High performance liquid chromatography (HPLC) has become an essential technique in pharmaceutical analysis for the separation, identification, and quantification of pharmaceuticals. Due to its high resolution and sensitivity, it is used for testing drug purity, monitoring degradation, studying pharmacokinetics, and examining drug stability. With recent advances in instrumentation, the technique has become more efficient in terms of separating multiple components and analyzing tricky chemical compounds. The use of HPLC in pharmaceutical analysis (https://www.formulationbio.com/analytical.html) has improved drug safety and efficacy, providing a valuable tool for pharmaceutical development and quality control. In conclusion, HPLC has become a critical tool for pharmaceutical analysis due to its high-resolution and unparalleled sensitivity in separating and quantifying complex chemical compounds.

In the past 30 years, gas chromatography (GC) has shown its unique application in pharmaceutical analysis. It is generally used for the inspection of residual solvents in various raw materials and preparations, and the qualitative and quantitative analysis of various volatile components. The chromatographic technique that has always occupied the mainstream position in drug analysis is HPLC, which has been widely used in the analysis and identification of metabolites. Depending on the mechanism of action, various HPLC techniques such as adsorption chromatography, partition chromatography (normal phase, reversed phase), ion exchange chromatography, affinity chromatography, gel filtration chromatography (also known as size exclusion chromatography) and gel permeation chromatography can all be used. There are a wide range of applications, of which the most widely used is still the reversed-phase mode (reversed-phase HPLC, RP-HPLC).

In recent years, new branches have been developed on the basis of partition chromatography, such as hydrophilic interaction liquid chromatography (HILIC) and hydrophobic interaction chromatography. As a variation of normal phase chromatography, HILIC uses a hydrophilic stationary phase, water and water-miscible organic solvents as mobile phases, and its application tends to increase. Its advantages are mainly reflected in the fact that it is especially suitable for separation of drugs and metabolites and compatibility with mass spectrometry.

The current research progress focuses on the use of monolithic columns or small-pore packing columns to reduce sample analysis time and increase sample throughput, that is, to achieve the purpose of "rapid" or "high-throughput" analysis. In an ultra-high pressure liquid chromatography (UPLC) system that can withstand about 1×108 Pa, chromatographic columns with a pore size of 1.5 and 3 mm can be used, which is more suitable for rapid analysis of complex drug samples. However, under ultra-high pressure conditions, the nonlinear heating and retention effects introduced by the compressibility of the liquid cannot be ignored, and special attention should be paid to this point during method development.

In addition, the introduction of a second organic additive into the mobile phase, used as ion-pairing reagents, micelles, or silanol masking reagents for the separation of extremely acidic and extremely basic substances has also received attention in HPLC method development. RP-HPLC can also be used to study the encapsulation and release of active ingredients in nanoparticles. For example, the RP-HPLC method with 0.1% trifluoroacetic acid as a mobile phase can be used to characterize the binding and release efficiencies of alginate-chitosan nanoparticles on insulin in a simulated gastrointestinal environment. There are also reports that 2D LC (such as normal-phase diol narrow-bore column-reversed-phase monolithic column) and multi-dimensional LC are used to separate and analyze drug mixtures or extracts, which can effectively improve peak capacity. The new chromatographic technology also includes the development of various new molecularly imprinted chromatographic columns and the column switching technology in in vivo drug analysis, which is used to achieve the purpose of fast pre-column switching purification.

In terms of detectors applicable to HPLC technology, the application of evaporative light scattering detectors (ELSD) in HPLC tends to increase, especially for the analysis of various antibiotics, natural products and phosphate esters. Other detectors used include differential detectors, fluorescence, chemiluminescence and electrochemical detectors, and charged aerosol detectors (CAD). ELSD, CAD and differential detectors are all universal detectors, generally used for the detection of substances containing no or little ultraviolet chromophores. Compared with ELSD, CAD has the characteristics of higher sensitivity, wide linear range up to 4 orders of magnitude, and constant signal response factor. These two detectors are mass-dependent detectors, which generate universal response signals. Therefore, the calibration curve obtained by using a reference substance is also applicable to other drugs and their impurities. This offers the possibility of accurate quantitation in the absence of controls or radiolabeled compounds. The common disadvantage of CAD and ELSD is that the mobile phase composition affects the detection signal, which can be solved by adding a mobile phase with the opposite gradient composition to compensate.

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