Detection of Tetrahydrocannabinol in Blood and Serum Using a Fluorescent Derivative and Thin-Layer Chromatography Joe A. Vinson,* Dinu D. Patel, and Arun H. Patel Chemistry Department, University of Scranton, Scranton, Pa. 785 10
A thin-layer chromatographic method Is described for the detection of As-tetrahydrocannabinol in blood and serum. The procedure utilizes a reagent, 2-p-chlorosulfophenyI-3-phenylindone, which reacts with the phenolic group to form a derivative. Following extraction and cleanup, the derivative is prepared and separated from reagent and naturally occurring compounds by thin-layer chromatography. The derlvative is then detected by spraying the thin-layer plate with an alkoxlde spray which produces a fluorescent spot visible under long wave ultraviolet light. The method can detect 0.2 ng/ml of tetrahydrocannabinolin 5 ml of serum and is suitable for routine screening.
Tetrahydrocannabinol (THC) occurs in plasma in very low concentrations (3-50 ng/ml) during its period of physiological activity. The peak value starts to rapidly decrease within 10 min after smoking (1, 2 ) . The physiological efects are most evident for a period of 2 to 3 h after smoking. The concentration of THC in plasma after 2 h is about 1-3 ng/ml(2). Thus, a method is needed which is capable of detecting THC down to a concentration of 1ng/ml. The method should be suitable for routine use, sensitive, specific, inexpensive, and preferably amenable to quantitative analysis. There are several methods currently available for the detection and analysis of THC. Gas chromatography (GC) is sensitive to about 300 pg/ml THC in plasma when a suitable derivative is made for electron capture or flame photometric detection (3-5). These GC methods require extensive cleanup in order to remove naturally occurring interferences. Bleeding from the column and natural interferences limit the selectivity and sensitivity of the method. Mass fragmentography used in combination with GC offers excellent sensitivity and selectivity (2, 6 ) . However, this method is too expensive for routine monitoring and requires considerable expertise. A promising approach to determining THC is the development of immunoassay techniques. A free radical immunoassay method has been developed for THC and its metabolites in urine (7). Another group has used radioimmunoassay for THC in plasma and urine (8). However, the detection limit in plasma is 8 ng/ml due to nonspecific interferences which are naturally present. Thin-layer chromatography (TLC) is the simplest and least expensive of the techniques and is thus suitable for routine use. Unfortunately, a previous report using TLC for THC in urine and blood as a lone method of detection could not be confirmed (9). Some success has been found using two-dimensional TLC as a preliminary separation technique for THC prior to injection in a mass spectrometer (10). But this method proved insensitive for THC in blood. The basic limitation of TLC is its lack of sensitivity. THC is only detectable with color-formingTLC spray reagents at the pg level. Dansyl derivatives of THC give fluorescent spots detectable at 0.5 ng ( 1 1 ) on a TLC plate, but the spots are very unstable to light and there are many other spots due to degradation of the derivative. Also, dansyl chloride is a very unselective reagent, thus limiting its utility. Dansyl derivatives have been used
recently to quantitate metabolites of THC in feces after considerable cleanup prior to high-pressure liquid chromatography (12). We wish to describe a TLC method suitable for routine use for the detection of THC in blood or plasma using a new derivatizing reagent, 2-p-chlorosulfophenyl-3-phenylindone, DIS-Cl(13).This reagent has been used for derivatizing amino acids (14),amino sugars (15),and vitamin B6 (16).The reaction of DIS-C1 with the phenolic group of THC is shown below:
DIS-THC
Treating the DIS-THC with methoxide on a TLC plate produces a fluorescent spot which is the basis for the detection method.
EXPERIMENTAL Extraction and Cleanup. Five ml of blood, serum, or plasma in a 20-ml screw-cpped culture tube was acidified to pH 4 with 0.8 ml of 2 N HCl and vortexed for 5 min with 7 ml of hexane/isoamyl alcohol (982).Two grams of ammonium sulfate were then added and the tube was vortexed for 3 min. A second extraction was made with 7 ml of hexane/isoamyl alcohol followed by centrifugation for 5 min a t 3000 rpm. The hexane layers were pooled and extracted two times with 3 ml of modified Claisen alkali (dissolve 3.7 g of potassium hydroxide in 20 ml of water followed by addition of 100 ml of methanol). The combined alkali was acidified with 0.3 ml of concentrated HCl. Upon standing, a precipitate of KCl forms. The precipitate was then completely redissolved by adding 1-2 ml of water to the solution and vortexing to ensure dissolution. This solution was then extracted two times with 7 ml of hexane. The hexane was evaporated at 70 "C under nitrogen to a small volume in a centrifuge tube and quantitatively transferred to a 0.3-ml Reacti-vial (Pierce Chemical Company) and evaporated to dryness. Dri-Film SC-87 from Pierce Chemical Company was used to silanize all glassware in the procedure. Derivatization, TLC, and Visualization. The residue from extraction was derivatized with 5 pl of a 1mg/ml acetonitrile solution of DIS-Cl (available from Polysciences, Inc., Warrington, Pa. 18976) and 5 pl of 0.2 M NaZC03. The reaction mixture was heated at 45 O C for 30 min in the closed Reacti-vial. The entire solution was then spotted on 5 X 20 cm or 20 X 20 cm Bakerflex IB2 Silica Gel Sheets (J. T. Baker Chemical Company) 2 cm from the bottom of the plate, using a stream of nitrogen and gentle heat from a hot plate to dry the spots. The plates were developed 15 cm from the origin in methanol/water (95:5) in a tank previously saturated in the developing ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
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Table I. RF Values for DIS-C1 and Drugs Which React with DIS-Cl
w
Natura Compourd
D15-THC
Orgin
L Figure 1. Representative TLC plate (A) DISCI and sodium carbonate heated for 30 min at 45 OC; (B) THC plus DIS-CI and sodium carbonate heated for 30 min at 45 OC; (C) Blank serum (5 mi) extracted and derivatized;(D) Serum (1 ml) obtained 2 h after smoking a cigarette containing 10 mg of AS-THC which was extracted and derivatized. The concentration of THC was 2 ng/ml as determined by GC-MS (Battelle Institute)
solvent for about 20 min. After evaporating the developing solvent in an air stream, the plate was sprayed with visualization reagent (saturated solution of 8 g of sodium metal in 100 ml of methanol and 8 ml of dimethyl sulfoxide).While still wet, the plates were examined under long-wave ultraviolet light. DIS-THC occurs as a yellow-green fluorescent spot at an RF of 0.40-0.46. RESULTS AND DISCUSSION
Figure 1shows a chromatogram of a reagent blank, reagent plus THC, serum blank, and an authentic serum sample taken 2 h after smoking 10 mg of A9-THC. No blank interferences occur although there is a fluorescent spot above DIS-THC at an RF of 0.53 due to a naturally occurring compound which is well separated from DIS-THC. As can be seen from Figure 1, DIS-THC in a biological matrix has a lower RF value than the standard DIS-THC. We have found that a better means of identifying THC is to use its hRF relative to the upper reagent spot (RF 0.83) which is visible to the naked eye as a yellow spot. The formula for hRF of DIS-THC is the following: distance DIS-THC spot from origin x 00 hRsm = distance reagent spot from origin The hRsTD for DIS-THC which is a constant, independent of development distance, plate saturation, and matrix, has a value of 50 f 3. The number of potential interferences in any method is very large due to the great number of drugs which could be ingested and to natural compounds present in the physiological fluid. Twenty-five common drugs including nicotine and caffeine were reacted with DIS-C1but gave no fluorescent spot on the TLC plate after spraying. This indicates that no derivative was formed. The following drugs do not react with DIS-C1: chloroquine, adrenalin, diazepam, diethylpropion, phenacetin, amphetamine, dextromethorphan, caffeine, cocaine, propoxyphene, oxazepam, quinine, meperidine, methadone, nicotine, codeine, methamphetamine, phenobarbital, barbital, salicylamide, secobarbital, thiopropazate, phenothiazine, promazine, and acetaminophen. All of the narcotics, amphetamines, tranquilizers, and psychoactives have nonreacting amine functional groups. Barbiturates with an imido functional group also do not react. Only phenols appear to 164
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Compound
RF
DIS-C1 Apomorphine 11-OH-AS-THC AS-THC 11-nor-9-carboxy-THC Pentazocine Morphine Levorphanol Bufotenine
0.76 (0.83degradation product) 0.56 0.63 (metabolite) 0.48
0.33 (metabolite) 0.27 0.22 0.12 0.10
react with DIS-C1 under the experimental conditions. However, common analgesics, containing phenolic groups, such as salicylic acid, salicylamide, and acetaminophen do not react. Table I shows the compounds which do react to give fluorescent spots with DIS-C1 after reaction followed by spraying with methoxide. As can be seen in Table I, good separation is obtained between THC, its major metabolites in blood, and other phenolic drugs. Morphine, a commonly abused drug, was spiked in blood at 10 ng/ml, a rather high level, and taken through the entire procedure. No fluorescent spot was seen at its RF value. Morphine was evidently not extracted at the acidic pH of 4 due to its being in a salt form at this pH. Apomorphine and pentazocine are dipolar, as is morphine, and therefore would not give fluorescent spots even if originally present. Also, phenolic metabolites of other basic drugs would be eliminated during the extraction procedure. It, therefore, appears that this method is highly specific. The detection limit of pure DIS-THC is 0.1 ng after development on a THC plate. The fluorescent spot is fairly unstable at subnanogram levels and should be viewed within a few minutes after spraying. This derivative can thus be seen at lower levels than the previously reported Dansyl-THC. An approximate spiked detection limit in serum is 0.2 ng/ml for a 5-ml sample. THC has easily been detected in authentic samples with a concentration of 0.4 ng/ml. Thus, this method is comparable in sensitivity to sophisticated instrumental methods utilizing GC or GC-MS, and is ideally suitable for routine screening. Current work is in progress in this laboratory toward utilizing this procedure for detection of THC and its metabolites in urine and saliva and for quantitation of these compounds in biological fluids. ACKNOWLEDGMENT
We acknowledge the technical support of J. T. Lewars 111, M. P. Snyder, J. A. Nebzydoski, and T. F. McNevin, Jr. We also thank J. R. Bryant of the Department of Health, Education, and Welfare, Public Health Service, National Institute of Mental Health for supplying samples of THC and its metabolites and R. Stillman of the National Institute of Mental Health and National Institute on Drug Abuse for supplying serum samples of subjects who have smoked marijuana. LITERATURE CITED ( 1 ) M. Galanter, R. J. Wyatt, L. Lemberger, T. B. Weingartner, T. B. Waugham, and W. T. Roth, Science, 176, 934 (1972). (2) S. Agurell, B. Gustafsson, B. Holmstedt, K. Leander, J.-E. Lindgren, I. Nilsson, F. Sandberg, and M. Asberg, J. fharm. Pharmacal., 25, 554 (1973). (3) E. R. Garrett and C. A. Hunt, J. fharm. Sci., 62,121 1 (1973). (4) D. C. Fenlmore, R. R. Freeman, and P. R. Loy, Anal. Chem., 45, 2331 (1973). (5) N. K. McCallurn, J. Chromafog.Sci., 11, 509 (1973). (6)J. J. Rosenfeld, 6. Bowins. J. Roberts, J. Perkins, and A. S.Macpherson, Anal. Chem., 46, 2232 (1974).
(7) M. Cais, S. Dani, Y. Josephy, A. Modiano, H. Genshon, and R . Mechoulam. FEBS Lett., 55, 257 (1975). (8)J. D. Teale, E. J. Forman, L. J. King, and V. Marks, Lancet, 553 (1974). (9) J. B. daSilva, Rev. Fac. Farm. Bioquim., 5 , 205 (1967). (10) W. W. Just, N. Filipovic, and G. Weiner, J. Chromatogr., 96, 189 (1974). (1 1) I. S. Forrest, D. E. Green, S. D. Rose, G. C. Skinner, and D. M. Tones, Res. Commun. Chem. Pathol. Pharmacol., 2, 787 (1971). (12) S. R. Abbott, A. Abu-Shumays, K. 0. Loeffler,and I, S.Forrest, Res. Commun. Chem. Pathol. Pharmacol., 10, 9 (1975). (13) Tsch. P. Ivanov, Monatsh. Chem., 97, 1499 (1966). (14) Ch. P. Ivanov, and Y. Vladovska-Yukhnovska, J. Chromatogr., 71, 11 (1972).
(15) Y. Vladovska-Yukhnovska, Ch. P. Ivanov, and M. Malgrand, J. Chromatogr., 90, 181 (1974). (16) I. Durko, Y. Vladovska-Yukhnovska,and Ch. P. Ivanov. Clin. Chim. Acta, 49, 407 (1973).
R~~~~~~~ for review M~~ 24, 1976. ~
~ October~ 20, 1976. This work was supported by a contract from the Insurance Institute for Highway Safety. The work was presented a t the 28th Meeting of the American Academy of FCW3nsic Sciences, Washington, D.C., February 1976.
Synthesis and Ion-Exchange Properties of Crystalline Stannic Vanadophosphate Mohsin Qureshi” and Ramesh Chand Kaushik Z. H. College of Engineering and Department of Chemistry, Aligarh Muslim University, Aligarh (U.P.) India
A new crystalline inorganic ion exchanger, stannic vanadophosphate, has been synthesized by treating vanadophosphoric acid with stannic chloride (sample 1). When 0.1 M solutions of sodium orthophosphate, sodium metavanadate, and stannic chlorlde are mixed in the volume ratio of 4:1:1, the amorphous stannic vanadophosphate (sample 2) is obtained. A comparative study of samples 1 and 2 has been made with respect to their ion-exchange behavior,the ir and x-ray spectra and Kd values. Three binary separations (I) in3+-Ga3+, (11) Mg2+-Ca2+, and (111) Mn2+-Ni2+ have been achieved using sample 1 in the column. in3+, Mg2+, and Mn2+ were removed with water, Ga3+ with 0.1 M HN03 while Ca2+ and Ni2+ were eluted with 1% NH4N03.
The properties of inorganic ion-exchange materials depend upon their chemical composition and also on their physical state. It is therefore instructive to prepare new inorganic ion exchangers and to compare their properties in the crystalline and amorphous states. Organic and inorganic salts of heteropoly acids have received considerable attention as ion exchange materials during recent years (1-4). The general formula of the acid is HmXY12040.nHz0where m = 3 , 4 , 5 and X may be As, Si, and P while Y is Mo, W, and V. The ionexchange properties of these materials have been found to be enhanced when two acidic groups are present to form salts of a mixed type. Ammonium tungstophosphate and ammonium molybdophosphate (1)are probably the first materials of this type which have been studied as ion exchangers. Stannic phosphate ( 5 )has long been recognized as a good ion-exchange material. The use of vanadates as ion-exchange materials has also been reported. They are, however, comparatively less stable and dissolve significantly in aqueous solutions. Titanium vanadate (6) which has been synthesized in these laboratories has been found specific for strontium and is stable at high temperatures. As pure vanadates appear to be unstable, it is possible that substances such as vanadophosphates may be more stable. As no studies have been made on stannic vanadophosphate as an ion-exchange material, a crystalline stannic vanadophosphate has been synthesized. Its ion-exchange properties have been studied and its value for some chemical separations explored.
EXPERIMENTAL Apparatus. A Bausch and Lomb Spectronic-20 Colorimeter, an
Elico pH meter model LI-10 (India), an electric temperature controlled “SICO” Shaker, Philips x-ray unit, and a Perkin-Elmer Model 137 spectrophotometer were used for spectrophotometry, pH measurement, shaking, x-ray, and ir studies, respectively. In the case of “Zn, 203Hg,134Cs,58C0, and llornAg,X counting was done on a scintillation counter, 0 counting was done in the case of z04Tland llsrnCd using a GM counter. Reagents. Stannic chloride pentahydrate (pure, Poland), sodium metavanadate (USSR), disodium orthophosphate (BDH), and phosphoric acid (BDH) were used. All other chemicals used were of analytical grade and istopes were supplied by BARC (India). Synthesis of Vanadophosphoric Acid. This acid was prepared by Wu’s method (7) by dissolving 100 g of sodium metavanadate in 450 ml of water adding 15 cm3of 85% H3P04 and 80 cm3of concd. HCl and boiling for 8 h, with a return condenser. The precipitate was filtered on a Buckner funnel and dried as much as possible. The precipitate was redissolved in 120 ml of H20 in a separatory funnel by adding about 70 ml of ether and 40 ml of HCl and shaking vigorously. After standing for a few minutes, the lowest layer which contained the complex was separated, transferred to a beaker, and dried on a steam bath. Synthesis of Stannic Vanadophosphate via Free Acid. Vanadophosphoric acid, 10 g, was dissolved in 850 ml of water and 150 ml of concd nitric acid. Then 0.1M SnC14 was added to this solution in the volume ratio of 1:l and a pale yellow precipitate was obtained. The precipitate was filtered and dried at 40 “C (sample 1). Synthesis of Stannic Vanadophosphate via Sodium Salt. The stannic vanadophosphate was synthesized by mixing 0.1M sodium orthophosphate and sodium vanadate with 0.1 M stannic chloride in the volume ratio 4:l:l.A yellow precipitate was obtained which was dried a t 40 “C (sample 2). Dissolution of Stannic Vanadophosphate. To determine the solubility of stannic vanadophosphate, 0.5 g of the exchanger was placed in a flask with 50 ml of the solution concerned for 6 h in a temperature controlled shaker. After removal of the undissolved material, tin, vanadium, and phosphorus were determined spectrophotometrically by phenylfluorone, hydrogen peroxide, and molybdenum blue methods, respectively. Ion-Exchange Capacity. The ion-exchange capacity of stannic vanadophosphate (samples 1 and 2) dried a t different temperatures was determined by the standard method using 1 M electrolyte solutions. pH titrations were performed by the method described earlier (8).The distribution coefficients (Kd) for inorganic ions were determined on both materials in those solvents in which the material was found stable. The loading of cations was less than 3% of the experimental ion-exchange capacity. The Kd values were calculated as described earlier (9). Chemical Composition. The exchanger (250mg) was dissolved in hydrochloric acid. The tin was precipitated by the basic acetate method (10) and titrated with potassium dichromate after prior reduction with lead metal. The phosphate was separated as magnesium ammonium phosphate (11) and determined gravimetrically. Vanadium was determined volumetrically after reduction with SO2. The results are shown in Table I. ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
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