Abstract
High performance liquid chromatography was used to develop and validate the detection of vitamin B12 in blood plasma sample. The mobile phase consists of a mixture of 0.025% trifluroacetic acid in deionized water and 30% methanol. The mixture used was adjusted to pH 2.9 and the flow rate was adjusted to 0.5 mL/min. The separation was achieved using C18 column maintained at 30ºC temperature and detection of vitamin B12 was conducted at maximum wavelength 230 nm.Video Clip of Methodology:Estimation of vitamin B12 in plasma by HPLC: 4 min 29 sec Full Screen Alternate
Highlights
Vitamin B12 is an essential water soluble micronutrient that plays an important role in regulation of brain functioning (Grotzkyj et al, 2012)
The deficiency of vitamin B12 may lead to various complications such as neurological abnormalities like peripheral neuropathy, autonomic dysfunction, psychosis, memory impairment, Cognitive decline etc (Roynolds et al, 2006)
Vitamin B12 in plasma, serum and urine can be measured by radioimmunoassay, enzyme-linked immunosorbent assay, microbiological assay, High Performance Liquid Chromatography (HPLC) (Stefova et al, 1997), capillary electrophoresis, mass spectroscopy, Raman spectroscopy (Mayer et al, 1973), Fourier transform Raman spectroscopy (Hancewicz and Petty, 1995), chemiluminescence (Song and Hou, 2003) and fluorescence quenching
Summary
Vitamin B12 (cobalamin and its derivatives) is an essential water soluble micronutrient that plays an important role in regulation of brain functioning (Grotzkyj et al, 2012). A deficiency vitamin B12 occurs when the level is less than 200 pg/mL (Shaik and Gan, 2013). Because of its very low concentration in plasma, it is very challenging to develop a method with good sensitivities and specificities. The available methods to estimate vitamin B12 in plasma are mainly based on microbiological assay which is time consuming, hazardous to the health of the operator and may lack specificity (Giorgi et al, 2012). Different analytical methods have been developed over recent years due to increasing demand of vitamins. Most of these methods were applied in food particle, drinks, and vitamin supplements and so on
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