Ten Years of Fentanyl-Like Drugs

Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

1 TEN YEARS OF FENTANYL-LIKE DRUGS:

2 A TECHNICAL-ANALYTICAL REVIEW 3 4 Gabriella Roda1, Francesca Faggiani1, Cristiano Bolchi1, Marco Pallavicini1, Michele Dei Cas2 5 1 Department of Pharmaceutical Sciences, University of Milan, Milan, Italy 6 2 Department of Health Sciences, University of Milan, Milan, Italy 7 8 Corresponding author: Michele Dei Cas, Department of Health Sciences, University of Milan, 9 Milan, Italy. tel.: +390250323274 email: [email protected]; 10 11 Keywords: fentanyl; designer opioids; fentanyl-like compounds; illicit fentanyls; mass 12 spectrometry 13 14 Abstract 15 Synthetic opioids such as fentanyl and its analogues are a new public health warning. Clandestine

16 laboratories produce drug analogues at a faster rate than these compounds can be controlled or

17 scheduled by drug agencies. Detection requires specific testing and clinicians may confront with a

18 sequence of severe issues concerning the diagnosis and the management of these contemporary

19 opioid overdoses. This paper deals with the methods for the biological sample treatment and the

20 methodologies of analysis that have been reported, in the last decade, in the field of fentanyl-like

21 compounds. From this analysis, it emerges that the gold standard for the identification and

22 quantification of 4-anilinopiperidines is LC-MS/MS coupled with liquid-liquid or solid-phase

23 extraction. In the end, the return to the scene of illicit fentanyls can be considered as a critical

24 problem which can be tackled only with a global multidisciplinary approach.

25

26 1. Introduction

27 Fentanyl is a potent μ-opioid agonist which has been used therapeutically, owing to its high lipid

28 solubility and potency (50-100 fold higher than morphine), as both an analgesic and anaesthesia

29 adjuvant. Fentanyl and its derivatives (Fig.1) such as sufentanil, alfentanil, remifentanil and

30 carfentanil belong to the class of 4-anilinopiperidines and exert their pharmacological action

1 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

31 through interaction with the -opioid receptor1. Their adverse effects are related to dose and include

32 respiratory depression, sedation, nausea, vomiting, constipation, pruritus, physical dependence, the

33 risk of addiction, bradycardia, and skeletal muscle rigidity2,3. Immediately after their introduction

34 into the market as drugs for the treatment of pain, there was a significant increase in abuse, off-label

35 and illicit uses4–6. Besides a series of illicit fentanyls (Fig.2), also known as non-pharmaceutical

36 fentanyls or designer fentanyls, have been developed causing a major health risks problem7–9. The

37 first far-reaching illicit use of fentanyl analogues (identified with the street names of China White

38 or Synthetic Heroin) was in California between 1979 and 198810. The composition of China White

39 is complex and non-standardizable. The scientific literature reported a mixture of p-fluorofentanyl,

40 alpha-methylfentanyl, 3-methylfentanyl, acetyl-fentanyl which may include heroin constituents11–14.

41 The danger related to fentanyl derivatives is due to 1) variability of compounds that may exist in a

42 given sample 2) the inconsistency of dosages 3) their potency 4) easy access by the consumer

43 through the deep web or black market14,15 and 5) fatal respiratory depression. In recent years, the

44 great spread of fentanyl-like compounds created a massive problem from either the social, health,

45 normative and analytical points of view. Hundreds of analytically confirmed deaths are synthetic

46 opioids-related within the past years. They are rising at a worrying rate in confront of those

47 produced by the misuse of natural and semi-synthetic opiates16.

48 In this paper, we have been focused on the problem of the identification and quantification of these

49 substances which is always very difficult because of the low dose administered and their chemical

50 heterogeneity. Thus, the instruments must be sensitive enough to detect them and equipped with

51 high resolution for the identification of unknown molecules17. The class of illicit fentanyls includes

52 a long list of fentanyl derivatives such as 1) methyl-analogues 2) W-series 3) acetyl-analogues 4)

53 butyr-analogues 5) thio-analogues 6) hydroxy-analogues 7) furanyl-analogues 8) benzyl-analogues

54 9) cyclo-analogues 10) acrylfentanyl 11) ocfentanil 12) fluoro-analogues and 13) other chemically

55 unrelated opioid agonists. In the latter significant examples are piperazine derivatives (such as MT-

56 45) and benzamide ones (such as AH-7921, U-47700 and U-50488). 2 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

57 2. Methods

58 A literature search was performed on Pubmed database and several governmental and institutional

59 websites, taking into consideration any analytical method of detection, extraction and determination

60 of fentanyl, fentanyl analogues, illicit fentanyl or designer fentanyls with particular focus on LC-

61 MS and GC-MS. The following sequence of keywords were used: (fentanyl OR illicit fentanyl OR

62 names of particular designer fentanyls) AND (analytical OR extraction OR detection OR method

63 OR determination OR analysis OR LC-MS OR GC-MS). In this review were included only those

64 articles that are more recent than ten years and had abstracts available in the English language. All

65 articles were then selected to determine their relevance in the framework of the present review.

66

67 3. Analysis of fentanyl-like compounds

68 3.1 Extraction techniques

69 The techniques that are most widely used to extract fentanyl-like compounds from

70 complex/biological matrices are solid-phase extraction (SPE) and liquid-liquid extraction (LLE).

71 SPE18–20 and LLE21–23 are reliable and widespread techniques used in the field of chemical-

72 toxicological analysis for the detection of the majority of toxic compounds. Several slightly

73 different protocols for fentanyls extraction were evaluated throughout the literature. In the main

74 text, the most significant methodologies will be discussed while a concise overview of variations

75 and alternative methods can be found in Tab.1.

76 SPE is currently the most widely used sample preparation technique used for chemical analysis in

77 different areas of interest24,25. The extraction process is based on the interaction of the analytes of

78 interest, dissolved in a liquid phase, with an adsorbent-solid phase. SPE is an attractive replacement

79 for LLE since it has some advantages. Compared to LLE, the SPE allows to 1) considerably reduce

80 the consumption of solvents 2) obtain higher recovery factor 3) achieve highly purified extracts and

81 4) can be extremely selective since it is possible to choose between a wide range of adsorbents and

82 solvents. SPE-screening methods are commonly performed at acidic conditions (pH<6) providing

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

83 the protonation of the amine group of the analytes. Moreover, the absorbent of cartridges can be

84 either reverse-phase26, cation exchange27,28, polymeric29 or mixed-mode30,31. In specific cases,

85 further steps were implemented prior SPE namely solvent protein precipitation27 or enzymatic

86 hydrolysis on urine samples1,28,32.

87 According to Eckart et al.26 serum, blood and tissue samples were purified for the detection of

88 different opioids using a Bakerbond SPE C18. Plasma was diluted with phosphate buffer (pH 6),

89 added in internal standard, centrifuged and applied to SPE. Otherwise, postmortem tissues were

90 homogenized in 0.9% saline solution and an aliquot applied to SPE. After washing, the alkaline

91 analytes were eluted with dichloromethane/isopropanol/ammonium hydroxide (40:10:2). Then

92 evaporated and reconstituted with acetonitrile/methanol/water (3:3:2).

93 LLE is the traditional method in which analytes, contained in a sample, are separated based on their

94 solubility in two different immiscible liquid solvents. Generally, the efficiency of an LLE process

95 can be strongly improved by modifying the distribution coefficient: acid and basic compounds

96 would prefer non-polar solvents at low (pH<6) and high pH (pH>8) respectively.

97 LLE-screening methods are generally performed at basic conditions (pH>8-9) providing the

98 deprotonation of the amine of the analytes. The extraction solvents, used on their own or in a

99 mixture, are diethyl ether, ethyl acetate, hexane, toluene, isoamyl alcohol, acetonitrile, acetone, ter-

100 butyl methyl ether and butyl acetate. In some cases, single-phase extraction was made using a

101 water-miscible mixture of solvents, in particular, methanol and acetonitrile33–36. Further steps can be

102 implemented coupled with LLE such as solvent protein precipitation37, enzymatic hydrolysis on

103 urine samples38,39 or back-extraction40,41.

104 Caspar et al.17 applied a LLE on blood diluted with saturated aqueous sodium sulfate solution and

105 then extracted with diethyl ether-ethyl acetate mixture (1:1). The mixture was shaken, centrifuged

106 and the upper organic extract was transferred. Sodium hydroxide and diethyl ether-ethyl acetate

107 mixture were added to the remaining liquid and again mixed and centrifuged. The upper solvent

108 phase was transferred and evaporated. 4 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

109 The analytes resolved in methanol + 0.1% formic acid (FA) in water.

110 Protein precipitation (PP) is used to eliminate protein-contaminants in samples. It can be used on its

111 own or coupled with other extraction techniques. Methods to precipitate proteins are salting out,

112 isoelectric precipitation, precipitation with miscible solvent, polyvalent metallic ions and others. In

113 order to easily recover fentanyl-like compounds, precipitation with acetonitrile, methanol and

114 ethanol were carried out42,43. Simplicity and rapidity are the main advantaged of this kind of method

115 making it appealing.

116 Rarely, other microextraction procedures were employed such as hollow fibre assisted liquid-phase

117 micro extraction44,45 (HF-LPME), surfactant assisted pulsed two-phase electro membrane

118 extraction46 (SA-PEME), dispersive liquid-liquid microextraction45,47 (dLLME) and

119 microextraction in packed syringe48 (MEPS). MEPS48 employed a low volume of samples reaching

120 low limits of quantification. dLLME45,47 is a straightforward and low-cost technique. The main

121 difficulty involves the removal of the extraction solvent cause the matrix is pelleted and placed at

122 the bottom of the tube. Due to its characteristics, it is not recommended for the extraction of

123 biological samples but can be employed for simple ones such as tap and river water. By contrast,

124 HF-LPME44,45 has different disadvantages such as the formation of air bubbles, time-consuming

125 extraction, low precision, the high cost of extraction fibres which are also fragile and have a limited

126 lifetime. All of those new microextraction methods represent a viable development of the traditional

127 LLE and SPE, though they remain the second choice.

128 The use of stable-isotope labelled as internal standard improve the quali-quantitative performances

129 since they share with the analyte most of the biological and physiochemical properties. Examples of

130 commercially available isotopically labelled internal standard are Fentanyl-D5, Acetyl fentanyl-

13 13 131 C6, Acetyl norfentanyl- C6, Norfentanyl-D5 and Sufentanil-D5.

132

133 3.2 Analytical techniques

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

134 As for extraction paragraph, in the main text the most significant methodologies of analysis will be

135 discussed while a concise overview of variations and alternative methods can be found in Tab.2.

136 Highly sensitive analytical methods are required for the detection of fentanyl-like compounds since

137 they are very potent, short-acting opioids and occur in low concentration. LC-MS/MS is indeed the

138 more popular analytical technique in the field of bioanalysis49–51 and also for fentanyl-like

139 compounds. Several papers reported a similar LC-MS/MS method: many of them share 1) the C18-

140 chromatographic column, 2) a gradient elution program, 3) ESI source operating in positive

141 ionization mode, 4) a targeted multiple reaction monitoring (MRM) scan mode and 5) the same

142 combination of mobile phases, commonly acetonitrile or methanol coupled with water or buffer.

143 The use of LC-MS compatible ammonium counter-ions (formate, acetate or hydroxide) in the

144 mobile phase has been implemented in different works, but its use seems not to be mandatory since

145 a negatively charged moieties are not present in fentanyls.

146 Fentanyls form stable protonated species in positive ion modes, and when undertaking collision-

147 induced dissociation, a common cleavages was described42,52. Fentanyl analogues, with a 4-

148 anilidopiperidine structure, in collision cell show a specific cleavage C-N between the piperidine

149 ring and the amide group commonly resulting in the peak of the carbocation m/z 188.10 which is

150 subsequently fragmented to m/z 105.06. The same fragmentation42 was observed for peaks with m/z

151 84.08 for norfentanyl and acetyl norfentanyl, m/z 268.17 for alfentanil, m/z 228.1233 for

152 remifentanil, m/z 238.1264 for sufentanil, m/z 246.17 for carfentanil and m/z 156.10 for N‐

153 methylcarfentanil. The other proposed main fragmentation42 corresponds to the degradation of the

154 piperidine ring due to the cleavage on both the C(2)‐N and C(6)‐N bonds of the ring.

155 Strayer et al.53 were able to detect 24 illicitly manufactured fentanyl analogues and metabolites in

156 whole blood at a low concentration ranged between 0.1-0.5 ng/ml using a validated assay on a 6420

157 triple quadrupole LC-MS/MS system (Agilent Technologies). Separation was achieved with a

158 Raptor biphenyl analytical column (150.0 mm x 3.0 mm, 2.7 μm) with a linear gradient between A:

159 10.0 mM ammonium formate and 0.1% FA in water and B: 0.1% formic acid in acetonitrile (ACN). 6 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

160 The elution program was the following: 10% B (0-2 min), 10-90%B (2-8 min), 90%B (8-8.5 min),

161 90-10%B (8.5-8.6 min) and held at 10% until 13.5 min. Electrospray ionization in a positive ion

162 scan mode and dynamic MRM scan function were applied. Unfortunately, according to the author,

163 separation and identification between isomeric species could not be achieved under these conditions

164 (e.g. butyryl fentanyl/isobutyryl fentanyl and para-fluorobutyryl fentanyl/4-fluoroisobutyryl

165 fentanyl). Another comprehensive analytical method was proposed by Fogarty et al.54 in which

166 fentanyl and 18 novel fentanyl analogues and metabolites were evaluated in peripheral blood by

167 LC-MS/MS. The instrumentation consisted of a Xevo TQ-S Micro coupled with an Acquity UPLC

168 (both from Waters). Chromatographic separation was achieved for all isobaric compounds using

169 Agilent Poroshell EC C-18 column (3.0 mm × 150 mm, 2.7µm) with a linear gradient between A: 5

170 mM ammonium formate (pH = 3) and B: 0.1% FA in methanol. The gradient was the following:

171 40-45%B (0-7 min), 45-90% (7-7.1 min), 90%B (7.1-8), 90-45%B (8-8.1) and held to 40% B until

172 9 min. The instrumentation was operated using positive ion electrospray in MRM mode. Such triple

173 quadrupole-based methods are highly sensitive and provide a robust quantification, although have

174 some drawbacks. The addition of new compounds usually needs a thorough mass spectrometry

175 (MS) optimization and the total number of monitored compounds is limited. Moreover, they also do

176 not allow the retrospective evaluation of MS data to identify formerly unknown or unexpected

177 compounds. The alternative is represented by high-resolution mass spectrometry (HR-MS) which

178 provides the contemporaneous nontargeted acquisition of precursor-ions and product-ions at both

179 high resolution and high mass accuracy. LC-HR-MS has emerged as a fundamental method for the

180 detection of novel synthetic opioids, Nevertheless, this technology is not sustainable in most

181 forensic laboratories. Hikin et al.1 developed a method for the detection and semi-quantitation of

182 synthetic fentanyl analogues using a data dependent approach in HR-MS. The instrumentation

183 comprised of a Thermo XRS Ultra High-Performance Liquid Chromatography system interfaced to

184 a Thermo Q Exactive Focus operating in heated positive ion electrospray mode. Chromatographic

185 separation was achieved on an Atlantis T3 column (Waters) using a gradient consisting of A: 0.1%

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

186 acetic acid and B: ACN containing 0.1% acetic acid. Full scan mode operated at a mass resolution

187 of 70,000 across a mass range of 50–750 amu whereas data dependent scanning was enabled

188 utilising an inclusion list of over 800 compounds. By contrast, Pedersen et al.55 presented a

189 UHPLC-TOF-MS method with data independent acquisition from m/z 50–1200 capable to

190 distinguish either common drugs of abuse as well as new designer drugs and hardly occurring ones.

191 GC-MS was the gold standard for the previous decade. It was appreciated for the untargeted data

192 acquisition coupled with library searching for the compounds detected in biological specimens.

193 Diagnostic ions of fentanyl analogues in GC/EI-MS are 1) M+ as base peaks for N-benzylated and

194 N-methylated analogues 2) M-91 coming from the elimination of the benzyl fragment and 3) ions

195 originated from the cleavages of the piperidine ring and the elimination of the propionyl group such

196 as m/z 146, 160, 164, 180 and 17756. Strano-Rossi et al.57 developed a rapid and sensitive method

197 for the simultaneous determination of alfentanil, sufentanil and fentanyl in urine. GC/EI-MS

198 analyses were performed in an Agilent 6890 Gas Chromatograph coupled with an Agilent 5973

199 mass selective quadrupole detector. The GC was equipped with a J&W 5% phenylmethylsyloxane

200 capillary column, 30m x 0.25 mm. i.d., 0,50 µm film thickness. The analysis was performed using

201 both full scan (m/z 50-500) and selected ion monitoring mode. A derivatization step with

202 pentafluoropropionic anhydride (PFPA) was proposed prior analysis in order to magnify the

203 intensity of nor-metabolites. Even though in many cases, fentanyls can be analysed in GC without a

204 derivatization step since they show inherently appreciable mass spectra. In addition, the use of

205 derivatizing agents is destructive for the stationary phase packaged in the chromatographic column,

206 therefore seem suitable to limit their use only when firmly required. However, due to lack of

207 sensitivity and extensive sample preparation GC-MS was displaced by targeted LC-MS/MS

208 methods15 . Occasionally LC-UV and GC-FID are still used.

209 Immunoassays are indeed becoming more successful. They are in continuous development trying to

210 obtain more and more analytical sensibility and also large selectivity for the highest number of

211 compounds. The strength of this method relies on the ease of use, so that not highly qualified staff 8 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

212 is required, and on its fast response. In general, immunoassays can be used by law enforcement to

213 check if people are under the influence of drugs, for example, while they are driving vehicles. In

214 any case after the immunoassay screening, more in-depth investigations are obviously

215 required. None of these drugs is revealed in standard urine opiate immunoassays. However, specific

216 commercial immunoassays for fentanyl58 and some designer fentanyl in urine are available: Thermo

217 DRI Fentanyl Enzyme Immunoassay, the ARK Fentanyl Assay homogeneous enzyme

218 immunoassay and the Immunalysis Fentanyl Urine SEFRIA Drug Screening Kit. These

219 demonstrated a good detectability of designer fentanyls in urine samples from authentic acute

220 intoxications compared to LC-HR-MS, as reference method59.

221 NMR and IR spectroscopy are reported only for the detection in powder from seized samples60,61. In

222 NMR most of designer opioids share signature signals attributed to piperidine protons and aromatic

223 protons in the regions 2.8 - 3.7 ppm, 4.5 - 5.0 ppm, and 7.0 - 8.0 ppm62,63. Raman can be

224 successfully applied in forensic science, to reveal the chemical identity of fentanyl-like compounds,

225 in a sensitive and straightforward approach64–67. In Tab.3 analytical characteristics of fentanyl-like

226 compounds are discussed and compared.

227

228 4. Conclusions

229 The work dealt with examining the fentanyl-related publications of the last years, between 2008 and

230 2018, reaching the maximum in the last two ones (Fig.3). From this research, it emerged that the

231 most common methods to extract and detect fentanyl-like compounds are SPE or LLE and LC-

232 MS/MS (Fig.4). In the past years, gas chromatography was the most common method, but it was

233 irredeemably replaced by the LC for its greater flexibility, accuracy and efficiency. In addition to

234 the classic chromatographic techniques, immunoassays methods have a good potential for

235 development since they can be used by non-qualified staff as first-step screening tests in the

236 toxicological survey.

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

237 The most common analysed matrices are blood or plasma and urine. Generally, the internal

238 standards used are the commercially available deuterated analogues. In our research, we realised

239 that many works published in the analytical field are American (39/100). Concerning the European

240 situation (42/100), only the countries situated in the north show a great interest. The Scandinavian

241 region, Belgium and Germany are the most active countries in this research area (Fig.5). To

242 conclude, we hope that in the coming years in Italy and throughout the rest of Europe more

243 attention will be placed on the fentanyl-issue. This fact, unfortunately, affects us as well as the rest

244 of the world, since the fentanyl emergency is a problem to be tackled on a global level.

245 246 Acknowledgements MDC was supported by the PhD program in Molecular and Translational Medicine of the 247 University of Milan, Milan. 248

10 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

249 References 250 251 1. Hikin, L.; Smith, P. R.; Ringland, E.; Hudson, S.; Morley, S. R. Forensic Sci Int 2018, 282, 179–183. 252 2. Kinshella, M.-L. W.; Gauthier, T.; Lysyshyn, M. Harm Reduct J 2018, 15, 64. 253 3. Ziesenitz, V. C.; Vaughns, J. D.; Koch, G.; Mikus, G.; van den Anker, J. N. Clin Pharmacokinet 254 2018, 57, 125–149. 255 4. Comer, S. D.; Cahill, C. M. Neurosci Biobehav Rev 2018. 256 5. Mars, S. G.; Rosenblum, D.; Ciccarone, D. Addiction 2018. 257 6. Jannetto, P. J.; Helander, A.; Garg, U.; Janis, G. C.; Goldberger, B.; Ketha, H. Clin Chem 2018. 258 7. Concheiro, M.; Chesser, R.; Pardi, J.; Cooper, G. Front Pharmacol 2018, 9, 1210. 259 8. Beardsley, P. M.; Zhang, Y. Handb Exp Pharmacol 2018. 260 9. Karila, L.; Marillier, M.; Chaumette, B.; Billieux, J.; Franchitto, N.; Benyamina, A. Neurosci 261 Biobehav Rev 2018. 262 10. Zawilska, J. B. Front Psychiatry 2017, 8. 263 11. Ramos-Matos, C.; Lopez, W. China White: Clinical Insights of an Evolving Designer Underground 264 Drug; 2015; Vol. 3. 265 12. Miller, J. M.; Stogner, J. M.; Miller, B. L.; Blough, S. Drug Alcohol Rev 2018, 37, 121–127. 266 13. Lozier, M. J.; Boyd, M.; Stanley, C.; Ogilvie, L.; King, E.; Martin, C.; Lewis, L. J Med Toxicol 2015, 267 11, 208–217. 268 14. Lucyk, S. N.; Nelson, L. S. Ann Emerg Med 2017, 69, 91–93. 269 15. Armenian, P.; Vo, K. T.; Barr-Walker, J.; Lynch, K. L. Neuropharmacology 2018, 134, 121–132. 270 16. Prekupec, M. P.; Mansky, P. A.; Baumann, M. H. J Addict Med 2017, 11, 256–265. 271 17. Caspar, A. T.; Kollas, A. B.; Maurer, H. H.; Meyer, M. R. Talanta 2018, 176, 635–645. 272 18. Roda, G.; Farè, F.; Dell’Acqua, L.; Arnoldi, S.; Gambaro, V.; Argo, A.; Visconti, G.; Casagni, E.; 273 Procaccianti, P.; Cippitelli, M. Pharm Anal ACTA 2015, 6, 1–5. 274 19. Gambaro, V.; Argo, A.; Cippitelli, M.; Dell’Acqua, L.; Fare, F.; Froldi, R.; Guerrini, K.; Roda, G.; 275 Rusconi, C.; Procaccianti, P. J Anal Toxicol 2014, 38, 289–294. 276 20. Guerrini, K.; Argo, A.; Borroni, C.; Catalano, D.; Dell’Acqua, L.; Farè, F.; Procaccianti, P.; Roda, G.; 277 Gambaro, V. J Pharm Biomed Anal 2013, 73, 125–130. 278 21. Procaccianti, P.; Farè, F.; Argo, A.; Casagni, E.; Arnoldi, S.; Facheris, S.; Visconti, G. L.; Roda, G.; 279 Gambaro, V. J Anal Toxicol 2017, 41, 771–776. 280 22. Farè, F.; Dei Cas, M.; Arnoldi, S.; Casagni, E.; Visconti, G. L.; Parnisari, G.; Bolchi, C.; Pallavicini, 281 M.; Gambaro, V.; Roda, G. Eur J Lipid Sci Technol 2018, 120. 282 23. Roda, G.; Arnoldi, S.; Dei Cas, M.; Ottaviano, V.; Casagni, E.; Tregambe, F.; Visconti, G. L.; Farè, 283 F.; Froldi, R.; Gambaro, V. J Anal Toxicol 2018, 42, 51–57. 284 24. Donnarumma, F.; Wintersteiger, R.; Schober, M.; Greilberger, J.; Matzi, V.; Maier, A.; Schwarz, M.; 285 Ortner, A. Anal Sci 2013, 396, 2629–2637. 286 25. FU, J.; CHU, J.; SUN, X.; WANG, J.; YAN, C. Anal Sci 2012, 28, 1081–1087. 287 26. Eckart, K.; Röhrich, J.; Breitmeier, D.; Ferner, M.; Laufenberg-Feldmann, R.; Urban, R. J 288 Chromatogr B Anal Technol Biomed Life Sci 2015, 1001, 1–8. 289 27. Moody, M. T.; Diaz, S.; Shah, P.; Papsun, D.; Logan, B. K. Drug Test Anal 2018, 10, 1358–1367. 290 28. Patton, A. L.; Seely, K. A.; Pulla, S.; Rusch, N. J.; Moran, C. L.; Fantegrossi, W. E.; Knight, L. D.; 291 Marraffa, J. M.; Kennedy, P. D.; James, L. P.; Endres, G. W.; Moran, J. H. Anal Chem 2014, 86, 292 1760–1766. 293 29. Boleda, M. R.; Galceran, M. T.; Ventura, F. J Chromatogr A 2007, 1175, 38–48. 294 30. Seither, J.; Reidy, L. J Anal Toxicol 2017, 41, 493–497. 295 31. Sofalvi, S.; Schueler, H. E.; Lavins, E. S.; Kaspar, C. K.; Brooker, I. T.; Mazzola, C. D.; Dolinak, D.; 296 Gilson, T. P.; Perch, S. J Anal Toxicol 2017, 41, 473–483. 297 32. Fleming, S. W.; Cooley, J. C.; Johnson, L.; Clinton Frazee, C.; Domanski, K.; Kleinschmidt, K.; 298 Garg, U. J Anal Toxicol 2017, 41, 173–180. 299 33. Odoardi, S.; Anzillotti, L.; Strano-Rossi, S. Forensic Sci Int 2014, 243, 61–67. 300 34. Bista, S. R.; Lobb, M.; Haywood, A.; Hardy, J.; Tapuni, A.; Norris, R. J Chromatogr B Anal Technol 301 Biomed Life Sci 2014, 960, 27–33. 302 35. Boumba, V. A.; Di Rago, M.; Peka, M.; Drummer, O. H.; Gerostamoulos, D. Forensic Sci Int 2017, 303 279, 192–202. 304 36. Lafreniere, N. M.; Watterson, J. H. Forensic Sci Int 2010, 194, 60–66. 11 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

305 37. de Bruijn, P.; Kuip, E. J. M.; Lam, M. H.; Mathijssen, R. H. J.; Koolen, S. L. W. J Pharm Biomed 306 Anal 2018, 149, 475–481. 307 38. Remane, D.; Montenarh, D.; Meyer, M. R.; Maurer, H. H. Ther Drug Monit 2014, 36, 257–260. 308 39. Gergov, M.; Nokua, P.; Vuori, E.; Ojanperä, I. Forensic Sci Int 2009, 186, 36–43. 309 40. Fort, C.; Curtis, B.; Nichols, C.; Niblo, C. J Anal Toxicol 2016, 40, 754–757. 310 41. Swanson, D. M.; Hair, L. S.; Rivers, S. R. S.; Smyth, B. C.; Brogan, S. C.; Ventoso, A. D.; Vaccaro, 311 S. L.; Pearson, J. M. J Anal Toxicol 2017, 41, 498–502. 312 42. Noble, C.; Weihe Dalsgaard, P.; Stybe Johansen, S.; Linnet, K. Drug Test Anal 2018, 10, 651–662. 313 43. Nosseir, N. S.; Michels, G.; Binder, P.; Wiesen, M. H. J.; Müller, C. J Chromatogr B Anal Technol 314 Biomed Life Sci 2014, 973, 133–141. 315 44. Fakhari, A. R.; Tabani, H.; Nojavan, S. Drug Test Anal 2013, 5, 589–595. 316 45. Saraji, M.; Khalili Boroujeni, M.; Hajialiakbari Bidgoli, A. A. Anal Bioanal Chem 2011, 400, 2149– 317 2158. 318 46. Zahedi, P.; Davarani, S. S. H.; Moazami, H. R.; Nojavan, S. J Pharm Biomed Anal 2016, 117, 485– 319 491. 320 47. Gardner, M. A.; Sampsel, S.; Jenkins, W. W.; Owens, J. E. J Anal Toxicol 2015, 39, 118–125. 321 48. Said, R.; Pohanka, A.; Andersson, M.; Beck, O.; Abdel-Rehim, M. J Chromatogr B Anal Technol 322 Biomed Life Sci 2011, 879, 815–818. 323 49. Song, A. Anal Sci 2016, 32, 645–652. 324 50. Ando, A.; Satomi, Y. Anal Sci 2018, 34, 177–182. 325 51. Nakazawa, H.; Iwasaki, Y.; Ito, R. Anal Sci 2014, 30, 25–34. 326 52. Helander, A.; Bäckberg, M.; Beck, O. Clin Toxicol 2016, 54, 324–332. 327 53. Strayer, K. E.; Antonides, H. M.; Juhascik, M. P.; Daniulaityte, R.; Sizemore, I. E. ACS Omega 2018, 328 3, 514–523. 329 54. Fogarty, M. F.; Papsun, D. M.; Logan, B. K. J Anal Toxicol 2018, 42, 592–604. 330 55. Pedersen, A. J.; Dalsgaard, P. W.; Rode, A. J.; Rasmussen, B. S.; Müller, I. B.; Johansen, S. S.; 331 Linnet, K. J Sep Sci 2013, 36, 2081–2089. 332 56. Ohta, H.; Suzuki, S.; Ogasawara, K. J Anal Toxicol 1999, 23, 280–285. 333 57. Strano-Rossi, S.; Álvarez, I.; Tabernero, M. J.; Cabarcos, P.; Fernández, P.; Bermejo, A. M. J Appl 334 Toxicol 2011, 31, 649–654. 335 58. Wang, G.; Huynh, K.; Barhate, R.; Rodrigues, W.; Moore, C.; Coulter, C.; Vincent, M.; Soares, J. 336 Forensic Sci Int 2011, 206, 127–131. 337 59. Helander, A.; Stojanovic, K.; Villén, T.; Beck, O. Drug Test Anal 2018, No. 10, 1297–1304. 338 60. Breindahl, T.; Kimergård, A.; Andreasen, M. F.; Pedersen, D. S. Drug Test Anal 2017, 9, 415–422. 339 61. Kanamori, T.; Iwata, Y. T.; Segawa, H.; Yamamuro, T.; Kuwayama, K.; Tsujikawa, K.; Inoue, H. J 340 Forensic Sci 2017, 62, 1472–1478. 341 62. Chambers, M.; Huang, L. Abstracts of Papers, 253rd ACS National Meeting & Exposition, San 342 Francisco, CA, United States, April 2-6, 2017; American Chemical Society, 2017; p 468. 343 63. Casale, J.; Mallette, J. Abstracts of Papers, 253rd ACS National Meeting & Exposition, San 344 Francisco, CA, United States, April 2-6, 2017; American Chemical Society, 2017; p 293. 345 64. Fedick, P. W.; Bills, B. J.; Manicke, N. E.; Cooks, R. G. Anal Chem 2017, 89, 10973–10979. 346 65. Leonard, J.; Haddad, A.; Green, O.; Birke, R. L.; Kubic, T.; Kocak, A.; Lombardi, J. R. J Raman 347 Spectrosc 2017, 48, 1323–1329. 348 66. Inscore, F.; Shende, C.; Sengupta, A.; Huang, H.; Farquharson, S. Appl Spectrosc 2011, 65, 1004– 349 1008. 350 67. Haddad, A.; Comanescu, M. A.; Green, O.; Kubic, T. A.; Lombardi, J. R. Anal Chem 2018, 90, 351 12678–12685. 352 68. Verplaetse, R.; Henion, J. Drug Test Anal 2016, 8, 30–38. 353 69. Elliott, S. P.; Hernandez Lopez, E. J Anal Toxicol 2018, No. 42, 41–45. 354 70. McIntyre, I. M.; Trochta, A.; Gary, R. D.; Wright, J.; Mena, O. J Anal Toxicol 2016, 40, 162–166. 355 71. Guerrieri, D.; Rapp, E.; Roman, M.; Druid, H.; Kronstrand, R. J Anal Toxicol 2017, 41, 242–249. 356 72. Coopman, V.; Cordonnier, J.; De Leeuw, M.; Cirimele, V. Forensic Sci Int 2016, 266, 469–473. 357 73. Cooreman, S.; Deprez, C.; Martens, F.; Van Bocxlaer, J.; Croes, K. J Sep Sci 2010, 33, 2654–2662. 358 74. Mahlke, N. S.; Ziesenitz, V.; Mikus, G.; Skopp, G. Int J Legal Med 2014, 128, 771–778. 359 75. Griswold, M. K.; Chai, P. R.; Krotulski, A. J.; Friscia, M.; Chapman, B. P.; Varma, N.; Boyer, E. W.; 360 Logan, B. K.; Babu, K. M. J Med Toxicol 2017, 13, 287–292. 12 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

361 76. Remane, D.; Wetzel, D.; Peters, F. T. Anal Bioanal Chem 2014, 406, 4411–4424. 362 77. Ghassabian, S.; Moosavi, S. M.; Valero, Y. G.; Shekar, K.; Fraser, J. F.; Smith, M. T. J Chromatogr 363 B Anal Technol Biomed Life Sci 2012, 903, 126–133. 364 78. Shaner, R. L.; Kaplan, P.; Hamelin, E. I.; Bragg, W. A.; Johnson, R. C. J Chromatogr B Anal Technol 365 Biomed Life Sci 2014, 962, 52–58. 366 79. Shanks, K. G.; Behonick, G. S. J Anal Toxicol 2017, 41, 466–472. 367 80. Yang, P.; Li, Y.; Li, W.; Zhang, H.; Gao, J.; Sun, J.; Yin, X.; Zheng, A. Drug Dev Ind Pharm 2018, 368 44, 953–960. 369 81. Koster, R. A.; Vereecke, H. E. M. M.; Greijdanus, B.; Touw, D. J.; Struys, M. M. R. F. R. F.; 370 Alffenaar, J. W. C. Anesth Analg 2015, 120, 1235–1241. 371 82. Clavijo, C. F.; Thomas, J. J.; Cromie, M.; Schniedewind, B.; Hoffman, K. L.; Christians, U.; 372 Galinkin, J. L. J Sep Sci 2011, 34, 3568–3577. 373 83. Helland, A.; Brede, W. R.; Michelsen, L. S.; Gundersen, P. O. M.; Aarset, H.; Skjølås, J. E.; Slørdal, 374 L. J Anal Toxicol 2017, 41, 708–709. 375 84. El Hamd, M. A.; Wada, M.; Ikeda, R.; Kawakami, S.; Kuroda, N.; Nakashima, K. Biomed 376 Chromatogr 2015, 29, 325–327. 377 85. Butler, D. C.; Shanks, K.; Behonick, G. S.; Smith, D.; Presnell, S. E.; Tormos, L. M. J Anal Toxicol 378 2018, 42, e6–e11. 379 86. Poklis, J.; Poklis, A.; Wolf, C.; Hathaway, C.; Arbefeville, E.; Chrostowski, L.; Devers, K.; Hair, L.; 380 Mainland, M.; Merves, M.; Pearson, J. J Anal Toxicol 2016, 40, 703–708. 381 87. Steuer, A. E.; Williner, E.; Staeheli, S. N.; Kraemer, T. Drug Test Anal 2017, 9, 1085–1092. 382 88. Takashina, Y.; Naito, T.; Mino, Y.; Kagawa, Y.; Kawakami, J. J Clin Pharm Ther 2009, 34, 523– 383 529. 384 89. Shoff, E. N.; Zaney, M. E.; Kahl, J. H.; Hime, G. W.; Boland, D. M. J Anal Toxicol 2017, 41, 484– 385 492. 386 90. Mohr, A. L. A.; Friscia, M.; Papsun, D.; Kacinko, S. L.; Buzby, D.; Logan, B. K. J Anal Toxicol 387 2016, 40, 709–717. 388 91. Ristimaa, J.; Gergov, M.; Pelander, A.; Halmesmäki, E.; Ojanperä, I. Anal Bioanal Chem 2010, 398, 389 925–935. 390 92. Poklis, J.; Poklis, A.; Wolf, C.; Mainland, M.; Hair, L.; Devers, K.; Chrostowski, L.; Arbefeville, E.; 391 Merves, M.; Pearson, J. Forensic Sci Int 2015, 257, 435–441. 392 93. Saari, T. I.; Fechner, J.; Ihmsen, H.; Schüttler, J.; Jeleazcov, C. J Pharm Biomed Anal 2012, 66, 306– 393 313. 394 94. Jeleazcov, C.; Saari, T. I.; Ihmsen, H.; Schüttler, J.; Fechner, J. Br J Anaesth 2012, 109, 698–706. 395 95. Liang, L.; Wan, S.; Xiao, J.; Zhang, J.; Gu, M. J Pharm Biomed Anal 2011, 54, 838–844. 396 96. Ramirez Fernandez, M. del M.; Van Durme, F.; Wille, S. M. R.; di Fazio, V.; Kummer, N.; Samyn, 397 N. J Anal Toxicol 2014, 38, 280–288. 398 97. Kim, T.; London, A.; Kharasch, E. D. J Pharm Biomed Anal 2011, 55, 487–493. 399 98. Takase, I.; Koizumi, T.; Fujimoto, I.; Yanai, A.; Fujimiya, T. Leg Med 2016, 21, 38–44. 400 99. Mochizuki, A.; Nakazawa, H.; Adachi, N.; Takekawa, K.; Shojo, H. Forensic Toxicol 2018, 36, 81– 401 87. 402 100. Martucci, H. F. H.; Ingle, E. A.; Hunter, M. D.; Rodda, L. N. Forensic Sci Int 2017, 283, e13–e17. 403 101. Malkawi, A. H.; Al-Ghananeem, A. M.; Crooks, P. a. AAPS J 2008, 10, 261–267. 404 102. Sinicina, I.; Sachs, H.; Keil, W. Drug Alcohol Depend 2017, 180, 286–291. 405 103. Quintana, P.; Ventura, M.; Grifell, M.; Palma, A.; Galindo, L.; Fornís, I.; Gil, C.; Carbón, X.; 406 Caudevilla, F.; Farré, M.; Torrens, M. Int J Drug Policy 2017, 40, 78–83. 407 104. Kudo, T.; Kimura, F.; Kudo, T.; Kudo, M.; Hirota, K. J Anesth 2013, 27, 615–617. 408 105. Strano-Rossi, S.; Bermejo, A. M.; De La Torre, X.; Botrè, F. Anal Bioanal Chem 2011, 399, 1623– 409 1630. 410 106. Tiscione, N. B.; Wegner, K. J Anal Toxicol 2017, 41, 313–317. 411 107. Snyder, M. L.; Jarolim, P.; Melanson, S. E. F. Clin Chim Acta 2011, 412, 946–951. 412 108. Cannaert, A.; Ambach, L.; Blanckaert, P.; Stove, C. P. Front Pharmacol 2018, 9, 1–5. 413 109. Fels, H.; Krueger, J.; Sachs, H.; Musshoff, F.; Graw, M.; Roider, G.; Stoever, A. Forensic Sci Int 414 2017, 277, 30–35. 415 110. Watanabe, S.; Vikingsson, S.; Roman, M.; Green, H.; Kronstrand, R.; Wohlfarth, A. AAPS J 2017, 416 19, 1102–1122. 13 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

417 111. Burke, M. J.; Soma, L. R.; Boston, R. C.; Rudy, J. A.; Schaer, T. P. J Vet Emerg Crit Care 2017, 27, 418 539–547. 419 112. Meyer, M. R.; Dinger, J.; Schwaninger, A. E.; Wissenbach, D. K.; Zapp, J.; Fritschi, G.; Maurer, H. 420 H. Anal Bioanal Chem 2012, 402, 1249–1255. 421 113. Krotulski, A. J.; Papsun, D. M.; Friscia, M.; Swartz, J. L.; Holsey, B. D.; Logan, B. K. J Anal Toxicol 422 2018, 42, e27–e32. 423 114. Berg, T.; Jørgenrud, B.; Strand, D. H. J Anal Toxicol 2013, 37, 159–165. 424 115. Cunningham, S. M.; Haikal, N. A.; Kraner, J. C. J Forensic Sci 2016, 61, 276–280. 425 116. Yonemitsu, K.; Sasao, A.; Mishima, S.; Ohtsu, Y.; Nishitani, Y. Forensic Sci Int 2016, 267, e6–e9. 426 117. Lu, C.; Jia, J.; Gui, Y.; Liu, G.; Shi, X.; Li, S.; Yu, C. Biomed Chromatogr 2010, 24, 711–716. 427 118. Blanco, M. E.; Encinas, E.; González, O.; Rico, E.; Vozmediano, V.; Suárez, E.; Alonso, R. M. Drug 428 Test Anal 2015, 7, 804–811. 429 119. Hisada, T.; Katoh, M.; Hitoshi, K.; Kondo, Y.; Fujioka, M.; Toyama, Y.; Ieda, H.; Gocho, S.; Nadai, 430 M. Biol Pharm Bull 2013, 36, 412–416. 431 120. Verplaetse, R.; Tytgat, J. J Chromatogr B Anal Technol Biomed Life Sci 2010, 878, 1987–1996. 432 121. Allibe, N.; Richeval, C.; Phanithavong, M.; Faure, A.; Allorge, D.; Paysant, F.; Stanke-Labesque, F.; 433 Eysseric-Guerin, H.; Gaulier, J. M. Drug Test Anal 2017, 10, 995–1000. 434 122. Scientific Working Group for the Analysis of Seized Drugs. No Title http://www.swgdrug.org/. 435 123. Palaty, J.; Konforte, D.; Karakosta, T.; Wong, E.; Stefan, C. Clin Biochem 2018, 53, 164–167. 436 124. Krotulski, A. J.; Mohr, A. L. A.; Papsun, D. M.; Logan, B. K. Drug Test Anal 2018, 10, 127–136. 437

14 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

438 Table 1 Extraction procedures used in different matrices for the isolation of fentanyl-like compounds; “screening” 439 means a method comprehensive of multiple fentanyl-like drugs

Fentanyl pH Extraction Matrix References analogues samples Acid hydrolysis + single- Screening 4 Hair 35 phase extraction Acid hydrolysis+ single- Fentanyl 4-6 Bone and bone marrow 36 phase extraction Automated on-line SPE1 Screening - Dried blood spot 68 Back extraction Carfentanil 11 Blood 69 dLLME2 Screening 9 Plasma, urine 45 Enzymatic hydrolisis+ Screening 9 Urine, blood 38,39 LLE3 Enzymatic hydrolysis + Screening 5 Urine 1,28 SPE Enzymatic U-47700 3-5 Urine 32 hydrolysis+SPE HF-LPME4 Screening 10 Biological samples 44,45 Blood, liver, urine, gastric content LLE Butyrfentanyl 12 70 and vitreous LLE Furanyl fentanyl 11 Femoral blood 71 LLE Ocfentanil 10 Tissues and seized drug 72 LLE Screening 10-12 Blood, plasma, urine, oral fluid 17,57,73–75 LLE Screening - Plasma 76 LLE/ SPE/ dLLME Fentanyl 11 Urine 47 LLE+ acid back- Acetyl fentanyl 11 Blood, liver, brain and urine 40 extraction LLE+ back extraction Screening 8.2 Blood, urine, vitreous 41 MEPS5 Remifentanil 5 Plasma 48 On-line SPE Screening 9.3 Plasma, urine 77,78 PP6 Carfentanil - Blood 79,80 PP Remifentanil 5 Blood and plasma 81 PP Screening - Blood, serum, plasma, DBS 42,43,82 PP+hSPE7 o-Fluorofentanyl - Serum, blood and urine 83 PP+LLE Remifentanil 11 Plasma 84 PP+LLE Screening 10 Plasma 37 PP+SPE Screening 6-7 Blood 27 SA-PEME8 Screening 4 Plasma, urine, breast milk 46 Single-phase extraction Fentanyl - Saliva and plasma 34 Single-phase extraction Screening 4 DBS 33 SPE Acrylfentanyl 6-7 Blood 85 SPE Butyrfentanyl 8-9 Post-mortem fluids and tissue 86,87 SPE Fentanyl 8.8 Plasma 88 SPE Screening 5-6 Blood, serum, tissue, urine 26,30,31,53–55,89,90 SPE Screening 9 Urine 78

1 SPE solid phase extraction 2 dLLME dispersive liquid-liquid microextraction 3 LLE liquid-liquid extraction 4 HF-LPME hollow fibre assisted liquid-phase micro extraction 5 MEPS microextraction in packed syringe 6 PP protein precipitaion 7 hSPE hybrid solid phase extraction 8 SA-PEME surfactant assisted pulsed two-phase electro membrane extraction 15 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl pH Extraction Matrix References analogues samples Blood, vitreous, bile, gastric SPE Screening 6 content, urine, brain, meconium 91,92 and liver tissues SPE Sufentanil 3 Plasma 93–95 SPE cation exchange Screening 5 Urine 96 SPE mixed mode Alfentanil 5 plasma 97

16 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

440 Table 2 Analytical techniques used to quali-quantitative determination of fentanyl-like compounds; screening means a method comprehensive of multiple fentanyl-like drugs

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues Varian CP-3800 GC (Palo Alto, CA, 0.01- sufentanil and CP-Sil8 fused-silica GC-FID11 - plasma and urine - 44 USA) equipped with an FID detector 0.07 alfentanil capillary column an Agilent 6890 GC (Agilent GC-MS12 Technologies, Inc., Wilmington, DE) - - Acetyl fentanyl blood - - 13 equipped with a 5973 MSD GC-MS an Agilent Technologies 7890A series gas 125 62.5 Acetyl fentanyl Post-mortem tissues RTX-1-ms column - 40 chromatograph coupled with a 5975C mass spectrometer GC-MS GC–MS (7890A/ 5975C; Agilent Technologies, Santa Clara, CA, 100 50 Acetyl fentanyl Blood, urine and vitreous Zebron ZB-5MS - 70 USA) GC-MS GC/MS analysis was performed on a GC/MS-QP2010 (Shimadzu, - - Acetyl fentanyl Blood and urine DB-5 ms column - 98 Kyoto, Japan) equipped GC-MS/MS13 Bruker 456-GC gas chromatograph connected to a SCION TQ mass 20 1 Acetyl fentanyl Blood and urine Rtx-5Sil MS - 99 spectrometer (Bruker Daltonics, (ng/g) (ng/g) Billerica, MA, USA) 6890 gas chromatograph with a 5973 GC-MS mass spectrometer from Agilent - - Acrylfentanyl seized capsule XTI-5 capillary column - 60 (Santa Clara, CA, USA) Agilent Technologies (Santa Clara, GC-MS CA, USA) 7890A/5975C Gas - 5 Acrylfentanyl peripheral blood DB-1MS column - 85 Chromatograph-Mass Spectrometer Agilent Technologies (Santa Clara, Alfentanil, J&W 5% 0.04- GC-MS CA, USA) 6890/5973 Gas 0.5 sufentanil and urine phenylmethylsyloxane - 57 0.08 Chromatograph-Mass Spectrometer fentanyl capillary column

9 LOQ limit of quantification express in ng/mL; in the case of screening method LOQ values are indicated as range among the substances studied 10 LOD limit of detection express in ng/mL; in the case of screening method LOD values are indicated as range among the substances studied 11 GC-FID gas-chromatography coupled to flame ionization detector 12 GC-MS gas-chromatography coupled to single quadrupole mass spectrometer 13 GC-MS/MS gas-chromatography coupled to triple quadrupole mass spectrometer 17 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues GC–MS (7890A/ 5975C; Agilent GC-MS Technologies, Santa Clara, CA, 100 50 Butyr fentanyl blood, vitreous and urine Zebron ZB-5MS - 70 USA) equipped GC–MS (Agilent Technologies, GC-MS - - Furanylfentanyl blood, vitreous and urine HP-5MS - 100 Santa Clara, CA, USA) Hewlett Packard 5890/6890N Series GC-MS 50 10 Fentanyl urine HP-5MS/ DB-1MS - 47 II GC with a 5971A/5973A MS An Agilent 6890 GC with an Agilent GC-MS 5973 MSD (Agilent Technologies, 4 1 Fentanyl Rabbit plasma HP-5MS 101 Wilmington, DE) GC-MS - - - Fentanyl Post-mortem tissues - - 102 CP-3800 gas chromatograph GC-MS connected to a 1200 L mass - - Methyl-derivatives powdered sample DB-5MS - 61 spectrometer (Bruker, Billerica, MA) an Agilent 7890B gas Chromatograph, coupled to a 5977A GC-MS quadrupole mass spectrometer - - Ocfentanil heroin samples HP-5MS - 103 detector (Agilent; Santa Clara, CA, USA) GC– MS (AUTOMASS GC–MS InerCap17MS capillary GC-MS - - Remifentanil blood - 104 system, JEOL, Tokyo, Japan) column GC-MS - - - Screening blood, vitreous and urine Rtx-5 capillary column - 41 an Agilent 7890 Gas Chromatograph coupled with an Agilent 5975A short GC column 5% GC-MS 5-200 2-100 Screening urine - 105 quadrupole mass selective detector phenyl methyl silicone (Agilent Technologies, Milano, Italy) Varian CP-3800 Alfentanil, GD-FID system (Palo Alto, CA, USA) 2-15 0.6-4.5 urine Chrompack CP-Sil 8CB - 46 sufentanil equipped with an FID detector ELISA kits purchased from Immunoassays - - Acrylfentanyl blood - - 85 Immunalysis (Pomona, CA) Blood and ELISA kits purchased from Immunoassays - 0.1 Fentanyl fresh/decomposed - - 36 Immunalysis (Pomona, CA) skeletal tissues

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues homogeneous enzyme immunoassay was performed on the Olympus Immunoassays 2 1 fentanyl urine - - 58 AU400e automated chemical analyzer Fentanyl ready-to-use (RTU) ELISA kits (Product #s 131519 and 131515) Immunoassays - 0.25-0.5 fentanyl blood and urine - - 106 were obtained from Neogen® (Lexington, KY) HEIA (Immunalysis Corporation, Pomona, CA) was performed on the Immunoassays 2 - fentanyl urine - - 107 Olympus AU480 analyzer (Beckman Coulter Inc., Brea, CA) Thermo DRI Fentanyl Enzyme Immunoassay, the ARK™ Fentanyl Assay homogeneous enzyme Immunoassays 0.5-1 - Screening urine - - 59 immunoassay, and the Immunalysis Fentanyl Urine SEFRIA Drug Screening Kit in-house developed opioid activity Immunoassays - - Screening Blood, urine and vitreous - - 108 reporter assay Hewlett- Packard 1090-II liquid Alfentanil, (A) sodium butane-1- chromatograph (now Agilent, Palo LC-UV14 1.1-5.5 0.4-1.9 fentanyl, plasma and urine C6 reversed-phase sulfonate in sulfuric 45 Alto, CA, USA) equipped with a sufentanil acid 30% UV-Vis diode array detector. (A) water+FA16 LC-HR-MS15 - - 1-2 Screening oral fluid and urine Zorbax Eclipse C18 75 (B) MeOH17+FA an Agilent Technologies 1200 series instrument AH-7921 and MT- Various body fluids and Zorbax Eclipse XDB-C8 (A) AmFo18+FA LC-HR-MS coupled to a TripleTOF - - 109 45 tissues column (B) MeOH+FA 5600 system (AB Sciex, Concord, Ontario, Canada)

14 LC-UV liquid-chromatography coupled to ultraviolet detector 15 LC-HR-MS liquid-chromatography coupled to a high-resolution mass spectrometer 16 FA formic acid 17 MeOH methanol 18 AmFO ammonium formate 19 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues an Agilent LC-HR-MS- (Waldbronn, Germany) 1100 HPLC Phenomenex Luna PFP (A) AmAc19 targeted instrument coupled to a Bruker - - Screening meconium 91 (2) (B) ACN20+FA method Daltonics (Bremen, Germany) micrOTOF a Waters Acquity UPLC (Waters, LC-HR-MS- Milford, MA) coupled to a Sciex U-47700 and (A) AmAc+FA - - urine Poroshell 120 EC-C18 32 DIA21 5600 TripleToF (Sciex, Framingham, metabolites (B) MeOH+FA MA) mass spectrometer set TF Q- Exactive Plus system equipped with a heated electrospray LC-HR-MS- ionization (HESI)-II source coupled (A) AmFo+FA 0.25 0.1 Screening plasma Accucore Phenyl-Hexyl 17 AIF22 to a ThermoFisher Scientific (TF, (B) MeOH+ACN+FA Dreieich, Germany) Dionex UltiMate 3000 HPLC Thermo Fischer Ultimate 3000 LC-HR-MS- Synergy Polar RP (A) AmFo+FA UHPLC system coupled to a Sciex - - Butyr fentanyl blood and urine 87 DDA23 column (B) ACN+FA 6600 QTOF system. Thermo XRS UHPLC system, interfaced to a Thermo Q Exactive LC-HR-MS- (A) AcA+water Focus mass spectrometer, operating - - Screening blood, urine and vitreous Atlantis T3 HPLC 1 DDA (B) AcA+ACN in heated positive ion electrospray mode Acquity UPLC system (Waters Corporation, Milford, USA) coupled 0.001- LC-HR-MS- Acquity UPLC BEH (A) water+FA to SYNAPT G2 (Waters - 0.005 Screening blood 55 DIA C18 (B) ACN MS Technologies, Manchester, UK) (mg/kg) TOF mass spectrometer

19 AmAc ammonium acetate 20 ACN acetonitrile 21 LC-HR-MS-DIA liquid-chromatography coupled to a high-resolution mass spectrometer operating in data independent acquisition mode 22 LC-HR-MS-AIF liquid-chromatography coupled to a high-resolution mass spectrometer operating in all-ion fragmentation mode 23 LC-HR-MS-AIF liquid-chromatography coupled to a high-resolution mass spectrometer operating in data dependent acquisition mode 20 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues ACQUITY UHPLC system from Waters Corporation (Milford, MA, 0.001- 0.0005- LC-HR-MS- ACQUITY UHPLC (A) AmFo USA) with Xevo G2‐ S QTOF 0.005 0.001 Screening blood 42 DIA HSS C18 column (B) ACN+FA (Waters MS Technologies, (mg/kg) (mg/kg) Manchester, UK) Agilent 1290 Infinity UHPLC LC-HR-MS- system with an Agilent (A) AmFo+FA - - Screening urine Acquity HSS T3 110 DIA 6550 iFunnel QTOF mass (B) ACN+FA spectrometer a Thermo Scientific Dionex Ultimate 3000 RSLC ultra high-performance liquid chromatograph (Idstein, blood and urine Acclaim RSLC 120 C18 (A) AmFo+FA LC-MS-LIT24 Germany) coupled to a Bruker - 0.1-0.5 Screening samplesand/or tissue 89 column (B) AmFo+FA+ACN Daltonics AmaZon Speed ion trap homogenates mass spectrometer (Bremen, Germany) a LTQ XLTM cardiac blood, gastric linear ion trap mass spectrometer (A)AmAc LC-MS-LIT - - Acetyl fentanyl contents and urine Hypersil Gold 98 (Thermo Fisher Scientific, Waltham, (B)MeOH detected MA, USA). (A) water+FA LC-MS-LIT - - - Fentanyl Goat blood HALO C18 111 (B) ACN +FA An Accela LC system (Thermo Fisher Scientific, TF, Dreieich, 3-methylfentanyl (A) water+FA LC-MS-LIT - 10 Rat urine TF Hypersil GOLD 112 Germany) coupled to the TF LXQ and isofentanyl in (B) ACN +FA LIT HPLC 1260 Infinity system coupled (A) AmFo+FA LC-MS/MS25 0.1-0.25 0.02-0.1 Screening blood Raptor biphenyl 53 to a 6420 triple quadrupole (B) ACN+FA

24 LC-MS-LIT liquid-chromatography coupled to linear ion trap mass spectrometer 25 LC-MS/MS liquid chromatography coupled to triple quadrupole mass spectrometer 21 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues An Agilent 1260 liquid chromatogram system couplet to 6460 triple quadrupole mass Poroshell EC-C18 (A) AmFo+FA LC-MS/MS spectrometer with a - 0.01-0.1 Screening blood and urine 30 (B) ACN+FA Jetstream electrospray source (Agilent Technologies, California, USA) an ABSciex QTrap 4000 tandem mass spectrometer (Darmstadt, Fentanyl and (A) AmFo+FA LC-MS/MS 1 - urine Zorbax Eclipse C18 76 Germany) coupled to a LC-20 norfentanyl (B) ACN+FA (Shimadzu, Jena, Germany). Shimadzu Nexera X2 30 AD UPLC (Shimadzu, Melbourne, Australia) 0.001 (A) AmFo+FA LC-MS/MS - Screening hair Kinetex C18 35 coupled to a Sciex 4500 Q-Trap (ng/mg) (B) ACN+FA (Sciex, Melbourne, Australia) Agilent 1290 Infinity system UPLC coupled to an Agilent 6460 (A) AmFo 33 LC-MS/MS triple quadrupole mass spectrometer 0.2-2 0.05-0.5 Screening dried blood sample Kinetex C18 (B) MeOH/ACN+FA (Agilent Technologies, Santa Clara,CA, USA) Agilent LC 1100 coupled to an (A) AmAc LC-MS/MS AB/MDS Sciex 3200 QTrap LC– 0.1-0.3 0.05-0.2 Screening blood and urine Gemini C18 39 (B) ACN+FA MS/MS Shimadzu Prominence HPLC system (Shimadzu USA Manufactoring Inc., Canby, OR, (A) AmAc LC-MS/MS - - Screening meconium Genesis C18 91 USA) couplet to a Sciex 3200 Q (B) ACN+FA TRAP LC-MS/MS System (Applied Biosystems, Concord, ON, Canada) a Waters (Milford, MA, USA) Acquity UPLC (A) AmFo LC-MS/MS - - Screening blood and urine Poroshell 120 EC-C18 113 I-Class coupled to a Xevo® TQ-S (B) MeOH+FA micro LC–MS-MS. a Waters Xevo TQD LC/MS mass spectrometer attached to a (A) AmFo+FA LC-MS/MS ACQUITY UPLC® System con- 1 - Screening blood, urine and vitreous Allure Biphenyl 92 (B) MeOH trolled by MassLynx software (Milford, MA) 22 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues a Nexera UHPLC system coupled to a LCMS-8050 mass spectrometer (A) AmFo+FA LC-MS/MS 0.1 - Screening dried blood spot Raptor Biphenyl 68 from Shimadzu (Marlborough, MA, (B) MeOH USA) An Agilent LC coupled to an API 4000 tandem quadrupole mass Plasma, blood and dried (A) water+FA LC-MS/MS 0.1-0.25 - Screening Eclipse XDB-C8 82 spectrometer as detector (Applied blood spot (B) MeOH Biosystems, Foster City, CA) UPLC–MS/MS system from Waters Chromatography B.V. (Etten-Leur, The Netherlands) consisted of a Fentanyl and (A) water+AmFo+FA LC-MS/MS 0.1-0.2 - plasma Acquity BEH C18 37 Waters Aquity UPLC Sample norfentanyl (B) MeOH+FA Manager coupled to a Waters TQ Detector. UHPLC was performed using an UPLC separation module (Waters, Fentanyl and (A) water+FA LC-MS/MS Milford, MA, USA) coupled to a 3 - urine BEH Phenyl 96 norfentanyl (B) MeOH+FA Quattro Premier tandem mass spectrometer (Waters) an Agilent 1100 series HPLC U-47700, U-50488 coupled to an Agilent 6430 triple (A) water+FA LC-MS/MS 1 - and Furanyl blood, urine and vitreous Zorbax Eclipse C18 90 quadrupole tandem mass (B) MeOH+FA Fentanyl spectrometer (Santa Clara, CA) Alliances HT 2795 LC system (A) water+FA LC-MS/MS coupled to a Quattro Premier mass 0.1-0.2 - Screening Plasma and urine XTerra MS C18 73 (B) ACN+FA spectrometer (Waters, Milford, USA) LC Symbiosis system coupled to mass spectrometry detection was Fentanyl and (A) water+FA LC-MS/MS carried out in ESI mode using 0.5 - plasma XTerra MS C18 77 metabolites (B) ACN+FA an API 5500 (AB-Sciex, Concord, Ontario, Canada) triple quadrupole LC Symbiosis system coupled to Analytes were detected using an 0.002- (A) water+FA LC-MS/MS - Screening urine XTerra MS C18 78 Applied Biosystems API 5500 Triple 0.04 (B) ACN+FA Quadrupole MS (Foster City, CA).

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues LC–MS/MS analyses were performed using an Agilent Technologies UPLC 1290 Serum, plasma and post- Zorbax Eclipse phenyl- (A) water+AmFo LC-MS/MS 0.1-2 0.02-0.6 Screening 26 coupled with a 6490 Triple Quad mortem tissues hexyl (B) ACN+FA from Agilent Technologies (Santa Clara, California, United States). API 4000 tandem mass spec- trometer (AB Sciex, Darmstadt, (A) LC-MS/MS Germany) interfaced to a binary LC 0.02-0.7 0.01-0.2 Screening plasma and urine Luna C18 74 MeOH/ACN/AmAc pump (series 1100, Agilent, Waldbronn, Germany). a Waters Xevo TQ-S Micro coupled (A) AmFo LC-MS/MS with a Waters Acquity UPLC 0.1 - Screening blood Poroshell EC C-18 54 (B) MeOH+FA (Milford, MA) a Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC system (Idstein, Germany) coupled (A) water+FA LC-MS/MS to a Thermo Scientific TSQ Vantage 0.1-1 - Screening blood and vitreous Kinetex F5 31 (B) ACN+FA triple quadrupole tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Waters Acquity UPLC system (Milford, MA, USA) coupled to a 0.01- blood, serum, plasma and (A) AmFo LC-MS/MS Waters TQD tandem mass spec- 0.05-0.5 Screening Zorbax RX-SIL 27 0.25 urine (B) ACN trometer (Waters Corp., Milford MA, USA) Accela UHPLC system coupled to a TSQ Quantum (A) AmFo+FA LC-MS/MS Access (Thermo- Fisher (TF) - - Screening plasma Hypersil Gold Phenyl 38 (B) ACN+FA Scientific, Dreieich, Germany) mass spectrometer Accela UHPLC coupled to a TSQ Quantum Access LC-MS/MS 2-15 - Screening plasma Hypersil Gold C18 (A) ACN+FA 43 MS (Thermo- Fisher Scientific, Dreieich, Germany)

24 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues An Acquity UPLC coupled to a Quattro Premiere Xe tandem mass (A) AmFo LC-MS/MS 0.14 0.044 Fentanyl blood Acquity BEH C18 114 spectrometer from Waters (Milford, (B) MeOH MA) Waters Acquity Ultra-Performance blood, liver, vitreous and (A) water+FA LC-MS/MS Liquid Chromatograph (UPLC) - - Acetyl fentanyl UPLC BEH T3 115 urine (B) ACN+FA coupled to a Waters TQ-D detector LC–MS was performed with a blood, urine and gastric (A)AmFo LC-MS/MS Shimadzu LC-MS8040 (Shimadzu - - Acetyl fentanyl Shim-pack FC-ODS 116 contents (B)MeOH Corpora- tion, Kyoto, Japan) a Waters Acquity UltraPerformance Liquid Chromatograph coupled to a Acquity UPLC BEH (A) water+FA LC-MS/MS 0.1 0.05 Acrylfentanyl blood 85 Waters Quattro Premier XE tandem C18 (B) ACN+FA mass spectrometer LC-20AC pumps (Shimadzu, Columbia, MD) interfaced with an API 3200 triple-quadrupole mass (A) water+AcA LC-MS/MS 0.25 - Alfentanil plasma Sunfire C18 97 spectrometer (Applied (B) ACN+AcA Biosystems/MDS Sciex, Foster City, CA) a Waters Xevo TQD LC/MS mass spectrometer attached to an Butyr fentanyl, blood, vitreous humor, (A) AmFo+FA LC-MS/MS ACQUITY UPLC System con- 1 - acetyl fentanyl and gastric contents, brain, Allure Biphenyl 5 µm 86 (B) MeOH trolled by MassLynx software acetyl norfentanyl liver, bile and urine. (Milford, MA) A Sciex 3200 QTRAP mass spectrometer coupled to an Agilent (A) water+FA LC-MS/MS 0.05 - Carfentanil blood C18 column 69 1260 LC system (Sciex, Cheshire, (B) ACN UK) was Waters Acquity UltraPerformance Liquid Chromatograph coupled to a Waters Acquity UPLC A) water+FA LC-MS/MS 0.01 - Carfentanil blood 79 Waters Quattro Premier XE tandem BEH C18 (B) ACN mass spectrometer. An Agilent6460 triple quadrupole A) water+FA LC-MS/MS mass spectrometer (Agilent 0.5 - Carfentanil Rat plasma Poroshell 120 SB-C18 80 (B) ACN Technologies, Santa Clara, CA)

25 of 34

Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues The HPLC system (Shimadzu, Japan) consisted of LC-20 AD LC-MS/MS 0.02 - Fentanyl plasma Capcell Pak C18 MG II (A)ACN+water+FA 117 coupled to an API 3000 (Applied Biosystems, USA) The HPLC system (Shimadzu, Japan) consisted of LC-20 AD 0.03- Fentanyl and (A) water+FA LC-MS/MS - Plasma and saliva Alltima C18 34 coupled to an API 3200 (Applied 0.04 norfentanyl (B) ACN+MeOH+FA Biosystems, USA) The LC system (Shimadzu, Kyoto, Japan) consisted of an LC-10AT pump coupled to Finnigan model (A) water+FA LC-MS/MS 0.05 - Fentanyl Plasma TSKgel ODS-100V 88 TSQ-7000 triple- (B) ACN +FA quadrupole (Thermo Fisher Scientific, Waltham, MA, USA) Alliance HPLC 2695 separation module (Waters, Milford, MA, USA) newborn pig plasma and (A) water+FA LC-MS/MS coupled to a tandemmass 0.2-0.25 - Fentanyl cerebrospinal fluid Luna C18 118 (B) ACN +FA spectrometer Quattro micro (Waters, samples Milford, MA, USA) LC Prominence (Shimadzu, Kyoto, Japan) coupled to API 4000 tandem (A) water+FA LC-MS/MS 0.05 - Fentanyl plasma Inertsil ODS-3 119 mass spectrometry system (AB (B) ACN +FA Sciex, Framingham, MA, U.S.A.) LC–MS/MS analysis was carried out using a Shimadzu system LC- 20ADXR (Shimadzu Prominence, 0.0025- 0.3-5 Fentanyl and (A) water+FA LC-MS/MS urine and whole blood Acquity C18 120 Antwerpen, Belgium) in combination 0.005 (pg/ml) norfentanyl (B) ACN +FA with a 3200 QTRAP (Applied Biosystems, Halle, Belgium)

A LC-MSMS (Waters, Acquity Fluorofentanyl Chiral column 83 LC-MS/MS - - serum (A) MeOH+FA+NH3 UPLC, Xevo TQ-S) isomers CHIROBIOTIC LC-30AD liquid chromatography system, (Shimadzu Scientific Instruments, Kyoto, Japan) equipped 0.01 Acquity UPLC BEH (A) AmFo+FA LC-MS/MS - Acrylfentanyl blood 71 with a Triple Quad 4500 System (AB (ng/g) Phenyl (B) MeOH+FA SCIEX Instruments, Concord, Ontario)

26 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues An Agilent 1100 series, HPLC chromatograph (Agilent Technologies, Palo Alto, USA) and (A) AmFo+FA LC-MS/MS - - Ocfentanil heroin sample Poroshell 120 EC-C18 103 an Esquire 3000 plus mass (B) ACN+FA spectrometer MRM (Bruker Daltonic GmbH, Bremen, Germany) The UPLC-MS/MS analysis was kidney, liver, stomach performed using a Acquity content (semi-solid), bile separations module coupled to the Acquity UPLC HSS (A) water+FA LC-MS/MS 2 - Ocfentanil and brain tissue and swab 72 Acquity TQD mass detector C18 column (B) ACN+FA of the mucous membrane equipped with ES interface (Waters of the nose Milford, MA, USA). LC Acquity system (Waters, blood, gastric content, Manchester, UK) coupled to an API ACQUITY HSS C18 (A) water+FA LC-MS/MS 0.05 0.01 Ocfentanil bile, vitreous and nose 121 4000 mass spectrometer (ABSciex, column (B) ACN+FA swabs Toronto, Ontario, Canada) An Accela LC (Thermo Scientific, Waltham, MA) coupled to a triple (A) water+FA LC-MS/MS 0.05 - Remifentanil plasma Kinetex C18 48 quadrupole mass spectrometer (TSQ (B) MeOH Quantum) All experiments were performed on an Agilent 6460A (Santa Clara, CA) (A) water+FA LC-MS/MS triple quadrupole LC-MS/MS 0.2 - Remifentanil whole blood and plasma 3-μm HyPURITY C18 81 (B) MeOH system, with a combined Agilent 1200 series LC system. The A Waters 2695 separation module (Waters Co., Milford, MA, USA) Chromolith Performance LC-MS/MS and a Quattro micro triple- 0.17 0.10 Remifentanil Rat plasma RP-18 monolithic (A)AmAc+ACN 84 quadrupole mass spectrometer column (Waters) A Waters Alliance HPLC system (Waters, Eschborn, Germany) LC-MS/MS 0.005 - Sufentanil plasma Kinetex C18 (A) water+ACN+TFA 93 coupled to a Waters Quattro Micro tandem mass spectrometer A Waters Alliance HPLC system (Waters, Eschborn, Germany) LC-MS/MS - - Sufentanil blood Kinetex C18 (A) water+ACN+TFA 94 coupled to a Waters Quattro Micro triple quadrupole mass spectrometer 27 of 34

Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

Fentanyl Detection Instrument LOQ9 LOD10 Sample Column Phases References analogues A Waters ACQUITY UPLC system coupled to a Micromass Quattro ACQUITY UPLC BEH LC-MS/MS 0.07 - Sufentanil plasma (A) ACN+water 95 Premier XE ES mass spectrometer C18 column (Waters Corp., Milford, MA, USA) 1260 Infinity LC coupled to a 6460 tandem mass spectrometer (A) AmAc+FA LC-MS/MS 1 - U-47700 urine Poroshell 120 EC-C18 32 (Agilent Technologies, Santa Clara, (B) MeOH+FA CA)

28 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

441 Table 3 Summary of analytical characteristics of fentanyl-like compounds

MRM Chemical Monoisotopic GC/MS base Analytes transition References formula mass peaks (ESI +) 2-Fluorobutyryl fentanyl C23H29FN2O 368.22639 277, 164, 207 369.2>299.1 42,122 2-fluorofentanyl C22H27FN2O 354.21074 263, 164, 207 355.3>105.1 27,122 2-Thiophenoyl Fentanyl C24H26N2OS 390.17658 111, 299, 256 - 122 3-methylfentanyl C23H30N2O 350.23581 259,160,203 351.5>202.4 73,122 4-ANPP C19H24N2 280.19394 146, 189 281.2>105.0 27,122 4-fluorobutyryl fentanyl C23H29FN2O 368.22639 277, 164, 207 369.3>188.1 27,122 4-fluoroisobutyryl fentanyl C23H29FN2O 368.22639 277, 164, 207 - 122 4-methoxyacetylfentanyl C22H28N2O2 352.21508 - 353.2>281.2 42 4-methoxybutyrfentanyl C24H32N2O2 380.24637 289, 176, 219, 290 381.2>311.2 42,122 4-methoxymethylfentanyl C24H32N2O2 380.24637 - 381.2>325.2 42 Acetyl Fentanyl C21H26N2O 322.20451 146, 231, 188 323.4>188.2 31,122 Acetyl norfentanyl C13H18N2O 218.14191 82, 83, 175 219.1>177.1 42,122 Acrylfentanyl C22H26N2O 334.20451 - 335.2>105.0 27 AH-7921 C16H22Cl2N2O 328.11091 126, 127, 173 329.1>284.0 122 Alfentanil C21H32N6O3 416.25359 - 417.3>268.6 73 Alpha-methylacetylfentanyl C22H28N2O 336.22016 245, 246, 91, 110 337.2>295.2 122 Benzoylbenzyl fentanyl C25H26N2O 370.20451 91,105, 265 - 122 Benzylfentanyl C21H26N2O 322.20451 - 323.2>267.2 42 C20H26N2O2S Beta-hydroxy-thiofentanyl 358.17150 245, 146 359.2>192.1 27,122

Beta-hydroxyfentanyl C22H28N2O2 352.21507 - 353.2>279.2 42 Butyr-fentanyl C23H30N2O 350.23581 146, 259, 189 351.1>105.0 27,122 Carfentanyl C24H30N2O3 394.22564 303, 304, 187, 105 395.4>335.3 31,122 Cyclopentyl fentanyl C25H32N2O 376.25146 285, 189, 146 377.2>281.2 42,122 Cyclopropyl Fentanyl C23H28N2O 348.22016 257, 146, 189, 69 349>188 122,123 Fentanyl C22H28N2O 336.22016 245, 146, 189, 202 337.2>188.5 73,122 Furanyl fentanyl C24H26N2O2 374.19942 95, 283, 240 375.2>188.2 31,122 Hexanoylfentanyl C25H34N2O 378.26711 287, 146, 189 - 122 Isobutyryl fentanyl C23H30N2O 350.23581 259, 146, 189 351.2>282.2 42,122 Lofentanyl C25H32N2O3 408.24129 - 409.2>353.2 42 Methylcarfentanil C17H24N2O3 304.17869 - 305.1>249.2 42 Mirfentanyl C22H24N4O2 376.18992 - 377.2>283.2 42 MT-45 C24H32N2 348.25654 257, 91 349.1>181.0 27,122 Norfentanyl C14H20N2O 232.15756 - 233.2>84.1 73 Ocfentanil C22H27FN2O2 370.20565 279, 45, 105, 176 371.2>299.2 42,122 Remifentanil C20H28N2O5 376.19982 - 377.1>317.0 73 Sufentanil C22H30N2O2S 386.20280 - 387.6>238.2 73 Thenylfentanyl C19H24N2OS 328.16093 - 329.1>273.1 73 THF Fentanyl C24H30N2O2 378.23072 287, 146, 71, 189 379.2>281.2 42,122 Thiofentanyl C20H26N2OS 342.17658 - 343.1>287.1 42 Trefentanyl C25H31FN6O2 466.24925 - 467.2>411.2 42 U-47700 C16H22Cl2N2O 328.11091 84, 125, 58, 71 329.2>284.1 27,122 U-49900 C18H26Cl2N2O 356.14221 112, 153 357.2>284.0 122,124 U-50488 C19H26Cl2N2O 368.14222 - 369.2>298.1 27 Valeryl fentanyl C24H32N2O 364.25146 146, 273, 189 365.2>188.2 27,122 442 443 444

29 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

445 446 Figure 1: Chemical structures of some clinical opioids: fentanyl (A), ocfentanil (B), 447 sufentanil (C), alfentanil (D), carfentanil (E), remifentanil (F) and mirfentanil (G) 448

30 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

449 450 451 452

453 Figure 2: Chemical structures of common illicit fentanyls: alpha-methylfentanyl (A), 3- 454 methylfentanyl or mefentanyl (B), MT-45 (C), AH-7921 (D) and U-47700 (E) 455

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Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 Figure 3: Trend of papers published in the last ten years in the field of fentanyl-like 474 compounds 475

32 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

476

477 478 Figure 4: Number of papers published in the last ten years in the field of fentanyl-like 479 compounds: comparison of extraction techniques (A) and analytical procedures (B) 480

33 of 34 Analytical Sciences Advance Publication by J-STAGE Received November 14, 2018; Accepted January 11, 2019; Published online on January 25, 2019 DOI: 10.2116/analsci.18R004

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 Figure 5: European distribution of papers published in the last ten years in the field of 508507 fentanyl-like compound

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