Determination of α-tocopherol in red bell pepper (Capsicum annuum) by

UPLC-DAD

1. Introduction

The human body is like a machine that, to function well, needs certain supplements. Vitamins are
one of the most essential elements for the body. They are a various group of organic molecules, and
each play a different but indispensable role in our body 1.

Vitamin E is one of this micronutrients of high importance for our metabolism. It is a plant-derived,
lipid-soluble substance 2. Vitamin E is the collective name for the eight naturally occurring forms,
four tocopherols (α-, β-, γ-, and δ-T) and the corresponding tocotrienols (α-, β-, γ-, and δ--T3),
which contain unsaturated side chains. α -tocopherol is the most abundant and active form of
vitamin E in the human body 3. All these homologs are synthesized by plants from homogen-tisic
acid 1.

Tocopherols act as extra and intracellular antioxidants to maintain homeostasis of labile metabolites
in the cell and tissue 4. They play a fundamental role in the prevention of radical formation in
biological systems like plasma, membranes and tissues, and enhances immune function and
prevents clots from forming in heart arteries 5. Vitamin E is essential to the central nervous system 1.

Plants and photosynthetic organisms are able to synthesize this micronutrient, since humans cannot,
our main source of vitamin E is dietary fruit and vegetables 6. Most plant-derived foods contain low
to moderate levels of this vitamin and red bell pepper (Capsicum annuum) has shown to be good
source 7. Earlier studies have shown that red bell pepper fruits contained noticeably higher amounts
of tocopherols than green ones 8.

An accurate determination of the nutrients content of vegetables is extremely important to

understand the relationship of dietary intake and human health 6. Therefore, the object of this work
was to determine the amount of α-tocopherol in red bell pepper (Capsicum annuum) by UPLC-
DAD.

2. Materials and methods

2.1 Extraction of α-tocopherol

A red bell pepper was purchased at a local market in Merida, Yucatan. Then it was sliced, dehydrated on a
drying oven (Felisa Fe-291ad) at 60 °C for 4 hours and blended. Afterwards, 5g of pepper´s powder was
weighed and mixed with 4ml of 99% ethanol. Afterwards, the sample was sonicated for 40 minutes in a
Branson 5800 Ultrasonic Cleaner. Finally, the sample was passed through a micro-filter to avoid the presence
of any solid particle on the sample.

2.2 Analysis of α-tocopherol by UPLC

The determination of α-tocopherol was realized with an UPLC Acquity H Class equipment (Waters, USA)
with a diode array detector and an Acquity UPLC HSS C18 column (100 Å, 1.8 µm, 2.1 x 50 mm) (Waters,
USA). Chromatographic conditions consisted of a mobile phase of acetonitrile:methanol (50:50) with a flow
speed of 0.5 mL min-1 with a column temperature set at 35°C and injection volume of 2 µL. The DAD
performed the measurement at 290 nm. This was quantified by external calibration prepared with standards of
α-tocopherol. The calibration curve was prepared in the range of 1 to 75 µg mL-1. The α-tocopherol was
identified in the samples with a comparison of the retention time of the standard.

3. Results and discussions

It is shown in Figure 1the α-tocopherol peak obtained from the red bell pepper that was chromatographed.
According to the results, the studied sample presented 5.892 ± 0.135 mg of α-tocopherol per 100g of sample.
According to the National Institute of Health (NIH) (2021), the daily recommended intake of vitamin E for an
adult is of 15 mg 9. Based on this information, by consuming 100g of red bell pepper in one day, an adult
could cover almost 40% of his daily recommended amount.

Figure 1. Chromatogram of red bell pepper.

The obtained results vary from what was reported by the United States Department of Agriculture (USDA) on
their Food and Nutrient Database for Dietary Studies (FNDDS) (2018), a database that is designed to provide
the nutrient values for foods and beverages of important foods in the US food supply, so it can be possible for
researchers to conduct enhanced analysis of dietary intakes. In the latest version of this document, there are
presented the nutrient values of 7,083 foods and beverages (6,286 foods and 797 beverages). On it, it is
reported that red bell pepper contains 1.58mg of vitamin E per 100g of edible portion 10; compared with the
5.892 mg of this vitamin obtained from red bell pepper, it is very noticeable the difference between this data,
being the amount obtained by the USDA almost 4 times smaller than the one obtained in this study.
Nevertheless, on the FNDDS it is not specified the method or procedure used for the determination of the
compound, nor the conditions of the sample.

On the other hand, Soon Ching and Mohamed (2001), studied the amount of alpha-tocopherol on 62 edible
tropical plants using the HPLC method, and reported that red Capsicum annum presented 29.07 ± 0.91 mg per
11
100g of edible portion of the sample . This cipher is considerably higher than the one obtained in the
present study, being almost 5 times bigger than the one we obtained. However, in their article, Soong Ching
and Mohamed specify that the plants they studied were fresh from the market or even picked fresh from the
plant, and the red pepper used for this study was purchased at a supermarket, almost one week before the
analysis. Furthermore, in this study, we used a different method to determine the presence of the vitamin, as
well as a different solvent, since they used a mixture of analytical grade hexane with ethyl acetate and
butylated hydroxytoluene.

Considering how alterative the content of this compound can be, there are a lot of variables that could have
caused the variations between the data obtained in this study and the one reported by the USDA and Soong
Ching and Mohamed. The contents and composition of tocopherols may vary markedly not only between the
plant species, but also within the same species, by the maturity and growing conditions of the plant like the
location, weather, growing season, and the state of the soil. Even after harvest, variation in the vitamin E
values can be caused by many factors including processing procedures, storage time and conditions and
sample preparation and variation in analytical methods 12.

4. Conclusions

Vitamin E is a lipid-soluble micronutrient with antioxidant activity and is exclusively obtained from the diet,
mainly from fruits and vegetables. In the present study we determined by UPLC-DAD the amount of the
active form of this vitamin (alpha-tocopherol) in red bell pepper (Capsicum annuum). The studied sample
presented 5.892 ± 0.135 mg of α-tocopherol per 100g. This data varies from the one reported in the FNDDS
of the USDA (1.58mg / 100g) and with the one reported by Soon Ching and Mohamed (29.07 ± 0.91 mg
/100g). This variation can be caused by many factors including the method of analysis and the extraction
solvent, or even the storage time. A new study is recommended, where more variables are as controlled as
possible.
5. References

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