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Mass Spectrometric Methods for Determination of Cannabinoids in Physiological Specimens

Mass Spectrometric Methods for Determination of Cannabinoids in Physiological Specimens

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This chapter describes the published mass spectrometric (MS) methods that have proven most effective for quantitative measurement of D9-tetrahydrocannabinol (THC) and its major metabolites in physiological specimens. Because determination of 11-nor-9-carboxy-D9-tetrahydrocannabinol (THCA) in urine continues to be the most frequently used indicator of marijuana use, the first portion of the chapter will discuss methods for measurement of THCA in urine. However, the major portion of the chapter is devoted to the most recent developments for measuring THC and its metabolites in other biological specimens including blood, plasma, meconium, oral fluids, hair, and other tissues. Tables 1–7 are designed to facilitate location of references describing analytical methods involving key components for analysis of cannabinoids in various matrices.
This chapter describes the published mass spectrometric (MS) methods that have proven most effective for quantitative measurement of D9-tetrahydrocannabinol (THC) and its major metabolites in physiological specimens. Because determination of 11-nor-9-carboxy-D9-tetrahydrocannabinol (THCA) in urine continues to be the most frequently used indicator of marijuana use, the first portion of the chapter will discuss methods for measurement of THCA in urine. However, the major portion of the chapter is devoted to the most recent developments for measuring THC and its metabolites in other biological specimens including blood, plasma, meconium, oral fluids, hair, and other tissues. Tables 1–7 are designed to facilitate location of references describing analytical methods involving key components for analysis of cannabinoids in various matrices.

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Published by: Ahmad Abdullah Najjar on Jul 28, 2008
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MS for Detection of Cannabinoids179
From:
Forensic Science and Medicine: Marijuana and the Cannabinoids
Edited by: M. A. ElSohly © Humana Press Inc., Totowa, New Jersey
179 
Chapter 8
 Mass Spectrometric Methods for Determination of Cannabinoidsin Physiological Specimens
 Rodger L. Foltz
1. I 
 NTRODUCTION 
This chapter describes the published mass spectrometric (MS) methods that haveproven most effective for quantitative measurement of 
9
-tetrahydrocannabinol (THC)and its major metabolites in physiological specimens. Because determination of 11-nor-9-carboxy-
9
-tetrahydrocannabinol (THCA) in urine continues to be the most fre-quently used indicator of marijuana use, the first portion of the chapter will discussmethods for measurement of THCA in urine. However, the major portion of the chap-ter is devoted to the most recent developments for measuring THC and its metabolitesin other biological specimens including blood, plasma, meconium, oral fluids, hair,and other tissues.Tables 17are designed to facilitate location of references describ- ing analytical methods involving key components for analysis of cannabinoids in vari-ous matrices.Analysis of THC and its metabolites in biological specimens has been reviewedby Lindgren
(1)
, Foltz
(2)
, Bronner and Xu
(3)
, Goldberger and Cone
(4)
, Cody andFoltz
(5)
, and Staub
()
.The selection of internal standards is an important factor in the development of quantitative assays involving MS. Because of the demand for effective internal stan-dards for MS analysis of THC and its major metabolites, a variety of deuterium-labeledanalogs have become commercially available. THC-d
3
, THCA-d
3,
and trideuterated11-hydroxy-
9
-tetrahydrocannabinol (HO-THC-d
3
) have often been used as internal
 
180Foltz
standards. However, cannabinoid analogs with more than three deuteriums (THC-d
6
,THC-d
9
, THCA-d
6
, THCA-d
9
, THCA-d
10
, and HO-THC-d
6
) are reported to be evenmore effective as internal standards
(7–10)
.
2. D
 ETERMINATIONOF 
THCA
 IN 
 RINE 
THCA is primarily excreted in urine as the ester-linked glucuronide conjugate.Consequently, the urine is most often subjected to mild alkaline hydrolysis to releasethe THCA
(11,12)
. Enzymatic hydrolysis using
-glucuronidase can also free the THCAfrom the conjugate, but the procedure takes considerably longer than alkaline hydrolysis
(13,14)
. After hydrolysis the urine is acidified and extracted by either liquid/liquid orsolid-phase extraction (SPE). A solvent mixture of hexane and ethyl acetate, typically7:1 (v/v), has been used most often for extraction of free THCA in urine
(11)
. A widevariety of solid-phase systems are also available for extraction of THCA in urine
(10,15 –24)
, and two research groups have selectively extracted THCA from urine by meansof immobilized antibodies
(8 ,25)
.THCA has two polar functional groups that must be derivatized prior to gas chro-matography (GC)/MS analysis. The carboxyl group and the phenolic group can bothbe derivatized by trimethylsilylation or by methylation. Trimethylsilylation is mostoften performed by adding
bis
-(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1%trimethylchlorosilane (TMCS) to the dried extract and heating at approx 70
C for 20minutes, followed by direct injection into the GC/MS system
(17,18)
. Methylation isgenerally performed by addition of methyl iodide in the presence of tetramethylam-monium hydroxide (TMAH) in dimethyl sulfoxide
(16,26 )
. Some investigators haveused propyl iodide when interference problems were encountered after derivatizingwith methyl iodide
(27 )
; others have used a perfluorinated anhydride and aperfluorinated alcohol
(10 ,24,28,29)
. The latter protocol can provide increased sensi-tivity, particularly when the derivatives are detected by negative ion chemical ioniza-tion mass spectrometry (GC/NCI-MS; ref.
28
). However, it is important to remove theperfluorinated anhydride reagent by evaporation prior to reconstitution and injectioninto the GC/MS because the anhydride tends to degrade the chromatographic column.Szirmai and co-workers compared five different methods for derivatization of THCA and two other acidic metabolites of THC in urine
(9)
. Two of the methodsinvolved esterification of the carboxylic acid group with diazomethane followed bytrimethylsilyation or trifluoroacetylation of the phenolic group; the other three meth-ods employed (1) BSTFA alone, (2) methyl iodide-TMAH, or (3) pentafluoropropionicanhydride (PFPA) and trifluoroethanol.Nearly all GC/MS assays for determination of THCA in urine employ fused silicacapillary columns with methyl silicone or 5% phenylmethylsilicone stationary phases.Electron ionization (EI) continues to be the dominant method for ionizing derivatizedTHCA. With EI-MS, each of the reported THCA derivatives yields at least three ionswith high relative intensities, an important benefit in forensic analyses.The first published liquid chromatography (LC)/MS assay for determination of THCA in urine employed positive ion electrospray ionization (ESI; ref.
30
). Underselected ion monitoring the protonated molecule ion (M + H)
+
at
m/z
345 was domi-nant and could be detected down to 2.5 ng/mL. Up-front collision-induced dissocia-
 
MS for Detection of Cannabinoids181
tion generated qualifying ions at
m/z
327 and 299, but their ion intensities were rela-tively low and thereby increased the lower limit of detection to 15 ng/mL. Signifi-cantly better sensitivity has been achieved by monitoring the (M – H)
ions for THCA(
m/z
343) and THCA-d
3
(
m/z
346) formed by ESI
(23)
.Weinmann and co-investigators
(21)
developed a very rapid LC/MS/MS assayfor THCA in urine using negative ion atmospheric pressure chemical ionization (APCI)in combination with selected-reaction monitoring. When subjected to collision-induceddissociation, the (M – H)
ion at
m/z
343 fragmented to abundant ions at
m/z
325, 299,and 245. The runtime took 6 minutes, and the lower limit of quantitation was 5 ng/mL.Investigators in the same laboratory reported using positive-ion turboionspray todetermine THCA and THCA glucuronide in urine by LC/MS/MS
(31)
.Skopp and Potsch used LC/MS/MS to study the stability of THCA and THCA-glucuronide in urine and plasma stored at temperatures of –20, 4, 20, and 40
C
(32)
.The analytes and their deuterated internal standards were ionized by turboionspray,and the protonated molecule ions collisionally dissociated to abundant product ions.Unfortunately, THCA and other cannabinoids are not as efficiently ionized byeither ESI or APCI as most other drugs. Nevertheless, the advantage of not having toderivatize an analyte prior to analysis is an inducement to utilize LC/MS rather thanGC/MS.Potential problems that can occur in determination of THCA in urine includeinterferences
(27,33)
, adsorptive losses during storage and extraction
(12 ,29,34–36 )
,and degradation of THCA as a result of adulteration of a urine sample
(37 )
.
3. D
 ETERMINATIONOF 
O
THER
 ANNABINOIDSIN 
 RINE 
Although detection of THCA in urine continues to be the primary method foridentifying recent use of marijuana, Manno and Manno and their co-investigators haveshown that THC and other metabolites of THC are also excreted in urine as glucu-ronide conjugates that are not, however, as easily hydrolyzed as THCA glucuronide
(38,39)
. THC and its hydroxylated metabolites are excreted in urine primarily as ether-linked glucuronide conjugates that do not undergo hydrolysis under alkaline condi-tions. Enzymatic hydrolysis using
-glucuronidase from
 Escherichia coli
at a pH of 6.8 is highly effective in cleaving ether-linked glucuronide conjugates. Manno et al.have used this method for quantitative analysis of cannabidiol, cannabinol, THC, andsix THC metabolites in plasma and urine. After enzymatic hydrolysis, they extractedthe cannabinoids with hexane:ethyl acetate (7:1), derivatized them with BSTFA, andanalyzed the products by electron ionization GC/MS. Analysis of urine samples bythis method proved useful for estimating the time of marijuana use
(14)
.GC/MS analysis for 11-nor-
9
-tetrahydrocannabivarin-9-carboxylic acid(THCVA) has been used to determine whether the presence of THCA in a subject’surine indicates the use of marijuana or is solely the result of the use of the prescriptiondrug Marinol
®
(synthetic THC; ref.
40
).
9
-Tetrahydrocannabivarin, a homolog of THC,is present in most marijuana and is metabolized in the body to THCVA
(41)
. BecauseTHCVA is a homolog of THCA, the two compounds behave very similarly duringextraction and derivatization but have different retention times and form abundantions that differ by 28 amu
(40)
.

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