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Compounds that are used in the synthesis of active
pharmaceutical ingredients (API), or reaction
byproducts that form during synthesis, have the
potential to remain as impurities in API. Some of
these compounds are potentially genotoxic impuri-
ties (PGIs) and may raise concern about cancer
and/or birth defects. Because of the toxicity of these
compounds, it is essential that they be controlled to
low levels in API after synthesis. In January of 2007,
the European Medicines Agency (EMEA) released
guidance on acceptable limits of PGIs in APIs
(Guideline on the Limits of Genotoxic Impurities
(EMEA/CHMP/QWP/251344/2006)). Developing
new methods for sensitive detection of impurities
is an increasingly active area of research across the
pharmaceutical industry.
Four structural classes of PGIs are discussed in this article. The first three classes, known collectively as sulfonate esters, include
mesylates, besylates, and tosylates (Figure 1). These alkylating sulfonic acid esters may form when sulfonic acid reacts with an alcohol
solvent. The first three classes are differentiated by the group that forms an ester with the sulfur: mesylates contain a methyl group,
besylates contain a phenyl (benzyl) group, and tosylates contain a toluene group. The fourth class of PGIs tested here, alkyl halides,
consists of short alkyl chains with halogen constituents. Since alkyl halides are polar and very volatile, they are not retained well on
thin film stationary phases. This can make analysis of a mixture of sulfonate esters and alkyl halides quite problematic.
Two options for the analysis of PGIs in API have been developed to meet different laboratory needs. The first option is a fast method
for the analysis of sulfonate esters on the Rxi®-5Sil MS column. The second option is a comprehensive method for the analysis of
both sulfonate esters and alkyl halides on the Rtx®-200 column. Both methods require very little sample preparation, which helps
increase laboratory productivity.
Option 1: Fast Analysis of Sulfonate Esters
Scientists from Merck, in collaboration with Restek, have developed a fast method for the analysis of sulfonate esters on the Rxi®-
5Sil MS column. The use of a thin film Rxi®-5Sil MS column allows for speedy analysis of these active compounds. Since the Rxi®-
5Sil MS column is very selective toward sulfonate esters, a fast oven program can be used to speed analysis. This method allows for
the analysis of selected sulfonate esters in less than 4.5 minutes. A linearity study performed by Merck shows that this method is lin-
ear for sample concentrations from 1ppm to 1,000ppm in API (Figure 2). Depending on the dose of API to the patient, it may be
necessary to detect levels of impurities as low as 1 ppm in order to meet EMEA requirements. The 1ppm spike represents the thresh-
old for toxicological concern (TTC) as set by the EMEA for PGIs.
Figure 2
Linearity of fast GC/MS analysis for selected sulfonate
esters.
Two Options for Analyzing Potential Genotoxic
Impurities in Active Pharmaceutical Ingredients
Laboratory needs for analyzing PGIs in API vary. Here we developed both a fast analysis of
sulfonate esters on the
Rxi®-5Sil MS column
, and a comprehensive method for both sulfonate
esters and alkyl halides on the
Rtx®-200 column
.
By Amanda Rigdon, Pharmaceutical Innovations Chemist, Rick Lake, Pharmaceutical Market Development Manager, Claire Heechoon*, Research Chemist, Roy Helmy*,
Ph.D., Research Fellow, Christopher Strulson*, Research Assistant, and Margaret Figus*, Research Chemist
*Merck & Co., Inc.
• 20 •
2008 vol. 3
Figure 1
Sulfonate ester PGIs. Differences between sulfonate
esters and alkyl halides make the analysis of mixtures challenging.
Pharmaceutical
Scientists fromMerck, in
collaboration with Restek,
have developed a fast
method for the analysis
of sulfonate esters on the
Rxi®-5Sil MS column.
800-356-1688 •
www.restek.com