Determination of Methadone and Metabolite (EDDP) in Urine Samples Using Solid Phase Extraction and GC–MS Analysis


The Application Notebook

The Application NotebookThe Application Notebook-09-01-2014
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Issue 0

Methadone is a commonly prescribed pain management type drug. As such, patients are monitored on a routine basis.

Methadone is a commonly prescribed pain management type drug. As such, patients are monitored on a routine basis. This application note presents a simple and effective method for the extraction and analysis of the parent compound as well as its primary analyte EDDP. The method utilizes mixed mode SPE for urine sample extraction followed by GC–MS analysis.

SPE Materials

Sample Preparation

1) To 1 mL of 100 mM pH 6 phosphate buffer in a sample tube add internal standards (methadone-d9, EDDP-d3). To this add 1 mL of urine and mix. Add an additional 2 mL of 100 mM ph 6 phosphate buffer and mix. Centrifuge this mixture for 10 min at 3000 rpm.

2) Condition the SPE cartridge with sequential additions of the following liquids in the order specified: 3 mL methanol, 3 mL DI water, 3 mL 100 mM pH 6 phosphate buffer. Draw each solvent through the cartridge (< 3 in. Hg) until it reaches the top of the sorbent bed; do not allow the cartridge to go dry between solvent additions.

Table I: Mass spec table

3) Load the previously prepared (step 1) clear sample onto the cartridge.

4) Wash the SPE cartridge with 3 mL of DI water, followed by 3 mL of 1 M acetic acid, and then with 3 mL of methanol. Apply full vacuum (> 10 in. Hg) and allow the column to dry for 5 min.

Figure 1: Calibration curves of methadone and EDDP

5) Elute the sample with 3 mL of a freshly prepared methylene chloride:isopropanol:ammonium hydroxide (78:20:2) mixture. The eluate is collected at 1–2 mL/min and then vortex mixed.

6) The extract is evaporated to dryness under nitrogen (< 40 °C). Then reconstituted with 100 µL methanol.


GC-MS: Thermo Scientific Trace 1300 GC–MS

GC column: Thermo Scientific TG1-MS; 30 m × 0.25 mm; 0.25 µm

Injector: 1 µL at 250 °C

Split Ratio: 10:1

Transfer Line: 280 °C

Carrier gas: Helium at a constant flow of 1.2 mL/min

Oven: Initial temperature at 50 °C, hold for 1 min; ramp at 30 °C/min to 320 °C, hold for 3.6 min


This simple and efficient method analyzes both methadone and its primary metabolite (EDDP) in urine samples. Recoveries for both compounds were found to be >90%, the method was linear from 5 ng/mL to 500 ng/mL. The limits of detection and quantification were found to be 1 ng/mL and 5 ng/mL, respectively. The GC–MS run time was less than 15 min.


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