6. AOACSPIFANMethods-2018Awards

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L in et al .: J ournal of AOAC I nternational V ol . 100, N o . 1, 2017  149

Figure 2. Representative calibration curve.

response occurs when biotin is injected at high concentration. To achieve accurate quantification, the method requires an appropriate sample size to ensure that the response (peak area) of the injected sample fits within the established calibration curve range. Based on the recovery data of Standard Reference Material (SRM) 1849a with different sample sizes, the suitable range of the calibration curve is 8.5–60 ng/mL for this method (Table 2). If the biotin concentration of the injected sample is >60 ng/mL, a further dilution with mobile phase is required. Precision . — All fortified and unfortified matrixes were freshly prepared and analyzed in duplicate on 6 days. The SMPRs require RSD r to be ≤6% for matrixes that contain >1 μg biotin/100 g reconstituted final product. The RSD r obtained from the SLV ranged from 0.5 to 3.0% and met the requirements. The intermediate RSD R was in the 1.3–4.5% range (Table 3). Accuracy . — A total of 11 representative SPIFAN matrixes were spiked with biotin dissolved in 0.5% ethanol. Fortified matrixes were spiked at either 100% or 50% of the previously determined biotin level, and placebos were spiked at either 150% or 50% of the previously determined biotin level in their corresponding fortified matrixes. The spiked sample was either stored at room temperature for 2 h or stored refrigerated for 24 h

to allow biotin to become incorporated into the sample matrix. The spiked samples were prepared and analyzed in duplicate on 3 days. In addition, SRM 1849a with different sample sizes was prepared and analyzed. The result was compared to the certificate of authenticity value. The SMPRs require recovery to be 90–110% for matrixes that contain >1 μg biotin/100 g reconstituted final product. The requirements were met for 10 of 11 tested SPIFAN matrixes. The mean spike recovery data ranged between 95 and 111% (Table 4). Recovery of SRM1849a met the requirement (Table 5). LOQ . — Biotin LOD and LOQ values were determined experimentally by spiking a very low level of biotin into placebos. Blank mean and SD were obtained from eight injections. LOQ = blank mean + 10 SDs The result was confirmed by the S/N method. A placebo was spiked with biotin at the estimated LOQ level and the peak S/N was 10. The LOQ was estimated to be 0.8 μg/100 g reconstituted final product for powder, assuming a 4 g sample was diluted to 50 mL; and 1.5 μg/100 g for RTF, assuming a 20 g sample was diluted to 50 mL. The biotin levels in all the SPIFAN matrixes are above the LOQ of this method.

Table 2. Suitable range of sample concentration after dilution

Calibration standard

HPLC-injected sample concn, ng/mL

SRM Product

Reference value, mg/kg powder

Sample size, g

Suitable range concn, ng/mL

Measured value, mg/kg powder

Recovery versus CoA, % a

Code

Level Blank

Concn, ng/mL

1849a

CLC-10b

1.99

0.1445 0.1532 0.2006 1.0020 1.0006 1.5177 1.5082 2.0006 2.0003

6.276 6.981 8.454 40.21 41.05 58.69 57.60 69.39 69.28

2.172 2.278 2.107 2.006 2.051 1.934 1.910 1.734 1.732

109 114 106 101 103 97.2 96.0 87.1 87.0

0

8.5–60

1 2 3 4 5 6 7

4.981 9.962 19.92 39.85 59.77 79.70 99.62

a  CoA = Certificate of Analysis.