Journal of Analytical Toxicology, Vol.
14, January/February 1990
Testing Human Hair for Drugs of Abuse. I. Individual
Dose and Time Profiles of Morphine and Codeine in
Plasma, Saliva, Urine, and Beard Compared to Drug-
Induced Effects on Pupils and Behavior*
Edward J. C o n e
Addiction Research Center, National Institute on Drug Abuse, RO. Box 5180, Baltimore, MD 21224
Abstract
The time course of appearance of morphine and codeine In
beard after single dose administration In two human subjects
was monitored by radiolmmunosesay and confirmed by gas
chromatography/mass spectrometry. Both morphine and
codeine appeared In beard approximately 7-8 days after
drug administration at a time when drug levels In urine,
plasma, and saliva were not detectable and drug-induced
effects had disappeared. Drug levels in beard appeared to be
dose-related suggesting that hair analysis can provide
evidence of time and degree of drug exposure.
Introduction
Testing human hair for drugs of abuse is a newly developing
science that offers the possibility of revealing an individual's
recent historical record of drug exposure beginning at sampling
time and dating back over a period of months to years (1). Since
scalp hair grows at a relatively constant rate of 1 cm/month,
it has been suggested that drug concentration along the hair shaft
reflects degree of drug exposure and the distance from the hair
segment being tested to the root is a measure of time elapsed
since exposure (2). A number of reports on the detection of drugs
of abuse in human hair have appeared for opiates (2-9), cocaine
(5,10-12), phencyclidine (13), methamphetamine (14,15), nico-
tine (14), and phenobarbital (16). Despite the interest in the
presence of drugs of abuse in hair, the two basic pharmacologic
principles regarding (1) the presence of a dose-response rela-
tionship between administered dose and hair drug level and (2)
the time course of drug appearance in hair, have not been
demonstrated in these studies. For accurate interpretation of
test results, it is critical that these principles be established for
drug excretion in hair. Obviously, many other questions that
have impact on the validity of hair test data also remain
unanswered. Some individual factors which could limit inter-
pretation are differences in hair growth rate, drug biotransfor-
mation rate, drug excretion rate, environmental contamination,
* A preliminary report of this study was presented at the Workshop on Hair Analysis for Drugs
of Abuse, National Institute of Justice, Washington, DC, January 9-10, 1989.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
hair treatments, and site of hair selection. In addition, many
analytical factors become increasingly important to consider due
to the unique protein matrix of hair, low drug or metabolite
concentration, limited sample availability, and the accuracy and
validity of the drug assay employed.
The present study was designed to determine if a dose-
response relationship exists between peak drug concentrations
in hair and dosage in human subjects who received single doses
of morphine or codeine. The second purpose was to define the
time "window" in which these drugs appear in hair after their
administration and the time relationship of this "window" to
drug effects and to drug levels in other biofluids. Beard (facial
hair) was considered a suitable alternate to head hair for this
study since it grows at a similar rate (1) and could be collected
on a daffy basis. Two male subjects were administered two doses
of morphine or codeine, and beard and biofluids along with
pharmacologic measures were collected. Beard and biofluids
were analyzed for morphine/codeine by radioimmunoassay
(RIA). The presence of the specific opiate in beard was con-
firmed by gas chromatography/mass spectrometry (GC/MS).
S u b j e c t s and M e t h o d s
Subjects
Two healthy male volunteer subjects (designated Subject O
and Subject T) with a history of intravenous heroin abuse
participated in the study. Informed consent was obtained and
all procedures were approved by the Francis Scott Key Institu-
tional Review Board. The subjects resided on a closed research
unit of the Addiction Research Center, National Institute on
Drug Abuse during the study. Subject characteristics are listed
in Table I.
Study protocol
Prior to induction into the study, the subjects were given
medical and psychological examinations and their morning
urines were tested for the presence of drugs of abuse by EMIT
d.a.u. This preprotocol period lasted for six days for both
subjects.
After medical clearance and obtaining informed consent, Sub-
ject O received single intramuscular doses of morphine sulfate
or placebo at weekly intervals in the following order: 20 mg,
1

10 mg, placebo. Subject T received single intramuscular doses
of codeine phosphate or placebo in the following order: 120 mg,
60 mg, placebo. The doses were administered under double-blind
conditions.
On the day prior to the first drug administration, both sub-
jects began collection of all urine specimens in 12-h pools (10:00
a.m. to 10:00 p.m. and 10:00 p.m. to 10:00 a.m.). They also
collected mixed saliva samples (stimulated with sour candy) at
the end o f each 12-h time period. In addition, the subjects were
instructed to shave their cheeks and neck area daily (prior to
10:00 a.m.) with a clean electric razor (Remington Microscreen
Rechargable Shaver, Model XLR-900) provided on a daily basis.
No liquid or powder preshave products were used. Collections
of biofluids and beard continued in this manner throughout the
study with the exception of more frequent collections of big-
fluids on test days.
On the test day, an intravenous catheter was placed in the
subject's arm vein at 8:00 a.m. Predrug control blood was col-
lected at 9:00 a.m. and 9:30 a.m., and the subject completed
his 12-h urine collection and saliva collection at 10:00 a.m. Other
control measures also were collected prior to 10:00 a.m. Drug
administration occurred at 10:00 a.m. Blood specimens were
collected at the following time intervals (hours) after drug ad-
ministration: 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 24.0.
Pupil diameter (Polaroid camera) end self-rated scores of drug
"high" (visual analog scale, VAS) also were measured at these
times. During the first 12 h after drug administration, individual
urines and saliva specimens were collected, if possible, to coin-
cide with the blood collection times. Thereafter, collection of
urine and saliva proceeded as described earlier. Following the
last test dose, specimen collection continued for 6 days.
Plasma, sallve, end urine analyses
Following specimen collection, samples were frozen until time
of analyses. Plasma, saliva, and urine were analyzed by RIA
(Coat-A-Count Morphine, Diagnostic Products Corp.). The an-
tibody in this RIA is selective for free morphine and demonstrates
minimal cross-reactivity with morphine metabolites (6.2% with
normorphine, 3.8% with morphine-3-glucuronide), but cross-
Table I. Subject Characteristics, Self-Reported Drug
History, and Urinalysis Upon Ward Admission
Drag Two week drag
Sub- Age Weight of use prior to Admission
Ject (years) (Kgs) choice admission urinalysis*
0 28 56.4 Heroin Heroin: 4 of 14 EMITd.a.u, positive
days ($10/day); for opiates (588/473),
cocaine: 4 of 14 cocaine(621/521),
days; marijuana: marijuana(537/493)
2 joints/day; EMIT d.a.u, negative
alcohol: 12 beersfor barbiturates,
amphetamine metha-
done, propoxyphene
benzodiazepines, PCP
T 37 78.6 Heroin Reported no drug EMiT d.a.u, positive
use for opiates (459/443),
cocaine (6651478)
EMIT d.a.u, negative
for barbiturates,
amphetamine, metha-
done, propoxyphene
benzodiazepines, PCP
* Enclosed values represent EMIT d.a.u, absorbance values for subject urine/low
calibrator,
Journal of Analytical Toxicology, Vol. 14, January/February 1990
reacts approximately equally with free codeine. The approximate
sensitivity of the assay for morphine is 0.6 ng/mL. The mean
accuracy =1: standard error of the assay for morphine controls
at 25-ng/mL concentration was 24.0 =t= 1.6 ng/mL. The within-
run coefficient of variation (CV) for controls was 6.0o70 (N =
30) and the between-run CV was 3.2% (N = 3).
Preparetlon of heir control specimens
Control beard was collected by a staff member who had
remained drug-free for over 4 weeks. The specimen was stored
frozen until used as the drug-free hair control. For the drug-
control hair, a methanolic solution of morphine, morphine
sulfate, codeine, or codeine phosphate (1.0 mL of 100.0 ng/mL
free-base concentration) was added to 100.0 mg of the control
hair in a flat bottom vial. The methanol was carefully evaporated
under nitrogen leaving the dried hair control specimen. The
drug-control hair was prepared fresh for each assay.
Hair enslyses
Dally beard collection was accomplished by exchanging razors
and careful removal of the chopped hair particles inside the
razor head. The hair particles were collected by removing the
head and tapping the contents onto glassine paper. Hair par-
ticles inside the razor were removed by brushing the razor
interior and cutter with a small brush. After collection, the razor
head and interior were cleansed with ethanol and dried with
an air jet for reuse. Daily hair collection weights for individual
subjects ranged from 10-25 mg.
Preliminary analytical work was directed toward developing
procedures for handling, incubation, and assay of small hair
specimens (5-15 mg). An initial wash of the specimen was ac-
complished by placing a weighed hair sample in a 1-mL dis-
posable filtration column (J.T. Baker Inc.) and inserting the
column in a vacuum manifold ('Baker'-10 SPE System).
Methanol (3 • 1 mL) was added to the filtration column and
allowed to remain in contact with the hair for 60 s before
applying vacuum. These "wash" extracts were collected,
evaporated, and analyzed by RIA. The filtration column was
removed from the manifold and cut to allow insertion of the
barrel body portion containing the filtration frit (and the washed
hair) into a 1-dram glass vial (3.7 mL). The column end was
sealed with capillary putty prior to insertion into the vial. A
small magnetic stirring bar was placed inside the column body
and an incubation medium (0.25 mL of 0.1M NaOH) was added
to the column. The vial was capped and placed in a water bath
on a magnetic stirrer. The temperature of the water bath was
adjusted to 60~ and the vial was incubated for 18 h. After cool-
ing, an aliquot (0.1 mL) was withdrawn and added to 0.1 mL
of potassium phosphate buffer (1.0M, pH 7.4) in a glass tube.
This neutralized extract was analyzed directly or stored frozen
for later analysis.
As an alternative means of analysis without the "wash" pro-
cedure, weighed hair specimens could be introduced directly into
l-dram glass vials containing 0.5 mL of 0.1M NaOH solution.
These specimens were incubated as before at 60~ with stirring
for 18 h. The cooled sample was neutralized with 0.5 mL of
1.0M phosphate buffer. This solution was suitable for assay
by RIA and for extraction and assay by GC/MS.
For GC/MS analysis, incubated hair specimens were filtered
through a l-mL filtration column. An aliquot of the filtrate,
with added internal standard (nalorphine), was placed on a l-mL
Clean-Screen DAU sample preparation column (Worldwide
Monitoring Corp.) which had been conditioned with methanol,
Journal of AnalyticalToxicology,Vol. 14, January/February1990
water, and 0. IM potassium phosphate buffer (pH 7.0). Vacuum
was applied and the column was washed with water, sodium
acetate buffer (pH 4.0) and methanol. The column was treated
with 2 x 1 mL of elution solvent (methylene chloride-2-pro-
panol (8:2) containing 2070 ammonium hydroxide) and the eluate
was collected in clean glass vials. The solvent was evaporated
under nitrogen and the residue was treated with pentafluoropro-
pionic anhydride at 60~ for 15 min. The excess derivatizing
reagent was removed under nitrogen, and toluene (0.025 mL)
was added. A portion of the derivatized extract was analyzed
by G C / M S (Hewlett-Packard Model 5970B MSD; HP-I col-
umn, 0.2 mm x 12 m, temperature-programmed from 150~
(3-min hold) to 250~ (2-min hold) at 20~ in the selected
ion mode. The ions monitored for morphine, codeine, normor-
phine, norcodeine, and nalorphine (internal standard) along with
their percent abundance and their respective retention times were
as follows: morphine, m/z 414 (100070), 119 (22070), 577 (13%),
7.7 min; codeine, m/z 282 (100070), 445 (41070), 7.8 min; nor-
morphine, m/z 355 (100%), 119 (98070), 709 (3070), 8.0 min; nor-
codeine, m/z 223 (100%), 119 (80070), 355 (73070), 577 (37%),
8.2 min; nalorphine, m/z 440 (100070), 603 (12070), 8.3 min. The
assay had a >i 85~ extraction efficiency for all components;
the limit of detection was approximately 5 ng with a S/N >110
for each analyte extracted from a hair specimen (5-50 mg).
Results
Recovery studies
The effectiveness of removing morphine or codeine from the
surface of facial hair by washing with methanol was assessed
with hair standards prepared with drug adsorbed onto the hair
specimen. Free-base morphine or codeine (1 ng/mL of hair)
which had been recently adsorbed on drug-free control beard
was quantitatively removed by three l-mL methanol washes.
Analysis of the sequential wash eluants by RIA indicated that
93.9~ of drug was recovered in the first l-mL eluant followed
by 5.50/0 and 0.6070, respectively. Analysis of "washed" drug-
control hair after incubation in sodium hydroxide solution, as
described, was negative for both drugs. Without the wash step,
morphine and codeine were recovered quantitatively from the
controls after incubation.
Quantitative studies of other common opioids and metabolites
provided information on the stability of these analytes in the
sodium hydroxide incubation procedure used for hair analysis.
Heroin and 6-acetylmorphine were quantitatively converted to
morphine during incubation as indicated by GC/MS analysis.
Both codeine and morphine remained unchanged in the pro-
cedure. Morphine-3-glucuronide appeared to be resistant to
hydrolysis under the incubation conditions since less than 20070
of equivalent morphine was recovered.
Away for morphine and codeine in hair
Both the wash and incubation steps adopted in this study were
designed to produce accurate handling procedures for small
quantities (5-10 mg) of hair specimens. Use of the filtration
columns and vacuum manifold for handling and processing
small samples allowed the convenience of collecting the initial
methanol wash to test for surface contamination. The "washed"
hair could then be incubated in sodium hydroxide solution for
near complete dissolution of the hair specimen. After incuba-
tion, an aliquot of the solution was withdrawn, neutralized
(pH 7.4), and analyzed by RIA and GC/MS (after further ex-
traction and derivatization steps). Recoveries of morphine or
codeine added to hair specimens were quantitative by RIA
analysis. Drug recoveries from the same specimens after extrac-
tion and analysis by G C / M S were i>85%.
Standard curves generated from different hair matrices were
compared to the manufacturer's urine-based standard curve to
determine if interferences were present in the hair extracts. A
variety of standard curves were generated. Morphine or codeine
standards were added to control hair and processed with and
without a "wash" step. In these tests, drug added prior to
washing was recovered completely in the wash fraction. Without
the "wash" step, drug was recovered after incubation. Both
the standard curves from the hair wash residues and the stand-
ard curves from the incubated hair specimens were similar to
the manufacturer's urine-based standard curve. Generally the
hair standard curves deviated from the manufacturer's standard
curve by less than 30~ Consequently, although matrix standard
curves were prepared with all hair drug assays, quantitative data
are reported based on the manufacturer's standard curve.
The sensitivity of the RIA procedure for morphine and
codeine in hair was determined by assaying standards containing
known amounts of drug added to drug-free hair. Drug-free hair
(10 mg) with and without 1.0 ng (total amount) of morphine
or codeine was incubated and processed without washing to a
final volume of I mL and assayed. The mean difference observed
in cpm's between the drug-free hair standards and the processed
hair standard was 3,001 cpm for morphine and 7,331 cpm for
codeine. For 2.0-ng standards, the mean difference from drug-
free hair standards was 8,315 cpm for morphine and 14,668 cpm
for codeine. For comparison, the mean :t: SE difference ob-
served in cpm's between the manufacturer's zero standard
(urine-based) and the 1.0-ng/mL morphine urine standard was
9,309 _+ 691 cpm (N = 5). Based on these observations and
the manufacturer's stated sensitivity of 0.6 ng/mL for free mor-
phine, an arbitrary lower limit of sensitivity (with a 10-mg hair
specimen) was established as 0.2 ng/mg for morphine and 0.1
ng/mg for codeine.
Confirmation of morphine and codeine in all hair extracts
was successful by GC/MS with selected ion monitoring. The
sensitivity of this assay was approximately 5.0 ng of standard
added to hair controls with subsequent processing, neutraliza-
tion, extraction, and derivatization. A major drawback in quan-
titation by GC/MS was recognized in the procedure due to rapid
build-up of acid residues on the capillary column from the
derivatization reagent despite attempts to eliminate excess
reagent. As a consequence, sensitivity declined over the course
of a batch analysis and quantitation was not attempted by
this method.
Profile of morphine and codeine excretion in beard and blofluids
and accompanying pharmacologic effects
The time courses of appearance and disappearance of
unmetabolized morphine in plasma, saliva, urine, and beard
for Subject O following 20-mg and 10-mg doses of intramus-
cular morphine and placebo are illustrated in Figure 1. Morphine
levels in plasma (Figure 1, Panel B) peaked early (0.25-0.5 h)
at concentrations of 150.2 ng/mL and 66.1 ng/mL, respectively,
after the high and low doses. Similarly, morphine levels in saliva
(Figure 1, Panel A) peaked at 0.5 h after dosing at 37.8 ng/mL
and 10.8 ng/mL, respectively. Thereafter, levels declined rapidly
in both biofluids and approached the level of the sensitivity
of the assay (0.6 ng/mL) by 24 h. Morphine levels in urine
 J o u m a l of Analytical Toxicology, Vol. 14, January/February 1990

(Figure 1, Panel C) rose f r o m nondetectable levels before drug
administration to peak concentrations of 2,423.5 ng/mL and
1,058.8 ng/mL, respectively, in the first voiding (approximately
1 h after drug) after the high and low doses. Levels then declined
rapidly and fell below 300 ng/mL within 24-36 h. Thereafter,
urine levels declined somewhat more slowly and approached
assay sensitivity levels in 3-6 days. Urine screening data (EMIT
d.a.u., Opiates) for Subject O also is shown in Figure 1, Panel
C (as positive ( + ) and negative ( - ) results) for the seven-day
period prior to entry in the study. These data show that the sub-
ject tested positive (I> 300 ng/mL) seven days earlier, but was
negative for opiates for four consecutive days immediately prior
to the study. During the period of the study, morphine levels
in beard were elevated (3.1 ng/mg) in the initial control sample
prior to first dosing and declined gradually over a period of
six days (Figure 1, Panel C). Thereafter, beard morphine levels
remained constant for two additional days followed by an
increase to a peak of 1.3 ng/mg over a period of three days
followed by another decline over a period of three days. A
further slight increase appeared to occur over the remaining
five days.
The presence of morphine in beard specimens from Sub-
ject O was confirmed by GC/MS with selected ion monitoring.
Figure 2 illustrates responses obtained from a morphine stand-
ard added to control beard (Panel A), drug-free control beard
(Panel B), response for morphine on day 0 (Panel C, pre-
morphine administration), and response for morphine on day
11 (Panel D, post-morphine administration). Additional con-
40
A)
30
.J
20
(.9
Z dose. The second voiding at 3.8 h (4,384.4 ng/mL) was the peak
10
0 and fell below 300 ng/mL within 36 h. Thereafter, urine levels
2O0
B)
:PLASMA :
lS0
=1
100
Z
8O
firming ions for morphine also were present in all assays allow-
ing unequivocal identification of morphine in beard specimens.
An absence of codeine was observed for all of Subject O's
specimens.
Pharmacologic effects during morphine and placebo dosing
of Subject O, together with urine and hair excretion data for
timed comparisons, are shown in Figure 3. The miotic effect
of morphine on pupils (Figure 3, Panel A) was evident im-
mediately (0.25 h) after the high and low doses; peak effect after
both doses was reached within 0.5-2.0 h. The effect declined
slowly and had not returned to baseline by 24 h. Pupillary
response after placebo administration was minimal. Subject-
rated responses for drug-induced "high" are shown in Figure
3, Panel B. Maximal effect was recorded immediately (0.25 h)
after the high morphine dose, but occurred later (1.5 h) after
the low dose. These effects on behavior diminished quickly and
were absent by 4 h. An absence of "high" was obtained after
placebo drug administration.
The time courses of appearance and disappearance of un-
metabolized codeine in plasma, saliva, urine, and hair for Sub-
ject T following 120-rag and 60-rag doses of intramuscular
administered codeine and placebo are illustrated in Figure 4.
Codeine levels (morphine equivalents) in plasma (Figure 4, Panel
B) peaked early (0.25-0.5 h) at concentrations of 272.4 ng/mL
and 212.4 ng/mL, respectively, after the high and low doses.
Codeine levels in saliva (Figure 4, Panel A) peaked at 0.5-0.75 h
after dosing at 307.6 ng/mL and 183.9 ng/mL, respectively.
Thereafter, levels declined rapidly in both biofiuids. By 24 h,
levels in both biofluids after the high and low doses were in
the range of 1.0-4.0 ng/mL. By 36 h, saliva levels were below
assay sensitivity. Codeine levels in urine (Figure 4, Panel C) rose
from nondetectable levels before drug administration to levels
of 3251.1 ng/mL and 1,797.3 ng/mL, respectively, in the first
voiding (approximately 1 h after drug) after the high and low
doses. This was the peak urinary concentration after the low
concentration after the high dose: Levels then declined rapidly
declined somewhat more slowly and approached assay sensitivity
levels in 3-4 days. Urine screening data (EMIT d.a.u., Opiates)
for Subject T also is shown in Figure 4, Panel C for the seven-

0
2500
c) HAIR 3.0
2OOO :''t ,,)cuo.~ ,L I J
,~ BL'-m URINE 2.0
~z lsoo

' I
u~ 1000
i ,
Z

0
. . . . 1T ~ 0.0
-1 Q 10 20

20 MG 10 MG

DAYS
PLACEBO
:=4 "-" I
6,e |,S ?*IB ?,S O.m , 9 9
Floure 1. Time profile of morphine (equivalents) in (A) saliva, (B) plasma,
and (C) hair (beard) and urine of Subject 0 following morphine administra- Figure 2. Selected ion monitoring of mlz 414 for morphine (penta-
tion (20 mg and 10 mg) and placebo administration. Panel C also in- fluoropmpionic anhydride derivative) in extracts of (A) morphine stand-
dicates daily EMIT d.a.u, test results for urinary opiates (+ = positive, ard added to control beard, (B) drug-free control, (C) Subject O's beard
- = negative, 300-ng/mL cutoff) for Subject 0 prior to entering study obtained on day 0 (see Figure 1), and (D) Subject O's beard obtained
protocol. on day 11.
Journal of Analytical Toxicology, Vol. 14, January/February 1990

day period prior to entry in the study. These data show that
the subject tested positive (>/300 ng/mL) seven days earlier,
but was negative for opiates for six consecutive days immediately
prior to the study. During the study, codeine levels in beard
were in the range of 0.0-2.0 ng/mg over the first seven days
(Figure 4, Panel C). Beard levels then increased to a peak of
7.6 ng/mg over the next 2 days and remained near that level
for 2 additional days. A n erratic decline followed over the next
3 days. A second increase to a peak of 5.9 ng/mg occurred over
a 3-day period followed by a final 3-day decline.
The presence of codeine in beard also was confirmed by
GC/MS with selected ion monitoring. Figure 5 illustrates
responses obtained from codeine standard added to control
beard (Panel A), drug-free control beard (Panel B), precodeine
beard on day 0 (note absence of response in Panel C), and
response for codeine on day 9 (Panel D, post-codeine admin-
istration). Additional confirming ions for codeine were present
in all assays allowing unequivocal identification of codeine
in beard specimens. Also, responses for morphine in these
specimens were minimal with only traces being detected.
Accompanying pharmacologic effects during codeine and
placebo dosing of Subject T, together with urine and hair ex-
cretion data for timed comparisons, are shown in Figure 6. The
miotic effect of codeine on pupils (Figure 6, Panel A) was evi-
dent immediately (0.25 h) after the high and low doses; peak
effect was reached after both doses within 0.75-1.0 h. The effect
declined slowly over the first 4-6 h and approached baseline
within 24 h. Pupillary response after placebo administration was
minimal. Subject-rated responses for drug-induced "high" are
shown in Figure 6, Panel B. An effect was recorded immediately
(0.25 h) after the high dose and was maximal at 0.5 h. The effect
declined somewhat slowly over the first 6 h after drug admin-
istration and was absent by 24 h. No effects were recorded on
the VAS scale after the low dose or after placebo administration.
Discussion
Interpretation of drug data from hair analysis must be founded
on established pharmacologic principles. Clinical and behavioral
implications arising from data interpretation require a back-
ground of knowledge regarding the dose-response relationship
of hair drug levels to effects and a knowledge of the time course
of appearance (and disappearance) of drug in hair. These data
are only available through carefully controlled drug studies,
preferably with human subjects, since most animal species
appear to be substantially different in hair growth rate and
pattern of distribution from humans,
Controlled studies on drugs of abuse are particularly necessary
since there is much uncertainty regarding identity and purity
of illicitly obtained drugs. Although a number of reports have
appeared regarding the identification of morphine in the hair
of heroin users (2-9), there is a paucity of data on morphine
or other opiates following the administration of known doses.
Puschel et ai. (3) repotted that a carcinoma patient who received
80 mg of morphine over 14 days had similar hair opiate levels

300 A) SALIVA

E
E
z-
_o
,<
Ilc
...
O
3

0
A) 9 PUPILS i

Z
J9 200

100

300

200
B)
i PLASMA

e) VAS O
Z
100

.J 40OO c)
0 :E
25O0 ~3 3000 HAIR
Z
c) 3.O H URINE 4
20O0
m-;--m URINE
Z
). u~ 2ooo
Z
O
1500
:o
2.0 1000 2
Z + ..... ~
1000 0
1.0
500 5") -10 0 10 20
+
0 ~0 t
-10 0 1o 2o 120 MG 6O MG PLACEBO

DAYS
20 MG 10 MG PLACEBO
Flguro 4. Time profile of codeine (morphine equivalents) in (A) saliva,
DAYS (B) plasma, and (C) hair (beard) and urine of Subject T following co-
deine administration (120 mg and 60 mg) and placebo administration.
Figure 3. Time profile of morphine effects on (A) pupils, (B) visual analog Panel C also indicates daily EMIT d.a.u, test results for urinary opiates
scale (VAS), a measure of drug "high", and (C) hair (beard) and urine (+ = positive, - = negative, 300-ng/mL cutoff) for Subject T prior
levels of morphine (equivalents). to entering study protocol.
 Journal of Analytical Toxicology, V o l . 14, J a n u a r y / F e b r u a r y 1990

by RIA as other patients who received as much as 530 mg of
morphine. In the same study, codeine administered to guinea
pigs was detected in hair, but the amount was variable and not
related to dose. Based on these findings, the authors concluded
that a dose-response relationship did not exist between the ad-
ministered opiate dose and hair drug level. However, the clinical
information provided was limited and the possibility was not
addressed that these patients had received other opiate therapy,
e.g., codeine. Furthermore, in their animal studies, it was noted
that surface washing of the hair prior to assay reduced opiate
content by as much as 50%, suggesting that substantial environ-
mental contamination had occurred. Their findings were further
limited by a lack of confirmation of their RIA analyses by a
specific methodology; thus, a general conclusion regarding a
dose-response relationship cannot be drawn from this study.
In the present study, opiate levels in beard were elevated in
the early specimens (Figure IC), presumably as a result of heroin
use within the previous 1-2 weeks. Beard opiate levels then
declined, followed by a new peak on the 1lth day after ad-
ministration of 20 mg of morphine. A second, barely discernible
peak appears to occur during the 17-19th day, presumably as
a result of the 10-mg morphine dose. A similar observation was
made following the administration of codeine (Figure 4C).
Opiate levels in beard peaked during the 9-12th day period after
administration of 120 mg of codeine with a second peak occur-
ring at the 16-17th day, presumably as a result of the 60-mg
dose of codeine. The presence of morphine in beard of subject O
and codeine in beard of subject T was confirmed by GC/MS
analyses. In addition, codeine was not detected by GC/MS assay
in subject O's beard specimens, and only traces of morphine
were detected in subject T's specimens. It is possible that codeine
excretion in sweat produced the early appearance of opiate levels
(approximately 2 ng/mL) in the heard of subject T (days 1-6,
Figure 4C). Overall, these findings suggest a time lag between
dose administration and first appearance in hair of approximately
7-8 days, likely as a result of the growth time necessary for the
hair shaft to emerge from the bulb area in the follicle to a height
above the skin surface sufficient for razor collection. After first
appearance, opiate levels peaked within 1-2 days and declined
over a similar time period. A diminished second peak, presum-
ably as a result of a second lower dose administration, is sug-
gestive of a dose-response effect in beard.
Generally, a dose-response relationship was observed within
24 hours of dosing with drug levels in biofluids, pupil measures,
and behavior for subjects O and T, with the exception of the
VAS measure ("high") for subject T (codeine). The peak
response on all measures occurred within the first 4 h of dosing.
Surprisingly, Puschel et al. (3) reported detection of codeine
in beard within 24 h of drug administration with peak concen-
tration occurring on the 3rd day and peakinl; of urinary codeine
on the 2rid day. Thus, a time lag of only one day occurred be-
tween peak urinary codeine and peak levels in beard. Although
Puschel et al. (3) offered no explanation for the early detection
of codeine in beard, as noted previously, it could be that this
early detection resulted from drug excretion from the sweat
glands surrounding the follicle. It is possible that "sweat solu-
tions" of drug bathing the growing hair strand allow drug entry,
albeit possibly at a lower concentration than that resulting later
from appearance of drug entrapped during the growth process.
Although the limited subject data in the present study preclude
generalization, the results are suggestive that a dose-response
relationship exists between drug levels in beard and dose and
that a lag Of seven days or more exists between time of ad-
ministration and dosing. Furthermore, it is likely that these prin-
ciples would hold for drugs in head hair as well since both hair
types grow at similar rates (1). If these conclusions are validated
by further controlled studies, test data from hair analysis would
reflect degree of drug exposure and segmental analysis could
provide approximate time windows of drug exposure. However,
further analytical refinements are needed in all phases of the
EE 2.5"
A)
1.5"
k-.
0
<
0.5-
Z
0
O
-0.5

15
B) VAS

10
E
E
5

0
.... ]
i,s~ B.) CCml~ ~ J J I h,.
1l m 2 1 e ~ 8
gee ,-I
4000 C)
SN
0 3000 HAIR .
Z
H URINE
2000 a
Z
1000 -

0 i L_ 0
4aeee1 -1 10 2
z~ao
P
P sI (aim s s m Qs i= 9
m
120 M G 6 0 MG PLACEBO
DAYS
Figure 5. Selected ion monitoring of m/z 282 for codeine (pentafluoropro-
pionic anhydride derivative) in extracts of (A) codeine standard added Figure 6. Time profile of codeine effects on (A) pupils, (B) visual analog
to control beard, (B) drug-free control, (C) Subject T's beard obtained scale (VAS), a measure of drug "high", and (C) hair (beard) and urine
on day 0 (see Figure 4), and (D) Subject T's beard obtained on day 9. levels of codeine (morphine equivalents).

6
Journal of AnalyticalToxicology,Vol. 14, Januanj/February1990
hair assay process. Standardization of the assay procedures for
screening and confirmation, establishment of appropriate cut-
offs, development of reliable quality control programs, and pro-
ficiency testing should precede use of hair testing for making
decisions regarding employment or liberty.
Acknowedgements
The author wishes to thank S.L. Dickerson, MT, and P.
Welch, LPN, for expert technical assistance.
References
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Manuscript received February 15, 1989;
revision received June 26, 1989.