Anal. Chem. 1989, 61,925-928
925
Selective Sample Preparatlon for Determination of 1l-Nor-9-carboxy-~'-tetrahydrocannabinolfrom Human Urine by Gas Chromatography with Electron Capture Detection J. M. Rosenfeld* and Yadu Moharir Department of Pathology, Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 325, Canada
S. D. Sandler Central Analytical Laboratorv, - . Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N3Z5, Canada Assessment of cannabis intoxication by determining 11-
nor-9-carboxy-A9-tetrahydrocannabinol(I) in urine requires analytical methods with high specificity and sensitivity (1-7). Such methods are frequently based on preparation of electrophoric derivatives by reaction at both carboxyl and phenolic groups (1,2,6);thus these products are detected in the general organic acid fraction of urine. The complexity of the problem is such that instrumentation alone cannot be relied upon to give valid results and attention must be given to sample preparation ( 4 , 5 ) . Partitioning by liquid/liquid extraction prior to derivatization ( I , 2,6) has been used to prepare a more selective isolate of urine. We considered selective derivatization as an alternative approach to improve specificity and simplify procedures of analysis. Selective derivatization of carboxyl and phenolic functionalities on the same organic molecule has been recently reported (8). Under very stringent conditions of temperature (-15 "C), reagent concentrations (minimal excess), and reaction times (0.5 min) the carboxyl moiety of compounds with phenolic and carboxylic acid groups could be selectively esterified with diazomethane and the phenolic group(s) subsequently derivatized with electrophoric reagents preparatory to gas chromatography/electron capture detection (GC/ECD) analysis. Methylation of I, even under mild conditions (9, IO), produced the dimethyl derivative rather than any products of selective reaction. Selective derivatization of I based on such reactions did not promise to produce a rugged procedure unless newer derivatization techniques were investigated. Derivatization on solid supports of XAD-2 has been used to determine A9-THC and two of its metabolites, 11-OHA9-THC and I, in plasma ( 7 , I I ) . These procedures produced 11, but the isolate contained many electrophoric derivatives arising from carboxylic acids in that fluid (11);this problem would be exacerbated in urine analysis where concentrations of organic compounds could be increased by 2 orders of magnitude relative to plasma (12). If on the other hand only the carboxyl or phenolic group were derivatized (Figure l ) , the product could be detected as a part of the carboxylic acid or phenolic fraction and these should be cleaner. In addition such selective derivatization would leave a polar group free; thus the reaction product would be more selectively retained on the column during chromatography facilitating removal of interferences. Heterogeneous reactions on XAD-2 or with phase transfer catalysis can be made selective by adjusting pH to two log units above the pKa of the acid which ensures quantitative ionization (12, 13). Thus in the p H range 7 to 9 only the carboxyl group of I would be ionized as the pKa of the phenol is above 10 and esterification and would be the predominant reaction. Control of pH, however, was unlikely to affect selectivity for reaction at the phenolic group of I since conditions sufficiently alkaline to ionize the phenol would also ionize the carboxyl group, and so both groups would react (7, 14).
Solid supported reactions on XAD-2 have been made selective for phenols in the presence of carboxylic acids at alkaline pH despite ionization of both functionalities (11). When hydroxylic solvents were used as diluents for PFBBr, yields of PFB esters decreased as the molecular weight of the alcohol increased, but yields of phenol ethers remained quantitative. Such selectivity of reaction improved specificity for determination of A9-THC in plasma since interferences were due primarily to plasma carboxylic acids. When A9-THC was determined from plasma, however, the phenolic and carboxylic functionalities were on separate molecules (11). In the case of I both functionalities were on the same molecule and it was unclear if the molecule would react as a phenol or a carboxylic acid or if both functionalities would be derivatized. Indeed it was uncertain if any reaction would occur as reactions on XAD-2 had not been studied for the analysis of urine where concentrations of organic compounds can be 10 to 500 times higher than in plasma (12).
EXPERIMENTAL SECTION Apparatus. Pentafluorobenzyl (PFB) derivatives of pure analytes were determined on a Hewlett-Packard (H-P) 5790 gas chromatograph (GC) equipped with a pulse linearized electron capture detector (ECD) and with a J&W fused capillary column DB-1,30 m X 0.321 mm with film thickness 0.25 mm. Detector output was monitored on a H-P 3390A recording integrator. Hydrogen was used as a carrier gas with a linear velocity of 62 cm at 180 "C; 10% methane in argon was the make-up gas maintained at a flow rate of 15 mL/min. Oven temperature was programed as follows: 190 to 245 "C at 20 "C/min, 2 min at 245 "C; 245 to 262 "C at 1 "C/min; 262 to 315 "C at 20 "C/min and 2 min at 315 "C. Retention time of product I11 was 15.32 min and that of product IV was 16.34 min. Mass spectra were obtained on a VG Micromass 7070 sector instrument with the same column and conditions used for GC/ECD. The ion source was maintained at 280 "C and at a vacuum of lo+ Torr. The ionizing voltage was 70 eV, the emission current was 20 mA, and the accelerating voltage was 3 kV. Ion trap mass spectra were obtained on a Finnegan MAT 700 ion trap mass spectrometer (IT-MS). The column was the same as that used for GC/ECD analysis. The ion source was maintained at 230 "C and at a vacuum of lo-* Torr. The ionizing voltage was 80 eV, and the emission current was 20 mA. Reagents. Pentdluorobenzyl bromide (PFBBr) was purchased from Caledon Laboratories, Georgetown, ON. The macroreticular resin, XAD-2, a cross-linked copolymer of styrene/divinylbenzene, was obtained from BDH Laboratories, Toronto, ON, and was cleaned and stored as previously described (9). Florisil was purchased from Supelco, Mississauga, ON. Trimethylchlorosilane (TMCS) and N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA) were purchased from Pierce Chemical Co., Rockford, IL. Disposable 5-mL tips used for packing Florisil was purchased from Fisher Scientific (Catalog No. 13-689-25A). Solvents were purchased from the usual commercial suppliers, such as Fisher, BDH, and Aldrich Canada. The analyte, I, and tritiated I (3H-I)with a specific activity of 0.021 mCi/mmol was provided by the National Institute of Drug Abuse (USA) under the auspices of the
0003-2700/89/036 1 -0925$01.50/0 0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 8, APRIL 15, 1989 COIPFB
Table I. Yield of Reaction Products as a Function of Reaction Conditions
A
OPFB
reaction
II
t
50f555f5 yield" of ester 0 yielda* of 1-0-PFB ether 0 yield of dibenzyl 0 0
PFBEr In
Trichloroethylene 0. IN NaOH
COaH
A J
OH
111
\CH COIH
MlPFB
A
I
9Of7 0 0
phenol selective 10f10 80 f 8 10 f 9
Average f RSD (n = 6). *Based on total radiolabel recovered. Based on recovery of radiolabel in specific fractions from a semipreparative column.
1l-nor.OCARBoXV.A*-THC
pH PFBBrln Trkhlomthy(em 8.5
carboxyl selective composition of aqueous Dhase PH 8.5/20% pH 7.4 pH 8.5 CHSCN
OPFB
OH
Iv
Flgure 1. Reactions of 11-nor-9-carboxy-As-THCon XADP.
Food and Drug Directorate of Canada. Glassware was treated with dimethyldichloroeilane as previously described (7,11,13). All glassware and plasticware were washed with methylene dichloride, methanol, and acetonitrile by soaking for 5 min in each solvent and then dried prior to use. Preparation of Solutions of I in Urine. Solutions of I in urine were prepared by adding varying amounts of analyte in 100 p L of ethanol to 40 mL of urine containing 8.0 mL of acetonitrile. A solution was also prepared with 90000 counts/min of 3H-I in 40 mL of urine containing 8.0 mL of acetonitrile. Carboxyl Selective Derivatization. Four milliliters of a solution containing 125 ng/mL of I in urine was transferred to 16 X 100 mm screw-cap vials containing 300 mg of XAD-2 prewetted with 200 mL of acetonitrile. Acetic acid (25 pL) was added to the mixture, the tube was sealed with a Teflon-lined screw cap and shaken on a mechanical shaker for 15 min. Urine was aspirated with a 23-gauge syringe needle. Resin was washed with 5 mL of 0.01 N hydrochloric acid and the liquid aspirated as before. The following solutions and volumes were added to the reaction vial: 4 mL of 0.1 M phosphate buffer at pH 8.5; 800 mL of acetonitrile; 150 mL of a solution of PFBBr in trichloroethylene (TCE) (1/9 (v/v)). The vial was sealed and the reaction mixture was shaken for 1 h on a mechanical shaker at room temperature. Resin was transferred to a 5-mL Pipetteman tip packed with silanized glass wool and washed with 5 X 5 mL of 0.1 N hydrochloric acid. Glass wool (1/2 cm) was packed on top of the resin to prevent floating of resin during subsequent work-up. This was then inserted into a second 5-mL Pipetteman tip packed with 1g of Florisil and topped with 0.5 g of anhydrous sodium sulfate. Material was eluted from the resin with the following solvents and volumes: three 10-mL portions of hexane/methylene dichloride (7/3 (v/v)), two 10-mL portions of methylene dichloride, two 10-mL portions of diethyl ether, and three 10-mL portions of diethyl ether/ethanol (9/1 (v/v)). Phenol Selective Derivatization. Four milliliters of urine containii I at varying concentrations or 4 mL of urine containing 7500 counts/min of 3H-I was transferred to a screw cap and the
adsorption carried out as described above. Four milliliters of 0.1 N sodium hydroxide was added and the mixture agitated on a Vortex Genie for 15 s. One hundred fiiy microliters of a solution of PFBBr in pentanol (1/9 (v/v)) was added to the reaction mixture and the vial was again sealed and shaken for 1 h on a mechanical shaker at room temperature. The reaction mixture was acidified with 1mL of 1N hydrochloric acid and was again shaken for 15 min to ensure penetration of hydrochloric acid into the pores of the XAD-2 resin. The reaction workup and chromatographic purification were identical with those used for the carboxyl-selective reaction. Recovery Studies. When the experiment was carried out with 3H-I to determine elution profiies and yield, all the fractions were collected in separate screw-cap tubes and evaporated under a stream of nitrogen. The residue was reconstituted in 300 pL of acetonitrile and 100 pL was transferred to counting vials and the amount of radiolabel was determined by liquid scintillation counting. After evaporation of remaining solvent (200 pL), reaction products were silylated with 50 pL of BSTFA/TMCS (85/15 (v/v)) at 65 OC for 1 h or overnight at 40 "C. Determination of I in Urine. For determination of concentrations of I in urine by GC/ECD the diethyl ether/ethanol eluatea were combined and evaporated under a stream of nitrogen. External standard pentafluorobenzyln-tetracosanoate (97 ng) was added. After evaporation of solvent, IV was silylated with 50 pL of TMCS/BSTFA (15/85 (v/v)) at 65 O C for 1 h or overnight at 40 "C.
RESULTS AND DISCUSSION At lower pH and with TCE as the diluent for PFBBr the yield was dependent both on the pH of the aqueous phase and on the presence of acetonitrile (7).At pH 7.4 only 50% f 6% (n = 6) of the radiolabel was recovered in the methylene dichloride and ether fractions; no radiolabel eluted with hexane/methylene dichloride (7/3 (v/v)) or with diethyl ether/ethanol(9/1 (v/v)). Although greater than 50% of the recovered material always eluted in the methylene dichloride, the relative amounts eluted in that solvent or in diethyl ether was variable. Gas chromatographic analysis showed material in the methylene dichloride and diethyl ether fractions to be identical. This elution profile suggested that the reaction product was of intermediate polarity, which had a consistent formation of 111. The low yield at pH 7.4 may have been due to incomplete ionization of the lipophilic acid adsorbed on the surface, but reaction at pH 8.5 only marginally improved recovery although the elution profile remained the same. Alternatively a low yield may have been due to low solubility of I, which did not permit efficient desorption and ionization. This may have been the case as in the presence of 20% acetonitrile and a t a pH of 8.5 the yield of radiolabel recovered in the methylene dichloride and diethyl ether increased to 90% 8% (n = 6) (Table I). When the reaction was carried out at 0.1 N NaOH with pentanol as the diluent, recovery of radiolabel was quantitative. The major product IV,recovered in 80% 5% (n = 6)
*
ANALYTICAL CHEMISTRY, VOL. 61, NO. 8, APRIL 15, 1989
100%
-
r
-+
19.06 rnin
41 5
50 %
Flgure 2. GUECD trace of an isolate of 4 mL of urine containing 125 ng/mL of I prepared without chromatographic cleanup; one microltter from 50 pL of final Isolate injected at attenuation 8; retention time of product I V is 15.32 min, and retention time for PFB-tetracosanoate (external standard) it is 19.06 min.
596
lo
371
15.32 min
927
525 A, I 300
Flgure 4. product.
.
..I I
I
I
400
I
I 500
I
600
Ion trap mass spectrum of 111, the carboxyl-selective
A
1-0-PFB9-COZH-THC
io0
60
161
40
30
-
299
20 -
IO -
'49
242
415
371 401
596
1% 1'
L."..
J
Figure 3. Mass spectrum of I V , the phenol-selective product.
yield, was eluted only in the diethyl ether/ethanol(9/1 (v/v)) fraction, suggesting a polar product which would be consistent with a PFB derivative containing a free carboxyl group. Minor products were recovered in the hexane/methylene dichloride (7/3 (v/v)) and in the methylene dichloride and diethyl ether fractions; these were subsequently characterized as I1 and 111, respectively, each comprising 10% of the yield. Control reactions were carried out at pH 8.5 and 0.1 N NaOH. Reaction conditions were identical except that 150 p L of diluent and no PFBBr was used. Underivatized I did not elute from Florisil with solvents used for recovery of products but could be recovered in a wash with acidified ethanol. The structure of I11 and IV were characterized by GC/MS and GC/ITMS analysis of the trimethylsilylated product. As noted below the analytical method was based on IV; consequently this compound was first characterized by the more classical magnetic sector instrumentation. The mass spectrum of IV showed a molecular ion a t m / z 596 and major ions at m / z 581; 479 (base peak) and 415 (Figure 2). This corresponded to fragmentation patterns previously reported for the silylated derivatives of 11-nor-9-carboxy metabolites of THC (15) and demonstrated reaction a t the phenol as the preferred pathway in the presence of pentanol. The GC/ITMS spectrum of IV was also determined because the high sensitivity of this technique would provide structural information a t low concentrations of analyte and this would be useful in forensic analysis. Such a spectrum was obtained on an isolate from 4 mL of urine containing 25 ng/mL of I. A similar pattern was found but with an (M + 1) ion present, the base peak at 581 and a major ion at 479. There appeared to be less fragmentation due to softer ionization conditions. The IT-MS spectrum of I11 demonstrated that the PFB ester of I was formed at pH 8.5. In this case the molecular ion was present as the base peak at 596 with ions at 581,415, and 371 (Figure 3). Again this fragmentation was consistent
15:32 min
ii.06
min
B
r' Flgure 5. GC/ECD traces of isolates prepared by phenol selective derivatization. Samples were (a) 4 mL of urine containing 25 ng/mL of I and (b) 4 mL of urine containing no analyte; prepared with a chromatographic cleanup; 1 pL from 50 pL of final isolate injected at attenuation 6.
with mass spectral data previously reported (6, 10, 15) for derivatives of I and confirmed derivatization at the carboxyl group. Selective derivatization coupled with semipreparative chromatography incorporated further specificity of determination into the sample preparation step. For instance, in the phenol-specific reaction procedure, derivatization selected for one group (e.g. the phenol) whereas chromatography selected for the other function, i.e. the carboxylic acid. Thus the sample preparation method produced a fraction containing phenolic carboxylic acids. Obtaining such a fraction was important as the isolate obtained by phenol selective derivatization with no chromatographic clean-up contained considerable impurities that could interfere with determination of I a t low concentrations (Figure 4). Incorporating the chromatographic step produced an isolate that was substantially free of interferences to the determination of I from urine (Figure 5a) and with a considerably reduced solvent front. While there was considerable variability in the traces, the phenol-selective isolate tended to be cleaner, particularly in the early part of the gas chromatographic trace, reflecting
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 8, APRIL 15, 1989
reduced yield for reaction of carboxylic acids which are major components of the biological matrix. Furthermore the linked reactor bed/Florisil column system could be washed with methylene chloride and diethyl ether prior t o elution of derivative. This more efficiently removed the small amount PFB esters that were formed (11). Finally these washes could also remove compounds that leached from plasticware (16) as well as other more lipophilic contaminants of the urine and glass matrix. For reasons described above, the analytical method was based on the phenol selective reaction. Attention to some details was necessary. Firstly, to ensure complete dissolution of I in urine, it was necessary, as others had reported (6), to add 20% acetonitrile to spiked solutions of analyte in that fluid. It was also essential to clean all plasticware by a brief wash with organic solvent prior to use to remove electrophoric material (16) which could compromise analytical specificity. A calibration curve with concentrations ranging from 12.5 to 100 ng of I per milliliter of urine was linear ( r = 0.999) with no interference detected in the blank (Figure 5b). A t a concentration of 25 ng/mL, the relative standard deviation was 8% ( n = 6) and at this concentration it was possible to obtain ion trap mass spectra to provide structural information. Samples from the Armed Forces Institute of Pathology (United States) were analyzed. The mean target concentrations of three samples were 54 ng and were found to be 46% 2%, which may have been due to prolonged storage in plastic containers at ambient pH (6) and/or due to adsorption onto solids present in urine.
*
ACKNOWLEDGMENT The ion trap mass spectra were provided courtesy of D. Woodard from Mann Testing Laboratories (Mississauga, ON, Canada) whose technical expertise and assistance are gratefully acknowledged. LITERATURE CITED Foltz, R. L. I n Advances in Analytical Toxicology; BaseR, R. C.. Ed.; Biomedical Publications: Forte City, CA, 1984; Vol. 1, p 133. Elsohly, M. A.; Arafatt, E. S . ; Jones, A. J. Anal. Toxicol. 1984, 8, 7. Elsohly, M. A.; Elsohly, H. N.; Jones, A,; Dlmson, P. A,: Wells, K. E. J. Anal. roxico/. 1983, 7 , 262. Schwarlz, R. H.; Hawks, R. L. J. Am. Med. Assoc. 1985,254, 788. Finkle, B. S. Clin. Chem. 1887,33, 138. Joern, W. A. J. Anal. Toxicol. 1987, 11 49. Rosenfeld, J. M.; McLeod, R.: Foltz, R. L.Anal. Chem. 1988, 58, 716. Lehtonen, K.; Ketola, M. J. Chromatogr. 1986,370, 465. Paul, D. B.: Mell, L. D.; Michell, J. M.; McKlnley, R. M. J. Anal. Toxicol. 1987, 7 1 , 1. Burstein, S.; Rosenfeld, J. M.; WHtstruck, T. Science 1972, 176, 422. Rosenfeld. J. M.; Osei-Twum, E.; Yeroushalmi, S.Anal. Chem. lS86, 58,3043. Guyton, a. C. Textbook of Medicalphysiology; W. 0. Saunders: Philadelphia, PA, 1966; p 407. RosenfeM, J. M.; Mureika-Russell, M.; Yeroushalmi, S. J. Chromatogr. 1886, 358,137. Gustavii, K.;Furangen, A. Acta Pharm. Suec. 1985,27, 295. Harvey, D. J.; Paton, W. D. M. I n Msrihuana Chemistry, Biochemistry and Ca/lu/ar Effects; Nahas, Gabriel G., Ed.; Springer-Verlag: Berlin, 1976; p 93. Junk, G. A.; Avery, M. J.: Richard, J. J. Anal. Chem. 1988,60, 1347.
RECEIVED for review October 12,1988. Accepted January 19, 1989. Support of this work by the National Institute on Drug Abuse of the United States (Grant DA03470-05) is gratefully acknowledged.
CORRECTIONS Micellar Induced Simultaneous Enhancement of Fluorescence a n d Thermal Lensing Chieu D. Tran and Timothy A. Van Fleet (Anal. Chem. 1988, 60,2478-2482). Equation 2 on page 2479 should read ~
E=
-P(dn/dT) 1.91Xk
Simultaneous E n h a n c e m e n t of Fluorescence a n d Thermal Lensing by Reversed Micelles Chieu D. Tran (Anal. Chem. 1988,60, 182-185). Equation 2 on page 182 should read
E=
-P(dn/dT ) 1.91Xk
