OMB Meeting Book_9-11-14

50 = total volume of Working Standard (mL) (e) Concentration of TMP in Calibration Standards (CS):

CS (µg/mL) = IS 1 25 where: IS = concentration of nucleotide in Internal Standard (μg/mL)

1 = volume of IS in Calibration Standard (mL) 25 = total volume of Calibration Standard (mL)

(f)

Concentration of nucleotides in Calibration Standards (CS):

CS (µg/mL) = S

25 where: WS = concentration of nucleotide in Working Standard (μg/mL) V WS = volume of Working Standard in Calibration Standard (mL) 25 = total volume of Calibration Standard (mL) (g) Determine the linear regression curve for the ratio of peaks areas (nucleotide/TMP; y-axis) vs. the ratio of concentrations (nucleotide/TMP; x-axis) for calibration standards and calculate the slope with the y-intercept forced through 0. (h) Interpolate the nucleotide contents in unknown samples from this calibration curve. (1) For powders: Nucleotide, mg hg -1 =

(2) For ready-to feed liquids: Nucleotide, mg dL -1 =

where: A NT

= nucleotide peak area in sample

A IS = TMP peak area in sample L = linear regression slope of calibration curve C IS

= concentration of Internal Standard added to sample (μg/mL)

V IS W S

= volume of Internal Standard added to sample (mL)

= weight of sample (g) 1000 = mass conversion of result (μg to mg) V S = volume of sample (mL) 100 = mass or volume conversion of result (g to hg or mL to dL)

J. Data Handling

Report results in mg hg -1 or mg dL -1 to 1 decimal place.

K. References

(1) Gill, B. D.; Indyk, H. E.; Kumar, M. C.; Sievwright, N. K.; Manley-Harris, M. A liquid chromatographic method for routine analysis of 5′-mononucleotides in pediatric formulas. J. AOAC Int . 2010, 93 , 966–973. (2) Gill, B. D.; Indyk, H. E.; Manley-Harris, M. Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry. Anal. Bioanal. Chem. 2013, 405 , 5311–5319. NOTE: Reference (2) added to method after publication of the First Action Method

Method Modifications

A number of changes to the First Action method have been implemented. In response to user feedback, an

ultracentrifugation cleanup step was incorporated into the sample preparation for starch-based products as

they tended to block the SPE cartridges without prior treatment. Extinction coefficients of nucleotides were

modified as part of work published after the method was endorsed as First Action method; resulting in a

more accurate quantitation of standards. The extinction coefficients were based on two different techniques,

Karl Fisher titration for nucleotides and gravimetric method for nucleosides (with molecular weight

adjustment) (1).

Collaborative Study Report: Method 2011.20 Nucleotides by HPLC-UV

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