA probes with radiographic detection. These techniques are extremely valuable for detecting clones or free DNA from a desired cell line, but they are not well-suited for quantitation. It is necessary to know from the outset what DNA sequence one hopes to detect in order that the proper RNA probes be obtained; DNA that does not contain the complementary sequence could be completely overlooked by hybridization. Further, the kinetics of hybridization are dependent on the effective solution volume, temperature, agitation, and the solvent used, leading to imprecision in the measurement. Chromatographic methods are somewhat immune to interferences because the components in the sample are separated prior to detection and quantitation. Gas chromatography, following hydrolysis and derivatization, has been used to quantitate DNA with a detection limit of 2 ng (12). High-performance liquid chromatography (HPLC) has been used to quantitate the bases liberated by hydrolysis of nucleic acids. HPLC/UV provides detection limits of 0.25-7.5 nmol (13) for the individual bases, while HPLC/fluorescence yields a detection limit of 1 pmol for derivatized adenine (14, 15). Recently, Yamamoto et al. (16) reported an HPLC method with electrochemical detection for the quantitation of guanine nucleotides in rat brain at the 0.5-pmol level. We report here a chromatographic method for quantitating nucleic acids down to the 50-pg level. The method is based on hydrolysis and quantitation of adenine (detection limit = 0.1 pmol) and guanine (detection limit = 0.05 pmol) by HPLC with electrochemical detection (HPLC/EC).
EXPERIMENTAL SECTION HPLC Apparatus. Chromatography was carried out with a Du Pont Model 8800 gradient liquid chromatograph operated in the isocratic mode. Samples were injected with a Micromeritics Model 725 automatic injector equipped with a 2O-wL sample loop. Separations were achieved on a 300 X 4.6 mm i.d. MicroBondapak C-18 reversed-phase column (10-pm porous support particles). The columm effluent was monitored electrochemically with a Bioanalytical Systems (West Lafayette, IN) LC-4A amperometric detector equipped with a glassy carbon electrode. All potentials specified in the text are vs. Ag/AgCl (3 M NaC1). Chemicals and Reagents. Nucleotides, nucleosides, and the corresponding bases were purchased from Nutritional Biochemicals Co. (Cleveland, OH). Calf thymus, salmon testes, E. coli, and human placental DNA were obtained from Pharmacia. Ribonuclease TB,bakers' yeast RNA, and theophylline were purchased from Sigma Chemical Co. All other chemicals were of analytical reagent grade. The mobile phase was a methanol/phosphate buffer mixture (1090 v/v), from which dissolved oxygen was removed by saturation with nitrogen, The phosphate buffer was prepared by adjusting 0.05 M Na2HP04to pH 7 with phosphoric acid. Sample Preparation. Approximately 1 mg of DNA from various sources was dissolved in 10.0 mL of 2 mM NaCl solution. The DNA concentration of each solution was determined by UV absorption at 258 nm, according to manufacturer specifications. Aliquots (10-50 pL) of the various DNA solutions were placed in a culture tube with a polytetrafluoroethylene-lined screw cap and hydrolyzed to the free bases by one of the following procedures. For complete hydrolysis of the DNA, 0.1 mL of 70% HC104 containing 50 nmol of theophylline (used as internal standard) was added to the sample, and the mixture was heated to 100 OC
V I D P l . VS A O / A I C I
IN1
Flgure 1. Hydrodynamic voltammograms for adenine and guanine at pH 4 and 7: ( 0 )guanine at pH 7, (0)adenine at pH 7, (V)guanine at pH 4, (0)adenine at pH 4. Conditlons are as follows: flow injection mode with 20-pL sample loop; flow rate, 1.0 mL/min; glassy carbon
electrode. for 1 h. Selective removal of all purine bases (17) was accomplished by use of 0.1 mL of 0.01 M acetic acid at pH 2.8, in place of the perchloric acid. After hydrolysis, the samples were diluted with phosphate buffer and injected directly into the column. For the determination of DNA in the presence of RNA, samples were first treated with RNase (1IU/mL) for 1 h at 37 "C. The enzymatic hydrolyzates were removed from the DNA solution using Du Pont Nensorb cartridges. The procedure specified by the manufacturer was used to separate and elute the DNA. After elution, the DNA was acid-hydrolyzed and chromatographed as described above. Standard solutions of the purine bases were prepared daily using the phosphate buffer. For guanine, the sample was first dissolved in 1mL of 0.01 M NaOH and then immediately diluted to volume with the phosphate buffer.
RESULTS AND DISCUSSION Hydrodynamic voltammograms of 100 pmol of adenine and guanine at pH 4 and 7 are shown in Figure 1. The data were generated by operating the detector a t various potentials in the flow injection mode (flow rate, 1.0 mL/min), and obtaining the response (peak height) corresponding to 20 pL of sample. The mobile phase was identical with that used for chromatography except that the pH of the phosphate buffer was adjusted to either 4.0 or 7.0 prior to addition of the methanol. It can be seen from the figure that the response for guanine is 2-fold greater than for adenine, and that the optimum conditions for detection of these compounds are pH 7 and an applied potential of 1.1V. The need for compatibility with silica-based columns precluded any investigation of the oxidation behavior under alkaline conditions. (Polymeric reversed-phase columns gave poorer separations.) Figure 2 illustrates the analysis of 80 ng of E . coli DNA. The lower trace represents the chromatogram obtained prior to hydrolysis. Samples were routinely chromatographed before hydrolysis to ensure that no free bases or other compounds were present that might interfere with the analysis. This initial chromatogram can also detect the presence of free nucleotides or nucleosides in the sample, which would lead to false high values for the nucleic acid content. The middle trace represents the chromatogram obtained after hydrolysis with perchloric acid. The upper trace corresponds to a standard mixture of adenine (66 pmol), guanine (74 pmol), and theophylline (50 nmol). The adenine and guanine peaks exhibit a slight amount of tailing. Therefore, peak height was chosen as the mode of amperometric quantitation. The amperometric response for both adenine and guanine was linear from 0.1 to 250 pmol. The relative standard deviations (3 determinations) at the 10-pmol level for adenine and guanine were 1.7 and 2.5%, respectively. For the 0.1-1
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
Table I. Purine Base Content
(X10-4
M) fluor-
HPLC/EC sample
adenine guanine A
calf thymus salmon testes
E. coli human placenta "Adenine
0.91 0.60 0.44 0.46
0.64 0.44 0.45 0.30
287
+
UV
escence (258 nm) G" A + G A+G
1.55 1.04 0.89 0.76
1.55 1.09 0.82 0.74
1.46 1.06 0.97 0.98
+ guanine.
~~
Table 11. Adenine-to-Guanine Molar Ratio sample calf thymus salmon testes
E. coli human placenta
adenine/guanine
lit. value
ref
1.40 1.37 0.99 1.51
1.35-1.50 1.35" 0.99-1.10 1.50
18, 19 20, 21 20, 22 23
" Averade of two reported values.
00
5
10 TIME ( m i d
15
20
Figure 2. Chromatograms for (A) 80 ng of E. coli DNA before hydrolysis; (B) 80 ng of E. coli DNA after hydrolysis In HCIO,; and (C) 66 pmol of guanine and 74 pmoi of adenine; (1) guanine, (2) adenine, (3) 50 nmol of theophylline (internal standard). Conditions are as follows: flow rate, 1.0 mL/min; applied potential, 1.1 V.
I Ilk
JWL. 5
10
15
TIM E ( m i d
Figure 3. Chromatogram representing the determinatlon of DNA near the detection limit: (1) guanine, (2) adenine. The original solution contained 1.0 ng/mL E . coli DNA before hydrolysis. The injection volume was 100 pL.
pmol-range, guanine gave a slope of 0.11 nA/pmol, an intercept of 0.05 nA, and a standard error of estimate of 0.002 nA, which leads to a detection limit of 0.05 pmol at the 95% confidence level. Adenine exhibited a slope of 0.06 nA/pmol, an intercept of 0.02 nA, and a standard error of estimate of 0.003 nA over the same range, giving a detection limit of 0.1 pmol. Figure 3 shows a chromatogram corresponding to the determination of E . coli DNA near the detection limit. A solution containingt l ng/mL of DNA was hydrolyzed at pH 2 by adding a minimum amount of concentrated HC104 and heating to 100 OC for 1 h. After hydrolysis, 100 pL of this solution was chromatographed to produce the trace shown in
the figure. The guanine and adenine peaks in the figure correspond to 100 pg total DNA. The chromatograms for hydrolyed DNA from the various sources investigated are shown in Figure 4. It can be seen from the figure that, in all cases, the peaks of analytical interest are well-separated from other components in the sample. The components represented by peaks 3 and 4 in the figure have not been positively identified; however, their retention times correspond to those of urea and glyoxylic acid, respectively. Based on retention time, and electrochemical and UV behavior, they cannot be any of the following: adenine or guanine nucleotides or nucleosides, ribose, deoxyribose, xanthine, or uric acid. Table I summarizes the quantitative information on DNA from the various sources. Solutions containing approximately 0.1 mg/mL DNA were prepared and quantitated by UV absorbance at 258 nm. These same solutions were diluted 100-fold and measured by ethidium bromide fluorescence,and diluted 104-foldand analyzed by hydrolysis and HPLC/EC. The fluorescence response values were normalized to the HPLC/EC value for calf thymus DNA. The concentration values for UV absorption were calculated on the basis that a 50 pg/mL DNA solution gives an absorbance of 1.0, and the m u s of an average base pair is 660 g/mol. It can be seen from the table that outstanding agreement is attained for the HPLC/EC and fluorescence values. The agreement with W absorption is not as good; however, this can easily be explained by the presence of UV absorbing impurities. The human placental DNA solution actually contained more sample (approximately 25% greater dry weight) than the others. Therefore, the relatively low amount of DNA found in this sample indicates the presence of substantial impurities. These impurities are not observed in the chromatogram (see Figure 4)because the detector is selective for those species that are electroactive. The determined adenine/guanine ratios for the four types of DNA studied are shown in Table II along with the literature values. These data, along with those in Table I, indicate that the DNA hydrolysis is complete, that no significant degradation of the purine bases occurs, and that components 3 and 4 in Figure 4 do not interfere in the determination. Four aliquots of each of the four different DNA samples were hydrolyzed and analyzed as described previously. The results and their relative standard deviations are shown in Table 111. The precision is approximately %fold poorer than for the adenine and guanine standards, owing to the hydrolysis step. The relative standard deviations compare well with those reported for other techniques (11,13).
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Table 111. Precision of the Assay for Acid-Hydrolyzed DNA adenine sample
nmol/mL
std dev
4.14 2.95 2.05 2.14
f0.30 10.10 f0.14 10.12
calf thymus salmon testes E. coli
human placenta
guanine nmol/mL std dev 3.01 2.05 2.03 1.37
f0.29 f0.14 f0.21 f0.13
A
+ Go
nmol/mL
std dev
RSD,b %
7.15 5.01 4.08 3.51
f0.41 f0.17 f0.25 f0.18
5.8 3.4 6.1 5.0
Adenine + guanine (4 determinations). *Relative standard deviation. 1
T
D
C
B
00
1..
1., -1, 5
10 TIME(min)
15
20
Flgure 4. Chromatograms for (A) 140 ng of human placenta DNA; (B) 90 ng of E. coli DNA: (C) 90 ng of salmon testes DNA, and (D) 110 ng of calf thymus DNA. Samples were hydrolyzed for 90 mln In 0.01 M acetic acid at 100 O C . The numbered peaks refer to (1) guanine, (2) adenine, (3) see text, (4) see text, (5) theophylline.
RNase was used to differentiate between RNA and DNA. Two mixtures of known DNA and RNA content and four samples that contained only DNA were subjected to enzymatic (RNase) hydrolysis, separation on Nensorb cartridges, and DNA quantitation by acidic hydrolysis and HPLCIEC. The recovery of DNA was 79.1 f 4.0% for the six samples. This value agrees well with that specified by the manufacturer (81%) for the Nensorb cartridges. RNA can be quantitated in the presence of DNA by processing one aliquot of the sample as described above and subjecting a second aliquot to direct acid hydrolysis and quantitation. After the DNA value is scaled by the observed recovery, the RNA content can be obtained by subtracting the DNA content from the value (RNA + DNA) obtained for the second aliquot (direct acid hydrolysis). Several different detection limits have been stated for this work. This was done to allow a meaningful comparison between this method and those that measure concentration (e.g., UV absorption). Since the purine bases are actually quantitated, it is appropriate to give their mass detection limits.
The mass detection limit for DNA (Le., 50 pg) can be obtained from these values. This is an optimal limit that could be achieved if no volume increase accompanied the hydrolysis step and no sample loss was incurred during injection. For comparison with optical methods, a concentration detection limit was also obtained by hydrolyzing 1 mL of a dilute DNA sample (see Figure 3). It should be pointed out that only 10% of this sample was injected into the chromatograph. This method involves quantitation of only purine bases because the pyrimidine bases are not electroactive below an applied potential of 1.2 V. All of the bases can be oxidized at 1.4 V; however, at this potential solvent oxidation leads to a large background current, and the electrode lifetime is shortened considerably. Quantitation of the purine bases is sufficient for all double-stranded DNA, even if it has been denatured to complementary single strands. As long as both complementary strands are hydrolyzed the base-pairing rules can be used to ascertain the pyrimidine base content. Further, this technique provides some qualitative information in that the adeninelguanine ratio is obtained. For RNA and native single-stranded DNA it is necessary to know the base composition in order to obtain accurate quantitation. However, this technique gives a response whenever any type of nucleic acid is present, unlike the hybridization technique, which only detects DNA containing the complementary sequence. Another advantage enjoyed by this method is the fact that the standards are purine bases, rather than DNA as is the case with all of the fluorescence assays. It is much easier to obtain pure, well-characterized bases than reliable DNA standards. Although this method is sensitive and accurate, there are one or two drawbacks that should be mentioned. First, the method requires a hydrolysis step. This step is time-consuming and can lead to imprecision (e.g., incomplete hydrolysis). The hydrolysis can also be considered an advantage in the case where proteins might interfere with other methods of analysis, through association with the nucleic acids. (Perchloric acid hydrolyzes proteins as well as the DNA.). Second, this method is unable to distinguish between denatured (i.e., cleaved or single-stranded) and native DNA. There may be occasions where the quantitation of only doublestranded DNA is important.
ACKNOWLEDGMENT The authors are grateful to Walter Holl, SK&F Labs, who performed the fluorescence measurements, and to Ken Joseph, SK&F Labs, for stimulating discussions.
LITERATURE CITED (1) Holden, M.; Pirie, N. W. Blochlm. Blophys. Acta 1955, 76, 317-321. (2) Burton, K. I n "Methods in Enzymology"; Grossman, L., Moidave, K., Eds.; Academic Press: New York, 1968; pp 163-166. (3) Burton, K. Blochem. J . 1956, 62, 315-323. (4) Croft, D. N.; Lubran, M. Blochem. J. 1965, 95, 612-620. (5) Le Pecq, J. 8.; Paoletti, C. Anal. Blochem. 1966, 17, 100-107. (6) Markovits, J.; Roques, B. P.; Le Pecq, J. B. Anal. Blochem. 1979, 94, 259-264. (7) Kapuscinski, J.; Skoczylas, B. Anal. Blochem. 1977, 83, 252-257. (8) Karsten. U.; Wollenburger, A. Anal. Blochem. 1977, 77, 464-470. (9) Neurath, A. R.; Strick, N. J. Vlfol. Methods 1983, 7, 155-166. (10) Southern, E. M. J . Mol. Blol. 1975, 98,503-517. (11) Steinman, C. R. Clin. Chem. (Winston-Salem, N . C . ) 1975, 27, 407-41 1. (12) Stadler, J. Anal. Biocbem. 1978. 86, 477-489.
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.
.
289
(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
U
Qinghaosu
Q292
8260
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
