Time-Course Mass Spectrometry Imaging for ... - ACS Publications

Apr 28, 2015 - ... Imaging for Depicting Drug. Incorporation into Hair. Tooru Kamata,. †. Noriaki Shima,. †. Keiko Sasaki,. †. Shuntaro Matsuta,...
0 downloads 0 Views 5MB Size
Download PDF
Editors' Highlight pubs.acs.org/ac

Time-Course Mass Spectrometry Imaging for Depicting Drug Incorporation into Hair Tooru Kamata,† Noriaki Shima,† Keiko Sasaki,† Shuntaro Matsuta,† Shiori Takei,† Munehiro Katagi,† Akihiro Miki,*,† Kei Zaitsu,‡ Toyofumi Nakanishi,§ Takako Sato,∥ Koichi Suzuki,∥ and Hitoshi Tsuchihashi∥ †

Forensic Science Laboratory, Osaka Prefectural Police Headquarters, 1-3-18 Hommachi, Chuo-ku, Osaka 541-0053, Japan Department of Legal Medicine & Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan § Department of Clinical and Laboratory Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan ∥ Department of Legal Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan ‡

S Supporting Information *

ABSTRACT: In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization− time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freezesectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2− 3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand’s growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.

H

more) strands in the resting stage, due to the growth rate variability of up to 40% (within subject)14 and inexact alignment of the strands in hair sampling.6 In earlier studies we have thus reported single hair analyses of drugs, such as methamphetamine (MA), by matrix-assisted laser desorption/ionization (MALDI)-imaging MS (IMS),15,16 as well as by strand-by-strand conventional sectional analysis by LC−MS/MS using one-pot micropulverized-extraction.12,17 The application of IMS to hair analysis for drugs has later been expanded to the detection of other drugs, such as cocaine,18,19 ketamine,20 and zolpidem.12,21 For the investigation of drug incorporation into hair, several experimental results have been reported about the time course of drug/metabolite concentration in daily shaved beard hair of

air is often compared to a tape recorder for drug use history. Hair drug test results have recently appeared more frequently in court, but the drug incorporation pathways into hair are still under much discussion among forensic toxicologists.1−3 Biosynthetic incorporation of the ingested drug directly from the bloodstream into the hair matrix cells in hair bulbs has been the popular explanation,4 but many reports have suggested the contribution of diffusion from skin tissue, sebum, and sweat.5−8 Hair analysis for drugs has conventionally been conducted by determining analytes in the extracts of hair specimens, primarily using gas chromatography/mass spectrometry (GC/MS)9,10 or liquid chromatography−tandem mass spectrometry (LC−MS/ MS).11,12 Analyses for a series of divided hair segments, from the root to the tip (typically cut into 2 cm segments using a tuft of more than 50 strands) can provide chronological information on drug intake.13 In a hair tuft, however, the time resolution is seriously deteriorated by the presence of 5−15% (or even © XXXX American Chemical Society

Received: March 12, 2015 Accepted: April 28, 2015

A

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Editors' Highlight

volunteers after a single dose of a drug/substance, such as methoxyphenamine (MOP),22 codeine,23 and ethanol.8 These reports discussed about drug incorporation based on the anatomy of the hair root and on the physiology of hair growth. However, unavoidable contamination of shaved powdered beard hair with skin scurf that may contain the drug, as well as variability of growth rate of each strand hair seems to obscure their results and its interpretation. This study was designed to depict the incorporation of ingested drug into scalp hair by IMS. Experiments were performed by selecting MOP, a nonregulated o-methoxy analogue of the potent illicit drug MA, as a model drug. MALDI-time-of-flight (TOF) IMS was performed in the TOF/ TOF mode on the longitudinal sections of single scalp hair shafts plucked with the roots intact, at regular time intervals, from five volunteers after a single oral administration of MOP. To effectively detect the drug inside the hair shafts, a sample preparation method that allows accurate longitudinal sectioning of single hair specimens was established and utilized. In addition, conventional sectional hair analysis was performed for each 1 mm segment of single hair specimens, using a validated LC−MS/MS procedure, to support quantitative aspects of IMS results. On the basis of these results, forensically crucial issues, including the site of drug incorporation into hair and time resolution in estimating drug use history, are investigated and discussed.

Information Figure S-1 illustrates lengthwise cutting of a single hair shaft by this method. Matrix Deposition. Digital images of the lengthwise-cut hair shafts were acquired using a flatbed scanner prior to matrix deposition. A 7 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in an acetonitrile−0.2% trifluoroacetic acid aqueous solution (1:1, v/v) was applied as a matrix solution. Multiple layers of matrix were automatically deposited onto the samples, over about 120 min, using an ImagePrep (Bruker Daltonics, Bremen, Germany). MALDI-TOF-MS Imaging. MALDI-TOF-IMS analyses were performed on the TOF/TOF mass spectrometer ultrafleXtreme (Bruker Daltonics), which utilizes a Smartbeam-II solid-state laser (wavelength, 355 nm; focus setting, “large” (actual diameter, about 90 μm); repetition rate, 1000 Hz). For the positive reflector ion mode, condition settings were as follows: accelerating potential, 19 kV; reflector voltage, 21 kV. External mass calibration was performed on the basis of [M + H]+ and [2M + H]+ ions of CHCA at m/z 190.050 and m/z 379.092, respectively. TOF/TOF data sets were acquired using collision induced dissociation (CID) under the following conditions: parent mass, m/z 180.1; fragment mass, m/z 149.1, for MOP. For imaging acquisition, a 100 μm raster width was selected and 500 individual spectra were acquired at a repetition rate of 1000 Hz and accumulated for each pixel. Image analysis and data visualization were performed using flexImage 4.0 software (Bruker Daltonics). Segmental Analysis by LC−MS/MS. Sample Preparation. Single hair specimens were individually analyzed as follows: A single hair shaft was wiped gentry with tissue paper moistened with distilled water. The hair was then divided into ten 1.0 mm segments from the root-side end, and the remaining peripheral portion was discarded. Each 1 mm segment was pulverized (5 min, 1500 strokes/min) and simultaneously extracted with 50 mM acetate buffer (pH 5.0, 0.2 mL) containing 500 pg/mL m-methoxy methamphetamine as internal standard (IS) in a disposable 2 mL Safe-Lock polypropylene tube (Eppendorf, Hamburg, Germany) applying ultrasonication for 30 min, followed by liquid−liquid extraction with 600 μL of chloroform−isopropanol (3:1, v/v). These processes were successively performed in the 2 mL plastic tube that also contained a round-bottomed-test tube-fit stainless steel bullet, using an “Automill TK-AM5” pulverizer (Tokken, Kashiwa, Japan). The organic layer was separated and evaporated to dryness under a gentle nitrogen stream after adding a mixture of hydrochloric acid ethanol solution (1:20, v/ v, 20 μL), followed by reconstitution with 100 μL of 10% methanol. LC−MS/MS Determination. LC−MS/MS was carried out on a Shimadzu Prominence series LC system linked to an AB Sciex QTRAP 5500 hybrid triple quadrupole linear ion-trap mass spectrometer equipped with an electrosplay ionization (ESI) interface in the selected reaction monitoring (SRM) mode. The analytical conditions were as follows: ESI ionization electrode, a hybrid electrode consisting of a stainless steel tip and PEEKsil (50 μm i.d.; Eksigent, Redwood, CA); ESI positive mode; precursor ions, protonated molecules (m/z 180.1 for both MOP and IS). Nitrogen was used as nebulizer and collision gases, and the operation parameters were set as described in the Supporting Information, Table S-1. LC separation was carried out using an InertSustain C18 semimicro column (50 mm × 1.0 mm i.d., 3 μm particles; GL Sciences, Tokyo, Japan). A volume of 10 μL of the extract solution

■

EXPERIMENTAL SECTION Single Hair Specimens. Five volunteers (four males in their 20s, 30s, 40s, and 50s and one female in her 30s, straight black haired Asian ethnic) who had not come into contact with MOP in the last 6 months were engaged in this study. They orally ingested the over-the-counter cough medicine “Asukuron” (Taisho Pharmaceutical, Tokyo, Japan) containing 50 mg of MOP hydrochloride, once in the morning (or 150 mg within 12 h, at several-day interval between administrations; e.g., every 7 days). All subjects washed their scalp hair with shampoo every night. Hair specimens were collected from the posterior vertex region, at appropriate intervals (e.g., on 0, 1, 2, 3 days and thereafter, 1, 2, 4 weeks after the final dose), by plucking out with the roots intact using tweezers. Hair was carefully plucked while pressing the head skin surface of the hair shaft with tweezers such that location of the head skin and the depth of hair root could be observed and noted. Oral and written informed consent was obtained from the volunteers prior to participating in this study. All protocols were approved by the ethics committee of Osaka Medical College. Sample Preparation for IMS. A single hair specimen was wiped gently with tissue paper moistened with distilled water. The hair was then cut into an appropriate length (∼30 mm) and affixed onto an ITO-coated glass slide to which a 5 mm width double-sided aluminum foil-based conductive adhesive tape (AL-25DC, Sumitomo 3M, Tokyo, Japan; applied with conductive acrylic adhesive material; total thickness 85 μm) had been attached lengthwise. The hair shaft was half-embedded into the conductive tape by pressing with an aluminum cubic block wrapped with a separate film. Several drops of distilled water were dripped onto the fixed hair and this was frozen on the freezing unit EF-22 (Nippon Microtome Laboratory, Osaka, Japan) at −20 °C. Freeze-sectioning was then carefully carried out using a rotary microtome RM-S equipped with a retraction system (Nippon Microtome Laboratory). Supporting B

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Editors' Highlight

(The imaging results for the remaining two volunteers are shown in the Supporting Information, Figure S-3.) IMS data were acquired by monitoring the MOP-specific MS/MS transition (from m/z 180.1, protonated molecule, to m/z 149.1, major product ion shown in Figure 1) in the TOF/TOF mode. Notable MOP-positive areas were observed both in the hair bulb and in the already matured hair fiber in the upper dermis zone (hair shaft under epidermis), for all of the five 24 h specimens (sampled 24 h after intake), though no notable signal was observed for any of the 0 h specimens (sampled immediately before intake). Because the difference in the detectability of MOP between the premature hair bulb and keratinized parts was anticipated, we next examined the qualitative aspect of the MALDI-IMS results by minute segmental hair analyses by LC−MS/MS. LC−MS/MS Analyses of 1 mm Segments of Single Hair Specimens. Segmental analyses of MOP were performed for each 1 mm sections of another 24 h and 7 d single hair specimens corresponding with those analyzed in the imaging study for three of the volunteers engaged in this study. Segmental analyses were conducted using a combination of an optimized LC−MS/MS procedure and a modified one-pot micropulverized-extraction method.12,17 The modification made for the micropulverized-extraction includes liquid−liquid extraction with chloroform−isopropanol (3:1, v/v), evaporation of the separated organic layer to dryness, and reconstitution with a minimum amount of 10% methanol. In achieving segmental hair testing approach, the use of such shorter single hair segments is detrimental to the sensitivity due to the reduced amount of analyte in each 1 mm segment. Therefore, the LC−MS/MS procedure was optimized by minimizing the inner diameters of the LC separation column (reduced to 1.0 mm from a common diameter of 1.5 mm), line tubes (commonly 0.13 mm, reduced to 0.05 mm), the electrospray ionization electrode (from a common diameter of 100 to 50 μm), and the flow rate (commonly 0.1 μL/min, reduced to 0.05 μL/min). Although the injection volume of sample extracts was set at 10 μL, these condition settings allowed for the injections of up to 30 μL sample solutions without any peak deterioration. In the micropulverizedextraction process, only a disposable 2 mL plastic vessel and a new clasher were used to avoid contamination and to maximize recovery. The optimized LC−MS/MS procedure which utilized the micropulverized-extraction process was validated for the determination of MOP in 1 mm segments of single hair shafts. The validation data was given in the Supporting Information, Table S-2. The lower limit of detection was 100 fg MOP/1 mm single hair. The amount of MOP was determined in each 1 mm section of the 24 h and 7 d single hair specimens, using the validated LC−MS/MS procedure. The results are shown in Figure 2B. A typical LC−MS/MS chromatograms obtained from a 1 mm segment of a single hair specimen from a volunteer (subject B, 2−3 mm segment, found to contain 80 pg MOP/1 mm single hair) is shown in the Supporting Information, Figure S-4. A significantly high concentration of MOP was detected in the 0− 1 mm segments of all three 24 h specimens. However, the intensity of MOP detected in the hair bulb region in the imaging of 24 h specimens appears to be weaker than those obtained by 1 mm segmental quantitation. This may be attributed to the difference in surface conditions of hair sections between the premature jelly hair bulb and the keratinized porous hair shaft sections. Except for the matter mentioned

prepared as mentioned above were automatically injected into the instrument. The analytes were chromatographed by linear gradient elution with (A) 10 mM ammonium formate buffer (pH 5) and (B) methanol, at a flow rate of 0.05 mL/min at a column temperature of 40 °C. A gradient was applied starting from 95% A/5% B, linearly increased to 5% A/95% B over 10 min, and held for 10 min.

■

RESULTS AND DISCUSSION Longitudinal Sectioning of Single Hair Specimens. Because most drugs have high affinities to melanin,1−3,6 the in vivo drugs deposition is thought to occur mainly in the melanin-rich cortex and the medulla, rather than in the cuticle junctions.24 Our previous experiments15,16 showed IMS of MA in hair from MA users was only successful when analyzing sectioned hair specimens rather than intact hair covered with repellent cuticle. This is probably due to the above-mentioned reason and better matrix deposition onto the porous hair section, which is crucial in MALDI-IMS. Therefore, in order to effectively perform drugs imaging within the hair shafts, we have established a technique that allows accurate longitudinal cutting of single hair shafts based on freeze-sectioning (Supporting Information, Figure S-1) using a specially customized microtome (equipped with micromanipulators for precise lengthwise cutting and a microscope to monitor the sectioning). A scanning electron microscope image of a longitudinally sectioned hair shaft prepared by this method is shown in the Supporting Information, Figure S-2. MALDI-TOF MS/MS for Detecting MOP in Hair and Its Imaging. Figure 1 shows the MALDI-TOF MS/MS spectra

Figure 1. MALDI-TOF MS/MS spectra for identifying MOP incorporated into hair. (A) MOP-positive and (B) MOP-negative areas of a single hair specimen from subject A who took 50 mg of MOP hydrochloride 14 days before sampling (spots A and B indicated in Figure 2A). (C) Spiked hair with MOP (found to contain 26 ng/mg hair by the conventional LC−MS/MS procedure). Precursor: m/z 180.1 (protonated MOP).

for identifying MOP in a single hair specimen from a volunteer who took 50 mg of MOP hydrochloride (subject A, plucked 14 days after ingestion) and a spiked hair sample containing MOP at 26 ng/mg hair (determined by a routine LC−MS/MS procedure). Figure 2A shows MALDI-TOF MS/MS images of MOP in the longitudinal sections of single hair specimens from three volunteers who orally ingested a single dose of 50 mg of MOP hydrochloride and thereafter sampled at regular intervals. C

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Editors' Highlight

Figure 2. (A) Time-course mass spectrometry imaging of MOP on the longitudinal sections of single hair specimens, plucked from subjects A−C who orally ingested a single dose of 50 mg of MOP hydrochloride. △ indicates the position of the scarp surface. (MALDI-TOF MS/MS spectra obtained at spots A and B indicated on the 14 day specimen of subject A are shown in Figure 1.) (B) Amount of MOP detected by LC−MS/MS in each 1 mm segment of single hair specimens from subjects A−C, plucked 24 h and 7 days after the single oral dose of 50 mg of MOP hydrochloride.

above, the intensity of MOP in the imaging results are well correlated with those of segmental quantitation. Although the specimens analyzed in Figure 2A and those in Figure 2B were identical but different hair strands, the same tendency was observed for the localization of MOP for three subjects tested here. Time-Course IMS of MOP Incorporation into Hair. We then looked into the time-course changes in the imaging results of Figure 2A with segmental quantitation data in Figure 2B. The amount of MOP concentrated onto the hair bulbs of the 24 h strands declined thereafter, and a corresponding

distinctive MOP-positive band, with much reduced intensity (reduced by 68−80%) and tailing, was observed(bulb-origin band) for all five volunteers. This result indicates that only a small portion of MOP initially concentrated onto the bulb becomes incorporated into the hair matrix. Additionally, higher drug concentration in the bulb leads to longer tailing, forming a 2−4 mm drug band. On the contrary, the other remarkable MOP-positive band observed in the upper dermis zone (about 2−4 mm from the hair papilla) of already matured hair fiber primarily formed a band of about 2 mm (dermis-origin band). Slight increases in their lengths by tailing were observed over D

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Editors' Highlight

the first 2 to 3 days for all five volunteers, suggesting that the incorporation of MOP at this region lasted for several days. This is probably related to the fact that drugs are generally detectable in sweat for several days after ingestion, which is much longer than in blood (typically within 1 day).25 However, no MOP-specific ion was detected in the hair shaft outside of the scalp surface (except for the region very close to scalp surface), for all of the 24 h specimens from five subjects. This result suggested that the incorporation of ingested MOP from sweat (by swelling of the hair shaft with sweat containing MOP) was of minor significance, at least in this experiment, which was performed in the spring (average temperature, 20 °C; average humidity, 61%; in Osaka, Japan). This was supported by the results of segmental LC−MS/MS determination for three subjects shown in Figure 2B with a detection limit of 100 fg of MOP in 1 mm single hair segment. No trace of MOP was detected in any of the 5−6, 6−7, 7−8, 8−9, and 9−10 mm segments of 24 h specimens nor in the 7−8 (except for subject A), 8−9, and 9−10 mm segments of the 7 d specimens. Figure 3 shows a schematic diagram of a scalp hair shaft indicated with incorporation sites of MOP. The drug delivered

quantification on days 8 to 10. On the basis of the assumed length of the hair root under the scalp skin (4−5 mm) and the average growth rate of beard hair (0.35 mm/day), they suggested that for beard hair the majority of EtG incorporation is in the upper part of the hair root, although the following drawbacks exist in their experiment: unavoidable contamination of shaved powdered beard hair with skin scurf that may contain EtG and variability of growth rate of each strand hair. Nakahara et al. have also reported similar experiments for MOP in daily shaved beard hair after a single dose of 250 mg of MOP.22 They detected MOP from day 1 through day 10. In consideration of a growth rate of 0.35 mm/day, this leads to a bandwidth of about 3.5 mm, but the same drawbacks seemed to obscure their results. The results from our study revealed the contribution of two comparable incorporation pathways in different regions, which lead to an approximate 6−7 mm length two-peak MOPpositive area as mentioned above. Time Resolution in Investigating Drug-Use History by Hair Testing. Finally, to examine the chronological resolution of the present hair drug analysis, we analyzed single hair specimens from volunteers who took MOP at various intervals. The MOP-positive areas did not separate into each administration at 3-day intervals (data not shown). Figure 4 shows the MS imaging results of MOP in hair specimens sampled from volunteers after oral administrations of MOP at

Figure 3. Schematic diagram of a scalp hair shaft indicated with incorporation sites of MOP.

into the tissue of the upper dermis, where numerous capillary loops of the papillary plexus are distributed for nutrients and oxygen delivery,26 may be the origin of the latter drug band as well as of drugs usually detectable in sweat. The bulb-origin and dermis-origin drug bands appeared to be connected with the insignificant MOP-positive area, which can be attributed to the overlap of the former and delayed drug incorporation into the latter as well as to drug incorporation in the lower dermis layer, forming a broad drug-positive area. This connected positive area moved toward the distal side thereafter, likely following the strand’s growth rate (typically around 0.4 mm/day6,13,14). Thus, a single oral administration of MOP typically generates an approximate 6−7 mm length two-peak MOP-positive area as shown in Figure 2B, though these lengths may somewhat vary depending on the individual growth rate of the hair strand and the depth of hair root. Schräder et al. determined ethyl glucuronide (EtG), a minor metabolite of ethanol, in daily shaved beard hair of three volunteers after single higher alcohol doses.8 At 9 h after the end of consumption, small concentrations of EtG were already detected for all volunteers and the concentrations increased to the maxima on days 2 to 4 then decreased to the limit of

Figure 4. Localization of MOP on the longitudinal sections of single hair specimens from volunteers C−E who orally ingested 150 mg of MOP hydrochloride/day, on three administration days scheduled at 7day and 11-day intervals. Specimens were sampled 14 days after the third administration day, by cutting at the level as close as possible to the scarp skin (indicated with ▽). D1 and B1 indicate possible dermisorigin and bulb-origin MOP positive areas generated by the ingestion of 150 mg of MOP hydrochloride (within 12 h) on the first day. B2, D2, B3, and D3 indicate those generated by the second and third 150 mg ingestion, respectively, at a 7-day interval between administrations. E

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Editors' Highlight

(2) Kintz, P.; Spiehler, V.; Negrusz, A. In Clark’s Analytical Forensic Toxicology; Jickells, S.; Negrusz, A., Eds.; Pharmaceutical Press: London, 2008; pp 153−190. (3) Scott, K.; Kronstrand, R. In Analytical and Practical Aspects of Drug Testing in Hair; Kintz, P., Ed.; CRC Press: London, 2006; pp 1− 23. (4) Baurngartner, W. A. In Forensic Application of Mass Spectrometry; Yinon, I., Ed.; CRC Press: London, 1995; pp 61−94. (5) Henderson, G. L. Forensic Sci. Int. 1993, 63, 19−29. (6) Pragst, F.; Rothe, M.; Spiegel, K.; Sporkert, F. Forensic Sci. Rev. 1998, 10, 81−111. (7) Potsch, L.; Skopp, G.; Moeller, M. R. Forensic Sci. Int. 1997, 84, 25−35. (8) Schräder, J.; Rothe, M.; Pragst, F. Int. J. Legal Med. 2012, 126, 791−799. (9) Villain, M.; Cirimele, V.; Kintz, P. Clin. Chem. Lab. Med. 2004, 42, 1265−1272. (10) Miki, A.; Katagi, M.; Zaitsu, K.; Nishioka, H.; Tsuchihashi, H. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 865, 25−32. (11) Vincenti, M.; Salomone, A.; Gerace, E.; Pirro, V. Bioanalysis 2013, 5, 1919−1938. (12) Shima, N.; Sasaki, K.; Kamata, T.; Matsuta, S.; Katagi, M.; Miki, A.; Zaitsu, K.; Sato, T.; Nakanishi, T.; Tsuchihashi, H.; Suzuki, K. Forensic Toxicol. 2015, 33, 122−130. (13) Nakahara, Y.; Shimamine, M.; Takahashi, K. J. Anal. Toxicol. 1992, 16, 253−257. (14) Pecoraro, V.; Astore, I. P. L. In Hair and Hair Disease; Orphanos, C. E., Happle. R., Eds.; Springer Verlag: Berlin, Germany, 1990; p 237. (15) Miki, A.; Katagi, M.; Kamata, T.; Zaitsu, K.; Tatsuno, M.; Nakanishi, T.; Tsuchihashi, H.; Takubo, T.; Suzuki, K. J. Mass Spectrom. 2011, 46, 411−416. (16) Miki, A.; Katagi, M.; Shima, N.; Kamata, H.; Tatsuno, M.; Nakanishi, T.; Tsuchihashi, H.; Takubo, T.; Suzuki, K. Forensic Toxicol. 2011, 29, 111−116. (17) Miyaguchi, H.; Kakuta, M.; Iwata, Y. T.; Matsuda, H.; Tazawa, H.; Kimura, H.; Inoue, H. J. Chromatogr., A 2007, 1163, 43−48. (18) Porta, T.; Grivet, C.; Kraemer, T.; Varesio, E.; Hopfgartner, G. Anal. Chem. 2011, 83, 4266−4272 DOI: 10.1021/ac200610c. (19) Musshoff, D. F.; Arrey, T.; Strupat, K. Drug Test Anal. 2013, 5, 361−365. (20) Shen, M.; Xiang, P.; Shi, Y.; Pu, H.; Yan, H.; Shen, B. Anal. Bioanal. Chem. 2014, 406, 1−4616 DOI: 10.1007/s00216-014-7898-1. (21) Poetzsch, M.; Steuer, A. E.; Roemmelt, A. T.; Baumgartner, M. R.; Kraemer, T. Anal. Chem. 2014, 86, 11758−11765. (22) Nakahara, Y.; Takahashi, K.; Konuma, K. Forensic Sci. Int. 1993, 63, 109−119. (23) Cone, E. J. J. Anal Toxicol. 1990, 14, 1−7. (24) Polettini, A.; Cone, E. J.; Gorelick, D. A.; Huestis, M. A. Anal. Chim. Acta 2012, 726, 35−43. (25) Kuwayama, K.; Yamamuro, T.; Tsujikawa, K.; Miyaguchi, H.; Kanamori, T.; Iwata, Y. T.; Inoue, H. Forensic Toxicol. 2014, 32, 235− 242. (26) Hallégot, P.; Peteranderl, R.; Lechene, C. J. Invest. Dermatol. 2004, 122, 381−386.

7- and 11-day intervals for three times. The positive areas barely separated into each administration day at 11-day intervals, for all six hair shafts tested (two shafts each for subjects C, D, and E, including those shown in Figure 4). For 7-day intervals, tricky overlaps of the possible bulb-origin band and the possible dermis-origin band of the next administration (7 days after the last intake) were observed for some specimens (subjects C and D, in Figure 4). The distance of these two types of bands generated by a single administration is typically 2.5−3 mm and scalp hairs typically growing at 2.5−3 mm/week causes this overlap. Thus, results indicate that the time resolution in estimating drug-use history is about 11 days, though it can be deteriorated due to various factors such as individual growth rate of the hair strand and the depth of the hair root.

■

CONCLUSION Hair has often been compared to a tape-recording that records drug use history. This study demonstrated that MALDI-IMS is a promising tool which allows for the direct detection and visualization of drugs incorporated inside the hair. Also, the method established here is useful in obtaining chronological information on drug exposure from a single hair. While we have demonstrated that single hair analysis is achievable, it should be noted that several hair shafts must be examined in real cases because some strands may be in the telogen phase, and the rate of growth varies among strands under the growing stage. A combination with different methodology, such as LC−MS/MS, is also recommendable in forensic purposes. The results of this study concerning drug incorporation into hair provide direct and visual evidence that there are two record heads to the tape recorder, which unfortunately cause overlap of the recordings and deteriorate chronological resolution of hair analyses down to 11 days or perhaps longer. These facts should be taken into account in interpreting the results of any hair analysis.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional figures and tables mentioned in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00971.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81 6 6268-1234 ext. 580. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors appreciate Dr. Takashi Nirasawa, Bruker Daltonics, for his useful suggestions and expertise. The authors also acknowledge financial support from the Japan Society for the Promotion of Science, Grants 26915013 and 24590065.

■

REFERENCES

(1) Huestis, M. A. In Drug Testing in Hair; Kintz, P., Ed.; CRC Press: London, 1996; pp 5−16. F

DOI: 10.1021/acs.analchem.5b00971 Anal. Chem. XXXX, XXX, XXX−XXX