ANALYTICAL

BIOCHEMISTRY

Gas

69, 1o- 15 ( 197.5)

Chromatographic

Determination

of Thymol

MATTHEW W. NOALL, VERNON KNIGHT, WILLIAM W. HARGROVE, AND BARRY W. ELLEDGE Departments Baylor

of Microbiology and Immunology College of Medicine, Houston,

and Biochemistry. Texas 77025

Received August 6, 1974; accepted May 30, 1975 Thymol in biological samples is analyzed by gas chromatography utilizing a 5% OV-25 column, a flame ionization detector, and eugenol as an internal standard. Samples are extracted with diethyl ether and analyzed without derivatization. The lower limit of detection of thymol in a sample is about 0.01 pg and quantitation is satisfactory in plasma at 0.05 pg/2 ml. Data was recorded digitally on magnetic tape and calculated off-line at a central facility.

Thymol was used formerly as an antihelminthic, germicide, and antifungal agent (I). More recently it has been shown to have a variety of effects on biological membranes. Seeman (2) has correlated the stabilization of the human erythrocyte membrane against hypotonic lysis with the mode of action of anesthetics. Thymol stimulates nonhormonally sensitive adenylcyclase of beef thyroid (3). The calcium accumulating ability of sarcoplasmic reticulum is reduced by thymol at moderate concentrations without inhibiting the calcium activated ATPase (4S). Recent observations in these laboratories have demonstrated the reduction in infectivity of the influenza virus on treatment with thymol (6). The antiviral properties of thymol and its membrane effects have prompted us to investigate its pharmacology and mode of action in lipidprotein systems. Because phenols are ubiquitous in biological systems, a selective method of analysis was needed. This report describes a gas chromatographic method with digital integration which is suitable for the determination of thymol in biological samples at low levels of concentration. Other reports on the analysis of thymol or related substances have lacked the necessary sensitivity (7,s). METHODS

Aliquots of plasma or serum, usually 2 ml each, are placed into 13 x 100 mm glass screw cap culture tubes. The samples are prepared for extraction by adding an excess of sodium chloride, about 0.6 g each, then capping and saturating with the salt. The salt facilitates the subsequent extraction through the salting-out effect on lipophilic organic substances in aqueous solution (thymof), by helping to break the ether10 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

THYMOL

11

DETERMINATION

water-plasma emulsion during centrifugation, and by decreasing the solubility of water in ether. The relatively dry ether extract on evaporation does not leave a water residue which would interfere with subsequent chromatography. To each tube there is added 2 ml of ether and 10 ~1 of an ethyl acetate solution containing a known quantity of eugenol as an internal standard (7). A convenient eugenol solution. which serves for many samples, is made by adding 5 ~1 of eugenol (to contain pipette) to 10 ml of ethyl acetate, resulting in a solution which contains 0.535 mg of eugenol/ml. After recapping, the samples are thoroughly extracted. Centrifugation for 30-40 min in a model HN International centrifuge at top speed usually yields a sample with about 1.8 ml of clear ether supernatant with protein and aqueous layers below. The ether layer is transferred with a Pasteur pipette into a special 10 x 50 mm i.d. conical tip sample tube.’ Occasionally, the serum-ether emulsion is only partially separated. In such cases, after removal of the clear ether, an additional 1 ml of ether is added and the sample is reextracted as above. The ether is evaporated with a very gentle stream of nitrogen and with the tip of the tube in a water bath at about 30°C. The tube is removed immediately as the last solvent is evaporated, IO-20 ~1 of ethyl acetate is added, the tube is closed with a cork stopper and the lipid residue is taken up in the solvent. Care must be exercised to avoid excess evaporation with the nitrogen with subsequent loss of sample. Aliquots of 1-3 ~1 are injected into the gas chromatograph for thymol analyses. Suspensions of human erythrocytes, erythrocyte ghosts, or solutions of hemoglobin were prepared as previously described (9). The samples and urine are prepared for thymol analysis by extraction and centrifugation as necessary as described above. Glucuronide conjugated thymol is hydrolyzed to free thymol and glucuronic acid with glucuronidase (Ketodase’). A 1- or 0.5-m] aliquot of urine is mixed with 0.5 ml of 0.5 M acetate buffer, pH 5.0, and 0.2 ml of Ketodase (1000 units of glucuronidase). Two drops of chloroform are added as preservative, the tubes are tightly capped to prevent loss of thymol and incubated overnight at 37°C. The samples are extracted and analyzed in a similar way. Plasma, 2 ml, is treated with 0.018 mequiv of 1 N hydrochloric acid (I 8 ,ul), 0.5 ml of the 0.5 M acetate buffer, 0.2 ml of Ketodase, 2 drops of chloroform, closed tightly, and incubated overnight. Extraction of the glucuronidase treated plasma as described above frequently results in considerable emulsion after centrifugation. These samples are therefore reextracted with a second l-ml aliquot of ether ’ Sample tube number r Ketodase is a product Morris Plains. NJ.

403 I, Glenco Scientific. of General Diagnostics

Inc., Houston, Texas. Division, Warner-Chilcott

Laboratories.

12

NOALL

ET AL.

and then analyzed. Because a preliminary extraction is not employed, the result will be the sum of the free plus glucuronidase liberated thymol. It is necessary to estimate the amount of eugenol to add to a sample because the thymol concentration can vary widely. Very low thymol concentrations have been analyzed successfully with a thymol to eugenol ratio of 1: 50. A desirable ratio is 1: 2. Urine from subjects treated with thymol has had as much as 300 pg of total thymol/ml. Gas chromatography is carried out in 4 mm X 6 ft glass U-tubes packed with 5% OV-25 precoated on 100/120 mesh Supelcoport, selected for low surface activity.3 Samples were chromatographed isothermally at 135°C and with a nitrogen carrier gas flow of 40 ml/min. A flame ionization detector was employed with a model 401 Cat-y vibrating reed electrometer loaded with a lo8 ohm resistor. The 25-mV output of the electrometer was used to drive a potentiometric strip chart recorder and a Vidar model 5200 digital magnetic tape data acquisition system.” The system’s digital voltmeter integrated the electrometer analog signal for 166.6 msec. The electrometer output was routinely digitized at the rate of 2 points per second. Analyses were calculated by the internal standard method off-line with an IBM model 7094 or CDC Cyber 73 computer system from the data stored on the magnetic tape using a program written in this laboratory for use with this equipment. RESULTS AND

DISCUSSION

A difficult problem in the analysis of thymol, especially at low concentration, is its volatility. Extensive evaporation of solvent with warming, common in many lipid extractions, will result in a sample with no remaining thymol. Two methods are used to overcome this problem. First, extraction of the sample is carried out with limited volumes of highly volatile diethyl ether. The ether is evaporated at near room temperature in a special tube. Experience suggests that the tube provides an optimum surface to volume configuration for the removal of the ether. Other lipids extracted from the sample lowers the vapor pressure of the thymol and assists in reducing losses. However, some thymol is carried off with the nitrogen. Second, compensation for thymol losses and quantitation are achieved by utilizing eugenol as an internal standard. Thymol and eugenol have very similar properties. Thymol has a molecular weight of 150.2, is aromatic, and has a highly hindered hydroxyl group while eugenol has a weight of 164.2, is aromatic, has a hydroxyl group vicinal to a methoxyl group, and a vinyl double bond. The volatility of the two substances is similar but they can be conveniently separated on a 5% OV-25 column with its moderate polarity. Under the specified conditions, thymol and eugenol are modestly lost from the sample during 3 Supelco, Inc., Supelco Park. Bellefonte, Pa. 4 Auto Data Inc., 265 North Whisman Road, Mountain View, Ca.

THYMOL

TABLE DETERMINATION

OF THYMOL

13

DETERMINATION

WITH

1

A CONSTANT

AMOUNT

OF EUGENOL”

pg Thymol Theoretical

Found

0.402 0.302 0.201 0.200 0.100 0.100 0.0520 0.0500 0.0251 0.0100 0.0100

0.404 0.301 0.197 0.197 0.098 0.098 0.0499 0.0480 0.0249 0.00970 0.00986

‘I Ethyl acetate.solutions each contained 0.535 wg of eugenol/pl and different amounts of thymol. Aliquots of 1 ~1 each were injected for analysis. Regression analysis showed: Intercept-O.0018 pg thymol; slope, 1.011; correlation coefficient. 1.000: standard error of estimate, 0.0003.

extraction and two substances Blank samples eugenol. Quantitative

evaporation to very nearly the same extent. Because the parallel each other in behavior quantitation is preserved. have not shown peaks in the positions for thymol or tests of thymol

determinations TABLE

RECOVERY

OF THYMOL

in the low range from

2 FROM

PLASMA”

Plasma sample no.

Added

pg Thymol found

1 2 3 4 5 6 7 8 9 10 11

0.050 0.050 0.10 0.15 0.15 0.20 0.20 0.30 0.30 0.60 0.60

0.057 0.060 0.094 0.139 0.152 0.178 0.178 0.268 0.290 0.598 0.5%

0 Different amounts of thymol and a constant amount of eugenol, 2.14 ,ug. were extracted from plasma. Aliquots. 3 ~1, of the extract concentrate were injected for analysis. Regression analysis showed: Intercept-O.0056 pg thymol, slope, 0.989. correlation coefficient 0.998. standard error of estimate 0.013.

14

NOALL

ET

AL.

TABLE 3 RECOVERY OF THYMOL FROM BIOLOGICAL SAMPLES~ Added (/-a)

Sample Krebs-Ringer Serum Urine Urine Urine Erythrocyte Erythrocyte Hemoglobin Erythrocyte Erythrocyte Erythrocyte

phosphate buffer, pH 7.4

30.0 0.18 IS. 120. 240. 30.0 150. 63.6 57.1 228.2 456.

suspension suspension solution ghost@ ghosts ghosts

Recovered (96) 102 103 97 98 100 100 95 99 90 97 95

t 2.8 (3) t 4.9 (3) 2 0.5 (2) 2 2.3 (2) k 0.9 (2) k 7.4 (6) k 0.9 (2) * 3.0 (2) i 1.0 (2) -c 2.7 (2) + 1.9 (2)

a Thymol was added to biological samples and after equilibration was extracted for analysis. The recovery is followed by the standard deviation and the number of determinations in parentheses. * Determined after 30 min of incubation, centrifugation, as the sum of ghost pellet and supernatant.

standard thymol-eugenol solutions are shown in Table 1. Regression analysis shows satisfactory performance of the system with standards down to 0.01 pg. Standards have no other interfering lipids and therefore the detection of thymol is optimized. However, simple standards without other protective lipid in the sample offer maximum opportunity to lose thymol and eugenol during evaporation. Considerable care must TABLE 4 PLASMA AND URINE THYMOL ANALYSES" Thymol (pgiml) Sample Plasma

Urine

Time (hr) 1.5 2.0 2.5 4.0 4 8 12 20

Free 0.33 0.51 0.59 0.11

10.5 44

158 22s

Total 1.05 1.78 1.75 0.38 26.3 196 302 390

Ratio, Glucuronide Free 2.2 2.5 2.0 2.4 1.5 3.4 0.91 0.75

a A volunteer was given thymol per OS at 0, 4, and 8 hr in amounts of 1. 0.5, and ).5 g, respectively. Plasma and urine were collected as indicated. The glucuronide thymol s the difference between the total and the free.

THYMOL

1.5

DETERMINATION

be exercised to prevent overevaporation. Table 2 gives recovery data from plasma samples. Low thymol levels provide a critical test and the range examined was 0.05-0.6 pg. Regression analysis showed satisfactory performance of the system. Below 0.05 ,ug some interferences were experienced from the extracted plasma lipid. Table 3 presents results of control samples drawn from investigations on the pharmacology of thymol. Thymol recoveries are satisfactory except for an experiment with erythrocyte ghosts. The processing of the ghosts involved centrifuging for 40 min in uncovered tubes. Lower recoveries were obtained. An application of the method in in viva experimentation is shown in Table 4. The procedure was satisfactory for levels of free and glucuronide liberated thymol which were encountered. The thymol levels encountered varied widely. For the higher urine values it was convenient to use in the procedure 0.5 ml of sample and 300 pg of eugenol for internal standard. The described method of thymol analysis is simple and requires no derivatization. It has been useful in studying the pharmacology of thymol and its interaction with cellular components. ACKNOWLEDGMENTS This study was supported in part by a Grant and USPHS Grant RR00954.

from

the Warner-Lambert

Research

Institute

REFERENCES I. Goodman, L. S. and Gilman. A. (eds.) (I 970) The Pharmacological Basis of Therapeutics, 4th Edition, p. 1037, Macmillan, New York. 2. Seeman, P.. and Weinstein, J. (1966) B&hem. Pharmucol. 15, 1737-l 752. 3. Wolff, J.. and Jones, A. B. (1970) Proc. Nat. Acud. Sri. USA 65, 454-459. 4. Greaser, M. L., Cassens, R. G., Hoekstra. W. G., and Briskey, E. J. (1969) Biochim. Biophys. Acta 193, 73-8 I. 5. Ogawa. Y. (I 970) J. Biochem. 67, 667-683. 6. Knight, V., Unpublished observations. 7. Zwaving, J. H. (1968) Pharm. Weebl. 103, 3-73-290. 8. Douglas. C. C. ( 1972) J. Ass. Off. Anal. Clrem. 55, 610-612. 9. Dodge. J. T.. Mitchell. C.. and Hanahan. D. F. (1963) Arch. Biochem. Biophy.s. 100, I16130.

Gas chromatographic determination of thymol.

ANALYTICAL BIOCHEMISTRY Gas 69, 1o- 15 ( 197.5) Chromatographic Determination of Thymol MATTHEW W. NOALL, VERNON KNIGHT, WILLIAM W. HARGROVE, A...
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