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For this MLT Study, it was difficult to predict the amount of filtrate that is needed for the SPE cleanup
as written in the protocol because the level of Vitamin B12 in the MLT samples were unknown to begin
with. The same is applicable to the final dilution volume.
A safety section can be added to AOAC 2011.11
We feel only safety concern, If we are working continuously on this method and handling of Potassium
cyanide on daily basis. what will impact of that on user health and ecosystem (post disposal)?
NO
The predominant safety concern is the use of KCN.
User Comments
Study Director Response
Due to the problems encountered with peak splitting using Zorbax Bonus RP column, it might be
considered to remove it from the alternatives. The stability of the column is not predictable and for a
routine laboratory that will be running the method there could be potential issues involved, such as
increased cost of consumables. Moreover, other laboratories that implement system suitability
requirements such as peak asymmetry may encounter issues meeting this requirement.
In your experience using the method, does the method perform according to the method’s applicability as written?
In your experience with the method, are there any safety concerns identified while using or regarding use of the method?
Do you have any information regarding the reproducibility of this method as written? If so, please specify and submit information.
Based on your experience with the method, are there any recommended changes to the AOAC First Action Method as written?
User Comments
Study Director Response
Yes
Yes, the method has performed as written, the mobile phase composition needed to be adjusted, which
is allowed in the method, rest all method parameters work well.
Yes but: We don’t always know the vitamin B12 concentration in products we have to analyze. So, we
can not always choose the volume of filtrate loaded onto SPE according the guidelines indicated in the
method. Are there any risks if the volume loaded is too high (interferences, lost of vitamin…)?
There is a risk of overloading the SPE cartridge if more than 80 mL of filtrate
from products containing high amounts of protein or hydrolyzed protein are
loaded onto the SPE cartridge. If the cartridge becomes overloaded, all of the
vitamin B12 in the sample will not be retained and results will be low.
Which is the elution protocol for samples collected in 5 ml volumetric flasks?
When samples are eluted from SPE cartridges into 5 mL volumetric flasks, the
acetonitrile concentration in the samples will be too high if samples are
immediately diluted to volume with water. Acetonitrile is removed from the
samples by heating the samples in a 95°C oven for ~1.5 hours along with a
small amount of potassium cyanide dissolved in pH 4.5 sodium acetate buffer.
Yes
We have used this method for routine analysis for reporting of Vitamin B12 results, in various
nutritional products and reproducibility of results is very good (getting an RSD ~5%, between day
precision)
None.
Fumes generated during mobile phase preparations (TEA and Formic Acid) and fumes from oven during A safety section can be added to AOAC 2011.11
Using the 550nm wavelength is more selective for B12
The chromatographic profiles were not always good. Is it necessary to use a detection wavelength at
550 nm or why 360 nm could not be used.
Include a note that the method (all preparations) should be carried out under a fume hood and ensure
that oven vent is connected to an exhaust system.
Study director can add a note recommending all sample preparation should be
performed in a fume hood. Internal environmental studies have indicated the
oven vent does not need to be connected to the exhaust system.
User Comments
Study Director Response
The SPE cartridges are not checked, there is no protocol in the method (efficiency, recovery)
Study director will provide instruction on how to evaluate solid phase extraction
None
