The concentration limit (in ppm) of a GTI in a drug substance can be calculated based on the expected daily dose to the patient
and the TTC value using the following equation:
The TTC concept should not be applied to carcinogens where adequate toxicity data are available from long-term studies and
allow for a compound-specific risk assessment.
Identification methods for GTIs.
GTIs can be identified by the following methods:
- Consulting a list of known genotoxins
- Determining whether an element has functional groups similar to those of known genotoxins
- Performing genotoxicity assays
- Using computer-based structure-activity relationship software programs (e.g., MultiCASE's Mcase, Accelrys's Topcat, or LHASA's
- Performing Ames bacterial mutagenicity testing after software provides a structural alert. Results from the Ames test are
considered more definitive than the in silico data.
Selection of the analytical technique
Lou et al. reported a method-development strategy for the control of GTIs (9–11). Analytical techniques can be selected by
dividing GTIs into two groups based on their volatility. High-performance liquid chromatography (HPLC) with UV detection should
generally be the first choice for nonvolatile GTIs because of the methods' simplicity and availability (12). However, HPLC
may not offer sufficient sensitivity for certain GTIs in low-level analysis. If GTIs offer insufficient UV response, ultraperformance
or ultra fast liquid chromatography (UPLC or UFLC) can be used because they have enhanced UV-detector sensitivity. When GTIs
lack chromophores, an evaporative light-scattering detector (ELSD) is the alternate choice. However, ELSD is limited in sensitivity
and dynamic range. Refractive index detectors and fluorescence detectors are alternate detectors used in HPLC. Because low-quantitation
limit establishment is challenging, coupling HPLC or UPLC with mass spectrometers (MS) significantly improves method sensitivity
and speed (13, 14). These detectors are selective and minimize issues caused by interference in the sample matrix, thus improving
data quality. However, these instruments are expensive and differ from vendor to vendor. Thus, transferring a method between
the developing and receiving laboratories, which may use instruments from different vendors, requires optimization of multiple
instrumental parameters (9).
Volatile GTIs, meanwhile, can be quantitated by gas chromatography (GC) with a flame ionization detector (FID) (14, 15). GC–FID
in direct and headspace injection modes is generally the preferred method, but this depends on the properties of GTIs and
sample matrices. An electron-capture detector (ECD) can be used when GTIs consist of halogens. Nitrogen–phosphorus detectors
(NPD) offer an additional tool for GTIs containing nitrogen or phosphorus atoms. However, the applications of ECD and NPD
are limited. GC–MS offers the most sensitive and selective detection and reduced background noise. The method also is less
prone to interferences for low-level analysis of GTIs (16, 17). If the GTIs are labile, do not possess chromophores, and have
reactive functional groups, they can be derivatized to form detectable species (e.g., hydrazine derivatizes with benzaldehyde
to form 1,2-dibenzylidenehydrazine) (14, 18). The derivatization reagent should be selected according to the functional groups
in the analyte. Derivatization helps in stabilization, incorporation of a unique structural moiety, enhancement of fluorescence,
ionization for mass detection, and volatization for GC.