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 interpret drug concentrations in hair, however, is uncertain, and there is conflicting evidence regarding the utility of hair analysis 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 positive 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 compliance 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 assessment 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 administration. Previous research demonstrated that OFLX could be detected in hair after therapeutic use and multiple-dose administration (50-55). Uemtasu et al. (50) first suggested the possible
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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 suggested 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 administration, 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 solutions of OFLX were used to fortify human hair at final concentrations 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 divided 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, approximately 10 hairs from each subject were segmented into 3.0-cm segments. Thus, each 3.0-cm segment (sample) consisted of approximately 10 hairs.
Ofloxacin concentrations in hair were determined by a modification of the method of Mizuno et al. (57). Internal standard (25 ng/mg DS-4632) was added to a l-rag sample from each segment. A Cahn TA4100 electrobalance was used to weigh specimens (+ 1.0% tolerance). Standards and quality control specimens containing hair fortified with known amounts of OFLX were included with all assays as described. Samples were completely 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 distilled 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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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 mobile phase; 50 IJL was injected for HPLC analysis. Plasma specimens 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 internal standard prior to degradation. Complete details of the modified method used for this study have been previously published (59).
LC conditions for eumelanin analysis. Quantitation of eumelanin 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 consisted 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 administration and whether its detection within the hair shaft followed 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 concentration. 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 beyond 3 cm during the study.
Although OFLX was readily measured in hair, a large interindividual variability in measured OFLX concentration was observed. Previous evidence has suggested that that hair pigmentation may be an important factor in the incorporation 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 concentrations of OFLX explain the variability in hair concentrations.
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 eumelanin 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 eumelanin 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 previously 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 reflect 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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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 concentration 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) incorporated 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 concentration in hair.
Our data also indicate, however, that eumelanin is not the only factor involved OFLX incorporation because all of the variability 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 eumelanin 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 eumelanin is unknown (62), the effect of potential differences between 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 pigmented 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 structure 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 location were then determined for each strand. The mean maximum OFLX concentration in the 2-ram hair segments (• 1 SD) ranged from 0.55 (• 0.25) to 6.74 (• 1.55) ng/mg. The maximum 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 centimeter 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 maximum 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 limited 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 between 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 administration. These discrepancies suggest that a more detailed examination of the variability in hair growth rates and the use of OFLX as a reference marker must be explored. Such an assessment is necessary to enable the toxicologist to provide more accurate estimates of potential drug exposure or use. The authors 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, regardless of hair color, and did not diffuse along the hair shaft independent 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
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, although 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 individuals, 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, indicating 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 programs, 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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Ofloxacin as a Reference Marker in Hair of Various Colors

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