Hi

Looks sidenafil is stable in room temperature 24 h (and 100h in 10 oC) see results of current study:

https://medwinpublishers.com/BEBA/BEBA16000113.pdf

https://innovareacademics.in/journal/ijpps/Vol6Issue5/9629.pdf

Maybe its problem with recovery or IS and deproteinisation step? I found some people used ice for preparation of acetonitrile before this step only " Nine milliliters of ice-cold acetonitrile was added" ....

https://watermark.silverchair.com/30-1-14.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAApswggKXBgkqhkiG9w0BBwagggKIMIIChAIBADCCAn0GCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMIHKlC0tAXs9C30H_AgEQgIICTr2cLHeNhj9_GQqUlnl4SOkWXDP-nVUY3bl-egvyVSr2KX-jHi7QNkaqdGuMpDoHz57OSdm-E8b9qrLEXdY7bWubmVDHHA5U1Q7hKoo69cV6Yl5MsXkoQk0d4PchOklzBgR4cO5dRE8I_V7a1ig7nxN4ezIRjXUABOjJS5Cv0vkP8qmuR-fyzlppCztICP342dCV_XU27F10neb1d89P_DiV7CNudFmzbLK5ObZ4r4Nsjzc-7ptT2Lrj1EXGzMQ-YWg2ZKIL5X8DlDiiEiwKFFqA2CYLICL3V5zXZ0hf-u_k1EN9-eeG_waAnRgUocQ6UjZFHOR9ctCe9G1UaafKYseVB7pNDhcm0Izut2WZOcqS1aC6fW7qnrfhCKn4TeIwPXSvT879gh3RtbQFOc7zhuiX5LpuiCEgDof6kfKHk9-Bn1Ewb3wZbmrfZrGrptT83eZCJ_LgfrK4GHtqhZuqP5J_b2JI66GaPR8fugbdtV8C4MDnRtysZZqkfWFHjXpqUdR13AB6m0dxkZzJp4m7yTnEcN_C5Lzbp40evKnb6fDuxhMSS0B4lTpcy9Pz2cRVBhDQmO3m0ztn8y6zGT83Z1zN7XnwEPOhi0r411ur91Jw9dl4zTcbG98nmMyL1SEg9w5pG-IUBZ20l2R12smWoI2Y809vx5bo9lqTN_2hddwc1uW91dA98o6V_0V2erY2hVmKjAyqx8L8h-mmOdSBPcgblhetSeTn8vZuBzF4qZ46Pqr4KLdEBAy3In4vkkdtuANBt96BT4V6hc6kfOop

Best regards

Tomasz

Hi Tomasz,

Many tahns for your time! But, in some subjects is not stable (please find the attached file, pag 1016), you need to work on ice, but I would like to know why.

Thanks a lot!!

Hi

If ice is a solution it means you slow down a chemical reactivity. If ice is the "gold standard" in the case of sildenafil then should be mentioned in more less all reports/publications ..... not sure about its current case. If metabolite rising but not sildenafil it's not effect of metabolism. From my point of view, the source of the problem could be the deproteinization method. It should be solved within validation (three freeze-thaw cycles). Your metabolite is quite lipophilic (XLogP3 = 1) so maybe it's the source of the problem that recovery for that metabolite levels rising because deproteinization could be different after multiple or single freezing? Maybe some kind of solution will be isotopically labeled N-desmethyl sildenafil as a second IS ..... for me it looks deproteinisation could be source of problem (need more attention inreltion to sample freezing) but maybe additionally second IS could be good solution to steady validation and the method....

Best regards

Tomasz

Dear Tomasz,

Thanks a lot. Why deproteinization could be different? Could you recommend any paper to read about this? They carried out the experiments at room temperatura and ice and the results are different.

Hi,

If deproteinisation in ice and in room temperature gives different recovery it means its not fully optimized step from chemical point of view. I dont know what deproinisation mixture was used in this case. From my experience its very critical step in method development and some times you need very composite deproteinisation mixture to provide stable and comparable day by day recovery and deproteinisation level. Some times for example within deproteinisation proces the drug is unhooked from proteins, but again connect with proteins. Im sure paper with full information about will not be available .... I think best way is checking each step of deproteinisation and optimize this steps using spiked blood samples after few freeze-thaw cycles and fresh ....

Best regards

Tomasz

Hi Tomasz,

Great. It has sense.

They used LLE (diethyl ether:dichloromethane, 70:30, v/v). For this situation, and with your experince, which extraction method could be better? SPE?

I am going to read and I will come back with you.

Thanks a lot for your time!

Hi

Sometimes LLE is easier than SPE. In case work with SPE some manual thinks could be very critical on effect of elution. Its the same situation like work with ... analytical column. Many factors have impact on elution profile but in case of SPE its not quite easy for optimization if you work manually. If you can work with automated SPE for example with Tecan or other solutions then its easier. For me LLE could be really good and not so expensive like SPE. Moreover LLE you can optimise in 2.0 mL Eppendorfs and work with 0.1-0.5 mL of deproteinisated sample for example. I worked many times with LLE in Eppendorfs volume and its works, easy to control, chip .... moreover if you want use freeze as a step to separate phases you can do it with LLE but not with SPE. Its very nice step when you want separate water phase you can mix many steps in one Eppendorf because Eppendorf could be centrifuged, SPE not ....for me LLE is more flexible, but some times mixing LLE and SPE is also nice way for sample preparation optimization .....

Best regards

Tomasz

Thanks!

This is not at all related to extraction method.....

Recently, I also faced the same issue. My method is LLE. Almost all the values for N-Desmethyl Sildenafil fails to meet the ISR criteria whereas 100% values are within acceptance for Sildenafil. After investigation, it was discovered that metabolite is not stable at ambient temperature in incurred sample. In ice cold water bath (below 10°C) there is no conversion.

No stability related issue was identified during method validation. The reason could be other fragile metabolite in incurred samples which is getting converted to N-desmethyl sildenafil at ambient temperature resulting in enhancement in metabolite concentration. The reaction/conversion stop when we use controlled temperature.