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www.rsc.org/methods Page 1 of 22 Analytical Methods

1 2 3 4 1 Application of Mixed-Mode Ultra High Performance Liquid 5 2 Chromatography to Forensic Drug Analysis 6 7 3 Cassandra Clyde, Samantha Blake, Stacey Obrien, Ihuoma Igwilo, and Ira S. 8 4 Lurie* 1 9 5 10 6 Methodology is presented for the analysis of drugs of forensic interest employing a single 11 7 12 column and the same solutions from two solvent reservoirs at different ratios for orthogonal 13 8 reversed phase chromatographic and hydrophilic interaction liquidchromatographic separations. 14 9 For the determination of the basic drugs in the SAMHSA-5 panel in urine, a 2.1 x 150 mm x 2.7 15 10 µm superficially porous dimethylpentafluorophenylpropyl (PFP) column was employed using 16 11 two acetonitrile-water-ammonium fomate solutions as A and B solvents for a binary pumpwith 17 12 18 time of flight mass spectrometric (TOF-MS) detection. Applicable to the analysis of seized 19 13 drugs, the same column and detector was used with acetonitrile-water-ammonium acetate 20 14 solutions as A and B solvents for the separation of 15 controlled “bath salts”. For both 21 15 applications, employing mixed mode chromatography minimized ion suppression and Manuscript 22 16 allowed the unique identification of each analyte. Solid phase extraction (SPE) performed on a 23 17 24 mixed mode MM1 column successfully recovered the solutes of interest with good recovery and 25 18 minimum ion suppression or ion enhancement was observed for the ultra high performance 26 19 liquid chromatography-TOF-MS analysis of the extracts. For the SPE sample preparation no 27 20 evaporation and reconstitution step was required, with the elution solvent directly compatible 28 21 with both the HILIC and RPC analysis on the same stationary phase. 29 22 30 23

31 1. Introduction Accepted 32 24 33 25 The SAMHSA-5 drug panel is used for workplace drug testing and includes the following five 34 26 35 drug classes: amphetamines (methamphetamine, amphetamine, MDMA, MDA, and MDEA), 36 27 cocaine (benzoylecgonine (BZE) a metabolite of cocaine), marijuana (carboxy-THC) a 9 37 28 metabolite of -THC, opiates (codeine, 6-monoacetylmorphine (metabolite of heroin), and 38 29 morphine), and phencyclidine (PCP) [1]. 39 30 The analysis of above drugs and metabolites in urine represents an analytical challenge. 40 31 Traditional techniques such as immunoassays [2] lack specificity which leads to confirmation by Methods 41 32 42 other techniques such as gas chromatography mass spectrometry (GC-MS) [2] and liquid 43 33 chromatography mass spectrometry (LC-MS) [3]. Using GC-MS can be problematic for 44 34 thermally labile, polar and non-volatile solutes. Therefore, derivatization could be required 45 35 which increases sample analysis time [4]. For LC-MS, which does not suffer from the above 46 36 limitations, sample preparation techniques such as liquid liquid extraction and solid phase 47 37 48 extraction (SPE) are commonly employed, as in GC-MS, to remove the solutes of interest from 49 38 the matrix. For LC-MS the use of solid phase extraction minimizes ion suppression and ion 50 39 enhancement effects, extends column life and minimize contaminants in the MS source [5, 6]. 51 40 Reversed phase chromatography (RPC) has been employed for most or all of the basic Analytical 52 41 drug classes of the SAMHSA-5 panel [7, 3]. Hydrophilic interaction liquid chromatography 53 42 (HILIC), a complementary technique to the widely employed reversed phase chromatography, 54 55 56 57 Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road, NW, Somers Hall, 58 Washington, DC 20007. E-mail: [email protected]; Fax: +1 202-242-5761; Tel:+1 202-994-3791 59 1 60

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1 2 3 43 offers certain advantages for the analysis of polar solutes, including drugs in urine [8]. This 4 44 5 includes the ability to provide an orthogonal separation mechanism which could resolve solutes 6 45 not separated under reversed phase conditions or provide an additional identification. In 7 46 addition, the use of the high organic content in the HILIC mobile phases is expected to produces 8 47 lower limits of MS detection for electrospray ionization (ESI) detection [8]. Using HILIC also 9 48 can mitigate the need for an evaporation and reconstitution step during solid phase extraction 10 49 (SPE) sample preparation [9]. Morphine and codeine have been analyzed using HILIC with solid 11 50 12 phase extraction [9, 10], with one of the studies [10] not employing an evaporation and 13 51 reconstitution step. Amphetamine, methamphetamine, MDA, MDMA and MDEA have been 14 52 analyzed by HILIC, with column switching used for sample preparation [11]. 15 53 “Bath salts” refers to synthetic drugs related to cathinone, an amphetamine-like stimulant 16 54 drug naturally occurring in the khat plant. These synthetic cathinones can produce euphoria and 17 55 18 increased sex drive, but adverse side effects include paranoia, agitation, and hallucinatory 19 56 delirium, psychotic and violent behavior [12]. These drugs marketed as “bath salts” to evade 20 57 detection by authorities typically take the form of a white or brown crystalline powder and are 21 58 sold in small packages labeled “not for human consumption” [12]. Compounds structurally Manuscript 22 59 similar to cathinone are synthesized to circumvent the controlled substances laws. Law 23 60 24 enforcement has countered by placing over 15 “bath salts” under temporary or permanent federal 25 61 control in the United States [13]. 26 62 Since new structurally similar compounds are created by slightly modifying the chemical 27 63 structure of a controlled substance, methodology to analyze these solutes should have the ability 28 64 to distinguish between similar solutes (analogs including positional isomers). Currently GC is 29 65 widely used for the analysis of synthetic cathinones [14] . However, this technique can be 30 66 problematic for highly polar cathinone derivatives which could require basic extraction and/or

31 Accepted 32 67 derivatization in order to obtain satisfactory chromatographic performance [14-16]. 33 68 Additionally, many cathinone derivatives undergo extensive fragmentation under EI (electron 34 69 impact ionization), and their molecular weight information is either missing or difficult to 35 70 determine [17]. In contrast liquid phase separation techniques, such as capillary electrophoresis 36 71 37 (CE), ultra high performance liquid chromatography (UHPLC) or high performance liquid 38 72 chromatography (HPLC) do not suffer from the above limitations, and therefore are well suited 39 73 for the analysis of ”bath salts” [14, 18-22]. In addition, CE-MS and UHPLC-MS which can

40 74 provide either low or high resolution molecular weight information due to its soft molecular Methods 41 75 fragmentation (electrospray ionization (ESI)) is well suited for the screening of the synthetic 42 76 cathinones [14, 17, 19-21, 23 Significant overlap in retention times existed for the separation of 43 77 44 “bath salts” using RPC [14]. For a more definitive compound identification based on retention 45 78 time, including minimizing the possibility that the retention time of a target solute matches the 46 79 retention time of a non controlled isomeric compound, and to facilitate quantitation, the use of a 47 80 complementary technique such as HILIC could help mitigate this situation [24]. 48 81 Mixed-mode chromatography, whereby a stationary phase exhibits both RPC and 49 82 50 HILIC properties is utilized, has been reported; e.g pentafluorophenyl (PFP) [25, 26], cystine and 51 83 cysteine bonded silica [27], steviol glycoside modified- silica [28], and a C18-Diol [29]. Solutes Analytical 52 84 examined using these stationary phases exhibited “U-shaped” retention behavior (retention 53 85 decreases and then increases with organic modifier concentration), which results in changes in 54 86 separation selectivity. For the PFP stationary phase, the selectivity changes are a result of both 55 87 ion exchange with surface silanol groups and simultaneous interaction with the bonded phase 56 57 58 59 2 60

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1 2 3 88 ligands via dispersion and polar interactions [25]. PFP columns have been used for both the RP 4 89 5 of abused drugs in urine [30] and 6 90 The use of 2.7 µm superficially porous (SPP) columns for ultra high performance liquid 7 91 chromatography (UHPLC), which allows for faster and/ or higher peak capacity separations than 8 92 conventional HPLC, is well suited for the analysis of drugs in urine and for the screening of 9 93 “bath salts” in seized drugs. 10 94 TOF MS detection with a good dynamic range, the capability for accurate mass 11 95 12 measurement over an entire selected mass range is a suitable alternative to traditional quadrupole 13 96 MS techniques, which provides unit resolution and lacks the ability to easily identify untargeted 14 97 compounds. This detection scheme was employed for HILIC separation of opiates [9] and the 15 98 RPC separation of synthetic cathinones [17, 21].. 16 99 UHPLC separation conditions for the basic drugs classes in the SAMHSA-5 drug panel 17 100 18 and synthetic cathinones, using both RPLC and HILIC, is investigated. This leads to creation of 19 101 methodology for both the analysis of the SAMHSA drugs in urine employing solid phase 20 102 extraction and the confirmation and/ or screening of bath salts via retention time . The analysis of 21 103 carboxy-THC which requires a separate solid phase extraction procedure is beyond the scope of Manuscript 22 104 this manuscript. 23 105 24 25 106 2. Experimental 26 107 27 108 2.1 Chemicals and reagents 28 109 29 30 110 The reference drug and metabolite standards and synthetic urine were obtained from Cerilliant

31 111 (Round Rock, TX, USA), while the synthetic cathinones were acquired from Cayman Chemical Accepted 32 112 (Ann Arbor, MI, USA). Agilent ESI electrospray ionization tune mix was acquired from 33 113 PerkinElmer (Shelton, CT, USA). LC/MS grade water, formic acid, acetonitrile and certified 34 114 35 ACS plus ammonium hydroxide were obtained from Fisher Scientific (Fairlawn, NJ, USA). 36 115 Buffers were prepared from stock solutions for sample preparation during the 37 116 solid phase extraction process and/or for use for buffer mixtures used as components of mobile 38 117 phases. Three stock buffers were prepared during this study including 200 mM ammonium 39 118 formate (pH~ 3), 200 mM ammonium acetate (pH~9), and 100 mM ammonium acetate 40 119 (pH~6.4). The 200 mM ammonium formate buffer was prepared by weighing out approximately Methods 41 120 42 12.6 g of ammonium formate into a 1 L volumetric flask, adding 900 mL of water and 25 mL of 43 121 formic acid, and filling to the mark with water to a total volume of 1 L [31]. The 100 mM 44 122 ammonium acetate stock solution was prepared by weighing out approximately 7.71 g of 45 123 ammonium acetate into a 1 L volumetric flask, adding water to the mark of 1 L and 10 drops of 46 124 concentrated acetic acid [32]. The 200 mM ammonium acetate buffer stock was prepared by 47 125 48 weighing out 15.4 g of ammonium acetate and adding it to 900 mL of water in a 1 L volumetric 49 126 flask, 8 mL of ammonium hydroxide was added to the flask, and the solution was mixed 50 127 thoroughly. Water was then added to the 1 L mark of the flask [32]. 51 128 Analytical 52 129 2.2 Instrumentation and Data Analysis 53 130 54 131 55 A UHPLC-TOF MS system consisted of a PerkinElmer Flexar 15 liquid chromatograph coupled 56 132 with a PerkinElmer Axion 2 time of flight-mass spectrometer (Shelton, CT, USA) were used for 57 133 the analysis of all extracted and neat samples. PerkinElmer Chromera version 3.4.1 and TOF MS 58 59 3 60

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1 2 3 134 driver version 6.1 was used for the overall instrument control, data acquisition, and processing. 4 135 5 The preliminary UHPLC chromatographic separations for the basic drugs in SAMHSA-5 panel 6 136 were performed using a PerkinElmer Brownlee SPP C18 and a Perkin Elmer Brownlee HILIC 7 137 column (150 mm x 2.1 mm, 2.7 µm) . , A PerkinElmer PFP column (150 mm x 2.1 mm, 2.7 µm) 8 138 was used for subsequent separations of both the SAMHSA analytes and the “bath salts” using a 9 139 dual chromatographic mode approach. The following parameters were used for ESI + TOF-MS 10 140 detection: the dry gas heater for nitrogen was set at 325°C with a gas flow of 14.0 l/min; 11 141 12 capillary exit voltage 90V; MS data was acquired in the full scan mode from 100-1000 m/z at 13 142 three spectra per second.. External calibration was carried out using the ESI tuning mix. Mass 14 143 spectrometric parameters for the various drug examined are given in Table 1. 15 144 A Perkin Elmer Rocker 115 V vacuum pump was used for the manifold set-up for the 16 145 SPE procedure that was developed. Several columns were experimented with in order to develop 17 146 18 the proper SPE method, including: Perkin Elmer Precise-Bed Technology® Supra Clean® mixed 19 147 mode (MM1) (N9306542) and MM2 (N9306549)), C18 (N9306478), weak cation exchange 20 148 (WCX) (N9306545), strong cation exchange (SCX) (N9306432), and Supra Poly® HLB 21 149 polymeric columns (N9306656 and N9306650)(Shelton, CT, USA) Manuscript 22 150 23 151 24 2.3 Chromatographic conditions for RPC and HILIC separations of basic drugs in the 25 152 SAMHSA-5 panel on a C18 and HILIC column 26 153 27 154 RPC conditions; column SPP C18; injection volume 1 µL; solvent A: 0.1% formic acid in water 28 155 and solvent B 0.1% formic acid in acetonitrile; flow rate 0.30 mL/min, and temperature 25 °C. 29 156 Mobile phase, initial conditions 5%B, 95% A and final conditions 42.2% B, 58.8% B. 6.5 minute 30 157 linear gradient to final conditions.

31 Accepted 32 158 HILIC conditions; column SPP HILIC; injection volume 1 µL; solvent 10 mM 33 159 ammonium formate in acetonitrile: water (9:1); flow rate 0.50 mL/min, and temperature 25 °C. 34 160 Mobile phase 100% B. 35 161 36 162 37 2.4 Chromatographic conditions for RPC and HILIC separations of basic drugs in the 38 163 SAMHSA-5 panel and “bath salts” on a PFP column 39 164

40 165 SAMHSA-5 basic drug panel RPC conditions; injection volume 1 µL; solvent A 10 mM Methods 41 166 ammonium formate in acetonitrile:water (1:9) and solvent B 10 mM ammonium formate in 42 167 acetonitrile:water (9.5:0.5); flow rate and temperature 0.3 mL/min. and temperature 25°C. 43 168 44 Mobile phase, initial conditions 100% A and final conditions 34% A and 66% B. 12 minute 45 169 linear gradient to final conditions. 46 170 SAMHSA-5 basic drug panel HILIC conditions; injection volume 2 µL; solvent A 10 47 171 mM ammonium formate in acetonitrile:water (1:9) and solvent B 10 mM ammonium formate in 48 172 acetonitrile:water (9.5:0.5); flow rate and temperature 0.3 mL/min. and temperature 25°C. 49 173 50 Mobile phase 5% A and 95% B run for 6 minutes. 51 174 “Bath salts” RPC conditions; injection volume 1 µL; solvent A 5 mM ammonium acetate Analytical 52 175 in acetonitrile:water (1:9) and solvent B 5 mM ammonium acetate in acetonitrile:water (9.5:0.5); 53 176 flow rate and temperature 0.3 mL/min. and temperature 25°C. Mobile phase, 15% A and 85% B 54 177 run for 11 minutes. 55 178 “Bath salts” HILIC conditions; injection volume 1 µL; solvent A 5 mM ammonium 56 179 57 acetate in acetonitrile:water (1:9) and solvent B 5 mM ammonium acetate in acetonitrile:water 58 59 4 60

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1 2 3 180 (9.5:0.5); flow rate and temperature 0.3 mL/m in. and temperature 25°C. Mobile phase, 100% B 4 181 5 run for 11 minutes. 6 182 7 183 8 184 9 185 10 186 11 187 12 13 188 14 189 2.5 Procedures 15 190 16 191 2.5.1 Sample preparation “bath salts” and retention factor (k) determination. The 17 192 18 standard mixture was prepared by adding 5 µL each of the individual standards (1.0 mg/mL in 19 193 methanol) into a solution containing 925 mL of a 1:9 mixture of the solvents A and B used for 20 194 the chromatographic separation. The For retention factor k determinations, the time of the void 21 195 volume was calculated from the first disturbance of the baseline after injection. Manuscript 22 196 23 197 24 2.5.2 Solid phase extraction procedure for SAMHSA panel in urine. Fresh urine samples 25 198 were prepared for each extraction. Samples were prepared using 500 L of synthetic urine, 26 199 spiking a given amount of drugs and bringing the volume to 1 mL with ammonium formate 27 200 buffer (200 mM) or acetonitrile. 28 201 As reported later in the manuscript, an MM1 performed best for the solutes of interest. 29 202 The optimized SPE procedure for the extraction of target solutes within the SAMHSA-5 drug 30 203 panel was as follows. An MM1 mixed mode column with a 1 mL void volume was conditioned

31 Accepted 32 204 with methanol (1 mL), followed by two consecutive washes of ammonium acetate buffer (100 33 205 mM, pH~6) (1 mL). The spiked synthetic urine sample (1 mL) was then added to the column and 34 206 allowed to elute through. The column was washed with water (1 mL), followed by ammonium 35 207 acetate buffer (100 mM, pH~6) (1 mL), and concentrated acetic acid (diluted 1:10) (1 mL). The 36 208 37 sample was then eluted from the column using an elution solvent composed of ammonium 38 209 hydroxide and acetonitrile (20:80, v/v), in two 1 mL washes. All washes were allowed to elute 39 210 through the column under positive pressure (between 5 and 15 psi).

40 211 Methods 41 212 2.5.3 UHPLC-TOF-MS assay validation procedures for the determination of the 42 213 SAMHSA panel in urine. Ion suppression, extraction recovery, and linearity were assessed as 43 214 44 previously reported [10, 33]. Two different samples were prepared in order to assess the amount 45 215 of ion suppression seen under both HILIC and RPC conditions. The first sample, considered the 46 216 “spiked urine sample” was made by spiking 1 L of each drug (1.0 mg/mL in MeOH of 47 217 amphetamine, methamphetamine, MDMA, MDEA, MDA, BZE, PCP, codeine, and morphine) 48 218 and 1 L of O6-monoacetylmorphine (1.0 mg/mL in acetonitrile) into a solution containing 49 219 50 500 L of synthetic urine and 490 L of acetonitrile. The second sample, or “neat sample” was 51 220 made by spiking the same amount of each drug as was used in the urine sample preparation into Analytical 52 221 990 L of acetonitrile. Both the urine and neat samples were taken through the entire extraction 53 222 process as noted in Section 2.5. These samples were analyzed under HILIC and RPC conditions. 54 223 Each of the two samples was injected four times (1 µL injection) and the amount of ion 55 224 suppression was calculated by dividing the average peak area of the urine sample by the average 56 225 57 peak area of the neat sample. 58 59 5 60

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1 2 3 226 The recovery for the individual analytes from solid phase extraction was determined 4 227 5 using HILIC. Two different samples were made in order to perform the recovery study. The first 6 228 was a “pre-spiked” urine sample, composed of 1 L of each drug (amphetamine, 7 229 methamphetamine, MDMA, MDEA, MDA, BZE, PCP, codeine, 6-monoacetylmorphine, and 8 230 morphine) in a solution of 500 L of synthetic urine and 490 L of ammonium formate buffer 9 231 (100 mM). A “post-spiked” urine sample was composed of 500 L of synthetic urine, 490 L of 10 232 ammonium formate buffer (100 mM), and 10 L of methanol. Both of these samples were 11 233 12 extracted using the solid phase extraction method described in Section 2.5. The elution solvent 13 234 (NH4OH/ACN 20:80 v/v) resulting from the extraction of the post-spiked urine sample was 14 235 spiked with 1 L of each drug (amphetamine, methamphetamine, MDMA, MDEA, MDA, BZE, 15 236 PCP, codeine, 6-monoacetylmorphine, and morphine). These two samples were analyzed under 16 237 HILIC conditions and injected four times each (1 µL injection). The percent extraction recovery 17 238 18 was determined by dividing with the peak area of the pre-spiked urine sample by the peak area of 19 239 the post-spiked urine sample. 20 240 The linearity of the assay was determined by spiking a post-extracted blank urine sample 21 241 with differing concentrations of each drug and analyzing them under RPC and HILIC conditions. Manuscript 22 242 The “blank urine sample” was composed of 500 L of urine, 400 L of ammonium formate 23 243 24 buffer (100 mM), and 100 L of methanol. This sample was extracted using the solid phase 25 244 extraction method described in Section 2.42, and the resulting elution solvent (NH4OH/ACN 26 245 20:80 v/v) was collected and used for the creation of a 10 g/mL standard stock solution. This 27 246 standard stock solution was created by spiking 10 L of each drug (amphetamine, 28 247 methamphetamine, MDMA, MDEA, MDA, BZE, PCP, codeine, 6-monoacetylmorphine, and 29 248 morphine) into 900 L of the elution solvent. Serial dilutions from the solid phase extraction 30 249 solvent were performed on this 10 g/mL stock solution in order to generate samples at drug

31 Accepted 32 250 concentrations of 10, 000, 2,000, 400, 200, 80, 40, 16, 8 and 3.3 ng/mL, respectively. These 33 251 samples were analyzed in triplicate under HILIC and RPC conditions (2 µL and 1 µL injections, 34 252 respectively). 35 253 Limit of detection (LOD) was estimated by measuring the respective signal-to-noise ratio 36 254 37 (S/N>3). 38 255 39 256 3. Results and discussion

40 257 Methods 41 258 42 3.1 UHPLC-TOF MS separation of SAMHSA basic drug panel solutes of interest 43 259 44 260 A reversed phase UHPLC-TOF MS separation of a neat standard solution of the target solutes 45 261 for the SAMHSA basic drug panel is shown in Figure 1. For this separation using a SPP C18 46 262 column and a 0.1% formic acid-acetonitrile gradient, co-elution exists between MDA, 47 263 48 methamphetamine and O6-monoacetylmorphine. Therefore depending on the relative 49 264 concentration of these solutes in urine ion suppression could exist and lead to a measured 50 265 reduced concentration of a target solute. The use of a complementary separation technique such 51 266 as HILIC UHPLC-TOF MS could resolve the problematic solutes. Analytical 52 267 A hydrophilic interaction UHPLC-TOF MS separation of a neat standard solution of the 53 268 above solutes is shown in Figure 2. Although for an isocratic separation using an acetonitrile- 54 269 55 ammonium formate mobile phase with a SPP HILIC column there is extensive overlap between 56 270 several of the solutes, MDA, methamphetamine and O6-monoacetylmorphine are now resolved. 57 58 59 6 60

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1 2 3 271 An easier approach would be to use a column with a PFP stationary phase that could be 4 272 5 used in both the RP and HILIC mode with the same solutions from the binary solvent reservoir. 6 273 For A and B solvents consisting of acetonitrile: water with ammonium formate at low and high 7 274 organic solvent concentration, it was possible to perform separations for the solutes of interest in 8 275 both the RP and HILIC mode. As shown in Figure 3, complementary separations are obtained as 9 276 evidenced by the significantly different retention order for the solutes of interest. All solutes are 10 277 fully resolved by a combination of both techniques. 11 278 12 13 279 14 280 15 281 16 282 17 283 18 3.2 Solid phase extraction of analytes of interest 19 284 20 285 In order to minimize ion suppression, ion enhancement and to preserve the cleanliness of the 21 286 column and the TOF source, it is necessary to remove components from urine such as creatine, Manuscript 22 287 creatinine and urea from the sample matrix. For this purpose solid phase extraction was utilized. 23 288 24 Various columns were tested for analyte recovery including the MM1 mixed mode (silica, 25 289 reversed phase/ strong cation exchange), the MM2 mixed mode (silica, reversed phase/weak 26 290 cation exchange), WCX (slica, weak cation exchange), C-18 (silica, reversed phase), SCX 27 291 (silica, strong cation exchange), HLB (polymer, reversed phase), and the Supra-Poly HLB 30 28 292 UM (polymer, reversed phase) [34]. Preliminary recovery experiments indicated that the HLB 29 293 and MM1 columns provided the best extraction efficiency. However the MM1 column, as 30 294 indicated by TOF MS detection, was the best at removing the natural products found in, and thus

31 Accepted 32 295 it was the column of choice for the developed SPE methodology. 33 296 34 297 The extraction solvent composed of ammonium hydroxide and acetonitrile (20:80, v/v) 35 298 was chosen not only to provide good recovery of the analytes of interest, but also to allow direct 36 299 37 injection of the solid phase extract into the PFP column under both RPC and HILIC conditions. 38 300 The presence of a high concentration of acetonitrile will maximize recovery by minimizing 39 301 hydrophobic interactions of the analytes of interest with the C18 moiety on the solid phase

40 302 extraction column. The presence of ammonia to maximize recovery is necessary to minimize ion Methods 41 303 exchange interactions of the basic analytes with the strong cation exchange sites of the solid 42 304 phase extraction material by increasing the basicity of the solutes and/or if ammonium ion is 43 305 44 present (depending on the apparent pH of the extraction solvent) acting as a competing ion. The 45 306 direct injection into a UHPLC system of the extract containing the analytes of interest would 46 307 avoid for the solid phase extraction an evaporation and reconstitution step, which would occur 47 308 when the extraction solvent contains a non-aqueous compatible organic solvent (e.g., methylene 48 309 chloride) is used. A combination of the mismatch of the solvent strength and apparent pH of 49 310 50 solid phase extraction solvent with the starting mobile phase for RPC and the isocratic conditions 51 311 for HILIC limited the injection volume to 1 and 2 µL, respectively. Analytical 52 312 53 313 54 314 55 315 56 316 57 3.3 Method validation 58 59 7 60

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1 2 3 317 4 318 5 Recovery, ion suppression and ion enhancement data for solutes of interest after solid phase 6 319 extraction is shown in Table 2. Good recovery after solid phase extraction is obtained for the 7 320 drugs and metabolites (78.1 ≤%recovery ≤102.0). For most solutes recovery losses (or gains) 8 321 could be explained in part by the %RSD of the peak areas of the pre and post extraction spiked 9 322 urine samples. For the most part minimal ion suppression and ion enhancement is obtained for 10 323 RPC and HILIC after solid phase extraction (87.6% ≤ ion suppression/ ion 11 324 12 enhancement ≤111.1%). For MDMA HILIC and O6-MAM RPC ion enhancement and ion 13 325 suppression of 120% and ion suppression of 80.2 % respectively is obtained. 14 326 Linearity and limit of detection (LOD) data for the drugs and metabolites of interest is 15 327 shown in Table 3 for both RPC and HILIC. For most solutes, using both chromatographic 16 328 systems linearity is obtained over two orders of magnitude (R 2 ≥0.99). Amphetamine, which 17 329 18 exhibited a poor detection response using HILIC, gave measurable peaks for linearity only at 19 330 2000 and 10000 ng/L. For all of the solutes of interest, except for HILIC amphetamine, the 20 331 LOD was within the SAMHSA guidelines for initial test and confirmatory test cutoff 21 332 concentrations [35]. Manuscript 22 333 Since the higher organic mobile phases in HILIC versus RPC would favor MS ionization 23 334 24 for the former technique it was of interest to compare LOQ for both chromatographic modes. 25 335 Taking in account different peak widths for both techniques and different injection sizes (see 26 336 Figure 3 and Table 3) only MDMA exhibits the a slightly lower limit of quantitation using 27 337 HILIC vs RP (~2X), while all other solutes. All other solutes give higher limits of detection 28 338 using the former technique (~2-15X). 29 339 30 340 3.4 UHPLC-TOF MS separation of synthetic cathinones

31 Accepted 32 341 33 342 RPC and HILIC separations of 15 controlled synthetic cathinones using a PFP column with the 34 343 same solutions from the binary solvent reservoir are shown in Figure 4. As per the SAMHSA 35 344 basic drug panel, complementary separations are obtained as evidenced by the significantly 36 345 37 different retention order obtained for the bath salts on both retention modes. As shown in Figure 38 346 4, most solutes are fully resolved using a combination of RPC and HILIC. Pentylone and 39 347 buphedrone which co-elute using RPC are resolved with a resolution of 0.9 using HILIC, which

40 348 would make the mixed mode approach in tandem with TOF-MS detection suitable for Methods 41 349 confirmation and/or screening of bath salts. Besides qualitative analysis, the mixed-mode 42 350 chromatographic approach would be useful for quantitative analysis of seized drugs where co- 43 351 44 elution of either ‘bath salts” or target solutes with adulterants could occur. 45 352 Since isocratic conditions for RPC and HILIC of synthetic cathinones were relatively 46 353 close in % Solvent B, it was of interest whether both separation mechanisms were indeed 47 354 operative for the mobile phases employed. A plot of k versus % Solvent B for “bath salts” 48 355 exhibits “U shaped” retention behavior with a minimum at 95% Solvent B (see Figure 5). For % 49 356 50 Solvent B lower than this minimum value, k increases with decrease in % Solvent B, indicative 51 357 of reverse phase behavior; while the opposite occurs for % Solvent B above the minimum value, Analytical 52 358 indicative of a HILIC mechanism. 53 359 54 360 3.5 Orthogonality of RPC and HILIC separations 55 361 56 57 58 59 8 60

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1 2 3 362 Although the RPC and HILIC separations of the SAMHSA basic drug panel and the synthetic 4 363 5 cannabinoids respectively appeared complimentary, it was of interest to measure the 6 364 orthogonality of both pairs of separations. RPC versus HILIC regression plots for both the 7 365 SAMHSA basic drug panel and the “bath salts” are shown in Figures 6 and 7, respectively. Both 8 366 pairs of chromatographic conditions for each of the applications are orthogonal as indicated by 9 367 the low R 2 values of 0.0839 and 0.3948, for the SAMHSA basic drug panel and the “bath salts” 10 368 respectively. The reason for significantly lower R2 values of the SAMHSA solutes versus the 11 369 12 synthetic cathinones is very complex. Solute type, buffer type and concentration, apparent pH, 13 370 and % acetonitrile can play a role in the differences in separation between RPC and HILIC. 14 371 15 372 4. Conclusion 16 373 17 18 374 A novel method is presented for the analysis of the SAMHSA basic drug panel in urine, and bath 19 375 salts in seized drugs, which increases accuracy of solute identification, and minimizes sample 20 376 preparation and or decreases sample analysis time. This rapid approach for enhanced separation 21 377 selectivity, which uses a single column and the same elution solvents for both RPC and HILIC, Manuscript 22 378 23 could be applicable to other classes of illicit drugs as long as they are amenable to the mixed 24 379 mode approach. 25 380 26 381 Acknowledgements 27 382 28 29 383 We would like to thank the Perkin Elmer Corporation for donating the UHPLC and solid phase 30 384 extraction columns, for loan of the solid phase extraction device and for providing financial

31 385 support for our laboratory thru GWU Proposal #13-04142. The authors also are appreciative to Accepted 32 386 Professor Nicholas Lappas of George Washington University for useful discussions. 33 34 387 35 388 36 389 37 38 390 References 39 391

40 392 [1] http://www.employee-drug-testing-ace.com/resources/glossary/define-samhsa-5. Methods 41 393 [2] S.-C. Tsai, M.A. ElSohly, T. Dubrovsky, B. Twarowska, J. Towt, and S.J. Salamone, 42 394 43 Determination of five abused drugs in nitrite-adulterated urine by immunoassays and gas 44 395 chromatography- mass spectrometry, J. Anal. Toxicol. , 1998, 22, 474-480. 45 396 [3] J. Feng, L. Wang, I. Dai, T. Harmon, and J.T. Bernert, Simultaneous determination of 46 397 multiple drugs of abuse and relevant metabolites in urine by LC-MS-MS, J. Anal. Toxicol. , 47 398 2007, 31, 359-368. 48 399 49 [4] F. Badoud, M. Saugy, and J.L. Veuthey, in: D. Guillame, and J.L. Veuthey (Eds), UHPLC in 50 400 Life Sciences, RSC Publishing, Cambridge, 2012, pp.283-315. 51 401 [5]. D. French, A. Wu, and K. Lynch, The challenges of LC-MS/MS analysis of opiates and Analytical 52 402 opioids in urine, Future Science , 2013, 5, 2803-2820. 53 403 [6] F. T. Peters, Recent advances of liquid chromatography-(tandem) mass spectrometry in 54 404 clinical and forensic toxicology, Clin. Biochem ., 2011, 44 ,54-65. 55 56 57 58 59 9 60

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1 2 3 405 [7] M. Concheiro, A. De Castro, O. Quintela, A. Cruz, and M. Lopez-Rivadulla, Determination 4 406 5 of illicit drugs and their metabolites in human urine by liquid chromatography tandem mass 6 407 spectrometry including relative ion intensity criterion, J. Anal. Toxicol ., 2007, 31, 573-580. 7 408 [8]. A.M. Ares, and J. Bernal, Hydrophilic interaction chromatography in drug analysis, Cent. 8 409 Eur. J. Chem. , 2012, 10, 534-553. 9 410 [9] M. Kolmonen, A. Leinonen, T. Kuuranne, A. Pelander, and I. Ojanpera, Hydrophilic 10 411 interaction liquid chromatography and accurate mass measurement for quantification and 11 412 12 confirmation of morphine, codeine and their glucuronide conjugates in human urine, J. 13 413 Chromatogr. B. , 2010, 878 , 2959-2966. 14 414 [10] D. French, A. Wu, and K. Lynch, Hydrophilic interaction LC-MS/MS analysis of opioids in 15 415 urine: significance of glucuronide metabolites, Future Science , 2011, 3, 2603-2612. 16 416 [11] C. Gorgens, S. Guddat, A-K. Orlovius, G. Sigmund, A. Thomas, M. Thevis, and W. 17 417 18 Schanzer, “Dilute-and-inject’ multi-target screening assay for highly polar doping agents 19 418 using hydrophilic interaction liquid chromatography/high accuracy mass spectrometry for 20 419 sports drug testing, Anal. Bioanal. Chem ., 2015, 407, 5365-5379. 21 420 [12] www.drugabuse.gov/publications/drugfacts/synthetic . Manuscript 22 421 [13] Title 21 United States Code Controlled Substances Act, Part B, Section 812. 23 422 24 [14] L. Li, and I.S. Lurie, Screening of seized emerging drugs by ultra-high performance liquid 25 423 chromatography with photodiode array ultraviolet and mass spectrometric detection, 26 424 Forensic Sci. Int. , 2014, 237 , 100-111. 27 425 [15] R.P. Archer, Fluoromethcathinone, a new substance of abuse, Forensic Sci. Int. , 2009, 185, 28 426 10-20. 29 427 [16] K. Tsujikawa, T. Mikuma, K. Kuwayama, H. Miyaguchi, T. Kanamori, Y.Iwata, and H. 30 428 Inoue, Identification and differentiation of methcathinone analogs by gas chromatography-

31 Accepted 32 429 mass spectrometry, Drug. Test. Anal. , 2013, 5, 670-677. 33 430 [17] D. Zuba, Identification of cathinones and other active components of “legal highs” by mass 34 431 spectrometric methods, Trends in. Anal. Chem. , 2012, 32, 15-30. 35 432 [18] S. Mohr, S. Pilaj, and M.G. Schmid, Chiral separation of cathinone derivatives used as 36 433 37 recreational drugs by cyclodextrin-modified capillary electrophoresis, Electrophoresis , 38 434 2012, 33, 1624-1630. 39 435 [19] M. Svidrnoch, L. Lnenickova, I. Valka, P. Ondra, and V. Maier, Ultilization of micellar

40 436 electrokinetic chromatography- tandem mass spectrometry employed volatile micellar phase Methods 41 437 in the analysis of cathinone designer drugs, J. Chromatogr. A , 2014, 1356, 258-265. 42 438 [20] G. Merola, H. Fu, F. Tagliaro, T. Macchia, and B.R. McCord, Chiral separation of 12 43 439 44 cathinone analogs by cyclodextrin-assisted capillary electrophoresis with UV and mass 45 440 spectrometry detection, Electrophoresis , 2014, 35, 3231-3241. 46 441 [21] K.. G. Shanks, T. Dahn, G. Behonick, and A. Terrell, Analysis of first and second 47 442 generation legal highs for synthetic cannabinoids and synthetic stimulants by ultra- 48 443 performance liquid chromatography and time of flight mass spectrometry, J. Anal. Toxicol. , 49 444 50 2012, 36, 360-371. 51 445 [22] S. Mohr, M. Taschwer, and M.G. Schmid, Chiral Separation of cathinone derivatives used Analytical 52 446 as recreational drugs by HPLC-UV using a CHIRALPAK® AS-H column as stationary 53 447 phase, Chirality , 2012, 24, 486-492. 54 448 [23] M. Moini, and C. M. Rollman, Compatibility of highly sulfated cyclodextrin with 55 449 electrospray ionization at low nanoliter/minute flow rates and its application to capillary 56 57 58 59 10 60

Page 11 of 22 Analytical Methods

1 2 3 450 electrophoresis/electrospray ionization mass spectrometric analysis of cathinone derivatives 4 451 5 and their optical isomers, Rapid Commun. Mass Spectrom. , 2015, 29, 304-310. 6 452 [24] C.R. Aurand, W.K. Way, and D.S. Bell, Highly efficient separation of bath salts using 7 453 hydrophilic interaction chromatography (HILIC), LC GC The Application Notebook Sept. 1, 8 454 2011. 9 455 [25] D.S. Bell, and A.D. Jones, Solute attributes and molecular interactions contributing to “U- 10 456 shape” retention on a fluorinated high performance liquid stationary phase, J. Chromatogr. A , 11 457 12 2005, 1073, 99-109. 13 458 [26] ] M. Jayamanne, I. Granelli, A. Tjernberg, and P-O. Edlund, Development of a two- 14 459 dimensional liquid chromatography system for isolation of drug metabolites, J. Pharm. 15 460 Biomed. Anal., 2010, 51, 649-657. 16 461 [27] L. Xu, T. Zhao, X. Guan, W. Tang, X. Liu and H. Zhang, Preparation, chromatographic 17 462 18 evaluation and comparison of cystine- and cysteine-bonded stationary phases, Anal. Methods , 19 463 2014, 6, 2205-2214. 20 464 [28] T. Liang, Q. Fu, A. Shen, H. Wang, Y. Jin, H. Xin, Y. Ke, Z. Guo, and X. Liang, 21 465 Preparation and chromatographic evaluation of a newly designed steviol glycoside modified- Manuscript 22 466 silica stationary phase in hydrophilic interaction liquid chromatography and reversed phase 23 467 24 liquid chromatography , J. Chromatogr. A , 2015, 1388, 110-118. 25 468 [29] Q. Wang, M. Ye, L. Xu and Z.-G. Shi, A reversed-phase/hydrophilic interaction mixed- 26 469 mode C18-Diol stationary phase for multiple applications, Analytica Chimica Acta , 2015 in 27 470 press. 28 471 [30] R. L. Fitzgerald, T.L. Griffin, Y-M. Yun, R.A. Godfrey, R. West, A.J. Pesce, and D.A. 29 472 Herold, Dilute and shoot: Analysis of drugs of abuse using selected reaction monitoring for 30 473 quantification and full scan product ion spectra for identification, J. Anal. Toxicol. , 2012, 36,

31 Accepted 32 474 106-111. 33 475 [30 34 476 [31] I. Lurie, Hydrophilic interaction chromatography of seized drugs and related compounds 35 477 with sub 2 µm particle columns, J. Chromatogr. A , 2011 , 1218, 9336-9344. 36 478 37 [32] Waters Corporation, Comprehensive Guide to HILIC: Hydrophillic Interaction 38 479 Chromatography, Waters Corporation , 2014, 1-72. 39 480 [33] B.K. Matuszewski, M.L. Constanze, and C.M. Chavez-Eng, Anal. Chem. , 2003, 75, 3019-

40 481 3030. Methods 41 482 [34] Perkin Elmer, Fast, easy efficient sample preparation, 42 483 http://www.perkinelmer.com/CMSResources/Images/44-134866FAR_Application 43 484 44 _Booklet_FINAL.pdf . 45 485 [35] http://www.samhsa.gov/sites/default/files/workplace/2010GuidelinesAnalytesCutoffs.pdf 46 486 47 487 48 488 Figures 49 489 50 51 490 Analytical 52 491 53 54 55 56 57 58 59 11 60

Analytical Methods Page 12 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 492 20 493 21 Manuscript 22 494 23 495 24 496 25 497 Figure 1. Extracted ion chromatogram (EIC) of a standard mixture of 2000 ng/mL each of (a) 26 498 morphine, (b) codeine, (c) amphetamine, (d) MDA, (e) methamphetamine, (f) O6-MAM, (g) 27 499 28 MDMA, (h) MDEA, (i) BZE, (j) PCP. RPC separation on a SPP C18 column. See experimental 29 500 section for UHPLC conditions. A and final conditions 42.2% B, 58.8% B. 6.5 minute linear 30 501 gradient of final conditions.

31 502 Accepted 32 503 33 504 34 35 505 36 506 37 507 38 508 39 509 40 510 Methods 41 42 511 43 512 44 513 45 514 46 515 47 48 516 49 517 50 51 Analytical 52 53 54 55 56 57 58 59 12 60

Page 13 of 22 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 518 21 519 Manuscript 22 520 Figure 2. EIC of a standard mixture of 2000 ng/mL each of the solutes a-j, whose identity is 23 521 shown in figure 1. HILIC separation on a SPP HILIC column. See experimental section for 24 522 UHPLC conditions. 25 523 26 524 27 28 525 29 526 30 527 31 528 Accepted 32 529 33 34 530 35 531 36 532 37 533 38 534 39 535 40 Methods 41 536 42 537 43 538 44 539 45 540 46 47 541 48 542 49 543 50 544 Analytical 51 545 52 53 546 54 547 55 548 56 57 58 59 13 60

Analytical Methods Page 14 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 549 23 550 24 551 25 552 26 Figure 3. EIC’s of a direct SPE extract of synthetic urine containing 2000 ng/mL each of the 27 553 solutes a-j, whose identity is shown in Figure 1. RPC separation (A) and HILIC separation (B) 28 554 on a SPP PFP column. See experimental section for SPE and UHPLC conditions. 29 555 30 556 31 557 Accepted 32 558 33 34 559 35 560 36 561 37 562 38 563 39 564 40 Methods 41 565 42 566 43 567 44 568 45 569 46 47 570 48 571 49 572 50 573 Analytical 51 574 52 53 575 54 576 55 577 56 578 57 579 58 59 14 60

Page 15 of 22 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 580 Manuscript 22 581 23 582 24 Figure 4. EIC’s of a standard mixture of 5000 ng/mL each of (a) pentedrone, (b) 3 - 25 583 fluoromethcathinone, (c) pentylone, (d) buphedrone, (e) butylone, (f) 4 -fluoromethathinone, (g) 26 584 methcathinone, (h) methylone, (i) 4 -methylethcathinone, (j) α-PVP, (k) α-PBP, (l) mephedrone, 27 585 (m) MDPV, (n) naphyrone, (o) 4 -MePPP. RPC separation (A) and HILIC separation (B) on a 28 586 SPP PFP column. See experimental section for UHPLC conditions. The standard mixture was 29 587 prepared by adding 5 µL each of the individual standards (1.0 mg/mL in methanol) into a 30 588 solution containing 925 mL of a 1:9 mixture of the solvents A and B used for the

31 Accepted 32 589 chromatographic separation. 33 590 34 591 35 592 36 593 37 38 594 39 595

40 596 Methods 41 597 42 598 43 599 44 45 600 46 601 47 602 48 603 49 604 50 51 605 Analytical 52 606 53 607 54 608 55 609 56 610 57 58 59 15 60

Analytical Methods Page 16 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 611 23 612 24 613 25 614 26 Figure 5. Plot of retention factor k of selected synthetic cathinones versus % solvent B (for a 27 615 mixture of Solvent A and Solvent B employed in separation of “bath salts”). See experimental 28 616 section for UHPLC conditions. 29 617 30 618 31 619 Accepted 32 33 620 34 621 35 622 36 623 37 624 38 625 39 626

40 Methods 41 627 42 628 43 629 44 630 45 46 631 47 632 48 633 49 634 50 635 Analytical 51 636 52 53 637 54 638 55 639 56 640 57 641 58 59 16 60

Page 17 of 22 Analytical Methods

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 642 23 643 24 644 25 26 645 Figure 6. Scatter plot of relative retention (RRT) HILIC versus RRT RPC for SAMHSA mix. 27 646 Retention times relative to amphetamine. See experimental section for UHPLC conditions. 28 647 29 648 30 649 31 650 Accepted 32 33 651 34 652 35 653 36 654 37 655 38 39 656 657

40 Methods 41 658 42 659 43 660 44 661 45 46 662 47 663 48 664 49 665 50 666 Analytical 51 52 667 53 668 54 669 55 670 56 57 58 59 17 60

Analytical Methods Page 18 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 671 19 672 20 21 673 Figure 7. Scatter plot of relative retention (RRT) HILIC versus RRT RPC for controlled bath Manuscript 22 674 salts. Retention times relative to methcathinone. See experimental section for UHPLC 23 675 conditions. 24 676 25 677 26 27 678 28 679 29 680 30 681 31 682 Accepted 32 683 33 34 684 35 685 36 686 37 687 38 688 39 689

40 Methods 41 690 42 691 43 692 44 693 45 46 694 47 695 48 696 49 697 50 51 698 Analytical 52 699 53 700 54 701 55 56 57 58 59 18 60

Page 19 of 22 Analytical Methods

1 2 3 702 Table 1- Mass spectrometric parameters 4 + 5 Solute Application Formula Mass MH 6 amphetamine SAMHSA panel C9H13N 136.1126 7 methamphetamine SAMHSA panel C10H15N 150.1283 8 MDA SAMHSA panel C10H13NO2 180.1025 9 MDMA SAMHSA panel C11H15N02 194.1181 10 MDEA SAMHSA panel C12H17N02 208.1340 11 PCP SAMHSA panel C17H25N 244.2065 12 morphine SAMHSA panel C17H19N03 286.1443 13 14 benzoylecgonine SAMHSA panel C16H19N04 290.1392 15 codeine SAMHSA panel C18H21N03 300.1600 16 O6-monoacetylmorphine SAMHSA panel C19H21N04 328.1550 17 methcathinone “bath salt” C10H13NO 164.1075 18 mephedrone “bath salt” C11H15NO 178.1232 19 buphedrone “bath salt” C11H15NO 178.1232 20 4-fluoromethcathinone “bath salt” C10H12FNO 182.0981 21 3-fluoromethcathinone “bath salt” C10H12FNO 182.0981 Manuscript 22 23 pentedrone “bath salt” C12H17NO 192.1388 24 4-methylethcathinone “bath salt” C12H17NO 192.1388 25 methylone “bath salt” C11H13NO3 208.0974 26 4’-methyl PPP “bath salt” C14H19NO 218.1545 27 α-PBP “bath salt” C14H19NO 218.1545 28 butylone “bath salt” C12H15N03 222.1130 29 α-PVP “bath salt” C15H21NO 232.1701 30 pentylone “bath salt” C13H17N03 236.1287

31 Accepted 32 MDPV “bath salt” C16H21NO3 276.1600 33 naphyrone “bath salt” C19H23NO 282.1858 34 35 703 36 704 37 705 38 706 39 707 40 708 Methods 41 709 42 43 710 44 711 45 712 46 713 47 714 48 49 715 50 716 51 717 Analytical 52 718 53 719 54 720 55 56 721 57 58 59 19 60

Analytical Methods Page 20 of 22

1 2 3 722 Table 2- Recovery, ion suppression and ion enhancement data for UHPLC RP TOF MS and 4 723 5 HILIC TOF MS separations of drugs and metabolites described in the experimental section 6 724 7 Drug Mode % Recovery % Ion Suppression/ 8 %RSD area pre- Ion Enhancement 9 extraction spiked, post %RSD urine drug 10 extraction spiked sample, neat drug 11 sample 12 amphetamine RPC 109.8 (9.1, 9.3) 13 14 amphetamine HILIC 92.2 (11.7, 9.1) 97.8 (4.5, 11.1) 15 methamphetamine RPC 101.8 (16.0, 3.8) 16 methamphetamine HILIC 96.1 (4.5, 6.5) 102.1 (3.3, 5.2) 17 MDA RPC 95.4 (3.9, 10.9) 18 MDA HILIC 83.9 (13.6, 6.8) 110.5 (5.3, 11.4) 19 MDMA RPC 98.2 (8.8, 9.8) 20 MDMA. HILIC 95.0 (5.8, 7.2) 120.0 (3.6, 6.2) 21 MDEA RPC 88.6 (6.2, 8.1) Manuscript 22 23 MDEA HILIC 94.1 (3.7, 4.5) 104.1 (2.4, 3.0) 24 PCP RPC 104.7 (16.8, 14.9) 25 PCP HILIC 78.1 (5.7, 4.1) 101.4 (3.1, 7.7) 26 BZE RPC 87.6 (7.9, 0.4) 27 BZE HILIC 102.0 (8.5, 7.5) 105.4 (7.0, 15.3) 28 morphine RPC 105.8 (3.5, 4.5) 29 morphine HILIC 97.8 (9.3, 10.4) 111.6 (7.9, 10.0) 30 codeine RPC 91.5 (10.8, 6.2)

31 Accepted 32 codeine HILIC 97.8 (8.4, 7.0) 110.8 (1.9, 11.0) 33 O6-MAM RPC 80.2 (26.6, 19.4) 34 O6-MAM HILIC 88.7 (7.3, 6.8) 100.3 (3.8, 9.3) 35 725 36 726 37 727 38 728 39 729

40 Methods 41 730 42 731 43 732 44 733 45 734 46 47 735 48 736 49 737 50 738 Analytical 51 739 52 740 53 54 741 55 742 56 743 57 58 59 20 60

Page 21 of 22 Analytical Methods

1 2 3 744 Table 3- Linearity data for UHPLC RP TOF MS and HILIC TOF MS separations of drugs and 4 745 5 metabolites described in the experimental section 6 746 7 Drug Mode Linearity R2 LOD Initial Test Confirmatory 8 range (ng/mL) Cutoff Test Cutoff 9 (ng/mL) Concentration Concentration 10 amphetamine RPC 40-10000 0.9984 13 500 250 11 amphetamine HILIC 500 250 12 methamphetamine RPC 16-10000 0.9976 5 500 250 13 14 methamphetamine HILIC 8-2000 0.9999 3 500 250 15 MDA RPC 80-2000 0.9990 27 500 250 16 MDA HILIC 200-10000 0.9999 67 500 250 17 MDMA RPC 40-10000 0.9992 13 500 250 18 MDMA HILIC 8-2000 0.9991 3 500 250 19 MDEA RPC 16-10000 0.9993 5 500 250 20 MDEA HILIC 8-2000 0.9951 3 500 250 21 Manuscript 22 PCP RPC 16-10000 0.9957 5 25 25 23 PCP HILIC 3.2-2000 0.9936 1 25 25 24 BZE RPC 16-10000 1.0000 5 150 100 25 BZE HILIC 40-10000 0.9997 13 150 100 26 morphine RPC 16-10000 0.9921 5 2000 2000 27 morphine HILIC 80-10000 0.9991 27 2000 2000 28 codeine RPC 40-10000 0.9989 13 2000 2000 29 codeine HILIC 80-10000 0.9966 27 2000 2000 30 O6-MAM RPC 16-10000 0.9996 5 10 10

31 Accepted 32 O6-MAM HILIC 16-10000 0.9997 5 10 10 33 34 35 747 36 748 37 38 39 749

40 Methods 41 750 42 43 44 751 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 21 60

Analytical Methods Page 22 of 22

1 2 3 Methodology is presented for the rapid analysis of important drugs of abuse using a single 4 5 extraction procedure and a single UHPLC column with orthogonal methods using different 6 combinations of the same solutions in the solvent reservoir. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30

31 Accepted 32 33 34 35 36 37 38 39

40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60