Our research focuses on the application of chromatography mass spectrometry in the field of nucleic acid, peptide protein, and CDT. The challenge of this experiment lies in the separation of multiple components of LNPs and determination of components with significant different concentrations and residual issues. The advantage of this protocol is that it achieves good separation, a wide linear range, and a current determination of LNPs at a low cost.
To begin, set up a high performance liquid chromatography, or HPLC system, coupled with an evaporative light scattering detector. Install the chromatographic column following the arrow direction on the column. Connect one end to the autosampler outlet, and the other to the evaporative light scattering detector inlet.
To prepare phase A, accurately measure and transfer 1, 387 microliters of triethylamine and dissolve it in 1000 milliliters of water. Mix well and adjust the pH to seven using acetic acid. To prepare Phase B, dissolve 1, 387 microliters of triethylamine in 1000 milliliters of methanol.
Mix thoroughly and adjust the pH to seven with acetic acid. Place phase A and phase B in the tray. Launch the LabSolutions software and open the realtime analysis window.
Click on File and select New to create a new method file, modify the liquid chromatography stop time to 15 minutes, and click Apply to All acquisition time. Then click on Pump and modify the pump parameters. Select the analysis mode as binary high pressure gradient, or BGE, and set the flow rate to one milliliter per minute.
Set the initial pump B concentration to 80%Modify the mobile phase gradient at three minutes to 80%at four minutes to 85%from 5.5 to 12 minutes at 100%and at 12.1 minutes, let pump be returned to 80%Now click on Column Oven and set the oven temperature to 55 degrees Celsius. Then click on ELSD to modify the drift tube temperature to 40 degrees Celsius and save the method. Click on Download and start up to start and equilibrate the instrument.
Precisely weigh 10 milligrams of each of the four lipid nanoparticle component standard substances separately. Dissolve each in one milliliter of methanol and vortex until completely dissolved to obtain stock solutions with a concentration of 10 milligrams per milliliter for each component. Using a pipette, accurately transfer 100 microliters of each stock solution into a sample vial.
Add 600 microliters of methanol and mix thoroughly to prepare a mixed intermediate solution one with a concentration of 1000 micrograms per milliliter. Now, accurately transfer 20 microliters of each stock solution into a sample vial. Add 920 microliters of methanol and mix thoroughly to prepare a mixed intermediate solution two with a concentration of 200 micrograms per milliliter.
Prepare a series of standard solutions at different concentrations by serial dilution. Next, using a pipette, accurately transfer 100 microliters of the sample into a sample vial. Then add 900 microliters of methanol and mix well to ensure a tenfold dilution of lipid nanoparticles and their dissociation from the nucleic acid drug.
Place the standard solutions and sample solutions into the autosampler of the liquid chromatograph. Click on realtime batch in the assistant toolbar of the realtime analysis window. Next, click New in the File menu to create a new batch table.
In the batch table, input the vial number, tray name, data file, and the injection volume of the standard and sample solutions. Select the saved method file and click save batch file in the File menu. Wait until the liquid chromatograph pressure and the baseline of the chromatogram are stable.
Then click on start real-time batch in the assistant toolbar to start data acquisition. For data analysis, open the LabSolutions software's browser window. Drag the standard solution data into the quantitative results view to establish a calibration curve.
Modify the data processing parameters by clicking on Edit in the method view. In the integration parameters window, change the integration algorithm to I-PeakFinder and set the baseline type to Base to Base. Then, in the identification parameters, change the identification method to band and set the default bandwidth to 0.1 minutes.
To modify the quantitative parameters, change the quantitative method to external standard. Set the number of calibration levels to seven. Then select the calibration curve type as exponentially and modify the compound settings.
Input the names and standard solution concentrations of the four lipid nanoparticle components. Double click on the peak apex to update the retention times. Click View to complete the modification of the data processing parameters.
Now, modify the sample type in the quantitative results view to standard calculate point. Set the levels of the standard solution from one to seven according to the concentrations. Once the calibration curves are established, click File in the menu bar and Save the method file.
Drag the sample data into the quantitative results view of the browser window. Observe the displayed concentration results of the four lipid nanoparticle components in the samples. The LNP standard solution analysis achieved baseline separation with a separation degree greater than 1.5 for the four components, and no residuals were observed in high concentration standard solutions.
The calibration curve for the four components exhibited correlation coefficients exceeding 0.999 within the concentration range of five to 250 micrograms per milliliter, indicating excellent linearity. The method showed high reproducibility, with a relative standard deviation for a retention time of less than 0.1%and a relative standard deviation for the peak area of less than 2%based on six repeated injections of 10 micrograms per milliliter standard solution. The analysis of actual LNP samples diluted 10 times with methanol showed component concentrations ranging from 150 micrograms per milliliter to 1, 500 micrograms per milliliter, achieving consistently high separation and sensitivity across multiple manufacturers.
