title: Improved ruggedness of an ion-pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells.
abstract: BACKGROUND
  Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents.
  
  
  METHODS
  An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity.
  
  
  RESULTS
  The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9.
  
  
  CONCLUSIONS
  By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey.
authors.affiliation: Bioanalytical Sciences Department, Research and Development, Bristol-Myers Squibb Co.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.forename: Guowen
authors.initials: G
authors.lastname: Liu
authors.forename: Yifei
authors.initials: Y
authors.lastname: Liu
authors.forename: Long
authors.initials: L
authors.lastname: Yuan
authors.forename: Dara
authors.initials: D
authors.lastname: Hawthorne
authors.forename: Jim X
authors.initials: JX
authors.lastname: Shen
authors.forename: Mausumee
authors.initials: M
authors.lastname: Guha
authors.forename: Anne
authors.initials: A
authors.lastname: Aubry
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008252:Macaca fascicularis; D009711:Nucleotides / Q000097:blood* / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000097:blood* / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000379:methods; D013936:Thymidine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry
chemical_list: C583040:BMS-986001; D007477:Ions; D009711:Nucleotides; D009930:Organic Chemicals; D018894:Reverse Transcriptase Inhibitors; D013936:Thymidine