6. AOACSPIFANMethods-2018Awards

219

Martin & Campos-Gim É

nez: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015  1699

Run in single-reaction monitoring mode. Monitor the transitions m / z 220.2 → 90.1 for PA, and m / z 224.2 → 94.1 for the isotope-labeled IS, between 0 and 2.1 min. Set collision energy at 14 V. The dwell time for each monitored transition is 0.1 s. The last two values are indicative and need to be checked and optimized for each instrument used. (d)  Identification .—MS detection in the single-reaction monitoring includes simultaneous detection of molecular ions corresponding to PA and labeled IS. The selected mass transitions are m / z 220.2 → 90.1 and m / z 224.2 → 94.1, respectively. (e)  Quantitation .—Calculate for each standard the peak area ratio between PA and IS. Establish a 5-point calibration curve (ranging from 0.16 to 2.4 ng on column) by plotting peak area ratio versus PA concentration. Calculate the linear regression. It is recommended to use a weighted regression curve (1/x). Calculate the slope (S) and the intercept (I). Calculate the PA concentration, w, in (mg/100 g) using the following equation: = ( ) × 1 × 3 × 100 × × 2 × 1000 where A= peak area ratio PA/IS in the test solution; I = intercept of the calibration curve; S = slope of the calibration curve; V 1 = volume of the of sample extract, in mL (= 50); V 2 = volume of the filtrate pipetted, in mL (= 1); V 3 = final volume of the test solution, in mL (= 10 ± 1); m = mass of the test portion, in g; 100 = conversion to 100 g basis; and 1000 = conversion from μg to mg. Collaborative Study Participanting laboratories received two practice samples. Laboratories set up the method described in this paper. Participants were asked to analyze each of the two practice samples in duplicate (two extractions from each reconstituted sample). Any deviation, such as necessity to substitute reagents, columns, apparatus, or instruments, was to be recorded and reported. Reporting to the Study Director was done electronically using a template. Laboratories were asked to give all areas obtained (both PA and labeled PA) for the standard curve as well as for the samples. Concerning the standard curve, participants were given the choice to either use linear regression or a weighted linear regression (with 1/x as weight). This decision was to be mentioned in the informatics template. Furthermore, different masses used during sample preparation Table 1. Results of practice samples for 14 laboratories ≤5% ≤15% Requirements (SMPR 2012.009) Mean, mg/100 g RSD r , % a RSD R , % b HorRat values Part 1

were to be reported. After review by the Study Director, results within a range of expected levels were used to identify the laboratories that had the capability to run the analysis successfully. The laboratories were thus qualified for the second part of the study. Part 2 All qualified laboratories received a second shipment containing 10 products in blind duplicates (i.e., 20 samples) for the collaborative study. The products came from a set of infant formula and adult nutritional products (i.e., SPIFAN kit) aimed to represent the whole range of commercially available products. Laboratories were asked to analyze all the samples (single extraction from each reconstituted sample) on 2 days (10 samples/day). Each sample was assigned to either day 1 or day 2. Results were transmitted to the Study Director via a similar electronic template as the one used in part 1. Statistical Evaluation After data collection, outliers were detected using Cochran’s and Grubbs’ tests. Average PA concentrations, SDs of repeatability (S r ), and RSDs of repeatability (RSD r ) were estimated from the blind duplicates in the collaborative study samples. The duplicates were assigned to be analyzed on the same day. SDs of reproducibility (S R ), RSDs of reproducibility (RSD R ), and HorRat (Horwitz ratio) values (RSD R /predicted RSD R ) were also estimated. Details on statistical analysis can be found in Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis of the Official Methods of Analysis of AOAC (9). Table 2. Results of collaborative study samples for 14 laboratories ≤5% ≤15% Requirements (SMPR 2012.009) n a Mean, mg/100 g RSD r , % RSD R , % HorRat values

Adult nutritional RTF high-fat b

14 2.07

2.9

7.0 0.69

SRM 1849a

14 6.96 14 5.91

2.0 2.8

5.1 0.60 4.9 0.57

Child formula powder Adult nutritional powder milk protein- based Infant formula powder soy-based Infant formula RTF milk-based

13 2.59

1.9

5.0 0.51

13 5.04

2.8

4.7 0.53

13 0.549

1.5

4.1 0.33

Adult nutritional powder low-fat

13 8.07

1.6

4.1 0.50

Adult nutritional RTF high protein

13 1.57

1.7

5.5 0.52

Infant elemental powder

Infant formula powder, milk-based Infant formula powder, soy-based

14 6.65

3.3

5.4 0.63

4.48

2.1

5.3

0.59

Infant formula powder part hydrolyzed soy-based

5.16

2.5

6.0

0.68

14 3.85

1.3

5.3 0.57

a  RSD r b  RSD R

is the RSD of repeatability.

a n = Number of laboratories (after removal of outliers). b  RTF = Ready-to-feed.

is the RSD of intermediate reproducibility.