Abstract:

Given the wide variety of fentanyl analogs, the test for this entire group tends to be crucial and particularly difficult since all fentanyl-like substances are listed as controlled substances in China. This study meticulously analyzed the fragmentation pathways and mechanisms of 29 fentanyl analogs and summarized the fragmentation pathways and features for the entire group of fentanyl analogs, thus providing a reference for related screening tests. Fentanyl, thiofentanyl, and sufentanil were selected as the representative compounds in this study, and the fragmentation mechanism of their fragment ions was interpreted. The general fragmentation rules for fentanyl analogs were summarized as well. The fragment ions of the three compounds formed by induced cleavage (i) came with high abundance ratios, such as fragment ions of m/z 188, 105, 194, 111, and 238, while the induced cleavage was due to the amide and piperidinyl groups. Moreover, the induction ability of amide group was significantly stronger than that of the piperidinyl group, and induced cleavage was the main fragmentation pathway for most of the fentanyl analogs. Furthermore, the fragment ions with m/z 281 and 287 for fentanyl and thiofentanyl were formed by loss of the propionyl group after single H rearrangement (rH). The fragment ions with m/z 216, 146, and 132 for fentanyl and thiofentanyl were formed by double H rearrangement (r2H). Although their abundance ratios were not high, they still had specificity and regularity. Elimination reaction (re) was also a very common fragmentation pathway for these compounds, leading to fragment ions with m/z 134 and 140. Phenylethyl substituents were more prone to the elimination reaction with a higher abundance ratio than thiophenethyl substituents. Compounds such as sufentanil with methoxy substituents at the piperidinyl para-position could produce a large number of fragment ions, which were more susceptible to the rH pathway and loss of methanol neutral molecules, leading to the formation of ions with m/z 355. Similarly, compounds such as remifentanil bearing a methyl formate substituent at the piperidine para-position also produced numerous fragment ions, which were more prone to the rH pathway to lose methyl formate or methanol neutral molecules and furnish fragment ions with m/z 317 or 345. Compounds containing hydroxyl substituents, such as β-hydroxyfentanyl and β-hydroxythiofentanyl, produce significant dehydration ions and formed fragment ions with m/z 335 (β-hydroxyfentanyl) and m/z 341 (β-hydroxythiofentanyl). A method based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for the qualitative and quantitative determination of the 29 fentanyl analogs was developed. Drugs and white powder samples were extracted by acetonitrile, as well as protein and milk beverage samples. Sugar-containing solids or powders, drinking water, fruit and vegetable drinks, health drinks, tea drinks, and alcohol samples were extracted by 10% acetonitrile aqueous solution. Following vortexing, centrifugation, and membrane separation, the target compounds were separated on a Kinetex C18 column (100 mm×2.1 mm, 2.6 μm) with gradient elution at a flow rate of 0.4 mL/min. The mobile phases were composed of acetonitrile and 0.08% formic acid aqueous solution. The target compounds were quantified by LC-QTOF-MS using an external standard method in positive ion mode. The 29 fentanyl analogs showed good linear relationships in the range of 1-20 μg/L, and the correlation coefficients were greater than 0.995. The limits of detection (LODs) and limits of quantification (LOQs) were 0.01 mg/kg and 0.05, respectively. The average recoveries were 85.2%-112.9% for hypoglycemic drugs, Lulu drinks, glucose powder, Zhenlu health drink and chocolate, with RSDs of 1.9%-19.8% (n=6). This method is rapid, simple, time-saving, highly sensitivity and stable, and it is applicable to a wide variety of samples. Hence, it is suitable for the identification, confirmation, and quantitative detection of the 29 fentanyl analogs in drugs, solids or powders containing sugar, beverages, drinking water, wine samples, etc.

Key words: liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), identification and confirmation, fragmentation mechanism, screening, fentanyl analogs