Analysis of d- and l-Amphetamine and Methamphetamine Enantiomers for High-Throughput Labs

• Separate d- and l- enantiomers without chiral columns and dedicated instruments.
• Simple precolumn derivatization and dilution sample preparation.
• Accurately distinguish and quantify licit vs. illicit methamphetamine.

Amphetamine and methamphetamine are psychostimulant drugs and occur as two enantiomers, dextrorotary and levorotary, as a result of their chiral center. The dextro-methamphetamine (d-isomer) form is highly abused and is typically found in illicit preparations. However, detection of abuse is complicated because consumption of over-the-counter and prescription medications may yield positive results if the analytical method used cannot distinguish between the enantiomers. Chiral separation of d- and l-methamphetamine and their metabolites d- and l-amphetamine can help determine whether the source was licit or illicit, but chiral columns can be expensive, may necessitate a dedicated instrument, and are not as broadly useful as ubiquitous C18 columns.

In order to provide labs with a high-throughput assay that effectively separates d- and l-amphetamine and methamphetamine enantiomers in urine without the use of a costly and specialized chiral column, we developed the LC-MS/MS method shown here on a standard reversed-phase Raptor C18 column. Our method employs a simple precolumn derivatization followed by dilution and results in a selective, specific analysis of d- and l-amphetamine and methamphetamine enantiomers that is free from sample matrix interferences. Separation was achieved within a total run time of seven minutes, and quantitation in urine was performed across a linear range of 50-5000 ng/mL. Validation across this range demonstrated that this method provides reliable analysis of d- and l-amphetamine and methamphetamine enantiomers in a workflow and time frame suitable for high-throughput clinical and forensic toxicology labs.

	Peaks	tR (min)	Precursor Ion	Product Ion 1	Product Ion 2
1.	l-Methamphetamine*	3.11	400.3	339.0	323.8
2.	d-Methamphetamine*	3.38	400.3	339.0	323.8
3.	l-Amphetamine*	4.16	386.1	325.0	308.0
4.	d-Amphetamine*	4.55	386.1	325.0	308.0

*Analytes are DNPA derivatives.

Column	Raptor C18 (cat.# 9304A12)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Guard Column:	Raptor C18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9304A0252)
Temp.:	35 °C
Standard/Sample
Conc.:	500 ng/mL in urine
Inj. Vol.:	10 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.5 40 60 5.00 0.5 40 60 5.01 0.5 10 90 5.50 0.5 10 90 5.51 0.5 40 60 7.00 0.5 40 60

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	HPLC
Sample Preparation	A 500 ng/mL standard (d- and l-amphetamines and methamphetamines) was prepared in pooled urine. Fifty microliters of the standard was aliquoted into a microcentrifuge tube. Ten microliters of a working internal standard (20 µg/mL (±)-amphetamine-D11 and (±)-methamphetamine-D11 in water) and 20 µL of 1M NaHCO3 was added and vortexed at 3000 rpm for 10 seconds. After vortexing, 100 µL of 0.1% (w/v) Marfey's reagent (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide) in acetone was added, vortexed, and heated at 45 °C for 1 hour. Samples were allowed to cool to room temperature before the addition of 40 µL of 1M HCl in water. The sample was then vortexed and evaporated to dryness under nitrogen at 45 °C. Samples were reconstituted in 1 mL of 40:60 water:methanol (v/v) and filtered using Thomson SINGLE StEP standard filter vials cat.# 25893 prior to analysis.
Notes	Thomson SINGLE StEP standard filter vials cat.# 25893 were used to produce this chromatogram, but have since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative.

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