title: Adduct formation in liquid chromatography-triple quadrupole mass spectrometric measurement of bryostatin 1.
abstract: Bryostatin 1, a potential anti-Alzheimer drug, is effective at subnanomolar concentrations. Measurement is complicated by the formation of low m/z degradation products and the formation of adducts with various cations, which make accurate quantitation difficult. Adduct formation caused the sample matrix or mobile phase to partition bryostatin 1 into products of different mass. Degradation of the 927 [M+Na](+) ion to a 869m/z product was strongly influenced by ionization conditions. We validated a bryostatin 1 assay in biological tissues using capillary column HPLC with nanospray ionization (NSI) in a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode. Adduct formation was controlled by adding 1mM acetic acid and 0.1mM sodium acetate to the HPLC buffer, maximizing the formation of the [M+Na](+) ion. Efficient removal of contaminating cholesterol from the sample during solvent extraction was also critical. The increased sensitivity provided by NSI and capillary-bore columns and the elimination of signal partitioning due to adduct formation and degradation in the ionization source enabled a detection limit of 1×10(-18)mol of bryostatin 1 and a LLOQ of 3×10(-18)mol from 1μl of sample. Bryostatin 1 at low pmol/l concentrations enabled measurement in brain and other tissues without the use of radioactive labels. Despite bryostatin 1's high molecular weight, considerable brain access was observed, with peak brain concentrations exceeding 8% of the peak blood plasma concentrations. Bryostatin 1 readily crosses the blood-brain barrier, reaching peak concentrations of 0.2nM, and specifically activates and translocates brain PKCɛ.
authors.affiliation: Blanchette Rockefeller Neurosciences Institute, 8 Medical Center Drive, Morgantown, WV 26505, USA. Electronic address: tjnelson@brni-jhu.org.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Nelson
authors.affiliation: Blanchette Rockefeller Neurosciences Institute, 8 Medical Center Drive, Morgantown, WV 26505, USA.
authors.forename: Abhik
authors.initials: A
authors.lastname: Sen
authors.affiliation: Blanchette Rockefeller Neurosciences Institute, 8 Medical Center Drive, Morgantown, WV 26505, USA.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Alkon
authors.affiliation: Blanchette Rockefeller Neurosciences Institute, 8 Medical Center Drive, Morgantown, WV 26505, USA.
authors.forename: Miao-Kun
authors.initials: MK
authors.lastname: Sun
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D054713:Bryostatins / Q000032:analysis* / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D051744:Protein Kinase C-epsilon / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D054713:Bryostatins; C046785:bryostatin 1; D051744:Protein Kinase C-epsilon
keywords: AD; Adduct; Alzheimer's disease; Aβ; Bryostatin 1; LC–MS; LLOQ; MTBE; Mass spectrometry; NSI; PKC; Paclitaxel; beta-amyloid peptide; lower limit of quantitation; methyl t-butyl ether; nanospray ionization; protein kinase C