title: Application of UPLC-MS/MS Method for Analyzing B-vitamins in Human Milk.
abstract: OBJECTIVE
  To determine ten B-vitamins in human milk by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
  
  
  METHODS
  The pretreated human milk samples were adequately separated and quantified within 11 min by UPLC-MS/MS with an Acquity UPLC HSS T3 column (2.1×100 mm, 1.8 µm). The mobile phase was a gradient of 2.5 mmol/L ammonium formate aqueous solution and acetonitrile at a flow rate of 0.35 mL/min. Stable isotope internal standards were used in the analysis, to correct for the method variability, including matrix and ionization effects. The homogenized human milk samples were deproteinzed using methanol, unknown contaminants were extracted with diethyl ether and hydrophobic phase was discarded. The analytes were monitored via ESI+ionization and detected in multiple reaction monitoring (MRM) with three acquisition functions.
  
  
  RESULTS
  Calibration curves ranged from 0.5-160 ng/mL (thiamin, riboflavin, biotin, nicotinic acid, pyridoxine, pyridoxamine, pyridoxal), and 2.5-800 ng/mL (pantothenic acid, FAD and nicotinamide) (R2=0.990-0.999). The relative recovery ranged from 80.1% to 120.2%; accuracy was determined to be 98.3% to 108.0%. Intra-day and inter-day variation were 3.4%-19.9% and 5.9%-18.1%, respectively. The limit of quantification (LOQ) for all vitamins was between 0.25 and 3 µg/L.
  
  
  CONCLUSIONS
  This method was successfully applied for simultaneous analysis of ten B-vitamins in human milk.
authors.affiliation: National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
authors.forename: Xiang Nan
authors.initials: XN
authors.lastname: Ren
authors.affiliation: National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
authors.forename: Shi An
authors.initials: SA
authors.lastname: Yin
authors.affiliation: National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
authors.forename: Zhen Yu
authors.initials: ZY
authors.lastname: Yang
authors.affiliation: National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
authors.forename: Xiao Guang
authors.initials: XG
authors.lastname: Yang
authors.affiliation: Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Research Center for Preventive Medicine, Beijing 100013, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Shao
authors.affiliation: Zhejiang Provincial Centre for Disease Prevention and Control, Hangzhou 310051, Zhejiang, China.
authors.forename: Yi Ping
authors.initials: YP
authors.lastname: Ren
authors.affiliation: Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Research Center for Preventive Medicine, Beijing 100013, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014803:Vitamin B Complex / Q000737:chemistry*
chemical_list: D014803:Vitamin B Complex
keywords: B-vitamins; Human milk; UPLC-MS/MS