Journal of Pharmaceutical Investigation

Vol. 41, No. 1, 25-30 (2011)

Development of Analytical Method of Biotin in Complex Drugs and

Dietary Supplements Using HPLC-UV
Yoonyoung Huh, Yun Pyo Kang, Yong Seok Choi, Jeong Hill Park and Sung Won Kwon †

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea
(Received January 10, 2011·Revised February 11, 2011·Accepted February 11, 2011)

ABSTRACT − Recently, Korean Food and Drug Administration (KFDA) has focused on developing quality control guide-
lines for all commercial products in Korea to enforce regulations, improve the quality control, and protect consumers by
developing prevalently used and efficient analytical tools to determine and quantify target compounds. Because the Korean
Pharmacopeia (KP) presents microbiological assays for biotin, which is laborious and time-consuming, this study is focused
on applying HPLC-UV to detect and quantify biotin in complex drugs and dietary supplements like multi-vitamin. Biotin
in complex drugs was extracted from methanol and analyzed using mobile phase with 10 mM potassium phosphate
(monobasic, pH=3.0) in distilled water and acetonitrile. Gradient condition was used to successfully detect and quantify
biotin within 20 minutes. Validation result for linearity was significant that average r was 0.999 (n=3) and its relative stan-
2

dard deviation (RSD) was 0.0578% which was less than 2%. Using this method, quantification of biotin in complex drugs
was completed successfully and recovery tests were finished that recovery percentage greater than 95% with relative stan-
dard deviation less than 2%.
Key words − Biotin, High performance liquid chromatography, Validation

Biotin is water-soluble vitamin B-complex and also called high performance liquid chromatography with an ultraviolet
vitamin H. Its chemical structure consists of tetrahydroim- detector (HPLC-UV), a fast and accurate qualitative and quan-
idizalone ring fused with a tetrahydrothiophene ring where titative analytical tool widely available in common analytical
valeric acid is attached to the tetrahydrothiophene ring (Fig. 1). laboratories. Even though there are other methods developed
Also, its major biological function is known as coenzyme for to analyze biotin using HPLC-UV (Chen et al., 2009; Durstl et
cell growth, the production of fatty acids, and the metabolism al, 1975; Ekpe and Hazen, 1998; Nandhasri et al., 1991; Nel-
of fats and amino acids (Nojiri et al., 1998; Yomota and son et al., 2006), most of them require complicated deriva-
Ohnishi, 2007). Moreover, biotin is daily required as other tization steps (Desbene et al., 1983) to prepare samples and its
vitamins participates in gluconeogenesis. Thus, its deficiency low yield due to incomplete reaction causes further problem
could be serious and even fatal (Aboul-Enein et al., 2004; with accurate determination of biotin in samples (Wilbur et al.,
Watanabe et al., 2005). 2000).
Recently, biotin has been included in various commercial Here, we report an easy and efficient method to quantify
products such as complex drugs and dietary supplements like biotin in various products accurately using HPLC-UV without
multi-vitamin. Therefore, solid and efficient method should be derizatization steps. Since its good analytical performance was
established in order to control and enforce regulations of prod- confirmed, it will potentially become a quality control guide-
ucts and protect consumers. However, since microbiological line for commercial products including biotin, and will provide
assays of biotin, the typical analytical method of biotin intro- low economic cost and simple procedures for easier approach
duced in the Korean Pharmacopeia, requires laborious and to be performed in common laboratories.
time-consuming works by growing and incubating cells; The
Korean Food and Drug Administration (KFDA) is currently Material and Experimental
searching prevalent, fast, efficient and accurate analytical
methods to determine biotin. In this aspect, KFDA focuses on Biotin standard solutions
Biotin standard solution was prepared by dissolving biotin
standard (Sigma Aldrich, MO, USA) in methanol (Duksan
†
Corresponding Author :
Tel : +82-2-880-7844, E-mail : [email protected] Chemicals, Ansan, Korea). Five different concentrations were
DOI : 10.4333/KPS.2011.41.1.025 prepared by making 120, 135, 150, 165, and 180 µg/mL (ppm).

25
26 Yoonyoung Huh, Yun Pyo Kang, Yong Seok Choi, Jeong Hill Park and Sung Won Kwon

formance. Since many precedents have not fully satisfied the

ultimate goal of their usage (Miwa et al., 2000), it was urgent
to establish an efficient and widely available method to detect
and quantify biotin in complex drugs. HPLC-UV was the best
method because it was the most extensively used analytical
instrument that provided accurate results with high sensitivity
(Aboul-Enein et al., 2004). The importance of this study is to
Figure 1. Chemical structure of biotin provide simple procedure to prepare samples to run exper-
iment within 30 min. and therefore, build a bridge that con-
All solvents used were of HPLC grade. nects scientific research and practical application of analytical
chemistry for administrative usage.
Samples preparation UV absorption wavelength to detect biotin was set at 205
Samples used for recovery tests were referred to as Drug A nm. Because biotin lacks chromophore, it does not show high
(500 µg/tablet), B (165 µg/tablet) and C (300 µg/tablet). All sensitivity to UV absorption (Miwa et al., 1985). There are
samples were multi-vitamins tablets and twenty of them were previously studied methods that support the weakness of UV
weighted and grounded. Twenty tablets for each drug were absorption by running additional derivatization in order to give
weighted and finely grounded. Then, the average amount was stronger chromophore to biotin. However, many of them cause
obtained and dissolved in methanol to make 150, 165, and 180 problems because performing reactions might produce low
ppm. All solutions were sonicated for 30 minutes (min.) and yield of finally derivatized products or even no product at all
centrifuged for 10 min. at 14,000 rpm. Finally, supernatants were (Miwa et al., 2000) and that will not accomplish the goal to
filtered using an Advantec membrane filter (0.45 µm, Toyo accurately quantify biotin in complex drug. Therefore, it was
Roshi Kaisha, Ltd., Tokyo, Japan) before injection to HPLC. determined to use UV detector without following precedents
but instead the lowest possible wavelength was chosen that
Method validation protocol for assays and impurities could still detect the target compound.
in tablets by HPLC The mobile phase used was 10 mM of potassium phosphate
The validation was carried out properly based on a protocol in water with pH of 3.0 and acetonitrile. The phosphate buffer
recommended by KFDA (The United States Pharmacopeia, and pH control in aqueous mobile phase increase selectivity
2002). and sensitivity of biotin peak. Methanol was not suitable for
this study because its UV cutoff is higher than 205 nm (Arm-
HPLC instrument and conditions strong and Carey, 1982), and so acetonitrile was used as
10 µL of a sample was introduced into a Phenomenex Luna organic mobile phase. Since developed analytical method
analytical column (C18, 150 × 4.6 mm, 5 µm) via Phenom- should be applicable to commercial products, such as complex
enex Security Guard column (C18, 3.0 × 4.0 mm) by a Waters drugs, the gradient mobile phase condition instead of the iso-
717 auto-sampler. Compounds in samples were separated by a cratic condition was used for the clear separation of biotin
mobile phase program between mobile phase A [10 mM potas- from other components and their complete elution from a col-
sium phosphate (mono basic) in water (pH=3.0)] and the umn in a short analytical time.
mobile phase B (acetonitrile) at a flow rate of 1.0 mL/min. In order to validate the analytical method, a validation pro-
using Waters 510 pump. Mobile phase B was started from 5% tocol recommended by KFDA was followed (The United
for 5 min. to 85% over 15 min. at a linear gradient and then States Pharmacopeia., 2002). It introduced three parts of the
back to 5% for 10 min. for equilibrium. Eluted compounds experiment: linearity, precision, and accuracy/system suitabil-
from the analytical column were observed at 205 nm through ity and in order to prove the linearity, five different con-
a Waters 486 UV detector and the column temperature was centrations were required, usually 80, 90, 100, 110 and 120%
kept at 25 C.
o
of standard solution. In this study, 150 ppm was determined
100%. Required relative standard deviation (RSD) in the pro-
Results and Discussion tocol is less than 2% in the case of three trials and the RSD for
linearity in this study satisfied the requirement (5.78×10 %).
-2

The purpose of this study was to develop a new analytical Then, precision was examined followed by system validation,
method for biotin in order to utilize in administrative per- intra-day validation, and inter-day validation. System valida-
J. Pharm. Invest., Vol. 41, No. 1 (2011)
 Development of Analytical Method of Biotin in Complex Drugs and Dietary Supplements Using HPLC-UV 27

Figure 2. Overlaid HPLC chromatograms of biotin standard

Table I. Slope, y-intercept, and r2 of calibration curves Table II. System validation (n=6) of the HPLC- UV method
generated by biotin standard solutions using a biotin standard solution (150 ppm)
Trial Slope y r2 Trial Peak Area
Intercept 1 6.76×10 5

1 4.83×10 3
-7.23×10 4
9.99×10 -1

2 6.76×10 5

2 5.04×10 3
-1.02×10 4
9.99×10 -1

3 6.71×10 5

3 4.51×10 3
-2.34×10 4
9.98×10 -1

4 6.73×10 5

Average 4.79×10 3
-3.53×10 4
9.99×10 -1

5 6.75×10 5

STDEV 1
2.63×10 2
3.27×10 4
5.77×10 -4

6 6.66×10 5

RSD (%)
2
5.78×10 -2

Average 6.73×10 5

STDEV: standard deviation

1

RSD: relative standard deviation

2
STDEV 3.92×10 3

RSD(%) 5.83×10 -1

tion requires six trials and intra- and inter-day validation need 1
STDEV: standard deviation
three trials. Results for system validation, intra-, and inter-day
2
RSD: relative standard deviation
RSD values were 5.83×10 %, 9.72×10 %, and 4.37×10 %,
-2 -2 -1

respectively. Finally, accuracy/system suitability test was done used for generating calibration curves and testing linearity. In
to confirm the accuracy of the method and applied to com- each trial, standard solutions with five different concentrations
mercial complex drugs. As a result, the recovery percentages were analyzed and total three trials were performed. As a
were calculated greater than 95% with RSD less than 2%. result, the average value of r greater than 0.999 and RSD less
2

than 2% were obtained (Table I).

Linearity
The biotin standard peak was observed at 14.4 min (Fig. 2) Precision and accuracy/system suitability
and its peak area from each biotin standard solution run was As a part of the validation of the analytical method, its repro-
Table III. Intra-day validation (n=3) of the HPLC-UV method using biotin standard solutions
Peak Area RSD (%) for
2

Trial each set

120 ppm 135 ppm 150 ppm 165 ppm 180 ppm
1 5.11×10 5
5.76×10
5
6.51×10 5
7.26×10 5
7.98×10
5
1.28
2 5.02×10 5
5.80×10
5
6.57×10 5
7.20×10 5
8.09×10
5
1.25
3 5.14×10 5
5.90×10
5
6.54×10 5
7.23×10 5
7.86×10
5
4.98×10 -1

Average 5.09×10 5
5.82×10
5
6.54×10 5
7.23×10 5
7.98×10
5
3.85×10 -1

STDEV 1
6.50×10 3
7.26×10
3
3.26×10 3
2.79×10 3
1.16×10
4
1.45
RSD (%)2
1.28 1.25 4.97×10 -1
3.85×10 -1
1.45 9.72×10 -1

STDEV: standard deviation

1

RSD: relative standard deviation

2

J. Pharm. Invest., Vol. 41, No. 1 (2011)

28 Yoonyoung Huh, Yun Pyo Kang, Yong Seok Choi, Jeong Hill Park and Sung Won Kwon

Table IV. Inter-day validation (n=3) of the HPLC-UV method repeatability. As shown in the Table III, the average RSD value
using biotin standard solutions was 0.972%. In this test, RSD for each set was gained by com-
Peak Area paring each concentration with its corresponding concentration
120 ppm 150 ppm 180 ppm obtained from calibration curve equation. Lower RSD values
Day 1 5.09×10 5
6.54×10 5
7.98×10 5
(the average RSD for each set was 0.972%) imply better accu-
Day 2 5.10×10 5
6.53×10 5
8.02×10 5 racy of our method. Finally, inter-day validation was per-
Day 3 5.05×10 5
6.50×10 5
8.05×10 5 formed by running three different concentrations per day for
Average 5.08×10 5
6.52×10 5
8.01×10 5
three consecutive days. Table IV illuminates the areas for dif-
ferent concentrations with RSDs.
STDEV 1
2.31×10 3
1.94×10 3
3.51×10 3

Recovery test was examined to determine the accuracy of

RSD (%)
2
4.55×10 -1
2.97×10 -1
4.37×10-1

the developed method and the system suitability for the anal-
STDEV: standard deviation
1

ysis of biotin in commercial products and three complex tablet

RSD: relative standard deviation
2

drugs were used to evaluate recovery and reproducibility of the

method. Each drug was diluted and extracted using methanol
ducibility was examined, first. System validation was per- to make 150, 165 and 180 ppm and each sample (n=3) was
formed by injecting a biotin standard solution of 150 ppm six membrane filtered to remove impurities. Satisfactory ranges
times consecutively and each area was compared to obtain for recovery test were from 95% to 105% and RSD value less
standard deviation and RSD. As presented in Table II, the RSD than 2% (The United States Pharmacopeia., 2002). Biotin
for system validation was confirmed as 0.583%, which was peaks for all three drugs appeared at same retention time as
less than 2%. Further, intra-day validation was carried out by Fig. 3 shows. Table V summarizes recovery percentages for
repeating one set of five different concentrations for three drug A, B and C. It was confirmed that all data accomplished
times and each RSD value was calculated to verify intra-day desired ranges of results. Since our precision and accuracy

Figure 3. HPLC chromatograms for complex drug A, B, and C

J. Pharm. Invest., Vol. 41, No. 1 (2011)
 Development of Analytical Method of Biotin in Complex Drugs and Dietary Supplements Using HPLC-UV 29

Table V. Recovery tests for drug A, B and C (n=3) using the Conclusions
HPLC-UV method
Averaged recovery (%) Averaged calibration
recovery (%)
In this study, a solid analytical method for biotin was devel-
oped and successfully completed while adhereing to the
Drug A method validation protocol set by the HPLC-UV. Therefore,
150 ppm 95.9 96.4 this method could potentially become a quality control guide-
RSD (%) 1
1.02 92.6×10 -2
line that could provide low economic cost and simple pro-
165 ppm 95.2 95.4 cedures for easier approach to be performed in common
RSD (%) 81.2×10 -2
74.2×10 -2
laboratories.
180 ppm 95.1 95.6
RSD (%) 69.6×10 -2
64.0×10 -2 Acknowledgements
Drug B
This research was supported by a grant 09102KFDA301
150 ppm 98.0 98.3 from the KFDA in 2009.
RSD (%) 1.61 1.46
165 ppm 95.2 95.3 References
RSD (%) 1.34 1.23
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J. Pharm. Invest., Vol. 41, No. 1 (2011)