AOAC SPIFAN Review Team (August 2016)

Validation of a LC-MS/MS method of Analysis of Nicotinic acid and Nicotinamide in infant formula and adult

nutritional samples

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Table 6: LOD and LOQ of the Method of total Vitamin B3

(niacin)analysis based on total nicotinic acid nicotinamide in a SPIFAN

sample

Vitamin

Total

Niacin

LOD

mcg/100g

LOQ

mcg/100g

195

Conclusions

–

A LC-MS/MS method for analysis of nicotinic acid and nicotinamide and calculation of

total niacin (vitamin B3) in SPIFAN samples was validated. The chromatographic part of the

method could separate nicotinic acid and nicotinamide in the samples from other water soluble

B vitamins and also from any matrix interferences. The calibration standards provided a good

linear relationship between concentration of the standards and their detector response for

nicotinic acid as well as nicotinamide with R

values > 0.999.

Accuracy of the method in estimation of total niacin (vitamin B3) was evaluated by

analysis of NIST SRM 1849, a milk based infant/adult nutrition formula, and NIST SRM 1546A a

meat sample. The method provided results which were within the NIST specifications for

vitamin B3 for both of analyzed NIST samples. The precision of the analysis of the replicates of

the samples analyzed on multiple days (3 days) in terms of % RSD was found to be 4.8% and

4.4% for infant/adult nutrition formula sample and the meat sample respectively.

The inter-day precision of the analysis of the replicates of the samples analyzed on multiple

days (2 days ) in terms of % RSD was found to be in the range of 3.5-6.0%. In some of the

samples % RSD obtained was slightly higher than 5% specified in the SMPR though the value

presented in the study are for the inter-day precision.

Spike recovery of nicotinic acid and nicotinamide was evaluated to further measure the

method accuracy. Nicotinic acid and Nicotinamide spike recoveries in a SPIFAN sample

averaged around 99.4 – 99.5%.

LOD and LOQ of the Method for analysis for total Vitamin B3 (niacin) analysis was

estimated in a reconstituted SPIFAN sample and was found to be 60 and 195 mcg/100

respectively.

VALIDATION REPORT

FOR ERP USE ONLY

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