AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Inject working standards approximately every 5 h of sample extraction analysis (e.g., 25 samples with analysis cycle time of 12 min) injected after the analysis of the last sample extract. Notes : Calibration curves must have a correlation coefficient, r 2 , of >0.990. Calibration curve residuals must be <15% for WS1 and <10% for WS2–WS5. H. Calculations and Results ( a )  Calculation of working standard concentrations .—Calculate the concentration of vitamin D in the working standards from the following equation: ,67' 9: where W = weight of vitamin D used to make the stock standard solution, in mg; and V ISTD = volume of ISTD used, in mL. ( b )  Standard curve calculation using linear regression and quantitation of vitamin D in samples .— ( 1 ) Use peak heights for both vitamin D and 2 H 3 -D for each working standard level to generate a linear regression line. ( 2 ) Calculate the regression by plotting peak height response ratios for each working standard vs vitamin D concentration. The response ratio is defined as the ratio of vitamin D peak height divided by 2 H 3 -D peak height. ( c )  Product calculation .— where C = vitamin concentration (ng/mL) from standard curve; V = volume (mL) of methanol to reconstitute extracts; and S = sample size in grams. References: J. AOAC Int . 96 , 1387(2013) DOI: 10.5740/jaoacint.13-176 J. AOAC Int . 95 , 583(2012) DOI: 10.5740/jaoacint.CS2011_13 > @ P/ PO 9 P/ P/ J QJ PJ P/ PJ: ' u P J ,67' P/ QJ u u u P 6 u 9& J ' 9LWDPLQ P NJ

( 10 ) Inject onto the equilibrated chromatographic system. G. Instrument Operating Conditions ( a ) UPLC conditions.—See Table 2012.11C . ( b ) Mass spectrometer conditions. — See Tables 2012.11D and 2012.11E . ( c )  UPLC analysis .—After verifying equilibration of the UPLC system, inject the working standards (WS1 to WS5) followed by a reagent blank, control sample, and sample extracts. Table 2012.11D. Mass spectrometer (Quattro Premier/Xevo) operating conditions Instrument parameter Setting Tune page file name Ionization mode APCI+ Corona 5.0 µA Cone voltage 20 Source temperature 130°C Desolvation temperature 500°C Desolvation gas flow 1100 L/h Cone gas flow 50 L/h Analyzer cell pressure ~2.0 e –5 Collision cell pressure ~2.0 e –3 Low mass 1 resolution 10.0 Low mass 2 resolution 10.0 Ion energy 1 0.5 Entrance lens –5 Collision energy 15 Exit lens 1 High mass 1 resolution 10 High mass 2 resolution 10 Ion energy 2 1.0 Multiplier 710

u u

AOAC SMPR 2011.004 J. AOAC Int . 95 , 292(2012) DOI: 10.5740/jaoac.int.11-0440

Table 2012.11E. Mass analysis parameters Compound

Retention time, typical, min Molecular ion (precursor) Product ion Dwell, s Cone voltage, V Collision energy, eV

Vitamin D 3

5.3 5.3 5.1 5.1

385.5 388.5 397.5 400.5

259.2 259.2

0.080 0.080 0.080 0.080

20 20 20 20

15 15 15 15

2 H

3 -Vitamin D 3

Vitamin D 2

69.1 69.1

2 H

3 -Vitamin D 2

© 2012 AOAC INTERNATIONAL