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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
2
The method does not include:
•
Example calculations
o
Height or area?
Calculations are based on sample area (MLT Step 3.3.12, page 8).
o
How the standard curve data is treated to create a calibration?
Injection sequence is
specified (MLT Step 3.3.11, page 8).
o
Sample calculation.
Sample calculation added (MLT Step 3.3.12, page 8)
•
System suitability
o
What sequence of injections should be used for calibrants and unknowns;
o
Performance criteria for standard curve (r) and read-backs.
o
These points are addressed in MLT Step 3.3.11, page 8.
The Study Director noted that a clarification was made to the original method, to one step of the sample
preparation, concerning elution from the immunoaffinity column:
•
Bio-02 (3/2016), page 3 Step 13: Introduce a reacti-vial and elute the analyte under gravity with
2mL methanol. Elute further with additional 1mL of methanol. Back flush at least 3 times when
eluting.
•
2016.02-MLT (3/2017), page 7 step 12, italics added: Introduce a reacti-vial and elute the analyte
under gravity with 2mL methanol. Elute further with additional 1mL of methanol. Back flush at least
3 times when eluting
and this can be achieved by gentle up and down motion of the syringe plunger
to maximize the elution
.
Method Safety Concerns:
None
Pros/Strengths:
•
The sample preparation is straightforward and uses conventional, widely practiced techniques
•
The quantification uses conventional
HPLC w UV detection.
•
The validation report documented similar analytical results for the majority of samples from a second
brand of immunoaffinity column; most samples produced results within ± 5%. The milk based IF
powder result was 10% lower - 3 mcg/100g powder, or c 0.4 mcg/100mL (4 mcg/L) as fed; although the
absolute difference is small, further investigation is warranted because of the commonness of this
matrix. (Table 5)
Cons/Weaknesses
•
Text needs to be added to describe system suitability and calculations.
Done, see above.
•
The values in the SLV are not consistently reported in the SMPR units (mcg/100g reconstituted)
The
MLT did not use the units of the SMPR either.
Supporting Data
•
General Comment:
The validation report stated that while a reagent blank and a matrix blank prepared from infant elemental
powder were devoid of interferences, the placebo (non-fortified) products child formula powder, adult
nutritional RTF high fat, and infant formula RTF milk based showed a response near the retention time of