Z-Drug and Metabolite Panel in Urine Using SPE Extraction and HPLC–MS-MS Analysis

June 1, 2016
Tina Fanning and Jody Searfoss, UCT, LLC

The Application Notebook

Issue 0

This application note describes the development of an analytical method for the sensitive and accurate determination of Z-drugs in urine using CLEAN SCREEN® XCEL I SPE columns and HPLC–MS-MS analysis. Chromatographic separation is achieved using a Selectra® DA column. Z-drugs or nonbenzodiazepines are a class of psychoactive drugs whose pharmacological actions are similar to those of the benzodiazepines and have shown efficacy in treating sleep disorders. Clean Screen® XCEL I columns were chosen because of their excellent capability to extract basic compounds while eliminating the need for time consuming column conditioning. This method reduces the amount of time needed to extract a panel of samples and minimizes solvent use for sample cleanup.


1. Sample preparation:

(a) To 1 mL of urine add 3 mL of 0.1% formic acid solution

(b) Vortex for 30 s

2. Apply sample to Clean Screen® XCEL I column

(a) Load sample directly to column without any preconditioning

(b) Pull sample through at a rate of 1–2 mL/min

(c) Dry column thoroughly under full vacuum or positive pressure for 1 min

3. Wash cartridge

(a) 1 × 3 mL 75:25 100 Mm acetic acid:MeOH

(b) 1 × 3 mL hexane

(c) Dry column thoroughly under full vacuum or positive pressure for 5–10 min

4. Elution

(a) 1 × 3 mL CH2Cl2/IPA/NH4OH (78:20:2)

(b) Evaporate to dryness/reconstitute in 100 µL of mobile phase


LC–MS-MS: Agilent 1200 Series Binary Pump LC System/ API 4000 QTRAP MS-MS

Column: UCT Selectra® DA LC column, 100 × 2.1 mm, 3 µm

Guard column: UCT Selectra® DA guard column, 10 × 2.1 mm, 3 µm

Injection volume: 10 µL

Mobile phase A: 0.1% formic acid in D.I. H2O

Mobile phase B: 0.1% formic acid in ACN

Column flow rate: 0.30 mL/min


Excellent recoveries ranging from 71% to 102% were obtained using UCT’s XCEL I extraction column (CSXCE106) and procedure in conjunction with the Selectra® DA HPLC column (SLDA100ID21-3UM) for the analysis of Z-drugs and their metabolites in urine. Insignificant loss of Zaleplon was observed using 3 mL 75:25 100 mM acetic acid:MeOH wash in this study. To correct for any residual recovery or matrix issues, the use of deuterated internal standards is strongly recommended.

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