The research work involve simultaneous determination of three drugs in a triple nanof0rmulated drug sample. The technique used was LC-MS-MS and the procedure used is as follows: 10mg of the nano-formulated samples were accurately weighed and suspended in 5.0ml of 0.5% glacial acetic acid in methanol. The suspended nano-formulated solution was sonicated for 3 minutes and centrifuged for 20 mins at a speed of 3000 rpm at 4°C. 20.0µl of the supernatant solution was transferred into 1.5ml Eppendorf tube and sieved further using 0.25µm filter. 100.0µl of internal standard with a concentration of 1µg/ml was added into a 20.0µl supernatant solution. The solution was further spiked with 100µl of 100ng/ml of drug A standard, 100µl of 300ng/ml of drug B standard and 100µl of 3000ng/ml of drug C standard and topped up to 1000µl using formic acid formate buffer. The buffer constituted of 0.5% formic acid in ammonium formate and 0.5% formic acid in acetonitrile in the 30:70, v/v. The supernatant was transferred into a 1.5 µl autosampler vial ready for LC-MS-MS analysis. 10. 0µL was injected into the chromatographic column.
My question is; assuming that the concentration of drug A is 98ng/ml from the LC/MS/MS data,
1. Should I use this concentration directly in calculating the percentage recovery?
2. Or is there a dilution factor that I need to consider?
3. How do I use the dilution factor in the calculation of % recovery?
You usually do not determine the exact amount/concentration of the analyte that went into the MS system. This is hard to predict because transferring analytes from the injector to the detector in the MS is not quantitative. There is always a loss of analytes in the system, mainly in the ion source - not every molecule in the electrospray enters the MS.
What you usually have to do is use an internal or external calibration standard. For example, you can construct a calibration curve for your analyte(s). For this, you have to prepare the samples the same way you will prepare your real sample. This way, dilution factors and analyte loss/gain cancel out. Since you know the starting concentrations of calibration curve standards, you can use that calibration curve to get the starting concentration of your analyzed sample.
- Badges
- Science topic