Obtainment from Cerilliant (Round Rock, TX) is amphetamine(AM), methamphetamine(MA), 4-Methyle-nedioxyamphetamine(MDA), 4-methylenedioxyme-thamphetamine(MDMA), N-Methyl-1-(3, 4-methyl-enedioxyphenyl)-2-butanamine(MBDB), 4-methylenedioxyethylamphetamine (MDEA) and MA-d5 at 1 mg· mL^-1. All reagents are of HPLC grade. Acetonitrile (ACN) and methanol (MeOH) were purchased from Fisher Scientific (Fairlawn, NJ). Formic acid (FA) was obtained from Spectrum Chemical MFG Corp. (Gardena, CA). Deionized water was generated with Elix, a Millipore water purification system (Millipore Corp., Molscheim, France).

The analytes were separated on a column (ACQUITY UPLC BEH Phenyl column; 100 mm× 2.1 mm, 1.7 μ m) that was held at 35 ° C. The mobile phase, consisted of ACN (eluent A) and 0.3 % FA (eluent B), was flowing in gradient at a rate of 0.40 mL/min. Immediately after sample injection, the ACN percentage was increased from the initial 10 % to 55 % over 3 min, and increased to 90 % in 1 min, then decreased to 10 % in 0.1 min with a post equilibration time of 1.9 min. The total run time was 6 min.

Ionization was achieved with electrospray in the positive ionization mode (ESI+). Nitrogen was used as the nebulization and desolvatation gas. The multi-reaction monitoring mode (MRM) was used, followed by the dependent scan, which was an enhanced product-ion scan. The two resulting transitions per analyte and respective setting for UPLC-MS/MS running were given in Table 1. The other main parameters were drying gas temperature of 500 ° C, source heater temperature at 300 ° C, nebulization gas flow in 650 L· h^-1, cone gas flow of 150 L· h^-1 and capillary voltage as 3.0 kV.

Table 1

Table 1

Table 1 Two resulting MRM transitions per analyte and corresponding settings for UPLC-ESI-MS/MS system (PI: parent ion; DI: daughter ion; CV: cone voltage; CE: collision energy) Analyte PI / (m/z) DI / (m/z) CV / V CE / V AM 136.200 91.1 20 20 119.0* 20 10 MA 149.961 90.912* 18 14 118.948 18 10 MDA 180.200 105.200 10 25 133.100* 10 20 MDMA 194.300 77.100 10 40 105.100* 10 25 MBDB 208.300 135.100 20 15 177.100* 20 10 MDEA 208.300 105.100 30 25 135.100* 30 25 * Quantitative ion

Table 1 Two resulting MRM transitions per analyte and corresponding settings for UPLC-ESI-MS/MS system (PI: parent ion; DI: daughter ion; CV: cone voltage; CE: collision energy)

Methanolic standard stock solutions (1 μ g· mL^-1) of each analyte were prepared from the commercially available methanolic solutions (1.0 mg· mL^-1). The working standard solutions were prepared from the stock solutions by dilution to the appropriate concentrations as needed. The internal standard (IS) of MA-d5 was prepared to produce its working solution (100 ng· mL^-1) in methanol. All the working solutions were stored at -20 ° C.

Human blank urine, used for development and validation of the proposed method, were obtained from volunteers without history of drug abuse, storing in refrigerator at -20 ° C.

The extraction of analytes in urine was performed with Oasis MCX solid phase extraction (SPE) cartridge (1 mL, 30 mg; Waters, USA). 2 mL of spiked urine sample (with 1 ng· mL^-1 MA-d5 as internal standard) was centrifuged for 5 min at 8000× g and the supernatant was loaded into an SPE cartridge which was preconditioned by the first addition of 1 mL methanol followed by 1 mL deionized water and allowed to flow freely under gravity. The SPE cartridge was washed with 1 mL of 10 % methanol in water. The columns were dried under vacuum for 10 min, then leaving the analytes to be eluted with 2 mL of 5 % ammonia water and 70 % hydrated methanol. The elution was submitted for UPLC-MS/MS analysis.

Selectivity was evaluated by analyzing ten separate sources of blank urine for background noise and identifying matrix interferences. Mean area measurements for the selected analyte ions were generated and analyzed to ensure that the matrices were free from interfering compounds. As shown in Fig. 1, no interfering peaks were observed in the extracts of blank urine samples, indicating that the assay was found to be selective for all tested compounds.

Fig.1

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	Fig.1 MRM chromatograms of extracted urine sample solutions (at 10 ng/mL) containing the six amphetamine-type drugs

Calibration standards (with 1 ng· mL^-1 MA-d5 as internal standard) for urine samples were obtained between 0.1 to 20 ng· mL^-1, depending on the sensitivity of the analytes. Daily calibration curves of the same concentration were prepared with each batch of validation and authentic samples. For all analytes, a linear weight (1/concentration) model was found to be the best and therefore used for the calibration curves. The regression equations and correlation coefficients of each analyte are listed in Table 2. The lower limit of quantification (LLOQ) and the limit of detection (LOD) in the MRM mode were defined as the concentrations with a signal-to-noise ratio of 10 and 3, respectively. The LLOQs corresponded to the lowest concentrations were used for the calibration curves with a signal-to-noise ratio of at least 10. The LODs reached 0.005~0.02 ng· mL^-1 for urine samples.

Table 2

Table 2

Table 2 Regression equations with correlation coefficients and LODs for the given analytes in urine samples Analytes Linearity/(ng· mL-1) Regression equations r2 LOD/(ng/mL) AM 0.1~20 y=4.93× 10-3x+5.39× 10-2 0.9987 0.02 MA 0.1~20 y=6.38× 10-2x+9.26× 10-2 1 0.02 MDA 0.1~20 y=3.7× 10-3x+7.1× 10-3 1 0.02 MDMA 0.1~20 y=1.08× 10-2x-4.7× 10-3 1 0.02 MBDB 0.1~20 y=2.35× 10-2x-6.95× 10-2 0.9998 0.005 MDEA 0.1~20 y=2.43× 10-2x-3.73× 10-2 0.9999 0.005

Table 2 Regression equations with correlation coefficients and LODs for the given analytes in urine samples

The concentrations for QC samples were 0.125, 1.25 and 12.5ng· mL^-1. They were selected to perform the accuracy and precision test. According to the procedure described in the preceding text, five QC samples’ replicates of low, middle and high concentration were run on three consecutive days. The concentrations of the analytes in the QC samples were calculated via the daily calibration curves. Precision for intra- and inter-day was calculated, given as relative standard deviation (RSD).

Extraction efficiencies and matrix effects were estimated in a post-extraction addition approach. Three sets of samples of five different blank matrices were prepared at three QC concentrations. Samples in set 1 consisted of neat standards. For preparation of the samples in set 2, blank urine samples were first extracted, as described previously. Then, the extracts were spiked with corresponding working standards and the IS. For preparation of set 3, blank urine samples as in set 2 were spiked with corresponding working standards and the IS at three concentrations. Thereafter, they were extracted through the previously-described extraction procedure. Extraction efficiencies were estimated by a comparison of the peak areas from the samples of set 3 with those from the corresponding samples of set 2. Matrix effects were estimated by a comparison of the peak areas from the samples of set 2 with those from the corresponding samples of set 1. Data were listed in Table 3. Intra- and inter-day precision was lay within the required limits of 15 % RSD (20% RSD at LLOQ) at all studied concentration levels. Process efficiencies were satisfying and matrix effects were in a non-disturbing range.

For estimation of stability of the processed samples under the conditions of UPLC-MS/MS analysis, three concentration levels of QC samples (n=3/each) were extracted. The resulting extracts at each concentration level were maintained at room temperature (+18 ° C) for 24 h before analysis. For the evaluation of stability after three freeze-thaw cycles, the QC samples were frozen at -20 ° C for 24 h. After this period, the samples were again thawed and frozen for 24 h and this process was repeated until the third thawing cycle when the QC samples were extracted and analyzed. The concentrations of the extracted analytes in the QC samples were calculated via the daily calibration curves. Stability of the analytes demonstrated that there were no significant differences from the reference concentrations.