The use of IHC in Subject C (a former addict) showed no positivity for morphine in the fingernail, while the UHPLC-HRMS analysis confirmed its absence in the fingernail and blood

The use of IHC in Subject C (a former addict) showed no positivity for morphine in the fingernail, while the UHPLC-HRMS analysis confirmed its absence in the fingernail and blood. of the scalp or pubic hair of the subjects was carried out using UHPLC-HRMS. The results suggest that IHC can be used to establish the site of accumulation of morphine in the nail matrix; for postmortem diagnosis; and that basic substances can be detected by UHPLC-HRMS. There are no previous studies on the use of IHC as a technique for forensic purposes in unconventional matrices, such as nails. in an ultracentrifuge. The supernatant was collected and evaporated; subsequently, the samples were resuspended with 50 L of phase B (Methanol + 1% formic acid) for chromatographic injection. Acid Hydrolysis and Extraction Procedure in Blood Matrix The acid hydrolysis and extraction procedure were carried out according to previous work [9]. Morphine is found in the blood as 3-glucuronide and 6-glucuronide forms after administration, so it was extracted by acid hydrolysis, detaching glucuronides to reveal the total amount of free morphine [10]. A total of 2 mL of blood was added with 200 L of chloridric acid and 250 ng of IS. The samples were incubated for 24 h at 55 C. The solid-phase extraction procedure followed the method used in our laboratory described in previous work [11]. Derivatization The eluted samples were completely evaporated and then derivatized with 20 L of MSTFA at 60 C for 20 min. One microliter of the derivatized sample was injected into the GC-MS. 2.2.3. Hr-LC and GC-MS Parameters Hair and Nail Analysis and Quantification by UHPLC-HRMS The Thermo Scientific Dionex Ultimate 3000 chromatographic system (UHPLC) coupled with Thermo Exactive Plus Orbitrap (HR-MS) was used for hair and nail analysis. The conditions applied for chromatographic analysis were as follows: the column used was Kinetex Biphenyl 2.6 m (50 2.1 mm) by Phenomenex; column flow was set at 0.4 mL/min. Phase A used H2O + 0.1% formic acid; Phase B used MeOH + 0.1% formic acid. The column temperature was set to 25 C. The elution gradient is shown in Table 1. Table 1 UHPLC elution gradient. thead th align=”center” valign=”middle” style=”border-top:solid thin;border-bottom:solid thin” rowspan=”1″ colspan=”1″ TIME /th th align=”center” valign=”middle” style=”border-top:solid thin;border-bottom:solid thin” rowspan=”1″ colspan=”1″ PHASE A (%) /th th align=”center” valign=”middle” style=”border-top:solid thin;border-bottom:solid thin” rowspan=”1″ colspan=”1″ PHASE B (%) /th /thead 0C0.59820.5C10010010C12010012C1398213C15982 Open in a separate window Scheme 60: For the identification of analytes, exact mass (EM) obtained from In Source Collision Induced Dissociation (50 eV) (In source CID), with an acceptance range of 5 ppm, and production (PI) were used. The values monitored for analytes were as follows: 286.14377 (EM) (PI: 201.09101, 229.08592, 183.08044) for morphine; 328.15433 (EM) (PI: 211.07540, 183.08040, 193.06480) for 6-MAM; 300.15942 (EM) (PI 215.10666, 243.10157, 199.07536) for codeine; 310.21654 (EM) (PI: 105.03349, 219.11683, 195.11683) for methadone; 287.19033 (EM) (PI: 234.12773, 249.15120, 186.12773) for EDDP; and 354.24276 (EM) (PI: 167.08553, 91.05423, 105.06988) for SKF ITGB8 (IS). RG7800 Blood Analysis and Quantification by GC-MS The GC-MS Polaris-Q was used for blood analysis. Analytical conditions were as follows: a capillary column (ZB 5 MS 30 m 0.25 mm 0.25 RG7800 m); helium as a carrier gas at a flow rate of 1 1.5 mL/min; a temperature program that started at 100 C for 1 min and was increased first to 220 C at 30 C/min for 1 min, and then to 320 C at 20 C/min for 6 min (total run time of 17 min); an injection volume that was 1 L in splitless mode; Full Scan mode, and a mass spectra range of 70C500. Subsequently, the RG7800 specific SIM layout for morphine, 6-MAM, codeine, methadone, and EDDP was applied. The ion values monitored for analytes were as follows: m/z 429 414 324 for morphine-TMS, m/z 399 340 287 for 6-MAM-TMS, m/z 371 178 196 for codeine-TMS, m/z 72 294 223 for methadone, m/z 276 277 262 for EDDP, and m/z 455.