cannabinol and Six Metabolites in Plasma and Urine Using GC-MS

Journal of Analytk al Toxicology, Vol. 19, September 1995

Cannabinoids in Humans. I. Analysis of Ag-Tetrahydrocannabinol and Six Metabolites in Plasma and Urine Using GC-MS Philip M. Kemp 1,2,', Imad K. Abukhalaf1,3, Joseph E. Manno 1,3,4, Barbara R. Manno 1,4,t, Dempsey D. Alford 1,3, and Gabi A. Abusada 1,4

Louisiana State University, 1Centerof Excellence for Clinical and Forensic Toxicology, Departments of 2pharmacology, 3Medicine, and 4Psychiatry Abstract This report describes a method for the quantitative analysis of Ag-tetrahydrocannabinol and six of its metabolites, 8cr Ag-tetrahydrocannabinol, 8~-hydroxy-A%tetrahydrocannabinol, 11-hydroxy-Ag-tetrahydrocannabinol, 80~,11-dihydroxy-A% tetrahydrocannabinol, 813,11-dihydroxy-A%tetrahydrocannabinol, and 11-nor-9-carboxy-A%tetrahydrocannabinol. In addition, the method was designed to detect cannabidiol and cannabinol, two naturally occurring cannabinoids. Plasma and urine samples were hydrolyzed with bacterial (Escherichia coh) ~-glucuronidase and extracted with hexane-ethyl acetate (7:1). Analysisand quantitation were performed by gas chromatography-mass spectrometry in the electron ionization mode coupled with selected ion monitoring. The cannabinoids were detected as their trimethylsilyl derivatives to enhance their chromatographic separation and massspectral characteristics. The linearity of the procedure was excellent for all of the compounds within the range tested (0-100 ng/m/), limits of detection ranged from 0.5 to 1.5 ng/mt in urine and from 0.6 to 2.1 ng/mt in plasma depending on the analyte.

Introduction

According to a recent survey (1), a significant proportion of the population of the United States uses marijuana. The prevalence of marijuana use and the passage of legislation regulating its use has mandated the development of analytical procedures for detecting Ag-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and its metabolic products in biological matrices. Forensic and clinical toxicologists are frequently called upon to interpret drug test results in order to provide information on time since marijuana use and the relationship between concentrations of drug or metabolite in biological fluids and possible physiological and psychological effects. Analytical procedures that will detect a number of cannabinoid metabolites at concentrations normally encounPresent address:Office ~f the Chief Medical Examiner, 9(11 N. Stonewall, Oklahoma City, OK 73117. f Author to whom correspondence should be addressed~

*

tered are necessary to establish these relationships and to conduct clinical research to relate cannabinoid effects to the detected concentrations in blood and urine. Thin-layer chromatography (2-4), radioimmunoassay (5,6), enzyme multiplied immunoassay technique (7,8), and fluorescence polarization immunoassay (9,10) are some of the analytical techniques currently available for the determination of marijuana use. Because of their inherent cross-reactivity with many cannabinoids and their metabolites, however, these methods do not provide specific information on the nature of the cannabinoids present in a biological sample. High-performance liquid chromatography (11,12) and gas chromatography (13,14) provide a higher degree of specificity, but they are not able to provide structural information to confirm the identification of cannabinoids with forensic validity. Only gas chromatography-mass spectrometry (GC-MS) provides the sensitivity, selectivity, and specificity necessary for confirmation of positive results generated by screening methods and for quantitative data needed in clinical studies on metabolic profiling and for evaluation of human performance (15,16). This paper reports a simple, sensitive procedure for the analysis of cannabidiol (CBD), THC, cannabinol (CBN), 8r (8cr 8[3-hydroxy-THC (8Lg-OHTHC), 11-hydroxy-THC (11-OH-THC), 8cr 11-dihydroxy-THC (8~, 11-diOH-THC), 8[3,11-dihydroxy-THC(8[3,11-diOH-THC), and 11-nor-9-carboxy-A9-tetrahydrocannabinol (THCCOOH). The procedure allows the resolution and quantitation of these cannabinoids in plasma or urine using a GC-MS system available in many laboratories.

Experimental Reagents

Cannabinoid standards were obtained from Research Triangle Institute (Research Triangle Park, NC) through the National Institute on Drug Abuse (NIDA). Deuterated (d3)THC and 11-OH-THC were obtained from NIDA to be used as internal standards. Deuterated THCCOOHwas obtained from

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285

~ournal of Analytical Toxicology, Vol. 19, September 1995

Alltech-Applied Science (Deerfield, IL). Bacterial 13-glucuronidase (Escherichia coli, type IX-A) was obtained from Sigma Chemical Co. (St. Louis, MO).N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from Pierce Chemical Co. (Rockford, IL). HPLC-grade methanol, hexane, acetonitrile, and ethyl acetate were obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ), Fisher Scientific (Pittsburgh, PA), and Burdick and Jackson (Muskegon, MI).

Hydrolysis and extraction Hydrolysis conditions were optimized to ensure maximal recovery of the cannabinoids and metabolites. A pH of 6.8 was recommended by the manufacturer (17) and was shown to be appropriate for opiates (18). Two variables, enzyme concentration and incubation time, were tested to optimize hydrolysis conditions. Three enzyme concentrations (1500, 5000, and 10,000 Fishman units) were added to 1 mL of urine collected 1 h after a single marijuana cigarette was smoked (3.58% THC). The recovery of 11-OH-THC was compared for the three enzyme concentrations, using the procedure for hydrolysis and extraction described below. Four incubation times were tested (0.25, 1.5, 4.0, and 16 h), using the optimum enzyme concentration. Recovery of 11-OH-THC was compared for each incubation time. Interpretation of the data from these recovery experiments resulted in the adoption of the final protocol described below. To 1 mL of plasma or urine in a 100- • 16-ram disposable glass culture tube (Fisher Scientific) was added 15 1JL of a methanol solution containing 2 ng/1JLTHC-d3, 11-OH-THC-d> and THCCOOH-d:~.To each tube was added 1.0 mL 0.1M potassium phosphate buffer (pH 6.8). If necessary, plasma specimens were adjusted to pH 6.8 with the dropwise addition of 50raM phosphoric acid, and urine was adjusted to pH 6.8 using 0.1N NaOH. The hydrolysis reaction was started with the addition of 200 pL of a 25,000-unit/mL solution of [3-glucuronidase (E. coli) in 0.1M potassium phosphate buffer (pH 6.8), giving a total of 5000 units added. The tubes were immediately capped with disposable polypropylene screw caps, gently vortex mixed for approximately 2 s, and placed in a 37~ (+1~ water bath (Multivap Analytical Evaporator; Organomation Assoc., Berlin, MA) for incubation overnight (approximately 16 h). Gentle vortex mixing was done to prevent the formation of a layer of foam on the top of the mixture, which was shown to interfere with the quantitation of cannabinoids in urine (19). Following incubation, the tubes were allowed to return to room temperature, and 2 mL acetonitrile was added to each plasma tube while vortex mixing (total vortex mixing time, 15 s). The samples were then centrifuged at 2800 rpm, and the supernatant (approximately 3.5 mL) was transferred to a clean, disposable 100- x 16-rnm glass culture tube where it was evaporated to approximately 2 mL under a stream of nitrogen. The remaining extraction for plasma then proceeded in a manner identical to that of urine. The liquid-liquid extraction was a modification of a procedure described elsewhere (15). For the extraction of the nonacidic cannabinoids or metabolites or both, the samples were made basic with 0.5 mL 2N NaOH. Four milliliters hexane-ethyl acetate (7:1) was added, 286

and the samples were gently mixed for 15 rain on a rotator (Nutator, catalog No. 1105; ClayAdams, Parsippany, NJ). The samples were then centrifuged for 5 rain at 2800 rpm. The solvent layer, containing THC, CBD, CBN, and the hydroxylated metabolites, was transferred to a clean 100- x 16-ram tube and evaporated to dryness using nitrogen and a heated water bath (60~ Care must be taken here to avoid transferring any of the aqueous phase with the solvent, as the moisture will interfere with derivatization. This nonacidic fraction was derivatized with the addition of 20 tJL BSTFAplus 1% TMCS, capped, and heated for 15 min in a 60~ water bath. The samples were removed from the water bath, allowed to cool to ambient temperature, and transferred to a 2.0-mL glass autosampler vial (Alltech, catalog No. 66002) equipped with a conical glass 100-tJL insert (Alltech, catalog No. 95201 ). The samples were capped (TFE-lined crimp cap, Alltech, catalog No. 73070), and 2 IJL BSTFA in 1% TMCS was injected on the GC-MS. For the extraction of the acidic fraction containing THCCOOH, the basic aqueous phase was acidified with the addition of 1.0 mL 1N HCI to adjust the pH to 4-5. Four milliliters hexane-ethyl acetate (7:1) was added, and the samples were gently mixed on the rotator for 15 min. Following centrifugation for 5 rain at 2800 rpm, the solvent layer was transferred to a clean culture tube, again avoiding any aqueous contamination. The solvent was evaporated to dryness with a stream of dry nitrogen and heat (60~ The extracts were derivatized with the addition of 20 IJL BSTFAplus 1% TMCS to the culture tube, capped, vortex mixed briefly, and heated for 15 rain in a 60~ water bath. Following removal from the water bath, the derivatized extracts were allowed to cool to room temperature. They were transferred to autosampler vials and capped, and 2 pL was injected on the GC-MS. The identification and quantitation of the analytes was performed using selected ion monitoring. The ions selected for this purpose were chosen from full mass spectral analysis of 200-ng/mL extracted standards of each cannabinoid. Two ions were chosen to monitor the presence or absence of each of the cannabinoids. A quantitative ion was selected on the basis of abundance and the absence of interfering peaks from the biological matrix used. Negative plasma and urine were spiked with 30-ng/mL deuterated internal standards and increasing levels of cannabinoid standards (0, 3, 5, 20, 50, and 100 ng/mL) for establishment of a calibration curve for each analyte. Negative plasma or urine was spiked with cannabinoids or metabolites or both (5, 50, and 100 ng/mL) and was used for calculating extraction recoveries and precision. Recoveries were determined by comparing the average peak-area ratios of the selected quantitative ions in extracted standards with areas of the ions found in unextracted standards. Blank plasma or urine was spiked with deuterated internal standard and extracted for determination of limits of detection (LOD) and quantitation (LOQ). The LOD was defined by this laboratory as the mean-area ratio (baseline noise:internal standard) plus 3 standard deviations. The LOQ was defined as the mean-area ratio plus 10 standard deviations. All statistical comparisons were conducted using the Student t test (20) for determination of significant differences (p < .05).

Journal of Analytical Toxicology, Vol. 19, September 199.5

Instrumentation Analysis of the cannabinoids took place using an HP 5890 gas chromatograph equipped with an HP-5 MS capillary column (for separation of the analytes), an injector with electronic pressure programming, and an HP 5972 mass selective detector. The column (HP-5 MS) was fused-silica coated with a 5% phenylmethylsilicone liquid phase (30 m x 0.25-mm i.d., 0.25-pm film thickness). High-purity helium (99.99%) was used for the carrier gas. The column head pressure was programmed with an initial pressure of 25 psi held for 0.5 rain, then decreased to 16 psi at a rate of 25 psi/rain, and finally maintained at this pressure for the duration of the analysis time. This resulted in a flow rate of 1.2 mL/rnin during the run. The final extracts, containing the trimethylsilyl derivatives, were injected (2 pL) in the splitless mode, and the splitless valve remained closed for 1.0 min. The heated temperature zones were as follows: injection port, 290~ transfer line, 280~ and ion source, 200~ The electron multiplier voltage was set 400 V above the tune value. The data system used was a Hewlett-Packard Vectra 486/33N. Data acquisition and analysis were performed with Hewlett-PackardHPG1034C MS ChemStation (DOS series) software. For the analysis of the nonacidic fraction containing the trimethylsilyl derivatives of CBD, THC, CBN,and hydroxylated metabolites, the column temperature was maintained at 150~ for 1 min after injection. The following temperature program was then initiated: 150-240~ at 30~ hold for 0.5 rain; 240-280~ at 3~ hold for 1.0 rain; and 280-300~ at 30~ hold for 1.0 min. The total time for the program was 20.5 min. For the analysis of the acidic fraction containing the trimethylsilyl derivative of THCCOOH, the column temperature was maintained at 200~ for 1.0 rain after injection. The temperature was then increased to 300~ at a rate of 30~

where it was held for 5.0 min. The total analysis time for this program was 9.33 min.

Results and Discussion Researchers are currently attempting to find a marker in biological fluids that would identify recent use of marijuana. The vast majority of analytical methodology developed for determining if a person has used marijuana has focused on the detection and quantitation of THC or THCCOOH, its major metabolite, in blood and urine (11,14,21,22). These compounds, however, have long terminal elimination half-lives and were shown to be present for days or weeks after cessation of drug use (23-25). A recent study (26) reported the development of two mathematical models for the prediction of time of marijuana use based on plasma concentrations of THC and the THCCOOH/THC ratio in plasma. Other metabolites, such as 11-OH-THCand 8[~,11-diOH-THC,received only limited attention (15,16,27,28). One of these reports (16) suggested that 813,11-diOH-THC is excreted in less than 24 h after smoking and may be useful for prediction of recent marijuana use. As investigators continue to study the patterns involved in production of metabolites by humans after inhalation or ingestion of marijuana, it is becoming increasingly clear that the complexity of the temporal relationship between marijuana and its effects will require a more complex analytical procedure than has previously been reported. The method reported here provides an analytical tool that reliably quantitates several cannabinoid metabolites. Cannabinoids are extensively metabolized, and some were found as glucuronide conjugates in blood and urine (29-31). For the analysis of the free compounds in plasma and urine, it 100

n=3

n=3 90

8o

80 11 - O H - T H C 7O 11-OH-THC

(ng/mL)

concentration

(ng/mL)

60

concentration

I r

60 5o

40,

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0;25

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3O 1500

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Versus 1500 units*

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115

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*p < .05, (+) = significant, (-) = not significant.

*p < .05, (+) = significant, (-) = not significant.

Figure1. Optimizationof enzymeconcentrationat 37~ Barsrepresent

mean 11-hydroxy-A9-tetrahydrocannabinol(11-OH-THC)concentrations (in nanogramsper mirri[iter)plusor minusstandarderrorofthe mean.

+

§

4-

4+

+

Figure2. Optimizationof hydrolysisincubationtime at 37~ Barsrepresentmean11-hydroxy-A9-tetrahydrocannabinol(11-OH-THC)concentrations (in nanogramsper milliliter) plus or minus standarderror of the mean.

287

Journal of Analytical Toxicology, Vol. 19, September 1995

is necessary to hydrolyze the glucuronide bond. Hydrolysisof cannabinoids has been performed enzymatically,using ~-glucuronidase, or with alkaline conditions. Bacterial [3-glucuronidase from E. coli (type IX-A)was chosen for this method because it produced a significantlyhigher recovery of free THC and 11-OH-THCwhen compared with Helix pomatia and basic conditions, and the recovery of free THCCOOH was not reduced (32). A pH of 6.8 was used according to the glucuronidase manufacturer's recommendations (17) and previous research (18) documenting this pH as that which shows the maximum reaction rate for this enzyme at 37~ Two variables were tested to further optimize hydrolysis conditions, enzyme concentration and incubation time. Three enzyme concentrations (1500, 5000, and 10,000 Fishman units) were added to I mL of urine that was collected 1 h after a single marijuana cigarette was smoked (3.58% THC). The results from the GC-MS analysis of 11-OH-THC are shown in Figure 1. The concentration of 11-OH-THC recovered using 5000 or 10,000 units of bacterial [~-glucuronidasewas significantly higher than when using 1500 units (p < .05). The difference between 5000 and 10,000 units was not significant.

Table I. Retention Times and Ions for Monitoring Trimethylsilyl Derivatives of Cannabinoids Retentiontime (min)

Compound

CBD* A9-THC CBNt 8mOH-THC 8[3-OH-THC t 1-OH-THC 8~,ll-diOH-THC 8[[],ll-diOH-THC THCCOOH

6.26 7.12 7.91 9.06 9.29 9,62 1038 11.65 5.75

Quantitativeion (m/z)

Qualitativeion (m/z)

390 386 367 384 343 37~ 472 369 371

458 371 382 343 384 474 457 459 473

* CBD = Cannabidiol. ~ CBN = Cannabinol.

Secondly, using 5000 units of enzyme, four incubation times were tested (0.25, 1.5, 4.0, and 16 h) (Figure 2). Comparedwith the other time intervals, the 16-h incubation time produced a significant increase in l l-OH-THC recovery (p < .05). The parameters adopted for this method were 5000 units [3-glucuronidaseand a 16-h incubation time. 350000 2 1~C A number of liquid-liquid and solid-phase 3 CIm tmknown 300000 4 techniques to extract cannabinoids from 5 Ik,-OH-TItC blood and urine were published (15,16,30, 6 M-Ot4-THC 250000 33,34). Manysolvent combinations that will ii Ila, 11-dK~4-THC 9 unlmown extract cannabinoids from biological 200000 matrices are possible, but a recent review 2 (35) found very few in the literature. The 150000 combination of hexane-ethyl acetate was found most often. Hexane-ethylacetate (7:1) 10 was chosen for the method describedhere as this combination was shown to adequately Pl extract THC, ll-OH-THC, and THCCOOH 0 .... ] 0 . , i , : , 1 ' ' . ' 1 " ] , 10.00 11.00 12.00 13.00 6.00 7.00 8.00 9.00 from urine (16,22). As Foltz et al. (15) suggested, other solvent combinations may exFigure3. Total ion chromatogram of the nonacidic cannabinoid fraction. tract a higher recoveryof the cannabinoids, but they also complicate the chromatogram with more interfering substances. As shown in Figures 3 and 4, trimethyl4500 silyl derivatization produced the necessary 1 mlmRm resolution between the cannabinoids. 4000 Pentafluoropropionyl, heptafluorobutyryl, 3500 and methyl derivatives were tested; how3OOO ever, none of these provided the necessary resolution of the analytes, particularly the 3500 cr and [3-mono- and dihydroxylated 3000 metabolites of THC. BSTFA in 1% TMCS 1500 was used in previous research (22,36) with excellent results and was, therefore, chosen 1000 1 for this method. It is compatible with most 500 injectors and columns and can be injected directly onto the GC with no cleanup steps. 5.00 $,50 6.00 4.50 7.00 7.50 ll.O0 Trimethylsilyl derivatives generally proT i m e (rain) duce abundant diagnostic ions (37). The Figure4. Total ion chromatogram of the acidic cannabinoid fraction. two ions used for identification and quanti4

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Journal of Analytical Toxicology, Vol. 19, September1995

Table II. tinearity of Analysis* Plasma Cannabinoid

r2 (SD)~

CBD* 0.998 (0.001) THC 0.999 (0.005) CBNw 0.999 (0.004) 8@-OH-THC 0.999 (0.005) 8~,-OH-THC 0.999 (0.005) 11-OH-THC 0.999 (0.005) 8(:(,11-diOH-THC 0.998 (0.001) 813,11-diOH-THC 0.999 (0.009) THCCOOH 0.999 (0.005)

Urine equation

r 2 (SD)

y = 1.35x- 0.01 y = 1.22x- 0.006 y = 5.49x- 0.03 y = 0.79x- 0.0059 y = 0.74x + 0.0001 y = 1.0x + 0.01 y = 0.37x + 0.002 y = 0.22x + 0.006 y = 0.98x + 0.009

0.998 (0.005) 0.998 (0.005) 0.999 (0.004) 0.999 (0) 0.999 (0.005) 0.999 (0.005) 0.999 (0) 0.999 (0) 0.999 (0.005)

* Concentrations of O, 3, 5, 20, SO, and 1O0 ng/mL were analyzed in triplicate. ~ SD = Standard deviation. CBD = Cannabidiol. w CBN = Cannabinol.

Table III. Efficiency of Cannabinoid Extraction from Plasma and Urine* Plasma Concentration Cannabinoid (ng/mL) % recovery CVt

Urine % recovery CV

CBD*

5.0 50.0 100.0

76.8 79.8 109.0

8.0 8.4 4.3

88.8 63.1 50.1

6.2 10.3 4.3

THC

5.0 50.0 100.0

92.7 72.5 90.3

11.4 2.3 3.1

101.7 81.5 75.2

3.1 4.0 7.4

CBNw

5.0 50.0 I00.0

59.3 74.5 86.8

7.9 4.7 2.6

65.4 64.9 109.9

5.4 3.9 6.5

8e-OH-THC

5.0 50.0 100.0

57.2 79.7 92.3

13.5 11.5 6.3

66.8 70.4 98.5

2.4 11.2 3.2

8[3-OH-THC

5.0 50.0 100.0

60.2 81.2 93.8

12.1 1.6 2.8

103.6 80.2 107.6

3.9 4.4 3.3

5.0 50.0 100.0

90.3 100.8 86.8

5.3 5.4 2.9

93.3 88.2 104.6

5.9 5.1 3.6

80~,11-diOH-THC

5.0 50.0 100.0

50.7 48.5 55.6

7.5 13.1 1.9

64.8 58.8 56.3

4.3 9.3 4.8

8~,11-diOH-THC

5.0 50.0 100.0

52.9 43.1 54.7

14.5 12.5 4.6

100.9 60.2 65.4

5.7 8.2 3.9

THCCOOH

5.0 50.0 100.0

58.4 61.2 110.5

4.3 6.9 2.9

61 .I 64.1 106.3

3.0 2.7 6.0

11-OH-THC

* n=3.

~" CV = Coefficient of variation. CBD = Cannabidiol. w CBN = Cannabinol.

equation y = 0.25x§ o.oo5 y = 0.27x+ o.o03 y=1.57x+O.01

tation in this method are shown in Table I along with retention times achieved under the stated conditions. Mechanisms leading to the formation of the ions were proposed previously (37). Exact masses (to 0.05 ainu) for each analyte were checked periodically to ensure maximum sensitivity of the method.

y= 0.57x-0.007

This method requires two separate extracts and two temperature programs.

y=0.65x-0.006 y= o.7sx-o.ool

Although it may be argued that these cannabinoids may be separated from the

y = 0.28x- 0.006 y = 0.23x- 0.004 y = 0.79x- 0.007

matrix into a single extract, a combination of factors made separation of the acidic and nonacidic fractions practical. The chromatographic separation of all nine cannabinoids from each other and any matrix interferences was a formidable task. Combining the acid and nonacidic fractions resulted in coeluting peaks that interfered with 8cr and THCCOOH. One of the coeluting substances (Figure 3, peak 4) had a base peak at rn/z 371 and a smaller ion at m/z 384 that interfered with 8~-OH-THC (Figure 3, peak 5). The other matrix compound (Figure 3, peak 9) had a basepeakat m/z 399 and smaller ions at m/z 311 and 473 that interfered with THCCOOH. The addition of the two-phase extraction producedthe necessaryresolution betweenTHCCOOH and the interfering substance (Figure 4). No further investigation was undertaken to identify these substances.Secondly, a commonly used cleanup step is to back extract the desired cannabinoids into aqueous acid or baseand reextract with solvent. Separation into the acidic and nonacidic phases in this method provides a "cleanup" step for each fraction. Linear regression analysiswas used to construct calibration curves for each of the cannabinoids. As shown in Table II, linearity was achieved and was reproducible over a range of 0-100 ng/mL of specimen. Day-to-daycorrelation coefficients ranged from 0.998 to 0.999 for both urine and plasma. Other studies

Table IV. Limits of Detection and Quantitation* Plasma(ng/mL) Compound CBDw THC CBN II 8cr 8~-OH-THC 11-OH-THC 8~,11-diOH-THC 8[~,11-diOH-THC THCCOOH *

Urine (ng/mL)

LOD~

LOQ*

LOD

LOQ

2.1 1.6 0.6 0.8 1.1 0.9 0.7 0.8 0.6

5.5 3.5 1.3 1.3 2.1 2.2 1.4 1.8 1.0

1.1 1.5 0.5 0.6 1.0 0.6 0.8 0.9 0.5

3.4 3.9 1.1 0.9 1.5 1.1 1.0 1.4 0.9

n=S.

~" LOD = Limit of detection; defined as the mean-area ratio plus 3 standard deviations. LOQ = Limit of quantitation; defined as the mean-area ratio plus 10 standard deviations. w CBD = Cannabidiol. II CBN = Cannabinol.

289

Journal of Analytical Toxicology,Vol. ]9, September1995

(15,21,22) demonstrated linearity over a broader range of concentrations for THC or THCCOOH, but levels of the hydroxylated and dihydroxylated metabolites tested in this method did not reach 100 ng/mL in urine or plasma following the inhalation of marijuana smoke (16,27). Recoverydata for each of the cannabinoids is shown in Table III. At concentrations of 5, 50, and 100 ng/mL (n = 3), percent recoveries ranged from 50.1 to 109.9 in urine and from 43.1 to 110.5 in plasma. The data also indicated the reproducibility of the method at each concentration by having coefficients of variation at 14.5 or less. The LOD and LOQ for each of the cannabinoids in plasma and urine are shown in Table IV. The limits were determined using extracted blank urine or plasma spiked with internal standards. A range of 0.5-1.5 ng/mL was achieved for the LOD in urine depending on the cannabinoid. For plasma, LODs ranged from 0.6 to 2.1 ng/mL. The LOQs for urine ranged from 0.9 to 3.9 ng/mL, and for plasma, they ranged from 1.0 to 5.5 ng/mL. In conclusion, the present report provides a sensitive and specific method for the analysis of THC, six metabolites, CBD, and CBN in urine and plasma. This method can be applied by forensic toxicologistsand researchers interested in ascertaining metabolic profiles from a single analytical procedure. Metabolite/parent and metabolite/metabolite ratios acquired through this method can be related to excretion times for the individual metabolites and may provide data for determination of time since ingestion of marijuana. In addition, the broad array of metabolites detected with this method may be used to determine the route of administration, as a different metabolic pattern was shown to occur in smoking versus oral comparisons (38,39).

Acknowledgment The authors gratefully acknowledge the support from NIDA Grant DA-05850.

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