Electrospray ionization mass spectrometry HPLC coupled

Chemical analysis of hair samples may also provide a method for examining chronic mycotoxin exposures. In 2003, Sewram et al. (2003) reported that human hair testing could be used to detect fumonisins. After extraction and clean up, high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS) was able to detect fumonisin Bi, fumonisin B2, and fumonisin B3 from human hair samples (Sewram et al., 2003). However, these were... 

Strege summarized the technique of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) in dereplication of natural products. In contrast to earlier electron impact ionization (El), ESI technique is applicable to virtually any ion in solution with a soft ionization process. A comparison of ESI with fast atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), atmospheric pressure chemical ionization (APCI) and other techniques demonstrates its superior sensitivity, compatibility and reliability when coupled with HPLC [51]. 

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. 

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... 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

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. 

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. 

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. 

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... 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

The synthesis of phosphopeptides is typically confirmed mass spectrometri-cally using either a MALDI (matrix-assisted laser desorption/ionizafion) or an ESI (electrospray ionization) source, and the peptide purity is determined by reversed-phase chromatography coupled to an UV detector (Figs. 1 and 2B). Whenever possible, phosphopeptide analyses should be complimented by mass spectra recorded in negative ion mode. For most biochemical applications it is necessary to purify the peptides by HPLC techniques (Fig. 2A). 

Electrospray ionization (ESI), a very powerful MS ionization mode, has been coupled with liquid chromatography becoming a popular tool in biomolecular analysis and drug analysis applications [1], Use of HPLC or ultra performance liquid chromatography (UPLC) combined with a mass spectrometry through the interface—ESI has become a powerful and routinely used analytical tool for many fields in scientific research, pharma industry, healthcare, and clinical diagnostic applications. 

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