The HPLC
system consisted of a Dionex (Germering, Germany) P 580
gradient pump, a Dionex Ultimate WPS-3000TSL analytical
autosampler with in-line split-loop injection and thermostat
and a Dionex RF 2000 fluorescence detector set at 445 nm
emission and 330 nm excitation wavelength with medium
sensitivity.
An autosampler designed for automated pre-column
derivatisation was used because of the need of a high
injection precision and an effective external needle wash
for eliminating carryover. Another autosampler tested
(Dionex ASI 100) yielded poor reproducibility. Vials
with OPA reagent and samples as well as vials for
preparation were stored in the autosampler at 15°C. For
derivatisation, 50 μl OPA reagent and 30 μl sample were
mixed in a preparation vial, and after 120 s reaction time,
15 μl of the indole derivates were injected.
The mobile phase consisted of two eluents. Eluent A was
a 97.8/0.7/1.5 (v/v/v) mixture of a water phase, methanol
and tetrahydrofuran (THF). The water phase contained
52 mmol sodium citrate and 4 mmol sodium acetate,
adjusted to pH 5.3 with HCl. Then, methanol and THF
were added. Eluent B consisted of 50% water and 50%
methanol (v/v). For degassing and sterilisation, both eluents
were filtered over 0.2-μm-pore size hydrophilic propylene
membrane filters. The mobile phase was delivered at a flow
rate of 1.5 ml min−1. The amino sugar separation was
performed isocratically, but a gradient was used for
cleaning the column after every run. Every run was starting
at an eluent A/B v/v composition of 93/7 for 19 min. A
linear gradient was run to reach 80% B after 3 min and
remaining isocratic for 3 min. A reverse gradient to 93/7
within 3 min was followed by 2 min isocratic run after
which the column is preconditioned for the next sample. It
is important to start the OPA derivatisation only at the
beginning of every run and not during column preconditioning
of the previous run. Otherwise, the equilibration
time is not long enough for a good reproducibility.