85 / 123
Collaborative Study Report: Method 2011.20 Nucleotides by HPLC-UV
Page 20
Lab 3: Sample prep centrifugation of the samples at 250 x g did not give a good separation between the two
layers. Samples were centrifuged at 2500 x g for 15 minutes.
Study Author:
Depending on the nature of the tubes used in this step higher centrifuge speed is
possible and desirable. Method modification to recommend “
≥ 250 × g”
Lab 4: The use of dry acetone (to dissolve PTAD) was not optimal. I am not convinced by magnesium
chloride as drying agent. When the PTAD/acetone solution was added in isooctane, I observed droplets like
PTAD/acetone solution was not miscible with isooctane but the derivatization seems to have worked
(totally?). The solution PTAD/acetone is fully miscible when I use regular acetone. We use this regular
acetone for the derivatization of the Vitamin D in our method and we do not have any problem in the
derivatization step. Is it really necessary to dry acetone when we use high quality acetone?
Study Author:
Agreed, this step was added in response to a bad batch of acetone and seems
unnecessary. This step was removed from method for other participants of MLT.
Lab 6: Sample prep completed steps in a hot block vessel instead of boiling tube, after adding water to hot
block vessel and inverting 10 times, total volume was transferred to 50 mL falcon tube for centrifugation…
preparation of PTAD solution was done in non-dried acetone,…final steps were completed in amber auto-
sampler vials.
Study Author:
Changes agreed to in advance due to limitations of equipment. Results do not seem
to have been compromised
.
Lab 9: Sample 4 (FPTE312) and sample 20 (DOMY545) are slurry and reconstitution was performed. The
sample weight input in this table was adjusted by dilution factor of reconstitution. During "step f" in the
procedure, isooctane layer of sample 4, 9, 11, 20 can't be separated at 250 g. The centrifuge speed was
adjusted to 3000 g to achieve separation.
Study Author:
As with Lab 3, a higher centrifuge speed is possible and desirable. Method
modification to recommend “
≥ 250 × g
”.
Conclusions
A multi-laboratory collaborative study of AOAC First Action 2016.05, an LC-MS/MS method for the analysis
of vitamin D
2
(ergocalciferol) and vitamin D
3
(cholecalciferol) in infant formula and nutritional products was
undertaken. The method was applied to the SPIAN kit and demonstrated acceptable precision..
Recommendations
On the basis of the results of this study, it is recommended that AOAC First Action 2016.05 be endorsed as
Final Action for the determination of vitamin D
2
(ergocalciferol) and vitamin D
3
(cholecalciferol) an
LC-MS/MS in fortified milk powders, infant formula, and nutritional products.
2016.05 (FEBRUARY 2017) VITD-18
MLT REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE