GENOTOXIC IMPURITIES: AN IMPORTANT REGULATORY ASPECT

Genotoxins are agents/carriers such as chemical or radiation that can cause the damage to DNA or chromosomal structure, thereby causing mutations and the process are called as genotoxicity. Identification and understanding of genotoxins at a primary stage of drug development would enable us to prevent the potential damage that can be caused by these genotoxic agents. Various regulatory agencies such as International Council for Harmonization and EMEA, USFDA, European Pharmacopeia guidance, guidance for oncology products provide guidelines to limits the level of impurities in drug substances and drug products. Nowadays, conventional protocol of isolation, various spectral analysis high-performance liquid chromatography (LC), Fourier transform infrared to on-line analysis using modern, sophisticated hyphenated tools, like gas chromatography-mass spectroscopy, LC-MS so on, as well as modern software based in silico drug designs are extensively used by industry, research, and development areas and also there is tremendous increase in publications in the literature involving their use. Our review article focused on the various regulatory guidelines, application of hyphenated tools, and in silico techniques in genotoxic impurity and degradation product profiling of small molecules. A brief explanation is made on possible pitfalls in the experimentation and data interpretation. From this review, it concluded that there are various countries having their own regulatory agencies and regulatory guidelines for drug approvals, which may be followed by applying new chemical entities the new drug application title (NDA) in new drug application as well as there are various conventional to modern software based techniques to quantification of genotoxic impurities.


Gupta et al.
data clarify the mechanism of GTIs. ICH Q3C (R4) specified these in class 2 solvents. The absence of any experimental data for evidence of GTIs mechanism but can be controlled "as low as reasonably practicable" accordance with ALARP principle. Limits of therapeutic toxic concentration (TTC) value of 1.5 g/day intake of GTI were considered under the acceptable limits [11].

PhRMA approach
It provides structural classification which consists of alerting functional groups. The presence of such structural moieties was known to be involved in mutation of DNA (Fig. 4).

PhRMA categorized impurities into five classes (Table 2) [12] USFDA
USFDA released draft guidance to address GTI issues which characterize and reduce the potential lifetime cancer risk associated with patient exposure to genotoxic and carcinogenic impurities. The recommended approaches include: • Prevention of genotoxic and carcinogenic impurity formation • Reduction of genotoxic and carcinogenic impurity levels (allowing a maximum daily exposure target of 1.5 g/day) • Characterization of genotoxic and carcinogenic risk and • Considerations for flexibility in approach to better support appropriate impurity specifications [13].

European Pharmacopoeial guidance
During revising or elaborating the monograph on GTI's, European Pharmacopoeia requires a pragmatic approach on GTIs. It says that the products that receive a marketing authorization after the issuance of General risk assessment methodologies and approaches for genotoxic and carcinogenic substances (2009).
The standard test battery for genotoxicity recommends the following for genotoxicity evaluation (Table 3) [19]   the EMEA guideline have to be evaluated for the presence of GTIs and this should be the basis for a new monograph [14,15].

Guidance for oncology products
The USFDA draft guidance states, "a TTC value higher than 1.5 g/day may be acceptable in certain situations such as human exposure will be short term, treatment which is used for life-threatening conditions having life expectancy is <5 years, conditions where impurities were known substance as well as human exposure will be much greater from other sources." The ICH S9 guideline on nonclinical evaluation for anticancer pharmaceuticals also states, for genotoxic impurities, several approaches have been used to set limits based on increase in lifetime risk of cancer but it is not appropriate for pharmaceuticals intended to treat patients with advanced cancer and if it is used justifications should be considered to set higher limits for that particular pharmaceutical' [16,17].

Risk assessment for genotoxic and carcinogenic substances
European Commission Health and Consumer Protection Directorate.

TECHNIQUES TO DETECT GENOTOXIC IMPURITIES
To analyze the impurities sensitive, selective, and robust analytical methods required to build rational and sufficient control strategies for GTIs. The selection of methodology for analyzing GTIs depends on target specifications, expected values for these impurities to comply with regulatory point of view. Therefore, a selected methodology should provide robust analytical data and the selected method can be used for routine testing burdens. Analytical techniques are selected based on genotoxic properties and the chemical properties of GTI's (given in Table 5) such as variety of structures, reactivities, and responsiveness to selected detection methods and potential matrix effects [21].
Quantitation of ppm level of GTIs presents in the pharmaceutical has many challenges to analytical chemist such as: • Appropriate analytical technique selection for method development depending on properties of a GTI (volatility, thermal stability, presence of a chromophore, hydrophobicity, etc.) • Feature of GTI such as reactive nature and stability of the GI must be reflecting during method development to provide evidence regarding requisite reproducibility and accuracy • Selection of sensitive analytical method as well as clinical dose and duration of the study must be taken into the consideration during method development.
Analytical method to be developed, include parameters such as choice of the detection technique (ultraviolet [UV], light scattering, electrochemical detection, mass spectrometry, etc.). Sample matrix challenges also interfering matrix components which are eliminated by: • Isolation of the analyte of interest by sample preparation • chromatographic resolution, or • Using a more selective detector.
A wide variety of analytical techniques can be used to analyze GTI. Due to their high structural diversity, complexity of the sample matrix, it is difficult to select a single ideal method. But ideally, no single approach is applicable to sort out all problems. As time passes out various conventional separations, hyphenated techniques, as well as software based in silico drug designs are widely used now.
Analytical laboratories in many pharmaceutical industry follows systematic strategies for GTI method development have 2 steps: • Evaluation of the volatility of the analyte to select chromatographic techniques • Selection of a detection techniques based on the properties of the analyte such as presence of a chromophore and presence of a halogen atom within the molecule.

High-performance liquid chromatography (HPLC)
Compounds which are nonvolatile and having aromatic or other structural features can be analyzed by HPLC with UV detection. The impurities and the API have structural similarity show excellent selectivity of the HPLC method and give accurate quantitation. Reversed-phase HPCL (RP-HPLC) also widely used now. For extremely polar analytes, hydrophilic interaction LC (HILIC) can be used to achieve sufficient retention. However, GTIs which enable to respond LC conditions or with a low response to UV detection, required chemical derivatization to increase the sensitivity of analysis by HPLC-UV method. Alternate to UV detection and to increase sensitivity-selectivity of low parts-per-million levels of GTIs, alternate detectors such as evaporative light scattering detectors or charged aerosol detectors may be used [22].
Chloramphenicol 2, 4-DNPH derivatization used to determine 4-nitrobenzaldehyde in injection formulations by HPLC-UV derivatization analysis method. For the extraction and concentrate, the derivatization products salt-assisted liquid-liquid microextraction technique was used, it also increased the sensitivity toward analysis. The complex nature of the concentrated derivatives, less volatility of the analyte hindered the analysis, therefore 4-NBA in drug was converted into 3-nitrophenylhydrazone (Fig. 5). 3-normal pressure hydrocephalus hydrochloride (HCl) had good sensitivity was selected for derivatization and quantitation of GTIs [23].

Dalfampridine
HPLC utilizing a HILIC Technique was applied to determine five potential genotoxic impurities (PGI's) in dalfampridine. It has sensitivity to detect impurities at low level up to 7.5 ppm for Impurity-I, Impurity-II, Impurity-III, Impurity-IV, and Impurity-V ( Fig. 6) [24].

Voriconazole
GTIs in the manufacturing process of voriconazole were detected, quantified and controlled by RP-HPLC method in combination with photo chemically induced fluorescence (PIF) detection for the analytical control of GTIs in the formulation. GTI properties were evaluated by PIF detector and utilized for its selective detection and sensitive quantification compared with the commonly used UV detection methods.
The aminosulfide and nitrosulfide used as raw material known to be potential GTIs were predicted to be mutagenic by DEREK Nexus version 2.0 and further confirmed by an Ames bacterial mutagenicity test (Fig.  7). Therefore, aminosulfide and nitrosulfide impurities were confirmed as genotoxic, need to quantify by accurate, sensitive, and quantitative method such as RP-HPLC method in combination with PIF [25].

Febuxostat
Hydroxylamine and its salts act as reducing agents in myriad organic and inorganic reactions. Hydroxylamine HCl reagent is used in the synthesis of febuxostat for febuxostat ethyl ester intermediate preparation from formyl febuxostat ethyl ester. From which hydroxylamine HCl found to be mutagenicity in the mouse lymphoma tk mutation assay. Therefore, hydroxylamine was determined and quantified by benzaldehyde derivatization procedure by HPLC. The derivatization mechanism is shown in Fig. 8 [26].

Linagliptin: (UPLC)
Linagliptin subjected to various conditions for degradation study, it was degraded at acidic condition and formed one unknown degradation product (impurity I) which was detected in HPLC. It was isolated, identified, and characterized by the spectral data (MS, MS/MS, 1D NMR, 2D NMR, and infrared spectrum) and finally impurity I was confirmed. The formed degradation product (impurity I) and another process-related impurity (impurity II) showed structural alerts of GTIs containing N-Acylated aminoaryl and alkyl halide, respectively, as a structural groups (Fig. 9). Hence, there is a need analyzed linagliptin by a rapid and sensitive method such as facile ultra-HPLC method was developed for simultaneous determination of these two PGI at trace level (ppm levels) in linagliptin [27].

MLN9708
MLN9708 is an investigational small-molecule proteasome inhibitor; undergo drug development studies done by Millennium: The Takeda Oncology Company in multiple clinical studies for the treatment of a broad range of human malignancies. MLN9708 drug initially evaluated for in silico studies for GTIs which includes starting materials, reagents, and intermediates used in the manufacturing process as well as for the process impurities and degradation products. Among them, 2,5-dichlorobenzoyl chloride (DCBC) which was a synthetic intermediate in the synthesis was reported to be mutagenic by DEREK Nexus version 2.0 and TOPKAT version 6.2 as an structural alert functional group such as acyl halide (Fig. 10). Due to multiple acid/base work-up steps in the manufacturing process, the risk of DCBC reported to be less but to assure that required to developed an accurate, sensitive, and quantifiable method. Therefore, it was an important to quantify the impurities in the view of regulatory point of view. Hence, the UHPLC/HRQMS method was developed, justified, and validated for the determination of DCBC in MLN9708 drug substance at low-level detection [28].

B. HYPHONATAED TECHNIQUES
Gas chromatography-mass spectroscopy (GCMS) GC/MS-a combination of two GC and MS, used to analyze complex organic as well as biochemical mixtures. GC can be used to separate volatile and semi-volatile compounds with high resolution. MS provide structural data of compounds such that they can be exactly identified and quantified, but cannot separate out. Therefore, combination of gas chromatography and mass spectrometry is used widely and is highly compatible techniques [29].

Valsartan
Valsartan tablets contain, N-nitrosodimethylamine (NDMA) as an impurity. This impurity is classified as a human carcinogen, which was incorporated into the finished product as manufacturing process related impurity of the drug substance. GC/MS headspace method was implied to detect the presence of NDMA in Valsartan drug substance and drug products. Depending on limit of detection LOD and limit of quantitation LOQ (as 0.05 and 0.3, respectively) the Valsartan tablets are recalled from the market (Fig. 11) [30,31].

Tolvaptan
Tolvaptan is synthesized using ethyl 4-bromobutyrate as a raw material in the intermediate manufacturing process of tolvaptan. It was reported as potential carcinogenic impurity as per its structure as primary alkyl halides. Hence, according to the ICH guidelines and regulatory point of view, there is a need to prove that the levels of ethyl 4-bromobutyrate were present below 1.5 μg/day based on the maximum daily dose (MDD) of the drug. According to the TTC approach of the ICH guideline, the limit for ethyl 4-bromobutyrate potentially genotoxic impurity is found to be 25 ppm for genotoxic impurities and based on the MDD, i.e., 60 mg/day of tolvaptan (Fig. 12) [33].
Chlorpheniramine/chlorpheniramine maleate 2-dimethylaminoethyl chloride HCl (DMC HCl) shows structural alert for genotoxic mutagenicity and carcinogenicity which was used as raw material in manufacturing of chlorpheniramine. Therefore, quantification of genotoxic impurity DMC HCl is important at ppm level in chlorpheniramine/chlorphenamine maleate. For these, GC-MS method was used and detection limit and quantification limit found to be 0.94 ppm and 3.75 ppm, respectively (Fig. 13) [34].

Dobutamine HCl
A sensitive GC-MS method for analysis of residues of methyl-p-toluene sulfonate ethyl p-toluene sulfonate and isopropyl p-toluene sulfonate genotoxic impurities in dobutamine HCl drug substance (Fig. 14) [35].

Fudosteine
In the synthesis process of fudosteine, 3-chloro-1-propanol used as raw material, it consists of 1,3-dichloropropane, 3-chloropropylacetate, and chloropropyl hydroxypropyl ether as residual GTIs. According to regulatory point of view, these residual genotoxic impurities should be within the limits <1.25 μg/g based on that a MDD of fudosteine is 1.2 g. Therefore, the dichloromethane and 5 N sodium hydroxide solutions were used to extract the impurities and levels of these impurities monitored and controlled by GC-MS with SIM mode, as well as helium as the carrier gas for greater resolved chromatographic method [36].

Zidovudine
Methyl methanesulfonate (MMS) is known to be carcinogenic and genotoxin and was a potential process impurity of zidovudine (Fig. 16). MMS is incorporated by international agency for research on cancer in Group 2A. MMS is possible formed because of reaction between methane sulfonyl chloride and methanol to form corresponding mesylate during the manufacturing process which was incorporated in the final product. Therefore, MMS in zidovudine drug substance as GTI was detected using a gas chromatography technique with mass spectrometer as a detector [38].

Folpet
Folpet is a widely used protective fungicide in Switzerland, which contains two impurities such as carbon tetrachloride (CCl4) and perchloro-methylmercaptan which was determined by headspace GC-MS (Fig. 17). CCl4 induces hepatic cell proliferation and DNA synthesis, also reported as mutagenic, induces aneuploidy and carcinogenic to humans (Group 2B) [39].

Candesartan cilexetil
Candesartan Cilexetil is an Angiotension II receptor antagonist synthesized with the use of 1 chloroethyl cyclohexyl carbonate, which is reported to be potential genotoxic impurity as per the EMEA guideline therefore certain limits was permitted based on daily dose basis and evaluation limit required was 0.49 µg/mL (i.e., 49 µg/g) (Fig. 18).
Therefore, most precise, accurate, and quantitative method such as GC technique with mass spectrometer as detector was selected [40].

LC MS
LC-MS is a versatile tool for the structural elucidation of impurities. It provides rapid, effective separation, and mass separations from the drug in the form of fragmented ions helps in characterization and structure elucidation of unknown impurities. The mass/charge ratio gives idea about molecular formulae. As LC-MS has high sensitivity and can detect impurities in femtogram level (1fg=10 −gi g) in bulk samples.
The structural elucidation data of LC-MS provides evidence of route cause for a rise of impurities which will further helps in controlling the impurity levels in the drug product. LC-MS/MS in the study helped in identifying genotoxic impurities in trace levels, reducing the cost per analysis, ultra-low limit of quantitation and obtaining high throughput results [41].

Rabeprazole
Rabeprazole contains 2 PRI originating from the route of synthesis of rabeprazole. The reported impurities were labeled as chloropropoxy analogue of rabeprazole and free base of chloro intermediate, as shown in Fig. 19. ICH-TTC provides certain limits based on the MDD of rabeprazole (120 mg), the permitted level of these impurities in rabeprazole API is 12.5 ppm/day and to quantify its trace level, LC-MS/ MS as a robust, precise, and accurate quantified method was selected with QbD principles. For the simultaneous examination numerical and categorical factors with UV detector, D-optimal experimental design was used [42].

Meropenem
Meropenem was evaluated for its PRIs and was reported three impurities were formed (318-BP, M9, S5) during synthesis of meropenem which was identified as PGIs (Fig. 21). Based on the meropenem MDD (6 g), 250 ppb/day level of these impurities in meropenem API is permitted [44] .

Atazanavir sulfate
Atazanavir sulfate synthesized using Tert-butyl 2-[4-(pyridine-2-yl) benzyl] hydrazine carboxylate (GTI-A) at early stage of drug synthesis (Fig. 22). However, it was reported as structural alert GTI; therefore, it is essential to control and prove that this material is not incorporated in final stage drug synthesis. For this, a sensitive LC-MS method selected for the determination of GTI-A, genotoxic impurity in atazanavir sulfate drug [45]. linearity, accuracy, precision, robustness, and stability. This method is able to detect the impurities in the presence of other impurities and main drug [46].

Gupta et al.
compound for genotoxic activity. Hence, it needs to identify as PGI present in a certain limit which was given by ICH and TTC for efavirenz API. To determined impurities, LC-MS/MS methods had been reported for trace level determination of AMCOL, which was a process-related impurity and degradation impurity of efavirenz also determined ( Fig. 24) [47].

Antipyrine
Phenylhydrazine used as raw material in the synthesis of antipyrine, but it was reported as a genotoxic impurity. Residual phenylhydrazine in antipyrine was determined and analyzed using inductively coupled plasma-mass spectrometry combined with two-dimensional LC (LC-ICP-MS), was employed. LC-ICP-MS method with iodo derivatization using mono-iodo derivatization reagent (3-iodophenyl isocyanate or 3-iodobenzoyl chloride) (Fig. 25). Monoiodo derivatizing agents as well as tri-iodo derivatization reagent (2,3,5-triiodobenzoyl chloride) also utilized to increase the sensitivity of residual phenylhydrazine in APIs toward analysis. By comparing selectivity and sensitivity and detection limits obtained from two reagents was evaluated with regards of how much no. of iodine atoms was replaced by two different derivatizing agent and hence liquid chromatography -inductively coupled plasma mass spectrometry (LC-ICP-MS ) methods used with 3-iodophenyl isocyanate or 2,3,5-triiodobenzoyl chloride were applied as a derivatizing agent for the quantitation of residual phenyl hydrazine in antipyrine [48].

Naproxen
Naproxen is synthesized by using 2-Butyl p-toluenesulfonate, a most important intermediate, but it was reported as an impurity found to be genotoxic (Fig. 26). The impurity present in naproxen was found to be nucleophilic alkylating agent which was acting as anticancer to treat several cancers. It is toxic toward normal cells (cytotoxic) leading to damage bone marrow. However, naproxen is used as NSAID to treat pain or inflammation induced due to the various conditions. Therefore, it was needed to determine 2-butyl p-toluenesulfonate in drug which was tedious to analyze by HPLC, GC. Therefore, trace levels, such levels such as 1 ppm of impurity in naproxen, were quantitatively determined using triple quad LCMS method [49].

ADVANCED SOFTWARE BASED DRUG DESIGN
In silico methods A few decades ago, mutagenic activity of drugs, or chemicals predicted depending on their chemical structure and potential reactivity toward DNA. Two types of in silico system, i.e., substructures known to be responsible for interacting with DNA or the fragment-based quantitative structure activity relationship paradigm based on the experimental data sets (e.g., results obtained in the Ames test). In silico systems are accelerated drug discovery and provide occupational safety processes in industry and also can be used for in-house risk assessment of potential GTIs genotoxicity testing, but required some expert knowledgeable person for interpretation and validation of the in silico predictions.   (Fig. 27) [50].

Levofloxacin
Levofloxacin contained descarboxy levofloxacin is an impurity which had insufficient toxic information about descarboxy levofloxacin impurity. In silico and in vitro methods were applied to risk assessment of impurity.
Derek-structure activity relationship software package was used for in silico study and the results reported that the levofloxacin contains quinoline as a structural alert impurity. It further evaluated by modified Ames test and by a chromosomal aberration test, using Chinese hamster lung cells. Both assays were conducted in the presence or absence of S-9 mix and their results were compared, on the basis of these assays, descarboxy levofloxacin could be controlled as a nongenotoxic impurity (Fig. 28) [51].

Montelukast
Montelukast evaluated for genotoxicological assessment considering regulatory approaches. RP-HPLC analysis was applied for the determination of impurities such as sulfoxide, cis-isomer, Michael adducts-I and II, methylketone and methylstyrene. However, sulfoxide impurity found to be present above the specified limits as well as the absence of toxicity data (Fig. 29). Therefore, computational program such as in silico mutagenicity prediction analysis done and further confirmed by miniaturizing bacterial gene mutation test, mitotic index determination and in vitro chromosomal aberration test w/ wo metabolic activation system. Leadscope and ToxTree programs as well as Ames MPF Penta I assay predicted sulfoxide impurity as non-mutagenic. Sulfoxide impurity was reported as dose-dependent cytotoxic in human peripheral lymphocytes. After that, sulfoxide impurity should be considered as non-mutagenic and can be classified as ordinary impurity according to the guidelines [52].

CONTROL STRATEGY FOR GTIS
A control strategy is planned for current product and process understanding that gives assurance that the process performance and product quality free of GTIs (ICH Q10). A control strategy includes the following options: • Controls on material attributes (including raw materials, starting materials, intermediates, reagents, solvents, and primary packaging materials) • Facility and equipment operating conditions • Controls implicit in the design of the manufacturing process • In-process controls (including in-process tests and process parameters) • Controls on drug substance and drug product (e.g., release testing) [53]. The FDA draft guidance was generally quite similar to EMEA guideline. Some notable exceptions are: • The FDA includes carcinogenic impurities (many carcinogens are non-genotoxic) • The FDA includes additional safety margins for pediatric populations.

Control strategy 1 -avoidance
According to the EMEA decision tree in the 2006 guideline, during production avoidance of side reaction and formation of reactive molecules which may act as GTIs can be a control strategy.
Example: Various reaction involved acids with alcohols and can produce various alkylating agents such as alkyl halides, esters of aryl sulfonic acids (besilates and tosylates and esters of sulfuric acid), and esters of alkyl sulfonic acids (mesilates such as ethyl mesilate (EMS) and m EMS) all are reported to be potential GTIs. Such cases can be avoided by substituting such acids with alternative acids so that production of GTIs avoided.

Control strategy 2 -adjust API process
It is more troublesome/difficult to adjust optimal chemical process of API which leads to formation of GTIs. But by applying some strategies, we can control over GTIs.
Examples are: • Placing GTIs early in the process so that the produced GTI will available longer duration for purging and will remain away from API forming step so that the GTI can be effectively controlled • Placing solid isolations strategically in the process such as crystallization for the purification of intermediates and APIs, it will help in reduction or remove crucial GTIs.

Control strategy 3 -demonstrate DTI threshold mechanism above TTC level
High levels of EMS, a potential GTI, lead to a recall of Roche's Viracept (nelfinavir mesilate) in 2007. It was due to improper cleaning of vessels with ethanol for longer duration of time and further deposition of EMS. This cause was eventually traced to a GMP failure in the manufacture of the API. While Roche's investigation on in vivo rodent toxicology studies with EMS and threshold of 2 mg EMS/kg for DNA damage was determined. It was four orders of magnitude higher than the 1.5 mg/day TTC as per EMEA guideline. Based on this finding, the EMEA accepted a higher TTC for EMS. It has been speculated that this result may ultimately provide a new approach to guiding risk management for genotoxic impurities in pharmaceuticals. This may be particularly true for monofunctional alkylating agents that react with DNA as soft nucleophiles through an SN2 mechanism in a similar fashion as EMS [54].

LIMITATIONS OF THE PRESENT REGULATORY SYSTEM TO TEST GENOTOXICITY
Nowadays, it is necessary to provide information regarding mutagenicity and carcinogenicity of a NCE. Different regulatory authorities have differences in protocol design and practices for NCE and hindered the drug discovery process as well as delay the marketing of the potential candidates (Tables 6 and 7).
As per regulatory point of view, the preclinical safety study has to be performed in each country as per that country's regulatory guidelines. These are time consuming, contain intensive processes and need a large number of animals for experimentation. Most of the country's guidelines are inadequate to draw a final accurate conclusion for the genotoxic potential of a NCE. Different pharmaceuticals, different design, protocols, and critical experimental evaluation as well as different guidelines by a various regulatory agencies will results in delayed in regulatory approval of new chemical entities (NCI). Lack of specific test system and test protocols as well as guidelines are devoid of recommendations for compounds, which are genotoxic, but seem to act by non-DNA targets. There are no specific recommendations on the threshold of different genotoxic and tumorigenic compounds and their organ specific effects when they are intended to use therapeutically.
The guidelines recommended by various regulatory authorities have four-test battery.

• A gene mutation in bacteria • A test for gene mutation in eukaryotic cells in vitro • In vivo test for genetic damage • A test for chromosome aberrations in mammalian cells in vitro.
When the results of all the tests in the four-test battery are uniformly negative, then the compound under study complies testing, but testing but if test results are not uniform then further experiments are suggested in the four-test battery [55,56].

CONCLUSION
In this article, we have presented a detailed regulatory guidance on GTIs and different analytical approaches for quantification GTIs in drug formulation. The above discussion shows that the conventional way of quantitation and structure elucidation of GTIs by spectral analysis to the use of modern hyphenated techniques are widely adopted currently by the pharmaceuticals. In case of hyphenated systems such as LC, and GC with MS systems reported to be widely used in current scenario. Many studies have been reported studies on in silico methods, but they need to confirm by further analysis by the other in vitro or hyphenated techniques. This review concluded that the determination and quantitation of GTIs by the use of accurate, sensitive, and quantitative methods will give safety regulatory framework to the novel drugs, pharmaceuticals, and human beings. Furthermore, some control strategy summarized so that the GTIs can be controlled at initial stage of drug production.