A fully automated high-throughput liquid-liquid extraction (LLE) methodology has been developed for preparation of biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation. The 96-well LLE plate is made of a 96-well filter plate filled with inert diatomaceous earth particles, allowing continuous and efficient extraction of analytes between the aqueous biological sample and the organic extraction solvent. Two carboxylic acid-based protease inhibitor compounds with high and low levels of plasma protein binding were chosen for the development and application of the automated methodology. The LLE extracts of the plasma samples of the two compounds were analyzed by high-performance liquid chromatography with electrospray (ESI) tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the negative ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized LLE method, a formate buffer solution was first loaded into a 96-well filter plate packed with inert diatomaceous earth material. Then crude plasma samples and a water-immiscible organic solvent, methyl ethyl ketone, were sequentially added to the LLE plate so that LLE would occur in the interface between the two liquid phases on the surface of individual particles in each well. The organic eluate containing extracted analytes was evaporated and reconstituted for LC-MS/MS analysis. This fully automated LLE methodology avoids several disjointed steps involved in a manual or semiautomated LLE method, leading to significantly reduced sample preparation time, increased sample throughput, and clean sample extracts for improved ESI-MS/MS detection. The automated LLE methodology is universal and can be employed for sample preparation of other biological fluids. The complete bioanalytical method, based on the automated LLE and fast gradient LC-MS/MS, was validated and successfully applied to the quantitative analysis of protease inhibitors in rat plasma.
High-grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We have developed potent synthetic analogues of a class of compounds known as phyllanthusmins, inspired by natural products isolated from Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its cytotoxic effects by inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition may be required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration where it significantly inhibited the growth of cancer cell lines in hollow fibers, as well as reduced tumor burden in a xenograft model. We demonstrate that PHY34 acts as a late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single agent against HGSOC This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating ovarian cancer. .
Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.
Acid-catalyzed intramolecular cyclization of S-cyclodecynone (1) under a variety of conditione givee bicyclo-[4.4.0]-1(6)-decen-2-0ne (6) as the only product. In a recent report, evidence for a reaction mechanism involving tranaannular triplebond participation with a polarized carbonyl group followed by attack of the original carbonyl oxygen on the developing vinyl cation to give an unstable oxete intermediate (4) was presented. Subsequently, several workers have euggested a mechanism involving acid-catalyzed enolization of 1, followed by the tranaennular attack of the enol double bond at the acetylenic function, as the first two step in an alternate procew for explaining this rearrangement. When 6-octyn-2-one (9) was treated with either mineral or Lewis acids, a mixture of l-acetyl-2-methyl-l-cyclopentene (12) and 2,3-dimethyl-2-cyclohexen-l-one (14) were produced. Experimenta have shown that the oxygen atoms in the etarting acelylenic ketone 9 are the same onee found in the cyclic products 12 and 14. A mechanism involving cyclization of enol intermediatea cannot account for the formation of 14. On the 0 t h hand, formation and rearrangement of oxete intermediatee demonstrata how both 12 and 14 are produced.The results from acid-catdyzd cyclization of 6-oc@n-2-one (9) are also compared to those reported for the eolvolysis of the taylata of 6-0ctyn-2-01 (18). of the NSF-ILI program (Grant No. USE8661276) for funds used in the purchase of the GC/MS system and the UT Martin Faculty Rmearch Office for its support of this work. -try No. 9,2439546-0; 12,3168-90-9; 14,1122-2@9; ethyl propionylacetate, 4949-44-4; vinyl ketone, 78-94-4; ethyl 50x0-2-propionylhexanoate, 55262-18-5; 2,3-epoxy-2,3-dimethylcyclohexanone, 35502-46-6.(19) Schreiber, J.; Felix, D.; Eechenmoeer, A.; Winter, M.; Gautachi, F.; schulte-ate, K. H.; Sundt, E.; Ohloff, G.; Kalvoda, J.; Kaufmann, H.; Weiland, P.; h e n , G. Helu. Chim. Acta 1967,50,2101.
developed a liquid chromatography-mass spectrometry technique for the quantitation of the soy-associated isoflavone phytoestrogens genistein and daidzein and their conjugates in human urine.Free isoflavones were extracted from 1 ml urine adjusted to pH to 6.0 and then extracted with 3 X 5 ml diethyl ether. "Total" isoflavone conjugates were measured by incubation of 1 ml of urine in 1 mol/L ammonium acetate, pH 6.0, with 100 units sulfatase (type V aryl-sulfatase from Helix pomatia, a preparation that contains 15^10 units of sulfatase activity/per milligram and 400-600 units/mg of /3-glucuronidase activity) at 37°C for 3 hours before ether extraction. These conditions were found to be sufficient to completely hydrolyze urine spiked with phenolphthalein glucuronide and 4methylumbelliferone sulfate. Glucuronide conjugates were measured by preincubation with 1000 units of jß-glucuronidase (type B-l from Arkansas Children's Nutrition Center, Little Rock, AR. bovine liver) before ether extraction. Sulfate conjugate values were calculated by subtraction of the free and glucuronide values from the "total" values. All the enzymes were purchased from Sigma Chemical Company (St. Louis, MO).Organic extracts were blown down to dryness and resuspended in 1 ml 50% methanol containing 10 mmol/L ammonium acetate and acetic acid (buffer A); 200 /xl was injected onto a Shimadzu SIL-10AXL high-performance liquid chromatography (HPLC) instrument fitted with a 15 cm x 4.6 mm X 3 /xm Supelcosil ABZ+Plus column. Isoflavones were separated using a 50:50 solution of methanol:10 mmol/L ammonium acetate, pH 5.0 (buffer A) and a 80:20 solution of methanol:10 mmol/L ammonium acetate, pH 7.0 (buffer B), with a linear gradient of 50% buffer B to 99% buffer B over 5 minutes, followed by a 9-minute isocratic elution in 99% buffer B. The compounds were detected by a PE Sciex AP-100 Mass Spectrometer interfaced with the HPLC via a heated nebulizer operated at 530°C with nitrogen as nebulizer gas and an after-column infusion of 50% aqueous ammonium hydroxide. Isoflavones were detected by positive-ion selective ion monitoring (SIM). Daidzein (mass ion, 255.2) eluted at 9.4 minutes, and genistein (mass ion, 271.2) at 10.9 minutes. Biochannin A 203
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