Ofloxacin as a Reference Marker in Hair of Various Colors
7
Journal of Analytical Toxicology, Vol. 27, April 2003 Technical Note I Ofloxacin as a Reference Marker in Hair of Various Colors Diana G. Wilkins 1, Atsuhiro Mizuno 2, Chad R. Borges 1, Matthew H. Slawson 1, and Douglas E. Rollins 1 1Center for Human Toxicology, Departmentof Pharmacologyand Toxicology, Room 490 Biomedical PolymersResearch Building, Universityof Utah, 20 South 2030 East,Salt Lake City, Utah 84112 and 2Phase 1 and Clinical Pharmacology Department, GlaxoSmithkline K.K., 6-15, Sendagaya4-chome, Shibuyaku, Tokyo 151-8566,Japan [Abstract It has been proposed that administration of a reliable marker substance to human subjects may enhance the ability to identify drug use and treatment compliance in drug treatment programs. The goal of this study was to determine if an oral dose of the antibiotic ofloxacin (OFLX) could be used as a "marker" substance to establish reference points with respect to time in hair of various colors. Male and female subjects (n = 32) between 18 and 40 years of age received 800 mg of OFLX as a divided oral dose on a single day. Subjects were restricted from cutting their hair or performing chemical treatments. Hair was collected (by cutting) before, and at weeks 4, 5, 6, and 7 after drug administration. Subjects were classified as having black (n = 5), brown (n = 13), blonde (n = 8), or red (n = 6) hair. Hair was segmented into 3.0-cm segments prior to digestion, extraction, and analysis by high-pressure liquid chromatography (HPLC). At 7 weeks, the mean OLFX concentrations (• 1 SD) in the first 3.0 cm of hair closest to the scalp were as follows: 30.6 • 8.5 ng/mg (black), 6.0 • 1.8 ng/mg (brown), 3.5 • 1.6 ng/mg (blonde), and 1.4 • 0.3 ng/mg (red). A similar pattern was found in hair collected at weeks 4-6. Quantitative eumelanin (EUM) hair concentrations for each subject were also determined for each subject via HPLC. A strong relationship between OFLX concentration at 7 weeks and EUM was noted (r2 adjusted = 0.728; p < 0.001). In six subjects, we also determined the intrasubject variability of OFLX incorporation into individual hair strands. Four strands from each subject were segmented into 2-mm segments and analyzed. OFLX appeared in segments #1-#10 at week 5 (the first centimeter of hair). OFLX appeared in segments #2-#20 at week 7 (the first and second centimeter of hair). The maximum OFLX concentration (the "band" of drug) and location was then determined for each strand. The maximum OFLX concentration was measured in segments #2-#5 at week 5 for all subjects (within the first centimeter of hair length). The maximum OFLX concentration was measured in segments #3-#8 at week 7 (within the first and second centimeter of hair). This was consistent with a growth rate of less than 1.0 cm/month, although considerable intersuhject variability was found. No significant axial diffusion of OFLX along the hair shaft beyond the first 3.0 cm of hair was noted. Despite a strong effect of hair color, these data suggest that OFLX may be a suitable marker substance for hair, allowing a subject to serve as their own "control". Future studies will explore whether drug use, treatment compliance, or recidivism in clinical drug-abuse studies can be determined with the aid of OFLX. Introduction The analysis of hair may be a useful adjunct specimen to plasma and urine for monitoring compliance and recidivism in drug treatment programs. It has been suggested that hair may serve as an historical record, or diary, of drug exposure (1,2). Improved methods for determining patient compliance over time are necessary because drug concentrations in plasma, urine, and saliva often reflect only the dosage taken within the last several days prior to sampling. Our ability to accurately in- terpret drug concentrations in hair, however, is uncertain, and there is conflicting evidence regarding the utility of hair anal- ysis for drug monitoring (3-31). There is evidence from small, controlled studies in humans and animals that the variability in measured hair concentrations of drug, given an equal dose, is af- fected by hair pigmentation (32--47). However, at least two re- ports have suggested that hair color does not play a role in the outcome of hair drug testing (48,49). These data are not nec- essarily contradictory, as the outcome of the drug test may be dependent upon the cutoff concentrations used to identify a pos- itive versus a negative specimen. Until these issues are clarified, the ability to interpret trends in quantitative hair data over time is limited. It has been suggested that the use of "marker" compounds may enhance the assessment of treatment compli- ance and recidivism by establishing known reference points (with respect to time) in hair, despite interindividual variability (45,50,51). These marker compounds may permit the assess- ment of illicit or therapeutic drug exposure in subjects partic- ipating in clinical treatment programs. Ofloxacin (OFLX) is a fluorinated carboxyquinolone antibiotic with approximately 98% bioavailability after oral administra- tion. Previous research demonstrated that OFLX could be de- tected in hair after therapeutic use and multiple-dose adminis- tration (50-55). Uemtasu et al. (50) first suggested the possible Reproduction (photocopying) of editorialcontent of thisjournalis prohibited withoutpublisher's permission. 149 Downloaded from https://academic.oup.com/jat/article-abstract/27/3/149/703818 by guest on 14 April 2019
Ofloxacin as a Reference Marker in Hair of Various Colors
Technical Note I
Ofloxacin as a Reference Marker in Hair of Various Colors
Diana G. Wilkins 1, Atsuhiro Mizuno 2, Chad R. Borges 1, Mat thew
H. Slawson 1, and Douglas E. Rollins 1
1Center for Human Toxicology, Department of Pharmacology and
Toxicology, Room 490 Biomedical Polymers Research Building,
University of Utah, 20 South 2030 East, Salt Lake City, Utah 84112
and 2Phase 1 and Clinical Pharmacology Department, GlaxoSmithkline
K.K., 6-15, Sendagaya 4-chome, Shibuyaku, Tokyo 151-8566,
Japan
[Abstract
It has been proposed that administration of a reliable marker
substance to human subjects may enhance the ability to identify
drug use and treatment compliance in drug treatment programs. The
goal of this study was to determine if an oral dose of the
antibiotic ofloxacin (OFLX) could be used as a "marker" substance
to establish reference points with respect to time in hair of
various colors. Male and female subjects (n = 32) between 18 and 40
years of age received 800 mg of OFLX as a divided oral dose on a
single day. Subjects were restricted from cutting their hair or
performing chemical treatments. Hair was collected (by cutting)
before, and at weeks 4, 5, 6, and 7 after drug administration.
Subjects were classified as having black (n = 5), brown (n = 13),
blonde (n = 8), or red (n = 6) hair. Hair was segmented into 3.0-cm
segments prior to digestion, extraction, and analysis by
high-pressure liquid chromatography (HPLC). At 7 weeks, the mean
OLFX concentrations (• 1 SD) in the first 3.0 cm of hair closest to
the scalp were as follows: 30.6 • 8.5 ng/mg (black), 6.0 • 1.8
ng/mg (brown), 3.5 • 1.6 ng/mg (blonde), and 1.4 • 0.3 ng/mg (red).
A similar pattern was found in hair collected at weeks 4-6.
Quantitative eumelanin (EUM) hair concentrations for each subject
were also determined for each subject via HPLC. A strong
relationship between OFLX concentration at 7 weeks and EUM was
noted (r 2 adjusted = 0.728; p < 0.001). In six subjects, we
also determined the intrasubject variability of OFLX incorporation
into individual hair strands. Four strands from each subject were
segmented into 2-mm segments and analyzed. OFLX appeared in
segments #1-#10 at week 5 (the first centimeter of hair). OFLX
appeared in segments #2-#20 at week 7 (the first and second
centimeter of hair). The maximum OFLX concentration (the "band" of
drug) and location was then determined for each strand. The maximum
OFLX concentration was measured in segments #2-#5 at week 5 for all
subjects (within the first centimeter of hair length). The maximum
OFLX concentration was measured in segments #3-#8 at week 7 (within
the first and second centimeter of hair). This was consistent with
a growth rate of less than 1.0 cm/month, although considerable
intersuhject variability was found. No significant axial diffusion
of OFLX along the hair shaft beyond the first 3.0 cm of hair was
noted. Despite a strong effect of hair color, these data suggest
that OFLX may be a suitable marker substance for hair, allowing a
subject to serve as their own "control". Future
studies will explore whether drug use, treatment compliance, or
recidivism in clinical drug-abuse studies can be determined with
the aid of OFLX.
In t roduct ion
The analysis of hair may be a useful adjunct specimen to plasma and
urine for monitoring compliance and recidivism in drug treatment
programs. It has been suggested that hair may serve as an
historical record, or diary, of drug exposure (1,2). Improved
methods for determining patient compliance over time are necessary
because drug concentrations in plasma, urine, and saliva often
reflect only the dosage taken within the last several days prior to
sampling. Our ability to accurately in- terpret drug concentrations
in hair, however, is uncertain, and there is conflicting evidence
regarding the utility of hair anal- ysis for drug monitoring
(3-31). There is evidence from small, controlled studies in humans
and animals that the variability in measured hair concentrations of
drug, given an equal dose, is af- fected by hair pigmentation
(32--47). However, at least two re- ports have suggested that hair
color does not play a role in the outcome of hair drug testing
(48,49). These data are not nec- essarily contradictory, as the
outcome of the drug test may be dependent upon the cutoff
concentrations used to identify a pos- itive versus a negative
specimen. Until these issues are clarified, the ability to
interpret trends in quantitative hair data over time is limited. It
has been suggested that the use of "marker" compounds may enhance
the assessment of treatment compli- ance and recidivism by
establishing known reference points (with respect to time) in hair,
despite interindividual variability (45,50,51). These marker
compounds may permit the assess- ment of illicit or therapeutic
drug exposure in subjects partic- ipating in clinical treatment
programs.
Ofloxacin (OFLX) is a fluorinated carboxyquinolone antibiotic with
approximately 98% bioavailability after oral administra- tion.
Previous research demonstrated that OFLX could be de- tected in
hair after therapeutic use and multiple-dose adminis- tration
(50-55). Uemtasu et al. (50) first suggested the possible
Reproduction (photocopying) of editorial content of this journal is
prohibited without publisher's permission. 149
D ow
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Journal of Analytical Toxicology, Vol. 27, April 2003
use of ofloxacin as an index of exposure. In that study, the OFLX
content was measured in hair collected from 14 subjects known to
have taken OLFX for treatment of bacterial infection. Although
exact doses and duration of therapy varied among these subjects, it
did demonstrate that OFLX moved outwards along the hair shaft after
administration. The authors sug- gested that the compound could be
useful in the future for testing patient compliance. Further
studies in humans and al- bino and pigmented rats by this same
group suggested that OFLX is excreted in a dose-dependent manner
and that the mechanism of excretion is closely linked with the
presence of melanin (56). However, only four human subjects with
black/grizzled hair were included in the study, and it was un-
clear to what extent OFLX would be incorporated in hair of other
colors.
As suggested by these early studies, administration of OFLX (as a
marker) at specific, but limited, time points during drug treatment
may establish a frame of reference with which to determine
concomitant use of other drugs. The purpose of this study was to
determine if the OFLX could be used as a "marker' substance to
establish reference points with respect to time in hair of various
colors. First, we hypothesized that OFLX can be readily detected in
human hair after single day of administra- tion, regardless of hair
color. Second, we hypothesized that OFLX will move along the hair
shaft in a pattern consistent with natural hair growth (not simple
diffusion along the shaft).
Materials and Methods
orthophosphoric acid were obtained from Sigma (St. Louis, MO).
(R)-9-Fluoro-2,3-dihydro-3-methyl- 10-(4-ethyl-l-piper-
azinyl)-7-oxo-7-hydroxy-pyridol[ 1,2,3,-de][1,4]benzoxazine-6-
carboxylic acid (DS-4632) for use as an internal standard was
graciously donated by Daiich Pharmaceutical Co. (Tokyo, Japan).
Chloroform and hydrochloric acid were obtained from Burdick and
Jackson (Muskegon, MI) and Mallinckrodt Chem- ical (St. Louis, MO),
respectively.
Standards and solutions Stock solutions of the OFLX (1 mg/mL) and
DS-4632 in-
ternal standard (0.5 mg/mL) were prepared in methanol and stored at
-4~ Daily working solutions of OFLX were prepared in methanol at
10.0 ng/mL, 100.0 ng/mL, and 1.0 IJg/mL. A se- rial dilution of the
DS-4632 stock solution was prepared, in methanol, to a final
concentration of 500.0 ng/mL. Working so- lutions of OFLX were used
to fortify human hair at final con- centrations of 0.5, 1.0, 3.0,
5.0, 10.0, 20.0, 30.0, and 50.0 ng/mg (standard curves). Positive
quality control specimens (5 and 30 ng/mg, final concentrations)
for verification of accuracy and precision were analyzed in
duplicate with each analytical batch. Separate stock solutions of
OFLX were prepared from reference materials for standards and
quality control samples.
Study protocol Written informed consent approved by the University
of Utah
was obtained from all study participants. All enrolled subjects
claimed no prior use of OFLX within at least the previous six
months. Subjects were housed in the Clinical Research Center at the
University of Utah Health Sciences Center at the time of OFLX
administration. Males and females (n = 32) between 18 and 40 years
of age received 800 mg of OFLX as an equally di- vided oral dose at
0800 and 2000 h. Plasma was collected 12 h after the last dose.
Hair was collected (by cutting) at the vertex region of the scalp
before and at weeks 4, 5, 6, and 7 after drug administration.
Subjects were visually classified as having black (n = 5), brown (n
= 13), blonde (n = 8), or red (n = 6) hair. Sub- jects were
restricted from cutting their hair or performing chemical
treatments for the duration of the study. Hair was stored at -20~
until analysis.
Analytical procedures Ofloxacin in hair of different colors. All
hair strands from
each subject were individually aligned root-to-tip. Then, ap-
proximately 10 hairs from each subject were segmented into 3.0-cm
segments. Thus, each 3.0-cm segment (sample) con- sisted of
approximately 10 hairs.
Ofloxacin concentrations in hair were determined by a mod-
ification of the method of Mizuno et al. (57). Internal standard
(25 ng/mg DS-4632) was added to a l-rag sample from each seg- ment.
A Cahn TA4100 electrobalance was used to weigh speci- mens (+ 1.0%
tolerance). Standards and quality control speci- mens containing
hair fortified with known amounts of OFLX were included with all
assays as described. Samples were com- pletely dissolved in 2 mL of
1N NaOH at 70~ for 20 rain. After cooling to room temperature, the
pH was adjusted to 9.0 with several drops of 6N HC1 and 1 mL of
phosphate buffer (pH 9.0). Digest solutions were extracted with
Bond-Elut Certify TM solid- phase extraction columns. Columns were
prewashed with dis- tilled water and methanol, and the digest
solution was applied to the column, followed by column rinses with
3 mL of distilled water, 2 mL of 0.1M acetate buffer (pH 4.0), and
5 mL of methanol. OFLX was then eluted with 5 mL of methylene
chlor- ide/isopropanol (80:20) containing 2% ammonium hydroxide.
Eluates were evaporated to dryness at 40~ in a water bath. Dried
extracts were reconstituted in 7501JL of mobile phase; 50 laL was
injected for high-pressure liquid chromatography (HPLC) analysis.
The limit of quantitation for OLFX (hair) in this procedure was 0.5
ng/mg of hair.
Ofloxacin distribution in individual hair strands and plasma. Two
different methods were used for the determination of ofloxacin in
"intact" versus "individual" hairs to increase our confidence and
ability to compare data to previous research (both methods had been
previously published and validated). For individual single strands,
hairs were individually aligned root-to-tip and each strand
segmented into 2-mm segments. OFLX concentrations in hair were
determined by a modification of the method of Mizuno et al. (57).
Internal standard (25 ng DS-4632) was then added to each 2-mm
segment. Standards and quality control specimens containing hair
fortified with known amounts of OFLX were included with all assays.
Samples were completely dissolved in 0.5 mL of 1N NaOH at 70~ for
10 rain. After cooling to room temperature, the pH was adjusted to
7.0 with 0.5mL of 1N HCl and 1 mL of phosphate buffer (pH
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Journal of Analytical Toxicology, Vol. 27, April 2003
7.0). Digest solutions were extracted with chloroform (5 mL) for 20
rnin, centrifuged at 1670 x g for 10 rain, and the organic phase
collected. Extracts were evaporated to dryness at 40~ in a water
bath. Dried extracts were reconstituted in 150 I~L of mo- bile
phase; 50 IJL was injected for HPLC analysis. Plasma spec- imens
were extracted and analyzed similarly, with the following
exceptions. Internal standard (500 ng) was added to 100 iJL of
plasma, 1 mL of 0.5M phosphate buffer (pH 7.0) was then added and
OFLX extracted into chloroform. The limits of quantitation for OFLX
in this procedure were 0.2 ng/2 mm hair segment or 0.2 ng/mL
plasma.
HPLr analysis ofOFLX. HPLC analyses were performed on a Waters
(Milford, MA) 600E multisolvent delivery system equipped with a
Waters 600 controller, Waters 717plus au- tosampler, and model 474
scanning fluorescence detector. Re- constituted extracts were
injected onto a Symmetry TM 5 tim C18 250 x 4.6-ram column. The
mobile phase consisted of acetoni- trile/0.025M orthophosphoric
acid adjusted to pH 3.0 with 40% tetrabutylammonium hydroxide
solution (6.5:93.5, v/v). The flow rate and excitation and emission
wavelengths were 1.0 mL/min, 290 nm, and 490 nm, respectively.
Peak-height ratios of OFLX to DS-4632 internal standard were
compared to a standard curve made from a series of standards
extracted con- currently with the specimens.
Eumelanin in hair. The degradation products pyrrole-2,3-di-
carboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA)
of eumelanin were measured by a modification of the method of Ito
and Wakamatsu (58). Five milligrams of hair was cut into 1-2-ram
pieces and degraded in 1 mL of 0.5M NaOH containing 80 IJL of 3%
H202 by heating in a boiling water bath for 20 rain. Pthalic acid
(40 nmol) was added as in- ternal standard prior to degradation.
Complete details of the modified method used for this study have
been previously pub- lished (59).
LC conditions for eumelanin analysis. Quantitation of eu- melanin
degradation products (PDCA and PTCA) and internal standard was
carried out with a Waters 600E HPLC system with UV detection at 280
nm. Samples (100 I~L) were injected onto a Phenomenex (Torrance,
CA) Luna 5 IJm C18 250 • 4.6- mrn column at a temperature of 55~
The mobile phase con- sisted of 0.01M potassium phosphate buffer
(pH 2.1) and methanol at a flow rate of 0.8 mL/min under the
following gra- dient: 98%/2% aqueous/organic ramped to 40%/60%
aqueous/organic over 14 rain, held for 6 rain, and returned
to
Table I. Ofloxacin and Eumelanin in Hair
Nalural Mean Ofloxacin Mean Plasma Mean Eumelanin Hair Color Gender
Hair Conc. (TW) Conc. at 12 h Concentration
(Self.Reporl) (M/F) (ng/mg) (rig/mr) (pg/mg)
Red 2 Males/4 Females 1.49 (+ 0.38)* 1394.0 (• 649.8) 1.68 (• 0.69)
Blonde 6 Males/2 Females 3.51 (• 1.68)* 1304.9 (• 296.5) 2.25 (+
0.33) * Brown 7 Males/6 Females 6.03 (• 1.81)* 1270.3 (• 298.4)
3.51 (• 0.95)* Black 2 Males/3 Females 30.64 (• 8.55)* 1291.0 (•
238.8) 10.92 (• 3.6)*
* Significantly different from all other hair colors at p <
0.05. Significantly different from brown and black hair only at p
< 0.05.
98%/2% over 5 min. Peak-height ratios of PDCA and PTCA to internal
standard were compared to a standard curve made from pure PDCA and
PTCA standards subjected to alkaline hy- drogen peroxide
degradation.
Statistical analysis Simple linear regression analysis, ANOVA, and
tests of sig-
nificance were performed with SPS$ (Statistical Package for the
Social Sciences, version 6.1, Chicago, IL) and DataDesk (version
4.0, New York, NY).
Results and Discussion
A suitable marker substance for use in clinical studies of drug
incorporation into hair would be one that is detectable after a
single dose, is present in high concentrations, and has minimal or
no adverse pharmacologic effects on the individual. It should also
move along the hair shaft in a predictable pattern, remaining as a
tight "band" to permit establishment of a "window of time" within
the hair. Ideally, the marker should also be one that has no
potential for passive exposure from the environment and limited
abuse liability. OFLX was selected for this study because it
appeared to meet most of these criteria and it had been previously
reported that administration of multiple oral doses OFLX to humans
was associated with large measur- able concentrations in hair
(50-55). These studies also strongly suggested that OFLX would be a
useful time marker for both clinical and forensic purposes.
In our current study, we proposed to investigate whether OFLX could
be detected in human hair after 1-day oral dose ad- ministration
and whether its detection within the hair shaft fol- lowed a
pattern consistent with normal hair growth. In partic- ular, we
were interested in whether the OFLX marker moved along the hair
shaft as a '%and" of drug, allowing us to estimate the time of
original exposure. Baseline hair specimens were collected just
prior to oral administration of OFLX. OFLX was not detected in
these pre-dose hair specimens, indicating that the subjects had
either not taken the drug, or had not previously ingested a
sufficient amount to produce a detectable concen- tration. At 7
weeks, the hair concentrations of OFLX for all subjects ranged from
a low of 0.84 to a high of 44.70 ng/mg in the first 3 cm of hair
closest to the scalp. Assuming an average
hair growth rate of approximately 1.0 era/month, we expected that
OFLX would not be detected beyond the first 3-cm of hair during
this study. Consistent with this expectation, OFLX was not detected
in any segments be- yond 3 cm during the study.
Although OFLX was readily measured in hair, a large interindividual
variability in measured OFLX concentration was observed. Previous
ev- idence has suggested that that hair pigmenta- tion may be an
important factor in the incor- poration of some drugs into human
hair (32-47). These studies support the hypothesis that drugs that
retain a positive "charge" at
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physiologic pH may be preferentially incorporated into melanin-
containing cells of the hair shaft. Of relevance to our current
study, Uematsu et al. (56) demonstrated significantly greater OFLX
concentrations in black versus white hairs from four subjects
described as having "grizzled" hair. To determine whether
differences in hair pigmentation contributed to the variability in
OFLX concentrations in our study, hair from each subject was
classified into one of four hair-color categories (black, brown,
blonde, and red). The initial classification was made on the basis
of self-report and confirmed by visual obser- vation by laboratory
staff. A summary of hair concentration data and hair color is
presented in Table I. At 7 weeks, the mean OLFX concentrations (• 1
SD) in the first 3 cm of hair closest to the scalp were as follows:
30.6 • 8.5 ng/mg (black), 6.0 • 1.8 ng/mg (brown), 3.5 • 1.6 ng/mg
(blonde), and 1.4 • 0.3 (red) ng/mg. Differences were statistically
significant (ANOVA, p <
A { ,
y2 (adjusted) = 0.728 p < 0.001
a SubJect
(IJglmg)
Figure 1. Ofloxacin versus eumelanin concentrations. The ofloxacin
concentration for hair from each subject (collected 7 weeks after
OFLX administration) versus their respective eumelanin
concentration.
9 .... ..,, ~.~,,,~.,~ ,~.:,~,,~ ~ .....
0.05, DataDesk) between all hair-color groups. We then evaluated
whether differences in plasma concentra-
tions accounted for the interindividual variability in OFLX hair
concentrations. Plasma OFLX concentrations (12 h) were not
significantly different between subjects with different hair colors
(see Table I). The mean OFLX plasma concentrations (• 1 SD) at 12 h
for subjects with black, brown, blonde, and red hair were 1394.0 •
649.8 ng/mL, 1304.9 • 296.5 ng/mL, 1270.3 • 298.4 ng/mL, and 1291.0
• 238.8 ng/mL, respectively. Based on the 12-h data, it is unlikely
that differences in peak plasma con- centrations of OFLX explain
the variability in hair concentra- tions.
Because the classification of hair color into categories is largely
a subjective assessment, we implemented a more ob- jective measure
of hair color with the use of quantitative eu- melanin
concentrations. Our laboratory has previously devel- oped an
improved quantitative procedure for the determination of eumelanin
concentrations in human hair (59). The method was used for analysis
of eumelanin in hair specimens in this current OFLX study (see
Table I). The mean measured eume- lanin concentrations (• 1 SD) for
subjects with black, brown, blonde, and red hair were 10.92 + 3.6
I~g/mg, 3.51 • 0.95 IJg/mg, 2.25 • 0.33 IJg/rng, and 1.68 • 0.69
IJg/mg, respectively. Differ- ences were statistically significant
between black hair and all other colors (ANOVA, p < 0.05,
DataDesk), as well as brown hair versus red and blonde hair. There
was no statistical difference in eumelanin concentration between
blonde and red hair. These data are consistent with trends in
eumelanin content previ- ously reported for black, brown, and
blonde hair based on the PTCA content of human hair, as determined
by Ito and Fujita (60). The data trends are also consistent for all
four hair colors with the spectrophotometric eumelanin
determinations of human hair performed by Ito et al. (61).
A positive relationship between OFLX concentration at 7
[ 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0
LocaUon from hair root (ran)
Figure 2. Ofloxacin distribution in 2-mm hair segments. Four
individual
hair strands (collected at week 7) from six subjects were each
segmented into 2-mm lengths and analyzed for OFLX. Error bars along
the x-axis re- flect the standard deviation of the range of
location (ram) from the hair root in which maximum OFLX
concentrations were detected. Error bars along the y-axis reflect
the standard deviation of the range of maximum OFLX concentration
measured in the four hair strands.
Table II. Variation in Growth Rate of Individual Hair Strands
Subject ID
Location of Maximum Estimated OFLX Concentration Growth Rate (Range
of Segments)* (cm/month) f
#1 #5-#8 0.61-0.98 (Female; Asian; Black Hair)
#2 #4-#5 0.49-0.61 (Mate; African-American; Black Hair)
#3 #5-#7 0.61-0.85 (Male; Caucasian; Brown Hair)
#4 #5-#7 0.61-0.85 (Male; Caucasian; Brown Hair)
#5 #4-#8 0.49-0.98 (Male; Caucasian; Blonde Hair)
#6 #3-#5 0.37-0.61 (Female; Caucasian; Blonde Hair)
* The range of segments in which the maximum OFLX concentration was
measured for four hair strands per subject.
t The estimated hair growth rate, based on the location of maximum
OFLX concentration and the time since OFLX administration (49 days;
see text for details).
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Journal of Analytical Toxicology, Vol. 27, April 2003
weeks and EUM was noted (Figure 1). The independent variable (EUM
concentration) accounted for approximately 72.8% of the variance in
the dependent variable (OFLX, r z adjusted = 0.728,p < 0.001).
The adjusted r z was selected to prevent an "in- flation" of r from
overfitting the data. Therefore, the concen- tration of eumeianin
in hair does appear to be associated with the amount of OFLX
incorporated into hair. Subjects with the darkest hair (confirmed
by eumelanin concentrations) incor- porated more drug than
individuals with lighter-colored hair. The effect of gender and age
were also evaluated; however, no as- sociation was observed for
these two variables with OFLX con- centration in hair.
Our data also indicate, however, that eumelanin is not the only
factor involved OFLX incorporation because all of the vari- ability
in OFLX concentration could not be explained by the subject's
eumelanin concentration. Some of the variability may be explained
by limitations of the analytical procedure for eu- melanin itself.
The results of the analytical procedure are based on the assumption
that tyrosinase-produced synthetic melanins are accurate and
reproducible models of in vivo biological melanins. As the exact
three-dimensional structure of eumel- anin is unknown (62), the
effect of potential differences be- tween synthetic and biological
melanins in this study cannot be determined.
Also, it is highly probable that there are other regions of
drug-binding to hair components that have yet to be eluci- dated.
It is known that basic drugs can be incorporated into non-pigmented
hair, albeit at lower concentrations than in pig- mented hair
(33--44). Despite basic structural similarities among all hair
types regardless of hair color, there do appear to be some
differences in the chemical and physical characteristics of ethnic
hair types together with considerable intra-ethnic variation
(63,64). Differences include such factors as the diam- eter of the
hair shaft, degree of medullation, curvature of the hair shaft,
crimp, cross-sectional shape, protein content, and follicular form,
to name a few. It is possible that differences in the
ultra-structure, morphology, and protein content and struc- ture
between hair types may play a lesser role in the binding of OFLX to
hair.
In six subjects, we determined the intrasubject variability of OFLX
incorporation into individual hair strands (Figure 2). Four entire
hair strands from each subject were segmented into 2-mm segments,
and each segment was analyzed for OFLX. The maximum OFLX
concentration (the '~and" of drug) and lo- cation were then
determined for each strand. The mean max- imum OFLX concentration
in the 2-ram hair segments (• 1 SD) ranged from 0.55 (• 0.25) to
6.74 (• 1.55) ng/mg. The max- imum OFLX concentration was measured
in segments #2--#5 at week 5 for the six subjects (within the first
centimeter of hair length). The maximum OFLX concentration was
measured in segments #3--#8 at week 7 (within the first and second
cen- timeter of hair). Assuming a hair growth rate of 1.0 cm/month,
and noting that the last hair specimen was collected 7 weeks after
OFLX administration, we hypothesized that OFLX would be detected
only in the first and second centimeters (the 20-mm closest to the
scalp) of hair of most subjects. As expected, our data demonstrate
that no significant axial diffusion of OFLX along the hair shaft
occurred during the period of time en-
compassed by this study. This suggests that normal hygienic
practices (e.g., regular hair washing) do not result in movement of
the OFLX along the hair shaft.
We also explored whether the time of OFLX administration could be
reasonably extrapolated from the location of the max- imum measured
OFLX concentration at week 7 (Table II). The range of location for
the mean maximum OFLX concentration for each of the six subjects
was as follows: Subject #1 (sag- ments #5--#8/~ 10-16 mm); Subject
#2 (segments #4-#8/~8--10 ram), Subject #3 (segments #5-#7/~10-14
turn), Subject #4 (segments #5-#7/~10-14 ram), Subject #5 (segments
#4--#8/~8-16 rnm), and Subject #6 (segments #3--#5/~6-10 ram). If
an estimated hair growth rate of 1.0 cm/month (or ~0.033 crrgday
for an average of 30 days) was assumed, then the maximum OFLX
concentration would be expected to be lo- cated at about 16 mm at 7
weeks (49 days after receiving the dose). However, as shown in
Table II, the estimated hair growth rate for individual hair
strands in these six subjects varied con- siderably and was
generally less than 1.0 cm/month. For the six subjects in this
study, a 3-cm hair segment represents a much longer 'Window of
time" (> 3 months) than would be predicted using an assumed 1.0
cm/month growth rate. The data from our study suggest that it may
not be possible to determine the use of another drug to within a
time frame of one month; however, our study was not designed to
specifically address this question. It should be also be noted that
these findings are based on a lim- ited number of subjects, as well
as a limited number of hair strands per subject. The wide range in
growth rate estimates for any single subject may be partly
explained by the stage of hair growth of the individual hair
follicles during the period of drug administration. For example,
anagen-phase (actively growing) hair may incorporate drug more
readily than hair in catagen- or telogen-phases of the hair growth
cycle (3).
The variability in growth rate in hair specimens from this study is
inconsistent with that obtained in earlier studies of multiple-dose
OFLX administration (51-53). These studies in- dicated an estimated
mean hair growth rate (• 1 SD) of 1.12 • 0.11 cm/month. However,
there are numerous differences be- tween the study designs that may
contribute to the differences observed between the studies. These
differences include method of hair collection (plucking versus
cutting), inclusion of hair bulb in hair length estimates, gender
of individuals studied, eth- nicity, hair color, OFLX dose, and
frequency of OFLX adminis- tration. These discrepancies suggest
that a more detailed ex- amination of the variability in hair
growth rates and the use of OFLX as a reference marker must be
explored. Such an assess- ment is necessary to enable the
toxicologist to provide more ac- curate estimates of potential drug
exposure or use. The au- thors are currently conducting a study
with controlled administration of OFLX and codeine, at various time
intervals, to determine whether OFLX can establish reference points
for the time of codeine use within the hair shaft.
OFLX was readily detected in the hair of all subjects, regard- less
of hair color, and did not diffuse along the hair shaft inde-
pendent of natural hair growth. This demonstrates that OFt,X, and
perhaps other fluoroquinolone antimicrobial compounds, can be
useful "time markers" in hair under certain conditions. As
discussed previously, these compounds are attractive for use
153
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Journal of Analytical Toxicology, Vol. 27, April 2003
as biomarkers because they are present in high concentrations in
hair, have minimal adverse pharmacologic effects, and have limited
abuse liability and potential for passive exposure from the
environment. However, OFLX is one of the most widely used
fluorquinolones for the treatment of infections (65). Pho-
totoxicity and hypersensitivity reactions have been reported, al-
though they are less likely to occur with single doses. Another
important limitation is that OFLX-resistant microorganisms may
emerge during fluoroquinolone therapy. Overuse and in- appropriate
use of fluoroquinolones can erode their clinical utility for the
treatment of future infections. Therefore, the benefit of a single
dose of OFLX, given to otherwise healthy in- dividuals, at
intervals of several weeks must be carefully weighed against the
potential risk(s) associated with antimicrobial use.
Ideally, the search for suitable alternatives for use in clinical
hair studies will continue.
Conclusions
OFLX was detected in human hair after a single therapeutic dose to
normal volunteers. Also, OFLX was only detected in the first 3.0 cm
closest to the scalp through week 7 of the study, in- dicating a
lack of axial diffusion of OFLX along the hair shaft. Hair color,
as determined by the total eumelanin content (in pg/mg), was
associated with the incorporation of OFLX into human hair. Despite
this significant effect of hair color, these data strongly suggest
that OFLX is a suitable marker substance for hair because it is
readily detected in hair of all colors. The use of compounds such
as OFLX may permit the evaluation of treatment compliance and
recidivism in drug treatment pro- grams, allowing a subject to
serve as their own "control". Fur- ther studies are needed to
determine whether OFLX can be used as a marker substance so that
determination of other drug use (such as illicit drugs) can be
determined.
Acknowledgments
This work was supported by National Institutes of Health Grant
DA09096.
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