Journal of Analytical Toxicology, Vol. 33, November/December 2009
Identification and Quantitation of Amphetamines,
Cocaine, Opiates, and Phencyclidine in Oral Fluid by
Liquid Chromatography–Tandem Mass Spectrometry
Dean Fritch1,*, Kristen Blum1, Sheena Nonnemacher1, Brenda J. Haggerty2, Matthew P. Sullivan3, and
Edward J. Cone4
1OraSure Technologies, Research and Development, Bethlehem, Pennsylvania 18015; 2DeSales University, Center Valley,
Pennsylvania 18034; 3Kutztown University, Kutztown, Pennsylvania 19530; and 4Johns Hopkins School of Medicine,
Department of Psychiatry and Behavioral Sciences, Baltimore, Maryland 21205
Abstract
Analytical methods for measuring multiple licit and illicit drugs
and metabolites in oral fluid require high sensitivity, specificity,
and accuracy. With the limited volume available for testing,
comprehensive methodology is needed for simultaneous
measurement of multiple analytes in a single aliquot. This report
describes the validation of a semi-automated method for the
simultaneous extraction, identification, and quantitation of
21 analytes in a single oral fluid aliquot. The target
compounds included are amphetamine, methamphetamine,
3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine,
pseudoephedrine, cocaine, benzoylecgonine, codeine, norcodeine,
6-acetylcodeine, morphine, 6-acetylmorphine, hydrocodone,
norhydrocodone, dihydrocodeine, hydromorphone, oxycodone,
noroxycodone, oxymorphone, and phencyclidine. Oral fluid
specimens were collected with the Intercept® device and extracted
by solid-phase extraction (SPE). Drug recovery from the Intercept
device averaged 84.3%, and SPE extraction efficiency averaged
91.2% for the 21 analytes. Drug analysis was performed by liquid
chromatography–tandem mass spectrometry in the positive
electrospray mode using ratios of qualifying product ions within
±25% of calibration standards. Matrix ion suppression ranged from
–57 to 8%. The limit of quantitation ranged from 0.4 to 5 ng/mL
using 0.2 mL of diluted oral fluid sample. Application of the method
was demonstrated by testing oral fluid specimens from drug abuse
treatment patients. Thirty-nine patients tested positive for various
combinations of licit and illicit drugs and metabolites. In conclusion,
this validated method is suitable for simultaneous measurement of
21 licit and illicit drugs and metabolites in oral fluid.
Introduction
The convenience of oral fluid collection under observed conditions provides a significant advantage over urine in drug
* Author to whom correspondence should be addressed: Dean Fritch, 150 Webster Street,
Bethlehem, PA 18015. E-mail: [email protected].
testing and monitoring programs (1,2). Without observed
collections, drug abusers are frequently motivated to “tamper”
with their urine specimens by various means such as substitution with clean specimens or fluids resembling urine and by
addition of various chemicals that are designed to either destroy drugs present or interfere with their measurement (1).
Drug users also have learned that prior to collection, consumption of excess fluids may successfully dilute drug concentrations in urine to undetectable levels (2). Although the
prevalence of tampering and dilution attempts in various urine
testing programs is difficult to establish, it is clear that such
techniques are broadly understood among illicit drug users and
a variety of commercial products and instructions are available
to assist drug users to avoid detection.
The growing popularity of oral fluid testing over the last
two decades has been made possible through improvements in
screening and confirmation technologies. Oral fluid specimens
typically contain drugs and metabolites at considerably lower
concentrations than in urine and are limited in volume generally to 1 mL or less. Thresholds for oral fluid are at least 10fold lower than urine, and methods must be validated to enable
reliable detection of recent drug use for the numerous classes
of abused drugs (3,4). These analytical challenges have been
met by use of sensitive screening methods such as ELISA and
by confirmation methods such as gas chromatography (GC)
and liquid chromatography (LC) coupled with tandem mass
spectrometry (MS) (5).
The requirements for accurate confirmation of multiple
drugs and metabolites at low nanogram-per-milliliter concentrations in limited volumes of oral fluid have been addressed by
use of various MS techniques. Several reviews have appeared
that describe inherent advantages and weaknesses of the technologies for analysis of oral fluid (4,6–8). Early confirmation
methods for oral fluid analysis for drugs of abuse were generally based on GC–MS and tandem GC–MS analysis (5,9–11).
More recently, LC–MS–MS has emerged as a preferred method
of analysis obviating the need for the production of thermally
stable volatile analytes required by GC–MS methods. Enhanced
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
569
Journal of Analytical Toxicology, Vol. 33, November/December 2009
sensitivity, simpler sample preparation, no required derivatization, and short analysis are among the advantages. Concheiro
et al. (12) reported a method for the determination of 23 illicit
and medicinal drugs and their metabolites of interest in oral
fluid specimens from drugged drivers by LC–MS–MS, although
this method did not include semisynthetic opiates such as hydrocodone and oxycodone or phencyclidine. A number of other
comprehensive methods for the analysis of illicit and medicinal
drugs and metabolites by LC–MS–MS have been recently reported (13–16), but these methods also did not include hydrocodone or oxycodone. Kala et al. (3) reported an LC–MS–MS
method for the determination of amphetamines, opiates, phencyclidine, cocaine, and benzoylecgonine in oral fluid. Opiates
detected by this method were codeine, morphine, hydrocodone,
hydromorphone, oxycodone, and oxymorphone and did not
include 6-acetylmorphine or dihydrocodeine.
The present study reports a single extraction procedure for
simultaneous confirmation testing of 21 licit and illicit drugs
and their metabolites in oral fluid by LC–MS–MS. The drugs,
with the exception of ∆9-tetrahydrocannabinol (THC), include
the standard panel of basic drugs proposed by the U.S.
Department of Health and Human Services (17) for federal
workplace drug testing. Additional drugs and metabolites
(pseudoephedrine, norcodeine, dihydrocodeine, 6-acetylcodeine, hydrocodone, norhydrocodone, hydromorphone,
oxycodone, noroxycodone, and oxymorphone) were included in
the method.
Materials and Methods
Materials
Ammonium hydroxide, isopropanol, hydrochloric acid,
HPLC-grade water, and methanol were purchased from VWR
(West Chester, PA). Methylene chloride was purchased from JT
Baker (Phillipsburg, NJ). Acetonitrile, ammonium acetate, and
formic acid were purchased from Sigma Aldrich (St. Louis,
MO). Phosphoric acid was purchased from EMD (Gibbstown,
NJ). Oral fluid diluent was supplied by OraSure Technologies
(Bethlehem, PA). Oral fluid diluent is a synthetic saliva matrix
designed to simulate the composition of dilute oral fluid in the
collector tube.
One-milliliter ampoules in methanol of either 100 µg/mL or
1 mg/mL of clonazepam, cotinine, dextromethorphan, diphenhydramine, doxepin, gemfibrozil, hydroxyalprazolam,
imipramine, lidocaine, norchlordiazepoxide, nortriptyline, pentobarbital, phenobarbital, THC, theophyline, cocaethylene,
morphine-3β-D-glucuronide, nalorphine, phentermine,
phenylpropanolamine, codeine, morphine, oxycodone, hydrocodone, hydromorphone, norhydrocodone, oxymorphone,
dihydrocodeine, norcodeine, methadone, noroxycodone, amphetamine, methamphetamine, pseudoephedrine, 3,4-methylenedioxyethylamphetamine (MDEA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine
(MDA), benzoylecgonine, phencyclidine, 6-acetylmorphined6, hydromorphone-d6, MDMA-d5, benzoylecgonine-d3, MDEAd5, morphine-d3, methamphetamine-d11, amphetamine-d11,
570
oxycodone-d6, phencyclidine-d5, hydrocodone-d6, MDA-d5, oxymorphone-d3, dihydrocodeine-d6, norhydrocodone-d3, noroxycodone-d3, and pseudoephedrine-d3 were purchased from Cerilliant (Round Rock, TX). One-milliliter ampoules in
acetonitrile of either 100 µg/mL or 1 mg/mL of cocaine-d3, 6acetylmorphine, 6-acetylcodeine, and cocaine were also purchased from Cerilliant. 6-Acetylcodeine-d6 was purchased from
Lipomed (Cambridge, MA). Ibuprofen, medazapam, naproxen,
norchlordiazepoxide, and procaine were purchased from Alltech (Nicholasville, KY). Loperamide, penicillin, quinidine,
quinine, and tolmetin were purchased from Sigma Aldrich.
Extraction solutions consisting of 50:50 methanol/water
(v/v) solution and 78:20:2 methylene chloride/isopropanol/
ammonium hydroxide (v/v/v) solutions were prepared daily. A
solution of 50 mM phosphoric acid was prepared using HPLCgrade phosphoric acid (85%) in deionized water. 1% HCl (v/v)
was prepared in HPLC-grade methanol. HPLC-grade methanol
(10%) in HPLC-grade water (v/v) was prepared fresh for
each run.
Specimens
Collection of oral fluid with the Intercept collection device
(OraSure Technologies) was performed according to manufacturer’s instructions. Briefly, the Intercept collection device
consists of a treated absorbent cotton fiber pad affixed to a
plastic stick and is packaged with a preservative solution (0.8
mL) in a plastic container. The Intercept collection device collects oral fluid with expected mean volumes of 0.4 mL (14). Because of the possible range of oral fluid volumes collected,
concentrations are reported in this study without correction
Table I. Cutoff Concentrations for the 21 Drugs and
Metabolites and Internal Standard Compositions
Analyte
Cutoff
Concentration
(ng/mL)
Amphetamine
Methamphetamine
MDMA
MDA
MDEA
Pseudoephedrine
Cocaine
Benzoylecgonine
Codeine
Norcodeine
6-Acetylcodeine
Morphine
6-Acetylmorphine
Hydrocodone
Norhydrocodone
Dihydrocodeine
Hydromorphone
Oxycodone
Noroxycodone
Oxymorphone
Phencyclidine
12.5
12.5
12.5
12.5
12.5
12.5
2
2
10
10
5
10
1
10
10
10
10
10
10
10
1
Internal
Standard
Amphetamine-d11
Methamphetamine-d11
MDMA-d5
MDA-d5
MDEA-d5
Pseudoephedrine-d3
Cocaine-d3
Benzoylecgonine-d3
Dihydrocodeine-d6
Dihydrocodeine-d6
6-Acetylcodeine-d3
Morphine-d3
6-Acetylmorphine-d6
Hydrocodone-d6
Norhydrocodone-d3
Dihydrocodeine-d6
Hydromorphone-d6
Oxycodone-d6
Noroxycodone-d3
Oxymorphone-d3
Phencyclidine-d5
Concentration
(ng/mL)
50
500
50
50
50
12
8
8
60
60
60
120
8
50
120
60
25
250
24
60
10
Journal of Analytical Toxicology, Vol. 33, November/December 2009
for dilution. Typically, approximately 0.4 mL of oral fluid is colmL of diluted sample at 2 mL/min. The columns were rinsed
lected on the collection device pad which results in a 1:3 dilusequentially with deionized water, 50 mM phosphoric acid,
tion when placed in the preservative solution.
50:50 methanol/DI water, methanol, and methylene chloride,
Authentic oral fluid specimens were collected under a proall at a flow rate of 12 mL/min. The columns were dried for
tocol approved by Institutional Review Board (IRB) review
2 min and eluted with 1 mL methylene chloride/
from drug treatment subjects at the Addiction Research Testing
isopropanol/ammonium hydroxide (78:20:2) at 0.5 mL/min.
Center, Division of Medical Services, Research and Information
After elution, 100 µL of 1% hydrochloric acid in HPLC-grade
Technology (New York, NY). Two oral fluid specimens were
methanol was added to each tube to prevent the loss of amcollected simultaneously (one on each side of the mouth). All
phetamines and then dried under nitrogen at 5–15 psi. The exspecimens were coded and contained no confidential informatracts were reconstituted with 200 µL of 10% HPLC-grade
tion about participants. Upon completion of collection, specimethanol in HPLC-grade water and transferred to autosampler
mens were shipped to OraSure Technologies for processing.
vials with inserts and capped for analysis. Before starting
The specimens were centrifuged at 1500 × g
for 10 min to recover the diluted oral fluid
Table II. Multiple Reaction Monitoring (MRM) Transitions and Conditions for
specimen in 16.8 × 67-mm Sarstedt
Drugs, Metabolites, and Their Deuterated Analogues
(Numbrecht, Germany) polypropylene
tubes. Each pair of simultaneously colPrecursor
Product Ions:
Retention
Declustering Collision
lected specimens was processed by comIon
Quant, Qual
Time
Potential
Energy
bining the specimens in a single container
Analyte
(m/z)
(m/z)
(min)
(v)
(v)
with mixing to ensure adequate volume
for testing. From this point on, the specAmphetamine
136.1
91.0, 118.9
3.12
36
23, 13
imens were maintained at 2–8°C.
Amphetamine-d11
147.0
98.0
3.15
36
23
Negative specimens were collected
Methamphetamine
150.1
91.0, 119.1
3.65
31
27, 15
using the Intercept collection device from
Methamphetamine-d11
161.0
98.0
3.68
31
27
five volunteers to determine percent reMDMA
194.0
163.0, 105.0
3.99
35
17, 33
covery from the collector, SPE recovery,
MDMA-d5
199.0
165.0
4.03
35
17
MDA
180.0
163.0, 105.0
3.46
27
14, 30
and to test for matrix effects during LC–
MDA-d5
185.0
168.0
3.49
27
14
MS–MS analysis.
Specimen preparation
Specimens were prepared for extraction
by addition of 3.8 mL of 50 mM phosphoric acid to a 13 × 100-mm glass
culture tube. Internal standards and a
200-µL aliquot of sample were added to
each corresponding tube. To blanks, 200
µL of oral fluid diluent was added in place
of the specimen. To prepare the calibration curve, calibration standards in
methanol corresponding in concentrations to 1-, 2-, 8-, and 16-times cutoff concentrations of each analyte were added
along with 200 µL of oral fluid diluent.
Table I lists the cutoff concentrations for
each analyte, internal standards, and their
respective concentrations. An unextracted
sample tube was prepared by adding
internal standard to elution solvent in a
12 × 75-mm glass culture tube and set
aside until after extraction. The tubes
were then vortex mixed.
Specimen solutions were then extracted
on the Caliper Life Sciences (Hopkinton,
MA) RapidTrace SPE workstation using
Varian SPEC DAU 30-mg SPE columns
(Lake Forrest, CA). The SPE columns
were conditioned with 0.5 mL of ACSgrade methanol, then loaded with the 4
MDEA
MDEA-d5
Pseudoephedrine
Pseudoephedrine-d3
Cocaine
Cocaine-d3
Benzoylecgonine
Benzoylecgonine-d3
Codeine
Norcodeine
6-Acetylcodeine
6-Acetylcodeine-d3
Morphine
Morphine-d3
6-Acetylmorphine
6-Acetylmorphine-d6
Hydrocodone
Hydrocodone-d6
Norhydrocodone
Norhydrocodone-d3
Dihydrocodeine
Dihydrocodeine-d6
Hydromorphone
Hydromorphone-d6
Oxycodone
Oxycodone-d6
Noroxycodone
Noroxycodone-d3
Oxymorphone
Oxymorphone-d3
Phencyclidine
Phencyclidine-d5
208.0
213.0
166.0
169.0
304.0
307.0
290.1
293.0
300.2
286.0
342.0
345.0
286.2
289.2
328.2
334.2
300.2
306.2
286.0
289.0
302.2
308.3
286.2
292.0
316.2
322.2
302.2
305.0
302.2
305.0
244.3
249.0
163.0, 105.0
163.0
148.0, 133.0
151.0
182.0, 150.0
185.0
168.2, 77.1
171.0
152.3, 115.2
268.0, 165.0
225.0, 165.0
225.0
152.1, 128.1
152.2
165.1, 211.2
165.1
199.2, 128.2
202.2
199.0, 171.0
202.0
199.0, 171.0
202.0
185.2, 157.1
185.2
241.3, 256.3
244.3
284.0, 227.0
287.0
284.0, 227.0
287.0
86.0, 90.9
96.0
4.82
4.84
2.65
2.66
6.44
6.49
2.91
2.92
2.74
2.57
5.43
5.45
1.4
1.4
3.29
3.31
3.53
3.55
3.27
3.29
2.57
2.59
2.11
2.12
3.22
3.35
2.91
2.92
1.74
1.74
7.31
7.34
35
35
30
29
45
45
51
51
56
50
60
60
56
56
51
56
56
56
56
60
65
67
51
56
41
41
49
48
50
47
26
26
17, 35
17
15, 28
16
35, 35
35
25, 73
25
83, 91
25, 60
35, 67
36
75, 75
69
53, 35
51
41, 77
41
37, 49
37
43, 57
44
41, 53
43
37, 35
41
22, 35
22
25, 37
26
47, 47
47
571
Journal of Analytical Toxicology, Vol. 33, November/December 2009
another extraction series, the RapidTrace SPE Workstation
was rinsed with methanol and water to minimize carryover.
Chromatographic conditions
Chromatography was performed with an Agilent (Santa
Clara, CA) model 1100 HPLC system using a Restek (State
College, PA) Allure PFP Propyl LC column (50 × 2.1 mm, 5
µm) with gradient elution. The mobile phases were 0.1%
formic acid, 2 mM ammonium acetate, and 2% acetonitrile in
HPLC-grade water (A) and 0.1% formic acid, 2 mM ammonium
acetate, and 10% HPLC-grade water in acetonitrile (B). The
column temperature was maintained at 30°C. The injection
volume was 20 µL, and the flow rate through the column was
800 μL/min. The mobile phase gradient was as follows: autosampler load 1 min, equilibration at 90% A for 0.5 min,
ramp to 65% A over 1.5 min, hold at 65% A for 2 min, ramp to
5% A in 1 min, ramp to 2% A in 1 min, hold at 2% A for 2 min,
then rapidly ramp back to 90% A, and re-equilibrate for 3 min
for a total time of 12 min per sample. The vial wash option was
used for the Agilent 1100 HPLC autosampler to minimize carryover by rinsing of the autosampler needle in a vial containing 50:50 methanol/water.
MS
Detection was performed on an API 3200 tandem MS
operating in positive electrospray mode (ESI) (MDS SCIEX,
Toronto, ON, Canada). The optimum conditions were as follows: curtain gas, 50 psi; collision-activated dissociation, 5 psi;
heated nebulizer temperature, 600°C; nebulizing gas, 50 psi;
and heater gas, 60 psi.
In order to establish the appropriate multiple reaction monitoring (MRM) conditions for individual compounds, solutions
of standards (Table I) in methanol/water (50:50, v/v) were infused into the MS, and the declustering potential (DP) and
collision energy (CE) were optimized for the different ions.
Data acquisition, peak integration, and calculation were interfaced to a computer workstation running the Analyst 1.5
software. The precursor ions, the corresponding product ions,
retention times, and DP and CE for the drugs, metabolites, and
their deuterated analogues are listed in Table II.
Each analysis required the ratio between the quantitation
and qualification product ion to be within ±25% of that established by the average response of the four calibration standards used each run. This ion ratio as well as a retention time
within ±2% of the average of the four calibration standards
were required to meet criterion for a positive result for each
analyte.
Method Validation
Linearity and interassay precision
Quantitation was performed by integration of the area under
the specific MRM chromatogram peak in reference to the integrated area of the corresponding deuterated analogue.
Freshly prepared oral fluid calibrators were generated by
spiking oral fluid diluent with methanolic standards at con572
Figure 1. Chromatograms of an extracted calibrator sample spiked at
cutoff concentrations for the 21 drugs and metabolites.
Journal of Analytical Toxicology, Vol. 33, November/December 2009
Table III. Interassay Precision of Cutoff Controls (±25%) and Signal/Noise (S/N) of Quantitative and Qualifier Ions for
Cutoff Calibrator Samples*
Analyte
Amphetamine
Methamphetamine
MDMA
MDA
MDEA
Pseudoephedrine
Cocaine
Benzoylecgonine
Codeine
Norcodeine
6-Acetylcodeine
Morphine
6-Acetylmorphine
Hydrocodone
Norhydrocodone
Dihydrocodeine
Hydromorphone
Oxycodone
Noroxycodone
Oxymorphone
Phencyclidine
Low Control
(–25%)
ng/mL
Interassay
Precision
(% CV)
%
Accuracy
High Control
(+25%)
ng/mL
Interassay
Precision
(% CV)
%
Accuracy
S/N,
Quant/Qual
Ions†
9.4
9.4
9.4
9.4
9.4
9.4
1.5
1.5
7.5
7.5
3.8
7.5
0.8
7.5
7.5
7.5
7.5
7.5
7.5
7.5
0.8
9.0
6.4
4.4
8.4
4.6
5.8
11.2
19.1
16.4
14.6
12.4
13.2
10.5
11.9
7.8
9.1
15.7
16.2
9.5
12.5
16.5
118.1
115
109.7
109.1
112.7
114.1
105.7
124.8
112
101.5
115.5
107.7
104.8
108.5
113.7
116.3
107.6
105.4
108.3
112.9
115.5
15.6
15.6
15.6
15.6
15.6
15.6
2.5
2.5
12.5
12.5
6.3
12.5
1.3
12.5
12.5
12.5
12.5
12.5
12.5
12.5
1.3
10.5
4.7
5.7
8.9
5.0
5.1
9.7
6.6
7.5
13.9
7.1
11.1
13.2
6.9
7.6
10.1
8.5
18.7
13.8
10.2
9.5
113.6
113.5
109.6
118.3
111.4
113.3
98.2
113.6
112
100.2
110
109.2
109.7
109.4
109.5
111.2
108.0
101.8
109.8
105.4
106.5
125 / 337
128 / 144
157 / 190
302 / 251
1150 / 1090
200 / 248
1010 / 244
292 / 286
301 / 219
89.8 / 77.2
1380 / 174
287 / 225
202 / 105
354 / 287
174 / 145
123 / 258
131 / 318
211 / 84.5
171 / 84.7
434 / 192
131 / 198
* The signal-to-noise ratio was measured by the LC–MS operating system software (Analyst 1.5).
† Signal-to-noise ratios for the quantitative and qualifier ions determined with cutoff calibrator samples.
centrations equaling 1-, 2-, 8-, and 16times cutoff concentrations. Linearity was
calculated by plotting peak-area ratios
using linear regression and forcing zero,
using 1/x weighting with Analyst version
1.5 software from Applied Biosysytems.
Linear response and LC–MS–MS carryover were assessed by running spiked oral
fluid diluent samples with methanolic
standards from 32-fold to 512-fold cutoff
concentrations with extracted blanks between each of these samples. The assay
was considered linear if the quantitative
response was within 20% of the target
range for the compound. Carryover was
noted when the extracted blank after the
standard was greater than the concentration of the limit of quantitation (LOQ) for
that analyte. Interassay precision was determined by running the low and high
controls (±25%) and elevated controls in
10 different runs. These controls were
prepared by spiking oral fluid diluent with
methanolic standards at the start of the
validation process and storing these
spiked controls at 2–8°C for the duration
of the validation, which was approximately 3 months.
Table IV. Interassay Precision and Accuracy of Quality Control Samples Prepared
at LOD and LOQ Concentrations
Analyte
Amphetamine
Methamphetamine
MDMA
MDA
MDEA
Pseudoephedrine
Cocaine
Benzoylecgonine
Codeine
Norcodeine
6-Acetylcodeine
Morphine
6-Acetylmorphine
Hydrocodone
Norhydrocodone
Dihydrocodeine
Hydromorphone
Oxycodone
Noroxycodone
Oxymorphone
Phencyclidine
Target
LOD
(ng/mL)
2.5
2.5
2.5
2.5
2.5
2.5
0.4
0.4
2
2
1
2
0.4
2
2
2
2
2
2
2
0.2
Interassay
Precision Accuracy
(% CV) (% of Target)
4.4
13.0
2.7
9.6
4.9
12.6
18.4
29.4
15.5
5.4
13.1
19.2
15.5
15.7
0.7
9.6
10.9
17.4
15.9
19.4
5.9
109.7
107.1
105.0
103.8
100.4
110.4
117.3
100.4
112.0
85.3
102.8
97.6
101.4
116.3
86.6
119.8
113.5
104.5
89.7
104.1
120.0
Target
LOQ
(ng/mL)
5
5
5
5
5
5
0.8
0.8
4
4
2
4
1
4
4
4
4
4
4
4
0.4
Interassay
Precision Accuracy
(% CV) (% of Target)
13.6
5.2
3.3
5.5
3.3
3.0
6.7
6.5
6.9
10.5
2.7
13.2
5.2
9.8
5.2
14.0
1.2
15.7
5.2
17.8
13.6
103.7
113.3
106.7
111.8
107.2
118.1
102.3
106.1
97.0
78.6
97.9
104.9
115.8
98.6
104.5
100.8
118.4
109.6
95.3
105.7
116.2
573
Journal of Analytical Toxicology, Vol. 33, November/December 2009
Limit of detection (LOD) and LOQ
Table V. Interassay Precision and Accuracy of Control Samples Prepared at
Elevated Concentrations, Upper Limit of Linearity (ULOL), and Carryover Limits
Analyte
Amphetamine
Methamphetamine
MDMA
MDA
MDEA
Pseudoephedrine
Cocaine
Benzoylecgonine
Codeine
Norcodeine
6-Acetylcodeine
Morphine
6-Acetylmorphine
Hydrocodone
Norhydrocodone
Dihydrocodeine
Hydromorphone
Oxycodone
Noroxycodone
Oxymorphone
Phencyclidine
Elevated
Control
(ng/mL)
Interassay
Precision
(% CV)
%
Accuracy
ULOL
(ng/mL)
Carryover
Limit
(ng/mL)
2500
2500
2500
2500
2500
2500
400
400
2000
300
200
2000
30
2000
2000
2000
2000
2000
400
2000
100
12
9.3
5.9
7.9
5.0
5.5
12.6
16.4
19.2
17
6.2
8.2
9.2
10.4
6.9
9.3
12.5
9.0
16.1
11.3
7.7
104.5
90.4
96.1
97.6
99.4
97.8
87.4
112.5
112.7
119.3
105.1
108.8
99.5
107.9
111
100.5
108.5
111.9
115.7
88.7
106.4
6400
1600
3200
6400
6400
6400
1000
1000
5000
5000
1000
5000
30
5000
5000
5000
5000
5000
2000
1200
250
6400
6400
6400
6400
3000
6400
250
1000
5000
5000
1000
5000
500
5000
5000
5000
5000
5000
2000
5000
100
Table VI. Extraction Recovery (%) and Matrix Effect
Analyte
Amphetamine
Methamphetamine
MDMA
MDA
MDEA
Pseudoephedrine
Cocaine
Benzoylecgonine
Codeine
Norcodeine
6-Acetylcodeine
Morphine
6-Acetylmorphine
Hydrocodone
Norhydrocodone
Dihydrocodeine
Hydromorphone
Oxycodone
Noroxycodone
Oxymorphone
Phencyclidine
574
% Recovery
from Intercept
Collection
Device
% SPE
Extraction
Recovery
% Matrix
Effect
84.2
86.3
84.7
83.5
85.8
88.9
73.6
96.3
99.2
82.6
75.0
83.2
75.9
86.2
86.2
84.3
73.3
85.2
93.4
73.9
87.7
84.8
86.0
86.2
85.0
88.7
84.6
87.7
92.3
91.0
92.4
102.1
91.2
94.8
91.9
84.0
90.6
90.7
91.5
98.2
90.2
111.8
–21.9
–26.1
–23.3
–13.1
–26.7
–27.8
–27.1
–25.3
–6.1
–9.0
–25.9
–6.9
1.8
2.5
–21.7
–10.6
7.9
–9.8
–18.2
5.3
–57.2
The LOD and the LOQ were administratively defined at 20% and 40% of cutoff
concentrations, respectively. The LOD
and LOQ were tested in triplicate over
three separate runs by spiking oral fluid
diluent with methanolic standards to
achieve these concentrations. The average
percent accuracy and percent CV of the
LOD and LOQ controls were calculated
across three runs. The LOD and LOQ both
required the ion ratio to be present within
the ±25% range that was established for
each analyte as well as the retention time
to be within ±2% of the average of the
retention time of the four calibration
standards.
Recovery and matrix effects
To test for recovery and matrix effects,
negative specimens were collected from
five drug-free volunteers using the Intercept collection device. The specimens
were centrifuged at 1500 × g for 10 min.
Oral fluid specimens with final concentrations of twofold cutoff concentration
(PRE) and negative oral fluid (POST)
specimens were prepared from the five
volunteer collections. The negative
specimens were spiked to a twofold
cutoff concentration after extraction and before being
dried under nitrogen. Five unextracted samples were also
prepared with a twofold cutoff concentration. The percent
recovery was calculated with the equation PRE × 100. The
––—–
POST
percent matrix effect was calculated with the equation
POST
__________
– 1 × 100. These equations were based on the
Unextracted
study by Chambers et al. (18).
(
)
Collection device recovery
For the study of drug recovery from the Intercept collection device, oral fluid specimens were collected from five
volunteers by expectoration into plastic tubes. Half of the
specimen from each volunteer was spiked to a 12-fold cutoff
concentration with each analyte. For each specimen, 400 μL
of the spiked oral fluid was added to an Intercept pad and
400 µL of matching negative oral fluid was added to a
second Intercept pad. The pads were stored overnight at
room temperature in 0.8 mL of preservative in the Intercept
collection device. The next day, the devices were centrifuged
at 1500 × g for 10 min. The matching negative Intercept
specimens were then spiked at fourfold cutoff concentrations to account for the threefold dilution of the spiked oral
fluid specimens. The specimens were analyzed, and the
percent recovery from the device was determined for each
analyte.
Journal of Analytical Toxicology, Vol. 33, November/December 2009
Selectivity
To determine selectivity, 10,000 ng/mL of clonazepam, cotinine, dextromethorphan, diphenhydramine, doxepin, gemfibrozil, hydroxyalprazolam, ibuprofen, imipramine, lidocaine,
loperamide, medazapam, naproxen, norchlordiazepoxide, nortriptyline, penicillin, pentobarbital, phenobarbital, procaine,
THC, quinidine, quinine, theophyline, tolmetin, cocaethylene,
morphine-3β-D-glucuronide, nalorphine, phentermine, and
phenylpropanolamine were added to a series of +25% controls.
The controls were then analyzed and the results were evaluated
for deviations in concentration greater than 20% of target concentration as well as ion ratios exceeding acceptable ranges.
Interferences due to common foods and beverages
A series of products were evaluated to test for possible interferences from common foods and beverages that may be present in residual amounts in the oral cavity. Orange juice, cranberry juice, antiseptic mouthwash, cough syrup, cola, coffee,
tea, baking soda, toothpaste, sugar, and hydrogen peroxide
were added directly to +25% control samples to determine if
there was any interference with identification (ion ratio effects) or quantitation. The solid materials were prepared in
water at a concentration of 100 mg/mL. Twenty microliters of
each solution was then added to 200 µL of +25% controls.
The controls were then evaluated for deviations greater than
20% of target concentration as well as ion ratios exceeding
acceptable ranges.
Results and Discussion
Calibrators, controls, and stability
Calibration curves were prepared daily for each analytical
batch. The calibrator samples contained all analytes over the
range of 1- to 16-times that of the cutoff concentrations. These
calibrators generated the quantitative data as well as defined
the ion ratio (±25%) and retention time (±2%) acceptance
criteria. Low (–25%), high (+25%), and elevated quality control
samples were included in each batch to determine batch acceptability.
The ±25% as well as the elevated quality control samples
were stored at 2–8°C, were analyzed periodically over the duration of the study (3 months). There was no significant change
in the control concentration for any of the analytes during
this period of time.
Method validation
The method was validated for precision, accuracy, sensitivity, specificity, linearity, and recovery in compliance with international guidance (19). A representative chromatogram of
an extracted calibrator sample containing the 21 analytes at
cutoff concentrations is shown in Figure 1. Analysis of extracts of individual analytes (one analyte and its deuterated analogue) showed no interferences in other MRM channels. The
cutoff concentrations utilized in this method (Table I), with the
exception of the amphetamines and the synthetic opiates and
their metabolites, when adjusted for buffer dilution are con-
sistent with the cutoff concentrations reported by Cone et al.
(11) and are similar, with the exception of phencyclidine, to the
confirmatory test cutoff concentrations proposed by SAMHSA
(17). Cutoff concentrations for the synthetic opiates and their
metabolites were established at values equal to morphine and
codeine (10 ng/mL), except for 6-acetylmorphine (1 ng/mL)
and 6-acetylcodeine (5 ng/mL).
Precision and accuracy were determined across a range of
concentrations. Data were collected over 10 separate runs for
each analyte. The interassay precision for the controls at ±25%
of the cutoff concentrations ranged from 4.6 to 19.1% (Table
III). Accuracy ranged from 98.2 to 124.8%. The signal-to-noise
(S/N) ratio of the quantifying ion and the qualifying ion (cutoff
control sample) for each analyte is also listed in Table III. The
signal to noise was measured by the LC–MS operating system
software (Analyst 1.5). The lowest S/N for any of the ions at the
cutoff concentration was the qualification ion for norcodeine
which was 77 at 10 ng/mL. The highest S/N was the 6-acetylcodeine quantitation ion which was 1380 at 5 ng/mL. However,
its qualification ion had a S/N of 174.
Precision and accuracy of LOD and LOQ control samples are
listed in Table IV. The LOD and LOQ concentrations were administratively established at 0.2 and 0.4 times the cutoff concentrations with the exception of 6-acetylmorphine. For 6acetylmorphine, the LOQ and the cutoff were both 1 ng/mL
and the LOD was 0.4 ng/mL. CVs for the LOD samples ranged
from 0.7 to 29.4%, and accuracy ranged from 85.3 to 120.0%.
Relative to LODs, the range of CVs for the LOQ samples were
lower and ranged from 1.2 to 17.8%, and the accuracy ranged
from 78.8 to 118.4%. Oxycodone and oxymorphone were the
only two analytes to have % CVs greater than 15% for both the
LOD and LOQ. However, these %CVs were still under 20%.
Considering that both LOD and LOQ specimens were required
to meet both retention time and ion ratio criteria, it became
apparent that the LOD samples demonstrated sufficient precision and accuracy to qualify as the LOQ for all compounds
other than 6-acetylmorphine. This observation was supported
by the strong S/N ratios demonstrated for each analyte at its respective cutoff concentration (Table III).
Precision and accuracy of elevated (highly concentrated)
control samples are listed in Table V. The %CVs for the elevated
quality control sample, which was in most cases at 200 times
the cutoff concentration, ranged from 5.0 to 19.2% (Table V).
The 6-AM elevated control concentration was 30 ng/mL because of the limited linearity for this compound. Table V also
lists the upper limit of linearity (ULOL) and carryover limits for
the analytes. For most analytes, the ULOL was at least 100
times the cutoff concentration. The only exception was 6-AM,
which was only linear to 30 ng/mL. Carryover was determined
by running extracted blanks between each calibrator from a
concentration of 30 times the cutoff concentration to a concentration of 500 times the cutoff to determine the concentration at which carryover occurred. This procedure analyzed
the total carryover, including contribution by the RapidTrace
SPE workstation as well as that contributed by the LC–MS–MS.
There were only three compounds, MDEA, cocaine, and PCP,
that demonstrated carryover limits below the established upper
linear range. Specimens that exceeded the ULOL or carryover
575
Journal of Analytical Toxicology, Vol. 33, November/December 2009
Table VII. Oral Fluid Concentrations of Drugs and Metabolites in Authentic
Specimens From Drug Treatment Patients*
Patient
Oral Fluid Analysis
1
1.7 ng/mL BZE†, 4.0 ng/mL PCP
2
4.5 ng/mL PCP
3
8.9 ng/mL PCP
4
45.3 ng/mL PCP
5
1.1 ng/mL COC, 24.7 ng/mL COD, 54.2 ng/mL MOR, 16.4 ng/mL 6-AM
6
1.1 ng/mL COC, 7.2 ng/mL COD, 15.4 ng/mL MOR, 5.7 ng/mL 6-AM
7
1.1 ng/mL COC
8
1.3 ng/mL COC, 11.8 ng/mL BZE
9
1.3 ng/mL COC, 4.1 ng/mL BZE
10
1.9 ng/mL COC, 5.8 ng/mL BZE
11
2.2 ng/mL COC, 37.4 ng/mL BZE
12
2.6 ng/mL COC, 3.8 ng/mL BZE
13
4.3 ng/mL COC, 4.8 ng/mL BZE
14
4.6 ng/mL COC, 31.1 ng/mL BZE, 1.8 ng/mL 6-AM
15
6.7 ng/mL COC, 58.2 ng/mL BZE
16
6.9 ng/mL COC, 5.0 ng/mL BZE
17
7.9 ng/mL COC, 54.5 ng/mL BZE
18
8.9 ng/mL COC, 5.4 ng/mL BZE
19
9.9 ng/mL COC, 28.7 ng/mL BZE
20
12.1 ng/mL COC, 18.0 ng/mL BZE
21
12.7 ng/mL COC, 14.4 ng/mL BZE
22
16.0 ng/mL COC, 57.1 ng/mL BZE
23
36.8 ng/mL COC, 247 ng/mL BZE
24
41.6 ng/mL COC, 426 ng/mL BZE
25
61.9 ng/mL COC, 168 ng/mL BZE, 7.7 ng/mL 6-AM
26
407 ng/mL COC, 56.5 ng/mL BZE
27
872 ng/mL COC, 263 ng/mL BZE
28
1040 ng/mL COC, 389 ng/mL BZE
29
1.1 ng/mL 6-AM
30
6.3 ng/mL COD, 7.6 ng/mL MOR, 1.6 ng/mL 6-AM
31
2.3 ng/mL BZE, 7.9 ng/mL COD, 3.3 ng/mL MOR, 2.4 ng/mL 6-AM
32
35.4 ng/mL MOR, 48 ng/mL 6-AM
33
38 ng/mL OC, 4.2 ng/mL NOC
34
124 ng/mL HC, 7.6 ng/mL NHC, 31.6 ng/mL DHC, 324 ng/mL OC,
25.2 ng/mL NOC
35
45.9 ng/mL COD, 28.9 ng/mL 6-AC, 1800 ng/mL MOR, 435 ng/mL 6-AM,
358 ng/mL OC, 2.4 ng/mL NOC
36
2.0 ng/mL BZE
37
2.5 ng/mL BZE
38
34.2 ng/mL BZE
39
35.6 ng/mL BZE, 4.5 ng/mL MOR
* Underlined oncentrations are between the LOD and cutoff concentrations of the method.
† Abbreviations: COC, cocaine; BZE, benzoylecgonine; COD, codeine; 6-AC, 6-acetylcodeine; MOR, morphine; 6-AM,
6-acetylmorphine; HC, hydrocodone; NHC, norhydrocodone; DHC, dihydrocodeine; OC, oxycodone; NOC,
noroxycodone; and PCP, phencyclidine.
576
limit were diluted and reanalyzed.
Percent recovery of drug/metabolites from the Intercept oral fluid collection device, extraction recovery, and
matrix effects are listed in Table VI.
Recovery from the device averaged
84.3% for all analytes with the lowest
recovery being 73% for hydromorphone. The SPE recovery averaged
91.2% and exceeded 84% for all analytes. The largest matrix effect was
observed for PCP at –57%. The amphetamines, cocaine, and benzoylecgonine had matrix effects of about –25%.
Nine opiates (oxycodone, dihydrocodeine, norcodeine, morphine,
codeine, 6-AM, hydrocodone, oxymorphone, and hydromorphone) had matrix effects within ±10%. There were
only three compounds that shared the
same internal standard in this method.
All others had their own matching
deuterated internal standard that
helped mask any potential matrix effects. The three compounds that
shared the same internal standard
were codeine, norcodeine, and dihydrocodeine. Deuterated (d 3 ) dihydrocodeine was selected for these opiates due to the similar retention time
between the three compounds. There
was no deuterated internal standard
available for norcodeine, and dihydrocodeine interfered with the deuterated (d3) codeine internal standard, so
the deuterated codeine internal standard was eliminated from the method.
The only compound, of the 29 tested
for specificity, that produced an interference problem was phentermine in
the analysis of methamphetamine.
When 10,000 ng of phentermine was
added to the +25% control, the quantification ion for methamphetamine
was increased resulting in failed ion
ratio criterion. Of the 11 food and beverage products added directly to the
+25% control material prior to extraction, none interfered with quantitative
accuracy or ion ratios of the analytes.
Authentic specimens
The validated method was applied to
the analysis of 73 oral fluid specimens
collected with the Intercept device
from patients in a drug treatment
center with IRB approval. The patient
specimens were also stored at 2–8°C
Journal of Analytical Toxicology, Vol. 33, November/December 2009
and were tested within 3 months of collection. A total of 39
specimens (53.4%) were positive for one or more drugs or
metabolites at concentrations exceeding LOD. The results of
the analysis for the 39 specimens are listed in Table VII. No amphetamines were detected in this group of patient specimens.
Twenty-six (35.6%) patients were positive for cocaine or benzoylecgonine. An additional four patients were positive for cocaine and/or benzoylecgonine at concentrations ≥ LOQ. There
were nine patients that tested positive in various combinations of “opiate” derivatives (morphine, codeine, 6-acetylmorphine, and 6-acetylcodeine). All nine of these opiate positives
were positive for 6-acetylmorphine, and three of these patients
were positive only for 6-acetylmorphine in the range of 1.1 to
7.7 ng/mL. Only one patient was positive for 6-acetylcodeine at
28.9 ng/mL. This same patient had a 6-acetylmorphine concentration of 435 ng/mL, the highest 6-acetylmorphine concentration observed in this population. One patient was positive for hydrocodone and its metabolites, norhydrocodone and
hydrocodol (dihydrocodeine). Three patients were positive for
oxycodone, and all three had concentrations of noroxycodone
above the LOD (2 ng/mL) of the method ranging from 2.4 to
25.2 ng/mL. Four patients (5.5%) were positive for phencyclidine in the range of 4.0–45.3 ng/mL.
Conclusions
A comprehensive assay for sensitive detection and measurement of 21 licit and illicit drugs and metabolites has been described that is suitable for testing a small volume aliquot of oral
fluid. The method was validated for linearity, accuracy, precision, specificity, sensitivity, and recovery. The method was successfully applied to analysis of oral fluid specimens obtained
from patients in drug abuse treatment.
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577