title: A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites.
abstract: BACKGROUND
  Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies.
  
  
  METHODS
  This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min.
  
  
  RESULTS
  The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of >0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of <15.0% on average.
  
  
  CONCLUSIONS
  The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States. Electronic address: bwei@cdc.gov.
authors: Binnian
authors: B
authors: Wei
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States. Electronic address: czf2@cdc.gov.
authors: June
authors: J
authors: Feng
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors: Imran J
authors: IJ
authors: Rehmani
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors: Sharyn
authors: S
authors: Miller
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors: James E
authors: JE
authors: McGuffey
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors: Benjamin C
authors: BC
authors: Blount
authors: Tobacco and Volatiles Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors: Lanqing
authors: L
authors: Wang
mesh_terms: D000470:Alkaloids / Q000378:metabolism / Q000652:urine; D000691:Anabasine / Q000378:metabolism / Q000652:urine; D053000:Analytic Sample Preparation Methods / Q000295:instrumentation / Q000379:methods*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015925:Cryopreservation; D004926:Escherichia coli / Q000201:enzymology; D005966:Glucuronidase / Q000378:metabolism; D006372:Helix, Snails / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D009538:Nicotine / Q000378:metabolism* / Q000652:urine*; D011725:Pyridines / Q000378:metabolism / Q000652:urine; D012371:Robotics; D012907:Smoking / Q000652:urine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D016482:Urinalysis / Q000379:methods*
chemical_list: D000470:Alkaloids; D011725:Pyridines; C012737:anatabine; D009538:Nicotine; D005966:Glucuronidase; D000691:Anabasine
keywords: Biomarker; Biomonitoring; High-throughput; Robotic; Tobacco exposure; Urinary metabolites