Navigation Links
Rapid Screening of Amphetamine Drugs in Urine by Positive Ion Electrospray LC/MS/MS

Z. Yang and S. Sadjadi, Varian, Inc.


Amphetamine drugs are often abused and misused in sports, in the work place, and by recreational users. Forensic, clinical, and doping laboratories are frequently asked to analyze for the presence of amphetamines in urine. Urine samples are most common because large sample volumes can be collected non-invasively. These drugs generally remain detectable in urine for two to three days longer than in blood. For most clinical and forensic applications, initial screening is done by immunoassay with presumptive positive samples confirmed by a second, more specific method such as gas chromatography/ mass spectrometry (GC/MS).

A simple and sensitive LC/MS/MS method is described below for high throughput identification and quantitation amphetamine drugs in urine. A rapid and effective solid-phase extraction (SPE) procedure using FocusTM was used to extract amphetamines from urine samples.


Varian ProStar 410 AutoSampler

Varian ProStar 210 Isocratic Solvent Delivery Modules

Varian 1200L LC/MS equipped with ESI source

Materials and Reagents

Standard solutions: 1.0 mg/mL (()-Amphetamine, ()-Methamphetamine, 1S,2R(+)-Ephedrine, ()-MDMA, ()-MDA and ()-MDEA), from Cerilliant Corp., Texas, USA.

Internal standard (IS) solutions: 0.1 mg/mL (()-Amphetamine-D5, ()-Methamphetamine-D5, 1S,2R(+)-Ephedrine-D3 HCl, ()-MDA-D5, ()-MDMA-D5 and ()-MDEA-D5), from Cerilliant Corp., Texas, USA.

All other chemicals are reagent grade or HPLC grade.

FocusTM Solid Phase Extraction Cartridges (Varian Part No. A5306021).

In-house vacuum or vacuum pump (Varian Part No. WL2012B01).

Vac Elut 20 Manifold with the standard Glass Basin (Varian Part No. 122345 05) and Collection Rack for 13 x 75 mm test tubes (Varian Part No. 12234507).

Sample Preparation

A 100 μL aliquot of a 500 ng/mL deuterated internal standards solution was transferred into individually labeled tubes (double blank tube was urine only). To each tube, a 1 mL of urine sample followed by 0.1 mL of 0.1 N KOH solution was added and mixed by vortex.

The mixture was loaded onto the sorbent bed of an activated 3 mL Focus cartridge pretreated with 1 mL of methanol followed by a 1 mL deionized water wash under gentle vacuum of1 to 2 in. Hg. Next, the sorbent bed was washed with 2 x 1 mL acetonitrile/water (10:90, v/v) under gentle vacuum.

The analyte was collected in a 2 mL autosampler vial by eluting with 2 x 100 μL elution solvent (acetonitrile/methanol/ water/formic acid (22:68:9:1, v/v) under gentle vacuum. The sorbent bed was then flushed with 600 μL of water under vacuum to wash off the elution solvent and dilute the sample for injection. A 10 μL aliquot was injected directly for analysis.

Results and Discussion

The LC method used a six-minute run cycle time with the first peak at 1.65 minutes and the last peak at 4.12 minutes (Figure 1). The two product ions for each analyte can be quantitatively analyzed at the level of 5 ng/mL in urine (Figure 2, about 50 pg on-column). This level is 50 times below the proposed drug cutoff levels published by the Substance Abuse and Mental Health Services Administration (SAMHSA).1 Eight concentration levels were used to generate the calibration curves for the standard. The linearity of the detector response and the response factor-Relative Standard Deviation (rf-RSD) are excellent (Table 1, Figure 3).

The recovery of the drugs from urine was > 85%. The eluent from the Focus cartridge can be injected dir ectly into LC/MS system without derivatization, evaporation, and reconstitute steps. The 96-well format Focus can be used for automation and high-throughput screening.

Only two product ions were used for this analysis because amphetamine and methamphetamine only give two intense product ions (Figure 4, Table 2) while ephedrine, MDA, MDMA, and MDEA produce multiple intense product ions (Table 2). Run-to-run retention time is very reproducible with a <1.4% RSD. Two product ions with a retention time match can be strong evidence for positive identification of amphetamine drugs (Figure 2). Both the urine double blank and the blank with IS show no interference of the analysis at low quantitation level (LQL). For the standard calibration curve, the LQL is 5 ng/mL and upper quantitation level (UQL) is 1000 ng/mL. This LC/MS/MS method is very sensitive and can be possibly adapted to other body fluid analysis for amphetamines, such as sweat and oral fluid which have confirmatory drug cutoff levels of 25 ng/mL and 50 ng/mL, respectively.


The LC/MS/MS method described in this application note is simple and sensitive. This method can quantitatively analyze amphetamine drugs at 50 times below the drug cutoff levels in urine. The Varian SPE and 1200L LC/MS/MS system demonstrated excellent performance for the urinalysis of amphetamines. The system can be a useful tool for forensics, clinical, and doping laboratories.


1. GuidelinesDraft4.htm



Page: All 1 2 3

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
3. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
4. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
5. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
6. Simple and Rapid Isolation of Plant RNA Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit
7. Rapid PCR* Using Active Tube Temperature Control On Thermos PCR Machines
8. Molecular Biology:Easy Dephosphorylation and Rapid DNA Ligation
9. Rapid detection of genomic duplications and deletions using the Agilent 2100 bioanalyzer
10. Rapid hit confirmation and early hERG liability testing using the IonWorks HT system
11. Rapidly Prepare Probes and Markers for Small RNA Analysis
Post Your Comments:

(Date:11/27/2015)... 27, 2015 ... of companion diagnostics is one of the ... with pharmaceutical companies and diagnostic manufacturers working ... . --> ... on global cancer biomarkers market spread across ...
(Date:11/25/2015)... -- 2 nouvelles études permettent d , ... entre les souches bactériennes retrouvées dans la plaque ... . Ces recherches  ouvrent une nouvelle voie ... efficace de l,un des problèmes de santé les ...    --> 2 nouvelles études permettent d ...
(Date:11/25/2015)... ... November 25, 2015 , ... ... of Black Aerospace Professionals (OPBAP) has been formalized with the signing of a ... leaders met with OPBAP leaders Capt. Karl Minter and Capt. Albert Glenn Tuesday, ...
(Date:11/24/2015)... ... , ... The United States Golf Association (USGA) today announced Dr. Bruce Clarke, ... annually since 1961, the USGA Green Section Award recognizes an individual’s distinguished service to ... Clarke, of Iselin, N.J., is an extension specialist of turfgrass pathology in the department ...
Breaking Biology Technology:
(Date:11/9/2015)... 9, 2015  Synaptics Inc. (NASDAQ: SYNA ), ... broader entry into the automotive market with a comprehensive ... pace of consumer electronics human interface innovation. Synaptics, industry-leading ... for the automotive industry and will be implemented in ... Europe , Japan , and ...
(Date:11/2/2015)... MENLO PARK, Calif. , Nov. 2, 2015 /PRNewswire/ ... to $9 million to provide preclinical development services to ... Under the contract, SRI will provide scientific expertise, modern ... a wide variety of preclinical pharmacology and toxicology studies ... --> --> The PREVENT Cancer Drug ...
(Date:10/29/2015)... Calif. , Oct. 29, 2015  The J. ... new report titled, "DNA Synthesis and Biosecurity: Lessons Learned ... the Department of Health and Human Services guidance for ... in 2010. --> ... it also has the potential to pose unique biosecurity ...
Breaking Biology News(10 mins):