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Chiral Separation on a C18 Column? Separation of d- and l- Amphetamines Part II

7 January 2020
  • Ravali Alagandula
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To continue my blog part 1 (Part 1: , where I have briefly discussed the importance of separating the d and l isomers to accurately identify the illicit isomer using an achiral method on a Raptor C18 column employing a pre-column derivatization technique. Today I’d like to discuss more about the matrix of interest, sample preparation and derivatization, chromatographic and mass spectrometric conditions.

Sample Preparation: 50 µL of calibration standard or QC sample prepared in analyte free pooled human urine (spiked in the range of 50-5000ng/mL) was aliquoted into a micro centrifuge tube. 10 µL 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) prepared in acetone was added, vortexed, and heated at 45 °C for 1 hour in a water bath. Samples were allowed to cool to room temperature before the addition of 40 µL of 1M HCL in water. The d- and –l amphetamines are now converted to DNPA derivatives. The sample was then vortexed and evaporated to dryness under nitrogen at 45 °C. Samples were reconstituted in 1 mL (20x dilution) of 40:60 water: methanol (v/v) and filtered using Thomson SINGLE StEP standard filter vials (cat# 25893) and then injected for LC-MS/MS analysis.

Matrix…. What Matrix? Currently urine is the sample of choice for pain management or toxicology labs for routine drug testing, because of its ease of sample collection and clean up as well. So we bought 9 lots of drug free human urine lots from Bio IVT, the samples were derivatized and analyzed on LC-MS/MS to check for any presence of endogenous Amphetamines and other significant interference peaks. All the urine lots were free from interferences or presence of amphetamines. Pooled urine was prepared from the analyte free 9 urine lots and was used for method development and validation. Surine was evaluated as well and no matrix peaks were evident and amphetamines were well separated.

Urine dilution factor? How do I decide which dilution factor was right for this analysis. Well, I derivatized both 100uL (10x) and 50uL (20x) pooled urine: 20x dilution gave best results with good sensitivity and less matrix effects compared to 10x and increasing dilution rate more than 20x decreased the signal sensitivity.

Sensitivity issues? One of our customer complained about poor signal intensity of the analytes even after derivatization, a chemists nightmare. I blame the derivatizing reagent here, it is very light sensitive and should be prepared fresh in acetone and stored in dark at 40C. However, the derivatized samples were found to be stable for at least 48 hours in the auto sampler rack, no significant fluctuations in signal intensity was noticed.

Can Cleaning the MS source help? Definitely, especially in this particular case because you are just diluting the urine sample. I clean my MS source once a week with water, Methanol and Acetonitrile after running hundreds of urine samples or any other biological matrix like whole blood, oral fluid etc..

Chromatography: Now that our samples are derivatized and ready for the analysis, I’d like to discuss more about the LC conditions. A Raptor C-18 100 x 2.1 mm, 2.7 µm column was utilized for the separation of the DNPA derivatives of Amphetamines and their respective deuterated internal standards in 7 minutes (Figure 1 & 2). Using a simple derivatization and dilution procedure along with a Raptor C18 column— good baseline resolution of the target compounds was obtained, allowing easy peak identification and quantitation.

Fig 1: Separation of d- and l- Amphetamine and Methamphetamines enantiomers in fortified Human Urine at 500ng/mL (TIC)

Fig 2: Separation of d- and l- Amphetamine and Methamphetamines enantiomers in fortified Human Urine at 500ng/mL (XIC)

Instrument Conditions

Analysis of amphetamines by LC-MS/MS was performed on a Shimadzu Prominence HPLC equipped with a SCIEX API 4000 MS/MS. Instrument conditions were as follows and analyte transitions are provided in Table 1.

Analytical column: Raptor C18 2.7 µm, 100 mm x 2.1 mm (cat# 9304A12)
Guard column: Raptor C18 EXP guard column cartridge (cat# 9304A0252)
Mobile phase A: 0.1% Formic acid in water
Mobile phase B: 0.1% Formic acid in methanol
Flow rate:    0.5 mL/min
Injection volume: 10 µL
Column temp.:  35 °C

Ion mode: Negative ESI

Table 1: Analyte Transitions for the Analysis of Amphetamines by LC-MS/MS.

Hopefully this workflow and some of the tips discussed above on both sample prep and chromatography and can help get you some good baseline separation and resolution of these enantiomers to identify the illicit isomer.

In my next blog I will share some interesting data that mimics the real urine sample concentrations containing very high and low concentrations of the legal and illegal isomers present in urine and optimization of derivatizing procedure. Stay tuned till next one….


  1. Newmeyer, N. M, Concheiro, M and Huestis, A. M. J Chromatogr A. 2014; 1358: 68–74.
  2. Foster, S. B and Gilbert, D. D. J Analytical Toxicology.1998; 22:265-9.

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