Fig 1.
Synthetic opioids belonging to the fentanyl family.
(a) Chemical structures of fentanyl, acetylfentanyl and their main metabolites, the potencies of each opioid relative to morphine are given in brackets; (b) commonly employed antidotes against fentanyl poisoning along with their circulation half-lives.
Fig 2.
Chemical modification of fentanyls.
(a) Reaction of fentanyl with 2,2,2-trichloroethoxycarbonyl chloride (Troc-Cl) to yield two unique, abiotic products that bear structural features of the original opioid (grayed boxes); (b) overall protocol to aid in the confirmation of fentanyl and norfentanyl in a biological sample by EI-GC-MS and targeted HR-LC-MS.
Fig 3.
Extraction and reaction of fentanyl with Troc-Cl when spiked in plasma at two concentrations (10 and 20 ng/mL).
(a) GC chromatogram of extracted plasma sample; (b) extracted ion (m/z = 245) chromatogram for fentanyl (rt = 32.8 min.) for low concentration spike; (c) extracted ion (m/z = 245) chromatogram for fentanyl for high concentration spike; (d) GC chromatogram of the reaction between extracted plasma sample and Troc-Cl; (e) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the low concentration sample showing no discernible peak while (f) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the high concentration sample shows a clear peak (rt = 33.03 min.) composed of Troc-norfentanyl.
Fig 4.
Mass spectral data associated with the reaction between fentanyl-spiked plasma matrices with Troc-Cl.
Mass spectra for (a) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (20 ng/mL) with Troc-Cl, note that the spectrum is complex and it is the result of an additional Troc-containing interference (Troc-norfentanyl peaks indicated in red); (b) Expansion of the molecular ion peak region for Troc-norfentanyl where the peaks arising from our product are highlighted in red and those from the Troc-containing interference are in gray; mass spectra for (c) extracted fentanyl when spiked at 10 ng/mL and (d) extracted fentanyl when spiked at 20 ng/mL; (e) mass spectrum for 2-phenylethyl chloride for the lowest fentanyl concentration spike and (f) for the highest fentanyl concentration.
Fig 5.
Extraction and reaction of fentanyl with Troc-Cl when spiked in urine.
(a) GC chromatogram of extracted urine sample; (b) extracted ion (m/z = 245) chromatogram for fentanyl (rt = 32.8 min.) for low concentration urine spike (5 ng/mL); (c) extracted ion (m/z = 245) chromatogram for fentanyl for high concentration urine spike (10 ng/mL); (d) GC chromatogram of the reaction between extracted urine sample and Troc-Cl; (e) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the low concentration urine sample (rt = 33.03 min.); (f) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the high concentration spiked urine sample.
Fig 6.
Mass spectral data associated with the reaction between fentanyl-spiked urine matrices with Troc-Cl.
Mass spectra for (a) extracted fentanyl when spiked at 5 ng/mL; (b) extracted fentanyl when spiked at 10 ng/mL; (c) mass spectrum of Troc-norfentanyl product arising from the treatment of fentanyl-spiked urine extract (10 ng/mL) with Troc-Cl; (d) expansion of molecular ion peak for Troc-norfentanyl; (e) 2-phenylethyl chloride, the second confirmatory by-product from the Troc-Cl reaction; (f) mass spectrum of 2-phenylethyl chloride containing interfering signals from other matrix components.
Fig 7.
Extraction and reaction of acetylfentanyl with Troc-Cl when spiked in plasma.
(a) GC chromatogram of extracted plasma sample; (b) extracted ion (m/z = 231) chromatogram for acetylfentanyl (rt = 32.3 min.) for low concentration plasma spike (50 ng/mL); (c) extracted ion (m/z = 231) chromatogram for fentanyl for high concentration plasma spike (200 ng/mL); (d) GC chromatogram of the reaction between extracted plasma sample and Troc-Cl; (e) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the low concentration plasma sample (rt = 32.6 min.); (f) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the high concentration spiked plasma sample.
Fig 8.
Mass spectral data associated with the reaction between acetylfentanyl-spiked plasma matrices with Troc-Cl.
Mass spectra for (a) extracted fentanyl when spiked at 50 ng/mL; (b) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (50 ng/mL) with Troc-Cl; (c) and 2-phenylethyl chloride, the second confirmatory by-product from the reaction; mass spectra for (d) extracted fentanyl when spiked at 100 ng/mL; (e) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (100 ng/mL) with Troc-Cl; (f) and 2-phenylethyl chloride for the highest concentration spike.
Fig 9.
Extraction and reaction of acetylfentanyl with Troc-Cl when spiked in urine.
(a) GC chromatogram of extracted urine sample; (b) extracted ion (m/z = 231) chromatogram for acetylfentanyl (rt = 32.3 min.) for low concentration urine spike (20 ng/mL); (c) extracted ion (m/z = 231) chromatogram for fentanyl for high concentration urine spike (100 ng/mL); (d) GC chromatogram of the reaction between extracted urine sample and Troc-Cl; (e) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the low concentration urine sample (rt = 32.6 min.); (f) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the high concentration spiked urine sample.
Fig 10.
Mass spectral data associated with the reaction between acetylfentanyl-spiked urine matrices with Troc-Cl.
Mass spectra for (a) extracted fentanyl when spiked at 20 ng/mL; (b) Troc-norfentanyl product arising from the treatment of fentanyl-spiked urine extract (20 ng/mL) with Troc-Cl; (c) and 2-phenylethyl chloride, the second confirmatory by-product from the reaction; mass spectra for (d) extracted fentanyl when spiked at 100 ng/mL; (e) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (100 ng/mL) with Troc-Cl; (f) and 2-phenylethyl chloride for the highest concentration spike.
Fig 11.
Analysis by HR-LC-MS of extraction and reaction of fentanyl with Troc-Cl when spiked in plasma.
(a) Total ion chromatogram (TIC) of extracted plasma sample; (b) extracted ion ([M+H+] = 337.2274 ± 5 ppm) for fentanyl (rt = 13.85 min.) for low concentration plasma sample (10 ng/mL); (c) extracted ion ([M+H+] = 337.2274 ± 5 ppm) for high concentration in plasma sample (20 ng/mL); (d) Total ion chromatogram (TIC) of reaction between extracted plasma sample with Troc-Cl; (e) extracted ion ([M+H+] = 407.0691 ± 5 ppm) for Troc-norfentanyl for low concentration spiked plasma sample; (f) extracted ion ([M+H+] = 407.0691 ± 5 ppm) for Troc-norfentanyl for the high concentration spiked plasma sample showing its appearance at rt = 15.91 min.
Fig 12.
Analysis by HR-LC-MS of extraction and reaction of fentanyl with Troc-Cl when spiked in urine.
(a) Ion chromatogram of extracted urine sample; (b) extracted ion ([M+H+] = 337.2274 ± 5 ppm) for fentanyl (rt = 13.85 min.) for low concentration urine sample (5 ng/mL); (c) extracted ion ([M+H+] = 337.2274 ± 5 ppm) for high concentration in urine sample (10 ng/mL); (d) Ion chromatogram of reaction between extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 407.0691 ± 5 ppm) for Troc-norfentanyl for low concentration spiked urine sample; (f) extracted ion ([M+H+] = 407.0691 ± 5 ppm) for Troc-norfentanyl for the high concentration spiked urine sample showing its appearance at rt = 15.91 min.
Fig 13.
Analysis by HR-LC-MS of extraction and reaction of acetylfentanyl with Troc-Cl when spiked in plasma.
(a) Total ion chromatogram (TIC) of extracted plasma sample; (b) extracted ion ([M+H]+ = 323.2118 ± 5 ppm) for acetylfentanyl (rt = 12.46 min.) for low concentration plasma sample (50 ng/mL); (c) extracted ion ([M+H]+ = 323.2118 ± 5 ppm) for acetylfentanyl for high concentration plasma sample (200 ng/mL); (d) Total ion chromatogram (TIC) of the reaction of extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 393.0534 ± 5 ppm) for Troc-norfentanyl for low concentration spiked plasma sample; (f) extracted ion ([M+H+] = 393.0534 ± 5 ppm) for Troc-norfentanyl for the high concentration spiked plasma sample showing its appearance at rt = 15.42 min.
Fig 14.
Analysis by HR-LC-MS of extraction and reaction of acetylfentanyl with Troc-Cl when spiked in urine.
(a) Total ion chromatogram (TIC) of extracted urine sample; (b) extracted ion ([M+H]+ = 323.2118 ± 5 ppm) for acetylfentanyl (rt = 12.46 min.) for low concentration urine sample (20 ng/mL); (c) extracted ion ([M+H]+ = 323.2118 ± 5 ppm) for acetylfentanyl for high concentration urine sample (100 ng/mL); (d) Total ion chromatogram (TIC) of the reaction of extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 393.0534 ± 5 ppm) for Troc-norfentanyl for low concentration spiked urine sample; (f) extracted ion ([M+H+] = 393.0534 ± 5 ppm) for Troc-norfentanyl for the high concentration spiked urine sample showing its appearance at rt = 15.42 min.