Toward the Interpretation of Positive Testing for Fentanyl and Its Analogs in Real Hair Samples: Preliminary Considerations

Abstract

The detection of new psychoactive substances (NPS) in hair has become extensively researched in recent years. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have appeared with increasing frequency in the illicit drug supply. While the detection of NSO in hair is now well documented, interpretation of results presents several controversial issues, as is quite common in hair analysis. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair was applied to 293 real samples. Samples were collected in the USA between November 2016 and August 2018 from subjects who had reported heroin use in the past year or had already tested positive to hair testing for common opiates. The range, mean and median concentrations were calculated for each analyte, in order to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. Over two-thirds (68%) of samples tested positive for fentanyl at concentrations between LOQ and 8600 pg/mg. The mean value was 382 pg/mg and the median was 95 pg/mg. The metabolites norfentanyl and 4-ANPP were also quantified and were found between LOQ and 320 pg/mg and between LOQ and 1400 pg/mg, respectively. The concentration ratios norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also tested as potential markers of active use and to discriminate the intake of fentanyl from other analogs. The common occurrence of samples positive for multiple drugs may suggest that use is equally prevalent among consumers, which is not the case, as correlations based on quantitative results demonstrated. We believe this set of experimental observations provides a useful starting point for a wide discussion aimed to better understand positive hair testing for fentanyl and its analogs in hair samples.

Targeted and untargeted detection of fentanyl analogues and their metabolites in hair by means of UHPLC-QTOF-HRMS

“…On the other hand, fentanyl itself is possibly potent enough to exclude the need of introducing further potentially lethal substances into the illegal market. Indeed, more investigation is still needed before a final interpretation is given for the low occurrence of carfentanil in hair testing [ 7 ].…”

“…Unlike blood and urine, hair analysis is increasingly used to detect long-term exposure to toxicologically relevant substances, as it offers a wide diagnostic window basically dependent on the hair length [4]. Hair analysis has been repeatedly used to ascertain past exposure and prevalence of different novel synthetic opioids (NSO) in the consumer's population [5][6][7][8][9][10][11][12][13][14]. The analytical methods currently available are generally based on the targeted detection of a limited and well-defined list of compounds to monitor, usually chosen on the base of the national or international reports, or alerts from national warning systems [15,16].…”

Hair analysis for New Psychoactive Substances (NPS): Still far from becoming the tool to study NPS spread in the community?

“…Generally, the analytical methodology consisted of the following steps: hair decontamination from external contaminants, hair digestion or pulverization and analytes extraction from a hair amount ranging between 10−100 mg. The hair decontamination procedures used were washing with: (i) organic solvents, such as methanol [ 20 , 21 , 23 , 24 , 32 , 34 , 43 , 44 , 47 , 50 , 51 , 53 , 54 , 56 , 58 , 60 ], ethanol [ 35 ], acetone [ 19 , 22 , [25] , [26] , [27] , 37 , 38 , 41 , 46 , 49 , 57 , 61 , 63 ], hexane [ 27 ], petroleum ether [ 41 , 49 ], dichloromethane [ 20 , 21 , 23 , 24 , [28] , [29] , [30] , [31] , 33 , 34 , 36 , 39 , 40 , 42 , 45 , 48 , [52] , [53] , [54] , [55] , [56] , [58] , [59] , [60] , 62 ], isopropanol [ 23 , 28 ], isooctane [ 38 , 52 ]; (ii) sodium dodecyl sulfate solution [ 35 , …”

“…Analytes’ extraction from hair, which followed hair digestion or pulverization, was achieved by either single step methanol extraction [ 20 , 25 , 27 , 43 , 44 , 47 , [49] , [50] , [51] , 53 , 56 , [58] , [59] , [60] , 62 ], or acidified methanol extraction [ 19 , 21 , 32 , 49 ], or ethanol extraction [ 41 ], or liquid-liquid extraction (LLE) with various mixtures of organic solvents [ 22 , 25 , 30 , 36 , 39 , 40 , 42 , 45 , 46 , 48 , 55 , 61 , 63 ], or extraction with aqueous buffers of organic solvents (methanol/ acetonitrile/ ammonium formate) [ 26 ], or (methanol/ acetonitrile/ trifluoroacetic acid (TFA)) [ 37 ] or (methanol/ TFA) [ 38 ] or (methanol/ acetonitrile/ ammonium acetate) [ 57 ], or solid phase extraction (SPE) on various cartridges [ 24 , 29 , 31 , 52 , 54 ] or mixed-mode solid phase extraction (MSPE) [ 33 , 34 ] or pressurized liquid extraction (PLE) [ 23 , 28 , 35 ], assisted in many cases by mild heat of the samples [ [19] , [20] , [21] , [22] , [23] , 25 , 26 , 29 , 34 , 36 , 39 , 40 , ...…”

“…Most of the reports presented the concurrent detection of several NPS from different chemical classes [ [19] , [20] , [21] , [22] , [23] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , 33 , 46 , 62 ] and others focused on the analysis of just one NPS class [ 24 , 32 , 34 , [43] , [44] , [45] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , 63 ]. Most of the reviewed methodologies used liquid chromatography coupled to low resolution mass spectrometry [ 21 , 22 , [24] , [25] , [26] , [27] , [28] , [29] , 31 , 33 , 35 , 36 , 39 , 41 , [43] , [44] , [45] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] ,…”