title: Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry.
abstract: We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA. Electronic address: Guodong.Zhang@pfizer.com.
authors.forename: Guodong
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Cardiovascular, Metabolic, and Endocrine Diseases Research Unit, Pfizer Global Research and Development, Cambridge, MA 02139, USA.
authors.forename: Annie D
authors.initials: AD
authors.lastname: Walker
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors.forename: Zhaosheng
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Han
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Blatnik
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors.forename: Rick C
authors.initials: RC
authors.lastname: Steenwyk
authors.affiliation: Biomarker Research, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Groeber
mesh_terms: D000227:Adenine Nucleotides / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D000227:Adenine Nucleotides; D007477:Ions
keywords: ADP; AMP; ATP; IP–HILIC–MS/MS; Nucleotides; Organic Acids