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Home , Alimemazine, Brallobarbital, Cisapride, Clomethiazole, Desmethylclozapine, Desmethylflunitrazepam

Screening of 300 in Blood Utilizing Second Generation Exactive Plus High-Resolution, Accurate Mass Spectrometer and ExactFinder Software Kristine Van Natta, Marta Kozak, Xiang He Thermo Fisher Scientific, San Jose, CA

FIGURE 2. Schematic diagram of the Exactive Plus high-resolution accurate FIGURE 5. ExactFinder processing method and database. FIGURE 7. LODs for around 490 compounds For targeted screening, ExactFinder uses parameters set in processing method to Overview mass benchtop mass spectrometer. identify and confirm the presence of compound based on database values. Figure 8 Purpose: Evaluate Thermo Scientific Exactive Plus high performance bench-top mass Compound LOD Compound LOD Compound LOD Compound LOD shows data review results for one donor sample. In this method, compounds were 1‐(3‐Chlorophenyl) 5 Deacetyl 5 5 Perimetazine 5 identified by accurate mass within 5 ppm and retention time. Identity was further spectrometer for screening of whole blood for forensic toxicology purposes. 10‐hydroxycarbazepine 5 5 5 Phenacetin 5 5‐(p‐Methylphenyl)‐5‐phenylhydantoin 10 Des(2‐hydroxyethyl) 5 LSD 5 Phenazone 5 confirmed by isotopic pattern and presence of known fragments. Methods: Whole blood samples were processed by precipitation with ZnSO / 6‐Acetylcodeine 100 Desalkylflurazepam 5 5 5 4 6‐Methylthiopurine 5 5 Maraviroc 5 5 Matrix effects were observed to be compound dependent and were generally within . Samples were injected onto an HPLC under gradient conditions and 6‐Monoacetylmorphine 5 5 MBDB 5 Phenylbutazone 5 7(2,3dihydroxypropyl) 5 50 MDEA 5 10 ±50%. Figure 9 shows peaks for in and blood to compare effect of dtdetec tdted on an Eti™Exactive™ Plus mass spec trome ter. RltResults were ana ldithlyzed with 7‐Aminoclonazepam 5 Desmethyldoxepin 5 MDMA 10 5 7‐Aminoflunitrazepam 5 Desmethylmirtazapine 5 10 5 matrix on signal intensity. Thermo Scientific ExactFinder software. 9‐hydroxy‐ 5 Desmethylnefopam 5 5 5 5 Desmethyltramadol 5 5 5 Results: Over 400 drugs were detected at LODs ranging from 5-100 ng/mL in whole Acenocoumarol 5 5 Mefloquine 5 5 5 5 5 Piroxicam 5 FIGURE 8. ExactFinder results page showing XIC chromatogram reconstructed blood. 5 5 Meperidine 5 5 Acetazolamide 5 10 10 50 wit h 5 ppm mass window (a), isotopic pattern (b) and fragment ion confirmation Ajmaline 5 5 Mepivacaine 5 5 (c). Fragment mass deviation is 1.5 ppm. Albuterol 20 5 5 5 Introduction Alfentanyl 5 Dibenzepine 5 10 Prazepam‐d5 5 Alfuzosine 5 5 5 5 5 Diltiazem 5 fumarate 50 Prednisone 5 Forensi c sci en tittists and dt toxi co log itists nee dtd to searc hfh for many differen t compoun ds in 10 5 Metenolone 20 Primaquine 5 Alpha‐hydroxy‐ 5 5 5 10 samples of human blood. Endogenous matrix components and the wide variety of 5 Dipyridamole 5 5 Procainamide 50 possible compounds make the task daunting. The second generation Exactive Plus 5 Disopyramide 5 Methandienone 100 5 Amidopyrine 5 100 5 5 high-resolution accurate mass spectrometer with fast polarity switching enables 5 5 10 Proguanil 5 5 5 10 10 identification of compounds in a wide chemical space with minimum interference from 5 Doxepine 5 Methoxsalen 5 5 endogenous compounds. Additionally, full scan data allows for retrospective analysis 5 Doxorubicin 5 Methoxyamphetamine 50 Prometryn 5 5 5 Methoxymethamphetamine 5 5 of data for previously unknown compounds. 5 Dropopizine 5 10 Propericiazine 5 5 5 Methylparaben 10 5 10 EDDP 5 5 5 Ampicillin 20 5 Methylprednisolone 10 5 Amprenavir 5 EMDP 5 Metobromuron 5 10 Methods FIGURE 3. Scan Parameters for FIGURE 4. Source Parameters Antipyrine 5 Enalaprilat 5 5 Pyrilamine 5 Apocodeine 5 20 5 5 Sample Preparation Exactive Plus Mass Spectrometer for APCI Probe. Aprindine 5 5 Metronidazole 5 Quinine 5 5 Esmolol 5 5 5 5 5 Mianserine 5 Raltegravir 5 Mix 50 µL of blood with 5 µL of internal standard solution containing and Parameter Value Parameter Value 5 5 Miconazole 20 Ramipril 5 5 Ethylamphetamine 10 Midazolam 5 5 -d9. Add 150 µL of 40mM ZnSO4 in 66% methanol and vortex 5 5 Milnacipram 5 5 immediately. Place samples in a –20°C freezer for 20-30 minutes. Centrifuge at 13,000 Full MS Sheath Gas 35 AZT 50 5 5 Riboflavin 5 5 Etodolac 5 Molsidomine 5 Rifabutin 5 rpm for 10 minutes. Transfer supernatant to and autosampler vial, cap, and inject Micro scans 1 Aux gas 15 Bendroflumethiazide 5 5 5 Risperidone 5 5 Etoposide 5 Mycophenolic acid 5 5 (a) (b) 20 µL onto HPLC system. Benzoylecgonine 5 5 5 Ropivacaine 5 Resolution 70,000 Sweep gas 1 5 5 5 Roxithromycin 5 Liquid Chromatography Betamethasone 5 Fenbufen 20 5 5 AGC Target 3e6 Discharge current 4 5 5 5 Salicylamide 50 The HPLC used is a Thermo Scientific Accela 600 pump with Accela™ open Bezafibrate 5 Fenofibrate 5 5 Saquinavir 5 Maximum IT 200 msec Capillary temp 320 5 5 N‐DemethylTrimipramine 5 5 autosampler. Mobile phases are 10 mM ammonium acetate in water (A), 0.1% formic 5 5 N‐Desmethyl 5 5 5 Finasteride 5 N‐Desmethyl Mephenytoin 5 5 acid in acetonitrile (B), and acetonitrile:1-propanol: (45:45:10) (C). The HPLC Scan Range m/z 120-1200 S-Lens RF Level 60 10 5 N‐Desmethylclobazam 5 5 ExactFinder utilizes novel algorithms for column used is a Thermo Hypersil GOLD, 5 µm, 100 x 3 mm run under the gradient 5 5 N‐ 5 5 background noise subtraction, peak AIF Vaporizer Temp 350 10 Flumethasone 5N‐Desmethyltrimipramine 5 Stanozolol 10 shown in Figure 1. Bupivacaine 5 20 N‐Desmethylvenlafaxine 5 5 BiBupropion 5 Flun itrazepam 5 NbillNebivolol 5 SfSufentan il 5 detection and integgqration that require no Micro scans 1 5 Flunixin 5 5 Sulfamethoxazole 5 user input. 5 5 Nelfinavir 5 Sulindac 5 Resolution 35,000 Butabital 5 Fluoxymesterone 5 Nelfinavir M8 5 5 (c) FIGURE 1. HPLC gradient method 5 5 Netilmicin 5 5 5 5 Nevirapine 5 Telmisartan 5 AGC Target 3e6 Cafeine 5 5 50 5 5 5 5 Terbinafine 5 Carbamazepine epoxyde 5 50 5 5 FIGURE 9. XIC of hydrocodone in water (a) and blood (b) showing matrix Start (min) Sec Flow (mL/min) %A %B %C Maximum IT 100 msec 5 5 5 Tetracaine 5 5 Glafenine 5 5 5 effects. 0.00 60 0.50 95 5 NCE 35.0 5 5 5 Theophylline 10 Theophylline‐7‐acetic 5 5 5 acid 5 101.0 660 0500.50 100 Scan Range m/m/z 80-1200 5 5 5 Thiopental 5 (a) (b) FIGURE 6. Chromatograms reconstructed with a 5 ppm mass window at 5 5 5 5 12.0 60 0.75 100 Parameters are the same for Chloramphenicol 10 Glyburide 50 5 20 5 ng/mL. 5 5 Norbenzoylecgonine 5 Thioridazine sulfone 5 positive and negative mode UCSF7 20 Haloperidol‐d4 5 10 5 13.0 30 1.00 100 RT: 2.78 - 10.67 SM: 5B RT: 2.91 - 10.11 SM: 5B 10 Heptabarbital 5 5 5 RT: 7.08 NL: 4.84E4 RT: 5.30 NL: 1.49E6 100 100 m/z= 409.1505-409.1545 m/z= 268.1894-268.1920 Chlorpheniramine 10 10 5 Ticlopidine 5 80 F: FTMS + p APCI corona 80 F: FTMS + p APCI corona 5 5 50 5 Full ms [120.00-1200.00] Full ms [120.00-1200.00] 13.5 60 2.00 95 5 60 60 Study Design AmlodipineMS ICIS UCSF7 Metoprolol MS ICIS UCSF7 10 5 Norethandrolone 50 Tinidazole 5 40 40 5 Hydrocodone 5 Norfloxacin 5 5 20 20 14.5 30 0.50 95 5 RT: 4.89 5 5 Norfluoxetine 5 Tolbutamide 5 To determine LOD for each compound, aliquots of whole blood were spiked with sets 0 0 RT: 4.92 NL: 2.60E7 RT: 6.40 NL: 1.79E6 100 100 Chlortalidone 5 Hydroquinine 5 Norfluvoxamine 5 Tolmetin 5 Conclusion m/z= 290.1372-290.1402 m/z= 361.1992-361.2028 of compound at 5, 10, 20, 50, and 100 ng/mL. Matrix effects were determined by 80 F: FTMS + p APCI corona 80 F: FTMS + p APCI corona Cilazapril 5 Hydroxychloroquine 5 5 5 Full ms [120.00-1200.00] Full ms [120.00-1200.00] 60 60 Trans‐2‐ MS ICIS UCSF7 MS ICIS UCSF7 processing a sample spiked in water in place of blood. 40 Benzoylecgonine 40 Prednisolone 5 HydroxyItraconazole 5 Nor‐LSD 5 Phenylcylopropylamine 10 . We have developed a screening method for over 400 compounds in whole blood RT: 6.01 Mass Spectrometry 20 20 Cinchonine 5 5 Normaprotiline 5 5 0 0 with LODs of 5-100 ng/mL. RT: 4.48 NL: 1.95E6 RT: 7.40 NL: 1.24E5 5 5 Normeperidine 20 5 Data Analysis 100 100 m/z= 195.0867-195.0887 m/z= 609.2777-609.2837 5 5 Normesuximide 5 5 80 F: FTMS + p APCI corona 80 F: FTMS + p APCI corona Full ms [120.00-1200.00] Full ms [120.00-1200.00] 5 5 10 50 Compounds are detected on a Exactive Plus high performance bench-top mass 60 60 . Matrix effects are compound dependent and ±50%. Chromatograms are reconstructed with a mass tolerance of 5 ppm. Compounds are MS ICIS UCSF7 Reserpine(+) MS ICIS UCSF7 5 5 5 5 spectrometer equipped with an Orbitrap™ mass analyzer. A schematic diagram of the 40 40 20 20 5 5 5 5 . Combination of high-resolution, accurate mass and fast polarity switching allows identified based on exact mass and retention time; confirmation is with fragments and 0 0 Clindamycin 50 Indometacin 5 5 5 RT: 4.28 NL: 7.59E5 RT: 7.45 NL: 5.00E2 Exactive Plus instrument is illustrated in Figure 2. 100 100 m/z= 177.1013-177.1031 m/z= 607.2631-607.2691 5 Irebsartan 5 5 Trimeprazine 5 isotopic pattern using ExactFinder™ software. Figure 5 shows the ExactFinder method 80 F: FTMS + p APCI corona 80 F: FTMS - p APCI corona for rapid and confident identification in whole blood matrix for forensic toxicology Full ms [120.00-1200.00] Full ms [120.00-1200.00] 5 Isocarboxazide 5 Noscapine 5 5 60 60 An atmospheric pressure chemical ionization (APCI) probe was used as an ion source. MS ICIS UCSF7 MS ICIS UCSF7 Clomipramine 5 5 5 Trimethoprim 5 used in this study along with the compound database. 40 40 Reserpine(-) purposes. 5 5 5 5 The instrument was oppgerating in alternatin gpg positive and ne gative full-scan and all-ion 20 20 0 0 Clop idogre l 5 KtKetoconazol e 10 OdOndanse tron 5 TtiTropatepine 5 RT: 5.30 NL: 2.24E5 RT: 7.70 NL: 3.24E6 100 100 fragmentation (AIF) mode with fast polarity switching at a resolution of 70,000 to m/z= 250.1789-250.1815 m/z= 280.1662-280.1690 5 Ketoprofen 5 Opipramol 5 5 80 F: FTMS + p APCI corona 80 F: FTMS + p APCI corona Cloxacillin 100 Ketorolac 5 5 Valsartan 50 Full ms [120.00-1200.00] Full ms [120.00-1200.00] Results 60 60 Acknowledgements 35,000 (FWHM) at m/z 200. MS ICIS UCSF7 MS ICIS UCSF7 5 5 5 Vecuronium 100 40 Desmethyltramadol 40 Tolbutamide-d9(+) RT: 7.17 Clozapine N‐oxide 5 50 5 5 20 20 RT: 5.04 RT: 6.46 RT: 8.30 RT: 9.32 5 Lacosamide 5 5 5 Over 400 different compounds were tested in this study: 487 were detected at 0 0 We would like to thank Bénédicte Duretz of Thermo Scientific in France for supplying The Exactive Plus has an S-lens for improved ion transmission. The performance and RT: 6.99 NL: 2.23E6 RT: 7.71 NL: 3.39E6 100 100 10 5 5 5 m/z= 278.1889-278.1917 m/z= 278.1516-278.1544 5 ng/mL, 39 at 10 ng/mL, 10 at 20 ng/mL, 18 at 50 ng/mL, and 7 at 100 ng/mL. Figure 80 F: FTMS + p APCI corona 80 F: FTMS - p APCI corona 5 LAMPA 5 5 10 compound pools and Sarah Shugarts of UCSF Hospital for supplying donor samples. robustness of the Automatic Gain Control (AGC) is improved using a C-Trap charge Full ms [120.00-1200.00] Full ms [120.00-1200.00] 60 60 MS ICIS UCSF7 Tolbutamide-d9(-) MS ICIS UCSF7 5 5 10 Vincristine 5 detector (CTCD). The higher-energy collisional dissociation (HCD) cell collects 6 shows a representative chromatogram. Fast polarity switching enabled identification 40 EDDP 40 Cotinine 5 10 Pancuronium 50 5 20 20 of both positively and negatively charged species in one analytical run. Average mass RT: 7.30 RT: 8.74 5 10 5 Voriconazole 5 fragment-rich spectra similar to those generated in the collision cell of a triple stage 0 RT: 7.37 NL: 1.34E7 0 4.13 NL: 1.88E8 For forensic toxicology use only. 100 100 5 5 5 5 m/z= 310.2149-310.2181 TIC MS UCSF7 quadrupole instrument. Relevant scan and source parameters are shown in Figure 3 accuracy was <3 ppm. Figure 7 shows the limits of detection of compounds in this 80 F: FTMS + p APCI corona 80 Cyclophosphamide 5 5 5 5 Full ms [120.00-1200.00] 5.28 TIC All trademarks are the pppropert y of Thermo Fisher Scientific and its subsidiaries. 60 60 10. 11 5 Lisinopril 100 5 5 MS ICIS UCSF7 4.93 9.67 Methadone 7.36 and 4. stdtudy. 40 40 3.75 4.49 9.11 Danazol 5 Lopinavir 5 5 5 6.41 7.24 This information is not intended to encourage use of these products in any manners that might infringe the 20 20 Dantrolene 100 5 5 10 0 0 intellectual property rights of others. 3 4 5 6 7 8 9 10 3 4 5 6 7 8 9 10 Dapsone 5 5 5 5 Time (min) Time (min) Darunavir 5 5 5 5