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Single Laboratory Validation Report for Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals
Jaudzems, Lahrichi, and Fuerer
–
January 2017
page 7/20
3.5.6 Derivatization (of samples, cystine standards, and amino acids standards)
Derivatization converts free amino acids into highly stable derivatives. Standards and
samples are derivatized following
the manufacturer’s
instruction as described below:
a. Preheat a heating block to 55 °C.
b. With a micropipette, add 70
μL of AccQ•Tag™ Ultra Borate buffer (reagent 1, see
section
3.2.2.1)to a clean 12 x 32 mm glass screw neck total recovery vial.
c. Add 10
μL of calibration standard
( 3.5.5), neutralized sample solution
(3.5.4), or
neutralized converted cystine standard
(3.5.4)to the vial.
d. Vortex mix briefly.
e. Add 20
μL of reconstituted AccQ•Tag™ Ultra reagent
( 3.2.2.2)to the sample vial.
f. Mix the solution
immediately
by pipetting up and down several times. Vortex mix
immediately for several seconds and tap the vial.
g. Let stand for 1 minute at room temperature.
h. Heat the vial in a heating block for 10 minutes at 55 °C (± 1 °C).
3.5.7 UHPLC separation
- Prime solvent lines for 5 min.
- Prime wash / sample syringes for 4 cycles.
- Allow the chromatographic system to stabilise before injecting standards and samples.
Make sure the system pressure and initial conditions are stable before performing
injections (around 9000 psi).
- Before starting a series of analyses, inject two blanks (water) to condition the column.
- Inject 1 µL of each derivatized calibration standards, and then inject 1 µL of derivatized
sample solutions. Perform single injections. Add a blank injection (water) at the end of
each calibration series.
- Perform UHPLC under the following conditions:
Column temperature:
50 °C
UV detector:
260 nm
Injection volume:
1
μL
Flow rate:
0.4 mL/min
Mobile phase A:
Eluent A
(3.4.1)Mobile phase B:
Eluent B
(3.4.2)Elution gradient:
Time
(min)
% A
% B
Curve
0.00
99.9
0.1
5.50
99.9
0.1
2
15.22
90.9
9.1
7
20.47
78.8
21.2
6
21.26
40.4
59.6
6
21.29
10
90
6
22.84
10
90
6
26.00
99.9
0.1
6
32.00
99.9
0.1
6
Operating conditions may vary depending on the apparatus. Follow the supplier’s
instructions.
AMINO-01 (FEBRUARY 2017)
SLV REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE