HPLC, coupled with electrospray ionization

Recombinant human growth hormone, tryptic digests Vydac 218TPB5 (Cl8), 5 pm, 300 A A 0.1% TFA/water, B 0.09% TFA/acetonitrile, gradient from 0 to 60% B 250 mm x 100 pm i.d. Electrically assisted capillary HPLC, coupling with electrospray ionization-mass spectrometry... 

Wang H, Feng F (2009) Identification of components in Zhi-Zi-Da-Huang decoction by HPLC coupled with electrospray ionization tandem mass spectrometry, photodiode array and fluorescence detectors. J Pharm Biomed Anal 49 1157-1165... 

Pluym et al. compared the use of CE to that of HPLC in chemical and pharmaceutical quality control. They stated that CE could be considered as a complementary technique to HPLC because of its large separation capacity, its simplicity, and its economical benefits. Jimidar et al. decided that CE offers high separation efficiency and can be applied as an adjunct in HPLC method validation. Mol et al. evaluated the use of micellar electrokinetic chromatography (MEKC) coupled with electrospray ionization mass spectrometry (ESI—MS) in impurity profiling of drugs, which resulted in efficient separations. 

Neuromuscular blockers can be analyzed using HPLC with fluorescence, ultraviolet (UV), or electrochemical detection (ECD). As an alternative to HPLC, GC with nitrogen—phosphorus detection (NPD) and LC coupled with electrospray ionization tandem mass spectrometry (ESI-MS-MS) have also been investigated as viable techniques to measure neuromuscular blockers (Table 10.2). 

High-throughput preparative HPLC coupled to electrospray ionization mass spectrometry (Chapter 17), which disposes upon a signal for collecting detected compounds of the defined molecular mass, is one of the highly promising new developments in this area. Such systems can be incorporated for synthesis purposes into the periphery of automated multicomponent systems, thus making a valuable contribution to the rationalization and quality enhancement of combinatorial synthesis processes. The combination of automated synthesis, purification and on-line instrumental identification (NMR, IR, MS) will become feasible in the near future, and as a matter of routine operation. Analytic methods of structure elucidation will then also be able to be combined with automated combinatorial chemistry. 

RP-HPLC is well suited for direct online coupling with electrospray ionization-mass spectrometry (ESI-MS) due to solvent compatibility with the electrospray ionization technique. 

Today, micro-LC is a valuable analytical tool for sample-constrained applications such as proteomics and bioanalysis. The microliter flow rates are ideally suited for direct, splitless coupling with electrospray ionization mass spectrometry (ESI-MS). Many benefits can be demonstrated from well-established HPLC theory, which allows for direct method transfer to micro-LC. 

Li, Y.-J., J. Chen, Y. Li, and P. Li. 2012. Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Biomedical Chromatography 26(4) 449-457. 

Much data on the structure of flavonoids in crude or semipurified plant extracts have been obtained by HPLC coupled with MS, in order to obtain information on sugar and acyl moieties not revealed by ultraviolet spectrum, without the need to isolate and hydrolyze the compounds. In the last decade, soft ionization MS techniques have been used in this respect, e.g., thermospray (TSP) and atmospheric pressure ionization (API). However, the most used methods for the determination of phenols in crude plant extracts were the coupling of liquid chromatography (LC) and MS with API techniques such as electrospray ionization (ESI) MS and atmospheric pressure chemical ionization (APCI) MS. ESI and APCI are soft ionization techniques that generate mainly protonated molecules for relatively small metabolites such as flavonoids. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

The detection and identification of phenolic compounds, including phenolic acids, have also been simph-fied using mass spectrometry (MS) techniques on-hne, coupled to the HPLC equipment. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces dominate the analysis of phenohcs in herbs, fmits, vegetables, peels, seeds, and other plants. In some cases, HPLC, with different sensitivity detectors (UV, electrochemical, fluorescence), and HPLC-MS are simultaneously used for the identification and determination of phenolic acids in natural plants and related food products.In some papers, other spectroscopic instmmental techniques (IR, H NMR, and C NMR) have also been apphed for the identification of isolated phenolic compounds. 

With electrospray ionization, a fine mist of highly charged particles is produced when a liquid flows from a capillary tube into a strong electrical field (3 to 6kV).In practice, electrospray ionization sources are often directly coupled with reversed phase HPLC or capillary columns. The ability to couple a liquid chromatograph with an electrospray ionization source and a mass spectrometer allows the online removal of salts and contaminants and the analysis of complex mixtures. Although different from MALDI, electrospray provides similar sensitivity and application to the analysis of large proteins. 

One of the most powerful techniques used in Upid analysis today is HPLC coupled with mass spectrometry (HPLC/MS). Several mass spectrometric ionization techniques, such as fast atom bombardment (FAB) [23], electrospray ionization (ESI) [29,30], ionspray ionization (ISI) [31], and atmospheric pressure chemical ionization (APCI) [22,30,32] have been used. By using HPLC/MS, one can get information on the molecular structure of the intact lipids, which helps differentiate molecular species within different lipid classes. By using tandem mass spectrometry (MS/MS), identification of molecular species of different sphingolipids can be achieved in an easier and more sensitive way. There are many other advantages of using MS, such as small sample size, minimal sample preparation, and lack of need for derivatization, speeds, and sensitivity. In the literature, sphingolipids of both animal and plant origin were analyzed by MS. 

HPLC coupled with UV/Vis [142], chemiluminescence [143] and electrochemical [144] detection is proposed to determine AA using a FIA system. Since CE in its modern form was first described by Jorgenson and Lukacs (1981), its application for the separation and determination of a variety of samples has been increasingly widespread because of its minimal sample volume requirement, short analysis time, and high separation efficiency. Nevertheless, only one method for AA determination involving a FIA system has been proposed [145]. Bhandari et al. [146] demonstrated the potential of automated FIA electrospray ionization tandem MS (ESTMS/MS) for the mass-spectroscopic... 

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) was introduced in the 1980s [1]. Today it has become a standard method for separation and characterization of nonvolatile compounds. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to ESI-MS is the method of choice for peptide and protein analysis, but also used for the characterization of contaminants, therapeutic drugs, and food additives [2-5], More than 75% of HPLC analyses are run on RP stationary phases, and a wide range of columns are available with various substituents of the silica matrix, base deactivation, endcapping, and column dimensions. 

As in HPLC, the coupling of MS detection with CE has provided an excellent opportunity for more selective analysis, but the much reduced flow rates, small injection volumes, limitations in the types of buffers used [since electrospray ionization (ESI) is used in capillary electrophoresis/mass spectrometry (CE/MS)], and need to... 

Electrospray ionization (ESI) is ideally suited as a detection technique for the online interfacing of liquid-phase separations (HPLC and CE) to MS, because it facilitates the transfer of analytes from the liquid phase of the HPLC or CE column to the gas phase of the MS. Also, it allows the detection of high molecular weight species, such as peptides. Three interface designs have been developed in the past 18 years for coupling CE with MS. The first CE-MS interface, coaxial sheath flow, was introduced by Smith and his group in 1987 (Olivares et al., 1987) and was improved upon in later work (Smith et al., 1988). Coaxial sheath flow is formed using two concentric metal capillaries, whereby the CE terminus and the makeup flow line are inserted into the... 

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