Results and Discussion
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).
Figure 4: Chemical structures of the test compounds.
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
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 µg and 2 µg 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.
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 µL injections).
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 µmol/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).