Impurities isolation

Figure 7.2 Unknown impurity isolation and identification workflow.

Nalidixic acid, the prototype of this family of drugs, was synthesized as the result of the discovery that an impurity, isolated during the preparation of the antimalarial chloroquine, had significant antibacterial activity. It was... 

Technical malathion (-95% pure) contains several impurities. Of the impurities isolated, four compounds have been shown to be important in insecticide toxicology (Figure 4.5). Umetsu et al. (1977) demonstrated that all of these impurities potentiated the toxicity of purified malathion in rats, with compounds C and D being more active. Further studies showed that these impurities inhibited serum malathion carboxylesterase and liver malathion carboxylesterase in vitro and in vivo in rats (Talcott et al., 1979), which would explain the potentiating activity observed with these impurities because these carboxylesterases... 

Talcott, R.E., Mallipudi, N.M., Umetsu, N., and Fukuto, T.R., Inactivation of esterases by impurities isolated from technical malathion, Toxicol. Appl. Pharmacol., 49,107,1979. 

In recent years, the combination of HPLC with NMR (LC/NMR) has become a reality, and the application of LC/NMR to trace level impurity identification has been demonstrated. ° LC/NMR experiments along with the development of higher sensitivity NMR probe technology can potentially reduce the need for trace level impurity isolation and sample enrichment. However, it is important to point out the following ... 

In this chapter the applications of TLC in a pharmaceutical environment such as method development and validation for drug substance and drug product, and impurity isolation and characterization by TLC are discussed. 

This test is done to rule out any TLC artifacts that can occur from the sample applied to the origin not moving at the same rate as the drug substance upon the first pass in the mobile phase. It is often utilized to rule out TLC procedure-related bands, as described later in this chapter (Impurity Isolation and Characterization by TLC). A plate is spotted, not streaked, with an appropriate amount of sample at a nominal concentration (typically 10-15 pL). This is done in several applications of about 3-5 units each to prevent diffusion. It is spotted on the lower left corner, at the origin, and this lane is labeled as first pass. The plate is developed in the normal fashion. Once dried, the plate is rotated 90° counterclockwise, another spot is applied in the lower left corner, labeled as second pass, and developed in the same manner. The result shows any TLC artifacts present in the second pass. 

This impurity isolation process (TLC —> HPLC) is commonly utilized in our laboratory. However, the process is tedious and lengthy for extraction from the silica plates, and timing must be coordinated such that the sample is immediately analyzed by the HPLC system to rule out any potential degradation. Sometimes, the band in question is very close to the drug substance or another band, making it difficult to extract for TLC —> HPLC. An alternative is to correlate an impurity by collecting the impurity from HPLC and reapplying it to the TLC system (HPLC — TLC). 

Ligure 20 summarizes how impurity isolation and characterization is performed by TLC. If the correlation experiment shows an impurity match between TLC and HPLC, usually HPLC is used to monitor this impurity. On the other hand, if no match is found, TLC will continue to be the primary quantification method for this particular impurity. 

This chapter briefly describes the various types of packing materials and equipment that can be utilized to isolate impurities from drug substances. It then goes into how to screen for a preparative HPLC method that could be used for impurity isolation and how to move from the screening phase of the project to develop an analytical-scale preparative method. The chapter concludes by giving rules on how to scale that preparative method up to any size column that will be used in the impurity isolation. 

Unstable compounds are problematic. A sample purified in the laboratory might have a short shelf-life and poor performance as a standard. Compounds altered by assays are also inconvenient. For example, substituted benzylic alcohols can dehydrate under acidic HPLC conditions, or carboxylic esters can hydrolyze in aqueous mobile phase. An impurity isolated from an active pharmaceutical ingredient as an organic salt of an organic compound poses two problems at once. The analyst must account for both the acid and the base. In the case of a toluenesulfonic acid salt of an aliphatic amine, two different methods of detection might be needed. The toluenesulfonic acid in a reverse-phase HPLC assay can by monitored by UV light, but the aliphatic amine, with no chromophore, must be measured by a different technique. 

Figure 7. Structure of the symmetric dimethacrylate trace impurity isolated from the SOCM product by HPLC.

The focus of this section will be on the process, techniques and tools by which low-level impurities (i.e., >0.1% level) are isolated for structure elucidation pm-poses. The collection of background information and a clear understanding of the customers needs are central to the success of the impurity isolation step. These factors will determine the isolation approach and strategy one chooses. The isolation step is a first step from which many downstream experiments depend. 

Analytical screens are performed with both reverse-phase RP-HPLC and SFC isolation techniques. Analytical SFC should be screened first unless instrumentation availability or project background specifics dictate otherwise. Screening achiral column bonded phases varying in polarity and functionality against different mobile-phase solvent choices is effective for identifying analytical methods for the purpose of impurity isolation. There are currently many unique achiral SFC bonded phase column choices commercially available (2-ethyl pyridine, diethyl amino, dinitrophenyl, pyridine urea, diol, cyano, etc.). SFC column choice provides the most impact in manipulation of relative selectivity for individual... 

Over the years a number of techniques and approaches have proved to be useful tools to successfully isolate low-level impiu ities and degradants. TLC is most useful when an impurity or degradant is identifiable by LC-MS and above the 1% level. In cases, where NMR analysis is essential for identification, semipreparative SFC, semipreparative HPLC, and flash chromatography are more suitable techniques. Please refer to the Handbook of Pharmaceutical Analysis, 1st ed. for a more in-depth explanation of TLC and flash chromatography s use for impurity isolation. As mentioned earlier, SPE and liquid-liquid extractions are at times incorporated into the process. These tools can quickly convert bulk supply materials into a more suitable form for SFC or HPLC injection. SPE is also a useful tool in dewatering and desalting final RP-HPLC isolated materials obtained from solvents containing mobile-phase additives. 

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