l
Allow all the tubes to cool at RT for 10 min and then
read each tube UV ABS at 335 nm, using as blank for
the spectrophotometer the 0 nmol/mL standard.
(d) Calculate a linear regression between concentration and
ABS for the standard and calculate on it the sample
amino groups concentration.
8. Free ADH quantification by RP-HPLC [21].
(a) ADH Calibration curve.
l
Prepare in labeled Eppendorf tubes the dilutions of
ADH 0.4 μg/mL standard solution as indicated in
Table 6.
(b) Sample/standard derivatization.
l
The derivatization procedure is applied to calibration
curve solutions and to samples.
l
Per each standard/sample, transfer two 100 μL aliquots in different Eppendorf tubes, labeling them
with their content name.
l
In a 50 mL Falcon tube, add in the following order and
stir after each addition:
– 4.840 μL of PBA solution 2.5 mM.
– 1.210 μL of pyridine 20% solution.
– 1.210 μL of EDC 2 M solution.
This solution must be prepared just before use.
l
To each 100 μL of solution to be derivatized, add
300 μL of the solution prepared.
l
Close the Eppendorf tube and incubate at 40
C for
60 min.
l
Dry them overnight (at least for 24 h) on centrifugal
evaporator at 60
C in order to remove DMSO and
Pyridine.
l
Redissolve each tube content in 400 μL of ACN 65%
solution (the dissolution time is about half an hour)
and vortex.
l
Centrifuge all Eppendorf tubes for 30 min at
12,000 RCF in order to remove insoluble salts.
l
Filter the upper liquid through a 0.2 μm nylon filter
into different HPLC vials labeling them with content
sample name.
(c) System setting:
l
Column System: HPLC in line filter—C18 column
connected in series.
294
Francesca Micoli et al.
Allow all the tubes to cool at RT for 10 min and then
read each tube UV ABS at 335 nm, using as blank for
the spectrophotometer the 0 nmol/mL standard.
(d) Calculate a linear regression between concentration and
ABS for the standard and calculate on it the sample
amino groups concentration.
8. Free ADH quantification by RP-HPLC [21].
(a) ADH Calibration curve.
l
Prepare in labeled Eppendorf tubes the dilutions of
ADH 0.4 μg/mL standard solution as indicated in
Table 6.
(b) Sample/standard derivatization.
l
The derivatization procedure is applied to calibration
curve solutions and to samples.
l
Per each standard/sample, transfer two 100 μL aliquots in different Eppendorf tubes, labeling them
with their content name.
l
In a 50 mL Falcon tube, add in the following order and
stir after each addition:
– 4.840 μL of PBA solution 2.5 mM.
– 1.210 μL of pyridine 20% solution.
– 1.210 μL of EDC 2 M solution.
This solution must be prepared just before use.
l
To each 100 μL of solution to be derivatized, add
300 μL of the solution prepared.
l
Close the Eppendorf tube and incubate at 40
C for
60 min.
l
Dry them overnight (at least for 24 h) on centrifugal
evaporator at 60
C in order to remove DMSO and
Pyridine.
l
Redissolve each tube content in 400 μL of ACN 65%
solution (the dissolution time is about half an hour)
and vortex.
l
Centrifuge all Eppendorf tubes for 30 min at
12,000 RCF in order to remove insoluble salts.
l
Filter the upper liquid through a 0.2 μm nylon filter
into different HPLC vials labeling them with content
sample name.
(c) System setting:
l
Column System: HPLC in line filter—C18 column
connected in series.
294
Francesca Micoli et al.