Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC - - Chromatography Online
Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC

LCGC Europe
Volume 25, Issue 11, pp. 600-611

Results and Discussion

Figure 4: Chemical structures of the test compounds.
Determination of D- and L-Lactic Acid: Lactic acid is an important metabolite produced in several biochemical processes such as anaerobic respiration or fermentation. In mammalian organisms there are two naturally occurring stereoisomers: the L(+)-lactic acid and its counterpart, D(–)-lactic acid, which is present in healthy individuals at around 1% (11). Detection of higher levels of the D enantiomer are indicators of bacterial activity in the intestinal tract or metabolic acidosis (patients with short bowel syndrome, severe gastroenteritis or consequences of jejunoileal surgery).

The determination of D- and L-lactic acid in food and clinical samples is therefore of great importance. Currently determination is performed by enzymatic techniques that are time-consuming and are of relatively low quantitative accuracy (12); GC after derivatization (13); and HPLC. The most widely used LC method is based on chiral ligand exchange chromatography with a copper containing mobile phase on Chiralpak MA (14). Its use is therefore limited to UV detection and so mass spectrometry (MS) detection cannot be used.

In this frame, QN AX and QD AX columns allow the enantioselective determination of lactic acid enantiomers using MS-compatible mobile phase systems and offer the possibility of reversing the elution order by switching from one column to the other [Figure 2(b)]. This latter option is not possible with the previously described applications of antibiotic-derived columns, such as Chirobiotic TAG (15).

The mobile phase was composed of an acetonitrile and methanol mixture containing 30 mM formic acid as the additive, and the pH of the mixture was adjusted to pH 4 by the addition of ammonia. This mobile phase provided separations as illustrated in Figure 2. The retention can be adjusted by the additive concentration yielding faster separations when the concentration is increased and longer retention at lower additive concentrations, while enantioselectivities remain nearly constant with such changes.

Table 1: Performance data for ultra-violet (UV) (230 nm) and electrospray ionization mass spectrometry selected reaction monitoring (ESI-MS SRM) detection using a QN AX column (5 L injections).
A preliminary method validation using HPLC UV at 230 nm revealed linearity over two orders of magnitude and allowed LOD (limit of detection) and LOQs (limit of quantifications) below 1 g and 2 g respectively, on column (Table 1). For many applications such as in clinical analysis, however, UV detection is not sensitive and selective enough. Owing to its volatile mobile phase constituents, the method is fully compatible with MS detection. Preliminary testing on hyphenation of the above developed enantioselective chromatography with tandem MS (QTrap 4000) showed that this method with QN AX and QD AX respectively is sensitive and selective enough for clinical applications.

Since lactic acid is a very small molecule, no intensive characteristic fragmentations, except for the transition m/z 89 > 43, could be found. Unfortunately, this transition did not yield an intensive signal. Thus, pseudo-molecular SRM with ion transition of m/z 89 > 89 (in negative ion mode) was selected for quantification, as it is more sensitive than the ion transition which was initially utilized as a qualifier. With these detection parameters a reasonably sensitive method could be defined. The preliminary calibration data and information on sensitivity are summarized in Table 1. It can be seen that the limits of quantitation for MS–MS detection are by a factor of about 100 lower than for UV detection and ranged in the low nanogram level (ca. 10 ng) on-column. The signal was linear over about 20–500 mol/L. Thus, the method is adequate and can be useful for clinical applications such as diagnosis of diabetic ketoacidosis (16) and other clinical applications in where Dlactic acid may serve as a diagnostic marker.

In addition other α-hydroxy carboxylic acids can be separated (for example, 2-hydroxy butyric acid or glyceric acid), thus, the follow-up analysis of a metabolic change or an enzymatic reaction could be aided by this type of column. This was demonstrated in the recently published article about the determination of D- and L-glyceric acid on a QN AX column (17).


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