AOAC Final Action Methods in 2017

J OSEPH ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 3, 2018 839

Matrix recoveries were carried out during the SLV at different levels of calibration range, and the recoveries were within 95 – 105%.

Table 4. Biotin practice samples

Biotin concentration, µg/100 g

Practice sample 1

Practice sample 2

Selectivity (Comparison with the LC with tandem MS method)

Lab No.

A

B

A

B

1 2 3 4 5 6 7 8 9

39.28 37.76 40.21 39.40 42.90 39.33 31.73 40.49 40.83 38.58 39.80 34.29 38.72

39.28 38.74 40.21 39.40 37.30 39.92 32.26 40.38 40.61 38.58 39.10 35.89 38.47

15.50 14.69 15.11 15.17 13.20 14.73 12.38 15.98 14.41 14.73 15.10 16.00 14.75

15.50 15.68 15.11 15.17 15.90 15.79 11.50 16.48 15.53 14.73 15.30 14.87 15.13

Selectivity/specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present. Typically, these might include impurities, degradants, matrix, etc. The selectivity of the method was confirmed by analyzing the SPIFAN matrixes using an LC with tandem MS (MS/MS) method by one of the laboratories participating in the MLT study (Laboratory 5). The method is a modified version of AOAC Method 2012.16 for pantothenic acid and uses an ultra-performance LC platform with triple-quadrupole MS. The biotin results by the LC-MS/MS method were closely comparable to the MLT results for the range of SPIFAN matrixes used in the study (Table 8). The SLV and the MLT data provide systematic scientific evidence for a simple, selective, accurate, and precise method as a potential candidate reference method for dispute resolution for the determination of total biotin in all forms of infant, adult, and/or pediatric formula. The method fully meets the intended purpose and applicability statement by complying with the requirements outlined in SPIFAN SMPR 2014.005. The method was applied to a cross-section of infant formula and adult nutritional matrix types, and acceptable precision and accuracy were established. The analytical platform is inexpensive, and the method can be used in almost any laboratory worldwide with basic facilities. The IAC cleanup extraction is the key step to successful analysis. Although an R-Biopharm Rh ˆ one IAC was used for the MLT, alternative IACs were compared during the SLV, with comparable results. The performance parameters of the method are compared with SPIFAN SMPR 2014.005, and the key points are summarized in Table 9. Summary and Conclusions

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10 11 12

Mean

RSD, %

7.75

6.17

7.14

8.19

value that is accepted either as a conventional true value or an accepted reference value. The established procedure for accuracy assessment is by analyzing samples with known concentrations such as Certified Reference Materials. The SPIFAN SMPR 2014.005 recommends NIST SRM 1849a for accuracy evaluation, and this SRM is included in the SPIFAN matrixes as blind coded duplicates. The NIST SRM 1849a results were all within the certified limits of biotin, confirming the accuracy of the method. It was noticed that one of the duplicate results of the National Institute of Standards and Technology reference sample was slightly low (185.41 µg/100 g) for Laboratory 7, but this was investigated as previously explained. The NIST SRM 1849a has a comparatively higher biotin content and is homogeneous; therefore, unlike other powder samples, it was not reconstituted with water, and 1 g powder was directly weighed for the analysis.

Table 5. Biotin MLT samples (day 1)

Biotin concentration, µg/100 g

MLT sample 1

MLT sample 2

MLT sample 3

MLT sample 4

MLT sample 5

MLT sample 6

Lab No.

A

B

A

B

A

B

A

B

A

B

A

B

1 2 3 4 5 6 7 8 9

32.96 39.47 33.38 35.41 37.49 29.09 33.45 27.85 34.96 33.78

31.42 38.48 33.21 34.62 34.70 35.06 32.33 30.34 37.95 34.23

68.94 81.71 80.37 81.88 84.88 77.44 76.96 75.70 81.26 78.79

72.48 83.53 81.62 80.43 79.87 81.29 75.88 79.96 81.41 79.61

3.45 4.33 4.03 3.79 3.84 4.43 4.29 3.66 4.33 4.02 8.68

3.88 4.49 4.22 3.74 4.37 4.05 3.96 4.00 4.43 4.13 6.37

70.84 78.33 69.58 71.65 77.67 74.44 57.51 72.86 74.23 71.90

73.68 68.43 72.92 70.80 74.07 74.98 59.17 70.76 74.41 71.02

24.42 27.42 26.25 28.39 26.12 29.29 24.82 25.36 27.94 26.67

24.78 28.91 27.38 24.24 25.93 32.95 24.66 30.22 26.37 27.27 10.72

46.79 51.02 46.18 44.15 47.15 41.88 39.25 39.91 42.89 44.36

43.44 48.20 45.42 44.03 43.55 47.62 41.15 45.41 47.41 45.14

Mean

RSD, % 10.89

8.04

5.95

4.24

8.53

6.94

6.31

8.57

5.16