International Journal of Engineering and Science Applications

ISSN: 2406-9833 @2015 PPs-UNHAS IJEScA

Validation of Reversed-phase High-performance Liquid

Chromatography Method for Determination of Alpha Tocopherol
in Corn Oil

Nursiah La Nafie1*, Syarifuddin Liong2, Leny Irawati3

1
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University
e-mail: [email protected]
2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University
e-mail: [email protected]
3
Department of Chemical Engineering, Polytechnic of Ujung Pandang
e-mail: [email protected]
*Corresponding author, e-mail: [email protected]
Abstract
Determination of alpha tocopherol in corn oil can be analyzed by using Reversed-Phase High Performance
Liquid Chromatography (RP-HPLC). This research aimed to validate RP-HPLC method for determination of
alpha tocopherol in corn oil. The method was based on linearity, precision, accuracy, limit detection (LOD) and
limit quantization (LOQ). The Reversed Phase High Performance Liquid Chromatography (RP-HPLC) method
includes the determination of the wavelength, the mobile phase composition and the flow rate of mobile phase,
then validation of each of the above methods. The maximum wavelength was found in 290 nm, composition of
mobile phase was 100% methanol, flow rate of mobile phase was 1,5 mL/min and retention time was 9,67 min.
The researched result indicated that the method of RP-HPLC had a linearity with the correlation coefficient of
0.999, a relative standard deviation (%RSD) for intra and inter-day precision was < 5%, accuracy/recovery was
95.69-100.99 %, detection limit was 0,43 mg/L, quantization limit was 0,55 mg/L and alpha tocopherol content
in corn oil was 40.06 ± 0.24 mg/L.

Keywords: Corn oil, alpha tocopherol, RP-HPLC, and validation method

1. INTRODUCTION Corn oil is used to decrease cholesterol

In the last decade, the attention of the oil levels in the blood because it contains
consumed by humans is increasing. Oil for polyunsaturated fatty acids. The content of
human consumption can be derived from fried phytosterol in corn oil also inhibits the
foods or consumed directly as an addition to absorption of cholesterol in the intestine. In
the dressing, salad and mayonnaise, which is addition, the corn oil is a source of tocopherols
better known as vegetable oil (vegetable oils). which act as antioxidants in the body [2].
Vegetable oils contain high polyunsaturated Tocopherol is a group of fat-soluble
fatty acids and tocopherol which is useful for vitamins contained in four vitamin different
body. Olive oil and corn oil are vegetable oil forms, namely alpha (α), beta (β), gamma (γ)
consumed frequently. Corn oil is the oil and delta (δ) tocopherol. α-Tocopherol is a
obtained from the processing of corn seeds component of vitamin E, which has highest
(Zea Mays) and had undergone a process of antioxidant activity, which serves to fight free
purification with or without the addition of radicals cause arthritis, liver diseases, the
other additives [1]. aging process, various types of cancer,

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cataracts, Alzheimer's disease and specificity, linearity, limit of detection, limit of
atherosclerosis [3]. quantization, range, precision, accuracy and
α- Tocopherol could not be synthesized by the perseverance [8].
body and is found in small quantities in a Tocopherol can be analyzed by High
variety of foodstuffs. Fat-soluble antioxidants Performance Liquid Chromatography (HPLC)
can generally be stored in the liver for long (normal phase or reversed phase) with isocratic
time, however α- tocopherol is actually stored or gradient elution system, using uv-vis,
in body fat and used very fast [3]. fluorescence or electrochemical detector [9]. In
Analysis of the compounds contained in normal phase HPLC, the mobile phase is n-
corn oil can provide nutrition information that hexane which is relatively easy to separate all
is naturally present in it such as fatty acids, forms of tocopherol while the mobile phase of
phenol compounds, sterols and tocopherols as the reversed phase HPLC is methanol or
well as synthetic antioxidants when added to acetonitrile which is rather difficult to separate
the production process. The matrix in corn oil the β and γ-tocopherol [10].
is quite complex so that different analytical HPLC is now the most widely method to
methods are used for identification and determine the concentration of α-tocopherol.
quantitative analysis, which is not likely to The advantages of the HPLC compared to
give different results [4]. spectrophotometry, fluorometry or gas
Validation method is a process used to chromatography. Parameters usually use for
confirm that the analytical procedures used for the study are speed, relatively free from
a particular test has the appropriate interference of impurities, the condition of
designation. The results of the validation non- destructive ends and a simple method to
method can be used to assess the quality, be applied in a very small sample. Analysis of
reliability and consistency of analysis α-tocopherol using HPLC has been done [3, 11
results[5]. The validity of a specific method of – 14]. The studies showed that HPLC had good
analysis that must be demonstrated in a linearity, precision, accuracy and sensitivity.
laboratory experiment using the same standard Based on those researches, the aim of
or sample to the test sample to be analyzed this research is to validate methods of analysis
regularly [6]. The analytical method should be of α-tocopherol in corn oil by reversed-phase
validated is a non-standard method, a method HPLC with the parameters of linearity,
designed, developed by the laboratory, the precision, accuracy, detection limit and the
standard method used outside the scope limit of quantization.
prescribed and standard methods were
modified [7]. The parameters should be tested
in the validation of analysis methods including

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2. MATERIALS AND METHODS wavelengths of 240, 290 and 360 nm, with a
A. Equipment flow rate of 1.5 ml/min and the mobile phase
This study used Ultra Fast Liquid composition of 100% methanol. After the
Chromatography instrument with detector maximum wavelength was obtained, the
Shimadzu SPD 20A Prominence UV VIS selection of a mobile phase composition was
Detector, LC 20AD pump system, Online done consecutively with 100% methanol,
Degasser DGU-20A3/DGU-20A5 methanol:water a ratio of methanol to water of
Prominence, and System Controller CBM - 100:1, 98:2 and 99: 1.
20A/20Alite Prominence to record and process After the maximum wavelength and
the data, column used was Shimpack VPODS optimal mobile phase composition being
(250x4,6 mm ID), Branson sonicator, Vortex obtained, the flow rate election was conducted
VM 300 and a set of glassware. under conditions of 1mL/min, 1.5mL/min and
B. Materials 2mL/min with a mobile phase of 100%
Materials used were methanol, 2- methanol.
propanol (Merck), syringe Whatman PTFE The standard curve was obtained by
filter Puradisc 13, α- tocopherol purely from injecting a standard at concentrations of 0.5, 1,
Sigma Aldrich, aquabidest and commercial 2, 4, 8, and 16mg/L, and then plotting the peak
corn oil. area against concentration. Linearity was
C. Methods determined from the correlation coefficient (r)
Standard of pure α-tocopherol (Mr = of the standard curve.
430.71 g/mol) was prepared with a Precision was determined by evaluating
concentration of 1000 mg/L in methanol. the area and the retention time. Standards were
Standard solution was then diluted to 100 analyzed at concentrations of 0.5, 1, 2, 4, 8,
mg/L and created a standard series with a and 16mg/L in intra-day and inter-day. For
concentration of 0.5; 1.0; 2.0; 4.0; 8.0; and intra-day measurement, five repetitions were
16.0 mg/L. Each standard solution was then conducted at the same day, while for inter
filtered using a filter syringe 0:45 μm. measurements, five times experiments were
0.25 mL of corn oil was dissolved with 2 mL performed a day for five consecutive days.
to 10 mL of propanol, was shaked for five From the results obtained %RSD was
minutes and then centrifuged at 3000 rpm for determined.
five minutes. Before injected into the HPLC Accuracy is defined as the percent
instrument, corn oil sample was filtered using recovery of α-tocopherol standard (spike)
syringe filter of 0.45 μm. added to a sample of corn oil. Recovery was
Optimization wavelength was done by done against 1, 8 and 16 mg/L α-tocopherol
the injection of the standard solution at standard. Concentration of spike samples (A),

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ISSN: 2406-9833 @2015 PPs-UNHAS IJEScA
the concentration of the sample (B) and the the determination of α- tocopherol. The best
spike concentration (C). mobile phase obtained from experiments at
The recovery percentage was calculated as: various compositions of the mobile phase was
[(A- B)] / C x 100% 100% methanol. The best flow rate of the
Limit of detection and limit of quantization mobile phase was 1.5mL/min.
were calculated based on the standard Based on the calculation, the linear
deviation of the reading of the blank or regression correlation coefficient obtained for
standard that is closest to the blank. α-tocopherol was 0.999 with the equation of y
LOD = X + 3 SD = 5469x + 558.7. This showed that the method
LOQ = X + 10 SD
for determining α- tocopherol using HPLC had
0.25 mL corn oil sample was pipetted, put into good linearity. The calibration curve is given
a 10 mL volumetric flask and diluted with 2- in Figure 1.
propanol to 10 mL, shaked for 5 minutes, and
filtered with a PTFE filter syringe of 0.45μm.
Samples were injected as much as 5
replicates into the HPLC apparatus with the
mobile phase, the flow rate and the wavelength
corresponding to the optimum conditions
obtained. The concentration of α-tocopherol in Figure 1. Calibration Curve of α-tocopherol
the sample was calculated based on the
In this study, α-tocopherol was detected in
regression equation of the calibration curve
9.67 minutes on the condition as follows: a
obtained.
wavelength = 290 nm, mobile phase
composition = 100% methanol and a flow rate
3. RESULTS AND DISCUSSION
= 1.5 mL/min. The linearity of the standard
The discussion of results includes
curve in the range from 0.5 to 16 mg/L
the determination of the maximum
resulted in a correlation coefficient of 0.999,
wavelength, the mobile phase composition and
which means that the linearity and sensitivity
the flow rate, the calibration curve as well as
of this method is good enough to be applied to
the determination of linearity, precision,
the analysis of α- tocopherol.
accuracy, the detection limit and the
Chromatogram of α- tocopherol for
quantization limit, along with the
precision determination can be seen in Figure
determination of α- tocopherol in corn oil.
2.
The results showed that the maximum
wavelength was 290 nm which was the best for

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Figure 2. Chromatogram of α-tocopherol for precision determination, a) repetition 1, b)

repetition 2, c) repetition 3, d) repetition 4, and e) repetition 5

It is clear that the method has good precision

because the peaks obtained are similar for the
5 repititions of measurements. The accuracy of
the method was determined by calculating the
recovery of the fortified corn oil samples
(sample spike method). The experiments were
performed at a concentration of 1, 4 and 8
mg/L with the result of 95.69, 99.53 and The limit of detection of α-tocopherol
100.99%,, respectively. This indicated that the using HPLC was 0:43 mg/L, whereas the limit
accuracy was in agreement with the specified of quantization was 0:55 mg/L with a range of
requirements (95-100%). The results of the 0.5-16.0 mg/L.
examination accuracy/recovery can be seen in The determination of α-tocopherol in
Table 1. corn oil can be seen in chromatogram of
Table 1. Results of recovery measurements Figure 3.

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uV

20000

17500

15000

12500

10000

7500

5000

2500

0.0 2.5 5.0 7.5 min

Figure 3. Chromatograms of (a) corn oil (a) and (b) α-tocopherol standard

The concentration of α-tocopherol corn oil 0.43 mg/L and 0.55 mg/L, respectively. This
obtained in this study was 40.60±0.24mg/L. In method is good enough to be applied as a
the analysis, α- tocopherol corn oil had the method of routine analysis in the laboratory. A
same retention time with the standard retention subsequent study is suggested to continue the
time of α- tocopherol. It is also seen that the α- research until calculation of estimated
tocopherol in corn samples have a peak and a measurement uncertainty.
base line that is quite good. It shows that this
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