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 . 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 . 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 . 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.
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
Previous articleMoreover, Division of Neurochemistry and Neuropathology participates in external quality control for indirect immunofluorescence and collection blot and was certified by Institut fur Qualitatssicherung, Germany (certificate QV 1111\15013, II/2015)Next article Based on the verified Abs for ERCs, this study demonstrated that ERCs were positive for CD29, CD90, and CD105, while negative for CD45 (Fig