Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology Maria C. Prieto Conaway, 1 Nicholas E. Manicke, 2 Marta Kozak 1 1 Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134 2 Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry. Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by ypaper p spray ionization/orbitrap mass spectrometry. High resolution and accurate mass used in full MS, MS 2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific TraceFinder 3. software used for data analysis. Results: Able to identify six drugs of abuse from dried blood spots at a 1 ng/ml level with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-1 ng/ml, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific Orbitrap Exactive family of mass spectrometers are ideally suited for coupling to paper spray ionization. Introduction Paper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography. Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-ms instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrap analyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS. In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrap analyzer is explored for its potential as a forensic toxicology screening tool. Methods Sample Preparation Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, Lampire Biologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v). Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 2 min and loaded into stackers that hold up to 4 cassettes. Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kv) induces electrospray from the sharp tip of the paper (Figure 1). The extraction solvent used in this work is 95/5 (v/v) methanol/water with 1 ppm acetic acid (ph 4.5). Mass Spectrometry The paper-spray source was coupled to either a Thermo Scientific Exactive Plus or a Thermo Scientific Q Exactive Orbitrap mass spectrometer. An automated experiment for drug screening consisted of 3 sec data collection, switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS). For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 14, resolving power (FWHM at 2). Normalized collision energy was 4 ev. All data acquisition used the Thermo Scientific Xcalibur sequences and contact closure trigger from the paper spray source. FIGURE 2. Full scan MS e 14, (FWHM at 2 due to matrix interference below. Resolving power f The [M+H] + ion is highligh RP 17,5 35, 7, 14, amphetamine 136.738 1 8 6 4 136.113 2 136.738 1 8 6 136.155 4 2 tive Abundance Relat 1 8 6 4 2 1 8 6 4 2 136.739 136.739 136.157 136.845 136.156 Data Analysis Thermo Scientific QualBrowser software from the Xcalibur platform was used for 136.1 spectra visualization. TraceFinder 3. software was used for the automated identification and confirmation in the targeted screening of drugs. 2 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBSspotted paper cassette electrospraying into mass spectrometer inlet. ell established even though a work used a Thermo Scientific c MS/MS transitions, full-ms id screening tools. Orbitrap analysis for high confidence ethod and retrospective data in addition to All Ion Quantitation Amitriptyline-spiked in blood 25 ng/ml using amitriptyline- Variability in terms of %RSD blood. Figure 6 displays amit sitive and fast Orbitrap screening tool. vine blood, Lampire sample integrity maintained by paper cartridges, dried under a t hold up to 4 cassettes. lysis and an applied high f the paper (Figure 1). anol/water with 1 ppm acetic Scientific Exactive Plus or ometer. f 3 sec data collection, e Plus MS) or full scan and /MS (Q Exactive MS). d fragmentation data were. Normalized collision energy r sequences and contact FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 7, and 14, (FWHM at 2), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below. Resolving power from 17,5, 35,, 7, and 14, top to bottom. The [M+H] + ion is highlighted by a red line. Mass accuracies 1-2 ppm. RP 17,5 35, 7, 14, amphetamine PCP codeine cocaethylene 136.738 1 8 6 4 136.113 2 136.738 1 8 6 136.155 4 2 tive Abundance Relat 1 8 6 4 2 1 8 6 4 2 136.739 136.739 136.157 136.845 136.156 244.2284 244.269 244.1 244.268 244.2269 244.2268 244.266 alibur platform was used for 136.1 244.15 244.2 244.25 for the automated identification 3.1592 3.1614 3.166 3.1433 3.2911 3.219 3.181 3.297 3.2186 3.1794 3.2912 3.168 318.13911391 318.1964 318.2667 318.1716 318.1916 318.1432 318.2648 318.1715 318.2286 318.1414 318.284 3.1431 3.291 244.2269 3.2185 3.34 318.1417318.197 318.2836 3.1 3.2 3.3 318.1 318.2 318.3 p Figure 5 shows accurate ma DBS sample containing a mi resolution for enhanced sign signal to noise is exceptiona demonstrated. FIGURE 3. TraceFinder 3. so targeted screening analysis identified d by exact value presence of two fragments fr Data collected with the Exac Thermo Scientific Poster Note PN64316-EN 1114S 3
Results Screening for drugs of abuse: resolving power, accurate mass for compound identification Figure 2 shows that high and ultrahigh resolving gp powers (7, and 14, FWHM at 2) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (7, and 14,, FWHM at 2). Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively identified from a dry blood spot sample. Screening for drugs of abuse at various concentrations A drug mixture of six compounds was analyzed at 1, 5, 1 and 25 ng/ml for forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work. This group of samples were detected by full scan MS down to 1 ng/ml levels (Figure 4) (14, resolving power; FWHM at 2). lving powers, e.g., 7, and tification of drugs from DBS h six drugs, four drugs shown and 14, top to bottom. curacies 1-2 ppm. Fragmentation and isotopic pattern matching for compound confirmation Accurate mass values were used for identification of screened drugs. Isotopic pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactive mass spectrometer can be used. Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated. Quantitation Amitriptyline-spiked in blood (1 5, ng/ml) yielded limits of quantitation (LOQ) of 25 ng/ml using amitriptyline-d 3 as internal standard (Figure 6). Variability in terms of %RSD (Std Dev/Mean*1) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots. FIGURE 3. TraceFinder 3. software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively identified d by exact values and confirmed by isotopic i pattern and the presence of two fragments from the AIF experiment (see Table). Data collected with the Exactive Plus mass spectrometer. 5 1 ng/ml 4 136.126 2 136.1246 136.11 136.12 136.13 4 2 4 2 15.1211 15.113 15.996 15.1211 15.999 15.113 15.1282 15.1363 15.1 15.12 15.14 FIGURE 5. DD MS/MS fra (FWHM at 2) in the signal to noise ratio (as c shown) is observed. Acc and 1-3 ppm, respectivel screening applications. Sample: mixture of six d Concentrations noted in a) Amphetamine 1 91.544 1 ng/ml 6 2 119.858 deine.1592 3.2911 3.219.1614 3.181 3.297 3.2186.166 3 3.1794 3.2912.168 cocaethylene 318.1964 318.13911391 318.2667 318.1716 318.1916 318.1432 318.2648 318.1715 318.2286 318.1414 318.284 Isotopic pattern match Simulation Experimental 91.544 1 5 ng/ml 6 119.858 2 1 1 ng/ml 6 91.543 2 119.857 9 1 11 12 c) PCP 1 86.966 159.1173 6 1 91.545 2 86.966 1 1 3.291 3.2185 3.34 3.2 3.3 318.1417318.197 318.2836 318.1 318.2 318.3 6 91.544 2 14.171 86.966 1 5 n 6 2 1 n 95.857 135.1171 2 1 15 2 4 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 14, resolving power (FWHM at 2) showing drugs detected down to 1 ng/ml. Sample contained six drugs analyzed from DBS. methamphetamine amphetamine PCP codeine cocaine cocaethylene 1 1 15.1282 1 244.267 1 3.164 1 34.1552 1 318.171 und confirmation screened drugs. Isotopic ent were used for drug MS/MS from a Q Exactive targeted DD MS/MS for a is acquired at ultra high at the higher resolution, the its of detection than its of quantitation (LOQ) of re 6). een <1 to 16% for drug in spots. w. Data processed in ix are positively c pattern and the e Table). er. 25 ng/ml 8 6 4 2 1 1 ng/ml 8 6 4 2 1 ng/ml 5 8 6 4 2 1 1 ng/ml 8 6 136.1222 136.1222 136.1247 136.1154 136.1222 4 136.126 2 136.1247 136.1246 136.11 136.12 136.13 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 15.1212 15.1211 15.1211 15.113 15.996 15.1211 15.999 15.113 15.1282 15.1282 15.1282 15.1363 15.1 15.12 15.14 8 6 15.1313 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 244.27 244.1916 244.2475 244.267 244.28 244.1915 244.247 244.267 244.28 244.1914 244.1914 244.266 244.2 244.2471 244.28 244.2471 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 4 2 3.1426 3.14 3.1426 3.1426 3.1661 3.1787 3.164 3.1661 3.163 3.1787 3.163 3.224 8 6 4 2 1 8 3.179 6 3.224 3.224 4 2 1 8 6 4 2 1 8 3.1661 6 3.187 3.15 3.2 4 2 34.1458 34.1552 34.1646 34.191 34.176 34.1973 34.1552 34.1377 34.1551 34.1373 34.1891 34.1763 34.15 34.2 8 6 4 2 1 8 6 4 2 1 8 6 4 2 1 8 6 2 318.1918 318.145 318.228 318.178 318.143 318.178 318.144 318.142 318.178 4 318.2284 318.1 318.2 FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 14, (FWHM at 2) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,5 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppm and 1-3 ppm, respectively) are used for the identification of compounds in screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel. a) Amphetamine b) Methamphetamine 1 91.544 NL: 1.78E7 1 91.544 NL: 5.86E6 1 ng/ml 1 ng/ml 6 6 119.859 119.858 136.736 2 2 15.128128 91.544 NL: 9.6E6 91.544 1 1 NL: 2.68E6 5 ng/ml 5 ng/ml 6 6 136.736 119.859 119.858 2 2 15.128 136.736 149.237 1 1 1 ng/ml NL: 1.18E6 91.544 1 ng/ml NL: 1.59E6 5 1 5 1 5 1 5 1 5 25 ng/ml 1 ng/ml 5 ng/ml 25 ng/ml 1 ng/ml Conclusion 8.191 8.199 8.1911 8.199 8.15 8.2 We have shown an easy to that shows extraordinary po forensic toxicology. Any combination of user req MS/MS, are allowed for the Accurate mass fragments (fr matching are required to con We have demonstrated feas expected concentrations are lower levels can be achieved High resolution and accurate by MS and nicely compleme dried d blood spots. Isotopic pattern match Simulation Experimental 6 91.543 2 119.857 9 1 11 12 13 c) PCP d) Cocaethylene 1 86.966 6 2 91.545 86.966 1 NL: 4.33E7 6 2 1 119.858 1 12 14 159.1173 6 1 ng/ml 1 ng/ml 82.653 2 15.917 244.264 318.174 NL: 2.66E7 1 NL: 6.9E7 Data collected in this screen MS event is always acquired The paper spray technique c 3. software provides a com References 6 6 91.544 5 ng/ml 82.653 5 ng/ml 2 2 15.917917 14.171 244.263 318.175 NL: 6.6E6 86.966 1 NL: 3.26E6 1 For forensic toxicology use on 245.1365 6 1 ng/ml 6 8.948 95.857 135.1171 15.337 1 ng/ml All trademarks are the property of Ther 2 213.11 2 15.917 This information is not intended to enco 318.174 intellectual property rights of others. 1 15 2 25 5 1 15 2 25 3 NL: 3.74E7 1. Manicke, N.; Yang, Q.; Wan Paper Spray Ionization for Q 3, 123-129. Thermo Scientific Poster Note PN64316-EN 1114S 5
3.224 1 6 3.187 3.2 4 2 1 8 4 2 34.1377 34.1373 34.1551 34.1891 34.15 34.2 34.1763 4 2 1 8 6 318.144 318.142 318.178 4 318.2284 318.1 318.2 esolving power of 14, tive detection. An enhanced resolving power, data not or and fragments (4-5 ppm tion of compounds in ur shown below. 2 FIGURE 6a. Full scan MS spectra for the [M+H] + ion of amitriptyline at various concentrations from DBS samples. Acquired at 7, resolving power. Mass accuracy 2-3 ppm 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 5 ng/ml 1 ng/ml 5 ng/ml 25 ng/ml 1 ng/ml 5 ng/ml 25 ng/ml 1 ng/ml Conclusion 8.1911 8.191 8.191 8.199 8.191 8.199 8.1911 8.199 8.15 8.2 8.2183 8.218 8.2182 8.218218 FIGURE 6b. Quantitative results for amitriptyline i t normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown. Area Ratio 9 8 7 6 5 4 3 2 1 Amitriptyline Y = -.519284+.1756*X R^2 =.9933 W: 1/X 1 2 3 4 5 ng/ml Level (ng/ml) % RSD We have shown an easy to use technique (no sample preparation, no chromatography) that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology. Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment. etamine NL: 1.78E7 Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3). 119.859 1 ng/ml 15.128128 NL: 9.6E6 We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high ( 1 ng/ml, Fig. 4). DD MS 2 data (Fig. 5) indicates lower levels can be achieved and this is part of ongoing investigations. 5 ng/ml 119.859 15.128 149.237 1 ng/ml NL: 1.59E6 High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from dried d blood spots. ne 119.858 12 14 NL: 6.9E7 Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired. The paper spray technique coupled with automated data processing using TraceFinder 3. software provides a complete solution for drug screening in forensic toxicology. 917 917 1 ng/ml 318.174 5 ng/ml 318.175 NL: 3.74E7 NL: 6.6E6 References 1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 211, 3, 123-129. For forensic toxicology use only. 1 ng/ml 917 318.174 2 25 3 All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64316-EN 1114S 6 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
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