6. AOACSPIFANMethods-2018Awards

5

1112 J oseph et al .: J ournal of aoaC I nternatIonal V ol . 99, n o . 4, 2016

( 3 ) Standard 3 (5.0 μg/100 mL) .—Dilute 500 μL intermediate standard to 10 mL with water. ( 4 ) Standard 4 (7.5 μg/100 mL) .—Dilute 750 μL intermediate standard to 10 mL with water. ( 5 ) Standard 5 (10 μg/100 mL) .—Dilute 1 mL intermediate standard to 10 mL with water. ( 6 ) Standard 6 (20 μg/100 mL) .—Dilute 2 mL intermediate standard to 10 mL with water.

Table 2016.02B. Gradient program

Flow rate, mL/min

Mobile phase A, %

Mobile phase B, %

Mobile phase C, %

Time, min

0.0

0.6 0.6 0.8 0.8 0.6 0.6

90 90

10 10

0 0

18.0 18.5 24.0 24.5 27.0

0 0

0 0

100 100

90 90

10 10

0 0

G. Chromatographic Conditions

J. Repeatability

(a) Mobile phase A .—0.1% phosphoric acid. (b) Mobile phase B .—100% acetonitrile. (c) Mobile phase C .—80% acetonitrile.

The difference between the results of duplicate portions of the same sample tested at the same sequence should not exceed 6% of the mean result.

(d) Column .—Kinetex Phenyl-Hexyl (Cat. No. 00F-4495-E0, Phenomenex, Torrance, CA), (150 × 4.6 mm × 2.6 μm × 100 Å). (e) Column temperature .—25 ± 2°C. (f) Retention time .—16 to 17 min. (g) Run time .—27 min. (h) Detector .—Photodiode Array Detector operating at 200 nm (spectrum scan 200–350 nm). (i) Injection volume .—100 μL. For Gradient program see Table 2016.02B . (a) Check system suitability by injecting Standard 3 five times. The RSD, % should be ≤2%. (b) Run the calibration standards at the beginning and end of the sequence (slope drift ≤2%). (c) The six-point calibration should give a correlation coefficient ≥0.997. (d) Test one in five samples in duplicate. The duplicates should be within the method repeatability. (e) Inject one of the calibration standards after every five sample injections. (f) Analyze a reference sample (e.g., National Institute of Standards and Technology Standard Reference Material 1849a) in duplicate. (g) Identification of biotin peak is based on absolute retention time. Spectrum scan can be used for peak purity confirmation if required. The chromatography software will automatically calculate the concentration of the sample in micrograms per 100 grams, provided the concentration of the standard in micrograms per 100 milligrams, sample weight (grams), and dilution are entered correctly. Manual calculation can be performed by using the following equation: ( ) ( ) ( ) µ = × × Biotin g 100 g Sample area volume in milliliters Slope sample weight in grams (The valid slope calculation is based on concentration on x -axis and area on y -axis.) Report results to three significant figures, using microgram-per-100-gram units or convert to other units as required. H. QC I. Calculation and Reporting

K. Reproducibility

The difference between the results of duplicate determinations tested on different days should not exceed 12% of the mean result.

L. Uncertainty of Measurement

Uncertainty of the method was calculated as 7%, using appropriate statistical procedure (square root of the sum of squares of the errors expressed as a percentage).

M. LOQ

The LOQ was calculated based on the lowest working standard and dilution factor, ( ) ( ) ( ) = × × = LOQ 1 100 20 50 0.1 mg 100 g 1 ppb where 1 = 1 μg/100 mL lowest standard, 100 = volume (milliliters), 20 = 20 g sample, 50 represents the volume (milliliters) loaded on immunoaffinity column, and 1 = final volume (milliliters). (1) Bonjour, J. (1991) Biotin in Handbook of Vitamins , L.J. Machlin (Ed), Marcel Dekker, Inc., New York, NY, 393–425 (2) Woollard, D.C., & Indyk, H.E. (2013) Biotin Analysis in Dairy Products in B Vitamins and Folates: Chemistry, Analysis, Function and Effects , V.R. Preedy (Ed) RSC Publishing, London, United Kingdom, pp 377–395 (3) Livaniou, E., Costopoulou, D., Vassiliadou, I., Leondiadis, L., Nyalala, J.O., Ithakissios, D.S., & Evangelatos, G.P. (2000) J. Chromatogr. A 881 , 331–343. doi:10.1016/S0021- 9673(00)00118-7 (4) Frappier, F. (1993) Biotin: Properties and Determination in Encyclopedia of food science, Food Technology and Nutrition , R. Macrae, R.K. Robonson, and M.J. Sadler (Ed); Academic Press, London, United Kingdom, pp 395–399 (5) Lahély, S., Ndaw, S., Arella, F., & Hasselmann, C. (1999) Food Chem. 65 , 253–258. doi:10.1016/S0308-8146(98)00185-X (6) IS EN 15607 (2009) Foodstuffs - Determination of D-Biotin by HPLC (7) Höller, U., Wachter, F., Wehrli, C., & Fizet, C. (2006) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 831 , 8–16. doi:10.1016/j.jchromb.2005.11.021 (8) Bitsch, R., Salz, I., & Hotzel, D. (1989) Int. J. Vitam. Nutr. Res. 59 , 59–64 References