Name: Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector (Video) | JoVE Journal
Uploaded: 2025-05-30T12:50:00.000+00:00
Duration: 8 min 54 s
Description: Learn quantitative analysis of lipid nanoparticle components in RNA delivery systems using HPLC with evaporative light scattering detection. This method achieves excellent separation, wide linear range, and cost-effective determination.

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5 minutes

Thank biopharmaceutical companies for supplying the LNP samples for the experiment, and gratitude to Shimadzu (Shanghai) Global Laboratory Consumables Co., Ltd for providing the chromatography column.

1. Preparation of instrument
<ol>
	<li>Instrument setup
	<ol>
		<li>Set up an HPLC system coupled with an ELSD.</li>
	</ol>
	</li>
	<li>Column installation
	<ol>
		<li>Install the chromatographic column (C4-300; 150 mm &#215; 4.6 mm I.D., 5 &#181;m), following the arrow direction on the column, with one end connected to the autosampler outlet and the other to the ELSD inlet.</li>
	</ol>
	</li>
	<li>Mobile phase preparation
	<ol>
		<li>Prepare the mobile phases: A phase, a 10 mM TEAA aqueous solution (pH = 7.0), and B phase, a 10 mM TEAA methanol solution (pH = 7.0).</li>
		<li>To prepare A phase, accurately weigh 1.01 g of triethylamine, dissolve it in 1000 mL of water, mix well, and adjust the pH to 7.0 with acetic acid.</li>
		<li>To prepare B phase, dissolve 1.01 g of triethylamine in 1000 mL of methanol, mix, and adjust the pH to 7.0 with acetic acid.</li>
	</ol>
	</li>
	<li>Method editing
	<ol>
		<li>Turn on the power switches for each module of the LC-ELSD and launch the LabSolutions software. Then, open the Realtime Analysis window.</li>
		<li>Click on File and select New to create a new method file. Modify the LC Stop Time in the method to 15 and click Apply to all Acquisition Time.</li>
		<li>Then, click on Pump to modify the pump parameters. Select the analysis mode as Binary High-Pressure Gradient (B.GE) and set the flow rate to 1.0 mL/min.</li>
		<li>Then, set the initial Pump B Concentration (%B) to 80% and modify the mobile phase gradient: at 3 min, %B is 80%, at 4 min, %B is 85% from 5.5 min to 12 min, %B is 100%, at 12.1 min, %B returns to 80%.</li>
		<li>Next, click on Column Oven and set the oven temperature to 55 &#176;C. Finally, click on ELSD to modify the drift tube temperature to 40 &#176;C. Save the method. 
		NOTE: The summary of parameters is shown in Table 1.</li>
	</ol>
	</li>
	<li>Start and equilibrate the instrument.
	<ol>
		<li>Click on Download and Startup to start and equilibrate the instrument.</li>
	</ol>
	</li>
</ol>
2. Preparation of standard solutions and samples
<ol>
	<li>Standard solutions preparation
	<ol>
		<li>Precisely weigh 10.0 mg of the four LNP component standard substances separately, dissolve in 1 mL of methanol, and vortex until completely dissolved to obtain stock solutions with a concentration of 10 mg/mL for each component.</li>
		<li>Using a pipette, accurately transfer 100 &#181;L of each of the four stock solutions into a sample vial, then add 600 &#181;L of methanol. Mix thoroughly to prepare a mixed intermediate solution 1 with a concentration of 1000 &#181;g/mL.</li>
		<li>Accurately transfer 20 &#181;L of each of the four stock solutions into a sample vial, then add 920 &#181;L of methanol. Mix thoroughly to prepare a mixed intermediate solution 2 with a concentration of 200 &#181;g/mL.</li>
		<li>Next, prepare a series of standard solutions at concentrations of 5 &#181;g/mL, 10 &#181;g/mL, 20 &#181;g/mL, 50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL, and 250 &#181;g/mL by serial dilution according to Table 2.</li>
	</ol>
	</li>
	<li>Samples preparation
	<ol>
		<li>Using a pipette, accurately transfer 100 &#181;L of the sample into a sample vial, then add 900 &#181;L of methanol and mix well. At this point, ensure that the LNP concentration in the sample is diluted 10-fold and the LNP is dissociated from the nucleic acid drug.</li>
	</ol>
	</li>
</ol>
3. Data acquisition
<ol>
	<li>Place the standard solutions and sample solutions into the autosampler of the liquid chromatograph.</li>
	<li>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.</li>
	<li>In the batch table, input the Vial#, Tray Name, the Data file, and the injection volume of the standard and sample solutions, and select the method file saved in step 1.4. Click Save Batch File in the File menu to save the batch file.</li>
	<li>Wait until the liquid chromatograph pressure and the baseline of the chromatogram are stable. Then, click on Start Realtime Batch in the assistant toolbar to start data acquisition.</li>
</ol>
4. Data analysis
<ol>
	<li>Establish calibration curves
	<ol>
		<li>Open the LabSolutions software&#39;s Browser Window and drag the standard solution data into the Quantitative Results View to establish a calibration curve. Next, modify the data processing parameters. Click on Edit in the Method View.</li>
		<li>In the Integration Parameters window, change the slope value to 10000. Then, in the Identification Parameters, change the identification method to Band and set the Default Bandwidth to 0.1 min.</li>
		<li>After that, modify the Quantitative parameters. Change the Quantitative Method to External Standard, and set the # of Calibration Level to 7. Select the calibration curve type as Exponentially. Next, modify the Compound.</li>
		<li>Input the names and standard solution concentrations of the four LNP components. Double-click on the peak apex to update the retention times.Finally, click View to complete the modification of the data processing parameters.</li>
		<li>Modify the Sample Type in the Quantitative Results View to Standard (Calc. Point). According to the concentrations, set the levels of the standard solution from 1-7. Once the calibration curves are established, click File in the menu bar and save the method file.</li>
	</ol>
	</li>
	<li>Quantitative analysis of samples
	<ol>
		<li>Drag the data of samples into the browser window&#39;s Quantitative Results View, which will display the concentration results of the four LNP components in the samples.</li>
	</ol>
	</li>
</ol>

<ol><li>Zong, Y., Lin, Y., Wei, T., Cheng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+nanoparticle+%28LNP%29+enables+mRNA+delivery+for+cancer+therapy%5BTitle%5D)+AND+%22Adv+Mater%22%5BJournal%5D)">Lipid nanoparticle (LNP) enables mRNA delivery for cancer therapy.</a> Adv Mater. 35 (51), e2303261(2023).</li>
<li>Vlatko, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-immunotherapy+application+of+LNP-mRNA%3A+Maximizing+efficacy+and+safety%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Non-immunotherapy application of LNP-mRNA: Maximizing efficacy and safety.</a> Biomedicines. 9 (5), 530(2021).</li>
<li>Eygeris, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemistry+of+lipid+nanoparticles+for+RNA+delivery%5BTitle%5D)+AND+%22Acc+Chem+Res%22%5BJournal%5D)">Chemistry of lipid nanoparticles for RNA delivery.</a> Acc Chem Res. 55 (1), 2-12 (2021).</li>
<li>Sun, D., Lu, Z. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+function+of+cationic+and+ionizable+lipids+for+nucleic+acid+delivery%5BTitle%5D)+AND+%22Pharm+Res%22%5BJournal%5D)">Structure and function of cationic and ionizable lipids for nucleic acid delivery.</a> Pharm Res. 40 (1), 27-46 (2023).</li>
<li>Rebecca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+nanoparticle+formulations+for+enhanced+co-delivery+of+siRNA+and+mRNA%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Lipid nanoparticle formulations for enhanced co-delivery of siRNA and mRNA.</a> Nano Lett. 18 (6), 3814-3822 (2018).</li>
<li>Maugeri, M., Nawaz, M., Papadimitriou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linkage+between+endosomal+escape+of+LNP-mRNA+and+loading+into+EVs+for+transport+to+other+cells%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.</a> Nat Commun. 10, 4333(2019).</li>
<li>Renee, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+PEGylation+on+LNP+based+mRNA+delivery+to+the+eye%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The effects of PEGylation on LNP based mRNA delivery to the eye.</a> PLoS One. 15 (10), e0241006(2020).</li>
<li>Benedicto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+phospholipid+chemistry+for+improved+lipid+nanoparticle+%28LNP%29+delivery+of+messenger+RNA+%28mRNA%29%5BTitle%5D)+AND+%22Biomater+Sci%22%5BJournal%5D)">Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).</a> Biomater Sci. 10 (2), 549-559 (2022).</li>
<li>Kamanzi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+visualization+of+lipid+nanoparticle+fusion+as+a+function+of+formulation+and+process+parameters%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Quantitative visualization of lipid nanoparticle fusion as a function of formulation and process parameters.</a> ACS Nano. 18 (28), 18191-18201 (2024).</li>
<li>Melissa, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+lipid+nanoparticles+for+the+delivery+of+nebulized+therapeutic+mRNA+to+the+lungs%5BTitle%5D)+AND+%22Nat+Biomed+Eng%22%5BJournal%5D)">Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs.</a> Nat Biomed Eng. 5 (9), 1059-1068 (2021).</li>
<li>Packer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mechanism+for+the+loss+of+mRNA+activity+in+lipid+nanoparticle+delivery+systems%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.</a> Nat Commun. 12 (1), 6777(2021).</li>
<li>Snyder, L. R., Kirkland, J. J., Dolan, J. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSnyder%2C+LR+%22Introduction+to+Modern+Liquid+Chromatography%22">Introduction to Modern Liquid Chromatography</a>. , John Wiley & Sons. Hoboken, NJ. (2010).</li>
<li>Zhang, L. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+mRNA-LNP+components+of+two+globally+marketed+COVID-19+vaccines+on+efficacy+and+stability%5BTitle%5D)+AND+%22NPJ+Vaccines%22%5BJournal%5D)">Effect of mRNA-LNP components of two globally marketed COVID-19 vaccines on efficacy and stability.</a> NPJ Vaccines. 8 (1), 156(2023).</li>
<li>Zhang, Y., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performance+comparison+of+different+ELSD+detectors+in+pharmaceutical+analysis%5BTitle%5D)+AND+%22Anal+Methods%22%5BJournal%5D)">Performance comparison of different ELSD detectors in pharmaceutical analysis.</a> Anal Methods. 7 (12), 4800-4807 (2015).</li>
</ol>

The authors have nothing to disclose.

All four components of LNP exhibit a relatively weak polarity. When analyzed using a C18 column, the target compounds demonstrate strong retention within the column, leading to the broadening of the phospholipids peak for strongly retained components. Furthermore, after analyzing high-concentration solutions, residuals accumulate at the head of the C18 column, which could compromise the accuracy of quantification. The chromatogram is shown in Figure 5.
To optimize...

Lipid nanoparticle (LNP) is one of the most advanced non-viral gene delivery systems, mainly used for the delivery of nucleic acid drugs, such as antisense oligonucleotides, small interfering nucleotides, mRNA, etc1,2,3. LNP is generally composed of four components: ionizable lipids, polyethylene glycol (PEG) modified lipids, phospholipids, and cholesterol. The key component is ionizable lipids, which are the decisive factor in the efficiency of nucleic acid drug delivery. The other three components can increase the stability of LNP and reduce the recognition of the immune system4,5,6.
The varying ratios of the four components in LNP can significantly impact the self-assembly process of the nanoparticles, affecting properties such as the encapsulation efficiency of the active pharmaceutical ingredient (API), its delivery efficacy, and the rate of release in vivo7,8,9. Therefore, it is essential to establish a quantitative analysis method for LNP components, thereby facilitating the synthesis of an optimal LNP delivery system10,11.
High-performance liquid chromatography (HPLC) is a contemporary analytical technique widely employed in pharmaceutical analysis and is esteemed for its high efficiency, sensitivity, and automation capabilities. HPLC encompasses various detectors, including conventional ultraviolet (UV) detectors, as well as general-purpose detectors such as refractive index detectors (RID), evaporative light scattering detectors (ELSD), charged aerosol detectors (CAD), and highly selective radio frequency (RF) detectors, among others12.
In the case of analyzing the four components of LNP, which lack distinct UV-absorbing groups and thus yield no response in UV detectors, the use of general-purpose detectors was considered. Although RID is one such option, it is not suitable for gradient analysis and offers relatively lower sensitivity compared to the other two general-purpose detectors; hence, it was not chosen12. Furthermore, while CAD detectors boast high sensitivity, their prohibitive cost renders them less appealing12.
Consequently, the ELSD detector was selected. This detector features a distinctive gain mode option - the &#39;wide&#39; mode - which enables simultaneous analysis of both high concentration and trace components, enhancing sensitivity and expanding the linear range.
This article employs an HPLC system coupled with ELSD, achieving efficient separation and quantitative analysis of the four components of LNP through optimization of chromatographic columns, gradient elution of mobile phases, and other conditions13.

<table><tbody><tr><td>Acetic acid</td><td>ANPEL Laboratory Technologies (Shanghai) Inc.</td><td>CAEQ-4-000319-0050</td><td></td></tr><tr><td>Balance</td><td>Shimadzu (Shanghai) Global Laboratory Consumables Co.,Ltd</td><td>AP135W</td><td></td></tr><tr><td>Column</td><td>Shimadzu (Shanghai) Global Laboratory Consumables Co.,Ltd</td><td>ShimNex WP C4(380-01235-74)</td><td></td></tr><tr><td>ELSD</td><td>Shimadzu (China) CO.,LTD</td><td>ELSD-LT III(228-65900-46)</td><td></td></tr><tr><td>LabSolutions Software</td><td>Shimadzu (China) CO.,LTD</td><td></td><td></td></tr><tr><td>LC</td><td>Shimadzu (China) CO.,LTD</td><td>LC40D XR</td><td></td></tr><tr><td>Methanol</td><td>ANPEL Laboratory Technologies (Shanghai) Inc.</td><td>CAEQ-4-003302-4000</td><td></td></tr><tr><td>Pipette-100 &amp;micro;L</td><td>Shimadzu (Shanghai) Global Laboratory Consumables Co.,Ltd</td><td>00-NAR-100</td><td></td></tr><tr><td>Pipette-1000 &amp;micro;L</td><td>Shimadzu (Shanghai) Global Laboratory Consumables Co.,Ltd</td><td>00-NAR-1000</td><td></td></tr><tr><td>TEA</td><td>ANPEL Laboratory Technologies (Shanghai) Inc.</td><td>CAEQ-4-012190-0250</td><td></td></tr><tr><td>Ultra-pure Water System</td><td>Millipore</td><td>ZIQ7000T0C</td><td></td></tr><tr><td>Vortex Mixer</td><td>ANPEL Laboratory Technologies (Shanghai) Inc.</td><td>VM-B</td><td></td></tr></tbody></table>

determining four components in a lipid nanoparticle rna delivery system by liquid chromatography combined with evaporative light scattering detector

Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector

This article presents a method for the analysis of lipid nanoparticle (LNP) components. LNP, serving as a pivotal vector for RNA-based drugs, primarily consists of cholesterol, PEG with modifications, ionizable lipids, and helper lipids. These components exhibit weak polarity, leading to strong retention and difficulty in separation using reverse-phase chromatography, as well as lacking distinct ultraviolet absorption characteristics. In order to address this challenge, a liquid chromatography system was coupled with an evaporative light-scattering detector (ELSD). By systematically adjusting the type of chromatographic column and optimizing the gradient elution program of the mobile phase, rapid and complete baseline separation of the four critical components was accomplished. Utilizing the latest ELSD technology enhanced detection sensitivity significantly and expanded the linear range of the method. Experimental results demonstrated that within a concentration range of 5 &#181;g/mL to 250 &#181;g/mL, the four components of LNP displayed excellent linearity with correlation coefficients all greater than 0.999, and the accuracy ranged from 94.2% to 108.0%. In precision experiments, the relative standard deviations of both retention time and peak area for a 10 &#181;g/mL standard solution were below 0.1% and 2%, respectively. When using this method to analyze different LNP samples, all components are successfully separated, and their respective contents are accurately quantified, highlighting the robust adaptability of this analytical approach.

The analysis of the LNP standard solution using this method achieved a separation degree greater than 1.5 for the four components, realizing baseline separation. Furthermore, no residuals were observed in high-concentration standard solutions, as depicted in Figure 1. A calibration curve was established within a concentration range of 5 to 250 µg/mL, exhibiting correlation coefficients all exceeding 0.999, indicating a broad linear range and excellent linearity. The calibration curve is illu...

Here, we establish a quantitative analysis method for four components in a lipid nanoparticle (LNP) RNA delivery system using high-performance liquid chromatography combined with an evaporative light scattering detector (ELSD). The method has good separation, high sensitivity, and high efficiency.

This article presents a method for the analysis of lipid nanoparticle (LNP) components. LNP, serving as a pivotal vector for RNA-based drugs, primarily ...

determining-four-components-lipid-nanoparticle-rna-delivery-system

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Learn quantitative analysis of lipid nanoparticle components in RNA delivery systems using HPLC with evaporative light scattering detection. This method achieves excellent separation, wide linear range, and cost-effective determination.

Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector (Video) | JoVE Journal

Determining Four Components in a Lipid Nanoparticle RNA Delivery System by Liquid Chromatography Combined with Evaporative Light Scattering Detector

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