BIOANALYTICAL TECHNIQUES – AN OVERVIEW
Practical considerations for robust and sensitive CE-MS coupling
CE has several advantages over HPLC, namely fast method development, low sample and solvent consumption, rapid and very efficient separations and, in the particular field of chiral separations, the use of expensive chiral stationary phases is not required. Nevertheless, problems of ruggedness in the on-line coupling of CE with MS often limit its application for quantitative purposes. Among others, this can be explained by variations in the migration times (MTs), due to fluctuations in the EOF and/ or the lack of thermo stating of the capillary part linking the CE instrument to the MS source. Furthermore, some parameters of the electro spray, the most common CE interface, have to be carefully adjusted to obtain stable CE-ESI-MS conditions, namely the sheath liquid composition and flow rate, the nebulizing gas pressure, and the capillary outlet position.
Composition and flow rate of the sheath liquid
Even if the composition of the sheath liquid greatly depends on the studied analyte, some general rules can be prescribed. An aqueous solution containing 50– 80% of a moderately polar organic solvent is often required to achieve the formation of a stable spray, due to a decreased surface tension. On the other hand, a higher organic solvent content gives rise to a higher response for most organic analytes, due to a more efficient desolvation of the compound as well as a better stability of the spray.
Nebulizing gas pressure
In an ESI interface, the nebulizing gas pressure, used to assist droplet formation and to obtain a stable spray, is a compromise between sensitivity and spray stability. It is well known that the application of the nebulizing gas gives rise to a reduced pressure at the capillary outlet. Therefore, separation performance (efficiency and resolution) and MTs can be decreased, as a result of the hydrodynamic flow generated in the CE capillary. This decrease of separation performance and MTs with increasing nebulizing gas pressure is illustrated in the work. It also appeared that the nebulizing gas pressure influences the detection sensitivity. Indeed, at the highest pressure value, the S/N ratio was 20% lower than at the lowest value due to higher noise level while the abundance of drug improved with increasing nebulizing gas pressure.
Capillary outlet position
To achieve reproducible CE-MS analysis, it is necessary to have a well-defined procedure to install a new capillary In the literature, several strategies have been reported. Very recently, Ohnesorge et al described a new procedure, based on the observation of a strong neostigmine carry-over effect . After the replacement of the capillary, the analysis of the buffer, instead of the sample, was performed. The signal resulting from the neostigmine carryover effect was adjusted to a high intensity level, selected as reference intensity level for the following capillary changes, assuming that the intensity of the carry-over signal is constant.
Most applications dealing with the bioanalysis of drugs in CE-MS were focused on serum and urine samples, although other biological fluids can be used, such as hair, cerebrospinal fluid (CSF), etc. Proteins, the main constituents of plasma, strongly adsorb onto the capillary wall and therefore adversely affect separation efficiency, resolution and MT. Urine contains inorganic ions and other endogenous compounds, such as urea, that can also interfere in the electrophoretic analysis. Moreover, the high ionic strength of urine is unfavourable to sample stacking, and therefore, peak broadening may be observed. Among sample preparation techniques, SPE and liquid/liquid extraction are efficient cleanup procedures which can also be applied to increase the analyte concentration. Nevertheless, these sophisticated sample preparation procedures are often tedious and time-consuming .
Figure 5: CE/MS instrument
This review is aimed in focusing the role of various bioanalytical techniques in pharmaceuticals and gives a thorough literature survey of the bioanalytical methods and instruments in drug analysis. This review also highlights the recent advancements of bioanalytical techniques. Among all the bioanalytical methods, LCMSMS method is widely used and vast number of research publications has been reported by LC/MS/MS method due to its better sensitivity and precision.
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