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27th July 2012 @ 01:20
PZQ was selected as a standard for monitoring the formation of MNR41 due to it's similarity to the product. PZQ was initially investigated at different concentrations to show if it had a linear relationship between concentration and peak area. Stock solution 1 was made by dissolving PZQ (0.006 g) in MeCN (1.8 mL) giving a concentration of 3333.33 ug/mL. Stock solution 1 (0.2 mL) was then dissolved in (1.6 mL) giving stock 2 a concentration of 370.4 ug/mL. Stock 2 (0.08 mL) + MeCN (0.4 mL) gave LCMS sample of 61.7 ug/mL. Stock 2 (0.07 mL) + MeCN (0.4 mL) gave LCMS sample of 55.2 ug/mL. Stock 2 (0.06 mL) + MeCN (0.4 mL) gave LCMS sample of 48.3 ug/mL. Stock 2 (0.04 mL) + MeCN (0.4 mL) gave LCMS sample of 33.7 ug/mL. Stock 2 (0.03 mL) + MeCN (0.4 mL) gave LCMS sample of 25.8 ug/mL. Stock 2 (0.02 mL) + MeCN (0.4 mL) gave LCMS sample of 17.6 ug/mL. Stcok 2 (0.01 mL) + MeCN (0.4 mL) gave LCMS sample of 9.0 ug/mL. Running the samples in the LCMS with the method 0to50ov30 Water, 0.1 % formic acid, acetonitrile, 0.1% formic acid measuring at 210 nm produced the following calibration plot. *This method was selected as it was the shortest run time where we could achieve baseline separation between the MNR41 and PZQ. [b]Testing The Work Up Conditions.[/b] Following the reaction by LCMS requires a mini workup. There is an area of doubt here as to how much standard and how much product is extracted in the single extraction. Therefore the following was conducted. MNR41 (56 mg) and PZQ (5 mg) was dissolved in MeCN (5 mL). Three aliquots (0.1 ml) were taken. Aliquot 1 - no further workup - just concentrated to dryness. MeCN (1.8 mL) added and 0.04 ml was taken and diluted with MeCN (0.3 mL) Aliquot 2 - EtOAc (0.2 mL) add and sodium hydrogen carbonate (0.2 ml) added and organics extracted roughly by pipette then concentrated to dryness. MeCN (1.8 mL) added and 0.04 ml was taken and diluted with MeCN (0.3 mL) Aliquot 3 - EtOAc (0.4 mL) add and sodium hydrogen carbonate (0.2 ml) added and organics extracted roughly by pipette then concentrated to dryness. MeCN (1.8 mL) added and 0.04 ml was taken and diluted with MeCN (0.3 mL) Aliquot 1 - Product peak area 27,287,433 - 93.0% PZQ peak area 2,044,010 - 7.0% [data]4730[/data] Aliquot 2 - Product peak area 14,938,837 - 92.4% PZQ peak area 1,235,799 - 7.6% [data]4732[/data] Aliquot 3 - Product peak area 20,056,141 - 92.4% PZQ peak area 1,642,434 - 7.6% [data]4734[/data] Conclusion - A single extraction does not extract all of the product or PZQ but it does extract then at the same ratio therefore this procedure can be used to calculate yield.
Attached Files
mnr41-7-PZQ_plot.PNG
mnr41-7_Aliquot 1.pdf
mnr41-7_Aliquot 2.pdf
mnr41-7_Aliquot 3.pdf