Estimation of Water-Soluble Vitamin B-Complex in Selected Leafy and Non-Leafy Vegetables by HPLC Method

Introduction

Vitamins play a significant role in our health; even though they only make up a small portion of the ingested food.¹ Vitamins are classified as fat-soluble vitamins and water-soluble vitamins.² Water‐soluble vitamins include vitamin B complexes and ascorbic acid such as vitamin C. The vitamin B complexes comprise of thiamine; B₁, riboflavin; B₂, niacin; B₃, pantothenic acid; B₅, pyridoxal; B₆, folic acid; B₉, and cyanocobalamin; B₁₂.³ They play essential roles by providing antioxidant properties, co-enzymatic functions, and other cellular and metabolic mechanisms in the human body.⁴

Leafy and non-leafy vegetables provide considerable amounts of water-soluble vitamins especially vitamin B complex. Regular intake of leafy and non-leafy vegetable helps the body to keep away the chronic diseases.⁵ Most of the B vitamins must regularly be replenished since any excess is excreted in the urine.⁶ In contrast, several health problems and nutritional disorders such as vitamin B deficiency anemia, weakness, appetite loss, depression, numbness and tingling in the arms and legs, muscle cramps respiratory infections, hair loss, eczema, poor growth in children and congenital disabilities are accompanied due to deficiency of B vitamins. Many researchers are studying the relationships between vitamin B intake and risk of developing certain diseases because of the importance of vitamin B complexes in human health.⁷

The concern of B vitamins for the welfare of human health has been increased over the past decades. Their increasing awareness has driven the today’s global health industry and pharmaceuticals market to produce a lot of vitamin B supplements, and fortified vitamin B enriched nutrients with particular concentration on their accurate assays and analytical methods.^3,8 The current knowledge for the analytical assays of B vitamins is based on spectroscopic, chromatographic, chemical, or microbiological techniques with various extraction procedures.⁹ Among the analytical methods, high performance liquid chromatographic method (HPLC) has been identified as a promising method for its accuracy, high sensitivity and added advantages for using the different detector.^3,9,10

Although high-performance liquid chromatography (HPLC) has been identified as a reliable chromatographic application to assess the vitamin B complexes in plant food-stuffs,³ efforts are continuing in search of accurate and reliable HPLC procedures especially when the analysis is performed in connection with a nutritional survey or establishing food composition tables¹¹. According to the type of food matrices and number of specific factors, estimation of the water-soluble vitamin B complexes is often a challenging and tedious task. Furthermore, apart from the matrix effects, the signal for each analyte of interest should be free from significant interferences, and of course, the detector used in HPLC method should be selective for the analyses of interest.¹²

Previously, we investigated the vitamin B profiles in indigenous leafy vegetables of Bangladesh using HPLC equipped with UV detector.¹³ The study was intended to report the water soluble vitamin B contents (B₂, B₃, B₅ and B₆) in five commnly consumed vegetables that were already absent from any published literature. As a continuation of research and given preferences for non-leafy vegetables along with others selected leafy vegetables, the current study was undertaken to determine the vitamin B-complex especially B₁, B₂, B₃, and B₆ using high-performance liquid chromatography (HPLC) with PDA detector. The accuracy and reliability of the HPLC method may provide valuable nutritional information of vitamin B complex in the food matrices of leafy and non-leafy vegetables and also supplement the current Food Composition Table for Bangladesh (FCTB) to calculate the habitual dietary intake and/or nutritional survey purposes.

Materials and Methods

Origin of Samples

The samples such as Jute leaves (Corchorus olitorious), Coriander leaves (Coriandrum sativum), Mint leaves (Mentha arvensis), Thankuni leaves (Centella asiatica), Cabbage (Brassica oleracea), Carrot (Daucus carota subsp. sativus), Ladies finger (Abelmoschus esculentus), Green chilies (Capsicum frutescens), Green tomato (Lycopersicon esculentum) and Ripe tomato (Lycopersicon esculentum) were collected from different local markets of Dhaka city, Bangladesh. After collection, the samples were immediately sent to the Institute of Nutrition and Food Science, Dhaka University where the analyses were performed.

Reagents and Solvents

HPLC grade acetonitrile and sodium-1-octanesulfonate were purchased from Merck, Germany. Orthophosphoric acid and glacial acetic acid were purchased from BDH, India. All the vitamin B (B₁, B₂, B_3, and B₆) standards were purchased from Sigma-Aldrich, Germany. Millipore direct system was used for distilled and deionized water.

HPLC Instrumentation

A high-performance Liquid Chromatographic system (Shimadzu-UFLC Prominence) equipped with an autosampler (Model-SIL 20AC HT) and PDA detector was used for the vitamin B analysis. The HPLC data were compiled and recorded using the LC-solutions software.

Preparation of Extraction Solutions

For preparing an extraction solution, precisely 50 ml of acetonitrile was mixed with 10 mL of glacial acetic acid, and the final volume was made up to 1000 mL with double distilled water.

Preparation of Buffer and Mobile Phase

For buffer preparation, 0.173 g of sodium 1- Octanesulfonate and 15.603 g of Sodium dehydrogenate phosphate was dissolved in double distilled water, and the volume was finally made up to 1000 mL with double distilled water. The pH was adjusted to 2.2 with orthosiphoric acid. To prepare the mobile phase, the buffer was filtered through a 0.45 µ membrane filter and degassed by using helium gas.

Preparation of Stock Standards Solution

The stock solution for standard vitamin B₁ (thiamine HCl) was prepared by dissolving 26.7 mg of thiamine hydrochloride in 25 mL of double distilled water. In the case of vitamin B₂, the stock solution for standard vitamin B₂ (riboflavin) was prepared by dissolving 6.9 mg of riboflavin in 100 mL of extraction solution. For vitamin B₃ (nicotinamide), the standard stock solution was prepared by dissolving 41.5 mg of nicotinamide in 25 mL of double distilled water. The standard stock solution for vitamin B₆ (pyridoxine) was prepared by dissolving 20.8 mg of pyridoxine hydrochloride in 25 mL of double distilled water.

Preparation of Samples for Vitamin B Analysis

The procedure for sample? preparation was followed as described by published literature¹³. Briefly, the exact amount of 10 g samples were weighed and homogenized. After that, the samples were transferred into a conical flask where 25 mL of extraction solution was already added. A shaking water bath operating at an ambient temperature of 70 °C for 40 min was used to sonicate the solution. After sonication, the sample was cooled down and finally filtered to make the volume of 50 mL with extraction solution. The extraction solution was again filtered with filter trips (0.45 µm) and 20 µl aliquots solution was injected into the HPLC by using auto-sampler.

HPLC Conditions

For the separation of B complex vitamins, an analytical reversed-phase C-18 column (STR ODS-M, 150 mm × 4.6 mm I.D., 5 µm, Shimadzu Corporation, Japan) was used. The mobile phase consists with a mixture of 100 mM sodium-phosphate buffer (pH 2.2) containing 0.8 mM sodium-1-octanesulfonate and acetonitrile in a ratio of 9:1 (v/v) at 40°C temperature. The flow rate was kept constant by 0.8 mL/min with a PDA detector in which the absorption rate was 270 nm. The calculations of B vitamins were determined from the peak area of respective chromatogram by using the following equation.

B vitamins (mg/100 g) = Concentration of standard X (Area of Sample/Area of Standard) X Dilution factor

Results and Discussion

HPLC Chromatographic Method

In this study, water-soluble B complex vitamins (B₁, B₂, B_3, and B₆) from some leafy and non-leafy vegetables commonly available in Bangladesh was carried out using an HPLC method equipped with PDA detector as a continuation from our previous work already published in elsewhere.¹³ Though the HPLC method is one of the accurate and established techniques for the estimation of B vitamins, the densitometric analysis performed by the PDA detector in the current study could be advantageous for the matric specific vegetables. Further, the solvent system used in the study produced a sharp and compact peak of the B complex vitamins using the validated RP-HPLC method with slight modification.¹⁴ The mobile phase of the solvent system consisted of a mixture of 100 mM sodium-phosphate buffer (pH 2.2) containing 0.8 mM sodium-1-octanesulfonate and acetonitrile in the ratio of 9:1 (v/v) at 40°C temperature producing a constant flow rate; 0.8 mL/min with the C-18 column and elution capacity of 270 nm absorption mode by using PDA detector. The retention times of B vitamins are presented in Table 1. Under the reversed-phase chromatographic conditions, the representative chromatograms of standards of vitamin B complexes (B₁, B₂, B_3, and B₆₎ along with 3D diagram are illustrated in Fig. 1. Fig. 2 shows the individual chromatograms and corresponding 3D diagram of vitamins B₁, B₂, B_3, and B₆ respectively at a wavelength of 270 nm. Furthermore, the chromatograms of different vegetable samples at a wavelength of 270 nm for PDA detector also presented in Fig. 3. For all individual vitamins, the method was found to be linear for the concentration range with a good correlation coefficient that already published in elsewhere.¹³

Table 1: Retention times of B vitamins standard at 270 nm.

Name	Injection volume mL	Concentration (µg/mL)	Retention time Rt (minutes)
Vitamin B1	30	60.02	4.11
Vitamin B2	30	35.19	8.03
Vitamin B3	30	10.00	3.48
Vitamin B6	30	5.90	3.68

 

Figure 1: Chromatograms of standard B1, B2, B3 and B6 (1A) and 3D diagram of the HPLC chromatograms (1B). Click here to view figure

Figure 2: Chromatograms of individual B vitamin standards (2A- B1, 2B-B2, 2C-B3 and 2D-B6). Click here to view figure

Figure 3: Chromatograms of different vegetables samples. Click here to view figure

 

Vitamin B₁ (Thiamin)

The contents of vitamin B₁ are presented in Table 2. Vitamin B₁ ranged from 0.00 to 0.19 mg/100 g of edible portion of vegetables in which the highest amount (0.19 mg/100g) of vitamin B₁ was estimated in Thankuni leaves (Centrella asiatica). The finding was higher than the value (0.09 mg/100g) as presented in Food Composition Table for Bangladesh (FCTB)¹⁵. The higher contents of vitamin B₁ observed under the current study might be the reason for different extraction techniques used for sample preparation though reversed-phase HPLC column was used in both studies. Besides, method validation with or without enzymatic steps could be another considerable point for extraction parameters when considering the nutritional standpoint of B vitamins for nutrition survey and derive a food composition table of daily recommended intake for the general population. The second highest content of B₁ was observed in ripe tomato which was 0.14 mg/100g. In contrast, carrot showed the least amount of B₁; 0.08 mg/100g which was doubled as the contents of carrot as provided in FCTB. However, no vitamin B₁ was detected in jute leaves as consistent with the result as showed by FCTB.

Table 2: Vitamin B₁ contents in analysed samples.

Bengali Name	English Name	Scientific Name	Mean contents of B1 (mg/100 g of edible portion)
Pat sak	Jute Leaves	Corchorus olitorius		0.00
Thankuni pata	Thankuni leaves	Centella asiatica		0.19
Gajor	Carrot	Daucus carota subsp. sativus		0.08
Dheros	Ladies finger/okra	Abelmoschus esculentum		0.10
Paka Tomato	Ripe tomato	Lycppersicon esculentum		0.14

 

Vitamin B₂ (Riboflavin)

Vitamin B₂ plays an essential role in cellular functions and nutrient metabolism. Although vegetables as a source Vitamin B₂ is not as rich as compared to milk and dairy products, people from low resource countries like Bangladesh and also vegetarians with access to a diversity of leafy and non-leafy vegetables can avoid the deficiency vitamin B₂.¹⁶ As shown in the Table 3, the highest amount of vitamin B₂; 0.25 mg/100 g was estimated in Thankuni leaves (Centrella asiatica) followed by the second most abundant amount of 0.10 mg/100 g in Carrot and Green Chilies respectively. The Food Composition Table for Bangladesh (FCTB)¹⁵ quantified 0.10 mg/100 g B₂ in Thankuni leaves which were lower than the current study. The variations of B₂ contents might be related to several factors. For example, analytical artifacts of vitamin B₂ determination have been identified in food samples in which sensitivity to UV light is an outmost important.¹⁰

Table 3: Vitamin B₂ content in analyzed samples.

Bengali Name	English Name	Scientific Name	Mean contents of B2 (mg/100 g of edible portion)
Pudina pata	Mint leaves	Mentha arvensis	0.06
Thankuni pata	Thankuni leaves	Centella siatica	0.25
Badhakopi	Cabbage	Brassica oleracea	0.00
Gajor	Carrot	Daucus carota subsp. sativus	0.10
Dheros	Ladies finger/okra	Abelmoschus esculentum	0.08
Kachamorich	Green chilies	Capsicum frutescens	0.10

 

Vitamin B₃ (Niacin)

The functions of vitamin B₃ in human health have already been reported.¹⁸ Thus regular consumption of leafy and non-leafy vegetables may supply adequate amounts of B₃ necessary to maintain these healthy body functions. The contents of vitamin B₃ analyzed by HPLC method are presented in Table 4. The highest amount of vitamin B₃ (0.59 mg/100 g) was estimated in Coriander leaves which were lower than the value (1.1 mg/100 g) as reported by Food Composition Table for Bangladesh (FCTB).¹⁵ In contrast, the lowest amount was observed in Carrot and Mint leaves; 0.04 mg/100 and 0.04 mg/100g respectively. However, no quantified amounts of niacin were observed in a case for Jute leaves and Ladies finger. The choice of extraction applied in the method could be the reason for the variation of vitamin B₃. However, enormous HPLC published procedures using different PDA, UV or fluorescence detectors reported varied levels of B₃ contents in vegetables and other foodstuffs. Notably, the extraction techniques should be carefully considered if the data is to be used for nutritional intake purposes ⁹. Besides, either the free form of B₃ in foodstuffs as nicotinic acid and nicotinic acid amide or exists in bound forms on food matrices are technological issues.¹¹

Table 4: Vitamin B₃ content in analyzed samples.

Bengali Name	English Name	Scientific Name	Mean contents of B3 (mg/100 g of edible portion)
Pat sak	Jute Leaves	Corchorus olitorius	0.00
Dhonepata	Coriander Leaves	Coriandrum sativum	0.59
Pudina pata	Mint leaves	Mentha arvensis	0.04
Thankuni pata	Thankuni leaves	Centella asiatica	0.13
Badhakopi	Cabbage	Brassica oleracea	0.08
Gajor	Carrot	Daucus carota subsp. sativus	0.04
Dheros	Ladies finger	Abelmoschus esculentum	0.00
Kachamorich	Green chiles	Capsicum frutescens	0.39
Kacha Tomato	Green Tomato	Lycppersicon esculentum	0.16
Paka Tomato	Ripe tomato	Lycppersicon esculentum	0.05

 

Vitamin B₆ (Pyridoxine)

Dietary vitamin B₆ acts as a coenzyme in the folate metabolism pathway and may have anti-carcinogenic effects such as prevention of colorectal cancer.²⁰ High consumption of vegetables and citrus fruit may improve folate status which intern may contribute to the prevention of cardiovascular diseases.²¹ The Table. 5 shows the contents of Vitamin B₆ of analyzed samples where the highest amount of vitamin B₆; 0.73 mg/100 g was estimated in Carrot followed by second highest contents in Thankuni leaves (Centrella asiatica) which was 0.13 mg/100 g of edible portion. The Food Composition Table for Bangladesh (FCTB)⁴ only reported the vitamin B₆ which was 0.133 mg/100 g in Carrot, but no reported values for Thankuni leaves. The fluctuations of vitamin B₆ contents in vegetables may be due to the different methods of extraction when applied to the same food matrix of vegetable and also vitamin B₆ determination procedures by HPLC and microbiological processes. Research conducted on different food matrix including vegetables reported that the highest values of total vitamin B₆ in food were achieved using 0.1 M HCl or 0.1 M sulphuric acid for autoclaving followed by b-glucosidase /acid phosphatase hydrolysis.²²

Table 5: Vitamin B₆ content in analyzed samples.

Bengali Name	English Name	Scientific Name	Mean contents of B6 (mg/100 g of edible portion)
Pat sak	Jute Leaves	Corchorus olitorius	0.00
Pudina pata	Mint leaves	Mentha arvensis	0.00
Thankuni pata	Thankuni leaves	Centella asiatica	0.13
Badhakopi	Cabbage	Brassica oleracea	0.00
Gajor	Carrot	Daucus carota subsp. sativus	0.73

 

Conclusion

The present study quantified the water-soluble vitamin B complex namely B₁, B₂, B_3, and B₆ in some selected leafy and non-leafy vegetables using HPLC method equipped with reversed-phase photodiode array detector (PDA). Though many factors may affect the simultaneous estimation of water-soluble vitamin B complex in different food matrices, the method used in the current study is already validated¹³ which is accurate enough to separate the compounds simultaneously. Apart from the methodological advantages, the result of vitamin B analysis in some selected leafy and non-leafy vegetables under current study will provide crucial supporting information which is believed to be absent in the current FCTB of Bangladesh. Precisely, the Food Composition Table for Bangladesh (FCTB)⁴ has been published to providing the nutritional information of Bangladeshi foods commonly consumed by the general population. However, the published FCTB selected some key foods and nutrients for establishing the database: lacking information to specific foods especially vegetables of different varieties and cultivars are prevalent. Since the demand for global and country-specific food composition database has been increasing over the past decades, the data derived from the current investigation serves as an important means to calculate dietary intake of B vitamins applicable during habitual food intake or nutritional survey or assessment.

Acknowledgements

We acknowledged the Institute of Nutrition and Food Science, and Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh for analytical support and assistance.

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