Early development HPLC

The increased use of IV-methyl carbamate insecticides in agriculture demands the development of selective and sensitive analytical procedures to determine trace level residues of these compounds in crops and other food products. HPLC is the technique most widely used to circumvent heat sensitivity of these pesticides. However, HPLC with UV detection lacks the selectivity and sensitivity needed for their analysis. In the late 1970s and early 1980s, HPLC using post-column hydrolysis and derivatization was developed and refined with fluorescence detection to overcome these problems. The technique relies on the post-column hydrolysis of the carbamate moiety to methylamine with subsequent derivatization to a fluorescent isoindole product. This technique is currently the most widely used HPLC method for the determination of carbamates in water" and in fruits and vegetables." " ... 

Options for multichannel LC-CD detection do exist.f " Stopped-flow accessories for commercial instruments are available that allow part of an eluted fraction to be taken off-line into a microcell placed in the regular sample compartment where data are measured in the normal way. The method still requires rapid scanning capabilities. Repeated injections and multiple scans can be averaged to improve the quality of the signal. A major deterrent to the progress in the early development of HPLC-CD detection was the lack of a dedicated instrument at a reasonable cost, the only option being a fully equipped CD instrument. 

Case Study HPLC Methods in Early Development 152... 

CASE STUDY HPLC METHODS IN EARLY DEVELOPMENT... 

However, use of multicolor REMPI (more than one laser emitting different wavelengths) should permit extension of the approach to analytes with more complex UV absorption behavior. Coupling of both conventional and capillary HPLC and also CEC to MS via the APLI source was reported (Constapel 2005 Eiroste 2005), with detection limits for polycyclic aromatic compounds in the low fmol range. This approach is still in its early development stage but appears to have considerable potential. 

Several useful articles and reviews on HPLC of peptides can be found in Refs. 38, 235, and 236, with Ref 237 representing a useful resource book in this area. For an extensive source of information on the early development of HPLC of peptides, the reader is directed to Ref 238. A comprehensive practical overview on this topic is supplied by Ref 239 the tatter publication also supplied much useful information on preparative approaches to HPLC purification, as well as an overview of micro- and narrow-bore HPLC of peptides. An excellent and current publication dealing with high-performance analysis of biomolecules [240] also offers a sound introduction to capillary electrophoresis (CE), an important complementary technique to conventional HPLC. 

However, compared with the traditional analytical methods, the adoption of chromatographic methods represented a signihcant improvement in pharmaceutical analysis. This was because chromatographic methods had the advantages of method specihcity, the ability to separate and detect low-level impurities. Specihcity is especially important for methods intended for early-phase drug development when the chemical and physical properties of the active pharmaceutical ingredient (API) are not fully understood and the synthetic processes are not fully developed. Therefore the assurance of safety in clinical trials of an API relies heavily on the ability of analytical methods to detect and quantitate unknown impurities that may pose safety concerns. This task was not easily performed or simply could not be carried out by classic wet chemistry methods. Therefore, slowly, HPLC and GC established their places as the mainstream analytical methods in pharmaceutical analysis. 

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