Preparative HPLC, isolation degradants

In terms of impact on the project, the N-oxide structural elucidation allowed for an appropriate specification of the degradant, and clinical time lines were not impacted. It is advised in all preparative HPLC isolations that an analytical or preparative scale reinjection is performed to clean up the analyte of interest from the salt. This can include washing the analyte by reversed-phase HPLC (preparative or analytical scale depending on isolate amount) with aqueous phase and ramping up the organic phase to elute the desalted analyte of interest. 

Sequence information for the remaining fragments was obtained by Edman degradation (see Section 5.3.1 above) after isolation of the individual peptides using preparative HPLC - the chromatographic resolution being sufficient to allow this, and thus enabled the complete sequence to be determined. 

For the synthesis of the D-C portion, two different concepts were followed either by modification of laminaribiose (166) [89] or by a stereospecific P, 1 ->3-glycosylation [20]. Laminaran is isolated from seaweeds or from Poria cocos Wolf, degraded by selective acetolysis, and the lower oligomers separated by preparative HPLC [90]. Following acetylation, the heptaacetyl laminaribiosyl bromide is prepared and transformed into the disaccharide glycal 167 by the classical approach in 93 % yield. The 2-deoxy-2-iodo-a-glycoside is formed by application of the NIS procedure after deprotection and subsequent 4,6-0-benzylidenation, the precursor 168 for the radical formation of the 6,6 -dibromo-6,6 -dideoxy derivative is at hand. This compound may be further reduced to methyl-3-0-(P-D-chinovosyl)-a-D-olivoside (169). 

The combination of solid-phase extraction (SPE) with HPLC analysis or preparative HPLC can be a valuable tool in concentrating and identifying degradation products. SPE can be a useful technique for the isolation and concentration of analytes from a complex mixture. Selection of the appropriate column depends on the properties of the API and the suspected degradants (70,71). Mixed mode columns having both non-polar and ion... 

Preparative HPLC is another convenient method for isolating degradants from excipient compatibility matrices (72,73). The peaks from stressed samples can be collected, the solvent removed with a rotary evaporator, and the remaining solution lyophilized to obtain purified compounds. The samples can then be analyzed by other methods such as mass spectrometry and nuclear magnetic resonance (NMR) in order to identify the molecular composition. 

Case Study A.3 Degradant Isolation Using Preparative HPLC, LC/MS, and NMR Characterization... 

Case Study A.4 Scaled-up Oxidative Degradation and Isolation Using Solid-Phase Extraction and Preparative HPLC... 

Case Study A.7 Scaled-Up Acid Degradation and Isolation Using Preparative HPLC Characterization by LC/MS, NMR, and IR Spectroscopy... 

Following the success of the hexanoate thioester feeding, we carried out similar experiments with H-labelled butyrate, pentanoate and heptanoate. Of these, the pentanoate results almost exactly mirrored those for hexanoate, the others showing only poor incorporation exclusively by prior degradation to acetate. The pentyl analogue (80) of norsolorinic acid was subsequently isolated by preparative hplc separation from the natural metabolite and fully characterised. Thus it appears that the NSA PKS can accept both pentanoate and hexanoate starters with comparable facility. 

Developing an isolation approach is an activity that is frequently overlooked or addressed as an afterthought. However, solubility and stability data may dictate the development of a chromatographic method that requires the elaboration of the isolation, that is, it is more complicated than a simple evaporation of the mobile phase. The development of the chromatographic process should be linked to and interactively codeveloped with the isolation. Ideally, the isolated impurity sample should not contain other compounds or artifacts, such as solvents, mobile-phase additives or particulate matter from the preparative chromatography, as they may interfere with the structure elucidation effort or adversely affect the stability of the impurity during the isolation process. Therefore, it is preferable to avoid or minimize the use of mobile-phase additives. However, should this prove to be impossible, the additive used should be easy to remove. The judicious choice of mobile phase in the HPLC process increases the ability to recover the compound of interest without or with minimum degradation. The most common... 

The columns are generally packed with silica gel. For the separation to be successful, the size of the silica gel particles should be 40-63 pm. A concentrated solution of the sample is prepared. The sample solution is applied at the top of the column, and the walls of the column are washed with a few milliliters of eluent. Solvent is added to the column, and air pressure is applied at a flow rate of 2 in./min to rapidly elute the desired impurity and/or degradant. Separation is based upon the differential interactions between the solute molecules and the adsorbent surface of the silica gel. Fractions are continuously collected and monitored by chromatographic techniques (HPLC with UV detection, GC, or TLC). The fractions containing the compound of interest are combined and evaporated to dryness. The isolated material is cleaned (post isolation cleanup, such as small-scale column or analytical HPLC reinjection, is essential) and submitted for LC-MS and NMR analysis. 

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