HPLC interfaced with mass

Yinon et al. (228) used an HPLC interfaced with a triple-quadrupole mass spectrometer by means of a particle beam for the identification of several azo dyes. Characterization of the dyes was achieved by observing typical fragment ions formed by cleavage of the N-C and C-N bond on either side of the azo linkage and/or cleavage of the N=N double bond with the transfer of two hydrogen atoms to form an amine. Sensitivity was observed to be two to three orders of magnitude worse than with thermospray ionization. 

Today, mass spectrometry offers an attractive alternative as a detector to HPLC. Newer techniques for linking HPLC systems with mass spectrometers directly via atmospheric pressure chemical ionization (APCI) and electrospray interfaces should see an expansion of this analytical tool in the analysis of confectionery fats, a field in which it has not yet been applied. Triacylglycerols... 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

A major application of HPLC interfaced with MS is in protein or peptide sequencing. This can be accomplished using a wide variety of chromatographic separations followed by different kinds of mass spectrometric characterizations. We will illustrate one example of such techniques to give an impression of the power and complexity of such analyses. A very large protein might consist of... 

Using RP-HPLC interfaced with ESI to an MS, it is possible to obtain a mass/ intensity map of all isoflavone metabolites in a single 20 min analysis. Analysis of isoflavonoid conjugate in serum/plasma samples requires initial extraction, but the conjugates can be measured intact either by capillary RP-HPLC-ESI-MS or on regular RP columns by HPLC-HN-APCI-MS. When it is only necessary to measure the total isoflavonoids and their metabofites in blood, hydrolysis can be performed directly in semm/plasma samples and flavonoids recovered by ether or ethyl acetate solvent extraction (Barnes et al., 1998). 

Reference has been made to the problems associated with the presence of highly involatile analytes. Many buffers used in HPLC are inorganic and thus involatile and these tend to compromise the use of the interface, in particular with respect to snagging of the belt in the tunnel seals. The problem of inorganic buffers is not one confined to the moving-belt interface and, unless post-column extraction is to be used, those developing HPLC methods for use with mass spectrometry are advised to utilize relatively volatile buffers, such as ammonium acetate, if at all possible. 

Currently, HPLC/fiuorescence is still the most common technique for the determination of residues of oxime carbamates. With the introduction of ESI and APCI MS interfaces, HPLC/MS analysis for oxime carbamates in various sample matrices has become widespread. However, for a rapid, sensitive, and specific analysis of biological and environmental samples, HPLC/MS/MS is preferred to HPLC/MS and HPLC/fiuorescence. With time, improved and affordable triple-quadrupole mass spectrometers will be available in more analytical laboratories. With stricter regulatory requirements, e.g., highly specific and conclusive methods with lower LOQ, HPLC/MS/MS will be a method of choice for oxime carbamates and their metabolites. 

Thermo SeparaAons Products HPLC system with an autosampler, solvent degasser and column heater interfaced to aFinnigan MAT 7000 mass spectrometer or equivalent insAumentation opAmized for the detection of azinphos-methyl. 

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

The first reported case of timesharing for a mass spectrometer9 involved the design of an Ionspray interface with multiple sprayers to support the analysis of effluents from multiple columns. This approach led to the development of a multiplexed electrospray interface (MUX)10 using an LC/MS interface and multiple (identical) sprayers linked to a HPLC system and a spinning screen to allow the output of only a single sprayer to enter the MS (Figure 4.5). The injections of the HPLC systems... 

When nano LC is combined with mass spectrometer detection, attamole detection can be achieved for low abundance components in biological fluids, drug metabolites, and natural products such as Chinese herb medicines. Nano LC-MS-MS has become an essential tool for complex biological and drug metabolite studies. Nano LC-MS presents two significant differences from conventional analytical HPLC (1) large enhancement factor for sample detection and (2) direct interface to MS without flow splitting. The enhancement in MS ion counts relative to a conventional 4.6 mm ID column is proportional to the ratio of the square of the column diameter ... 

Perhaps the most mechanically complex solution ever developed for uniting HPLC with mass spectrometry was the moving belt interface [54]. The heart of this system was a mechanically driven continuous belt (analogous to an escalator or moving walkway) to which the HPLC eluent was applied. The majority of the mobile phase was evaporated by a heat source (ideally hot enough to vaporize the solvents but not to... 

Bayliss and co-workers [10] combined ultra-high flow rates, parallel LC columns, a multiplex electrospray source, and mass spectrometric detection for the rapid determination of pharmaceuticals in plasma using four narrow bore (50 mm x 1 mm, 30 pm Oasis HLB) or capillary (50 mm x 0.18 mm, 25 pm Oasis HLB) HPLC columns with large particle sizes (to avoid high system back-pressure) in parallel with a multiple probe injector and a MUX MS interface. Small sample aliquots were injected directly into the system without sample pre-treatment procedure, obtaining very low limits of quantification (from 1 to 5 ng/mL). 

HPTLC is a very fast and convenient assay to separate samples components and is often used in Organic Chemistry and in Synthetic approach. Unknown substances, after different display assay, were generally scraped off from the TLC/HPTLC plate, diluted into a tube and transferred into the MS system for structural elucidation and characterization. Now, a TLC-MS interface was developed by CAMAG, which can semi-automatically extract zones of interest and on-line direct them into any brand of a HPLC-MS system. The TLC-MS interface is connected by two fittings to any HPLC instrument coupled with mass spectrometer, without other system configuration adjustments or mass spectrometer modifications. By this way, the unknown substances can be directly extracted from a TLC/HPTLC plate, eluted and resolved by HPLC system and sensitive and selective mass spectrometric signals are obtained within a minute per substance zone [33],... 

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