**4. Analytical approaches**

As discussed above, GIs possess unwanted effects and their contamination levels should be controlled. To achieve this, pharmaceutical R&D should employ robust and sensitive analytical methods for supporting drug development and monitoring the levels of GIs. In addition, analytical methods that are capable of measuring trace GIs must be employed to monitor the outcome of GIs during chemical synthesis. In recent years, manufacturers have developed sensitive methods for analyzing various GIs. In this context, conventional HPLC/UV methods are the first option for GIs analysis; however, these methods are often inadequate for the accurate determination of analytes at trace levels, depending on the properties of the analytes and sample matrices. Some of the challenges in the analytical determination of GIs in pharmaceuticals at trace levels include the diverse structural types of GIs, the unstable or chemically reactive nature of GIs, and an extremely high level of API as contaminant (Bai *et al.*, 2010; Liu *et al.*, 2010).

#### **4.1 HPLC methods**

In general, non-volatile GIs are analyzed by HPLC separation techniques, among which reversed phase HPLC (RPLC) is the most widely used separation mode (Elder *et al.*, 2008a; Liu *et al.*, 2010). A simple isocratic RPLC method has been employed for the determination of four genotoxic alkyl benzenesulfonates (ABSs) viz. methyl, ethyl, *n*-propyl, and *iso*propyl benzenesulfonates (MBS, EBS, NPBS, and IPBS) in amlodipine besylate (ADB). The RPLC is also applicable for sulfonate impurities with phenyl moiety such as methyl (MTs), ethyl (ETs) and *iso*propyl tosylates (ITs), methyl (MBs), ethyl (EBs), butyl (BBs) and isopropyl besylates (IBs) (Raman *et al.*, 2008).

Epoxides/hydroperoxides were analyzed using HPLC, and simple RPLC methods employing direct analysis (no sample preparation) were used for some of them. Yasueda *et al.* (2004) described an HPLC method for the determination of loteprednol impurities including a minor photolytic epoxide degradation product. Lacroix *et al.* (1992) reported an HPLC method for the determination of related substances, including the epoxide impurity of nadalol. A rapid resolution HPLC method was used for separating and quantifying the related impurities of atorvastatin, including two epoxide impurities atorvastatin epoxy

Genotoxic Impurities in Pharmaceuticals 399

190 nm.

nm.

Aryl hydrazones E-Aryl hydrazones HPLC with a 5 µm ODS stationary phase (Merck

nm (DAD).

Carbidopa Hydrazine Derivatization using benzaldehyde, followed by LLE.

detection at 305 nm.

detection at 252 nm.

detection at 305 nm.

Copovidone Hydrazine Derivatization using benzaldehyde, followed by LLE.

nm.

Hydrazine (1) Derivatization using benzaldehyde. HPLC

with no operating conditions reported. (2) LSE, followed by derivatization using benzaldehyde

HPLC with a 10µm cyanosilyl stationary phase (R) at 30◦C. Mobile phase: pH 3.0 phosphate buffer and sodium octane sulphonic acid in water/acetonitrile (740/260, v/v). Flow rate 2.0 ml/min; detection at 210

LiChrospher) at 25◦C. Mobile phase: 1mM pH 6.0 phosphate buffer with 2 mM EDTA and methanol (40/60, v/v). Flow rate 1.0 ml/min; detection at 200–400

HPLC with a 5 µm phenyl hexyl stationary phase (Phenomenex Luna) at 25 C. Mobile phase: water and acetonitrile (50/50, v/v). Flow rate 0.3 ml/min. Positive and negative ion mode ESI with ion trap analyzer in SIM mode (M + H ion). Range 50–1000 m/z. Voltage 4

HPLC with a 5µm ODS stationary phase (Altima C18 or Hypersil ODS). Mobile phase: aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min;

HPLC with a 4 µm ODS stationary phase (NovapaK C18). Mobile phase: pH 4.8 10mM phosphate buffer and acetonitrile (450/550, v/v). Flow rate 1.0 ml/min;

HPLC with a 5µm ODS stationary phase (Altima C18 or Hypersil ODS). Mobile phase: aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min;

Derivatization using benzaldehyde, followed by LLE. HPLC with a 5µm ODS stationary phase (R type). Mobile phase: aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min; detection at 305

kV, capillary temperature 250 C.

at lower temperatures. HPLC with no operating conditions reported. Detection at

**Impurities Method details** 

**Active Potential Ingredient (API)** 

Azelastine Impurity A:

Celecoxib Intermediate I: 4-

Dihydralazine sulphate

hydrazine benzene sulphonamide

Hydrazine (impurity B)

benzohydrazide, impurity B: 1 benzoyl-2-[(4RS)-1 methylhexahydro-1Hazepin-4yl] diazane

API (general method)

dihydroxy and atorvastatin epoxy diketone. The limit of detection (LOD) and limit of quantitation (LOQ) for atorvastatin epoxy dihydroxy and atorvastatin epoxy diketone were 0.025 and 0.075 g/ml, and 0.026 and 0.077 g/ml, respectively (Petkovska *et al.*, 2008). Kong *et al.* (2001) determined two epoxide terpenoid impurities (actein and 27-deoxyactein) in a traditional Chinese herbal preparation (*Cimicifuga foetida* L.). Subsequently, they compared the HPLC results with evaporative light scattering detection (ELSD) with UV detection and found that the ELSD was significantly more sensitive. Sample pretreatment was performed prior to analysis owing to the complexity of the matrix. For the two epoxides the on-column sensitivity using UV detection was found to be 606 and 880 ng, respectively, whereas the sensitivity using ELSD was 40 and 33 ng, respectively. Using the optimized extraction procedure (methanol/water, 80/20 v/v) the levels of the two analytes were detected to be 3.44±0.02% and 1.42±0.01%, respectively.

A more common method for the analysis of alkylating impurities is by RPLC and MS detection; however, HPLC/UV methods are also carried out successfully for alkylating impurities. Valvo *et al.* (1997) reported an HPLC/UV method for the separation of 13 impurities of verapamil; this method is claimed to be superior to both the existing pharmacopoeial methods for verapamil. Using this method, the LOD and LOQ were found to be 0.01% (0.05 g/ml) and 0.02% (1.0 g/ml), respectively. Also, the method was found to be sensitive to pH and mobile phase composition; however, it was in contrast to the findings of previous studies insensitive to stationary phase changes.

Hydrophilic interaction liquid chromatography (HILIC) seems complementary to RPLC for the retention and separation of small molecule polar analytes, and has thus gained increasing attention recently. Good retention can be achieved for more polar analytes, which is not possible on RPLC columns. In the hydrazine group, the HILIC method was used in addition to the HPLC/UV and HPLC/MS methods (Elder *et al.*, 2010c; Liu *et al.*, 2010). An Indian research group reported the development and validation of a stability indicating HPLC method for the determination of the anti-tuberculosis drug, rizatriptan, and its degradation products, including a hydrazone impurity (Rao *et al.*, 2006). Hmelnickis *et al.* (2008) used an HILIC method with different polar stationary phases (silica, cyano, amino, and the zwitterionic sulfobetaine) to separate six polar impurities, including 1,1,1-trimethylhydrazinium bromide, and demonstrated that HILIC was a useful alternative to reverse phase or ion chromatography (IC). Elder *et al.* (2010c) reported a table summarizing the various HPLC methods that were used in the literature for a wide range of drugs (Table 4).


dihydroxy and atorvastatin epoxy diketone. The limit of detection (LOD) and limit of quantitation (LOQ) for atorvastatin epoxy dihydroxy and atorvastatin epoxy diketone were 0.025 and 0.075 g/ml, and 0.026 and 0.077 g/ml, respectively (Petkovska *et al.*, 2008). Kong *et al.* (2001) determined two epoxide terpenoid impurities (actein and 27-deoxyactein) in a traditional Chinese herbal preparation (*Cimicifuga foetida* L.). Subsequently, they compared the HPLC results with evaporative light scattering detection (ELSD) with UV detection and found that the ELSD was significantly more sensitive. Sample pretreatment was performed prior to analysis owing to the complexity of the matrix. For the two epoxides the on-column sensitivity using UV detection was found to be 606 and 880 ng, respectively, whereas the sensitivity using ELSD was 40 and 33 ng, respectively. Using the optimized extraction procedure (methanol/water, 80/20 v/v) the levels of the two analytes were detected to be

A more common method for the analysis of alkylating impurities is by RPLC and MS detection; however, HPLC/UV methods are also carried out successfully for alkylating impurities. Valvo *et al.* (1997) reported an HPLC/UV method for the separation of 13 impurities of verapamil; this method is claimed to be superior to both the existing pharmacopoeial methods for verapamil. Using this method, the LOD and LOQ were found to be 0.01% (0.05 g/ml) and 0.02% (1.0 g/ml), respectively. Also, the method was found to be sensitive to pH and mobile phase composition; however, it was in contrast to the findings

Hydrophilic interaction liquid chromatography (HILIC) seems complementary to RPLC for the retention and separation of small molecule polar analytes, and has thus gained increasing attention recently. Good retention can be achieved for more polar analytes, which is not possible on RPLC columns. In the hydrazine group, the HILIC method was used in addition to the HPLC/UV and HPLC/MS methods (Elder *et al.*, 2010c; Liu *et al.*, 2010). An Indian research group reported the development and validation of a stability indicating HPLC method for the determination of the anti-tuberculosis drug, rizatriptan, and its degradation products, including a hydrazone impurity (Rao *et al.*, 2006). Hmelnickis *et al.* (2008) used an HILIC method with different polar stationary phases (silica, cyano, amino, and the zwitterionic sulfobetaine) to separate six polar impurities, including 1,1,1-trimethylhydrazinium bromide, and demonstrated that HILIC was a useful alternative to reverse phase or ion chromatography (IC). Elder *et al.* (2010c) reported a table summarizing the various HPLC methods that were

3.44±0.02% and 1.42±0.01%, respectively.

of previous studies insensitive to stationary phase changes.

used in the literature for a wide range of drugs (Table 4).

**Impurities Method details** 

Allopurinol Hydrazine Derivatization using benzaldehyde, followed by LLE.

HPLC with a 5 µm cyanosilyl stationary phase (R type) at 30 C. Mobile phase: 2-propanol/hexane (5/95, v/v).

Flow rate 1. 5 ml/min; detection at 310 nm.

Flow rate 0.4 ml/min; CLND detection.

Develosil 100 Diol-5(Nomura), (3) 5 µm TSK-Gel Amide-80 (Tosoh Bioscience) and (4) 5 µm Zorbax NH2 (Agilent) at different column temperatures (10–60 C). Mobile phase: TFA/water/ethanol (0.1/30/70, v/v).

Hydrazine HPLC with (1) 5 µm ZIC HILIC (SeQuant), (2) 5 µm

**Active Potential Ingredient (API)** 

API (general method)


Genotoxic Impurities in Pharmaceuticals 401

detection at 305 nm.

detection at 305 nm.

Povidone Hydrazine Derivatization using benzaldehyde, followed by LLE.

254 nm.

quinone.

ODS)

Hydrazine Derivatization using benzaldehyde, followed by LLE.

rate 1.0 ml/min; detection at 310 nm.

HPLC with a 5µm ODS stationary phase (Altima C18 or Hypersil ODS). Mobile phase aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min;

HPLC with a 5 µm ODS stationary phase (R type). Mobile phase acetonitrile/water (400/600, v/v). Flow

HPLC with a 5 µm ODS stationary phase (Altima C18, Hypersil ODS). Mobile phase aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min;

HPLC with 5 µm ODS (Nucleosil C18) and an isocratic mobile phase consisting of a mixture of methanol (A) and pH 3.0 10 mM phosphate buffer containing 5 mM 1 heptane sulphonic acid and 2 mM EDTA (B) in a ratio of 49/51, v/v. Flow rate 0.9 ml/min; detection at 297 and

HPTLC with a silica gel 60 TLC plate (Merck) with a chloroform/methanol/water (80/20/2.5, v/v/v) mobile phase. Examined using Scanner II (Camag) at 330nm for 25-desacetyl rifampicin and 490 nm for rifampicin

HPLC with 10 µm silyl and 10µm nitrile stationary phases (Micro Pak Si-10 and MicroPak CN, respectively) and anisocratic mobile phase consisting of a mixture of chloroform and methanol of varying proportions. Flow

HPLC with direct injection (DI) onto a 3 µm ODS stationary phase (Hypersil ODS) at 25 C and an isocratic mobile phase consisting of a mixture of pH 7.4 50 mM phosphate buffer and acetonitrile (64/36, v/v).

Alternatively, a 10 µm ODS stationary phase (Hypersil

HPLC with a 5 µm L1 ODS stationary phase at 25 C and a gradient mobile phase consisting of varying mixtures of mobile phase A (pH 6.8 phosphate

buffer/acetonitrile, 96/4, v/v) and mobile phase B (pH 6.8 phosphate buffer/acetonitrile, 45/55, v/v or 55/45, v/v). Flow rate 1.5 or 1.0 ml/min; detection at 238 nm. Three L1 columns were evaluated: 1: Zorbax XDB, 2: Shim-pak CLC ODS and 3. Nucleosil EC 120-5.

rate 0.2–0.7 ml/min; detection at 334 nm.

Flow rate 1.4 ml/min; detection at 240 nm.

**Impurities Method details** 

**Active Potential Ingredient (API)** 

Nitrofurazone Impurity A: Bis-

Rifampicin Hydrazones:

Rifampicin Hydrazones:

Rifampicin Hydrazones:

Rifampicin, isoniazid, pyrazinamide

FDC

[(5-nitrofuran-2- yl) methylene] diazane

Hydrazine, isoniazid

rifampicin quinone and 25-desacetyl rifampicin

rifampicin quinone

rifampicin quinone, 25-desacetyl-21 acetyl-rifampicin, 25-desacetyl-23 acetyl-rifampicin

Hydrazones: rifampicin quinone,

desacetyl rifampicin, isonicotinyl hydrazone

Nitrofural, nitrofurazone and nitrofuroxazide

Pyridoxal isonicotinoyl hydrazone


Hydralazine Hydrazine Derivatization using benzaldehyde, followed by LLE.

nm.

Isoniazid Hydrazine HPLC-MS using negative electrospray ionization ESI

under daylight.

detection at 305 nm.

HPLC with a 5µm ODS stationary phase (Hypersil ODS). Mobile phase: acetonitrile/THF/pH 2.6 10mM dibutyl aminephosphate (15/5/80, v/v/v). Flow rate

HPLC with a 5 µm ODS stationary phase (Altima C18 or Hypersil ODS). Mobile phase aqueous 0.03% EDTA and acetonitrile (300/700, v/v). Flow rate 1.0 ml/min;

HPLC with a 10 µm cyanopropyl stationary phase and a mobile phase consisting of a mixture of pH 3.5 10 mM acetate buffer and acetonitrile (95/5, v/v). Flow rate

HPLC with a 5µm ODS stationary phase (Zorbax XDB Eclipse C18). Mobile phase water and acetonitrile (960/40, v/v). Flow rate 0.5 ml/min; detection at 252

with a Bruker Daltonics ToF. TLC with a silica gel F254 TLC plate with a water/acetone/methanol/ethylacetate (10/20/20/50, v/v) mobile phase. Visualization using dimethyl aminobenzaldehyde solution; examination

HILIC with a 3 µm silica stationary phase (Atlantis HILIC silica, Alltima HP silica, and Spherisorb silica), 5 µm cyano stationary phase (Discovery cyano), 3 µm amino stationary phase (Hypersil APS-1), and 5 µm sulfobetaine stationary phase (ZIC-HILIC) at 30 C. Mobile phase acetonitrile and 0.1% formic acid in water. Flow rate 0.2 ml/min with positive ion mode ESI detection at 20–35 kV using a triple quadra pole MS.

HPLC with a 10µm ODS stationary phase (Waters µBondapak) at room temperature. Mobile phase: acetonitrile/5 mM SDS/phosphoric acid (150/850/0.45, v/v/v). Flow rate 2.0 ml/min; detection at 220 nm.

and detection wavelength not specified.

1.5 ml/min; detection at 254 nm.

**Impurities Method details** 

morpholino methyl-

nitrofurfurylidene amino)-oxazolidin-

Hydralazine hydrazone

hydrazine

nicotinyl-2- lactosyl

isonictonic acid-N´- (pyridyl-4 carbonyl) hydrazide (II), isonictonic acidpyridine-4 ylmethylene hydrazide (III), isonictonic acid ethylidene hydrazide) (IV)

**Active Potential Ingredient (API)** 

Hydralazine tablets

Ebifuramin Impurity III: (+)-5-

Isoniazid Impurity I: 1-

Isoniazid Hydrazine (I),

Mildronate Impurity 2: 1,1,1-

trimethyl hydrazinium bromide

3-(5-

2-one


Genotoxic Impurities in Pharmaceuticals 403

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 two-

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

dimensional GC (Figure is reproduced from David *et al.*, 2010).


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