Inverted Dispersive Liquid–Liquid Micro Extraction of Nicotinic Acid from Human Plasma and its Determination by High-Performance Liquid Chromatography

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Extractionand determination of nicotinic acid from human plasma was performed using inverted dispersive liquid-liquid microextraction and HPLC. The parameters affecting extraction recovery such as type and volume of extracting and disperser solvents, pH of sample solution, salt addition and extraction time were optimized. Optimal extraction conditions were: 150μL tributyl phosphate as extraction solvent, 400μL methanol as disperser solvent, and pH of sample = 4.5, concentration of NaCl = 3M, without effect of extraction time. Under the optimal conditions a linear range of 30-1000 ng mL-1 (R² = 0.9994) was obtained. Limit of detection, the extraction recovery and preconcentration factor were 10 ng mL^-1, 68% and 53 respectively. The method was successfully applied for the extraction and determination of nicotinic acid in human plasma sample.

Nicotinic acid (niacin or pyridine-3-carboxylic acid, Figure 1) is a water soluble B-complex vitamin present in many foods including fish, milk and green vegetables. The deficiency of nicotinic acid results in pellagra, affecting the skin and central nervous system. High-dose of nicotinic acid may cause thickening of the retina and increase the level of uric acid in the blood. Thus, determination of nicotinic acid in human plasma is very important for health.

The analytical techniques, such as flow injection analysis, thinlayer chromatography , liquid chromatography-mass spectrometry, capillary chromatography, and high-performance liquid chromatography were used for determination of nicotinic acid in human plasma. However, many of analytical techniques may need to preconcentrate target compounds before analysis. Preconcentration methods such as drop-to-drop solvent microextraction, solid phase extraction and Reactive Extraction are difficult and time consuming. Hence, simple and rapid preconcentration method is required to extract nicotinic acid from human plasma. Dispersive Liquid-Liquid Microextraction (DLLME) is a mode of Liquid- Liquid Extraction (LLE) in smaller level, which in comparison with the LLE method, its consumption of organic extracting solvent and environmental contamination significantly lower, and the obtained preconcentration factor is much higher. DLLME employs a mixture of a high-density extracting solvent and water miscible polar disperser solvent. In DLLME After a rapid injection of an appropriate mixture containing extracting and disperser solvents into the aqueous sample a cloudy state is formed. The contact area between the extracting solvent and the sample solution is very large. Thus the extraction equilibrium is achieved rapidly. After centrifugation, the extracted phase is settled at the bottom of the conical test tube. In 2009, Farajzadeh et al. designed inverted dispersive Liquid-Liquid Microextraction (IDLLME). IDLLME is invert of DLLME because in IDLLME the extracting solvent is lighter than water; therefore the separated phase was collected at the top of the sample solution. Thus in the present work, IDLLME-HPLC is used for the extraction and determination of nicotinic acid in plasma sample. This method is based on the extraction of nicotinic acid from plasma sample into an organic solvent. Parameters affecting extraction recovery of nicotinic acid, such as selection types and volume of extracting and disperser solvents, pH of sample solution, salt addition and extraction time were optimized.

Nicotinic acid was purchased from Sigma–Aldrich (Steinheim, Germany). HPLC grade (Methanol, acetonitrile, acetone), sodium hydroxide, hydrochloric acid, and sodium chlorid were obtained from Merck (Darmstadt, Germany). Xylene, n-hexan, toloen, dodecane, tributyl phosphate and 1-octanol were obtained from Aldrich (Milwaukee, WI, USA). Water used was double distilled deionized which purchased from Power Plant in sari city. Stock solution of nicotinic acid (1.0 mg/L) was prepared in methanol and stored in the dark at 4oC. Noted to the LC chromatogram this solution was stable for two months. Working standard solutions were diluted with deionizer double distilled water at concentration of 100.0 ngmL-1 when ever needed.