Extraction of Loperamide and N-Desmethyl Loperamide from Blood Followed by LC–MS/MS Analysis

Loperamide is an over-the-counter antidiarrhoeal drug that has been considered to be safe when used as directed. However, as the opioid epidemic continues to ravage our nation there has been an increasing number of reported cases of loperamide overdose. This application note outlines a simple three-step method for the extraction of loperamide and its main metabolite, N-desmethyl loperamide, from blood. UCT’s Clean Screen® XCEL I column provides users with the same level of sample clean-up as traditional SPE while allowing the elimination of timely conditioning and wash steps.

Procedure

Sample Pretreatment

• To 1 mL blood sample add 3 mL acetate buffer pH 5 and appropriate amount of internal standard.

• Vortex samples for 30 s to mix.

SPE Procedure

• Apply samples to SPE tubes without any preconditioning.

• Allow samples to flow through the column at a rate of 1–2 mL/min.

• Wash samples with 2 mL of D.I. H₂O.

• Wash SPE column with 2 mL of 98:2 methanol–glacial acetic acid.

• Dry column for 5 min at full vacuum or pressure.

• Wash samples with 2 mL of hexane.

• Dry column for 10 min at full vacuum or pressure.

• Elute compounds with 2 mL of 78:20:2 DCM–IPA–NH₄OH.

• Collect eluate at a rate of 1–2 mL/min.

• Evaporate to dryness at < 50 °C.

• Reconstitute sample in mobile phase.

Instrumental

LC–MS/MS: Thermo Scientific™ Dionex™ 3000 (LC) TSQ Vantage™ (MS/MS)
Column: UCT Selectra® DA HPLC Column 100 × 2.1 mm, 1.8-μm
Guard Column: UCT Selectra® DA Guard Column 10 × 2.1 mm, 1.8-μm
Injection Volume: 1 μL
Mobile Phase A: D.I. H₂O + 0.1% formic acid
Mobile Phase B: Acetonitrile + 0.1% formic acid
Column Flow Rate: 0.30 mL/min

Results

Conclusion

This application note effectively outlines a simple three-step method for the extraction of loperamide and its main metabolite, N-desmethyl loperamide, from blood. UCT’s Clean Screen® XCEL I column provides users with the same level of exceptional sample clean-up as traditional SPE while allowing the elimination of timely conditioning and wash steps in addition to a concentration step. Sample extracts are analyzed via UHPLC–MS/MS utilizing UCT’s Selectra® C18 1.8 µm analytical column.

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