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Single Lab Validation of a Method for the Determination of β‐
Galactooligosaccharides in Infant Formula & Adult Nutritionals / Sean Austin (NRC,
Lausanne)
25 Jul 2016
CONFIDENTIAL
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4.
PROTOCOL FOR MW DETERMINATION OF GOS BY UHPLC‐FLD‐MS
Sample preparation:
The same sample preparation is the same for both quantitative analysis and for the MW assignment
of signals, thus follow the normal sample preparation procedure (the same vial prepared for the
quantitative determination of GOS can thus also be used for the analysis of MW assignment).
Alternatively one may prepare a sample using only the GOS ingredient. In case the mass
spectrometer has insufficient sensitivity it is possible to prepare a sample having ten times greater
GOS concentration for the purposes of peak identification only (in this case it is recommended to
use the GOS ingredient).
Mass Spectrometer Set‐up:
In addition to the UHPLC‐FLD instrument, an API 4000 QTRAP (Applied Biosystems) mass
spectrometer detector was attached via a Triversa Nanomate (Advion) to work in nanoelectrospray
mode. The UHPLC flow was split at the exit of the LC column, approximately half of the flow (0.3
mL/min) goes to the FLD, and the remaining 0.3 mL/min is directed to the nanomate source). The
nanomate further splits the flow down to about 600 nL/min for introduction to the mass
spectrometer. The following describes the setup of the MS system in our lab. The mass
spectrometer settings in other labs should be optimised locally.
NOTE: Do not connect the FLD and the mass spectrometer in series. In general the FLD cells cannot withstand the
generated backpressure
LC parameters:
Use the same LC conditions as described in the quantitative method
Triversa Nanomate source parameters:
Method type: LC Chip Coupling
Trigger acquisition when input singnal received: Yes. Signal: DigIn 1
Voltage: 1.40 kV
Polarity: Negative
Output contact closure signal: MS Relay
Duration: 2.5s
Always use new nozzle:No
VALIDATION REPORT
FOR ERP USE ONLY
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