Norbuprenorphine in Human Hair After Multiple Doses. Diana G. Wilkins 1,', ... For this study, it was assumed that the mean hair growth rate was 1.0 cm/month.
lournal of Analytical Toxicology, Vol, 23, October 1999
A RetrospectiveStudy of Buprenorphineand Norbuprenorphine in Human Hair After Multiple Doses Diana G. Wilkins 1,', Douglas E. Rollins 1, Angelique S. Valdez 1, Atsuhiro Mizuno 1, Gerald G. Krueger 2, and Edward J. Cone 3
ZCenter for Human Toxicology,Departmentof Pharmacologyand Toxicology, Universityof Utah, 20 S 2030 East,Room490, Salt LakeCity, Utah 84712;2Division of Dermatology,School of Medicine, Universityof Utah, Salt Lake City, Utah 84172; and 3IntramuralResearchProgram,National Instituteon Drug Abuse, Baltimore,Maryland 21146
Abstract
Introduction
The analysisof hair has been proposed as a tool for monitoring drug-treatment compliance. This study was performed to determine if buprenorphine (BPR) and norbuprenorphine (NBPR) could be detected in human hair after controlled administration of drug and to determine if segmental analysis of hair was an accurate record of the dosing history. Subjects with dark hair (six males, six females) received 8 mg sublingual BPR for a maximum of t 80 days. Single hair collections were made once after BPR treatment and stored at -20~ until analysis. Hair was aligned scalp-end to tip and then segmented in 3-cm sections. For this study, it was assumedthat the mean hair growth rate was 1.0 cm/month. Deuterated internal standard was added to hair segments(2-20 mg of hair) and digested overnight at room temperature with 1N NaOH. Specimenswere extracted with a liquid-liquid procedure and analyzed by liquid chromatography-tandem massspectrometry. The limits of quantitatinn for BPR and NBPR were 3 pg/mg and 5 pg/mg, respectively, for 20 mg of hair. BPR and NBPR concentrations were highest for all subjects in hair segmentsestimated to correspond to the subject's period of drug treatment. With one exception, NBPR was present in higher concentrations in hair than was the parent compound. BPR concentrations in hair segments ranged from 3.1 pg/mg to 123.8 pg/mg. NBPR concentrations ranged from 4.8 pg/mg to 1517.8 pg/mg. In one subject, BPR and NBPR were not detected in any hair segment. In some subjects, BPR and NBPRwere detected in hair segmentsthat did not correspond to the period of drug treatment, suggestingthat drug movement may have occurred by diffusion in sweat and other mechanisms.The data from this study also indicate that there is a high degree of intersubject variability in measured concentration of BPR and NBPR in hair segments, even when subjects receive the same dose for an equivalent number of treatment days. Future prospective studies involving controlled drug administration will be necessaryto evaluate whether hair can serve as an accurate historical record of variations in the pattern of drug use. * Addresscorrespondenceto DianaG. Wilkins, Ph.D.,Centerfor HumanToxicology,20 South 2030 East,Room490, Universityof Utah, Saltlake City, Utah84112.
A potential use of hair analysis for drugs is the monitoring of treatment compliance and recidivism. Hair may serve as a historical record of drug exposures (1-3). It has been proposed that knowing the rate of hair growth in an individual and the location of the drug along the long axis of the hair shaft will allow determination of the approximate time of drug use. Treatment compliance would be indicated by the detection of therapeutic drug in hair segments corresponding to periods of therapeutic-drug treatment. Noncompliance in a drug-treatment program would be determined by gaps in the detection of the therapeutic drug in hair segments predicted to contain drug. Drug recidivism would be detected by finding an abused drug in a hair segment that corresponds to a supposed abstinence period. Improved methods for determining patient compliance over time would be very useful to clinicians because drug concentrations in plasma, urine, and saliva often reflect only the dosage taken within the last several hours to days prior to sampling. However,there are conflicting data regarding the utility of hair analysis for drug monitoring (4-23). In the United States, buprenorphine (BPR) is a drug currently under investigation for the treatment of heroin addiction and is of interest for compliance-monitoring purposes. BPR is a p opioid receptor partial agonist reported to produce minimal withdrawal symptoms, low potential for overdose, long duration of action, and the ability to block heroin effects (24). BPR has been demonstrated to be metabolized to buprenorphineglucuronide and partly to N-dealkylated product, norbupenorphine (NBPR) (25,26). Measurement of these drug metabolites in hair may be useful to determine that an actual ingestion, rather than external contamination of the hair from the environment, has occurred (1). If hair analysis is to have utility for compliance- or recidivism-monitoring purposes, then certain fundamental principles of drug disposition in hair, such as the predictable movement of a drug such as BPR along the hair shaft, must be demonstrated.
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Journal of Analytical Toxicology, Vol. 23, October 1999
This study was performed to determine if BPR and NBPR could be detected in human hair after controlled administration of drug and to determine if segmental analysis of hair was an accurate record of dosing history. Analysis of hair segments was performed to determine if the presence of drug in individual segments correlated with the period of drug treatment.
Experimental
Chemicals and reagents for analytical methods BPR, NBPR, and BPR-d4 reference materials were obtained from Radian Corp. (Austin, TX). NBPR-d4was obtained from Isotec Inc. (Miamisburg,OH). Separate lot numbers and weighings of reference materials from Radian or Isotec of BPR and NBPRwere used for preparing quality-control samples for accuracy evaluation. Methanol, n-butyl chloride, and acetonitrile (high-performance liquid chromatography [HPLC] grade) were obtained from Burdick & Jackson Co. (Muskegon, MI); nitrogen was obtained from Mountain Alrgas, Inc. (Salt Lake City, UT). All other reagent-grade chemicals were obtained from Mallinckrodt Chemical Works (St. Louis, NO). All drug solutions were
prepared in HPLC-grade methanol or distilled water (MilliQ| Milford, WI), as necessary. HPLC-gradesolvents were used in extraction procedures. Hair experiments Collection of hair. l~velve healthy volunteers with a history of drug use participated in an outpatient treatment study conducted at the Division of Intramural Research, NIDA.Medical screening and psychological testing were performed to ensure that subjects were healthy prior to participation in the study. All subjects provided informed consent and were paid for their participation. Six males and six females with dark hair received 8 mg sublingual BPR daily for up to 180 days. A single lock of hair was collected from each subject (see Table I) and stored at -20~ until analysis. Hair was collected based upon subject availability. Therefore, some subjects had specimens collected during the treatment interval and others after completion of treatment. Segmentation of hair. Hair was aligned scalp-end to tip and segmented in 3-cm sections. Itwas assumed that the mean hair growth rate was 1.0 cm/month. Therefore,it was estimated that a single 3-cm segment corresponded to an approximately three-month time frame. The first seven segments (or 21 cm) closest to the scalp were analyzed for BPR and NBPR.Hair segments beyond the first 21 cm of hair length were not analyzed in this study.
Table I. Subject Demographic and Treatment Information Numberof Date of Subject Age treatment Treatment hair ID Ethnicity* Gender (years) days periodf collection A
C
F
27
180
B
C
M
35
180
C
C
F
26
180
D
C
M
25
180
7/5/8912/31/89 3/24/899/19/89 11/I/894/29/90 10/5/88-
Hair length (cm)
4/5/90
> 21.0
4/2/90
7.0
4/2/90
> 21.0
5/14/90
> 21.0
11/14/89- 4/3/90 5/12/90 1/20/89- 4/16/90 7118189 7 / 1 9 / 8 9 - 4/30/90 12/5/89 9/20/894/9/90 1/30/90 11/22/89- 5/14/90 2/4/90 5/30/895/7/90 8/4/89 5/28/895/7/90 7/6/89 11/1/894/6/90 12/I 0/89
7.4
4/2/89 E
C
M
32
180
F
C
F
38
180
G
C
M
39
135
H
A
F
32
133
I
C
F
26
74
J
C
M
31
67
K
C
M
35
42
L
C
F
32
40
* Abbreviations: C = Caucasian, A = African American. f "Treatment period" refers to the period of time across which buprenorphine was administered.
410
13.3 10.9 > 21.0 > 21.0 12.2 11.4 > 21.0
Liquid chromatography-tandem mass spectrometry of BPR and NBPR in hair
Hair specimens were analyzed using a previously described liquid chromatographytandem mass spectrometry (LC-MS-MS) survivor ion procedure (27). Briefly, 5 ng/mg of deuterated internal standard was added to 20 mg of hair. Specimens were solubilized (digested) overnight at room temperature in 2 mL of 1N NaOH. The following day, the pH was adjusted to 10.5 and digests buffered with 1 mL of sodium bicarbonate buffer. Digests were then extracted with n-butyl chloride/acetonitrile (4:1, v/v), evaporated to dryness, and reconstituted in 75 IJL of water/acetonitrile (2:1) prior to analysis. Extracts were analyzed on a Finnigan TSQ~M7000with atmospheric pressure ionization (27,28). Chromatographic separation was achieved on an Alltech C8 Solvent Miser| column (2.1 x 150 ram) with an isocratic mobile phase of H~O/MEOH/ACN (25:30:45) containing 1% formic acid. The MS was operated in MS-MS mode with a collision energy of-25 eV and 2.5 retort argon collision gas pressure. Both Q1 and Q2 were set to monitor ions at rn/z 468 (BPR) and 414 (NBPR) (27). Standards and quality-control specimens were concurrently extractedwith all assays and prepared by fortifying drug-free human hair with known amounts of BPR and NBPR. Be-
Journal of Analytical Toxicology, Vol. 23, October 1999
cause it is unknown whether fortified hair standards and quality controls adequately evaluate the effectivenessof the digestion process (i.e., release of incorporated drug from hair shaft structures), additional samples were included in some assays. These samples consisted of hair from rats who had received intraperitoneal BPR once per day for five days and were used to verify reproducibility of the assay. Quantitative data for these specimens have been previously published (29). Also, quality-control specimens consisting of human hair fortified with BPR (only) were prepared to assess whether BPR was converted to NBPRduring digestion, extraction, and analytical procedures. Data from these quality-control specimens indicated that no conversion (degradation) of BPR to NBPR occurred during these procedures. The limits of quantitation for BPR and NBPRby LC-MS-MS were 3 pg/mg and 5 pg/mg, respectively, for 20 mg of hair. The assay was linear for both BPR and NBPR to 50 ng/mg. Intraassay precision was 12% at 10 pg/mg (BPR) and less than 10% at 100 pg/mg, 1 ng/mg, and 25 ng/mg (BPR and NBPR).
Results Table I presents the available information for subjects with respect to demographic and treatment information. Males (n = 6) and females (n = 6) ranging in age from 26 to 39 years participated in the study. One African-Americansubject and 11 Caucasian subjects were enrolled, and all subjects had dark brown to black hair. Six subjects received 8 mg sublingual BPR for the maximum of 180 days, and six subjects received BPR for less than 180 days. A single lock of hair was collected at least six months after initiation of BPR treatment as indicated in Table I. The column labeled "Treatment period" refers to the period of time across which sublingual BPR was administered. Comparison of the dates of treatment and the date of hair collection demonstrates that at the time of hair collection, some subjects had not received BPR for several months and other subjects were still actively receiving BPR. Detailed data regarding hygiene and chemical treatments for each subject were not available at the time of collection. Figures 1A-1Lpresent the segmentation data obtained from each of 12 subjects participating in the study. Three-centimeter segments of hair were analyzed, and quantitative data for BPR and NBPR were obtained for each segment. Bar graphs were constructed so that hair segment 1, closest to the scalp at the time of collection, is closest to the origin. Based on the dates between which treatment occurred (see Table I), the predicted tocation of drug is indicated immediately below the bar graph. In 5 of the 12 subjects (A, B, C, H, and J), the first 3 cm of hair closest to the scalp was not available for analysis. This first segment had been previously consumed in other drug analyses and insufficienthair remained for the analysis of BPR. These unavailable hair segments are indicated in Figures 1A-1Las "NA." The data represented in the bar graphs in Figure I demonstrate that both BPR and its metabolite NBPRwere detected in 11 of 12 subjects who were known to have received BPR therapy. In these subjects, BPR and NBPR concentrations were
greatest in hair segments corresponding to the period of active drug treatment. However, in 9 of 12 subjects (A, B, C, D, E, G, H, I, and K), parent drug or metabolite was also detected in hair segments that did not correspond to the period of drug treatment. After multiple 8-rag sublingual doses of BPR, hair concentrations of BPR ranged from 3.1 pg/mg to 123.8 pg/mg. NBPR concentrations ranged from 4.8 pg/mg to 1517.8 pg/mg. With one exception (B), NBPR was always present in higher concentrations in individual hair segments than was the parent compound. One subject (F) did not have BPR or NBPR in any hair segment, despite the known dosing history. A high degree of intersubject variability in measured concentration of BPR and NBPRwas observed, even when subjects received the same 8-rag dose for an equivalent number of treatment days.
Discussion BPR or NBPRwas detected in 11 of 12 subjects who had received controlled 8-rag doses of sublingual BPR in a treatment setting. The order of magnitude of our quantitative results (picograms-per-milligram range) for these 11 subjects is consistent with those previously reported by other investigators (30,31). The highest concentrations of BPR and NBPR were detected in hair segments estimated to correspond to the period of active drug treatment (see solid bars under bar graphs in Figure 1). In contrast to earlier reports, however, we found that the NBPR metabolite was detected in hair in higher concentrations than was the parent BPR compound. When both BPR and NBPR were present in hair segments corresponding to periods of active drug treatment, a ratio of at least 1:3 was observed. Only 1 of these 11 subjects (B) did not demonstrate this ratio for the period of active drug treatment. It has been suggested that at least one of the major routes of drug incorporation into hair is via distribution from plasma into growing hair cells (1,2). Recent data from Kuhlman et al. (32) demonstrated that NBPR accumulates in plasma after chronic administration of BPR and that the plasma concentrations exceed those of the parent BPR compound. The mean steady-state BPR plasma concentration after daily administration of sublingual BPR for 21-35 days was 0.8 ng/mL. The mean steady-state NBPR plasma concentration was 1.10 ng/mL. These findings may help explain the ratio of parent drug and metabolite obtained in hair specimens from our study. The inconsistency between the data in our study and those of previous investigators may be explained by the difference in sublingual BPR doses received by subjects. The subjects in our study may have received substantially higher daily sublingual doses of BPR than the subjects in the earlier reports. There may also have been a difference in the number of days of drug treatment prior to collection of hair specimens. Because the previous reports did not include specific information for each subject with respect to doses received or time of hair collection, we are unable to evaluate these differences further. Additional studies on the pharmacokinetics of BPR and its
411
Journal of Analytical Toxicology, Vol. 23, October 1999
metabolites with respect to hair and plasma are necessary to elucidate the pattern and quantitative distribution of BPR and NBPR into hair. Another possible explanation for our finding of higher NBPR concentrations is related to the stability of specimens upon prolonged storage. Our data demonstrate that BPR and NBPR can be readily detected in individual hair segments despite Subject
prolonged periods of storage. However, the locks of hair in this study were stored at -20~ for up to seven years prior to analysis, and there are no published studies on the effect of storage conditions on the stability of BPR and its metabolites in hair. It is unclear to what extent this may have affected our quantitative results. Our laboratory is currently conducting a prospective study of sublingual BPR administration, with spec-
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Figure 1. Bar graphs of buprenorphine and norbuprenorphine concentrations in individual hair segments after multiple doses. Lightly hatched bars indicate measured hair concentrations of buprenorphine. Densely hatched bars indicate measured hair concentrations of norbuprenorphine. The intersection of the x and y axes indicate the date of hair collection; therefore, the end of the hair closest to the scalp is closest to the origin. The predicted location of drug in the available hair specimen is indicated immediately below the bar graph.
412
Journal of Analytical Toxicology, Vol. 23, October 1999
imens analyzed for BPR and NBPRafter a few weeks of storage. Although that study is still in progress, data for the first four subjects indicate that the distribution and quantitative values of BPR and NBPR are consistent with those observed in this retrospective study (33); NBPR hair concentrations are consistently greater than those of the parent compound. These data indicate that degradation of BPR to NBPR due to pro-
longed storage is an unlikely explanation for our findings. Hair specimens in this study were not washed immediately prior to analysis. Subjects did, however, perform normal hygienic practices such as hair washing over the weeks and months encompassed by this study. It is possible that the lack of a laboratory-based hair-washing procedure may have contributed to our finding of higher NBPR concentrations than
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Figure 1. (continued) Bar graphs of buprenorphine and norbuprenorphine concentrations in individual hair segmentsafter multiple doses. Lightly hatched bars indicate measured hair concentrations of buprenorphine. Densely hatched bars indicate measured hair concentrations of norbuprenorphine. The intersection of the x and y axes indicate the date of hair collection; therefore, the end of the hair closest to the scalp is closest to the origin. The predicted location of drug in the available hair specimen is indicated immediately below the bar graph.
413
Journal of Analytical Toxicology,Vol. 23, October 1999
BPR in hair, assuming that there is a differential effect of removal between BPR and NBPR. Because of this possibility, a very brief evaluation was conducted on the effect of two different wash procedures on quantitative results of NBPR in human and rat hair. Wash condition A consisted of 2 mL of methanol; wash condition B consisted of 2 mL dry isopropanol, followed by 2 mL of phosphate buffer (pH 5.6). Hair specimens were washed under one of each of these conditions until no more BPR or NBPR could be detected in the wash solution. Depending on the wash condition, BPR and NBPR concentrations in human hair decreased from 12.9% to 100.0%. Rat hair specimens showed a similar pattern of decrease. NBPR concentrations in human hair remained higher than those of BPR. Therefore, although the inclusion of a wash procedure may have decreased the overall quantitative results, it is unlikely to be an explanation for the high NBPR concentrations. Nine of 12 subjects (A, B, C, D, E, G, H, I, and K) had BPR or NBPR in hair segments that did not correspond to the period of drug treatment. There are two probable interrelated explanations for these findings. First, a major assumption was made in estimating the hair growth rate at 1.0 cm/month for the preparation of these bar graphs. According to available literature, hair growth rates for scalp hair can actually vary between 0.6 and 3.36 cm/month (34,35). Because of this variability, it is possible that the hair segments immediately before (indicating more slowly growing hairs) and immediately after (indicating more rapidly growing hairs) those predicted for the period of drug treatment may contain some drug or metabolite. We then re-examined our data, excluding these hair segments, to determine which subjects would continue to have segmentation results that were inconsistent with the dosing history. In this case, five subjects (A, C, D, H, and I) still had BPR or NBPR detected in hair segments that did not correspond to the periods of active drug treatment. These data suggest that a second explanation is also involved.Drug movement may occur in hair via diffusion in sweat and by other mechanisms, such as movement of compounds within the hair shaft as a result of normal hygienic practices. Regardless of the mechanism involved, the detection of parent compound or metabolite in hair segments that do not correspond to the period of drug treatment is of concern. Finally, a high degree of intersubject variability in measured concentrations of BPR and NBPR was observed in 3-cm hair segments, even when subjects received the same 8-rag dose for an equivalent number of treatment days. We cannot totally eliminate the possibility that BPR was ingested by some subjects from sources outside the residential unit, particularly after discharge from treatment. However, there were no "takehome" medication privileges during the study, and the availability of outside sources of BPR was highly unlikely in the United States at the time of the study. The large variability in measured hair concentrations might also be due to the differences in the length of time between which BPR administration was discontinued and hair specimens were collected (see Table I). Also, there may have been subtle differences in the collection process with respect to the distance from the scalp (millimeters) at which hair was cut. In addition, animal studies have suggested that the deposition of BPR into hair is greater
414
in pigmented than nonpigmented hair (29). It is possible that the variable melanin content in dark-colored human hair from subjects in our study may have influenced the deposition of buprenorphine into hair. As suggested by many authors, other biological, physiological, and environmental factors may also play a role in affecting the amount of drug incorporated into hair (1,2,36,37). Other probable sources of variability include differences in hair structural characteristics, drug metabolism, contribution from sweat, or other routes of drug incorporation into hair. The high degree of variability in BPR and NBPR concentrations in hair segments will make it difficult to interpret time- and concentration-relationships in human hair.
Conclusions The data from this retrospective study demonstrate that BPR and NBPR can be detected in human hair segments after an 8-mg sublingual dose with metabolite concentrations greater than those of parent compound. The highest concentrations of BPR and NBPR were detected in hair segments estimated to correspond with a period of active drug treatment. However, BPR and NBPR were also detected in hair segments that did not correspond to periods of drug treatment, suggesting that drug movement may occur in hair via diffusion in sweat and by other mechanisms. The limitations of this retrospective study preclude a definitive explanation for these observations. Future prospective studies involving controlled drug administration will be necessary to evaluate whether hair can serve as an accurate historical record of variations in the pattern of drug use. Such studies will be also be needed to determine if hair analysis can be useful for monitoring treatment compliance.
Acknowledgments The authors wish to thank Dr. John Laycock and Mr. Alan Spanbauer for their valuable assistance in the analysis of buprenorphine specimens. This research was supported by NIDA Grant No. DA09096.
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Manuscript received March 4, 1999; revision received May 21, 1999.
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