30 chromatography • spectroscopy


Fig. 3. Overlay of CD spectra of phosphate-citrate buffers from pH2.2 to pH8.


the protein; a histogram of the ratio of positive to negative peak-heights would give a semi-quantitative picture and highlight conformational changes in a concentration- independent way. For quantitative results, however, some modelling of the data is required.


Te presence of two isosbestic points indicates that a three-state model would be appropriate to describe the data. Global fitting of the model to the data yielded end-point spectra and the concentration profiles of the three species. Te pKa values were calculated to be 2.34 for the E- and F-form and 4.44 for the F- and N-form equilibria respectively.


Te concentration profiles can be used to determine the onset of conformational change.


Te conformational stability (contour) map for the three-state model as a function of pH is shown in Fig. 4. Te region of conformational stability (pH>5.8) of the calculated spectra is well-defined, unambiguous and needs no expert interpretation.


Conclusion A new automated method to measure and analyse the conformational stability of proteins has been described. Te model protein BSA was used in this study


Fig. 4. Model conformational stability map over an extended pH range


but automated CD spectroscopy could be equally well applied to study the relative conformational stabilities of bio-therapeutic candidates in response to changing pH, ionic strength, ligand binding and other variables. Te improvement in reproducibility, productivity and sample economy brought to CD by automation give it the qualities needed to help


References: 1


identify promising bio-therapeutic candidates at the pre-formulation phase of product development.


Enter 30 or ✔ at www.scientistlive.com/elab


David Gregson is Managing Director and Lindsay Cole is Senior Applications Scientist, Applied Photophysics Ltd, Leatherhead Surrey, UK. www.photophysics.com


Carter D C and Ho J X, Structure of Serum Albumin pp153- 203, Advances in Protein Chemistry, Vol.45, Lipoproteins, Apolipoproteins and Lipases, Academic Press 1994, Ed. Verne Schumaker. ISBN: 0-12-034245-6.


Identifying and confirming illicit drugs in their salt form


Thermo Fisher Scientific has developed a method for the identification, confirmation and quantitation of illicit drugs and their salt forms. This unique method combines high-performance liquid chromatography with mass spectrometry (HPLC/MS) to provide accurate simultaneous separation and detection of cations and anions in illicit drug salt forms. The technique enables drug


salt forms to be precisely identified and confirmed by two independent parameters - retention time and mass spectral signature, allowing forensic drug chemists to reliably analyse seized materials. The method also enables


pharmaceutical drug QA/QC analysts www.scientistlive.com


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and violence and inappropriate and extended use of any of these drugs can lead to devastating health outcomes.. Illicit drugs are often encountered in


the form of salts and identification of the specific counterion present may provide valuable information about the source


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screen and identify drugs, drug precursors and new drug derivatives are crucial to law enforcement efforts to curb the black market in controlled substances. The new Thermo Scientific HPLC/ MS method is an alternative to other techniques currently being used to analyse illicit drugs and their salt forms. This method enables simultaneous determination of cations and anions with exceptional sensitivity, detection and accurate identification of a wider range of compounds without requiring derivitisation, which in turn provides simultaneous qualitative and


quantitative information on drug salt forms. In this method, HPLC/MS analysis is performed on a Thermo Scientific Accela UHPLC system coupled to a Thermo Scientific MSQ Plus Mass Detector, a sensitive and fast scanning single quadrupole mass spectrometer. The Accela UHPLC system offers


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For more information, visit www.thermoscientific.com


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