Rapid And Reproducible Bioanalytical Method Development And Validation Services For Biotech!

LC-MS/MS has become one of the most popular drug analysis technique given rapid method development on the platform. Liquid Chromatography with tandem mass spectrometry relies on their combined strengths. While liquid chromatography has excellent separation capabilities, triple quadrupole mass spectrometry offers nuanced selectivity and sensitivity.

These methods use a UV detector to analyze samples and measure their absorption ability for analyte identification. The ultraviolet (UV) detector measures the absorbance of monochromatic light of a fixed wavelength in the UV or visible wavelength range against a reference beam and relates the magnitude of the absorbance to the concentration of an analyte in the eluent passing through a flow cell contained within the instrument. The HPLC UV method is preferable for solutions that have an unknown molecule count. Similarly, drugs with analytes suitable for UV detection have good chromophore due to unsaturated bonds, aromatic groups, or functional groups containing heteroatoms.  Although in some instances, this technique may be necessary, analysts prefer LC-MS/MS method development over UV, as detection can be time-consuming and transition to Clinical samples difficult due to inconsistent interferences in plasma from subject to subject.

HPLC/FL method development can be extremely swift due to the selective nature of this technique. Here, it’s possible to detect even a single analyte molecule in the flow cell. Put differently, fluorescence sensitivity can be 100x more than that of the UV detector for some specific compounds. This is typically an advantage in the measurement of fluorescent species in samples.

Enzyme-linked immunosorbent assay is a method utilized for specific proteins. The technique captures an antigen on a solid surface which is then complexed to an antibody. The detection is achieved by assessing the activity of the enzyme.  Although there has been a notable shift towards using LC-MS/MS analysis for large molecules, it is still quite challenging to achieve the same sensitivity levels as with ELISA.  That said, method development with ELISA can be tricky as other endogenous substances in the biological matrices interfere with analyte detection.

When it comes to analytical method development, scientists have several critical choices to make from a variety of sample preparation techniques, chromatographic conditions, and detection systems. We make every effort to develop a robust and straightforward method that can be used frequently for producing reliable results. The first step in method development is to select a suitable sample clean-up procedure such as simple protein precipitation. Other available options include liquid extraction or solid phase extraction. In some case, it may be necessary to use an even more involved procedure such as chemical derivatization. Once a proper sample clean-up technique is decided, suitable chromatographic conditions are selected next, followed by the determination of an appropriate detection system. Although older detection systems such as ultraviolet (UV) and fluorescence are still in use, mass spectrometry has emerged as the detection system of choice over the last couple of decades due to its high selectivity.