21
Figure 1. Schematic protocol for sample preparation using Microlute™ CP SCX 96 well plate.
results. This creates longer lead times for testing, expensive re-tests and an increase instrument maintenance time.
This application note demonstrates how an SCX mixed-mode SPE method combined with UHPLC-MS/MS can analyse 12 natural, semi-synthetic and synthetic opioid drugs and metabolites from a sample of human urine in a reliable and reproducible way.
Compounds:
Morphine, oxymorphone, noroxycodone, hydrocodone, norhydrocodone, O-desmethyl-cis-tramadol, norfentanyl, cis-tramadol, meperidine, fentanyl, EDDP, methadone. All compounds were purchased from Cerilliant in their 1 mg/mL format.
Sample Preparation:
A stock of all opioids (3 µg/mL) was prepared in methanol. For the preparation of the urine samples, 12.5 mL of pooled source mixed gendered blank human urine was spiked with 625 µL of opioid stock solution. Blanks were prepared by spiking 12.5 mL of urine with 625 µL of methanol. The samples and blanks were both diluted 1:1 with 1% formic acid in water and vortexed for one minute to homogenise the solutions.
For the mixed-mode SPE, each well of the Microlute™ CP SCX 30 mg 96 well plate (Cat no. PSCX030P-001) was conditioned with 1,000 µL of methanol then equilibrated with 1,000 µL of water. Each pre-treated sample and blank was loaded onto the plate in full. Once loaded, 1,000 µL of 0.1% formic
Experimental LC Conditions:
LC system Column
Vanquish Horizon
Thermo Scientific™ Hypersil GOLD™ aQ, 3 µm, 100 x 4.6 mm
Column temp. 30°C Injection volume 4 µL Flow rate
acid in water was used to wash each well, followed by 1,000 µL of 0.1% formic acid in methanol. Each well was eluted with 2 x 400 µL of 45:45:10 methanol/acetonitrile/ TEA. The eluent was evaporated under N2
to dryness using the MiniVap® Gemini (Cat no. 500234) using the 96 needle head with straight needles (Cat no. 229036) at room temperature, taking approximately 45 minutes. Samples were reconstituted in 100 µL of starting mobile phase (0.1% formic acid in water). Post spike standards were prepared by reconstituting the blank solution wells with 100 µL of opioids standard solution (1500 ng/mL) diluted in starting mobile phase (0.1% formic acid in water). The same procedure was also performed on a commercially available loose filled 96 well plate (Figure 1).
MS Conditions: MS system
Ionisation Mode Acquisition Mode Polarity
1 mL/min
Mobile phase A 0.1% Formic acid in water Mobile phase B 0.1% Formic acid in methanol
Gradient
Time (min) A% 0.0 3.0 8.0 9.0 9.1
15.0
100 85 0 0
100 100
B% 0
15
100 100 0 0
Spray Voltage Sheath gas Aux gas
Sweep gas
Ion transfer tube temp. Vaporiser temp. Cycle time
Q1 resolution Q3 resolution CID gas
TSQ Altis H-ESI MRM
Positive +3500 V 60 arb 2 arb 2 arb
380°C 350°C
0.3 seconds 0.4 FWHM 0.4 FWHM 1.5 mTorr
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