Comparative analysis of LC-MS and LC-MS/MS

LC-MS can obtain total ion chromatograms by collecting mass spectra. Because electrospray is a soft ionization source, there are usually few or no fragments. There are only quasi-molecular ions in the spectrum, so it can only provide molecular weight information of unknown compounds, not structural information. It is difficult to use for qualitative analysis, but can be used for quantitative analysis. However, if the single-stage MS does not use a soft ionization source, but EI, there will be fragment peaks, which can provide molecular structure information.

LC-MS/MS uses tandem mass spectrometry to obtain both molecular ion peaks and fragment ion peaks, so it can be used for qualitative and quantitative analysis.

A basic similarity is that their application areas are all suitable for liquid phase applications.

The outstanding feature of mass spectrometry is: it has quality information itself, and it can be qualitatively based on this quality information or provide some basis for qualitative (other qualitative instruments are also needed). Secondly, mass spectrometry itself also has a separation function, that is, separation according to mass. If the liquid phase is separated once, then LC-MS will be separated twice, and LC-MS/MS will be separated three times, and LC-MS3 will be separated four times. Second… (Level 3 or higher is the characteristic of ion trap mass spectrometry).

The difference between LC-MS and LC-MS/MS (or MSn) is:

If the result you care about is the main component in your sample, and it is the target that you already know, such as quality control, organic synthesis, look at the pure product, part of the pesticide analysis work, and the guidance of the lead compound in the drug synthesis (synthesis) One shot in one pot to see if the synthesis is correct) etc., you can use LC-MS. Because it is cheap and easy to operate. Even if the liquid phases of some things are not separated, as long as you are concerned about the principal components, the effect will be small. You can adjust the liquid phase conditions, look at the extracted ion map directly after completing the scan, or do SIM. Using LC-MS/MS in these fields is overkill, and spending so much money is a waste.

And if the result you care about is:

(1) For unknown things, a shot of LC-MS cannot be qualitative, and more fragmentation information is needed;

(2) is the trace component in the mixture.

Then you must use LC-MS/MS: LC-MS/MS can give you more fragment information to help you qualitatively; LC-MS/MS can reduce background noise, so that the trace component spectra are not affected. The interference of abundant substances; LC-MS/MS can reduce a lot of background noise, greatly improve the sensitivity of your compound, and improve the quantitative results.

How does LC-MS-MS use internal standard for quantitative analysis

Q1: I am now using a triple quadrupole LC-MS/MS to quantitatively analyze the target, but there is no standard product. How should the internal standard method be operated specifically?

Q2: According to the data, first prepare a mixture of the component to be tested and the internal standard sample in a certain weight ratio for chromatographic analysis, measure the peak area, and draw the relationship curve between weight ratio and area ratio. This curve is the standard curve. If this is the case, there is no What about standard products?

method one:

(1) Quantitative analysis with internal standard method also requires the standard of the analyte. If there is no standard substance of the analyte, its content cannot be accurately measured because it is impossible to draw a standard curve.

(2) The content of the analyte cannot be accurately measured without the standard product of the analyte, but the relative content of the analyte can be measured by adding an internal standard, and the number of the analyte in each sample and the difference multiple can be compared.

(3) The quantification by LC-MS-MS generally uses the MRM scanning method. If you don’t have a standard product of the analyte, you cannot establish and optimize the mass spectrometry parameters of the MRM scanning. In this way, you can only use the FULL SCAN or SIM scanning method. . Without the standard product of the analyte, you cannot accurately obtain the retention time of the analyte on the LC. If your sample contains a compound with a mass similar to that of the analyte, the chromatographic peak you get will not be very pure. Interfere with your determination of which is your test compound.

Method Two:

If there is no standard product, only the area is used for relative quantification. Then choose the SIM mode of LC-MS/MS (the sample has less impurities, choose a molecular weight with an isotope abundance of 100%, and increase or decrease by 0.2). The change in the amount caused by the investigation factors is relatively small. Choose one substance as the recovery rate indicator (added before the pretreatment of the reaction solution), and one substance as the internal standard (after pretreatment, before the sample injection test in the liquid phase vial). Add a quantitative internal standard substance to the sample to be tested, and then perform the test).

Can LC-MS perform quantitative analysis?

Q1: How does LC-MS perform quantitative analysis?

Q2: What is the qualitative spectrum produced by mass spectrometry in LC/MS? Since mass spectrometry is used for quantification, what is the use of the spectrum produced by the UV detector in the chromatogram itself?

Q3: There are SIM and full scan for LC/MS, so which one is generally used in pharmacokinetic tests? Especially when analyzing drugs in vivo, what is the mass spectrum of internal standard method used for quantification?

Mass spectrometry quantitative analysis generally uses LC-MS/MS instead of LC-MS. Look at the mass-to-charge ratio and estimate the molecular weight. UV can be used for quantification, but when there are interference peaks of ultraviolet absorption, the quantification is not accurate. LC-MS/MS uses precursor/product ions for quantification. Relatively speaking, it has much less interference and is more accurate. It is widely used in PK/TK. Only the quantitative spectra of LC-MS/MS are here, for reference only!

The 183.1/141.3 channel is an internal standard, and the other two are compounds.

The widely accepted method of mass spectrometry quantification is MS/MS quantification. This quantification is often achieved by three-stage quadrupole or ion trap mass spectrometry. The reason why MS/MS is required is that many compounds have the same quality. When using the first dimension, namely single-stage mass spectrometry, MS to quantify, it will also lack specificity, especially for complex matrices like blood. The second dimension of MS (ie MS/MS) can provide only ① fracture in most cases. Combining the specific precursor ion mass and the only fragment ion information can selectively monitor the quantified compound.

How to perform quantitative analysis by LC-MS/MS

Q1: I want to use LC-Q-TOF-MS for metabolomics, because the sample is a body fluid containing multiple compounds. I want to perform a relative quantification after adding an internal standard to determine the compound whose content has changed. How to quantify ?

Q2: It seems inappropriate to use the total ion current graph. If the peak area is used for relative quantification, which graph is more appropriate? Do I have to use standard products for qualitative determination?

LC-MS-MS quantitative analysis uses SIM or MRM mode. The accepted MRM is more accurate. SIM uses Q to selectively filter selected molecular weights. However, there are many compounds with the same molecular weight, so if the sample is slightly complicated, SIM is basically not suitable.

MRM selects parent ions and product ions. Use Q to filter the precursor ions, q to bombard the precursor ions, and TOF to filter the product ions so that the product ions are detected by the detector. With the same molecular weight, interfering ions with different product ions produced after bombardment are eliminated. Therefore, MRM mode is a more reliable LC-MS quantitative analysis method. MRM detection is an ion pair.

The selection principle of the internal standard is that the structure is basically similar to that of the compound to be tested. The structure is the same or similar, mainly to satisfy that the efficiency of being ionized is similar to that of the sample. This is why many choose to use this compound or deuterated compounds of this type of compound. The selection of the internal standard also requires that its molecular weight does not overlap with the substance to be tested (it is also necessary to avoid the m/z of the isotopic peak of the compound as much as possible), and it can be separated from the substance to be tested.

If using MRM, the samples are separated, then theoretically each peak on the total ion current diagram represents a substance. Using the MRM mode at the same time, if the molecular weights of the samples do not overlap with the product ions, even the compounds that are not separated in the column can be considered separated in the MS.

There are several peaks in the liquid mass reported in the literature. MRM detects dozens of substances, which means that multiple substances escaped from the chromatographic column at the same time. Although the peak time is the same, the peak of each compound should be displayed in the software, but the report will be abbreviated due to limited space.

For qualitative purposes, standard products are required, and the retention time needs to be consistent.

For quantification, a standard curve (peak area ratio and concentration) needs to be drawn first.