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A selective and sensitive LC-MS/MS method for the simultaneous determination of twopotential genotoxic impurities in celecoxib



Impurity profiling is now receiving critical attention from regulatoryauthorities. For trace level quantification of potential genotoxic impurities(PGIs), conventional analytical techniques like high-performance liquidchromatography (HPLC) and gas chromatography (GC) are inadequate; consequently,there is a great need to apply hyphenated analytical techniques to developsensitive analytical methods for the analysis of pharmaceuticals.


A selective and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed for the simultaneous determination of(4-sulfamoylphenyl)hydrazine hydrochloride (SHH) and(4-methyl-acetophenone)para-sulfonamide phenylhydrazine hydrochloride(MAP) PGIs in celecoxib active pharmaceutical ingredient (API). The LC-MS/MSanalysis of SHH and MAP PGIs was done on Symmetry C18 (150 mm ×4.6 mm, 3.5 μm) analytical column, and the mobile phase used was5.0 mM ammonium acetate-acetonitrile in the ratio of 30:70(v/v). The flow rate used was 0.7 mL/min. Triplequadrupole mass detector coupled to positive electrospray ionization operated inmultiple reaction monitoring (MRM) mode was used for the quantification of SHH andMAP PGIs. The method was validated as per International Conference onHarmonization (ICH) guidelines and was able to quantitate both SHH and MAP PGIs at1.0 ppm with respect to 10 mg/mL of celecoxib.


The proposed method was specific, linear, accurate, precise, and robust. Thecalibration curves show good linearity between the concentration range of 0.06 and7.5 ppm for both SHH and MAP PGIs. The correlation coefficient obtained was>0.9998 in each case. The method has very low limit of detection (LOD) andlimit of quantification (LOQ). The obtained LOD and LOQ values were 0.02 and0.06 ppm, respectively, for both SHH and MAP PGIs. For both the PGIs,excellent recoveries of 95.0% to 104.0% were obtained at a concentration range of0.06 to 3.0 ppm. The developed method was also applied to determine the SHHand MAP PGIs in three formulation batches of celecoxib.


The proposed method is simple and accurate and is a good quality control tool forthe simultaneous quantitative determination of SHH and MAP PGIs at very low levelsin celecoxib during its manufacturing.


The presence of potential genotoxic impurities (PGIs) even in smaller quantities mayaffect the efficacy and safety of pharmaceutical products. Impurity profiling is nowreceiving critical attention from regulatory authorities. The different pharmacopoeiassuch as BP (British pharmacopoeias), USP (United States pharmacopoeias), IP (Indianpharmacopoeias), and so on, are slowly incorporating limits to the allowable levels ofimpurities present in the active pharmaceutical ingredients (APIs). Celecoxib is one ofthe most popular non-steroidal anti-inflammatory drug (NSAID) and a selectivecyclooxygenase-2 (COX-2) inhibitor used to treat osteoarthritis, rheumatoid arthritis,acute pain, painful menstruation, and menstrual symptoms (Dembo et al. [2005]). Celecoxib also reduces the number of colon and rectum polyps in patientswith familial adenomatous polyposis (Clemett and Goa [2000]; Silverstein et al. [2000]). It is chemically named as4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzene sulfonamide, and thechemical structure of celecoxib is shown in Figure 1.(4-Sulfamoylphenyl)hydrazine hydrochloride (SHH) and(4-methyl-acetophenone)para-sulfonamide phenylhydrazine hydrochloride (MAP)are the two important key intermediates used in the synthesis of celecoxib, which areidentified as PGIs in finished pharmaceutical substances due to their electrophilicfunctional groups (Ashby and Tennant [1988]; Muller et al. [2006]). Several analytical methods have been used to determine celecoxibconcentrations in human plasma with various analytical techniques such ashigh-performance liquid chromatography-UV (HPLC-UV), liquid chromatography-massspectrometry (LC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS)(Jalalizadeh et al. [2004]; Zarghi et al. [2006]; Emami et al. [2006]; Chow et al. [2004]; Stormer et al. [2003]; Abdel-Hamid et al. [2001]; Werner et al. [2002]; Bräutigam et al. [2001]), and few methods have been reported for the determination of impurities incelecoxib using HPLC and LC-MS/MS (Satyanarayana et al. [2004]; Rao et al. [2006]; Jadhav and Shinqare [2005]). Ideally, many conventional analytical instruments in pharmaceuticalindustry such as HPLC with UV detection and GC with flame ionization detector (FID)detection should be employed as the standards in the first attempt for PGIs analysis.However, there are some drawbacks with the abovementioned techniques because HPLCretention times can vary, and some methods are needed to characterize the impurities online when the impurity standards are not available (Hsieh and Korfmacher [2006]; Lee and Kems [1999]). Therefore, for accurate determination of PGIs at trace levels, theabovementioned techniques are inadequate; consequently, there is a great need to developbetter analytical methods for the analysis of such PGIs in pharmaceutical industries. Asa result, various kinds of hyphenated chromatographic techniques and methodologies havebeen explored as useful approaches.

Figure 1
figure 1

Chemical structure of celecoxib.

Based on the threshold of toxicological concern (TTC) of 1.5 μg/person/day,the impurity concentration in celecoxib must not exceed 7.5 ppm considering theworst case scenario where 200-mg daily dose of celecoxib is applied. To the best of ourknowledge, no analytical method for the simultaneous determination of SHH and MAP PGIsin celecoxib has been reported in the literature. Therefore, in the present study, wehave developed a simple LC-MS/MS method that can quantify two PGIs in celecoxib atpermitted levels. The method was validated as per ICH guidelines in terms of limit ofdetection (LOD), limit of quantification (LOQ), linearity, precision, accuracy,specificity, robustness, and solution stability. The developed method was also appliedto determine SHH and MAP PGIs in three formulation batches of celecoxib.


Chemicals and reagents

All chemicals and solvents were of analytical grade. HPLC grade acetonitrile andammonium acetate were purchased from Merck (Mumbai, India). Formic acid,trifluoroacetic acid, and methanol were obtained in their highest grade from SD finechemicals limited (Mumbai, India). Reference substances of SHH, MAP, and celecoxibwith the highest purity (>99.0) were obtained from Sigma-Aldrich (St. Louis, MA,USA). High-purity Milli-Q water was used with the help of Millipore Milli-Q pluspurification system (Bedford, MA, USA).

Preparation of stock and standard solutions

A stock solution of celecoxib (10 mg/mL) was prepared by dissolving appropriateamount in the methanol. A stock solution of mixture of PGIs (SHH and MAP) at1.0 mg/mL was also prepared in methanol. The diluted stock solution(0.01 mg/mL) was prepared by diluting 1.0 mL of the 1.0 mg/mLsolutions to 100 mL with methanol. Then, 0.1 μg/mL diluted stocksolution was prepared by diluting 1.0 mL of 0.01 mg/mL diluted stocksolution to 100 mL with methanol. The working standard solution was prepared byweighing accurately 100 mg of celecoxib into 10-mL volumetric flask and made thesolution up to the graduation mark after adding 10 μL of0.1 μg/mL diluted stock solution to give 10 ng/mL and 10 mg/mL ofPGIs with respect to celecoxib which corresponds to 1.0 ppm of PGI contaminationrelative to the drug substance. The PGI samples for validation at 0.06-, 0.5-, 1.0-,3.0-, 5.0-, and 7.5-ppm concentrations relative to the drug substance were preparedin the same manner using 0.5 μg/mL of diluted stock solution. Theconcentration of the standard solutions and samples was optimized to achieve adesired signal-to-noise ratio (S/N) and good peak shape. All the standards weresonicated well and filtered through 0.22-μm membrane filters before theanalysis.

Chromatographic conditions

All chromatographic experiments were carried out on a HPLC consisting of LC-20ADbinary gradient pump, SPD-10AVP UV detector, SIL-10HTC autosampler, and a column ovenCTO-10ASVP (Shimadzu, Switzerland) system coupled with MS/MS (Applied BiosystemsSciex API 4000 model, Rotkreuz, Switzerland). The analytical column used was SymmetryC18 (150 mm × 4.6 mm, 3.5 μm). The mobile phase flowoperated in isocratic mode using 5.0 mM ammonium acetate-acetonitrile in theratio of 30:70 (v/v). The flow rate of the mobile phase was set at0.7 mL/min, and the column oven temperature was maintained at 40°C. Theinjection volume was 10 μL. All the solutions were filtered through0.22-μm nylon filter before the analysis.

Mass spectrometer

The MS/MS system used was an Applied Biosystems Sciex API 4000 triple quadrupole massspectrometer with electrospray ionization (ESI) probe operated in positive polarity.Multiple reaction monitoring (MRM) mode was selected for the quantification of SHHand MAP PGIs, and the data acquisition and processing were conducted using theAnalyst 1.5.1 software. Typical operating conditions were as follows: ion sprayvoltage 5,500 V, source temperature 410°C, declustering potential (50 and55 V), entrance potential (10 and 10 V), collision energy (25 and20 V), respectively, for both SHH and MAP PGIs. The curtain gas flow, ion sourcegas 1, and ion source gas 2 nebulization pressure were maintained as 25, 30, and35 psi, respectively. Electrospray ionization in positive MRM mode was used forthe quantification of SHH and MAP PGIs at their transition ion pairs of m/z188.2→99.2 (protonated) and m/z 304.2→209.2 (protonated),respectively. Celecoxib was monitored with its transition ion pair m/z382.2→214.1 (protonated).

Method validation

To demonstrate the feasibility of the newly developed method, validation wasperformed in relation to specificity, linearity, LOQ, LOD, accuracy, precision,robustness, and solution stability. These parameters were validated in agreement withthe ICH guidelines.

The linearity was performed by diluting the impurity stock solution to the requiredconcentrations. The solutions were prepared at six concentration levels between 0.06to 7.5 ppm for both SHH and MAP PGIs and were subjected to linear regressionanalysis with the least squares method. Calibration equation obtained from regressionanalysis was used to calculate the corresponding predicted responses. Systemprecision of the mass spectrometric response was established by injecting sixindividual preparations of the standard solution. The method precision was evaluatedby spiking each analyte and determining the percent relative standard deviation(%RSD). LOD and LOQ were evaluated by considering the impurity concentration thatwould yield S/N ratios of 3:1 and 10:1, respectively. The precision of LOD and LOQvalues were experimentally verified by six injections of standard solutions of thecompounds at the determined concentrations. Recoveries of SHH and MAP PGIs in spikedsamples were studied at three different concentration levels, viz. 0.06, 1.5, and3.0 ppm. At each concentration level, three independent sample preparations wereinjected, and the percentage recoveries were determined by comparing theconcentration of the spiked sample obtained with the concentration of the spikingstandard. The robustness of the method was evaluated by changing mobile phase flowand column temperature, and the stability of the impurities in the sample solutionwas evaluated by analyzing spiked sample solution at different time intervals at roomtemperature.

Results and discussion

Optimization of sample preparation

Sample preparation is an important part in the pharmaceutical impurity analysis,because matrix effects in trace analysis were enlarged, causing loss of sensitivity,abnormal recovery, and analyte instability. Different diluents were evaluated withrespect to chromatographic efficiency. Solubility of both celecoxib and impuritieswere good in methanol. Good response and proper peak shapes were obtained for boththe impurities when methanol was used as the diluent. Good recoveries (95.0% to104.0%) were also observed for both SHH and MAP PGIs when methanol was used as adiluent. Therefore, methanol was employed as the diluent throughout the analysis.

Column selection and separation

The present method was developed by testing different stationary phases to achievegood separation of the impurity peaks from drug substance peak. It is important toachieve proper separation among the two PGIs and celecoxib, because of similarchemical structure of two PGIs and celecoxib. In order to obtain a short analysistime, various analytical columns like Kromasil C18 150 mm × 4.6 mm,3.5 μm (Altmann Analytik, Munich, Germany), Hypersil BDS C8 150 mm× 4.6 mm, 3.5 μm (Altmann Analytik), Symmetry C18 150 mm× 4.6 mm, 3.5 μm (Waters, Milford, MA, USA), and Zorbax Rx C8150 mm × 4.6 mm, 3.5 μm (Agilent Technologies, Inc., SantaClara, CA, USA) were evaluated. The tested columns were checked under the sameconditions; with the Kromasil C18 and Zorbax Rx C8 columns, the peaks of impuritieswere overlapped with celecoxib peak. The resolution between celecoxib and impuritieswere poor with Hypersil BDS C8 column. On Symmetry C18 column (150 mm ×4.6 mm, 3.5 μm), the separation and responses for both the impuritiesand celecoxib were found good. On this column, the analytes were well retained andseparated from each other and from the drug substance. This separation is achieveddue to polar group technology that ‘shields’ the silica residual silanolsurface from highly basic analytes; this reduced silanol activity for the symmetrycolumn significantly improved the peak shape and resolution. Different compositionsof mobile phases using 10 mM ammonium acetate and 5.0 mM ammonium acetatewith acetonitrile were tested; finally, good separation and response were observed ata ratio of 5.0 mM ammonium acetate-acetonitrile (30:70, v/v).Both isocratic and gradient elution modes were evaluated. Isocratic elution wasobserved to be more efficient in achieving optimum separation of impurities from eachother with respect to drug substance peak. The column was thermostated at 40°Cto avoid any shift in retention time. Retention times of SHH and MAP PGIs wereobserved at 3.08 and 4.02 min, respectively. Peaks were well separated from thedrug substance peak (5.79 min).

Optimization of MS-MS parameters

Selection of a detection method is also the most important part of pharmaceuticalimpurity analysis. From the instrument simplicity and availability, first, we haveevaluated with HPLC-UV and GC-FID. However, on these techniques sufficientsensitivity for the trace level analysis of SHH and MAP PGIs was not achieved. Inview of this, a sensitive and specific mass LC-MS/MS technique in MRM mode wasevaluated for the quantification of SHH and MAP PGIs in celecoxib drug substance.Then, the possibility of using electrospray ionization (ESI) source under positiveion detection mode was evaluated during the early stage of method development. Thesignal intensity in positive mode was much higher than that in negative mode.Further, the method development was carried out with ESI source operated in positivepolarity mode. The ion source parameters were optimized to get proper response. Therepresentative mass spectra of SHH, MAP, and celecoxib are shown in Figure 2.

Figure 2
figure 2

Representative mass spectra of SHH, MAP PGIs, and celecoxib.

Method validation

In order to prove that the method is capable of its intended use, the newly developedmethod for the quantification of SHH and MAP PGIs in celecoxib drug substance wasvalidated according to the international guidelines (Vijaya Bhaskar Reddy et al. [2013]; ICH [2005]).

Limit of detection and limit of quantification

The method validation was started by injecting 1.0-ppm concentration of individualsolutions of SHH and MAP PGIs of each with respect to the drug substanceconcentration of 10 mg/mL and determining their S/N ratios. Now, to evaluate LODand LOQ values, their concentrations were reduced sequentially such that they yieldS/N ratios as 3:1 and 10:1, respectively. Each predicted concentration was verifiedfor their precision by preparing the solutions at predicted concentrations andinjected each solution six times for analyses. The LOD and LOQ values calculated formS/N ratio was found to be 0.02 and 0.06 ppm, respectively. It is noteworthy thatthe LOD values for both the impurities were below the required concentration limit(7.5 ppm) for PGIs in celecoxib (Table 1).

Table 1 The precision at LOD and LOQ concentrations of SHH and MAP PGIs


By MRM, the linearity of SHH and MAP PGIs was satisfactorily demonstrated with asix-point calibration graph between 0.06 and 7.5 ppm with respect to a sampleconcentration of 10 mg/mL. The calibration curves were produced by plotting theaverage of triplicate PGI injections against the concentration expressed inpercentage. The slope, intercept, and correlation coefficient values were derivedfrom linear least squares regression analysis. The correlation coefficient obtainedin each case was >0.9998. The corresponding linearity data is presented inFigure 3. The results indicated that an excellentcorrelation existed between the peak areas and the concentrations of impurities.

Figure 3
figure 3

Linearity plot of SHH and MAP PGIs at 0.06- to 7.5-ppm concentrationlevels.


The precision of the method was evaluated at two levels, viz. repeatability andintermediate precision. Repeatability was checked by calculating the %RSD of sixreplicate determinations by injecting six freshly prepared solutions containing1.0 ppm each of the mixture of impurities on the same day. The same experimentswere done on six different days to evaluate the intermediate precision. As reportedin Table 2, %RSD values were lower than 3.0% for both theimpurities; this confirmed an adequate precision of the developed method.

Table 2 Intra-day and inter-day precision of SHH and MAP PGIs at1.0-ppm concentration

Accuracy and specificity studies

When three pure and formulation sample solutions of 10 mg/mL of celecoxib wereinjected, impurities were not at all detected in them. Hence, recovery studies by thestandard addition method were performed to evaluate accuracy and specificity.Accordingly, the accuracy of the method was determined by spiking at (LOQ) 0.06-,1.5-, and 3.0-ppm concentrations separately to three batches of pure and formulationsolutions of celecoxib (10 mg/mL). Each determination was carried out threetimes. The recovery data is presented in Table 3, and thecorresponding chromatogram is shown in Figure 4.Satisfactory recoveries of 95.3% to 98.6% for 0.06 ppm, 98.6% to 101.4% for1.5 ppm, and 103.4% to 103.6% for 3.0 ppm were obtained. At such lowconcentrations, these recoveries and %RSDs were satisfactory. The specificity of themethod was established by injecting blank celecoxib (tablet) solution and celecoxibspiked with two PGIs. It was observed that the common excipients used in the tabletswere not interfered at the retention times of any PGIs and drug substance. Thecorresponding specificity chromatogram is shown in Figure 5. The developed method was also successfully applied for thedetermination of SHH and MAP PGIs in three different batches of celecoxib. In twobatches, the PGIs were not detected. In one of the batches of celecoxib, only MAP wasobserved; however, its concentration was below the specification. The correspondingchromatogram is shown in Figure 6.

Table 3 The recovery data of SHH and MAP PGIs at three different concentrations
Figure 4
figure 4

Recovery chromatogram of SHH and MAP PGIs at LOQ concentration.

Figure 5
figure 5

Specificity chromatogram of celecoxib spiked with SHH and MAP PGIs.

Figure 6
figure 6

Representative chromatograms of (a) blank and (b) formulation sample ofcelecoxib.


The robustness of the method was studied with deliberate modifications in the flowrate of the mobile phase and the column temperature. The optimized flow rate of themobile phase was 0.7 mL/min, and the same was altered by 10% of its flow, i.e.,from 0.63 to 0.77 mL/min. The effect of column temperature on resolution wasstudied at 38°C and 42°C (altered by 2°C). However, the mobile phasecomponents were held constant as described above. As reported in Table 4, the %RSD in both the cases does not exceeded 3.0%, whichdemonstrates the robustness of the method.

Table 4 Robustness data of SHH and MAP PGIs at LOD and LOQ concentrations

Solution stability

Stability of SHH and MAP PGIs in methanol was checked by keeping them in anautosampler and observing the variations in their peak areas. From the stabilityresults, we found that SHH and MAP were stable up to 48 h. The correspondingdata is presented in Table 5.

Table 5 Solution stability data of SHH and MAP PGIs at LOQ concentration


The proposed method is a direct LC-MS/MS method for the separation and quantification ofSHH and MAP PGIs in celecoxib drug substance. The method utilized MRM mode for thequantitation, which provided the better sensitivity. The method was fully validated andpresents good linearity, specificity, accuracy, precision, and robustness, and it isalso found to be simple, sensitive, selective, cost effective, and stability indicating.The LOD and LOQ of the method were found very low, as 0.02 and 0.06 ppm for bothSHH and MAP impurities. The proposed method was successfully applied for thedetermination of the two PGIs in three formulation batches of celecoxib. The methodpresented here could be very useful to monitor SHH and MAP PGIs in celecoxib during itsmanufacturing.


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One of the authors Dr. A. Vijaya Bhaskar Reddy is highly grateful to the UGC (BSR),Government of India, New Delhi for financial assistance in the form of an award ofMeritorious Research Fellowship (RFSMS), and the authors are also thankful to SipraLabs Limited, Hyderabad for supporting this work.

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Authors’ contributions

VBR designed the experiment, carried out the experiment, and contributed in framing thearticle. V assisted during the method development and analysis using LC-MS/MS. Massisted during the analysis and contributed in framing the article. All authors readand approved the final manuscript

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Vijaya Bhaskar Reddy, A., Venugopal, N. & Madhavi, G. A selective and sensitive LC-MS/MS method for the simultaneous determination of twopotential genotoxic impurities in celecoxib. J Anal Sci Technol 5, 18 (2014).

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  • LC-MS/MS
  • Method validation
  • Genotoxicity
  • Ionization
  • Quantification