6. KDO quantification by semicarbazide/HPLC-SEC method.
OAg samples are analyzed by HPLC-SEC after derivatization with semicarbazide to quantify α-ketoacid present at the
reducing end. This reaction is performed as a slight modification of the semicarbazide assay for α-ketoacid
determination [20].
(a) Calibration curve standard solution.
l
Using water, prepare in labeled tubes, in duplicate, the
dilutions of KDO ammonium salt 40 μg/mL standard
solution as indicated in Table 4.
l
Vortex all the standard dilution tubes for few seconds.
(b) Sample preparation.
l
Each sample is analyzed in duplicate.
l
Label two 1.5 mL Eppendorf tubes with sample name
and two 1.5 mL Eppendorf tubes with sample
name + “blank.”
l
Gently mix the sample in order to homogenize the
content. Dilute the sample with water to an appropriate concentration (to fit the calibration curve) and with
a pipette transfer 100 μL of the diluted sample in each
labeled Eppendorf tube.
(c) Sample/standard derivatization.
l
With the automatic pipette add 100 μL of semicarbazide solution to each sample/standard tube. Close the
tubes. Vortex all tubes for few seconds.
l
With the automatic pipette add 100 μL of water to each
blank sample tube. Close the tubes. Vortex all tubes for
a few seconds.
l
Keep the tubes in a 50
C preheated bath for 50 min.
l
Chill the tubes in a 2–8
C fridge for 15 min.
l
Vortex all the tubes for few seconds.
l
Transfer samples, blank samples, and standards in prelabeled HPLC vials.
l
Place the vials in the HPLC autosampler prechilled at
4
C.
(d) Analysis setting.
l
Column System: PW-XL guard—G3000 PWXL columns connected in series.
l
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
UV detector acquisition channel: 252 nm ABS.
292
Francesca Micoli et al.
OAg samples are analyzed by HPLC-SEC after derivatization with semicarbazide to quantify α-ketoacid present at the
reducing end. This reaction is performed as a slight modification of the semicarbazide assay for α-ketoacid
determination [20].
(a) Calibration curve standard solution.
l
Using water, prepare in labeled tubes, in duplicate, the
dilutions of KDO ammonium salt 40 μg/mL standard
solution as indicated in Table 4.
l
Vortex all the standard dilution tubes for few seconds.
(b) Sample preparation.
l
Each sample is analyzed in duplicate.
l
Label two 1.5 mL Eppendorf tubes with sample name
and two 1.5 mL Eppendorf tubes with sample
name + “blank.”
l
Gently mix the sample in order to homogenize the
content. Dilute the sample with water to an appropriate concentration (to fit the calibration curve) and with
a pipette transfer 100 μL of the diluted sample in each
labeled Eppendorf tube.
(c) Sample/standard derivatization.
l
With the automatic pipette add 100 μL of semicarbazide solution to each sample/standard tube. Close the
tubes. Vortex all tubes for few seconds.
l
With the automatic pipette add 100 μL of water to each
blank sample tube. Close the tubes. Vortex all tubes for
a few seconds.
l
Keep the tubes in a 50
C preheated bath for 50 min.
l
Chill the tubes in a 2–8
C fridge for 15 min.
l
Vortex all the tubes for few seconds.
l
Transfer samples, blank samples, and standards in prelabeled HPLC vials.
l
Place the vials in the HPLC autosampler prechilled at
4
C.
(d) Analysis setting.
l
Column System: PW-XL guard—G3000 PWXL columns connected in series.
l
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
UV detector acquisition channel: 252 nm ABS.
292
Francesca Micoli et al.