Rapid Analysis of Amphetamines in Biological Samples

June 1, 2011
Michael Rummel

Jeff Layne

Matthew Trass

Phenomenex, Inc.

The Application Notebook

The Application Notebook, The Application Notebook-06-01-2011, Volume 0, Issue 0

This study demonstrates a faster method of analysis when coupling a simplified and effective SPE procedure with a core-shell HPLC column and LC–MS-MS detection.

This study demonstrates a faster method of analysis when coupling a simplified and effective SPE procedure with a core-shell HPLC column and LC–MS-MS detection.

Recently, the Substance Abuse and Mental Health Services Administration (SAMHSA) has lowered the amphetamine drug class cutoff level and has added MDMA, MDA, and MDEA to the panel. As a result, there will undoubtedly be an increase in positive result conformational testing, placing additional stress on the toxicology laboratory. We demonstrate how a specialized SPE sorbent and method, together with a high efficiency core-shell HPLC column and LC–MS-MS can decrease analysis time and increase sample throughput.

Experimental Conditions

Urine samples were pretreated and cleaned up using Strata™-X-Drug B solid phase extraction tubes. (It is important to note that neither a conditioning or equilibration step is required using this specialized SPE sorbent and method.) After diluting samples by a factor of 20 to bring the concentration into a suitable range for analysis, samples were injected onto the LC–MS-MS.

Analyses were performed using an HP 1100 LC system (Agilent Technologies, Palo Alto, California) with an upper pressure limit of 400 bar, equipped with an API 3000™ LC–MS-MS detector.

All analytes were present at a concentration of 10 ng/mL each and are listed in order of elution.

Column: Kinetex® 2.6 μm XB-C18 100 Å

Dimensions: 50 x 2.1 mm

Mobile Phase:

A: 5 mM Ammonium formate with 0.1 % Formic acid

Flow Rate: 0.4 mL/min

Detection: API 3000™ MS/MS, ESI negative (ESI-)

Sample: 1. D11-Amphetamine; 2. Amphetamine; 3. D14-Methamphetamine; 4. Methamphetamine; 5. D5-MDA; 6. MDA; 7. D5-MDMA; 8. MDMA; 9. D5-MDEA; 10. MDEA

Request a copy of Technical Note TN-1096 for complete method details.


The Strata-X-Drug B SPE procedure consists of only 5 steps, a load, two washes, dry, and an elution step, compared to the 9 step procedure called for by the traditional SPE method. The new Strata-X-Drug B resulted in a 7 min time savings and 11 mL solvent savings.

Figure 1 demonstrates the ability of the Kinetex 2.6 μm XB-C18 columns to rapidly screen amphetamines while providing efficient conformational results. Complete analysis is completed in less than 3 min with an average peak width of 0.147.

Figure 1: Rapid analysis of amphetamines by LC–MS-MS using a Kinetex XB-C18 2.6 μm, 50 × 2.1 mm.

LC analysis was completed at operating pressures less than 400 bar and may therefore be used on any HPLC system without the need for specialized ultra-high pressure equipment.


A rapid cleanup and analysis for amphetamines was developed using Strata-X-Drug B SPE and a Kinetex XB-C18 HPLC column which can dramatically improve the efficiency of toxicology laboratories while simultaneously reducing cost due to solvent consumption. These time and solvent savings can be multiplied to provide substantial savings when running multiple samples in a high capacity laboratory.

Phenomenex, Inc.

411Madrid Avenue, Torrance, CA 90501

tel. (310) 212-0555, fax (310) 328-7768

Website: www.phenomenex.com