Co-eluting components

As indicated above, the effect of the main peak on the ionisation of co-eluting components must not be ignored. One must not imagine that MS/MS is a cure-all for this situation. Suppression in the ion source will reduce the number of analyte ions produced and thus the sensitivity of any MS method. 

The resulting protocol of the coupling of HPLC with an MS/MS system, using an ion-spray interface, provides a means for sensitive and specific analysis of several BAs. Chromatographic separation allows the differentiation of isomeric forms. The MRM mode provides the necessary specificity even in the case of co-eluting components, thanks to the different transitions monitored this is evidenced by extracting the specific reconstructed ion chromatogram (RIC). The RIC traces of a BA standard mixture are shown in Fig. 5.4.13. 

Matrix Effects. The matrix effects (ion suppression or ion enhancement) can compromise the selectivity and sensitivity of LC/MS/MS methods for the determination of drug concentrations in biological matrices. One common matrix effect is ion suppression due to co-eluting components that can affect the ionization efficiency of the analyte of interest. The matrix effects are major causes for errors in precision, accuracy, linearity, and reproducibility of the quantitation methods based on LC/MS/MS [104,108,114-118]. It is critical to overcome such effects in quantitative bioanalysis by LC/MS/MS. 

HPLC peak 4, Iso-4, in Figure 19-6 corresponded to the putative reduced IFN-a-2b containing four free cysteine sulfhydryls Mr = 19,269Da). It was expected to have an MW that was 4Da higher than that of the target protein. The mass spectrum of peak 4 revealed that this symmetrical HPLC peak actually consisted of two co-eluting components. The MW of one of the compo-... 

Co-elution of peaks is also not a serious problem with DADs because the spectra obey the Beer-Lambert law. The data computer, therefore, can examine the spectra across a peak for proportionality and yield a peak purity measurement Since each co-eluting component contributes to the total absorbance at each wavelength, the changes in absorbance with retention time reflect the compositional changes. Software can assess these changes in the increase or decrease at specific wavelengths, and mathematically extract the contributions of each component. When recombined for aU wavelengths, individual spectra of each component are obtained [13]. These can then be compared to spectral Hbraries for identification (similar to the treatment of any of the spectra of any peak). 

Last but not least ACDLabs have MS capability in their SpecManager product (http //www.acdlabs.com). Their MS module reads single spectra or GC/(LC)/MS data files and can be used for the subsequent analyses including ESI LC/MS minor components recovery, GC/MS separation of co-eluting components as well as structure/substructure formula generation from mass and ion data (24, 25]. 

To summarize the situation with regard to matrix effects (suppression or enhancement of ionization efficiency by co-eluting components), it is always advisable to be aware of their importance in any proposed analytical method and to minimize them as far as possible. Use of an appropriate isotope-labeled standard in conjunction with matrix matched calibrators should give reliable results if all other appropriate precautions are taken (see Section 8.5.2), hut it is important to investigate the possibility of relative matrix effects (Matuszewski 2003) if it is suspected that the matrix in the analytical sample might differ appreciably from that used to make the matrix matched calibrators. 

Ion suppression Ion suppression occurs in the early stages of the ionization process in the LC-MS interface, when a co-eluted component influences the ionization of the analyte. This is why proper chromatographic separation is always beneficial even with highly selective detectors such as MS or tandem mass spectrometry (MS/MS). It is important to realize that LC-MS/MS methods are just as susceptible to ion suppression effects as single LC-MS techniques because the advantages of MS/MS begin only after the ion formation. 

The evaluation of these mass chromatograms allows the exact determination of the detection Hmit using the S/N ratio of the substance-specific ion produced by a compound. With the SIM/MID mode, this ion would be detected exclusively, but a complete mass spectrum for confirmation would not be available. In the case of complex chromatograms of real samples, mass chromatograms offer the key to the isolation of co-eluting components so that they can be integrated perfectly and quantified. 

True spectral deconvolution of the data. Even if there is no available constant background for subtraction, AMDIS extracts clean spectra. The extraction of closely co-eluting components is possible even for analytes that peak within a single scan of each other in a wide range of each component s concentration. 

The application of mass spectrometry in the analysis of polyphenolic compounds in biological samples produced excellent results. The coupling of liquid chromatography with mass spectrometry allows for the determination of accurate molecular masses, making it possible to predict the stmcture of an unknown compound. UHPLC-MS (20,22) was used to improve the selectivity of methods, and UHPLC-MS/MS (21,23,24,29,30,32-34) was used to reduce the interference caused by co-eluted components of the analyzed mixture, to track the mass fragmentation of the analytes, and to aid the elucidation of molecular stmctures. 

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