The term drugs of abuse (DOA) can be applied to a wide variety of natural and synthetic compounds prevalent in human culture. Many rapidly pass through the human body, while others can persist for extended periods. Screening for traces of these has become common place by analyzing human urine, either by LC–MS or by GC–MS through derivitization of the parent compounds.

Solid phase extraction (SPE) provides an ideal extraction media for DOA compounds, as it can be performed with small sample amounts with rapid turnaround. The utilization of automated SPE platforms (EconoTrace SPE, FMS Inc.) can greatly assist production laboratories generate large volumes of samples in relative short periods of time.

Consumables

• 150 mg 6 cc DOA mixed mode SPE cartridges
• Acetonitrile, LC–MS grade or equivalent
• Methanol, LC–MS grade or equivalent
• Toluene, pesticide grade or equivalent
• HPLC grade water
• 6N HCL
• Ammonium hydroxide
• Native and labeled DOA standards acquired from Cambridge Isotopes Laboratories.

Sample/Reagent Prep

1. 2 mL urine samples diluted to 5 mL DI water, pH adjusted to 2 with HCL.

SPE Procedure

1. Condition cartridges with 5 mL methanol at 2 mL per min

2. Condition cartridges with 5 mL DI water at 2 mL per min

3. Sample loaded across cartridge at 2.5 mL per min

4. Cartridge washed with 5 mL water

5. Cartridge washed with 5 mL methanol

6. Cartridges dried with nitrogen for 20 min

7. Cartridges eluted with 4 mL 3:2 methanol – acetonitrile with 5% ammonium hydroxide

8. Cartridges allowed to soak for 3 min

9. Cartridges eluted with 3 mL 3:2 methanol – acetonitrile with 5% ammonium hydroxide

10. Remaining elution solvent N₂ purged to collection vials

SuperVap

1. Preheat temperature: 35 °C

2. Evap mode: 8 psi nitrogen with sensor

3. Extracts reduced to 1 mL volume

SuperVap Vial Evaporator

At 1 mL, GC vials removed from SuperVap tubes and transferred to vial evaporator. Extracts taken to dryness at 2 psi, ambient temperature. Extracts reconstituted with recovery standard in initial mobile phase.

Analytical Conditions

Waters Acquity H-Class UPLC
Column: Waters BEH C₁₈, 2.1 × 100 mm, 1.7 µm
Column temperature: 30 °C
Solvent A: 0.1% formic acid in MilliQ water
Solvent B: 0.1% formic acid in acetonitrile

Xevo TQD mass spectrometer conditions:

Ionization mode: ESI+ and ESI-
Acquisition mode: MRM
Capillary voltage: 1.5 kV
Collision energy (eV): optimized for individual compounds
Cone voltage (V): optimized for individual compounds
Data: acquisition and analysis using Mass Lynx v.4.1 software

Conclusions

Extraction efficiencies for DOA compounds in human urine matrix showed excellent precision using the mixed mode cartridge. Between replicates, RPDs were below 15% for all analytes with most falling below 10%. With the easy operation, limited sample prep and excellent performance the FMS, Inc. EconoTrace SPE demonstrates to be an ideal fit for DOA screening of human urine.

FMS, Inc.
580 Pleasant Street, Watertown, MA 02472
tel. (617) 393-2396, fax (617) 393-0194
Website: www.fms-inc.com