Quantitation and Validation of 34 Fentanyl Analogs from Liver Tissue Using a QuEChERS Extraction and LC–MS-MS Analysis

2021 ◽  
Author(s):  
Joseph Cox ◽  
Kylea Mathison ◽  
Colby Ott ◽  
Joseph DelTondo ◽  
James C Kraner ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liver Tissue ◽  
Mass Spectrometry Analysis ◽  
Forensic Sciences ◽  
High Concentration ◽  
Spectrometry Analysis ◽  
Fentanyl Analogs ◽  
Ionization Suppression ◽  
High Concentrations ◽  
Overdose Deaths

Abstract Since 2013, drug overdose deaths involving synthetic opioids (including fentanyl and fentanyl analogs) have increased from 3,105 to 31,335 in 2018. Postmortem toxicological analysis in fentanyl-related overdose deaths is complicated by the high potency of the drug, often resulting in low analyte concentrations and associations with toxicity, multidrug use, novelty of emerging fentanyl analogs and postmortem redistribution. Objectives for this study include the development of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and subsequent liquid chromatography–mass spectrometry/mass spectrometry analysis, validation of the method following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 requirements and application to authentic liver specimens for 34 analytes including fentanyl, metabolites and fentanyl analogs. The bias for all 34 fentanyl analogs did not exceed ±10% for any of the low, medium or high concentrations and the %CV did not exceed 20%. No interferences were identified. All 34 analytes were within the criteria for acceptable percent ionization suppression or enhancement with the low concentration ranging from −10.2% to 23.7% and the high concentration ranging from −7.1% to 11.0%. Liver specimens from 22 authentic postmortem cases were extracted and analyzed with all samples being positive for at least one target analyte from the 34 compounds. Of the 22 samples, 17 contained fentanyl and metabolites plus at least one fentanyl analog. The highest concentration for a fentanyl analog was 541.4 μg/kg of para-fluoroisobutyryl fentanyl (FIBF). The concentrations for fentanyl (n = 20) ranged between 3.6 and 164.9 μg/kg with a mean of 54.7 μg/kg. The fentanyl analog that was most encountered was methoxyacetyl fentanyl (n = 11) with a range of 0.2–4.6 μg/kg and a mean of 1.3 μg/kg. The QuEChERS extraction was fully validated using the ASB Standard 036 requirements for fentanyl, metabolites and fentanyl analogs in liver tissue.

Quantitation of Fentanyl and Metabolites from Liver Tissue Using a Validated QuEChERS Extraction and LC–MS-MS Analysis

2020 ◽  
Author(s):  
Joseph Cox ◽  
Alex Train ◽  
Avery Field ◽  
Colby Ott ◽  
Joseph DelTondo ◽  
...  
Keyword(s):  
Matrix Effects ◽  
Linear Range ◽  
Mass Spectrometry Analysis ◽  
Forensic Sciences ◽  
Opioid Overdose ◽  
Spectrometry Analysis ◽  
Analytical Scheme ◽  
Target Drug ◽  
Ionization Suppression ◽  
High Concentrations

Abstract According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were −10.0% and 1.4% for fentanyl, −2.1% and −0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from −8.5% to −19.9% for the low concentrations, −3.6% to −14.7% for the medium concentrations and 1.5% to −16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5–100 and 4-ANPP had a linear range of 0.4–80 μg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3 μg/kg with a mean of 149.2 μg/kg (n = 10). The range of norfentanyl concentrations were 1.9 to 50.0 μg/kg with a mean of 14.1 μg/kg (n = 10). The range of 4-ANPP concentrations were 3.2 to 23.7 μg/kg with a mean of 7.5 μg/kg (n = 7).

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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