Simultaneous Determination and Stability Analysis of Ten New Psychoactive Substances including Synthetic Cathinones, Phenethylamines, and Ketamine Substitutes in Urine Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Knowing the stability of drugs is important to ensure accurate and reliable results of drug concentrations. This study evaluated the stability of ten new psychoactive substances (NPSs) in urine and methanol/water at different storage temperatures. Quantitative analyses were performed using liquid chromatography-tandem mass spectrometry. Three replicates of each storage condition were analyzed at day 0 and after 7, 14-, 30-, 60-, and 90 days with storage at +25°C, +4°C, and -20°C. For each analyte, the percent difference at each time interval from day 0 was calculated for each storage condition. Para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), deschloroketamine (DCK), and 2-fluorodeschloroketamine (2-FDCK) were stable in urine, even when stored for 90-day periods at various temperatures. For synthetic cathinones, the concentrations declined over time at room temperature (+25°C) in urine but were relatively stable in methanol solvent with 0.1% formic acid. The significant degradation was found at +25°C, and the most excellent stability was shown by samples stored at -20°C. Phenethylamines (PMA and PMMA) and ketamine substitutes (DCK and 2-FDCK) were relatively more stable than synthetic cathinones (mephedrone, butylone, pentylone, ephylone, 4-MEAPP, and eutylone).

Figure 1

Total ion chromatogram of the analytes. The total ion chromatogram (TIC) of (a) 10 new psychoactive substances and (b) four internal standards in this study. Mephedrone: 4-methylmethcathinone; ephylone: N-ethylpentylone; 4-MEAPP: 4-methyl-α-ethylaminopentiophenone; PMA: para-methoxyamphetamine; PMMA: para-methoxymethamphetamine; DCK: deschloroketamine; 2-FDCK: 2-fluorodeschloroketamine; MDA-D5: 3,4-methylenedioxyamphetamine-D5.

Figure 2

The stability of six synthetic cathinones in different matrices and temperatures. The stability of (a) 4-methylmethcathinone (mephedrone), (b) butylone, (c) pentylone, (d) eutylone, (e) N-ethylpentylone (ephylone), and (f) 4-methyl-α-ethylaminopentiophenone (4-MEAPP) in 50% methanol/water (50% MeOH), 50% methanol/water with 0.1% formic acid (50% MeOH in 0.1% FA), and urine at −20°C, 4°C, and 25°C.

Figure 3

The stability of phenethylamines and ketamine substitutes in different matrices and temperatures. The stability of (a) para-methoxyamphetamine (PMA), (b) para-methoxymethamphetamine (PMMA), (c) deschloroketamine (DCK), and (d) 2-fluorodeschloroketamine (2-FDCK) in 50% methanol/water (50% MeOH), 50% methanol/water with 0.1% formic acid (50% MeOH in 0.1% FA), and urine at −20°C, 4°C, and 25°C.

Figure 4

Comparison of the stability of the analytes in urine at different temperatures. Stability of the 10 analytes in urine at (a) frozen temperature (−20°C), (b) refrigerated temperature (4°C), and (c) room temperature (25°C). The targeted concentrations for mephedrone, butylone, pentylone, eutylone, ephylone, 4-MEAPP, PMA, and PMMA are 50 ng/mL. The targeted concentrations for DCK and 2-FDCK are 25 ng/mL. Mephedrone: 4-methylmethcathinone; ephylone: N-ethylpentylone; 4-MEAPP: 4-methyl-α-ethylaminopentiophenone; PMA: para-methoxyamphetamine; PMMA: para-methoxymethamphetamine; DCK: deschloroketamine; 2-FDCK: 2-fluorodeschloroketamine.