HPLC interfaced with mass spectrometry

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). 

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. 

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... 

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... 

Direct interfacing of hplc with mass spectrometry is a coupled or hyphenated technique similar to GC-Mass Spectrometry (p. 108) which provides structural information on separated sample components. Its development has been slow because ofdifliculties inherent in removing the liquid mobile phase whilst allowing only the analytes to pass into the mass spectrometer, particularly as reverse phase hplc often employs mobile phases containing aqueous... 

Mass spectrometry is an extremely versatile detection system for gas chromatography. Interfacing an HPLC system to a mass spectrometer is a much more difficult task, however. Describe the major reasons why it is more difficult to combine HPLC with mass spectrometry than it is to combine GC with mass spectrometry. 

AP Bruins. Developments in interfacing microbore HPLC with mass spectrometry (a review). J Chromatogr 323 99—111, 1985. 

Another way to speed up HPLC that does not interfere with the existing gradient separation method is by parallel operation of several HPLC columns. The development in this direction started a couple of years ago when fast gradient separation was first combined with mass spectrometry-based detection. Parallel column operation was achieved by a single pumping system and a splitter tee that transferred the gradient flow onto two HPLC columns. The effluent of the two columns was simultaneously sprayed into a modified ion spray interface of a quadrupole mass spec-trometer. From the overlay chromatogram both desired and previously known compounds were identified after their molecular ions were filtered from the total ion current (TIC). In this first system, however, it was difficult to enhance the parallelization, and the detection system created a bottleneck. ... 

CE provides a complementary approach to HPLC separation. It is performed in several different formats, including capillary zone electrophoresis, miceller electrokinetic chromatography, capillary gel electrophoresis, capillary isoelectric focusing, isotachophoresis, and capillary electrochromatography. Of these formats, capillary zone electrophoresis is the most popular separation technique. The most successful coupling of CE with mass spectrometry is achieved via an ESI interface. The three most practical designs are sheathless interface, sheath-flow interface, and liquid-junction interface. 

SEC suffers from poor resolution and low sensitivity [5], while GC is limited by the high molecular weight and polar nature of many antioxidants and light stabilisers, which are designed to be reactive and so decompose when exposed to heat [9]. HPLC the most widely used instrumental method also has limitations [10-12]. HPLC lacks a simple sensitive universal detector that is compatible with all liquid mobile phases. UV or fluorescence detectors, which are commonly used, require that additives have a chromophoric moiety, while the universal refractive index detector only functions under isocratic conditions. As a result, Vargo and Olson have coupled HPLC with mass spectrometry (MS) for this type of application by using a moving belt interface [13]. 

The coupling of liquid chromatography with mass spectrometry (LC/MS) has also been intensively investigated since the early 1980s. but so far without complete success. The new ionization techniques, especially ion spray, made the coupling of HPLC with MS to a routine technique. The decreasing pricesfe.g. of ion trap MS instruments open the use of these instruments in routine HPLC analysis. For more details see-> Mass Spectrometry. In GC/MS it is relatively easy to remove the mobile phase, or to limit and control its effects on the ionization source, but this has proven to be much more difficult in LC/MS. A variety of interfaces has been developed for that purpose ... 

The total taxane (TTAX) concentration, a sum of the concentrations for CT-2103, TXL, and TXL-metabohtes was determined from scintillation counting of the plasma or tissue sample homogenates. Extractable taxanes, including TXL and organically extractable TXL metabolites, were determined by scintillation counting of ethyl acetate extractions of the plasma, tumour, liver, and spleen samples. Plasma and tissue TXL concentrations were also determined by HPLC/radiometric analysis of the extracts. Metabolites were identified by HPLC followed by mass spectrometry on a Quattro It (Micromass, Manchester, UK) triple quadrupole mass spectrometer fitted with an electrospray orthogonal Z spray ion interface operating in the positive ion mode. ... 

The introduction of liquid samples into the mass spectrometer has proved especially difficult. However, the advent of electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) have overcome this problem and revolutionized the interfacing of HPLC with mass spectrometry. This success has pointed the way toward a major change in the mass spectrometric analysis of vitamin D, its analogs, and their metabolites. A disadvantage at the present time is that ionization using ESI and APCI is less efficient than the EI(+) used in GC-MS, but the immediate advantage is that it obviates the need for derivatiza-tion. 

Of these techniques, gas chromatography coupled with mass spectrometry has become of such importance that it merits a separate treatment, and this forms the subject of Chapter 7. As the interface between the GC column and the mass spectrometer and the need to prepare derivatives of high molecular weight may limit the resolution attainable by GC alone, silver ion chromatography and HPLC in the reversed-phase mode especially are important complementary techniques. They permit isolation of simpler fractions, more readily analysed by GC-MS and other techniques. It should, however, be noted... 

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