When performing mass spectrometry analysis of lipid classes using a lipidomics standard like the EQUISPLASH product from Avanti Polar Lipids, adding the standard to your test sample serves as an internal standard for quantification. This approach helps account for variations that can occur during sample preparation, extraction, and analysis. Here's how it works:

1. Internal Standard Principle:An internal standard is a compound that is added to both your standard samples and your test samples in a known amount. It's chemically similar to the analytes of interest but should be easily distinguishable in the mass spectrometry analysis. By adding a known amount of the internal standard, you can correct for variations that might occur during sample preparation and analysis.

2. Adding the Internal Standard:When you add the EQUISPLASH lipidomics standard to your test sample during the extraction process, it becomes a reference compound with a known concentration that you control. This internal standard will undergo the same extraction and analysis steps as your sample, which helps compensate for any losses, variations, or biases introduced during these steps.

3. Quantitative Data Analysis:To obtain quantitative data from your mass spectrometry analysis, you'll follow these steps:

• Measure the peak areas (or peak heights) of both your analytes of interest and the internal standard in your mass spectrometry data.
• Calculate the ratio of the peak area of your analytes to the peak area of the internal standard for each lipid class.

The rationale behind this is that the internal standard's known concentration serves as a reference point. If the extraction and analysis are consistent, the ratio of the analyte's peak area to the internal standard's peak area should be proportional to the ratio of their concentrations.

4. Calibration Curve:To convert the peak area ratio to quantitative concentration, you'll need to create a calibration curve. This involves analyzing a series of standard solutions with known concentrations of the lipid classes using the same extraction and analysis procedures. The calibration curve plots the concentration of the internal standard against the measured peak area ratio.

5. Quantification:Using the calibration curve, you can then determine the concentration of your lipid classes in your test samples based on the peak area ratio. The formula to calculate the concentration is derived from the linear relationship shown in the calibration curve.

By adding the internal standard and utilizing a calibration curve, you can obtain quantitative data for your lipid classes in your test samples, even considering any variations introduced during sample preparation and analysis. This internal standard approach enhances the accuracy and reliability of your quantitative results. Subhadip Kundu

Thank you for your elaborated explanation. I am realy grateful to you.

To get the calibration curve, would I have to analyse the standard sample by different dilution in the Mass spectrometry ?