Disposition of Hydrocodone in Hair - Journal of Analytical Toxicology

Journal of Analytical Toxicology, Vol. 30, July/August 2006
Disposition of Hydrocodone in Hair
Christine Moore'>, Michael Feldman-, Edward Harrison>, Sumandeep Rana', Cynthia Coulter', David Kuntz-,
Alpana Agrawal', Michael Vincent', and James Soares'
llmmunalysis Corporation, 829 Towne Center Drive, Pomona, California 91767 and2LabOne, Salt Lake City, Hayes Building,
Unit C, 2282 South Presidents Drive, West Valley City, Utah 84120
I Abstract I
Theuse of prescription drugs, including syntheticopiates,is
increasing in the U.S., withemergency room reports showing a
dramatic rise in prescription opiate abuse. As part of an ongoing
study, the hair of admitted opiate users wasanalyzed for
hydrocodone and hydromorphone, as wellas codeine,morphine,
and 6-acetylmorphine in order to determineifthere wasany
correlationbetweenself-reported frequency of opiate intakeand
the concentration of drug detected in hair. Thehairswere
confirmed using gaschromatography-mass spectrometry following
screening byenzymelinked immunosorbent assay(ELISA).
Twenty-four hair specimens collectedfrom volunteers showed the
presence of hydrocodone (130-15,933 pg/mg); four of those also
contained hydromorphone (59-504 pg/mg). Thespecimens were
alsoanalyzed for morphine, codeine,and 6-acetylmorphine. Hair
specimens from five self-reported codeineusersshowed
concentrations of hydrocodone between592 and 15,933 pg/mg.
Inaddition,codeinewas presentat concentrations of 575-20,543
pg/mg, but neither morphine nor hydromorphone were present in
any of those hair specimens. Though the analysis of someopiates
in hair has been previously published, this is the firststudywhere
the hydrocodone and hydromorphone concentrations have been
measured following self-reported opiate intake.
Introduction
Prescription opioids such as Vicodin'", Lortab" (hydrocodone),
and Dilaudid'? (hydromorphone) are becoming increasingly
abused in the U.S. Data from the 2003 Drug Abuse
Warning Network, which collects information on drug-related
episodes from hospital emergency departments in metropolitan
areas, aswell as from Medical Examiner offices, listed opiates
or opioids as present in 20,830 cases ofemergency room (ER)
visits because ofovermedication. Non-medical useof opioids
accounted for 17% ofER visits. Various surveys indicate an increase
in the use of prescription opiates, with hydrocodone
being among the drugs showing the mostpronounced trends
ofincreasing abuse (1). In a recent study ofprescription drug
* Author to whom correspondence should be addressed. E-mail cmoore@immunalysis.com.
abuse in Miami, 36% of ecstasy users also reported hydrocodone
use (2).
Procedures for the detection of these drugs in urine have
been published (3,4) and there are several publications regarding
the incorporation ofopiates intohair (5,6), including
studies employing the analysis ofhair to prove heroin intake
and/or distinguish heroin use from morphine or codeine ingestion
(7,8). As far back as 1994, Nakahara etal. (9) described
the importance ofthe efficient extraction ofopiates from hair,
comparing five extraction methods. Other procedures for the
determination ofdrugs used in the treatmentofheroin addiction,
such as methadone (10), l-a-acetylmethadol (11), and
buprenorphine (12), in hairhave also been reported. However,
we found only one publication specifically describing the detection
of hydrocodone and hydromorphone, as well as oxymorphone
and oxycodone simultaneously inhuman hairusing
gas chromatography-mass spectrometry (GC-MS) (13). The
paper describes the methodology ofanalysis, but has noresults
from the analysis of samples where drug intake history was
available.
Materials and Methods
Subjects
Ourstudy enrolled subjects from the Drug andAlcohol Recovery
Team (DART) in Fullerton, Orange County, Southern
California andwas approved underImmunalysis Institutional
Review Board (IRB # 2004-05-001). Subjects were made aware
of the purpose of the study and gave informed consent. Although
information ondrug use, including time oflast use, frequency
of use, ethnicity, age, gender, and hair color were
recorded for each subject, names, addresses, or other identifying
information were not collected on the interview sheet.
Complete anonymity was established during the sample collection
and laboratory testing procedures. Each subject provided
a hair specimen taken from the vertex portion of the
head at the time ofinterview. The subjects were asked ifthey
took opiates, not specifically heroin, codeine, or other synthetic
opiates. None of the subjects reported having a pre-
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 353

scription for opiates. Hair was transported to thetesting facility
andstored at room temperature.
Experimental
Hairscreening kitswere obtained from Immunalysis Corporation
(Pomona, CAl. The Opiates Direct ELISA Kit (Catalog
# 207-0480) was used for screening the hair specimens and
was used according to the manufacturer's instructions. For
confirmatory procedures, deuterated morphine-d-, codeine-dj,
hydrocodone-dj, hydrornorphone-dj, andfi-acetylrnorphine-dj
as internal standards aswell as the corresponding unlabelled
drug standards were obtained from Cerilliant (Round Rock,
TX). Solid-phase mixed-mode cation exchange-hydrophobic
phase extraction columns (CSDAU020, 200 mg) were obtained
from United Chemical Technologies Inc. (Bristol, PAl. The
derivatizing agent, N,O-bis-trimethylsilyl-trifluoroacetamide
(BSTFA) containing 1% trimethylchlorosilane (TMCS), was
obtained from Pierce Chemical Co. (Rockford, IL). All solvents
were high-performance liquid chromatography grade or
better, and all chemicals were American Chemical Society
grade.
Screening assay
The Immunalysis Opiates Direct ELISA kit is based upon
the competitive binding of enzyme-labeled antigenand unlabeled
antigen to antibody in proportion to their concentration
in the reaction well. Hair was cut into small segments
(3-5 mm), andan aliquotof10mgwas weighed. The
hair was washed briefly with methanol (2 mL/10 min), the
solvent was decanted, andthe hair was allowed to dry. To the
hairs, 0.025M phosphate buffer (pH 2.7,0.5 mL) was added;
the tubeswere capped and incubated at 60°C for 2 h. Phosphate
buffer (O.5M, pH 9.0, 50ul.)was added to neutralize the
acid environment, and the liquids were transferred to corresponding
clean glass tubes. Astandard curve consisting ofa
drug-free negative hair specimen, a drug-free hair specimen
spiked at 100pg/mg ofmorphine, anda drug free hair specimen
spiked at 400 pglmg of morphine, as well as a cut-off
calibrator at 200 pglmg morphine were included in duplicate
in every batch. All specimens, calibrators, and controls were
diluted 1:5 by adding 400 ul, ofphosphate buffer saline (PBS),
with 0.1 % bovine serum albumin (pH 7.0) to 100 ul, of extract.
An aliquot ofthe diluted hair extract (20 ul.) was then
added to the individual microplate well, and the screening
assay was performed according to the manufacturer's instructions.
Theassay performance was optimal when20-60
pg of morphine were placed into the microplate well. A
sample size of20ul, ofthe diluted hairextract gave good separation
at the screening cut-off concentration of 200 pg of
morphine equivalents permilligram ofhair. The assay showed
broad spectrum cross reactivity withother opiates, including
codeine (200%), hydrocodone (93%), 6-acetylmorphine
(83%), hydromorphone (81%), oxycodone (21%), and oxymorphone
(20%).
Efficiency of screening extraction
The extraction efficiency of the screening assay was determined.
Three hair specimens already confirmed for the pres-
354
Journal of Analytical Toxicology, Vol. 30, July/August 2006
ence ofopiates were selected and analyzed according to the described
procedure. After thefirst extract solution was removed,
the hairwas rinsed, dried, andre-extracted for 1h (Extract 2).
The solution was removed, anda third extraction was carried
out (Extract 3).The extracts were screened as described or, if
necessary, further diluted intothe linear range ofthescreening
assay.
Confirmatory assay
Specimens showing inhibition lower thanthat ofthe cut-off
calibrator (200 pglmg) were considered to be presumptively
positive for opiates andwere carried forward to confirmation
using GC-MS, operating in electron impact mode.
Aseparate aliquot of hair was weighed out (10 mg). Low
(100 pg/mg), medium (200 pg/rng), and high (500 pglmg)
controls were also prepared. The internal standard solution
contained deuterated morphine-dj, codeine-dj, acetylmorphine-d,
(6-AM), hydromorphone-dj, and hydrocodone-d, in
acetonitrile (200 nglmL). The samples were incubated in acetone
(1.5 mL) for5 minat roomtemperature to remove gels,
sprays, or extraneous cosmetic treatments on the hair, and
thenthe acetone was discarded. Internal standard (50 mL) was
added to each calibrator, control, or hair specimen at a concentration
of1000 pglmg. Methanol (3 mL) was added, andthe
samples were allowed to sonicate (2 hI70°C). The methanol
was decanted and evaporated to dryness. To the remaining
hair, O.1M hydrochloric acid (1.5 mL) was added, and the
tubeswere capped andincubated overnight at 55°C. The acid
was decanted into the corresponding dried methanol tube,
and O.1M phosphate buffer (pH 6.0; 3 mL) was added. Solidphase
mixed mode extraction columns (CleanScreen
CSDAU0133) were placed into a vacuum manifold. Each
column was conditioned withethyl acetate (2 mL), methanol
(2 mL), and O.1M phosphate buffer (pH 6.0; 2 mL). The samples
were allowed to flow through the columns, then the
columns were washed with deionized water (1 mL), O.1M hydrochloric
acid (1 mL), methanol (1 mL), andethyl acetate (1
mL). The columns were allowed to dry underpressure (15 psi,
5 min). The drugs were finally eluted using freshly prepared
ethyl acetate/2% ammonium hydroxide (98:2, 3 mL). Theextractswere
evaporated to dryness undernitrogen and reconstituted
in ethyl acetate (25 ilL) and BSTFA-l%TMCS (25 ilL).
Thetubes were capped andheated at 70°C for20min.The extracts
were transferred to autosampler vials for analysis by
GC-MS. Controls andcalibrators were included in each batch
as described.
Analytical procedure (GC-MS)
Forconfirmation ofthepresence ofopiates, anAgilent Technologies
6890 GC coupled toa 5975 mass selective detector operating
in electron impact mode was used for analysis
(GC-MS). The GC column was a DB-5 MS (0.25-mm i.d., 0.25­
11m film thickness, 15-m length, J&W Scientific, an Agilent
Company), and the injection temperature was 250°C. The injection
mode was splitless. The oven was programmed from
100°C for 0.5 min, ramped at 40°C/min to 230°C, thenramped
at 5°C/min to 250°C. The transfer line was held at 280°C, the
MS source was 230°C, andthe quadropole at 150°C. The ions

Journal of Analytical Toxicology, Vol. 30, July/August 2006
monitored were as follows: deuterated codeine and hydrocodone
m/z 374 (237); unlabelled codeine and hydrocodone
m/z371, (234, 196); deuterated morphine and hydromorphone
Table I. Extraction Efficiency for ELISA Screening
Procedure
Screening Result: Morphine Equivalents
Sample Extract Extract Extract Total
# 1 h 2h 3h value
259 pglmg 974 301 217 1492
% Extraction 65.2% 20.1% 14.5% 100%
260 pglmg 2207 222 126 2555
% Extraction 85.2% 9.4% 5.3% 100%
361 pglmg 1359 140 54 1553
% Extraction 87.5% 9.0% 3.4% 100%
Mean 79.2% 91.7%
CV(%) 15.4% 6.3%
Table II. Extraction Efficiency for Confirmatory
Procedure
GC-MS
Result Codeine Morphine 6-Acetylmorphine Hydrocodone
Sample 106
Day 1 2799 3036 1089 1828
% Extraction 92.2% 94.1% 90.2% 83.3%
Day 2 235 188 117 364
% Extraction 7.7% 5.8% 9.7% 16.6%
Sample 187
Day 1 20,543 - - 15,852
% Extraction 99.2% - - 99.5%
Day 2 164 - - 74
% Extraction 0.8% - - 0.4%
Sample 225
Day 1 339 1379 1758 764
% Extraction 100% 100% 100% 92.9%
Day 2 - - 58
% Extraction 7.0%
Sample 266
Day 1 19,489 - - 15,933
% Extraction 98.8% - - 99.3%
Day 2 217 - - 108
% Extraction 1.2% - - 0.7%
Sample 300
Day 1 2197 2501 932 1763
% Extraction 93.1% 94.4% 92.8% 83.5%
Day 2 161 147 72 346
% Extraction 6.9% 5.6% 7.2% 16.5%
Sample 340
Day 1 6918 - - 5814
% Extraction 100 - - 100
m/z 432 (417); unlabelled morphine andhydrornorphone m/z
429 (414, 401); deuterated 6-acetylmorphine m/z 402 (343);
unlabelled 6-AM m/z 399 (340, 287). The qualifying ions are
shown inparentheses. All the drugs were well separated by retention
time. Specimens were considered positive for theopiate
drugs if present above the limit of quantitation of the assay
(50 pg/mg).
Efficiency of confirmatory extraction
The extraction efficiency of the method was established.
Six hair specimens containing high concentrations of opiates,
relative to the 200 pg/mg screening cut-off, were selected
andanalyzed according to the extraction protocol. The
remaining hair was dried and extracted again; the hair
remaining from that was subjected to a third extraction
procedure. The extracts were confirmed using the GC-MS
procedure described.
Results and Discussion
Method validation
GC-MS. The precision ofthe assay was determined by analyzing
hair specimens containing codeine, morphine, and
6-acetylmorphine at concentrations of50, 100,200,500, and
1000 pg/mg. The interday precision ofthe assay at 200 pglmg
for codeine, morphine, and 6-acetylmorphine was 2.20%,
2.97%, and2.27%, respectively, (n = 6). The intraday precision
was 2.37%, 2.78%, and 0.53% for codeine, morphine, and
6-AM, respectively, (n = 5). The limit of quantitation of the
system was 50pglmg for all analytes; thecorrelation coefficient
for all calibration curves was greater than r 2 = 0.99, and the
upper limit oflinearity was 1000 pg/mg,Specimens exceeding
the upper limit were either re-extracted using less sample
volume or diluted so the analytical value lay within the calibration
curve parameters.
Extractionefficiency. The extraction efficiency ofboth the
screening procedure andtheconfirmatory extraction methods
were determined. The screening method was efficient, with
Table III. Overall Extraction Recovery
Specimen Codeine Morphine 6-Acetylmorphine Hydrocodone
106 92.2 94.1 90.2 83.3
187 99.2 99.5
225 100 100 100 92.9
266 98.8 99.3
300 93.1 94.4 92.8 83.5
340 100 100
Mean 97.2 96.1 94.3 93.08
extraction
recovery
(%)
CV(%) 3.68 3.45 5.38 8.53
355

an average extraction recovery of79.2% ofmorphine equivalents
inthefirst hourofincubation. After thesecond hour, the
mean recovery was 91.7% with a coefficient of variation of
6.3% (n =3). The confirmatory procedure was also extremely
efficient with recoveries for all four opiates over 93% (n =6).
The results areshown in Tables I-III.
Specimens
Overall, 24hairspecimens tested positively for hydrocodone
or hydromorphone.
Hydrocodone. Hydrocodone was present in all24synthetic
opiate-positive samples. Eight ofthe subjects did not admit to
opiate use ofany kind. Five subjects reported specifically to the
Table IV. Frequency of Opiate Intake in Specimens Positive for Hydrocodone
Journal of Analytical Toxicology, Vol. 30,July/August 2006
ingestion ofcodeine, andallprovided hairsamples that tested
positively for hydrocodone and codeine (it should be noted
that oneofthe subjects admitted hydrocodone intake in additionto
codeine). Three individuals reported hydrocodone use
inaddition toheroin or codeine intake, and all were positive for
hydrocodone in the hair.
The self-reported drug intake of the subjects is shown in
Table IV along with theanalytical results. Hydrocodone was detected
in all hair samples following admitted codeine intake,
but, notably, morphine was not detected in measurable concentrations.
Hydromorphone. Foursamples contained hydromorphone
above the limit of quantitation (Table III). Two were from
Self-Reported Opiate Intake Concentration Detected in Hair(pg/mg)
Drug Frequency of use Codeine Morphine 6-AM HYC' HYM
Heroin 4 times/week 841 2006 1622 130
Heroin 2 times/week 285 734 667 135 -
Heroin 3 times/week - 570 831
MTD 1time/day 154
Heroin 1time/month - - - 155
Heroin 14times/week 5743 15,206 7623 161 504
Heroin 7 times/week 1958 9160 9925 202 268
NA - - - - 224
Opiates 3 times/week 192 - - 263 -
Heroin 4 times/week 399 684
HYC 3000 mg/day 289 373
NA - - - - 477
Codeine 500 mg/day 6516 - -
HYC 750 mg/day 592
NA - - - - 669
Heroin 1time/week 339 1379 1758 764
Heroin 1 time/week 2197 2501 932
MTD 1time/day 1763
Heroin 1times/month
HYC 1time/week
MTD 1 time/day 2799 3036 1089 1828
NA - 100 190 81 2287 -
NA - 157 - - 2974
NA - 113 244 102 3110 82
Codeine 3000 mg/day 575 - - 3150 -
NA - 100 199 73 3156 59
Codeine 2000 mg/day 851 - - 4019 -
NA - 6918 - - 5814
Codeine 1000 mg/day 20,543 - - 15,852
Codeine 500 mg/day 19,489 - - 15,933
, Abbreviations; 6-AM, 6-acetylmorphine; HYC, hydrocodone; HYM, hydromorphone; MTD, methadone;and NA, no admission of opiate intake.
356

Correlation
There appeared to be no correlation between amount of
drug ingested and the concentration of any drug or drug
metabolite detected in hair. Such a correlation has been reported
for codeine, when the codeine levels arenormalized to
the melanin (phaeomelanin and eumelanin) content of the
hair. Unfortunately, theamount ofhairneeded tocarry outthe
determination of total melanin, eumelanin, screening, and
confirmation of each hair was impractical in our study. According
to the method ofKronstrand et al. (26),50 mgofhair
was necessary for total melanin determination and30mgfor
eumelanin. Our study required 10 mgfor screening and 20mg
for confirmation, requiring a total of 110 mgofhairpersubject,
assuming no repeats were necessary.
Because melanin is predominantly responsible for hair coloration,
we assessed theaverage opiate concentration based on
thehair color ofsubjects inthestudy (Figure 2). There was only
one red-haired subject and only one blonde subject positive for
morphine and 6-acetylmorphine; thus, these haircolors were
notincluded intheaverage values reported. Overall, grey-haired
subjects had thehighest average concentration ofcodeine (5069
pglmg) and hydrocodone, (6337 pg/mg; x = 54years) in hair.
The mean age ofblack-haired subjects was 48.8 years, and they
showed the highest average concentration ofmorphine (6144
pg/mg) and 6-acetylmorphine (2931 pg/rng). However, the
number ofsubjects in each haircolor class was limited; therefore,
nodefinitive conclusion can bedrawn regarding haircolor
and opiate concentration. The age ofthesubjects ranged from
26 to 62 years, butonly two were younger than42 years.
Conclusions
In summary, hydrocodone can be readily detected in hair
specimens. Following admitted codeine use, all hair samples
contained hydrocodone, and none showed thepresence ofmorphine,
6-acetylmorphine, or hydromorphone. In two of the
cases, thehydrocodone concentration was significantly higher
thanthecodeine. There appeared tobenocorrelation between
self-reported opiate intake and concentration in hair.
Acknowledgments
We aregrateful to Ms. Toby Evans for facilitating subject access
at DART and to Russell Munford (Immunalysis Corporation)
for the collection ofallthe specimens. Many thanks are
due to Michelle Nguyen and Erma Abolencia for all the
immunoassay testing ofhairsamples at Immunalysis Corporation.
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Manuscriptreceived February 9,2006;
revision received April 3, 2006.
359