AOACSPIFANMethods-2017Awards

ampos

iménez

artin

ournal of

AOAC I

nternational

100, N

1, 2017

141

(c)

System suitability test

.—Equilibrate the chromatographic

system for ≥0.5 h. Inject a working standard solution of ascorbic

acid at least six times and check the peak retention times and

response (peak height or area). Ensure orotic acid and isoascorbic

acid are fully resolved from ascorbic acid by injecting separate

standard solutions of each compounds (prepared as stated for

ascorbic acid). If the acids are not resolved, decrease the pH of the

mobile phase to 5.0 or increase the amount of acetonitrile used.

(d)

Calibration

.—Perform single injections of working

standard solutions, as a minimum at the beginning and end of

each analytical series. Establish the calibration curve (seven

points) by plotting peak response (height or area) vs ascorbic

acid concentration, perform linear regression, and calculate the

slope and intercept of the calibration curve.

(e)

Analysis

.—Perform single injections of sample solutions.

(f)

Identification

.—Identify the ascorbic acid peak in the

chromatograms of the sample solutions by comparing it with

the retention time and UV spectrum of the corresponding peak

in the standard solution.

H. Calculations

Calculate the concentration of vitamin C in mg ascorbic

acid/100 g expressed in as-is ready-to-feed (RTF) products—or

as reconstituted powder for powder samples—as follows:

A I V V

S m V

100

1000

1 3

(

)

− × × ×

× × ×

where

is the response (height or area) of the ascorbic acid

peak obtained for the sample solution,

is the intercept of the

calibration curve,

is the slope of the calibration curve,

the weight of the test portion in grams (2.0 g),

is the volume

of the test solution (volume used to dissolve the test portion) in

milliliters (10 mL),

is the volume used in the sample dilution

(1.0 mL), and

is the volume of the final sample dilution

(10 mL).

Note

: If results expressed in the powder sample are needed,

use the reconstitution rate for the calculation C× (225/25).

I. Collaborative Study Protocol

Part 1.—

All participant laboratories received two practice

samples and were asked to analyze each of them in duplicate

(two extractions from each reconstituted sample). Any deviation

from the written method was to be recorded and reported. Results

were communicated to the Study Director using the electronic

template provided with the protocol. The participants were asked

to report final vitamin C results, peak responses for standard

curves and samples, and the different masses used during sample

preparation. After review by the Study Director, results within a

range of expected levels (average ±2×

) were used to identify

the laboratories that qualified for part 2 of the study.

Part 2.—

All qualified laboratories received a second shipment

containing 20 coded products, corresponding to 10 products

in blind duplicates. The samples were a set of infant formula

and adult nutritional products, representing a wide range of

commercially available products. The laboratories were asked

to analyze all the samples (a single extraction from each

liquid or reconstituted powder) on 2 days (10 samples/day).

Each sample was assigned to either days 1 or 2. The blind

duplicates were assigned for analysis on the same day. Results

were communicated to the Study Director using an electronic

template similar to the one used in part 1.

Part 3.—

Two of the samples [infant formula RTF (milk-based)

and adult nutritional powder (low-fat)] failed to meet acceptance

criteria during part 2. Due to the questionable integrity of the

samples (some laboratories reported product spoilage) and

the fact that the samples had reached their expiration date,

new products representing the same type of matrix were sent

to a subset of 12 laboratories for analysis following the same

protocol. Results were transmitted to the Study Director using

the same electronic template as in parts 1 and 2.

J. Statistical Evaluation

After data collection, outliers were detected using Cochran’s

and Grubbs’ tests. Average concentrations,

, and RSD

were

estimated from the blind duplicates.

, RSD

, and Horwitz

ratio (HorRat) values (i.e., RSD

/predicted RSD

) were

also estimated. Details on statistical analysis can be found in

Official Methods of Analysis

“

Appendix D

Guidelines for

Collaborative Study Procedures To Validate Characteristics of

a Method of Analysis

” (7).

Results and Discussion

Part 1

Twenty-six laboratories initially agreed to participate

in the collaborative study. Two laboratories dropped out

during part 1 as a result of issues related to the availability of

resources. The remaining 24 laboratories set up the method

as described in the protocol. Fourteen laboratories used the

UHPLC conditions as described in the protocol, whereas the

remaining 10 used previously published HPLC conditions

(5). No differences could be found between the provided

results in either condition, and thus evaluation was performed

combining all results.

During method setup, it was brought to the attention of the

Study Director that there was a need to establish suitability

testing to ensure proper chromatographic separation between

ascorbic, isoascorbic, and orotic acids. This suitability testing

was added to the method as presented in this paper.

One laboratory did not receive the practice samples due to

customs restrictions. The laboratory qualified for part 2 by using

results from the reference samples.After data compilation, average

concentrations and

and

were calculated. Another laboratory

reported single results and was not included in the statistical

evaluation, although it qualified for part 2. Two laboratories

reporting data above or below the average (±2×SD) were flagged

as possible outliers and informed accordingly. Nevertheless, the

two laboratories were accepted to continue because their results,

although questionable, were still within ±3×SD.

Part 2

Of the 24 laboratories providing results for practice samples

and qualified to continue to part 2, two did not receive the

full collaborative study set due to customs restrictions. The

224