Determination of Folic Acid in Mint Vegetable, by High-Performance Liquid Chromatography

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1 Determination of Folic Acid in Mint Vegetable, by High-Performance Liquid Chromatography Rahmatallah Rahimi a, *, Neda Goodarzi b, * a Department of chemistry, Iran university of Science and Technology (IUST) b Department of chemistry, Faculty of Science and Engineering, Islamic Azad University, Saveh branch, Saveh, Iran [email protected], [email protected] Recevied date: June 8, 2011 Abstract We have applied a quick and convenient method for determining folic acid (FA) excreted in mint vegetable. Sample-treatment entails SPE with Sep-Pak C 18 (0.9 g) cartridge. The water-soluble vitamin were analyzed by HPLC on a Discovery C mm 4.6mm column with 0.1 mol.l -1 KH 2 PO 4 (ph 7.0), methanol, 90:10, as mobile phase (0.7 ml min -1 ) in isocratic mode. Keywords: Mint analysis, High-performance liquid chromatography (HPLC), Folic acid, Solid phase extraction (SPE). 1. Introduction Folates are a group of B vitamins required for the synthesis of DNA and RNA, which are primary events for cellular replication and growth. They have received considerable attention in recent years because of their health benefits regarding prevention of neural tube defects, coronary heart diseases and colon cancer. 1,2 Folate is the general term including folic acid (pteroylglutamate, PteGln) and poly-γglutamyl conjugates with the biological activity of folic acid. Folates present a diverse array of compounds that vary by oxidation state of the pteridine ring structure, one-carbon moieties carried by specific folate, and the number of conjugated glutamate residues on the folate. 3 These vitamins cofactors are essential for the synthesis of purines and pyrimidines and in the production of methionine from homocysteine. 4 Previous studies have shown that storage of foods by freezing does not seem to affect the concentration of folate in spinach, potatoes and broccoli. 5,6 On the other hand, leaching of 31

2 Org. Chem. J. 2010, 1, Determination of Folic Acid in mint Vegetable folate during boiling, has been shown to be substantial, 7 whereas other forms of processing such as oven baking and microwave cooking have been found preferable in terms of folate retention. 8,9 Therefore, potential losses of folates during processing and storage need to be considered when evaluating dietary intake. Numerous methods have been used for the determination of FA including electrochemical sensors, spectrophotometry, 20 flow injection chemiluminescence, 21,22 fluoimetric, 23 high-performance liquid chromatography (HPLC) with ECD, 24 Several HPLC methods were published to determine folates in biological fluids and foods after solid phase extraction (SPE). 25,26 In this report we have developed a convenient HPLC method coupled with a solid phase extraction for determination of water soluble vitamin B 9 (Folic Acid) in mint vegetable. The structure of folic acid is given in Fig 1. H 2 N N 3 2 OH 4 1 N 9 10 N CH 2 NH N O C NH COOH C CH 2 CH 2 COOH H Chemical Formula: C 19 H 19 N 7 O 6 Exact Mass: Molecular Weight: Fig 1. Structure of Folic Acid 2. Results and Discussion The isocratic separation was modified by increasing the concentration of KH 2 PO 4 from 0.05 mol. L -1 to 0.1 mol. L -1, reducing the solvent flow rate from 1.0 to 0.7 ml. min -1. In addition, KH 2 PO 4 content was reduced from 99% to 90%, and so methanol content was increased from 1% to 10%. Figure 2 shows a typical chromatogram obtained from a mint vegetable sample when the 0.1 mol. L -1 KH 2 PO 4 (ph 7.0), methanol, 90:10, was used as mobile phase. When the proportion of methanol was more than 10%, Retention time (min) Fig 2. Separation of folic acid in mint vegetable by HPLC 32

3 folic acid eluted in less than 14 min (within 5 min) but resolution was very poor. As seen from Table 1 linearity and detection limits, six determinations of the same mint vegetable sample were performed using the same reagents and apparatus to evaluate the method precision in mint vegetable. The precision of the method for determination of folic acid in mint vegetable is shown in table 2. Table 1. Linearity of standard curves and detection limits for the folic acid Vitamin Linear range (mg. L -1 ) R 2 Detection limit(mg. L -1 ) Folic acid Table 2. Precision of method for determination of folic acid in mint vegetable Vitamin Mean ± S. D. a (mg. L -1 ) a Mean ± standard deviation Folic acid 2.31 ± Experimental 3.1. Materials The mint vegetables used for the developing of the analytical procedure were collected from Borujerd farm in the city of Borujerd Iran Equipment HPLC analysis was performed with using Agilent 1200 HPLC system and an Agilent 1200 UV detector. The HPLC column used was a reversed - phase Discovery C mm 4.6mm, 5µm (Agilent Technologies, USA). Shaker (IKA R WERKE- OS5 Basic) Chemicals and standard solutions Methanol (HPLC grade), NaOH, K 2 HPO 4 and KH 2 PO 4 (HPLC grade), folic acid standard and all other chemicals were purchased from Merck (Darmstadt Germany). Water used in all the experiments was doubly distilled and deionised. Standard solutions were prepared by dissolving folic acid in 0.1 M phosphate buffer (ph 7.0). 33

4 Org. Chem. J. 2010, 1, Determination of Folic Acid in mint Vegetable 3.4. Sample preparation The samples (3 g) were extracted with 50 ml of extraction 0.1 mol.l -1 phosphate buffer ph 7.0 and 0.1% (v/v) 2-mercaptoethanol, The mixture was shaken for 30 min in a rotational shaker, and centrifuged at 3500 rpm for 15 min. The supernatant was filtered through a 42 µm Millipore filter (Bedford, MA, USA) before chromatography analysis SPE (Solid-Phase Extraction) Mint vegetable consists of many components that cause chromatographic interferences with vitamins. For this reason the sample treatment proposed consists of SPE with Sep-Pak C 18 (900 mg) cartridges (Waters) that enable separation of waters-soluble vitamins and remove most of the interfering components. The stationary phase was flushed with 5 ml methanol and 5ml water deionised to activate the stationary phase, the sample extract was passed through with a flow-rate of 2 3 drops/s. The sample was eluted with 5mL NaOH (0.005 mol. L -1 ) ph Prior to HPLC analysis, all samples were filtered through a 42 µm Millipore filter (Bedford, MA, USA) and then injected in to the chromatograph Methods The column elute was monitored with photodiode-array detector at 282 nm for folic acid. The mobile phase was filtered through at 0.5 µm membrane and degassed by sonication before use. The mobile phase was 0.1 mol. L -1 KH 2 PO 4 (ph 7.0), methanol, 90:10. The flow rate was 0.7 mol. min -1. The column was operated at room temperature. 4. Conclusion A new method is proposed for separation of water-soluble vitamin folic acid. SPE proved an effective tool for isolation of the water- soluble vitamins and HPLC enabled rapid, accurate, and determination of the compounds with recovery ranging from 102%. Acknowledgement Islamic Azad University, Saveh branch is gratefully appreciated. References and Notes 1. Selhub, J.; Rosenberg, I. H. Folic acid. In: Ziegler EE, Filer LJ (eds) Present knowledge in nutrition. ILSI: Washington DC, 1996,

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