Analysis of Drugs of Abuse by

GC/MS using the Ultra Inert Inlet

Liners with Wool

Application Note
Forensic Toxicology

Authors Abstract
Limian Zhao, Bruce Quimby With efficient deactivation on glass wool, the Ultra Inert liners with wool provide
Agilent Technologies, Inc. excellent inertness, homogeneous sample mixing and evaporation, non-volatile
2850 Centerville Road residue trapping, and column and detector protection for drugs of abuse screening.
Wilmington, DE 19808
USA Introduction
GC inlet liners are the centerpiece of the inlet system where the sample is vapor-
ized, mixed with the carrier gas, and introduced to the capillary column. Inlet liners
with wool are widely used because the wool promotes homogenous sample mixing
and better quantitation. Wool provides a large surface area which aids the vaporiza-
tion of liquid samples. It also acts as a trap to collect non-volatile residue in the
sample, thus protecting the GC column from the negative impact of sample matrix.
Wool liners also reduce sample loss by preventing sample droplets from reaching
the bottom of the inlet before vaporization. Agilent MS certified liners with glass
wool provide excellent performance for general application purposes. However, for
specific applications of active compounds analysis, liners with superior inertness
are required to achieve the most reliable results.

GC/MS screening methods are important in toxicology laboratories. With

the con-tinuing emergence of new drugs and toxins, the list of target compounds to
be screened can number in the hundreds. For those compounds that are compatible
with GC, using GC/MS in full-scan mode with electron impact ionization (EI) is well
suited for the task [1]. Samples for screening usually require minimal sample prepa-
ration, or even no clean-up, to preserve target analytes. However, heavy-matrix sam-
ples, such as plasma or urine extracts, deteriorate the performance of the analytical
column and detector, resulting in short column life and frequent MS source mainte-
nance. Therefore, it is beneficial to use inlet liners with wool to protect the entire
GC/MS system.
However, if liners with wool are poorly deactivated, they can eluting compounds, and contain different categories of drugs
cause the adsorption or decomposition of target analytes for including amphetamins, alkaloids, and benzodiazpines.
basic drugs of abuse. As shown in Figure 1, those drugs usu- Figure 1 shows the chemical structures for some of the ana-
ally contain hetero atoms, which strongly interact with the lytes. All liner tests were conducted using a GC/MS system
free silanol groups (Si-OH) in glass [2]. The resulting com- with simultaneous collection of scan and SIM data. A
pound adsorption and decomposition causes chromatographic 5 µg/mL standard was used for chromatographic evaluation.
problems such as broad or distorted peaks, tailing peaks, A 500 ng/mL standard (10× dilution) was used to assess the
ghosting phenomena, and low responses. Liners with glass repeatability of injections over 50 injections.
wool magnify these negative effects due to the large surface
area of glass wool and difficulty of complete deactivation. A
properly and efficiently deactivated inlet liner with glass wool
Experimental
is imperative for satisfactory chromatography with accurate
and reproducible responses for these forensic/toxicology
Chemicals and Reagents
applications. The Agilent GC/MS Forensic Toxicology analyzer checkout
mixture standard (p/n 5190-0471) was used to evaluate the
Agilent’s Ultra Inert liner deactivation process significantly performance of Ultra Inert liners with glass wool. HPLC grade
improves the efficacy and robustness of glass wool deactiva- Toluene and Methanol was purchased from Honeywell B&J
tion. The surface area is deactivated thoroughly. For the first (Muskegon, MI, USA), and Acetonitrile (AcN) was purchased
time, liners with glass wool can analyze basic drugs of abuse from Sigma-Aldrich (St Louis, MO, USA). An Internal Standard
using GC/MS. (IS) was purchased from AccuStandard (New Haven, CT,
USA), containing 0.5 mg/mL of Acenaphthene-D10,
The liners with wool were evaluated using Agilent Phenanthrene-D10, Triphenylphosphate, Chrysene-D12, and
Forensic/Toxicology analyzer checkout standards, including Perylene-D12 in Acetone.
28 popular and difficult basic drug compounds. These
compounds cover the retention range from early to late

Amphetamine Nicotine MDEA Ph

Phencyclidine
lidi

Cocaine Oxycodone Temazepam C

Codeine
d i

Diacetylmorphine (Heroin) Nitrazepam Strychnine

Figure 1. Chemical structure of selected basic drugs.

2
Solutions and Standards Table 2. Flow Path Supplies

The original checkout standard sample was made in a 90/5/5 Vials Amber screw cap (p/n 5182-0716)
Toluene/MeOH/AcN solution. A 90/5/5 Toluene/MeOH/AcN Vial caps Blue screw cap (p/n 5182-0717)
blank solvent mixture was prepared by combining 90 mL of Vial inserts 150 µL glass w/ polymer feet (p/n 5183-2088)
Toluene, 5 mL of MeOH and 5 mL of AcN, and was used as Septum Advanced Green Non-Stick 11 mm
reagent blank. The 5 µg/mL original standards were directly (p/n 5183-4759)
used for injection, and were diluted 10 times with blank sol- Ferrules 0.4 mm id, 85/15 Vespel/graphite
vent to 500 ng/mL solution. 4 µL of IS stock solution was (p/n 5181-3323)
spiked to 1 mL of standard solution, when necessary, to gener- O-rings Non-stick liner O-ring (p/n 5188-5365)
ate a concentration of 2 µg/mL for IS in the sample. Capillary Purged Ultimate Union (p/n G3182-61580)
Flow Technology Internal nut (p/n G2855-20530)
Instrumentation SilTite metal ferrules, 0.10-0.25 mm id
(p/n 5188-5361)
All testing was done on an Agilent 7890A GC system equipped
Inlet seal Gold plated inlet seal with washer
with a 7683B autosampler and a 5975C MSD. (p/n 5188-5367)
Table 1 lists the instrument conditions. Table 2 lists flow path Inlet liners Agilent Ultra Inert deactivated single taper splitless
consumable supplies. Table 3 list the Selected Ion Monitoring liner with wool (p/n 5190-2293)
(SIM) conditions for 28 target analytes. Table 3. SIM acquisition conditions used for 28 basic drug compounds by
GC/MS
Table 1. Instrumental conditions for Agilent GC/MS system used for basic
drug compounds test
Collection
GC Agilent 7890A Series
RT window
Autosampler Agilent 7683B, 5 µL syringe (p/n 5181-5246), Analytes (Peak no. on chromatogram) SIM * (min) (min)
1 µL injection volume
Amphetamine (1) 44, 91 1.77 1.4 – 2.7
Preinj solvent A (90/5/5 Toluene/MeOH/AcN) washes: 1 Phentermine (2) 58, 134 1.96
Sample pumps: 3 Methamphetamine (3) 58, 91 2.08
Postinj solvent B (90/5/5 Toluene/MeOH/AcN) washes: 3
Nicotine (4) 84, 133 3.06 2.7 – 3.6
Carrier gas Helium, constant pressure
Methylenedioxyamphetamine (MDA) (5) 44, 135 3.92 3.6 – 5.0
Inlet Splitless mode: 280 °C Methylenedioxymethamphetamine (MDMA) (6) 58, 135 4.27
Purge flow 50 mL/min, switched mode, hold for 0.75 min Methylenedioxyethylamphetamine (MDEA) (7) 72, 135 4.57
Inlet pressure 18.7 psi (RT locked) during run, 1.0 psi during back flush Meperidine (8) 71, 247 5.63 5.0 – 7.0
Phencyclidine (9) 200, 242 6.49
RT locking Proadifen (SKF-525a) @ 8.569 min
Methadone (10) 72, 57 7.72 7.0 – 8.9
Oven profile 100 °C for 0.5 min, to 325 °C at 20 °C/min, hold 2.5 min
Cocaine (11) 182, 82 8.10
Post run 2 min at 325 °C Prodifen (SKF-525a) (12)** 86, 99 8.57
Capillary Flow Purged Ultimate Union (p/n G3182-61580) Oxzepam (13) 239, 267 8.73
Technology used for back flushing the analytical column and inlet Codeine (14) 299, 162 9.01 8.9 – 9.5
Aux EPC gas Helium plumbed to Purge Ultimate Union Lorazepam (15) 239, 274 9.08
Diazepam (16) 256, 283 9.22
Aux pressure 4 psi during run, 75 psi during back-flushing
Hydrocodone (17) 299, 242 9.29
Analytical column DB-5MSUI, 15 m × 0.25 mm, 0.25 µm (p/n 122-5512UI) Tetrahydrocannabiol (18) 231, 314 9.36
Connections Inlet to Purged Ultimate Union (p/n G3182-61580) Oxycodone (19) 315, 230 9.63 9.5 – 10.4
Restrictor Inert Fused Silica tubing, 0.65 m × 0.15 mm Temazepam (20) 271, 273 9.87
(p/n 160-7625-5) Flunitrazepam (21) 312, 286 9.96
Diacetylmorphine (Heroin) (22) 327, 369 10.02
Connections Between Purged Ultimate Union and the MSD
Nitrazepam (23) 253, 206 10.62 10.4 – 11.6
MSD Agilent 5975C inert with performance electronics Clonazepam (24) 314, 286 10.94
Vacuum pump Performance turbo Alprazolam (25) 279, 308 11.32
Mode Scan/SIM
Tune file Atune.u Varapamil (26) 303, 304 12.03 11.6 – 14.0
EM voltage Atune voltage Strychnine (27) 334, 335 12.18
Transfer line temp 300 °C Trazodone (28) 205, 70 12.96
Source temp 300 °C * Ions in Bold were quantifiers, and the other ions were qualifiers.
Quad temp 150 °C
** Prodifen was used for the RT locking.
Solvent delay 1.4 min
Scan mass range 40 – 570 amu

3
A back-flushing system was used because it shortens analy- Chromatographic performance
sis times for samples that contain high-boiling matrix interfer- The adsorption or decomposition of basic drug compounds
ences, reduces column head trimming, and reduces frequency may cause various chromatographic problems including
of MSD source cleaning [3,4]. The instrument configuration is broad, distorted peaks, peak tailing, ghost peaks, and loss of
similar to the configuration shown in Figure 1B in the previ- sensitivity. All of these problems were observed in liners tests
ous setup [4], except no retention gap was used for this appli- using the checkout standard. Peak shape problems usually
cation. Retention time locking (RTL) was used to eliminate occurred for early eluting compounds, such as Phentermine,
recalibration of individual retention times and timed events Methamphetamine, MDA, and MDMA. The late eluting com-
such as SIM groups [5]. pounds, such as Temazepam, can disappear due to the loss of
sensitivity. Figure 2 shows problematic chromatograms
Results and Discussion obtained using similar liners compared to chromatograms
obtained using Ultra Inert liners with wool. As seen in
The purpose of these tests was to evaluate the Ultra Inert Figure 2, with 5 ng on column, other liner deactivations cause
deactivated liners with wool for screening analysis of drugs of chromatographic problems such as broad or distorted peaks
abuse by GC/MS. The Agilent Forensic Toxicology analyzer and significant loss of response. However, the corresponding
checkout standard was used for the evaluation (Table 3). The chromatograms with Agilent Ultra Inert deactivated liners
feasibility of using Ultra Inert liners with wool was deter- show better peak shape and typically higher responses.
mined by chromatographic evaluation, liner to liner repro- Figure 3 shows a full chromatogram of 5 ng checkout stan-
ducibility, and multi-injections repeatability. In parallel, liners dard on column using Agilent Ultra Inert splitless liner with
with wool from multiple sources were tested for comparison. wool by GC/MS. Figure 3 shows that Ultra Inert liners with
Restek Base gooseneck splitless liner with wool
wool provide the best chromatogram for all of analytes tested,
even though there is small peak tailing or broadening
A B observed for certain compounds. Six replicates of Ultra Inert
liners were tested, each providing similar chromatographic
Restek IP SemiVolatile gooseneck splitless liner with wool
performance, indicating excellent liner to liner reproducibility.
A B The satisfactory chromatograms obtained by Ultra Inert liners
demonstrate that the Ultra Inert liner deactivation process
Restek Sky gooseneck splitless liner with wool provides sufficient liner and glass wool inertness to prevent
A drugs of abuse from adsorption and decomposition.
A B

1. Amphetamine, 2. Phentermine, 3. Methamphetamine, 4. Nicotine, 5. MDA,

Agilent Ultra Inert single taper splitless liner with wool (p/n 5190-2293) 6. MDMA, 7. MDEA, 8. Meperidine, 9. Phencyclidine, 10. Methadone, 11. Cocaine,
2 3 4 7 12. SKF-525a, 13. Oxazepam, 14. Codeine, 15. Lorazepam, 16. Diazepam,
6
17. Hydrocodone, 18. Tetrahydrocannabinol, 19. Oxycodone, 20. Temazepam,
1 21. Flunitrazepam, 22. Heroin, 23. Nitrazepam, 24. Clonazepam, 25. Alprazolam,
5
26. Verapamil, 27. Strychnine, 28. Trazodone.

12

10
Agilent Ultra Inert single taper liner with wool (p/n 5190-2293)
21
22 25 9
17
20 24 7 21
19 23
16 26
2 4 11
6 18
3
15

8 14 20 22 27
C B 1 25
19 23
Restek Siltek gooseneck liner with wool 24
28
5
13

2.00 4.00 6.00 8.00 10.00 12.00

Figure 2. Chromatographic problems for drug of abuse compounds shown
on GC/MS SIM chromatograms when using other equivalent
Figure 3. Chromatogram of forensic toxicology analyzer checkout standard
liners and their comparison with chromatograms obtained by
(5 ng checkout standards on column) using Agilent Ultra Inert
Ultra Inert liners with wool. See Table 3 for peaks identification
single taper splitless liner with wool (p/n 5190-2293) by GC/MS.
and Table 1 for instrument conditions. 5 ng checkout standards on
See Table 1 for instrument condition. Satisfactory peaks shape
column. A) Broad or distorted peak, B) ghosting shoulder,
achieved for all of analytes
C) poor sensitivity

4
Liner to liner reproducibility Table 5. Deactivation stability: 50 injections repeatability (%RSD) for
Agilent Ultra Inert deactivated liners with wool (p/n 5190-2293)
To quantitatively evaluate the liner to liner reproducibility, six for all of tested basic drug compounds with 0.5 ng of standard on
Ultra Inert liners from four different lots were tested. column. (n = 3)
5 µg/mL and 500 ng/mL samples spiked with 2 µg/mL IS
RSD (%) over RSD (%) over
were used. Twelve sensitive compounds were selected for
Compound 50 injections Compound 50 injections
evaluation. The Response Factors (RFs) were calculated for
Amphetamine 0.3 Lorazepam 20.9
each concentration level. The average RF values were evalua-
Phentermine 1.1 Diazepam 3.7
tion criteria for the liner to liner reproducibility test. See Methamphetamine 1.5 Hydrocodone 3.7
Table 4. The results show excellent liner to liner performance Nicotine 2.3 Tetrahydrocannabinol 8.5
consistency with less than 7% RSD, except for Temazepam MDA 3.7 Oxycodone 22.2
with 11.7%, across six liners from four different lots. MDMA 2.2 Temazepam 59.9
MDEA 2.0 Flunitrazepam 8.7
Table 4. Liner to Liner Reproducibility: 12 sensitive basic drug compounds
Meperidine 1.9 Heroin 10.7
average RF (5 µg/mL and 500 ng/mL) and RSD values for six
Phencyclidine 15.6 Nitrazepam 11.2
replicates of UI deactivated liners with wool (p/n 5190-2293) *
Methadone 3.4 Clonazepam 12.0
Liner 1 Liner 2 Liner 3 Liner 4 Liner 5 Liner 6 Mean Cocaine 7.8 Alprazolam 13.1
Compounds (Lot 1) (Lot 1) (Lot 1) (Lot 2) (Lot 3) (Lot 4) RF RSD Prodifen 4.4 Verapamil 15.4
Methamphetamine (3) 0.875 0.876 0.882 0.940 0.955 0.904 0.905 3.8 Oxazepam 20.4 Strychnine 11.0
MDMA (6) 0.807 0.789 0.783 0.848 0.874 0.841 0.824 4.4 Codeine 20.5 Trazodone 23.6
Phencyclidine (9) 0.494 0.510 0.494 0.488 0.509 0.521 0.503 2.5
Cocaine (11) 0.636 0.645 0.647 0.637 0.660 0.668 0.649 2.0
As shown in Figure 2, when an inefficient deactivated liner
Oxazepam (13) 0.050 0.055 0.052 0.055 0.062 0.057 0.055 7.6
Codeine (14) 0.096 0.098 0.095 0.090 0.099 0.102 0.097 4.2 was used the response of Temazepam (5 ng on column) can
Oxycodone (19) 0.073 0.071 0.070 0.076 0.082 0.080 0.075 6.5
almost disappear. Compared to other similar liners, Agilent
Temazepam (20) 0.101 0.121 0.115 0.088 0.096 0.104 0.104 11.7 Ultra Inert liner with wool generated highest RF for
Heroin (22) 0.097 0.099 0.096 0.095 0.100 0.102 0.098 2.7 Tempazepam, which is clearly shown in Figure 4. This indi-
Nitrazepam (23) 0.038 0.032 0.037 0.034 0.037 0.036 0.036 6.3 cates that Agilent Ultra Inert liners with wool provide the best
Clonazepam (24)0.035 0.035 0.034 0.032 0.034 0.033 0.034 3.5 inertness compared to competitor’s equivalent liners.
Trazodone (28) 0.061 0.065 0.064 0.058 0.060 0.064 0.062 4.4
Temazepam Response Comparison between UI liners
Peak AreaAnalyte × ConcentrationInternal Standard and other equivalent liners
* RF =
Peak AreaInternal Standard × ConcentrationAnalyte 100

80
Injection repeatability and deactivation stability
Multi-injection repeatability and deactivation stability were
tested by continuously injecting 1 µL of 0.5 µg/mL standard 60
Relative RFs

samples for 50 injections. Data was collected and RF values

were calculated every 10 injections. RSD values were calcu- 40
lated over 50 injections. Table 5 shows the RSD value for all of
the basic drug analytes with 0.5 ng on column.
20
A 0.5 ng on column concentration was used for this repeata-
bility test since low level concentrations show greater devia-
0
tion contributions than high concentration samples. Higher Restek Restek Restek Restek
responses of analytes could hide some deviation impact and UI wool SV wool Sky wool Base wool Siltek wool
generate better repeatability. Twenty-two of 28 analytes have Figure 4. Sensitive compound response (Temazepam) comparison for Ultra
excellent repeatability for 50 injections of standard solution Inert liner with wool (p/n 5190-2293) and other equivalent liners.
with less than 20% RSD. 5 of 28 analytes have relatively high RF calculation was based on the average RF of 0.5 ng and 5 ng
RSD (between 20 – 25%), but still should be acceptable at the standard on column. Ultra Inert liner average RF value was set to
100% and other liners average RF values were scaled.
level of 0.5 ng on column. Temazepam is a very difficult com-
pound and extremely sensitive to the liner inertness.

5
The response of Temazepam decreased with more samples Agilent Ultra Inert liners with wool provide excellent inert-
were injected, thus generated high RSD over injections. This ness for forensic and toxicology screening. The benefits
phenomenon was observed for all of the liners tested, and provided by liners with wool such as homogeneous sample
the response decrease can be even worse for other liners. mixing and evaporation, non-volatile residue trapping, and
When Temazepam is a target analyte and the interested con- column and detector protection, are gained without com-
centration is at ppb level, it is strongly recommend that an promise of chromatography or sensitivity of active analytes.
Ultra Inert liner with wool should be used for no more than Ultra Inert liners with wool are an excellent choice for
30 samples. screening analysis for drugs of abuse.

Real matrix sample analysis

References:
Whole blood extracts prepared for GC/MS analysis were sup-
plied by NMS Labs (Willow Grove, PA). The whole blood was [1] B. Quimby, “Improved Forensic Toxicology Screening
prepared with a single step liquid/liquid extraction into a sol- Using A GC/MS/NPD System with a 725-Compound
vent, evaporated to dryness, and reconstituted in toluene at DRS Database”, Agilent Technologies publication 5989-
1/10th volume. Figure 5 shows the chromatogram of 2 ppm 8582EN.
matrix spiked sample using Agilent Ultra Inert liner with wool
by GC/MS, which is satisfactory for both early eluted com- [2] N. Seyhan and D.C. Ege, “Organic Chemistry”, Health
pounds’ peak shape and late eluted compounds’ sensitivity. and Company, 1984, pp. 124-136.
There are some minor interference peaks from matrix [3] M. Szelewski and B. Quimby, “New Tools for Rapid
showing up. Pesticide Analysis in High Matrix Samples”, Agilent
Technologies publication 5989-1716EN
Conclusion [4] P. Wylie and C. Meng, “A Method for the Trace
Analysis of 175 Pesticides Using the Agilent Triple
Agilent Ultra Inert liners with wool have shown excellent Quardrupole GC/MS/MS”, Agilent Technologies
inertness for the analysis of basic drugs of abuse. Ultra Inert publication 5990-3578EN
liners with wool provide satisfactory chromatography for the
selected popular and difficult basic drug compounds. The [5] V. Giarrocco, B.Quimby, “Retention Time Locking:
liner to liner performance shows excellent reproducibility Concepts and Applications”, Agilent Technologies
with an average of 5% RSD for these active compound RF publication 5966-2469EN.
values. With efficient and robust deactivation of the wool,

A. TIC

2.00 4.00 6.00 8.00 10.00 12.00

12
10

17 B. SIM
9
7
4
11
16 21 26
www.agilent.com/chem
6 15 18
23 8
14 22 25 27
1 20
5 19 23 24 28
13

2.00 4.00 6.00 8.00 10.00 12.00

© Agilent Technologies, Inc., 2011
Figure 5 Chromatogram of forensic toxicology analyzer checkout standard Printed in the USA
(2 ng on column) with whole blood matrix using Agilent Ultra March 25, 2011
Inert single taper splitless liner with wool (p/n 5190-2293) by 5990-7596EN
GC/MS. Refer to Table 1 for instrument condition, and Table 3 for
peaks identification. A) Full scan chromatogram, B) SIM chro-
matogram. Satisfactory peaks shape and response achieved for
all of analytes.
 Detection of Cannabinoids in Oral Fluid Using
Inert Source GC/MS
Application

Forensic

Authors Introduction
Christine Moore, Sumandeep Rana, and Cynthia Coulter Tetrahydrocannabinol (THC) is the active ingredient
Immunalysis Corporation in marijuana, administered via
829 Towne Center Drive smoking. While THC is the main psychoactive
Pomona, CA 91767 ingredient in the marijuana plant, other reports
USA have shown that some of the effects may be in com-
bination with at least one other constituent of the
plant, cannabidiol (CBD). Various cannabinoids
Abstract have been analyzed in plasma, blood, and urine,
but their detection in the more esoteric matrices,
Oral fluid is being considered as an alternative to urine in such as sweat, oral fluid, and hair, has only
many forensic arenas. In general, the concen-tration of recently been addressed.
drugs in oral fluid is much lower than in urine, so
sensitive extraction and analytical procedures are Oral fluid is becoming increasingly popular as a
required. Tetrahydrocannabinol (THC) is the active ingre- specimen for the detection of drugs at the roadside
dient in marijuana. Since it is generally smoked, the and in workplace testing. Several publications have
constituents of the plant material, as well as the active reported the presence of THC in saliva using vari-
ingredient, may be present in oral fluid specimens ous collection devices. However, the presence of
collected for the purposes of drug testing. An ana-lytical other cannabinoids, such as cannabinol (CBN) and
procedure for the simultaneous determination of the cannabidiol (CBD) in the marijuana plant material,
pyrolytic precursor ∆9-tetrahydrocannabinolic acid A and therefore possibly in the oral fluid sample col-
(THCA-A, 2-carboxy-THC), tetrahydrocannabinol (THC), lected, has not been reported previously and may
cannabinol (CBN), and cannabidiol (CBD) in human oral be of importance for screening and confirmatory
fluid specimens using an Agilent 5975 GC/MS with an assays. Further, ∆9-tetrahydrocannabinolic acid A
inert source is presented. The method achieves the (THCA-A, 2-carboxy-THC) is the main pyrolytic
required sensitivity for the detection of tetrahydro- precursor to tetrahydrocannabinol. The decarboxy-
cannabinol (THC), cannabinol (CBN), cannabidiol (CBD), lation of 2-carboxy-THC to the active THC during
and the pyrolytic precursor 2-carboxy-THC in oral fluid smoking converts only approximately 70% of the
specimens taken from a habitual marijuana smoker. precursor to the active form, so the potential pres-
While these drugs have been detected in other matrices, ence of 2-carboxy-THC in oral fluid specimens was
the increasing utility of saliva for drug analysis makes considered. While blood and urine are more com-
development of laboratory procedures necessary and monly used for these test profiles, oral fluid is
timely. increasing in popularity as an alternative matrix
due to its ease of collection, difficulty of adulter- instrument is sufficiently sensitive to meet the
ation, and improving sensitivity of analytical proposed regulations, using only 1 mL of the total
techniques. One of the main issues with the quan- specimen. However, it should be noted that if alter-
titation of drugs in oral fluid is the difficulty of nate collection devices that collect much smaller
collection in terms of specimen volume. Many of volumes of oral fluid are used, then a Deans switch
the currently available devices do not give an indi- microfluidic mechanism may need to be used to
cation of how much oral fluid is collected, thereby achieve the necessary sensitivity.
rendering any quantitative results meaningless
without further manipulation in the laboratory. Standards and Reagents
Further, devices incorporating a pad or material
• Tri-deuterated THC for use as an internal stan-
for the saliva collection do not always indicate how dard as well as unlabeled THC, CBN, and CBD
much of each drug is recovered from the pad
were purchased from Cerilliant (Round Rock,
before analysis, again calling into question any TX). 2-carboxy-THC was purchased from
quantitative result. The drug concentration
Lipomed (Cambridge, MA).
reported is dependent on the collection procedure
used. • Trace N 315 solid phase extraction columns
were purchased from SPEWare (San Pedro,
This work employed Immunalysis Corporation’s CA).
QUANTISAL oral fluid collection device, which col-
lects a known amount of neat oral fluid. The effi- • The derivatizing agent, N,O-Bis (trimethylsilyl)
ciency of recovery of the drugs from the collection trifluoroacetamide + 1% trimethylchlorosilane
pad into the transportation buffer was determined (BSTFA + 1% TMCS), was from Pierce
in order to increase confidence in the quantitative (Rockford, IL).
value. The extracts were analyzed using a standard
Internal Standard Concentration
single quadrupole Agilent GC/MS 6890-5975
instrument, with a limit of quantitation of THC 40 ng/mL
0.5 ng/mL.
Sample Preparation for Chromatographic Analysis
Experimental • 1 mL Quantisal specimen (equivalent to
0.25 mL of oral fluid)
Oral Fluid Collection Devices
• Add internal standard (40 ng/mL)
Quantisal devices for the collection of oral fluid • Add 0.1 M sodium acetate buffer (pH 4.5; 1 mL)
specimens were obtained from Immunalysis
Corporation (Pomona, CA). The devices contain a • Condition SPE columns: methanol (0.5 mL),
collection pad with a volume adequacy indicator, 0.1 M acetic acid (0.1 mL)
which turns blue when one milliliter of oral fluid • Add samples
(± 10%) has been collected. The pad is then placed
into transport buffer (3 mL), allowing a total speci- • Wash columns:
men volume available for analysis of 4 mL (3 mL • Deionized water:0.1 M acetic acid
buffer + 1 mL oral fluid). This is specifically advan- (80:20; 1 mL)
tageous in cases where the specimen is positive for
more than one drug and the volume of specimen • Deionized water:methanol (40:60; 1 mL)
available for analysis may be an issue. The oral • Dry columns under nitrogen (30 psi; 2 min).
fluid concentration is diluted 1:3 when using
Quantisal collection devices, and drug concentra- • Elute: hexane:glacial acetic acid (98:2; 0.8 mL)
tions detected were adjusted accordingly. Since • Evaporate to dryness under nitrogen
4 mL of specimen is available for analysis, the
single quadrupole Agilent GC/MS 6890-5975

2
GC/MS Conditions Derivatization
Instrument: Agilent 6890 GC 5975 MSD; inert
source; 220/240V oven Reconstitute in ethyl acetate (30 µL); add BSTFA
+1% TMCS (20 µL); transfer to autosampler vials;
Detection mode: Electron impact
cap; incubate (60 °C/15 min).
Column: DB-5 MS, 0.25 mm id, 0.25-µm film
thickness, 15-m length
Injection temperature: 250 °C
Results and Discussion
Purge flow: 50 mL/min for 1 min One of the issues associated with oral fluid analy-
Carrier gas: Helium sis is recovery of drug from a collection pad if a
device is used. Extraction efficiency of the collec-
Injection mode: Splitless tion system for these drugs was determined. Six
Injection volume: 2 µL synthetic oral fluid specimens fortified with all the
Mode of operation: Constant flow at 1.5 mL/min cannabinoids at a concentration of 4 ng/mL were
prepared. The collection pad was placed into the
Transfer line: 280 °C
samples until 1 mL had been collected, as
Quadrupole: 150 °C evidenced by the blue volume adequacy indicator
Ion source: 230 °C incorporated into the stem of the collector. The
pad was then transferred to the Quantisal buffer,
Dwell time: 50 ms
capped, and stored overnight to simulate
Oven program: 125 °C for 0.5 min; ramp at 40 °C/min transportation to the laboratory. The following
to 250 °C; hold 1.3 min ramp at day, the pads were removed with a serum separa-
70 °C/min to 300 °C tor, and an aliquot of the specimen was analyzed
Retention times: Deuterated THC: 4.27 min; as described. The amount recovered from the pad
THC 4.28 min; cannabidiol 3.88 min; was compared to an absolute concentration (100%)
cannabinol 4.61 min; 2-c-THC where drug was added to the buffer and left
5.66 min overnight at room temperature without the pad,
then subjected to extraction and analysis.

Ions Monitored THC CBD CBN 2-carboxy-THC

Mean drug 89.2 ± 9.0 71.9 ± 19.1 79.7 ± 7.8 78.2 ± 11.8
Drug Ions monitored recovery (%)
THC Deuterated (d3) 374.3, 389.3;
Unlabeled THC 371.2, 386.2, 303.1 GC/MS Method
CBN 367.3, 382.2, 310.1 The analytical methods were according
CBD 390.1; 301.2 to standard protocols, whereby the limit of
2-carboxy-THC 487.3, 488.2, 489.2 quantitation, linearity range, correlation, and
intra- and inter-day precision were determined
Quantitative ions in bold type
via multiple replicates (n = 6) over a period of
four days.

Analyte LOQ (ng/mL) Linear equation Correlation r2 Ion ratio range (%)
THC 0.5 y = 0.0266x + 0.00273 0.998 386/371:69.7–104.5
303/371:44.0–66.0
CBN 0.5 y = 0.138x + 0.0022 0.999 382/367:7.4–11.2
310/367:5.7–8.5
CBD 1 y = 0.0271x + 0.00178 0.998 301/390:17.1–25.7
2-carboxy-THC 1 y = 0.0571x + 0.0195 0.998 488/487:31.7–47.5
489/487:11.0–16.6

3
 THC CV (%) CBN CV (%) CBD CV (%) 2-c-THC CV (%)
Concentration Intra Inter Intra Inter Intra Inter Intra Inter
1 ng/mL 0 4.8 5.26 15.3 7.07 6.08 5.73 15.2
2 ng/mL 0 2.53 2.21 2.41 2.82 3.12 10.3 8.3
4 ng/mL 1.39 1.46 5.96 4.20 4.08 4.52 7.03 8.5
8 ng/mL 0.68 1.77 4.66 5.58 1.66 6.84 2.99 2.25

Precision: Inter-day (n = 4) and intra-day (n = 6) subject smoked (5 min), then at intervals of

precision for the determination of cannabinoids in 30 minutes and 1, 2, 12, 24, 36, and 48 hours after
oral fluid. smoking. Parent THC was detectable at concentra-
tions well above over 2000 ng/mL in the
Specificity: Commonly encountered drugs were 5-minute and 30-minute samples, apparently due
extracted and analyzed at high concentrations and to excessive oral cavity contamination by THC. The
found not to interfere with the assays. parent drug was detected for 24 hours, and 2-car-
boxy-THC was identified for up to 16 hours after
Authentic Specimens
intake. Cannabidiol was detected only in the speci-
The method was applied to specimens taken from mens from 5 minutes and 30 minutes after smok-
an authentic user. The subject willingly consented ing and at a concentration of 5 ng/mL. Cannabinol
to sample collection; he had been a marijuana was measurable for only 2 hours (Figure 1).
smoker for over 20 years. For the purpose of this
An extracted ion chromatogram of the sample
study, he remained marijuana free for five days
collected 1 hour after smoking is presented in
before smoking. The initial specimen was negative
Figure 2. The extracted ions for cannabidiol were
for the four cannabinoids. Samples were collected
not included since there was no CBD present in
almost immediately after the
the specimen.

Figure 1. Cannabinoids in oral fluid following marijuana smoking.

4
Conclusions
The procedure described is suitable for the
routine detection and confirmation of THC, CBN,
and 2-carboxy-THC in oral fluid using the
Quantisal oral fluid collection device and an
Agilent single quadrupole GC/MSD.

THC 104 ng/mL

2-carboxy-THC 31 ng/mL

CBN 4.1 ng/mL

Figure 2. Oral fluid specimen collected 1 hour after marijuana smoking.