**4.2 GC methods**

GC methods are commonly used for the analysis of several volatile small molecule GIs. Some examples include the liquid injection technique and the headspace sampling technique. Liquid injection is prone to contamination in which injection of a large amount of non-volatile API can accumulate in the injector liner or on the head of the GC column, which can cause a sudden deterioration in method performance. Headspace injection, on the other hand, is desirable because it minimizes potential contamination of the injector or column by avoiding the introduction of a large quantity of API (Liu *et al.*, 2010).

David *et al.* (2010) proposed a method selection chart (Figure 4) containing GC or LC methods, both in combination with a single quadrupole mass spectrometer as detector. These methods applied for a wide range of analytes including sulphonates, alkyl halides, and epoxides.

Nassar *et al.* (2009) developed a GC/MS method for residual levels of EMS in a mesylate salt of an API crystallized from ethanol. The method was capable of detecting EMS down to levels of 50-200 ppb. Subsequently, extraction techniques were developed for eliminating or reducing matrix related interference. Thus, Colon and Richoll (2005) surveyed liquid–liquid extraction (LLE), liquid phase micro-extraction (LPME), solid phase extraction (SPE), and solid phase micro-extraction (SPME) coupled with GC/MS and single ion-monitoring (SIM). Using these approaches, they developed limit tests (5 ppm) for some alkyl aryl esters of sulfonic acids.

Similar attempts were made for reducing or eliminating the matrix effect for alkylating agents as well. In all these procedures, a specific physical property of the analyte not shared by the matrix was utilized, e.g. low boiling point and/or in the presence of halide atom (Elder *et al.*, 2008a).

detection at 225 nm.

Table 4. Various HPLC methods used for a wide range of drugs; Abbreviations: DAD: diode array detection; EC: electrochemical detection; ESI: electrospray ionization; FDC: Fixed Dose Combination; HILIC: hydrophobic interaction liquid chromatography; LLE: liquid liquid extraction; LSE: liquid solid extraction; MS: mass spectroscopy; ODS: octadecyl silyl; SDS: sodium dodecyl sulphate; SIM: single ion monitoring; ToF: time of flight (Elder *et al.*, 2010c). The use of water as sample diluent could pose a limitation for this separation technique, especially when high water content is required for dissolving the drug substance or the

GC methods are commonly used for the analysis of several volatile small molecule GIs. Some examples include the liquid injection technique and the headspace sampling technique. Liquid injection is prone to contamination in which injection of a large amount of non-volatile API can accumulate in the injector liner or on the head of the GC column, which can cause a sudden deterioration in method performance. Headspace injection, on the other hand, is desirable because it minimizes potential contamination of the injector or column by

David *et al.* (2010) proposed a method selection chart (Figure 4) containing GC or LC methods, both in combination with a single quadrupole mass spectrometer as detector. These methods applied for a wide range of analytes including sulphonates, alkyl halides,

Nassar *et al.* (2009) developed a GC/MS method for residual levels of EMS in a mesylate salt of an API crystallized from ethanol. The method was capable of detecting EMS down to levels of 50-200 ppb. Subsequently, extraction techniques were developed for eliminating or reducing matrix related interference. Thus, Colon and Richoll (2005) surveyed liquid–liquid extraction (LLE), liquid phase micro-extraction (LPME), solid phase extraction (SPE), and solid phase micro-extraction (SPME) coupled with GC/MS and single ion-monitoring (SIM). Using these approaches, they developed limit tests (5 ppm) for some alkyl aryl esters of

Similar attempts were made for reducing or eliminating the matrix effect for alkylating agents as well. In all these procedures, a specific physical property of the analyte not shared by the matrix was utilized, e.g. low boiling point and/or in the presence of halide atom

avoiding the introduction of a large quantity of API (Liu *et al.*, 2010).

HPLC with a 5 µm nitrile stationary phase (Zorbax SB-CN) at 25 C and a gradient mobile phase consisting of varying mixtures of pH 3.4 10 mM phosphate buffer, acetonitrile, and methanol. Flow rate 1.0 ml/min;

HPLC with a 5 µm ODS stationary phase (R type) and a gradient mobile phase consisting of varying mixtures of pH 7.5 diethyl aminephosphate buffer and methanol.

Flow rate 2.0 ml/min; detection at 270 nm.

**Impurities Method details** 

hydrazinophenyl) methyl-1,2,3 triazole

Impurity C (desacetyl vinblastine hydrazide)

formulated drug product (Liu *et al.*, 2010).

**Active Potential Ingredient (API)** 

Vindesine sulphate

**4.2 GC methods** 

and epoxides.

sulfonic acids.

(Elder *et al.*, 2008a).

Rizatriptan Impurity I: 1-(4-

GC methods were rarely used for the analysis of epoxides/hydroperoxides, as compared to other impurities, owing to the size of molecule and the volatility properties within this group (Elder et al., 2010b). Klick (1995) used a GC method for the determination of residual levels of a chlorohydrin and the corresponding epoxide impurities in almokalant. Other literatures give an account of GC–MS methods for the analysis of volatile components in traditional Chinese herbal medicines (Yu *et al.*, 2007; Guo *et al.*, 2003).

Fig. 4. Method selection chart for analyzing genotoxic impurities with GC/LC; 1AP-ES/APCI: atmospheric pressure electrospray ionization/ atmospheric pressure chemical ionization; 2 If the analyte has sufficient vapor pressure in water or other low volatile solvent; 3 SHS: static headspace; 4 SPME: solid-phase micro-extraction; 5 DHS: dynamic headspace; 6 HILIC: hydrophobic interaction liquid chromatography; 7 derivatization-RPLC: reversed phase HPLC with precolumn derivatization; 8 Back-flush (CFT): capillary flow technology based back-flushing; 9 Deans 2DGC (CFT): capillary flow technology based twodimensional GC (Figure is reproduced from David *et al.*, 2010).

For the hydrazine group the normal flame ionization detection (FID) in GC analysis is not appropriate because these compounds possess no carbon atoms (Elder *et al.*, 2010c). A GC

Genotoxic Impurities in Pharmaceuticals 405

based methods, in comparison to other separation methods. The problem of limited sensitivity of CE methods can be solved either by the use of detection methods with sensitivity higher than UV absorption or by pre-concentration of the analytes (Jouyban and

Alternatively, the structure of the molecule as well as its properties can be altered to enhance detectability which in turn will help to achieve the desired sensitivity. This is especially true for GIs that lack structural features for sensitive detection (Bai *et al.*, 2010; Liu *et al.*, 2010). A number of general approaches could be considered, some of which are explained below:

This method is generally used for stabilizing reactive GIs and for introducing a detection specific moiety for enhanced detection, i.e. chromophore for UV. Also, this method sometimes produces a single compound for several GIs; thus, it becomes non-specific which can be considered as an advantage in determining a group of structurally related compounds (Liu *et al.*, 2010). Bai *et al.* (2010) introduced a chemical derivatization method for analyzing two alkyl halides and one epoxide. The objective of the three derivatization reactions is to generate a strong basic center by introducing an amine functional group. All three derivatization products are good candidates for electrospray ionization (ESI)-MS

Owing to their structural features, several analytes are not amenable to atmospheric pressure ionization methods, such as the ESI method. Alkali metal ions such as Li+, Na+, and K+ can form complexes with some organic molecules in the gas phase; this fact could be

The matrix deactivation approach is a chemical approach to stabilize unstable/reactive analytes. It is based upon the hypothesis that the instability of certain GIs at trace level is caused by the reaction between the analytes and reactive species in the sample matrix. Thus, controlling the reactivity of the reactive species in the sample matrix would stabilize the

As an example the alkylators are reactive unknown impurities which possess mainly nucleophilic characteristics. Their reactivity can be attenuated by either protonation or scavenging approaches. Sun *et al.* (2010) reported a matrix deactivation methodology for improving the stability of unstable and reactive GIs for their trace analysis. This approach appears to be commonly applicable to techniques like direct GC–MS and LC–MS analyses,

The concept of using structural alerts to predict potential genotoxic activity for identied impurities is now well established; however, the concordance between such alerts and biologically relevant genotoxic potential (in the context of genotoxic impurities) could be

Kenndler, 2008).

**4.5 Enhancing methods** 

**4.5.1 Chemical derivatization** 

**4.5.2 Coordination ion spray-MS** 

unstable/reactive GI analytes (Liu *et al.*, 2010).

or coupled with chemical derivatization as well.

**5. Genotoxicity prediction** 

**4.5.3 Matrix deactivation** 

owing to the high proton affinity or the permanent charge.

used as a solution for the analytes subjected previously (Liu *et al.*, 2010).

procedure involving the formation of a benzalazine derivative was developed for monitoring the residual levels of hydrazine in hydralazine and isoniazid APIs, tablets, combined tablets, syrups, and injectable products in which nitrogen selective detection was used (Matsui *et al.*, 1983).

In addition, Carlin *et al.* (1998) adapted a previously published method for monitoring a benzalazine derivative using GC with electron capture (EC) detection. The LOQ was 10 ppm and the method was linear over the range of 10-100 ppm. The inter-day residual standard deviation (RSD) based on six measurements at analyte levels of 10 ppm was 15%; however, this improved slightly at increased analyte concentrations of 25 and 100 ppm, to 9.5% and 11.3%, respectively.

Nevertheless, non-volatile API does not partition into the headspace and therefore does not enter the GC system; as a result, headspace injection becomes the preferred choice whenever possible (Liu *et al.*, 2010).
