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

24 April 2020
  • Ravali Alagandula
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To continue my blog parts 1 & 2 (Part 1:, Part 2: , where I have discussed the importance of separating the chiral 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 sample derivatization procedure and optimization of the derivatization conditions, which is the main step for separation of the chiral compounds on a reverse phase C18 column.

When it comes to sample derivatization, we think about how can we get the best derivatization conditions for our workflow. As a chemist, we want a procedure that’s simple, fast and reproducible with maximum derivatization efficiency and easy to replicate in other labs.

In the current published methods, samples like urine and oral fluid employ 2 steps sample preparation: first the biological samples are cleaned up using solid phase extraction, followed by derivatizing the sample to separate the chiral amphetamines and methamphetamines to detect the presence of illicit isomer.

Novelty of this application: So, how is this work different from the published or existing  procedures for the separation of chiral compounds on a reverse phase column? Here, we employ one step sample prep procedure: that is direct derivatization of urine sample without any cleanup, followed by evaporation and reconstitution with the starting mobile phase of the LC-MS/MS gradient conditions, where the reconstitution serves as dilution of the urine sample, to minimize the matrix effects. Sample centrifugation followed by filtration using filter vials also helps in mitigating the matrix effects.

Marfey’s Reagent: FDAA is 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide, also called Marfey's reagent. This reagent reacts with primary amines of the enantiomers or chiral compounds and convert them into diastereomers and are detected using UV and MS/MS in liquid chromatography methods. FDAA derivatives of D-isomers exhibit strong intramolecular bonding, which reduces their polarity relative to the corresponding L-isomer derivatives. Consequently, the D-derivatives are selectively retained on reverse phase columns and elute much later than corresponding L-derivatives.

Sample Derivatization Procedure: 50 μL of calibration standard or QC urine sample was aliquoted into a microcentrifuge 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. 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) and then injected.

Optimization of Derivatization Procedure: In order to obtain the best sensitivity and to achieve maximum derivatization, a series of experiments were performed using a 4000 ng/mL d- and l-amphetamines and d- and l-methamphetamines sample prepared in water. To determine the optimal derivatization conditions, various incubation times and derivatizing reagent volumes were assessed. For the length of the incubation time experiment, samples were incubated at 45 °C with 100 µL 0.1% (w/v) Marfey's reagent for 15, 30, 45, 60, or 90 minutes. For the volume experiment, varying volumes of 0.1% w/v Marfey's reagent (25, 50, 100, or 200 µL) were added to the samples, followed by incubation at 45 °C for 60 minutes. All the samples were subjected to the sample preparation procedure previously described and all conditions were evaluated in quadruplicate.

As shown in Figure 1, the highest peak responses were obtained using 100 µL of 0.1% (w/v) Marfey’s reagent with a 60-minutes incubation time at 45 °C. Longer incubation times showed decreased peak area responses for both amphetamines and methamphetamines diastereomers. Therefore, 60-minutes incubation time was selected for this method.

Figure 1

Figure 2. depicts that increased concentrations of the derivatizing reagent negatively affects the analytes sensitivity, 100 µL of 0.1% (w/v) Marfey's reagent incubated at 45°C for 60 minutes resulted in the highest signal response compared to both higher and lower levels of the Marfeys reagent for all the analytes.

Figure 2.

Incubation: Water Bath or a hot plate? I would recommend using a water bath, as the temperature distribution was found to be even to the sample tubes from the water compared to an oven or a hot plate.

Concentration of derivatizing reagent: Do not to use the derivatizing reagent in excess concentrations, excess derivatizing reagent in the samples can negatively affect the analyte signal sensitivity and mass spectrometer ion source.

Always prepare the reagent fresh in Acetone and store in a dark place. Based on the above discussed results, these optimized derivatization conditions for both derivatizing reagent volumes and incubation times were used for all subsequent experiments and method validation in urine.

Click on the link for more details on the full application note: Analysis of Amphetamines by LC-MS/MS for High-Throughput Urine Drug Testing Labs

Stay tuned for the part 4 of this blog series....


  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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