Adduct formation in liquid chromatography-triple quadrupole mass spectrometric measurement of bryostatin 1

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 869 m/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 1 mM acetic acid and 0.1 mM 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 1 μl of sample. Bryostatin 1 at low pmol/l concentrations enabled measurement in brain and other tissues without the use of radioactive labels. Despite bryostatin 1ʹs 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 0.2 nM, and specifically activates and translocates brain PKCɛ.