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Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

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Merck Reseurcb Labwatwies, R80Y-340, PO Box 2000, Rahay, New Jersq 07065ABsma.-The isolation and characterization of calbistrin A Illwere complicated by the compound's instability. The instability was caused by light, which led to a complex degradation of 1. T h e M U of the light instability waselucidated by forming the dimethyl acetal derivative 2, which isomerized upon exposure to light, leading to an equilibrium mixture of 2 and 3.Alkaline conditions also caused decomposition of 1 , leading to the formation of 4.Three independent research teams appear to have isolated calbistrin A in approximately the same time frame (1-3). The antifungal agent L-702,285 117 was isolated here from asolid fermentation ofAspergillusversicoIor(ATCC 74035, MF2664)(4,5)andgiven the trivial name versilumen (1). Versilumen is now referred to as calbistrin A for the sake of clarity. The isolation and characterization of calbistrin A were complicated by the compound's instability. Initial isolation efforts were characterized by a low overall recovery of calbistrin A. It was discovered that the instability was caused by light, which led to a complex degradation of 1. The isolation, structure elucidation, and biological characterization were performed in near darkness in order to maintain calbistrin A's integrity. The nature of the light instability was elucidated using the dimethyl acetal 2, which isomerized upon exposure to light leading cleanly to an equilibrium mixture of 2 and 3. Independent of the light-catalyzed instability, alkaline conditions also caused decomposition of 1, leading to the formation of 4 (Figure 1).Success in isolating as well as biologically and chemically characterizing calbistrin A depended upon minimizing the decomposition of the compound during manipulations. The influences of a number of experimental parameters were investigated in order to understand the origins of the instability. These parameters included temperature, light, pH, solvent, compound concentration, and oxygen. The results of these studies indicated that light and p H were the two major factors leading to compound degradation and that temperature, compound concentration, and the presence of oxygen did not contribute to the instability. Solvent effects were found to be dependent on the presence of light.The key role played by light was evident when solutions of calbistrin A remained stable at room temperature when stored in the dark. This observation was independent of the solvent used, the concentration ofcompound and whether the solution was exposed to air. In contrast, the same solutions decomposed when exposed to ambient laboratory light. Radical scavengers such as BHA (0.2%), Tinuvin 144 and 622 LD, and Chimassorb 944 FL (200 p,g/ml) did not ameliorate the effects of light. Reversed-phase hplc analysis indicated different light-catalyzed decomposition profiles depending upon whether a protic or an aprotic solvent was used (Figure 2). For example, exposure of a MeOH solution of calbistrin A to light resulted in the partial disappearance o...