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

Table 2011.07D. Uncertainty: Milk-based infant formula Analyte Unit Median CV iR

RSD (Rec.)

Standard uncertainty a

Expanded uncertainty b

µ g/100 g

Vitamin A

475

4.8

0.033

28

5.8

55

11.6

a  Standard uncertainty: = b  Expanded uncertainty: U = 2 * u, which gives a level of confidence of approximately 95%. 2 c RSD RiVC 2 ) ( + ) (Re * corrected Median u

( c )  Internal control plan .— QC samples .—Include QC samples (certified, in-house reference samples, or spiked samples) in every series of analysis and analyze in duplicate. Spiking experiments .— Verify recovery rate by spiking samples. Calculate recovery rate (Rec.) on the samples using the following equation:

hydrolysate with a glass rod and then transfer it into an empty cartridge. This improves the dispersion and absorption of the liquid throughout the filling material. In this case, it is not necessary to wait 15 min before starting the elution step. ( c )  Elution .—( 1 ) Elute with 100 mL n -hexane with BHT after 15 min. ( 2 ) Collect eluate into a 250 mL pointed-bottom amber glass flask. ( 3 ) Stop elution once all n -hexane has eluted, but no later than 30 min after all the n -hexane has been absorbed by the cartridge filling material (packing). Note: In some cases the saponified sample solution is not completely retained by the extraction cartridge even when using 15 mL instead of 20 mL. The problem may be solved by using a smaller quantity of potassium hydroxide during saponification, e.g., 4 g instead of 7 g or by adding 1 g potassium dihydrogen phosphate to the 20 mL saponified sample solution before loading the extraction cartridge. ( d )  Evaporation .—Evaporate solvent under reduced pressure at 30–40°C. Apply a nitrogen stream to evaporate the last few milliliters. Transfer residue quantitatively into a 5 mL amber glass volumetric flask by means of small portions of n -hexane. Make up to the mark with n -hexane. Filter through a 0.22 μm membrane filter (solution is ready to be injected in the UPLC system). Perform further dilutions in n -hexane to be within the calibration ranges (retinol 0.6–3.0 μg/mL) if needed. ( 1 )  Chromatography.— ( a )  Chromatographic conditions.—See Table 2011.07E . ( b )  Instrument check test .—Allow the chromatographic system to equilibrate for at least 15 min for UPLC before injecting standards and samples. Make sure the system pressure is stable and there are no leaks. Inject the lowest point of the calibration curve at least three times and ensure the stability of the system, repeatable response, and retention time before starting a series of analyses. Note: Coefficients of variation should not be higher than 2% for retention time and peak response. Table 2011.07E. Chromatographic conditions for UPLC Parameter Condition Analytical column Acquity UPLC HILIC 1.7 μm, 2.1 × 100 mm

C - C Rec. n

s =

100 x

C a

where C s = concentration of vitamin in the spiked test portion, n = concentration of vitamin in the nonspiked test portion, C a = concentration of vitamin A added to the test portion. Recovery rate should be higher than 90%. ( 2 )  Operating procedure and determination .—( a )  Sequence setup.— Set up (in duplicate) 4 μL of each of the working standard solutions [ see D ( c )( 3 )] . Inject, sequentially, 4 μL of the reagent blank and test solutions. Include a working standard or a QC sample every 6–8 samples to monitor system stability. ( b )  Calibration .—Calculate average of peak area (or height) and standard deviation in the series of analysis. Construct a calibration curve by plotting the peak area (or height) of vitamin A (all- trans retinol) in each of the working standard solutions against concentration in micrograms per milliliter. Calculate the slope (S) and the intercept (I) by linear regression. ( c )  Identification .—Identify the all- trans retinol, 13- cis retinol peak on the sample chromatogram by comparison with the retention time of the corresponding peak in the standard solutions. See example chromatograms in Figures 2011.07A and B . ( 3 )  Calculations and results .—( a )  All-trans retinol .—Calculate the mass fraction, w, of all- trans retinol (in micrograms per 100 g sample) by using the following equation: C where m = mass of the test portion, in g (10.0 for powders and 30.0 for liquids), A s = area (or height) of the all- trans retinol peak in the sample chromatogram, S = slope of the calibration curve, I = intercept of the calibration curve, V 0 = volume in which the saponified solution has been diluted, in mL (100.0), V 1 = aliquot of the saponified solution introduced in the cartridge, in mL (20.0 or 15.0 if the saponified solution is not retained in the cartridge), V 2 = final volume of the test solution, in mL (5.0). ( b )  13-cis Retinol .—Calculate the mass fraction, w, of 13- cis retinol, in micrograms per 100 g sample, by using the following equation: 100 V m S V V )I - A( w 1 2 s retinol -all 0 × ×× × × = trans

Column temperature

25°C

Mobile phase

A: 1% 2-propanol in n -hexane

Flow rate

1.2 mL/min

Injection volume Injection mode

4 μL

Partial loop (5 μL loop)

1830 V V )I - A(

1680 V m S × × × 2 ×

0

w

100

=

s

UV detection

326 nm 2 pts/s

cis

retinol -31

× ××

1

Data rate

where m = mass of the test portion, in g (10.0 for powders and 30.0

© 2011 AOAC INTERNATIONAL