LC-MS can acquire a total ion chromatogram by acquiring mass spectra. Since electrospray is a soft ionization source, there are usually few or no fragments, and there are only quasi-molecular ions in the spectrum, so it can only provide molecular weight information of unknown compounds, but not structural information. It is difficult to use for qualitative analysis, but it can be used for quantitative analysis. However, if single-stage MS does not use a soft ionization source, but EI or the like, there will be fragment peaks that can provide molecular structure information.
LC-MS/MS adopts tandem mass spectrometry, which can obtain both molecular ion peaks and fragment ion peaks, so it can be used for qualitative and quantitative analysis.
A fundamental similarity is that their fields of application are all applicable to liquid phases.
The outstanding feature of mass spectrometry is that it has mass information itself, which can be used for qualitative or qualitative basis (other qualitative instruments are also required). Secondly, mass spectrometry itself also has a separation function, which is separation according to mass. If the liquid phase is separated once, then LC-MS is separated twice, while LC-MS/MS is separated three times, and LC-MS3 is separated four times. Secondary…. (Level 3 and above are the characteristics 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 you already know, such as quality control, look at the pure product after organic synthesis LC-MS can be used for some full analysis of pesticides, guidance of leading compounds in drug synthesis (one injection in one pot to see if the synthesis is correct), etc. Because it is cheap and easy to operate. Even if the liquid phase of some things is not separated, as long as you care about the main component, it has little effect. You can adjust the liquid phase conditions, or directly look at the extracted ion map after completing the scan, or do SIM. LC-MS/MS in these areas is overkill, and spending so much money is a waste.
And if the result you care about is:
(1) For unknown things, a single injection of LC-MS cannot be qualitative, and more fragment information is needed;
(2) is a trace component in the mixture.
Then you have to use LC-MS/MS: LC-MS/MS can give you more fragmentation information and help you to characterize;
LC-MS/MS can reduce background noise, so that the spectrum of trace components is not disturbed by abundant substances;
LC-MS/MS can reduce a lot of background noise, making your compounds much more sensitive and quantifying better.
1. How does LC-MS-MS use an internal standard for quantitative analysis?
Q: I currently use triple quadrupole LCMSMS to quantitatively analyze the target, but there is no standard. How does the internal standard method work? The data says that a certain weight ratio of the mixture of the component to be tested and the internal standard sample is prepared for chromatographic analysis, the peak area is measured, and the relationship curve between the weight ratio and the area ratio is made. This curve is the standard curve. If this is the case, there is no standard product. How to do?
Method 1: 1) Quantitative analysis by the internal standard method also requires the analyte standard. The content of the analyte cannot be accurately measured without the analyte standard, because the standard curve cannot be drawn.
2) The content of the analyte cannot be accurately measured without the standard substance of the analyte, but the relative content of the analyte can be measured by adding an internal standard, and the amount of the analyte in each sample and the difference multiple can be compared.
3) Quantification by LC-MS-MS generally adopts the MRM scanning method. If you do not have the analyte standard, you cannot establish and optimize the mass spectrometry parameters of the MRM scanning, so you can only use the FULL SCAN or SIM scanning method. Without the analyte standard, you cannot accurately obtain the retention time of the analyte on the LC. If your sample has a compound with a similar mass as the analyte, the chromatographic peaks you get will not be very pure, and will Interfere with the compound you determine to be your test compound.
Method 2: If there is no standard product, only the area is used for relative quantification. Then select the SIM mode of LCMSMS (sample impurities are relatively few, select the molecular weight with the isotopic abundance of 100%, and increase or decrease by 0.2). The change in the amount caused by the investigation factor is relatively small, and one substance is selected as the recovery indicator (added before the pretreatment of the reaction solution), and the other is the internal standard substance (after pretreatment, before sample injection into the liquid phase vial for testing, in A quantitative internal standard is added to the sample to be tested, and then the test is performed).
2. Can LC-MS perform quantitative analysis?
How does LC-MS perform quantitative analysis? Excuse me, what is the qualitative spectrum from mass spectrometry in LC/MS, and since mass spectrometry is used for quantification, what is the use of the spectrum from the UV detector configured in the chromatography itself? LC/MS has SIM and full scan, so which one is generally used in pharmacokinetic tests? Especially in the analysis of in vivo drugs, what does the mass spectrum of the internal standard method for quantification look like?
Mass spectrometry quantitative analysis, generally using LC-MS/MS, does not use LC-MS, look at the mass-to-charge ratio, and infer the molecular weight. UV can be used for quantification, but when there are interference peaks of UV absorption, the quantification is inaccurate. LC-MS/MS uses parent ions/product ions for quantification. Relatively speaking, it has much less interference and is more accurate. It is widely used in PK/TK. There are only LC-MS/MS quantitative spectra here, for reference only!
Channel 183.1/141.3 is the internal standard, the other two are compounds.
A widely accepted method of mass spectrometry quantification is MS/MS quantification. This quantification is often achieved by triple quadrupole or ion trap mass spectrometry. The reason for requiring MS/MS is that many compounds have the same mass. There is also a lack of specificity when using the first dimension, single-stage mass spectrometry, for quantification, especially for complex matrices like blood. MS in the second dimension (i.e. MS/MS), in most cases, is able to provide unique breaks. Combining the specific precursor ion mass and the unique fragment ion information, the quantified compound can be selectively monitored.
3. How does LC-MS-MS perform quantitative analysis?
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 compounds whose content changes. How to quantify? It seems inappropriate to use a total ion current map. If the peak area is used for relative quantification, which map is more suitable? Do you have to use a standard to qualify?
LC-MS-MS quantitative analysis uses SIM or MRM mode. The accepted MRM is more accurate. SIM selectively filters selected molecular weights using Q. But there are many compounds with the same molecular weight, so if the sample is slightly complex, SIM is basically not suitable.
MRM selects precursor 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. Interfering ions with the same molecular weight and different product ions after bombardment are excluded. Therefore, MRM mode is a more reliable LC-MS quantitative analysis method. MRM detection is transition.
The selection principle of the internal standard is that the structure is basically similar to that of the test compound. The structure is the same or similar, mainly to meet the ionization efficiency similar to that of the sample. This is why many opt to use this compound or deuterated compounds of this type. The selection of the internal standard also requires that its molecular weight does not coincide with the substance to be tested (it also needs 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 chromatogram represents a species. Also using MRM mode, if the molecular weights of the samples do not overlap with the product ions, even compounds that are not separated in the column can be considered separated in MS.
There are several peaks in the liquid and mass reported in the literature, and dozens of substances are detected by MRM, that is, multiple substances run out of 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 the limited space.
Qualitatively, a standard product is required, and the retention time needs to be consistent.
For quantification, a standard curve (peak area ratio and concentration) needs to be made first.
