A selective and sensitive LC-MS/MS method for the simultaneous determination of twopotential genotoxic impurities in celecoxib
© Vijaya Bhaskar Reddy et al; licensee Springer. 2014
Received: 29 September 2013
Accepted: 8 January 2014
Published: 13 March 2014
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.
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.
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.
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).
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
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. ; ICH ).
Limit of detection and limit of quantification
The precision at LOD and LOQ concentrations of SHH and MAP PGIs
LOD (peak area)
LOQ (peak area)
LOD (peak area)
LOQ (peak area)
Intra-day and inter-day precision of SHH and MAP PGIs at1.0-ppm concentration
SHH (peak area)
MAP (peak area)
Accuracy and specificity studies
The recovery data of SHH and MAP PGIs at three different concentrations
Accuracy at LOQ level (n = 3)
Amount added (ppm)
Amount recovered (ppm)
Accuracy at 100% level (n = 3)
Amount added (ppm)
Amount recovered (ppm)
Accuracy at 150% level (n = 3)
Amount added (ppm)
Amount recovered (ppm)
Robustness data of SHH and MAP PGIs at LOD and LOQ concentrations
Column oven temperature (°C)
%RSD at LOD
%RSD at LOQ
%RSD at LOD
%RSD at LOQ
Solution stability data of SHH and MAP PGIs at LOQ concentration
Theoretical concentration (ppm)
Percent recovery (n = 3)
At 0 h
98.2 ± 0.94
96.2 ± 1.24
At 12 h
101.4 ± 1.13
95.6 ± 0.91
At 24 h
102.6 ± 0.82
96.4 ± 0.88
At 48 h
99.8 ± 1.45
100.4 ± 1.36
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.
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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