Early development HPLC method

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." " ... 

The first consideration when investigating HPLC method development protocols is the chemical structure of the analyte, in particular, the presence of functional groups capable of interacting with the stationary phase and containing or in the vicinity of the stereogenic elements [79]. Since the natural target of macrocyclic antibiotics is the A-acyl-D-alanyl-D-alanine terminus (see Section 2.1), the early choice of suitable substrates for this kind of CSPs was that of amino acids [45]. However, it turned out that the macrocyclic CSPs were very successful not only in amino acids enantioresolution, but also in the separation of a wide variety of different structures. 

Sometimes when extractions are performed to remove undesired byproducts, the concentration of the desired product in the organic and aqueous layers are also determined. The concentration in the organic layers are deemed the most important (contains the desired product), and the concentrations in the aqueous layers are determined later to ensure mass balance and overall yield of the reaction. The aqueous layers are usually enriched with the undesired by-products and are good samples to use during the development of the HPLC method in the early stages of the synthetic development. The pH of these layers is usually checked as well to ensure that the proper amount of acid or base has been added to the reaction mixture either to quench the reaction or to drive the desired product into the organic layer. 

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. 

As emphasized in Section ILA, a proactive approach to HPLC method development should involve purposeful degradation at the early stages of development with the key degradation samples used in the method development process. See Figure 6 for an overview of the role of purposeful... 

Whereas the other separation methods have been demonstrated to also provide the requisite performance for release and stability testing for select drug substances and drug products, more typically the techniques are applied as supportive methods for HPLC during early-phase development and in niche areas during late-phase development. Because each separation method provides a different mechanism of separation to HPLC, utilization in early-phase development can be used to confirm specificity of HPLC methods. In later phases, both SFC and CE have shown applicability to chiral separations, and GC remains as the unique technique for the determination of residual solvents. 

Case Study HPLC Methods in Early Development 152... 

This chapter reviews the use of HPLC in pharmaceutical analysis from drug discovery to quality control. The focus is on HPLC analysis of drug substances (DS) and products (DP) such as assay for potency, purity evaluation, and dissolution testing. A case study of the various HPLC methods used during early clinical development illustrates the versatility of this technique. Detailed descriptions of HPLC applications in pharmaceutical development and LC/MS analysis in drug discovery and bioanalytical studies can be found elsewhere.1-6 The regulatory aspects in pharmaceutical testing are covered in Chapter 9. 

CASE STUDY HPLC METHODS IN EARLY DEVELOPMENT... 

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