Two-dimensional Liquid Chromatography For The Determination Of Pyridoxal 5’-phosphoric Acid,4-pyridoxic Acid And Pyridoxal In Pig And Mice Plasma

Vitamin B6,existing in blood,tissues and organs,is an indispensable micronutrient that directly or indirectly participates in a number of metabolic processes through the form of coenzyme factors and can maintain the normal biochemical reactions at a very low level.The changes in the content and ratio of Vitamin B6 can influence overall physiology.It is important to reveal the relationship between illness and the changes in the content of Vitamin B6 by monitoring the level of B6 in organisms.At present,high performance liquid chromatographyfluorescence detection(HPLC-FLD),liquid chromatography-mass spectrometry(LC-MS),and other methods were mainly used to detect the high levels of pyridoxal 5’-phosphate(PLP),4-pyridoxic acid(PA),and pyridoxal(PL)in plasma.In this study,we firstly established a method to detect PLP,PA and PL simultaneously by the use of two-dimensional liquid chromatography-ultraviolet detector(2D-LC-UV).2D-LC consists of a first-dimensional phenyl hexyl column(Aston TM? SPY)and a second-dimensional reverse phase chromatography column(Waters Summetry? C18).In the first-dimensional mobile phase methanol: 20 m M ammonium acetate(5:95),the compounds enter the one-dimensional column for preliminary enrichment and separation,and then in the second-dimensional mobile phase methanol: acetonitrile: 20 m M ammonium acetate(9:6:85)under the conditions of transfer to a two-dimensional column for separation again and enter UV for detection,adding 25% ammonium dihydrogen phosphate(20 m M)to improve the chromatographic peak shape.The flow rate is 1 m L/min.This method was used to optimize the pretreatment conditions of mouse plasma samples,and verify 2D-LC system performance evaluation of this method,as well as methods of specificity,limit of detection,limit of quantification,standard curve,precision,accuracy and stability.The method was verified by science,and the method was applied to the plasma samples of pigs and rats.The results shown that the plasma was firstly precipitated with trichloroacetic acid and then derivatized with semicarbazide in the dark,which was simpler,faster,and has stronger impurity removal ability.The sample analysis time was 8 minutes,and the maximum injection volume was 400 μL.The methodological investigation results were great.The limit of detection of PLP,PA and PL were0.1,0.2,4 nmol/L,and the quantitative ranges were 0.2～400,0.4～400,0.2～2000 nmol/L,respectively.The method had good precision,accuracy and stability.The 2D-LC-UV method was successfully applied to the plasma samples of pigs,mouse,and rats.The PLP,PA,and PL in pig plasma were 197～431,3～15,82～185 nmol/L,respectively.The PLP,PA and PL in mice plasma were 131～256,10～15,and 25～130 nmol/L,respectively.The PLP,PA,and PL in rat plasma were 151～300,9～15,21～36 nmol/L,respectively.The PLP were higher than PA and PL,pig and mouse plasma PL was higher than PL,rat plasma PA was higher than PL.