Development and Validation of a Headspace Gas Chromatographic (HS-GC) Method for Determination of Residual Solvents in Nitazoxanide API

Controlling residual solvents in the drug substances or active pharmaceutical ingredients (API) is mandatory to the speci ied limits as per the International Conference on Harmonisation (ICH) Q3C guidelines. Residual solvents in pharmaceuticals are mostly determined by Gas Chromatography with Headspace. A simple and sensitive headspace gas chromatographic (HS-GC) method has been developed for the determination of Acetone, Dichloromethane andCyclohexane inNitazoxanideAPI. The separationof analytes was achieved with DB – 624 (30 m length, 0.53 mm inner diameter and 3.0μm in ilm thickness) capillary column. Dimethyl formamidewas used as a diluent. Nitrogenwas used as carrier gaswith 3.0mL/minutes and Flame ionisation detector (FID) for detecting analytes. The oven temperature was set at 60◦C for 5minutes at initial and programmed at a rate of 20◦C perminute to a inal temperature of 240◦C for 2 minutes. Run time was 16 minutes, and total GC cycle time was 25 minutes. The spilt ratio used as 1:20 to get optimum peak response. The developed method was validated as per the ICH guidelines for speci icity, accuracy, precision, linearity, range, the limit of detection, the limit of quanti ication and robustness. The results of validation were indicated no interference, good recoveries, precise, linear, rugged and robust method, suitable for the determination of residual solvents in Nitazoxanide API for research and routine quality control laboratory.


INTRODUCTION
Several organic volatile solvents or chemicals are used in the manufacturing of drug substances, excip-ients and drug products. They are also used to increase the inal yield, to enhance the purity or to change the physical form such as polymorphic form and solubility. These solvents or chemicals do not have any therapeutic activity but maybe toxic for humans if consumed more than permitted daily exposure (PDE) (Sitaramaraju et al., 2008). It is necessary to remove them, but some solvents remain in small quantities in the inal product. These small quantities of organic solvents remain in the inal product is known as residual solvents. Determination of these residual solvents from drug substances, excipients and drug products is a dif icult and challenging task. Headspace gas chromatographic technique is most suitable and used for the determination of residual solvents. The acceptance limit for residual solvents is set following the toxicity of solvents and speci ied in the international conference on harmonisation Q3C guidelines (ICH, 2016;Harold et al., 1997).
Literature survey revealed that several methods by UV -spectroscopy (Pandey, 2009;Gandhi et al., 2008), visible spectroscopy (Narayana and Manohara, 2007) and liquid chromatography (Malesuik et al., 2009;Kumar et al., 2009;Narayan and Mahendra, 2007) are reported for quantitative estimation (assay) of nitazoxanide in bulk alone and combination with other drugs. During the synthesis and puri ication process of nitazoxanide API, Acetone, Dichloromethane and Cyclohexane were used. This work aimed to develop and validate a simple and sensitive headspace gas chromatographic (HS-GC) method for simultaneous determination of Acetone, Dichloromethane and Cyclohexane in Nitazoxanide API. Acetone, Dichloromethane and Cyclohexane belong to class 3, 2 and 2 respectively. The speci ications as per international conference on harmonisation Q3C guidelines are tabulated in Table 1.

MATERIALS AND CHEMICALS
GC reference standards of Acetone, Dichloromethane (Methylene dichloride) and cyclohexane were procured from Biosolve Chimie, France. Dimethyl formamide (N, N-Dimethyl Formamide) (Supra Solv, GC grade) was procured from Merck Millipore, India. Nitazoxanide API sample was received as gift samples from Suven Life Sciences Limited, Hyderabad, India.

METHOD
The method was developed and validated on Agilent Technologies gas chromatograph (Model No. 7890B) and a headspace sampler (Model No. 7697A) equipped with lame ionisation detector (FID) using Empower 3 software. The separation of analytes was achieved with DB -624 (30 m length, 0.53 mm inner diameter and 3.0 µm in ilm thickness) capillary column. The chromatographic parameters were optimised, and optimised chromatographic conditions are shown in Table 2.

Blank
Use diluent as blank.

Preparation of Standard solution
Weigh accurately about 500 mg, 60 mg, 388 mg of Acetone, Dichloromethane and Cyclohexane reference standards respectively into a 100 mL volumetric lask having about 25 mL of diluent. Mix and make up to volume with diluent. Transfer 5.0 mL of above solution into a 100 mL volumetric lask and dilute to volume with diluent and mix well. Transfer 2.0 ml of the above solution into six different headspace vials and seal properly.

Preparation of Sample solution
Weigh and transfer accurately about 100 mg of Nitazoxanide API sample into a headspace vial. Add 2.0 mL of diluent, dissolve and seal the vial properly.

Preparation of System Suitability solution
Use the standard solution to check the system suitability.

Procedure
Inject blank (1 injection), and standard solution (6 injections), sample solution (1 injection) into the chromatograph and record the peak response of eluting peaks using the chromatographic and Headspace parameters.

Acceptance criteria for System Suitability
The resolution between Acetone and Dichloromethane peaks from the irst standard injection from system suitability should be not less than 3.0.
The relative standard deviation (RSD) of area response for Acetone, Dichloromethane and Cyclohexane peaks between the six replicate injections of the standard should be no more than 10 %.

Method validation
Validation of the developed method was conducted as per United States Pharmacopoeia general chapter <1225> (USP, 2018a) and International Conference on Harmonization Q2 (R1) (ICH, 2005) guidelines.

System suitability
System suitability was evaluated under United States Pharmacopoeia general chapter <621> (USP, 2018b). System suitability of the method was established by injecting blank and standard solution for system suitability, calculated the resolution between Acetone and Dichloromethane peaks from irst standard injection from system suitability and the relative standard deviation (RSD) of area response for Acetone, Dichloromethane and Cyclohexane peaks from the six replicate injections of the standard solution. The acceptance criteria for resolution between Acetone and Dichloromethane peaks was not less than 3.0 and % RSD for area response of Acetone, Dichloromethane and Cyclohexane peaks were not more than ten from six replicate injections of the standard solution.

Speci icity
The speci icity of the method was established by injecting blank in triplicate, standard solution, test solution, test solution spiked with analytes at the speci ication level, Acetone reference standard solution at the speci ication level, Dichloromethane reference standard solution at speci ication level and Cyclohexane reference standard solution at the speci ication level. The chromatograms were evaluated for any interference at the retention time of Acetone, Dichloromethane and Cyclohexane peaks.

Precision
The precision of the method was evaluated by injecting six test sample preparations spiked with Acetone, Dichloromethane and Cyclohexane reference standards at 100% speci ication level. % relative standard deviation of six test sample preparations spiked with analytes was calculated. Intermediate precision of the method was also evaluated using different analyst, different day, different instrument and different column by injecting six test sample preparations spiked with analytes prepared as same for precision. The acceptance criteria for individual precision % RSD was not more than 5.0, and for 12 preparation results was not more than 7.0.

Accuracy (Recovery)
Recovery study was performed to evaluate the accuracy of the method by spiking method. Recovery study was done by spiking Acetone, Dichloromethane, and Cyclohexane reference standards into the test sample in the concentration of LOQ, 50%, 100% and 150% level of the proposed speci ication concentration. The recovery samples were prepared in triplicate for 50% & 100% level and six preparations for LOQ & 150%. Injected the prepared recovery samples in the optimised experimental conditions. % recovery of Acetone, Dichloromethane and Cyclohexane peaks were calculated for all the levels. The acceptance criterion for recovery of Acetone, Dichloromethane and Cyclohexane analytes was 80.0 to 120.0% and % RSD for six recovery results at LOQ, and 150% was not more than 5.0.

Limit of detection (LOD) and limit of quantitation (LOQ)
LOD is the lowest amount of analyte that can be detected, but not necessarily quanti iable, and LOQ is the lowest amount of analyte that can be quantitated with acceptable precision and accuracy. The LOD and LOQ were established by injecting a known concentration of serial dilutions of Acetone, Dichloromethane and Cyclohexane under the stated experimental conditions. The LOD and LOQ were established from the Slope and STEYX by plotting the linearity curve of concentration versus area response. LOD and LOQ were estimated by using the following formulae: Where σ = STEYX of response and S = slope determined from the linear plot.

Linearity
The linearity of the method was established for Acetone, Dichloromethane and Cyclohexane from LOQ to 150% of the proposed concentration using six calibration levels (LOQ, 25, 50, 100, 125 and 150% of the targeted concentration). The reference standards were used to prepare calibration levels. The calibration curves for Acetone, Dichloromethane and Cyclohexane, were plotted for each level as concentration versus peak area response. The results of linearity were evaluated by regression analysis.

Robustness
Robustness of the method was determined with deliberate changes in the method conditions from the optimised inal conditions. Injected blank, standard solution, test sample and spiked test sample solution in each robustness conditions and evaluated the system suitability.
For robustness study, the following parameters were considered such as (i) Change in initial oven temperature 60 • C ±5 • C (55 • C and 65 • C) and (ii) Change in nitrogen gas low rate 3.0 mL/min ±10% (2.7 and 3.3 mL/min).

Solution stability
Solution stability was established for the standard solution and test sample preparations. Bench-top (controlled room temperature) stability was established by injecting standard solution and test sample at regular interval for 48 hours. Solution stability was established by calculating the similarity factor for the standard solution against a new standard and % difference for a test sample from the initial value.

System suitability
System suitability of the method was evaluated through resolution between Acetone and Dichloromethane peaks from standard solution and the % RSD of area response for Acetone, Dichloromethane and Cyclohexane peaks from the six replicate injections of the standard solution. The system suitability results were found well within the prede ined acceptance criteria. The results are presented in Table 3.

Speci icity
The speci icity of the method was performed to check blank interference and con irm the identity         of the analytes. The chromatograms con irm (Figures 2,3,4 and 5) no interference at the retention time of Acetone, Dichloromethane and Cyclohexane peaks peak due to blank.

Precision
The precision of the method was evaluated by injecting six test sample preparations spiked with Ace-   Table 4.

Accuracy (Recovery)
The accuracy of the method was evaluated by calculating the recoveries at LOQ, 50%, 100% and 150% level of the targeted speci ication concentration. The mean % recoveries for Acetone, Dichloromethane and Cyclohexane at LOQ (n=6), 50% (n=3), 100% (n=3), and 150% (n=6) were found within the acceptance criteria. The % RSD at LOQ was found 4.7, 4.6 and 3.7 respectively for Acetone, Dichloromethane and Cyclohexane. At 150% level the % RSD for Acetone, Dichloromethane and Cyclohexane was found 2.5, 2.8 and 2.4 respectively. The recoveries and precision at LOQ and 150% were found within the acceptance criteria. The results of accuracy are presented in Table 5.

Limit of detection (LOD) and limit of quantitation (LOQ)
The LOD and LOQ were established from the Slope and STEYX by plotting the linearity curve of concentration versus area response for Acetone, Dichloromethane and Cyclohexane. The LOD for Acetone, Dichloromethane and Cyclohexane was found 170, 25 and 118 ppm respectively. The LOQ was found 514, 76 and 359 ppm respectively for Acetone, Dichloromethane and Cyclohexane. The results are presented in Table 6.

Linearity
The linearity of the method was established for Acetone, Dichloromethane and Cyclohexane from LOQ to 150% of the target speci ication concentration by plotting concentration versus peak area response. The method was found linear for Acetone, Dichloromethane and Cyclohexane with correlation coef icient 0.998, 0.998 and 0.999 respectively. The linearity results are tabulated in Table 7. Chromatograms at LOQ and 150 % are shown in Figures 6 and 7. Linearity graphs of Acetone, Dichloromethane and Cyclohexane, are shown in Figures 8, 9 and 10.

Robustness
The robustness of the method was determined by deliberately changing the initial oven temperature and nitrogen gas low rate. Evaluated the system suitability results at each robustness condition and were found within the acceptance criteria.

Solution stability
Solution stability of the standard solution and test sample solution was established and found to be sta-ble for 48 hours on bench-top (controlled room temperature).

DISCUSSION
As per ICH Q3C, it is mandatory to estimate and control residual solvents used for synthesis, crystallisation and puri ication of drug substances or API. Several trials were taken on HS-GC to optimise the column dimensions, carrier gas low, oven temperature, detector temperature, gradient programme, split ratio and standard & test concentrations to achieve good peak shape and better retention and resolution of Acetone, Dichloromethane and Cyclohexane peaks. The developed method was very sensitive and straightforward with a shorter run time.
The developed method was validated as per the current method validation guidelines and found suitable.

CONCLUSIONS
A method was developed for the simultaneous estimation of Acetone, Dichloromethane and Cyclohexane in Nitazoxanide API. The developed method was validated as per ICH Q2 and USP <1225> guidelines for system suitability, speci icity, precision, accuracy, LOD & LOQ, linearity and robustness. The method validation results were found meeting the acceptance criteria for all parameters. The proposed method is simple, sensitive, selective, accurate and robust for quantitative estimation of Acetone, Dichloromethane and Cyclohexane in Nitazoxanide API by HS-GC and can be used for routine analysis in quality control and research laboratory.