25
AB Clonazepam-d4
4.08 Alprazolam-d5 4.42 4.33 4.39 0
3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.08
Lorazepam 4.38 4.01 4.31 3.78 3.85 3.94 3.97 4 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.27 4.18 4.44 4.48 Time 0 4.10 4.20 4.30 4.40 4.50 4.60 Time Flunitrazepam 0 4.10 4.20 4.30 4.40 4.42 4.50 4.60
standards and high QC samples were prepared in blank urine and diluted as appropriate. Each calibrator and QC sample contained all the analytes in the panel. All analytes are listed in Table 1, along with retention times and MS transitions parameters, including cone voltages and collision energies.
Sample pretreatment: 200 µL of urine was added to individual wells of an Oasis®
MCX
µElution Plate, along with 20 µL of internal standard (IS) solution (250 ng/mL), 200 µL of 0.5 M ammonium acetate buffer (pH 5.0) and 2 µL of β-glucuronidase (Sigma Aldrich, P. vulgate, 85k units/mL). The entire plate was incubated at 50°C for 1 hr. and then quenched with 200 µL of 4% phosphoric acid (H3
PO4 Figure 2. Chromatographic separation of key analyte pairs on the CORTECS UPLC C18 + 1.6 µm column.
A. Clonazepam-d4 contributes to the lorazepam MRM but is baseline separated on this column. B. Alprazolam-d5 at 4.33 minutes is baseline separated from flunitrazepam at 4.42 minutes.
).
METHOD CONDITIONS LC conditions: LC system Column
Column temp. Sample temp.
Injection volume Flow rate
Mobile phase A (MPA) Mobile phase B (MPB) Gradient:
Waters ACQUITY UHPLC I-Class (FL) Waters CORTECS® 30˚C 10˚C 5 µL
UHPLC C18 0.5 mL/min.
0.01% Formic acid in MilliQ water 0.01% Formic acid in acetonitrile (ACN)
Initial conditions were 90:10 MPA:MPB. The percentage of
MPB was increased to 50% over 5 minutes, ramped up to 95% by 5.25 minutes, held at 95% for 0.75 minutes and returned to 10% over 0.1 minute.
MS conditions: MS system
Ionisation mode Detection
Desolvation Temp Desolvation Flow Capillary voltage Collision energy Cone voltage
Data management: MS software
Quantification software ( Area A Area B )x100%
RESULTS AND DISCUSSION: Chromatography
Xevo® TQ-S micro ESI positive
MRM (transitions optimised for individual compounds, Table 1) 500°C
1000 L/Hr 0.5 kV
Optimised for individual compounds (See Table 1) Optimised for individual compounds (See Table 1)
All test compounds are listed in Table 1, and representative chromatograms are shown in Figure 1. Table 1 also lists the retention times (R.T.) and MS conditions of all compounds. The selectivity and high efficiency of the CORTECS UHPLC C18
+ Column, 1.6 µm, 2.1 x 100 mm
SPE Extraction: Pretreated samples were drawn into the sorbent bed by vacuum. All samples were subsequently washed with 200 µL of 0.02 N hydrochloric acid (HCl), followed by 200 µL of 20% MeOH. After washing, the plate was dried under high vacuum (~ 15 inch Hg) for 30 s. Samples were eluted with 2 x 25 µL of 60:40 (v/v) acetonitrile (ACN):MeOH containing 5% strong ammonia solution (Fisher, 28-30%). All samples were then diluted with 100 µL of sample diluent (2% ACN:1% formic acid in MilliQ water). The final concentration factor of the sample was 1.3 (200/150). A graphical workflow of the extraction procedure is shown in Figure 1.
+ 1.6 MassLynx® TargetLynx™
Analyte recovery was calculated according to the following equation: %Recovery=
Where A =the peak area of analyte in an extracted sample and B = the peak area of analyte in an extracted matrix sample in which the compounds were added post-extraction.
µm solid-core column enables the baseline separation of all potentially interfering peaks. Two key pairs are shown in Figure 2. While clonazepam-d4 (320>274.1) (R.T. 4.08 min) generates a slight contribution to the primary lorazepam MRM (323>277), the two peaks are baseline separated. Even at the lower limit of quantitation (LLOQ) (0.5 ng/mL), the clonazepam internal standard (IS) does not interfere with lorazepam and does not affect peak quantification. Another critical pair is alprazolam-d5 and flunitrazepam. In this case, flunitrazepam (314.1>239.2) makes a contribution that can be seen in the MRM trace of alprazolam-d5 (314.1>210.1). However, the baseline separation of these peaks ensures that even at the upper limit of quantification (ULOQ) (500ng/mL) the baseline separation prevents flunitrazepam from affecting the integration and quantification of the alprazolam IS.
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%
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