Fluorometric Method for the Determination of Lapachol in Serum Joe M. Finkel and S . D. Harrison, Jr.' Southern Research Institute, 2000 Ninth Aue., South, Birmingham, Ala. 35205
LAPACHOL (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) was first identified by Hooker (1) and synthesized by Fieser (2). Lapachol and related naphthoquinones have shown antimalarial activity (3) and an influence on oxidative phosphorylation (49, thus preventing the oxidative synthesis of adenosine triphosphate. Lapachol has also shown antitumor activity against Walter-256 tumors (5). Although the literature contains methods for readily converting lionfluorescent quinones into highly fluorescent compounds, quantitative methods have not been reported for the determination of lapachol. This may be due in part to the presence of hindering substituents in the 2 and 3 positions of the 1,4-naphthoquinone (6) that prevent many of the usual reactions of quinones. This paper describes a fluorometric method based upon conversion of lapachol under partial vacuum to a fluorescent product by reduction with sodium hydrosulfite. Because the fluorescent product is sensitive t o oxygen, it is extracted into benzene and determined spectrophotofluorometrically in a sample cell from which air is excluded. EXPERIMENTAL
Apparatus. Fluorescence measurements were made with an Aminco-Bowman spectrophotofluorometer and X-Y recorder (American Instrument Co., Inc.). The original cell holder, base plate, bafRes, and mounting screws were removed from the cell compartment of the spectrophotofluorometer and replaced with a modification of the Aminco phosphoroscope. The cell holder, without the motor-shutter assembly, was attached to the bottom of the cell compartment with four retaining screws. The lower opening of the cell holder was closed with a metal plate. Light baffles were inserted between the corners of the cell holder and the corners of the cell compartment. The Dewar holder assembly was mounted o n the top of the cell compartment. The cover assembly was placed directly o n top of the Dewar holder assembly. The cell holder was fitted with 3-mm slits, the cell compartment with 4-mm slits, and the photometer multiplier shutter with a 4-mm slit. The sensitivity control setting was 40. Clear, fused quartz tubes, 11 mm X 100 mm (Genera! Electric Co.), were used as sample cells. The cells were screened for uniformity by filling each cell with a dilute solution of quinine sulfate in 0.1N sulfuric acid and rotating it through 360" in the cell holder. If the variation in fluorescence intensity of the solution (as the cell was being rotated) was greater than 2%, the cell was discarded. The cell was aligned in the cell holder by upper and lower guides made of Styrofoam. The depth of the cell in the cell holder was fixed by the thickness of the lower guide so that the sample would be in the path of the exciting radiation. 1
Present address, University of Indiana, Bloomington, Ind.
(1) S. C. Hooker,J. Chem. Soc., 69, 1355 (1896). (2) L. F. Fieser, J . Amer. Chem. Soc., 49, 857 (1927). (3) W. B. Wendel, Fed. Proc., 5, 406 (1946). (4) J. L. Howland, Biochim. Biopkys. Acta, 77, 659 (1963). (5) P. K. Nayak, D. Molins, F. J. Carleton, and R. K. Morrison, Fed. Proc., 27, 532 (1968). (6) S. Udenfriend, "Fluorescence Assay in Biology and Medicine," Academic Press, Inc., New York, N. Y.,1964, p 276.
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Ultraviolet absorption measurements were made with a Cary Model 14 spectrophotometer. Reagents. Lapachol (NSC-11905) was obtained from Cancer Chemotherapy National Service Center. Standard solutions from 0.03 to 1.0 pg/ml were made from a stock solution of lapachol dissolved in 0.1N sodium hydroxide (1 mg/100 ml). The reducing solution contained 1.5 g of powdered sodium hydrosulfite (NanS204)in 10 ml of water and must be prepared immediately before use. About 10 to 20 ml of fluorometric grade benzene was purged prior to its use for 10 min with dry nitrogen to remove any dissolved oxygen. Fluorometric grade ethyl acetate (American Instrument Co.) was used to extract lapachol from serum. To avoid volume changes during extraction, the ethyl acetate was saturated with water. Serum was obtained from Parke, Davis & Co. Procedure. ANALYSISOF SOLUTIONSOF LAPACHOLI N WATER. The sample cell, sealed with a rubber serum cap, was evacuated for 2 min by inserting through the cap a No. 22 needle on a Leur-Lok glass syringe connected to a vacuum pump. The various reagents were added to the evacuated sample cell from 1-ml Luer-Lok glass syringes with No. 22 needles. First, 1.0 ml of lapachol standard solution and 0.1 ml of the reducing solution were added to the sample cell. The mixture was shaken very briefly by hand, and then 1.0 ml of benzene was added. The mixture was shaken for 1.0 min on a super-mixer (Cole-Parmer Instrument & Equipment Co.) and centrifuged for 2.0 min. Care must be taken so that benzene does not make contact with the rubber cap, otherwise a high background fluorescence will be observed in the samples. The sample cell was inserted into the modified cell holder of the spectrophotofluorometer, and the sample was excited at a wavelength of 340 nm. The maximum fluorescence emission was observed at 410 nm. A water blank containing no lapachol was also carried through the same procedure. The relative fluorescence intensities of the lapachol standard solutions were corrected by subtracting the average value obtained for water blanks from that of each of the standards. ANALYSISOF SOLUTIOSSOF LAPACHOLEXTRACTED FROM SERUM. One-half milliliter of lapachol standard solution, 0.5 ml of serum, 4.0 ml of water, and two drops of 0.3N hydrochloric acid were mixed in a centrifuge tube. The aqueous solution was extracted with 6.0 ml of ethyl acetate saturated with water. The aqueous and organic phases were shaken gently by hand, stirred for 1 min on a supermixer, and centrifuged for 4 min. Four milliliters of the organic phase (two-thirds of the total extracting solvents added) was transferred to a sample cell. The extract was evaporated almost to dryness by blowing dry nitrogen into the sample cell while it was immersed in a 40 "C water bath. The temperature of the sample should not exceed 40 " C because of possible decomposition of lapachol. The sample cell was removed from the water bath and connected to a Rinco vacuum evaporator to remove the last traces of solvent. The sample cell was sealed with a rubber serum cap and evacuated for 2 min with a vacuum pump. The residue was redissolved in 1.0 ml of 0.01N sodium hydroxide solution. The solution was then reduced, extracted with 2.0 ml of benzene, and measured fluorometrically as described in the previous section.
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RESULTS AND DISCUSSION
Initially, lapachol was reduced t o a fluorescent product by tin and stannous chloride in hydrochloric acid. When the reduced product was extracted into benzene, a stronger fluorescence was noted, but this fluorescence immediately decayed. The use of benzene purged with nitrogen for the extraction step slowed the rate of decay. This result indicated that the oxygen may have been causing the decrease in fluorescence intensity; therefore, a system was developed in which the reduction, extraction, and fluorescent measurements were done in a sample cell from which air was excluded. Fluorescence Data. Relative fluorescence intensities after reduction with sodium hydrosulfite are given in Table I for solutions containing various concentrations of lapachol in water. I n these analyses, 1 ml of lapachol standard was reduced and extracted into 1 ml of benzene. The solutions were excited at a wavelength of 340 n m and the maximum fluorescence was measured a t 410 nm. The values in the second column are the averages of four determinations for each standard and are corrected for an average background interference of 0.84 obtained from water blanks. The background values were constant within 5 %. The relationship between the relative fluorescence intensity and the concentration of lapachol was linear in the range investigated. The limit of detection was 0.03 pg/ml of lapachol in water. To determine lapachol in serum, two changes were made in the basic fluorometric method. First, the sample size of lapachol standards was reduced from 1.0 ml to 0.5 ml to better approximate biological samples. Second, the volume of benzene used in the extraction step after reduction of lapachol was doubled. Occasionally a film will collect at the interface of the aqueous and benzene layers and partially block the path of exciting radiation or fluorescence emission. An increase in the volume of extracting solvent reduces the film formation and gives a larger area that can readily be centered in the optical path of the spectrophotofluorometer. Because of the above changes, the relative fluorescence intensity of lapachol in water (Table I) is approximately four times that of lapachol in serum (Table 11). Recovery of Lapachol from Serum Solutions. Based on the above changes, the relative fluorescence intensities of lapachol standard solutions and similar concentrations of lapachol extracted from water and from serum were examined. Aqueous solutions and serum solutions of lapachol were extracted with ethyl acetate; the extracts were evaporated t o dryness; and the residual lapachol was reduced, then extracted with benzene. The fluorescence intensities of lapachol under these conditions were compared with aqueous solutions of lapachol that were not extracted with ethyl acetate. After corrections were made for the ethyl acetate aliquot, the ethyl acetate extracting procedure gave recoveries of lapachol equal to 92 f 10 %. These results are summarized in Table 11. As further confirmation of the efficiency of the ethyl acetate extracting procedure, a higher concentration of lapachol (10 pg/ml) was extracted and the concentration was determined by ultraviolet absorption spectrometry. The recovery of lapachol was 102 + 3 %. These results are shown in Table 111. The efficiency of the ethyl acetate extraction is dependent on the p H of the solution. The p H of the solutions must be adjusted between 6.0 t o 6.5 with dilute hydrochloric acid. Concentrations of lapachol in serum as low as 0.1 pg/ ml can be detected. The relationship between the relative fluorescence intensity and the concentration of lapachol is linear.
*
Table I. Relative Fluorescence Intensity of Lapachol after Reduction with Sodium Hydrosulfite and Extraction with Benzene Concn of lapachol Relative fluorescence standard, pg/mP intensity in benzene* 0.9 30.95 0.7 24,04 0.5 15.99 0.3 10.92 0.1 3.17 0.09 2.96 0.07 2.06 0.05 1.56 0.03 0.96 Sample size of 1.0 ml extracted with 1.0 rnl of benzene, * Corrected for background fluorescence of 0.84 obtained from water blanks. Table 11. Relative Fluorescence Intensities of Lapachol Solutions Relative fluorescence intensitv in benzene* No serum Concn of present; no lapachol extraction No serum present; 0.5 ml of serum standard, with ethyl initially extracted initially extracted pg/mP acetate with ethyl acetate with ethyl acetate 1.0 9.85 5.59 (8.39)c 5.36 (8.04) 0.5 4.30 2.72 (4.08) 2.68 (4.02) 0.1 0.87 0.55 (0.83) 0.60 (0.90) Blank 0.48 1.00 1.59 a Sample size of 0.5 ml extracted with 2.0 ml of benzene, * Corrected for background fluorescence values obtained from corresponding blanks. c Value in parentheses is the relative fluorescence intensities of total concentration of lapachol in the sample obtained by dividing the corrected relative fluorescence intensity by the ethyl acetate aliquot factor of 0.67. Table 111. Maximum Absorbance Values of Lapachol as a Standard Solution and as an Extract from Water and Serum Ethyl acetate Extract Extract L x standard from water from serum 274 nm 0.595 0.630 0,629 281 nm 0.605 0.639 0.639 330 nm 0.130 0.127 0.127 This fluorometric method can be applied to the determination of other fluorescent compounds that are sensitive to oxygen. The advantages of this technique are that the compound is not transferred or diluted after the initial addition of the reagents t o the evacuated sample cell, and the reduction, extraction, and analysis are carried out in the same sample cell. ACKNOWLEDGMENT
The authors are indebted t o L. €3. Mellett, William J. Barrett, and Kenneth T. Knapp for their constructive criticism in the development of this work and in the preparation of this manuscript. RECEIVED for review November 1, 1968. Accepted July 7, 1969. Work supported by Contract No. PH43-65-654 with Chemotherapy, National Cancer Institute, National Institutes of Health.
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