Liquid-liquid extraction technique is used to extract the drug from plasma. No interference was observed in plasma which was purchased from the hospital. When the pharmacokinetic study was done in humans, the freshly prepared plasma obtained from the human volunteer and extracted with the same procedure produced interference. The blood was drawn before the drug was administered to subject and then plasma was separated by centrifuge technique. The blood samples were from female volunteers. I used HPLC with a fluorescence detector (excitation 202 nm, emission 310 nm). Any help is appreciated.
Did you use the same anti-coagulant on both the occasions. Different anti-coagulants give a varied interference during bioanalysis. Also, the interference during fluorescent detection could be attributed to hemolysis, if any, and variation in LLE.
I used heparin as anti-coagulant during blood collection from volunteer. During method development, plasma prepared in hospital was purchased. I have not found heparin showing fluorescence property without derivatization. LLE steps were fixed for both occasion.
You can try plasma from different sources to check whether you still get the same interference. As per FDA, one needs to check matrix interference from six different sources.
As long as you have standardized the LLE conditions and the anticoagulant used as what Rohitash mentioned, so it has nothing to do with the collected plasma. Therefore, I believe the plasma that you purchased is purified from certain proteins or plasma components. However, I suggest to use the volunteers plasma to develop the method and run your experiment because this is simulating the reality and reflecting the actual picture of plasma in HPLC, try to change the excitation-emission wave lengths using the volunteers plasma.
The retention time of the drug is varying? If it is varying, your problem is analytical.
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