Rapid Enantiomeric Excess Determination of D- and L-Proline Using Electrospray Ionization–Mass Spectrometry

The enantiomeric composition of proline is commonly determined using conventional analytical methods such as HPLC, GC, and CE. Due to the relatively long analysis times of these methods, high-throughput requirements often cannot be fulfilled. A method based on parallel kinetic resolution and electrospray ionization–mass spectrometry (ESI-MS) with pseudo-enantiomeric mass-tagged auxiliaries has been developed that enables enantiomeric excess determination of proline in approx. 2 min per sample. The electrospray ionization–mass spectrometry method is fully automated, including sample pretreatment using liquid handlers and software-based data processing.

Determination of proline

Chiral amino acids have great impact on many physiological processes in plants, animals, and humans. One of the most important amino acids is proline, since it is incorporated in many biochemical pathways and exhibits neurotoxic effects. In the 1970s, studies were performed that showed that L-proline caused amnesia in chicks.1,2 Studies also showed that D-proline led to convulsions and death in chicks, although no significant amnesiac effect was seen.3 More recent research based on experiments with chicks showed that L-proline influences the stress-induced dopamine and serotonin metabolism,4 and that both enantiomers can induce sedative and hypnotic effects.5 These applications in the field of neurology research show the importance of the determination not only of the total proline concentration, but also of the differentiation between the D- and L-enantiomers and determination of enantiomeric excess. A number of studies investigated the determination of D-amino acids in mammals using HPLC,6–10 GC, CE, and other analytical techniques.11,12 Most of these techniques require long analysis times as well as cost-intensive columns and materials. In the face of the increasing cost of today’s health-care systems, the development of cost-effective, rapid analytical methods is required to ensure not only cost reduction but also an increase in the number of patients who can be screened.

Determination of D-/L-proline using parallel kinetic resolution

Figure 1 - Reaction equation for derivatization of D- and L-proline.

Mass spectrometry is a fast, reliable method for the analysis of complex mixtures. The parallel kinetic resolution method enables differentiation between enantiomers using slight differences in their reaction behavior with other chiral compounds, and has been used for the analysis of secondary alcohols, primary and secondary amines,13,14 carboxylic acids, amino alcohols, amino acid esters, and natural compounds.15,16 The chiral mixture of two enantiomers in a variable ratio is derivatized with two mass-tagged pseudoenantiomeric auxiliaries. A suitable mass difference of the auxiliaries causes four reaction products with two characteristic masses. The ratio of these masses is related to the enantiomeric ratio of the chiral substrates and is used for enantiomeric excess calculation. A classic derivatization agent used in amino acid analysis is Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA): Marfey’s reagent.17,18 As a corresponding auxiliary with a mass difference of 14 and opposed chiral constitution, Nα-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (D-FDLA) can be used, whereby reaction products are formed with characteristic masses of m/z = 395.14 and m/z = 409.16 (Figure 1).


Chemicals and reagents

D- and L-proline (both ≥99%), acetone (≥99%), and formic acid (~98%) were obtained from Sigma Aldrich (Steinheim, Germany). Nα-(2,4-dinitro-5-fluorophenyl)-D-valinamide (D-FDVA) and Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) (both ≥98%) were obtained from Fluka (Buchs, Switzerland). Nα-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (D-FDLA) and Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) (both 98%) were obtained from ABCR (Karlsruhe, Germany). Methanol (HPLC gradient grade) and hydrochloric acid (37%) were from Roth (Karlsruhe, Germany), and sodium bicarbonate (≥99.5%) was from AppliChem (Darmstadt, Germany).

Sample preparation

A stock solution of each enantiomer (50 mmol/L) was prepared by dissolving the crystalline proline in 3 mL hydrochloric acid (1N), neutralizing with 1N sodium bicarbonate, and filling up to 10 mL with ultrapure water. For experiments conducted within the following few days, 2 mL was filled in separate vials and stored at +2 °C (35.6 °F). The remaining solutions were frozen at –18 °C (–0.4 °F). Prior to the derivatization, the stock solution was diluted (1:50, v/v) to a concentration of 1 mmol/L with ultrapure water. The auxiliary solution contained L-FDVA and D-FDLA dissolved in acetone, each with a concentration of 2.5 mmol/L. For the calibration, the diluted proline solutions were used to prepare five mixtures with defined enantiomeric excesses of +100, +50, 0, –50, and –100 ee%. The derivatization was performed in 1-mL GC vials with screw caps (Agilent Technologies, Waldbronn, Germany) as well as in 96-well master blocks with a well volume of 500 μL (Greiner-BioOne, Essen, Germany). Fifty microliters of the chiral substrates, 100 μL of the auxiliary solution, and 20 μL of sodium bicarbonate (1 M) were added and mixed in a thermo shaker (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 750 rpm for 1 hr at 20 °C. Subsequently, 10 μL hydrochloric acid (2 M) was added and the samples were mixed for 5 min more. Finally, 320 μL of methanol was added and the samples were mixed for a few seconds.

Instrumentation and analysis parameters

The mass spectrometric analyses were carried out on an Agilent LC-MS system with the following units: G1379B  vacuum degasser, G1312B  binary pump, G1367C high-performance automated liquid sampler, and G1969A time-of-flight mass spectrometer (TOF-MS) with an electrospray ion source (electrospray ionization, ESI). Five microliters of the sample solution was injected with prior needle wash. A methanol–water mixture (90:10, v/v) containing 0.1% formic acid was used as mobile phase for the sample injection at a flow of 0.35 mL/min. The TOF-MS was operated in negative ion mode with the following parameters: nitrogen as nebulizer and drying gas, 35 psig nebulizer pressure, 10 L/min drying gas flow, 300 °C drying gas temperature, 4000 V capillary voltage, 215 V fragmentor voltage, 60 V skimmer voltage, and 250 V octupole voltage. Data acquisition, extraction of the peak areas of the required masses, and integration of these peak areas were performed using MassHunter Data Acquisition and MassHunter Qualification software (Agilent Technologies). Enantiomeric excess calculation and visualization of the results were realized with the software module “Chiral MS” self-implemented for these special tasks. This module was implemented using Microsoft Office Excel 2007 with Visual Basic 6.5 (Microsoft Corp., Redmond, WA).

Results and discussion

Figure 2 - Mass spectrum of the derivatives of a proline racemate.

The ratio of the characteristic m/z values of the derivatives was used for subsequent enantiomeric excess determination. The method was tested under various conditions during the sample preparation. Furthermore, a number of validation procedures were performed, and finally compared with a conventional analysis technique. Figure 2 shows a mass spectrum with the characteristic masses m/z = 394.14 and 408.16 of the detected [M–H] ions for a proline racemate.