Table of Contents Table of Contents
Previous Page  219 / 258 Next Page
Information
Show Menu
Previous Page 219 / 258 Next Page
Page Background

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

Part 1

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

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 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

Infant formula powder,

milk-based

4.48

2.1

5.3

0.59

Infant formula powder,

soy-based

5.16

2.5

6.0

0.68

a

 RSD

r

is the RSD of repeatability.

b

 RSD

R

is the RSD of intermediate reproducibility.

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

2.0

5.1 0.60

Child formula powder

14 5.91

2.8

4.9 0.57

Adult nutritional

powder milk protein-

based

13 2.59

1.9

5.0 0.51

Infant formula powder

soy-based

13 5.04

2.8

4.7 0.53

Infant formula RTF

milk-based

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

14 6.65

3.3

5.4 0.63

Infant formula powder

part hydrolyzed

soy-based

14 3.85

1.3

5.3 0.57

a

n

= Number of laboratories (after removal of outliers).

b

 RTF = Ready-to-feed.

219