Reversed-phase liquid chromatography column is not the mainstream of LC-MS/MS analysis
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The practical strategy for LC-MS/MS analysis is very different from HPLC! The selectivity of the LC-MS/MS analysis method is the most important!
Chromatography companies can't simply use off-the-shelf reversed-phase liquid chromatography cartridges as 5cm x 2.1mm short columns for LC-MS/MS columns, but virtually all chromatography companies do this. Such reversed-phase liquid chromatography LC-MS/MS columns have three distinct weaknesses:
(1) Many polar compounds are difficult to retain and the mass spectrometry sensitivity is poor.
Many important substances, such as the anticancer drug DTIC, the anti-pain drug tetrodotoxin, the pollutant melamine, the Chinese herbal medicine glycopeptide, etc. are very polar and are not retained on the reversed-phase liquid chromatography column. There are also many compounds that are relatively polar and elute from a reversed phase column using a small amount of methanol or acetonitrile. In drug metabolism studies, many compounds were observed to be hydrophobic, but the metabolites were hydrophilic. The methanol or acetonitrile content is low, the degree of ionization is low, and the mass spectrometry sensitivity is poor. The so-called aqueous C18 column has no value in LC-MS/MS analysis.
(2) Columns packed with 2.1 mm internal diameter using conventional reversed-phase chromatography cartridges are often trapped in the "trap" of the Carryover Effect in LC-MS/MS and LC/MS applications.
Another major problem in LC-MS/MS and LC/MS applications is the Carryover Effect. The memory effect means that after the injection, a further blank is observed and the compound peak is still observed for the same retention time. The side effects of the memory effect are obvious: it may artificially increase the MRM signal of the next sample, affecting the precision and accuracy of the quantitative analysis. The US FDA Bioanalytical Regulatory Guidances limit the memory effect of the highest calibration standard to no more than 20% of the 20% minimum calibration standard MRM signal.
Since most chromatographic companies use off-the-shelf reversed phase liquid chromatography cartridges to simply mount 5 cm x 2.1 mm short columns as LC-MS/MS columns, in many cases the memory effect is strong and the calibration standard curve must be artificially shortened. , or have to enter a needle or even a two-needle blank to minimize the memory effect after the injection, and then proceed to the next sample. All of this severely reduces production efficiency.
(3) Peak tailing or distortion Because the reactive silanol groups are not completely blocked, many basic compounds have significant peak tailing on the reversed-phase LC columns of almost all chromatography companies. This peak shape tail is an important source of memory effects.
On the other hand, excessive blocking using incorrect methods results in poor separation of some acidic and amphoteric compounds, especially peak distortion and splitting.
Chrom-Matrix believes that the first choice for LC-MS/MS columns and chromatographic methods is Hydrophilic Interaction! Hydrophilic chromatography uses acetonitrile as a weak solvent and water as a strong solvent to completely resolve the poor sensitivity of mass spectrometry, memory effect And other issues. The hydrophobic compound first flows out, followed by a hydrophilic compound. It not only completely solves the problem that the polar compound is difficult to retain, but also analyzes the hydrophobic compound and also improves the mass spectrometry sensitivity of the hydrophobic compound. The peak shape tailing problem has also been completely solved. In addition, the acetonitrile extract does not need to be evaporated and redissolved, saving a lot of time. On the other hand, reversed phase LC-MS/MS columns and chromatographic methods are still important, not only for important neutral compounds such as the anticancer drug paclitaxel, the anti-immune organ implants FK 506 and rapamycin. It is necessary to use reversed-phase LC-MS/MS columns and chromatographic methods for lipid-lowering drugs, and because most customers are accustomed to reversed-phase LC-MS/MS columns and chromatographic methods. Even such a reversed-phase liquid chromatography cartridge must be processed to fit a 5 cm x 2.1 mm short column for use as an LC-MS/MS column. Chrom-Matrix has developed the InnovationTM Reversed-Phase Liquid Chromatography Pack to maximize the blocking of reactive silanol groups by supercritical fluid technology, which solves the problem of peak tailing of basic compounds and then eliminates them by special treatment. The memory effect.
Chrom-Matrix has successfully developed three hydrophilic LC-MS/MS columns and five reversed-phase LC-MS/MS columns, and has successfully developed hundreds of LC-MS/MS applications for customers. Since its inception, Chrom-Matrix has won the US FDA, the US Department of Energy, the US Department of Agriculture, the International Anti-Drug Organization, many European and American pharmaceutical groups, third-party testing organizations, universities and research institutions, etc. with a generation of ideas, technologies and products. Customer sincere welcome.
Chrom-Matrix's goal is to help customers develop, validate and apply advanced, correct, accurate, realistic, rock-solid, yet sensitive, fast, and inexpensive LC-MS/MS quantitative methods.