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(
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.
G. Chromatographic Conditions
(a)
Mobile phase A
.—0.1% phosphoric acid.
(b)
Mobile phase B
.—100% acetonitrile.
(c)
Mobile phase C
.—80% acetonitrile.
(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
.
H. QC
(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.
I. Calculation and Reporting
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.
J. Repeatability
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.
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).
References
(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 , 59–64Table 2016.02B. Gradient program
Time, min
Flow rate,
mL/min
Mobile
phase A, %
Mobile
phase B, %
Mobile
phase C, %
0.0
0.6
90
10
0
18.0
0.6
90
10
0
18.5
0.8
0
0
100
24.0
0.8
0
0
100
24.5
0.6
90
10
0
27.0
0.6
90
10
0
5