Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Assay for Quantifying Fentanyl and 22 Analogs and Metabolites in Whole Blood, Urine, and Hair

Abstract

Recently, synthetic opioid-related overdose fatalities, led by illicitly manufactured fentanyl and analogs, increased at an alarming rate, posing a global public health threat. New synthetic fentanyl analogs have been constantly emerging onto the drug marked for the last few years, to circumvent the laws and avoid analytical detection. Analytical methods need to be regularly updated to keep up with the new trends. In this study, we aimed to develop a new method for detecting the newest fentanyl analogs with a high sensitivity, in whole blood, urine, and hair. The method is intended to provide to clinical and forensic toxicologists a tool for documenting consumption. We developed a comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantifying fentanyl and 22 analogs and metabolites. Urine samples were simply diluted before injection; a liquid-liquid extraction was performed for blood testing; and a solid phase extraction was performed in hair. The chromatographic separation was short (8 min). The method was validated with a high sensitivity; limits of quantifications ranged from 2 to 6 ng/L in blood and urine, and from 11 to 21 pg/g in hair. The suitability of the method was tested with 42 postmortem blood, urine, or hair specimens from 27 fatalities in which fentanyl analogs were involved. Average blood concentrations (±SD) were 7.84 ± 7.21 and 30.0 ± 18.0 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively ( n = 8), 4.08 ± 2.30 μg/L for methoxyacetylfentanyl, ( n = 4), 40.2 ± 38.6 and 44.5 ± 21.1 μg/L for acetylfentanyl and acetyl norfentanyl, respectively ( n = 3), 33.7 and 7.17 μg/L for fentanyl and norfentanyl, respectively ( n = 1), 3.60 and 0.90 μg/L for furanylfentanyl and furanyl norfentanyl, respectively ( n = 1), 0.67 μg/L for sufentanil ( n = 1), and 3.13 ± 2.37 μg/L for 4-ANPP ( n = 9). Average urine concentrations were 47.7 ± 39.3 and 417 ± 296 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively ( n = 11), 995 ± 908 μg/L for methoxyacetylfentanyl, ( n = 3), 1,874 ± 1,710 and 6,582 ± 3,252 μg/L for acetylfentanyl and acetyl norfentanyl, respectively ( n = 5), 146 ± 318 and 300 ± 710 μg/L for fentanyl ( n = 5) and norfentanyl ( n = 6), respectively, 84.0 and 23.0 μg/L for furanylfentanyl and furanyl norfentanyl, respectively ( n = 1), and 50.5 ± 50.9 μg/L for 4-ANPP ( n = 10). Average hair concentrations were 2,670 ± 184 and 82.1 ± 94.7 ng/g for fentanyl and norfentanyl, respectively ( n = 2), and 10.8 ± 0.57 ng/g for 4-ANPP ( n = 2).

Hair testing for 3-fluorofentanyl, furanylfentanyl, methoxyacetylfentanyl, carfentanil, acetylfentanyl and fentanyl by LC–MS/MS after unintentional overdose

“…From the present case study, it seems that Salamone's findings would probably reflect occasional exposure to fentanyl and furanylfentanyl rather than low rate of incorporation into the keratin matrix because the concentrations in the second hair analysis clearly demonstrated high rate of incorporation of the two compounds into the hair. Moreover, Busardò et al [27] have also reported high concentrations of fentanyl in hair (mean 2670 ± 184 pg/mg, n=2), confirming our findings.…”

“…Overall, at least 28 deaths were reported across Europe and the United States during 2017, largely in a home environment, which showed that in at least some of these cases the poisoning was so severe that the poisoned persons were unable to call for help [36]. Methoxyacetyl norfentanyl, a metabolite of methoxyacetylfentanyl was not screened in our method, but as reported by Busardò et al [27], this metabolite is not a suitable biomarker of methoxyacetylfentanyl consumption, and was not detected when methoxyacetylfentanyl was taken, whether in blood or urine.…”

“…4-ANPP is both a precursor and a metabolite of several fentanyl analogs. It is a major metabolite of fentanyl [27], furanylfentanyl [41], methoxyacetylfentanyl [42] and a minor metabolite of acetylfentanyl [41]. However, in previous studies, reported 4-ANPP hair concentrations were lower than those of parent drugs, making it an inefficient and nonspecific marker of consumption [27].…”

“…Even if this approach remains scarce and requires some particular skills, it deserves to be considered as a direct and long-term indicator of drug exposure. Some prevalence and analytical data regarding new psychoactive substance (NPS) study by hair testing in high-risk populations are now available [21][22][23][24][25][26][27].…”

Development and validation of liquid chromatography‐tandem mass spectrometry targeted screening of 16 fentanyl analogs and U‐47700 in hair: Application to 137 authentic samples

“…To our knowledge, only a few targeted screening methods for NSOs identification and quantification in hair have been published so far 19–22 . In this study, we describe the development and validation of a targeted LC–MS/MS assay for the detection and quantification of 16 fentanyl analogs and metabolites, and one non‐fentanyl derivative (U‐47700) in hair, and demonstrate its suitability in routine practice by the screening of 137 authentic hair samples collected from high‐risk populations in Paris and its suburbs.…”