Single Hair Analysis of Small Molecules Using MALDI-Triple

Oct 7, 2014 - Optimized MRM transitions (Supporting Information Table 2) were included in the MALDI-MS method, and the sample preparation used was the...
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Single Hair Analysis of Small Molecules Using MALDI-Triple Quadrupole MS Imaging and LC-MS/MS: Investigations on Opportunities and Pitfalls Michael Poetzsch, Andrea E. Steuer, Andreas T. Roemmelt, Markus R. Baumgartner, and Thomas Kraemer* University of Zurich, Institute of Forensic Medicine, Department of Forensic Pharmacology & Toxicology, Winterthurerstrasse 190/52, CH-8057 Zurich, Switzerland S Supporting Information *

ABSTRACT: Single hair analysis normally requires extensive sample preparation microscale protocols including time-consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDIMSI) was shown to be an alternative tool in single hair analysis, but still, questions remain. Therefore, an investigation of MALDI-MSI in single hair analysis concerning the extraction process, usage of internal standard (IS), and influences on the ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to a single hair, and hair strand LC-MS/MS results were performed, and the performance was compared to LC-MS/MS single hair monitoring. The MALDI process was shown to be independent from natural hair color and not influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semiquantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy, and cheap sample preparation is necessary, whereas LC-MS/ MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation, and both are suitable for consumption monitoring of drugs of abuse with a high time resolution.

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possible, and the results were used for case interpretation in forensic sciences.5,14 In the meantime, single hair analysis performed by GC/MS or LC-MS15,16 and matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) was developed. MALDI-MSI is the only imaging method which can determine the identity and distribution of drugs and their metabolites in biological matrices in one run without using labeling techniques. MALDI-MSI has proven to be a suitable tool for analyzing different molecule classes in biological samples and has shown its usefulness for pharmacokinetic studies for multiple drugs and their metabolites in tissue samples.17−20 Consequently, MALDI-MSI was also applied to the detection of DOAs in a single hair. Cocaine and tilidine consumption monitoring was performed using a triple quadrupole mass spectrometer, overcoming the problem of having single hairs in different growth states in one particular hair lock usually used for LC-

air analysis has gained importance in forensic sciences, ecotoxicology, work place drug testing, archeology, and other disciplines during the past decade.1−4 Hair as a matrix for a drug of abuse (DOA), and medication monitoring is unique in that information about the medication/drug consumption behavior is possible over a much longer time period compared to blood and urine.5,6 Small molecules are absorbed and distributed via the blood to the hair shaft, where the incorporation takes place into the hair matrix. The relative quantification of drugs in segmented hair strands can help to characterize the consumption behavior and amount taken.7,8 Hair analysis therefore provides a noninvasive option for biomonitoring with high analyte stability. DOA monitoring in the early days of hair analysis required relatively large amounts of hair. Separation and detection was mainly performed by gas chromatography coupled to mass spectrometry (GC/MS).9−11 Mass spectrometer developments and the coupling with liquid chromatography (LC-MS) led to improvements in hair analysis.1,8,12,13 The required hair sample amount could be reduced, and segmentation of hair locks became feasible. Consequently, a time related monitoring of drug intake by analyzing small segments of hair locks became © XXXX American Chemical Society

Received: August 25, 2014 Accepted: October 7, 2014

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MS.21,22 Head hair life cycle consists of different phases. The anagen phase is the active hair growth phase in which the incorporation of substances from the bloodstream takes place followed by the catagen phase as a transition state for 2−3 weeks. Hair life ends with the telogen phase lasting two to six months before drop out. About 20% of hairs are in the telogen phase and consequently represent a different time frame than hairs in the anagen or catagen phase.23 Preliminary results for cocaine and cannabinoids were published using a MALDIFourier transform mass spectrometer.24 The detection of methamphetamine in a single hair by applying MALDI timeof-flight mass spectrometry and MALDI Fourier transform ion cyclotron resonance mass spectrometry (FTICR) after complex sample preparation was shown by Miki et al.25 True to the motto “a picture is worth a thousand words”, MALDI-MSI was often claimed to be the future of single hair analysis, providing easy and straightforward sample preparation as well as segmentation of a single hair in the micrometer range. However, fundamental investigations for MALDI-MSI application to single hair analysis are still missing. The aim of this study was to examine these essential questions and thereby enable the reliable application to hair analysis. An important issue applying MALDI-MSI is the missing chromatographic separation of analytes of interest and other molecules, e.g., from the biological matrix. Therefore, particular attention has to be paid to ion-suppression/enhancement effects. Basic DOAs are trapped in vesicles (melanocytes) in the hair shaft and are therefore in close contact to the biomolecule melanin, which is also responsible for hair coloration.26 Substructural similarity of melanin to the routinely used MALDI matrix α-cyano-4hydroxycinnamic acid (CHCA) is conspicuous. MALDI laser energy might therefore also be absorbed by the chromophore of melanin27 and lead to differences in the analyte ionization processes, depending on the subject’s natural hair color. In this work, the influence of melanin on the ionization process was investigated. It should also be clarified whether extraction of analytes out of the hair shaft during the spraying/drying of matrix is a prerequisite for detection. Influences on the ionization by hair shaft components might cause irreproducible results. Therefore, intra and inter single hair ion enhancement or ion-suppression varieties caused by the hair shaft and its biomolecules were tested. The use of internal standards is highly recommended for the relative/absolute quantitation of small molecules in samples consisting of different compartments (e.g., whole body mice imaging) for the compensation of differences in ionization yield.28 The pros and cons of internal standard use in single hair analysis were evaluated in this study. Taking these basic results into account, a systematic investigation of MALDI-MSI for (semi-) quantitation in authentic single hair and hair snippets was performed by correlation to our routine LC-MS/MS method. For the first time, LC-MS/MS and MALDI imaging were compared for the usefulness for segmental single hair analysis with high time resolution. Single dose and multiple dose administration protocols were addressed with respect to reasonable segmentation and time resolution, sensitivity for detection of single dose administration, and the correlation between the two methods. Questions concerning segmentation and interpretation of highly resolved drug consumption monitoring were addressed. Furthermore, possibilities of triple quadrupole MALDI-MSI for screening for a limited number of analytes were tested.

Article

EXPERIMENTAL SECTION Chemicals and Reagents. Zolpidem (Zol), zolpidem D9 (Zol-D9), cocaine (COC), benzoylecgonine (BEC), norcocaine (NCOC), morphine (Mor), methylenedioxy-N-methylamphetamin (MDMA), and methadone (Met) were obtained from Cerilliant (Molsheim, France). Water, acetonitrile (ACN), α-cyano-4-hydroxycinnamic acid (CHCA), trifluoroacetic acid (TFA), synthetic melanin, methanol, dimethylsufoxide (DMSO), and sodium hydroxide (NaOH) were obtained from Sigma-Aldrich (Buchs/Switzerland). Acetone and nhexane were obtained from Merck (Darmstadt, Germany). All chemical solvents used were of high performance liquid chromatography (HPLC) grade. CHCA was cleaned up by recrystallization in 70% ACN/30% water before usage. Hair Specimens. DOA positive head hair samples were collected in our Center for Forensic Hairanalytics. Anonymized subjects were included in the trial after they tested positive for drugs, in an attempt to regain their driver’s license (Supporting Information Table 1). Head hair strand samples were collected by cutting close to the scalp in the posterior vertex region, and an average segmentation of 3 cm in length was performed for routine LC-MS/MS analysis, according to abstinence control requirements. Single hairs were randomly selected from the obtained subject’s hair strands for MALDI-MSI. For the evaluation of detection of a single dose administration in a single hair by MALDI-MSI and LC-MS/MS, seven volunteers (26−49 years, 5 male, 2 female) took one 10 mg Zol tablet. The volunteer’s head hair samples were collected before and 1 month after the single dose intake. Hairs collected before were screened and confirmed to be negative by our routinely used LC-MS/MS method. Sample Preparation. Hair samples were washed successively with 15 mL of water, 10 mL of acetone, and 10 mL of nhexane by shaking the plastic tubes for 2 min during each wash step and dried at room temperature. MALDI-MS images of intact hair were acquired after fixation of five single hair shafts of the same subject on the MALDI plate (OPTI TOF 384 well insert 123 × 81 mm, AB Sciex, Darmstadt, Germany) using a double-sided adhesive tape. Images of 5 mg hair snippets of one subject were acquired after fixation on the MALDI plate using a double-sided adhesive tape. Both times, a MALDI matrix (10 mg/mL α-CHCA solution; 0.1% TFA/ACN 50:50) was applied in multiple layers to the MALDI plate by manually spraying using a thin layer chromatography (TLC) sprayer (Corning Pyrex, Tewksburg, USA). MALDI-MS. Imaging experiments were performed on a Flashquant Workstation (AB Sciex, Darmstadt, Germany) fitted with a high repetition laser (Nd:YAG, λ = 355 nm, elliptic shape 100 × 200 μm). MALDI-MS images were acquired in MS/MS mode using positive ionization. Source operation conditions were: continuous mode (1 mm/s); raster pitch, 100 μm; laser energy, 40 μJ; laser frequency, 1000 Hz. MS conditions were: unit resolution; vacuum gauge q2, 4.8 × 10−5 Torr (nitrogen as collision gas). Transitions and optimized MS parameters are shown in Supporting Information Table 2. Data were acquired with Analyst 1.4.2 software and Flashquant software (AB Sciex, Darmstadt, Germany). Raw data conversion from wiff files to image files was performed with dedicated software provided by Markus Stoeckli (Novartis, Basel) and AB Sciex. Tissue View Software (v1.1, AB Sciex) was used for image processing. Unambiguous identification by enhanced product ion spectra was performed in positive B

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before MALDI matrix application to improve possible extraction. Internal Standard (IS) Use. For evaluation of the usefulness of internal standard, 10 ng/mL Zol D6 was added to the MALDI matrix solution and transitions for the internal standard were included into the MALDI-MS method. MALDI-MSI had been acquired of five intact single hair samples from each concentration level (Supporting Information Table 1). The limit of detection was determined and compared to samples without internal standard use. The homogeneity of internal standard over the sample was evaluated. Comparison of MALDI-MSI and LC-MS/MS Results. Quantification Using MALDI-MSI and Correlation to Hair Strand LC-MS/MS Results. ROIs were placed over the intact single hair shafts of five single hairs of the same subject, corresponding to the segment analyzed by LC-MS/MS hair strand analysis.29 The average background noise of negative areas with the same size was subtracted, and average ROI intensities of the corresponding five single hair segments were correlated to LC-MS/MS results. ROIs were placed over the hair snippets of the same subject. Average background noise of negative areas with the same size was subtracted, and average ROI intensities were correlated to LC-MS/MS results. Consumption Monitoring of Single/Multiple Zolpidem Intake Applying MALDI-MSI and LC-MS/MS. Single dose detection images of five single hairs of each volunteer collected one month after a single dose of Zol intake were acquired by MALDI-MSI and high throughput micro LC-MS/MS. First 1.5 cm, which correspond to the time window of single dose intake, was evaluated for positive Zol signals. Images of authentic routine hair samples were acquired, and self-declared consumption behavior was evaluated. Furthermore, segmental hair strand and segmental single hair analysis were performed, and results were compared to MALDI-MSI images for evaluation of time resolution. Screening in Single Hair Using MALDI-MS/MS. Authentic head hair samples that tested positive for Mor, COC, BEC/ NCOC, MDMA, or methadone by LC-MS/MS were included in the trial. Different hair concentrations were chosen. Optimized MRM transitions (Supporting Information Table 2) were included in the MALDI-MS method, and the sample preparation used was the same as that for Zol. One negative hair sample was tested as a blank sample. After imaging five single hairs of one subject, enhanced product ion scans were acquired at previously positive screened segments. Qtrap settings are described above. Concentrations for positive results and limits of detection were evaluated.

ionization mode with a scan rate of 1000 Da/s and a linear ion trap fill time of 120 ms applying Q0 trapping. Collision energy was set to 35 eV with a spread of ±15 eV and nitrogen as collision gas. The laser was controlled manually with a discrete laser burn time of 2 s at previously positive imaged regions. LC-MS/MS. Hair strand analysis was performed employing our routine procedure and an LC-MS/MS method for benzodiazepines.29 Segmental single hair analysis was performed using a high throughput micro LC-MS/MS method in segments of 5 mm of length for routine samples and 1 mm for the single dose administration study: Hair strands were decontaminated as described for hair strand analysis. Five randomly chosen single hairs were fixed on a graph paper using a transparent double adhesive tape. Five mm segments were cut with a scalpel and transferred to an auto sampler vial insert (target polyspring inserts, 300 μL, Thermo Scientific, Rockwood, USA). 50 μL of a mixture of eluent A/B (85:15) (eluent A: 10 mM formiate buffer pH 3.5; eluent B: 0.1% (v/v) formic acid in ACN) was added including Zol D6 (1 ng/mL) as internal standard. Inserts were centrifuged at 11 180g to ensure the transfer of the hair snippets into the mobile phase. Extraction was performed for 15 h at room temperature. Analysis was performed on a Qtrap 4500 (AB Sciex) fitted with a Turbo V ion source operated in ESI mode and coupled with an Eksigent 200 micro LC-system. MS operation conditions were: gas 1 = 50 psi and gas 2 = 60 psi (both nitrogen); ion spray voltage = 5500 V; ion source temperature = 150 °C. The analysis was performed in MRM (multireaction monitoring) mode with three transitions for Zol and one transition for the deuterated internal standard Zol D6, respectively (Supporting Information Table 3). Gradient elution was performed on a pentafluorophenyl (PFP) micro LC column (50 × 0.5 mm, 2.7 μm 90A). The gradient profile was: 0−0.4 min: eluent A 70%; 0.4−1.8 min: linear decrease to eluent A 30%; 1.8−2 min eluent A 30%; 2−2.2 min eluent A increase to initial conditions eluent A 70%; the flow rate was 30 μL/min at all times. Total micro LC run time was 2.2 min. Special Investigations on MALDI-MSI Issues. Influence on Ionization Process. For the investigation of the influence of melanin on the ionization process, a Q1 survey scan of dark brown Zol containing hair was acquired without the application of CHCA as MALDI matrix only using laser desorption ionization (LDI). Furthermore, a MALDI-MS image of a mixture of Zol (100 ng/mL) and synthetic melanin without the application of CHCA was acquired. Ion-enhancement/suppression experiments were performed by acquiring images of six different Zol negative hair samples after spraying a solution containing 1000 ng/mL Zol (water/ methanol 50:50) to a final Zol concentration of 0.125 ng/mm2 using a robotic sprayer (TM-sprayer, HTX technologies, USA). After this, a solution of 10 mg/mL CHCA MALDI matrix was sprayed using the TLC sprayer. Averaged regions of interest (ROIs) intensities with the same dimensions were compared along the hair shaft and in between six different hair shafts. To investigate whether the extraction of Zol from the hair shaft is necessary for ionization and positive drug detection by MALDI-MSI, 5 authentic hair samples (3000 pg/mg Zol) were sprayed several times with the MALDI matrix solvent mixture (0.1% TFA/ACN; 50 passes) before actual MALDI matrix application (for sprayer conditions, see the Supporting Information). Furthermore, 0.1% (v/v) HCl in MeOH, usually used for hair strand extraction in our routine lab, was applied

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RESULTS AND DISCUSSION Special Investigations on MALDI-MSI Issues. Influences on Ionization Process. Different physiological incorporation rates of DOAs into the hair shaft, depending on hair color, have already been described.26 Basic DOAs are trapped in vesicles (melanocytes) in the hair shaft and are therefore in close contact to the biomolecule melanin. Melanin has a broad UV/ vis light absorption range (Supporting Information Figure 1) including UV absorption at the MALDI laser wavelength (355 nm). Substructure similarity to routinely used MALDI matrix α-cyano-4-hydroxycinnamic acid (CHCA) is conspicuous (see Supporting Information Figure 4). MALDI yield for small molecules might therefore also be enhanced depending on the natural hair color. C

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ionization process after the hair decontamination procedures described above. Cosmetic treatment with oils or silicon based shampoos might have an influence on MALDI matrix crystallization and thus on ionization processes. Therefore, proper decontamination of hair is essential for MALDI analysis of hair. Internal Standard Use. The usage of internal standard increased the limit of detection for Zol dramatically from 270 to 1700 pg/mg, probably caused by ion-suppression. In contrast to tissue samples, the hair matrix can be considered as a homogeneous matrix with the same influence on ionization yield (ion enhancement/suppression) over the full hair length (see above). Therefore, the renunciation of internal standard use is reasonable. All quantitation experiments were finally performed successfully without the use of an IS. The application of an internal deuterated standard spiked in the MALDI matrix solution should compensate influences in sample preparation and ionization processes, e.g., when applying MALDI-MSI to tissue sample analysis. The IS is equal to different ionization behavior in different tissues such as liver, lungs, muscle, etc. and allows relative or absolute quantification. However, single hair samples have a very small surface and can be considered as a homogeneous sample matrix. Preliminary investigations have shown a rather inhomogeneous distribution of the IS over the hair sample when manually applied together with the MALDI matrix solution. This was especially critical when analyzing hair with very low analyte concentrations and only a few data points over the hair sample. A pixel size of 100 μm in combination with four MRM transitions (two per analyte) results in a required cycle time of 100 ms. A hair diameter of 80 μm results in one pixel for all analytes. Thus, a little inhomogeneity in the distribution of the internal standard may cause high effects on results. Comparison of MALDI-MSI and LC-MS/MS Results. Quantification Using MALDI-MSI and Correlation to Hair Strand LC-MS/MS Results. Correlations were performed with authentic Zol consumer hair samples with a concentration range available from our routine cases to meet experimental expectations (Supporting Information Table 1). Aqueous drug soaked hair samples do not offer the same experimental properties compared to authentic consumers’ hair since analytes are only incorporated into the outer hair matrix but not into the melanocytes like after physiological incorporation. A disadvantage of authentic samples is of course the limited sample amount and concentrations available. MALDI-MS to LC-MS/MS correlations were performed for intact single hair as well as for hair snippets. ROI intensities of images acquired of hair snippets fixed on the MALDI plate showed a linear correlation to the hair snippets from the same hair batch measured by LC-MS/MS (Figure 4a). Background subtraction for eliminating the influence of Zol negative areas on the average of ROI intensities was essential for correlation. Using intact single hairs instead of hair snippets combines an easy and straightforward sample preparation with the possibility of quantitative monitoring. ROIs were placed over the intact single hairs, and background was subtracted. Segment size was set to the same length as those for hair strand analysis by LCMS/MS, and ROIs were placed as close as possible to the single hair. Averaged ROI intensities of the averaged 5 single hairs of the same concentration were in good correlation to routinely obtained LC-MS/MS results (Figure 2b).

The summation of 70 laser desorption ionization (LDI) Q1 survey scans of zolpidem (Zol) containing hair samples (same source conditions applied to Zol imaging) showed no signals of the precursor of protonated Zol or possible adducts (data not shown). Mixtures of Zol and synthetic melanin showed no ionization of Zol molecules even at high concentrations of Zol. Furthermore, authentic hair samples were measured under the described imaging conditions without CHCA application. Images were negative at all Zol concentration levels (70− 3000 ng/mg) even after applying higher laser energy power. These results indicate that melanin or other hair structures do not induce ionization processes during MALDI analysis at least in the case of Zol. Therefore, MALDI ionization processes of Zol and most likely other small DOAs are independent from the subject’s hair color. Extraction experiments using the matrix solvent mixture should prove that Zol has to be extracted out of the hair shaft for ionization and positive drug detection by MALDI-MSI. A robotic sprayer allowed for reproducible extraction from the hair shaft (for the conditions, see the Supporting Information). Signal intensity increased for the chosen hair sample after extraction with the matrix solvent mixture (Figure 1). After

Figure 1. (a) MALDI-MS image of 5 single hairs without extraction; (b) MALDI-MS image after extraction using the robotic TM-sprayer with 0.1% TFA/ACN (50:50); (c) MALDI-MS image (overlay with optical image and zoomed in) after extraction using the robotic TMsprayer with 0.1% TFA in MeOH showing provoked delocalization.

applying 0.1 M HCl in MeOH, usually used for Zol extraction of whole hair strands in our routine LC-MS/MS method, a gain in signal intensity was detectable but strong delocalization effects could be observed. Provoked delocalization in the longitudinal and vertical direction additionally proved that extraction of the analytes from the inner part of the hair shaft seems to be essential for ionization processes. When using external calibration, suppression effects may increase limits of detection whereas ion enhancement will lead to higher calculated concentrations. Both effects may occur in MALDI analysis of a single hair but should be reproducible and not depend on the subject’s hair. External application of the drug on the single hairs by homogeneous robotic spraying and comparison of intra/inter hair shaft signal intensities were a useful experimental setup for corresponding investigations. Intra hair variation of the MALDI signals was ±9.4% along the hair shaft, and inter hair variation was ±10.2% tested with six different single hairs. The small standard deviation along the same hair shaft indicates that one subject’s hair sample can be considered as a homogeneous matrix, and therefore, almost identical effects on ionization can be expected. Also inter hair variations of different subjects had little influence on the D

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A concentration of 70 pg/mg Zol could not be detected either in hair snippets or in intact single hairs. The lowest concentration of authentic hair samples detected by MALDIMS was 270 pg/mg as determined by LC-MS/MS. The LOQ was therefore assumed to be between 70 and 270 pg/mg. Comparing the two approaches, snippets showed higher average intensities caused by a higher sample amount whereas the single intact hair approach showed a higher slope caused by the lower amount of background noise area in the ROIs. Consumption Monitoring of Single/Multiple Zolpidem Intake Using MALDI-MSI and LC-MS/MS. In hair analysis, absolute statements about drug ingestion must be given with caution because of different incorporation rates depending on hair color,30−32 external and environmental contamination,22 or cosmetic hair treatments. Nevertheless, quantitative hair analysis has gained an important role in assessing classification of drug intake or abstinence control in different fields of forensic toxicology. Single dose detection and its correlation to the time of consumption is an important task in hair analysis. MALDI-MS imaging was expected to combine the benefit of an easy sample preparation with sensitive time-resolved monitoring. Single hair and hair strands collected one month after a single dose intake of 10 mg Zol were investigated using MALDI-MSI and LC-MS/MS. LC-MS/MS concentrations of the corresponding hair strand segments ranged from 1.1 to 10.8 pg/mg hair (median = 3.8 pg/mg hair). LC-MS/MS single hair analysis showed positive signals for Zol in segments in 4 out of 5 hairs applying a segmentation of 1 mm (Supporting Information Figure 3). MALDI-MS images were negative for the corresponding segments with a segmentation of 100 μm. Considering a hair diameter of 80 μm and a hair density of 0.633 g/cm3, the maximum amount of Zol which can be desorbed is about 11 fg. These calculations are based on the assumption that the whole Zol amount is in one segment of 100 μm. The theoretical time resolution of MALDI-MS would be around 7 h considering a laser path of 100 μm and an average hair growth rate of 1 cm per month. Physiological incorporation and fixation into the hair matrix is a process of a few days.33 Smaller segmentation and therefore a higher time resolution seem to not be reasonable, even if analytical methods like MALDI-MSI theoretically offer this possibility. This can also be seen in the results from single hair LC-MS/MS (Supporting Information Figure 3). Zol was detected in multiple single hair 1 mm segments 30 days after a single

Figure 2. (a) Calibration snippets: left, optical image; right, MALDIMS images of different zolpidem concentrations, correlation of routine LC-MS/MS results and ROI intensities. (b) Calibration of a single hair: ROIs over 5 single hairs of the same concentration, correlation of routine LC-MS/MS results and ROI intensities.

Figure 3. (a) MALDI-MS image of case No. 3 showing localized high zolpidem concentrations in segments of approximately 0.8 cm length; (b) LCMS/MS segmental single hair analysis of the same subject with 5 mm segmentation (H = hair No.; aver. = average). E

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Figure 4. (a) MALDI-MSI segmental single hair analysis of subject No. 6; (b) LC-MS/MS segmental single hair analysis of the same subject (H = hair No.; aver. = average).

intake of 10 mg of Zol in 4 out of 5 single hairs. The negative result for one hair out of the 5 implicates that this hair was in the telogen growth phase, thus representing a time before the intake of Zol. In the Zol positive hairs, Zol was not sharply located in only one segment of 1 mm length. Also, the distance of the concentration maximum from the cut differed among the single hairs. This can mainly be explained by displacement of the single hairs during sample preparation and can be minimized by experienced people.34 In conclusion, time resolution in hair analysis cannot be better than several days regardless of the method applied. In a previous work, hair strand analysis results in 1 cm segments were compared with MALDI-MSI results in the corresponding extended 1 cm ROIs.21 With better analytical methods available, single hair LC-MS/MS in smaller segments could be compared. Figure 3a shows the MALDI-MS image of five single hairs of routine sample number 3 with a full hair length of 3.5 cm. The LC-MS/MS hair strand concentration of Zol determined in a hair segment of 3.5 cm length was 880 pg/ mg Zol. MALDI-MSI showed distinct Zol positive segments of only 0.8 cm of length and about 1 cm away from the cut. LCMS/MS (Figure 3b) showed Zol containing segments applying a segmentation of 5 mm with a maximum concentration at 15− 25 mm from the cut. The average of the five single hairs illustrates the limits of hair strand analysis due to the loss of resolution compared to single hair analysis. In hairs H2−H5, a distinct peak can be observed. H1 shows a completely different picture, indicating the hair is in a different growth state (transition from anagen to telogen phase in the time between Zol consumption and sample collection). The average (being comparable to a hair strand) shows no distinct peak. With both methods, positive segments showed a shift in distance from the cut. Furthermore, shifts of single hairs during sampling and handling might have occurred. Taking both facts into account, at least five single hairs have to be analyzed for quantification and interpretation. The small single hair segmentation performed with MALDI-MSI and LC-MS/MS analysis provides

important information for the interpretation of consumption behavior. Furthermore, it may be helpful for the differentiation of external contamination from actual intake of a drug, as already proposed in ref 22. In case No. 3, the volunteer had declared an abstinence of 9 months at the time point of hair sample collection. Applying our routinely used method for abstinence control with large hair strand segments of 3.5 cm, the final assessment was “moderate consumption in the last 3.5 months”. Taking into account the additional information from MALDI-MSI and LCMS/MS single hair analysis, the interpretation was different: “intake of Zol approximately 4−6 weeks before sample collection followed by abstinence”. Figure 4a illustrates five single hairs after chronic self-declared consumption of 5 mg Zol per day (Supporting Information). The result obtained from hair strand analysis was 3000 pg/mg Zol. The final assessment after routine analysis was “intense consumption over the last 3.5 months”. Both MALDI-MSI and LC-MS/MS single hair analysis showed a peak of Zol consumption corresponding to about 6−8 weeks before sample collection. MALDI-MSI and LC-MS/MS results of single hair analysis were in good agreement taking the physiological variability into account. Both examples prove the usefulness of segmental single hair analysis for proper judgment of consumption behavior. MALDI-MSI and LC-MS/MS single hair analysis showed comparable results. MALDI-MSI has the advantage of a fast and straightforward sample preparation which is an important aspect for routine lab implementation. Segmentation of five single hairs in the millimeter scale for LC-MS/MS analysis needs hours for sample preparation and extraction. Even if it is possible to use high throughput micro LC systems with very short run times, costs accrue mainly with the very timeconsuming sample preparation. MALDI-MSI sample preparation takes about 1 h for 5 single hairs and is therefore the cheaper and more straightforward method. LC-MS/MS showed a much higher sensitivity for segmental single hair analysis with the instruments used. The amount of Zol on column for LCF

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Author Contributions

MS/MS and the theoretical desorption for MALDI-MSI was calculated to be the same. Screening in Single Hair Using MALDI-MS/MS. Cocaine, BZE/NCOC, methadone, and MDMA were found to be positive in hair sample segments in typical concentration ranges. Morphine and MAM were negative in all hair samples and concentrations with the used standard sample preparation and MALDI matrix application method. Morphine and MAM contain acidic phenolic and basic amino structures, which lead to inefficient ionization of the precursor. The present method could detect cocaine in hair segments with concentrations down to 1800 pg/mg and was therefore more sensitive than that described in previous methods. Methadone showed positive signals even at concentrations of 850 pg/mg, and it can therefore be used for monitoring in methadone maintenance treatment. Several considerations have to be made when establishing a screening method using triple quadrupole linear ion trap technology. First, positive segments have to be detected for later confirmation. This was done by first including the transitions of the analytes of interest into our imaging method. The more transitions are included into the method, the higher is the pixel size; or, if pixel size is fixed, the smaller is the dwell time causing loss in signal-to-noise ratio. Therefore, only case relevant transitions should be included. Concentrations in positive segments are very low and require a relatively long fill time of the trap and if possible to be in combination with Q0 trapping. Our suggestion is to perform screening for DOA with routine LC-MS/MS in hair strands followed by MALDI-MSI for the positive drugs in single hairs offering much better time resolution. If high sensitivity is required, LC-MS/MS detection should be applied.

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors want to thank Markus Stöckli and Dieter Staab for their support.

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CONCLUSION MALDI-MSI proved to have great potential in modern single hair analysis. Interpretation of consumption behavior is much improved over conventional LC-MS/MS. MALDI ionization processes are independent from the subject’s natural hair color, and ionization enhancement/suppression effects of intra/inter hair samples are reproducible. Therefore, MALDI-MSI can also be performed without the use of an internal standard, thus increasing sensitivity. Zolpidem hair concentrations obtained from validated routine LC-MS/MS methods showed good semiquantitative correlation to MALDI image intensities in single hair and hair snippets. MALDI-MSI is superior to LCMS/MS analysis when a fast, easy, and cheap sample preparation is necessary whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation and are both suitable for consumption monitoring of DOAs with a high time resolution.

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ASSOCIATED CONTENT

S Supporting Information *

Descriptions of extraction sample preparation of hair samples, schemes, figures, and tables mentioned in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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dx.doi.org/10.1021/ac503193w | Anal. Chem. XXXX, XXX, XXX−XXX