ABSTRACTObjective: To develop an accurate, precise, and linear gas chromatographic-mass spectrometry selective ion monitoring (SIM) method for quantitativeestimation of methane sulfonyl chloride (MSC) as an impurity in itraconazole (ICR) active pharmaceutical ingredient (API) at ppm level and validatedas per International Council of Harmonization (ICH) guidelines. Methods: This method used in SIM mode mass selective detection was developed and validated for the trace level analysis of an impurity.Chromatographic separation of MSC was achieved in 5.45 minutes and m/z value was 79 on SIM mode, ZB-5 ms 30 m Ã— 0.25 mm Ã— 0.25 Âµm column,using helium carrier gas with 1.0 ml/min.Results: The method was linear for MSC in ICR 1.90-7.5 Âµg/ml, respectively. The coefficient of correlation (r) for the MSC was better than 0.999. Thelimit of detection and limit of quantification (LOQ) obtained were 0.44 and 1.32 Âµg/ml. The method was fully validated, complying Food and DrugAdministration, ICH, and European Medicines Agency guidelines. Furthermore, verified precision, accuracy, LOQ precision, LOQ accuracy, ruggedness,and robustness.2Conclusion: The methods were successfully validated to determination and quantification of MSC in ICR API. Hence, the method holds good for theroutine trace analysis of MSC in ICR and various pharmaceutical industries as well as academics.Keywords: Methane sulfonyl chloride, Itraconazole, Gas chromatography-mass spectrometry, Method development, Method validation

Durga Babu, Mannem

K, Surendrababu

M, Kishore

English

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Vol 9, Issue 5, 2016Online - 2455-3891 Print - 0974-2441DEVELOPMENT AND VALIDATION OF A GAS CHROMATOGRAPHY-MASS SPECTROMETRY WITH SELECTED ION MONITORING METHOD FOR THE DETERMINATION OF TRACE LEVELS OF METHANE SULFONYL CHLORIDE AS AN IMPURITY IN ITRACONAZOLE ACTIVE PHARMACEUTICAL INGREDIENTMANNEM DURGA BABU*, SURENDRABABU K, KISHORE MDepartment of Post-graduate Chemistry, SVRM College (Autonomous), Nagaram, Andhra Pradesh, India.  Email: m.durgababu1989@gmail.comReceived: 13 May 2016, Revised and Accepted: 24 May 2016ABSTRACTObjective: To develop an accurate, precise, and linear gas chromatographic-mass spectrometry selective ion monitoring (SIM) method for quantitative estimation of methane sulfonyl chloride (MSC) as an impurity in itraconazole (ICR) active pharmaceutical ingredient (API) at ppm level and validated as per International Council of Harmonization (ICH) guidelines. Methods: This method used in SIM mode mass selective detection was developed and validated for the trace level analysis of an impurity. Chromatographic separation of MSC was achieved in 5.45 minutes and m/z value was 79 on SIM mode, ZB-5 ms 30 m × 0.25 mm × 0.25 µm column, using helium carrier gas with 1.0 ml/min.Results: The method was linear for MSC in ICR 1.90-7.5 µg/ml, respectively. The coefficient of correlation (r2) for the MSC was better than 0.999. The limit of detection and limit of quantification (LOQ) obtained were 0.44 and 1.32 µg/ml. The method was fully validated, complying Food and Drug Administration, ICH, and European Medicines Agency guidelines. Furthermore, verified precision, accuracy, LOQ precision, LOQ accuracy, ruggedness, and robustness.Conclusion: The methods were successfully validated to determination and quantification of MSC in ICR API. Hence, the method holds good for the routine trace analysis of MSC in ICR and various pharmaceutical industries as well as academics.Keywords: Methane sulfonyl chloride, Itraconazole, Gas chromatography-mass spectrometry, Method development, Method validation.INTRODUCTIONNumerous analytical methods for the determination of pharmaceuticals and their metabolites in aqueous solutions have been described in the literature. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS (GC-MS) are the most widely used techniques [1]. An MS is typically utilized in one of two-ways: Full scan or selected ion monitoring (SIM). The typical GC-MS instrument is capable of performing both functions either individually or concomitantly depending on the setup of the particular instrument. The primary goal of instrument analysis is to quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum in SIM certain ion fragments are entered into the instrument method, and only those mass fragments are detected by the MS. The advantages of SIM are that the detection limit is lower since the instrument is only looking at a small number of fragments (e.g., three fragments) during each scan [2]. SIM mode the MS is “targeting a limited mass range,” the number of scans across the peak has increased resulting in better peak shape. This is an easy solution for getting better quantitation for early eluting peaks. Inspect the ions obtained for the peak in full scan mode and use at least one of the ions in SIM to obtain a better scan rate [3].Methane sulfonyl chloride (MSC) (Fig. 1) is an organosulfur compound with the formula CH3SO2Cl. It is a colorless liquid that dissolves in polar organic solvents but is reactive toward water, alcohols, and many amines. During the manufacturing process of itraconazole, formation of MSC is possible due to residual methanol available in the manufacturing process and may also be formed due to thermal interaction in the presence of methanol.MSC is a potential genotoxic impurity in itraconazole (ICR) drug substance as it was the part of the synthesis process. As per the International Conference on Harmonization Guidelines from European Medical Agency, the genotoxins were to be limited to 1.5 µg/day [4,5]. MSC is having chloro as a functional group with aliphatic chain as per the guideline it is a genotoxic alerting compound. Sensitive method for the analysis of ICR as genotoxic impurity was not available. While developing method at such a low-level, interferences due to drug substance as well as other process impurities and degradation products were the major problems in achieving specificity. Hence, based on published general strategies for genotoxic impurities and the threshold of toxicological concern, MSC was evaluated in ICR drug substance.ICR (Fig. 2) is a classical member of the triazole class and is an important drug in our arsenal to treat fungal infections because it exhibits broad-spectrum antifungal activity [4-7]. ICR, (+-)-ics-4[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl] methoxy] phenyl]-1-piperazinyl] phenyl]-2,4-dihydro-2(1-methylpropyl)-3H-1,2,4-triazol-3-one, is an orally active triazole antifungal agent which demonstrates broad-spectrum activity against a number of fungal species including dermatophytes. It has been demonstrated that GC-MS method offers several advantages over high-performance LC (HPLC) method including better sensitivity, specificity, and higher throughput. This paper presents a highly specific and sensitive GC-MS method for the MSC in ICR active pharmaceutical ingredient (API) as per International Council of Harmonization guidelines [11]. This approach eliminated the time-consuming liquid-liquid extraction used in HPLC-ultraviolet method, increased the detection limit, and greatly reduced sample processing and instrument acquisition time. Thus, the paper reports an economical, simple, and accurate GC-MS method for MSC in ICR.Research Article404Asian J Pharm Clin Res, Vol 9, Issue 5, 2016, 403-407 Babu et al. METHODSApparatusGC-MS analysis was carried out on GC-MS-QP 2010 plus system (Shimadzu) having GC-MS Solutions software, an analytical balance (XS 205 from Mettler Toledo) and autopipette (100 µL - 1000 µL from Eppendorf) were used. The GC-MS experimental conditions for MSC content in ICR as shown in Table 1.Chemicals and reagentsMSC was purchased from Sigma-Aldrich, Fluka, Acros Organics. Dichloromethane was procured from Rankem (HPLC grade). Pure sample of ICR was obtained from Local Research Laboratory.Preparation of standard solutionDiluentDichloromethane is an organic compound with the formula CH2Cl2. This colorless, volatile liquid with a moderately sweet aroma is widely used as a solvent. Although it is not miscible with water, it is miscible with many organic solvents. Dichloromethane was selected as the standard and sample diluent because of its ability to dissolve a wide variety of substance.Preparation of standard stock solutionWeighed accurately 50 mg of MSC in 50 ml of volumetric flask dissolve and make up diluents (1000 µg/ml). Transfer 1.0 ml of above solution into a 100 ml volumetric flask make up with diluents (10 µg/ml).Preparation of standard solution (1.87 µg/ml)Transfer 3.74 ml from standard stock solution into a 20 ml volumetric flask and make up to the mark with the same diluents to get a standard solution (standard solution was prepared with respect to sample concentration).Preparation of sample solutionWeighed accurately 10.0 g of the ICR API into 20 ml of volumetric flask, add 10 ml of diluents mix well then makeup with the same diluents (final concentration is 500 mg/ml of API).RESULTS AND DISCUSSIONMethod optimizationThe various genotoxic impurities are present in API is the foremost prerequisite for successful method development in GC-MS. The successful method development should result in a fast, simple, and time efficient method that is capable of being utilized in a manufacturing setting. Following were the stepwise strategies for the method development in our case.Column selectionThe primary goal of column selection was to resolve a genotoxic impurity which is formed during the synthesis and manufacturing of ICR API. Several columns were initially investigated to finalize a single method for the separation and quantitation of solvent. Wall-coated capillary columns of various brands with a variety of phases and dimensions have been investigated, e.g. column A is ZB-624 (30 m length, 0.32 mm i.d. with a stationary phase of 6% cyanopropyl phenyl and 94% dimethyl polysiloxane film of 1.8 µm) and column B is ZB-5 MS (30 m length, 0.25 mm i.d. with a stationary phase of 5% cyanopropyl phenyl and 95% dimethyl polysiloxane film of 0.25 µm). In the above two columns, the response was found to be comparatively lower, and peak shapes were found to be satisfactory in column B. Finally, column B is proved to be the best column that could fulfill all the needs of the GC-MS method, i.e., higher sensitivity and shorter runtime.MS analysisAs per the analysis conducted by GC-MS and the retention times of MSC was in the range 5.0-6.0 minutes, respectively. As per the MS of MSC, the fragments were observed at m/z 79. The spectrum of MSC the analytes match to the reference spectrum of NIST. The MS and reference MS of MSC shown in Fig. 3.Method validationThe method validation was done by evaluating specificity, repeatability, linearity and range, accuracy, limit of detection (LOD) and limit of quantitation (LOQ), LOQ - repeatability, LOQ-accuracy, ruggedness, and robustness.SpecificityThe ICR API sample was spiked with MSC, and sample was chromatographed to examine interference, if any, of the residual solvent peaks with each other. The retention time for standard MSC 5.45 minutes, respectively. The chromatograms of blank, standard MSC, and ICR API were as shown in Fig. 4.RepeatabilityThe MSC was prepared at 1.87 ppm absolute with respect to sample concentration and injected in six replicates. The relative standard deviation (RSD) (n=6) values obtained for the area of MSC is 40,452. Fig. 1: Chemical structure of methane sulfonyl chlorideFig. 2: Chemical structure of itraconazoleTable 1: GC-MS experimental conditionsColumn ZB-5MS, 30 m×0.25 mm ID×0.25 µmInjector temperature 150°CCarrier gas HeliumCarrier gas flow 1.0 mL/minSplit ratio 5:00Oven program Initial temperature 40°C hold time 4.0 minutes raise 20.0°C/min up to 200°C hold time 13.00 minutesTotal run time 25.0 minutesInjection volume 1.0 µlIonization source EIElectron energy 70 EvSource temperature 280°CInterface temperature 260°Cm/z fragment 79Solvent cut time 3.0 minutesDetector voltage 0.92 KVStart time 3.01 minutesEnd time 8.0 minutesGC-MS: Gas chromatographic-mass spectrometer405Asian J Pharm Clin Res, Vol 9, Issue 5, 2016, 403-407 Babu et al. Table 2: Repeatability data for MSCNumber of injections MSC areaInjection 1 38,014Injection 2 40,212Injection 3 43,256Injection 4 40,125Injection 5 39,851Injection 6 41,256Average area 40,452Standard deviation 1731Percentage of RSD 4.28RSD: Relative standard deviation, MSC: Methane sulfonyl chlorideThe % RSD for MSC peak area response of standard six injections should be not more than 15% as per United States Pharmacopoeia [12]. The data of repeatability were as shown in Table 2.LinearityThe linearity of the method was determined by making injections of standard MSC at five concentration levels from 50% to 200%. Three replicates were performed at each level. The calibration curves were obtained with the average of peak area ratios of three replicates. The correlation coefficient (r2) value for MSC was found to be higher than 0.999, and the calibration curves were linear within the range. These results revealed an excellent linearity. The linearity values for the MSC as shown in Table 3 and linearity graph is shown in Fig. 5.Fig. 3: (a) Mass spectrum (MS) of methane sulfonyl chloride (MSC) and (b) reference MS of MSC in NISTbaFig. 4: Typical chromatograms of (a) Blank (dichloromethane), (b) methane sulfonyl chloride, (c) itraconazole active pharmaceutical ingredientcba406Asian J Pharm Clin Res, Vol 9, Issue 5, 2016, 403-407 Babu et al. Table 3: Linearity data for MSCS. No. Concentration level (%)Run-I areaRun-II areaRun-III areaAverage area1 50 24,122 23,999 24,911 24,3442 75 37,528 37,241 37,002 37,2573 100 54,851 54,113 53,989 54,3184 150 85,521 85,426 85,422 85,4565 200 110,826 110,852 111,001 110,8936 Correlation  coefficient (r2)0.999MSC: Methane sulfonyl chlorideTable 4: Accuracy data for MSCS. No. Sample+50% areaSample+100% areaSample+150% area1 21,230 42,452 61,6382 21,290 42,136 61,8513 21,546 42,546 61,251Average area 21,355 42,378 61,580Percentage recovery105.58 104.76 101.49Standard average area40,452MSC: Methane sulfonyl chlorideFig. 5: Linearity graph of methane sulfonyl chlorideTable 5: Linearity data for MSC at LOQ concentrationS. No. Concentration (%) Area1 50 (1.9 µg/ml) 24,3442 75 (2.8 µg/ml) 37,2573 100 (3.75 µg/ml) 54,3184 150 (5.6 µg/ml) 85,4565 200 (7.5 µg/ml) 110,893Correlation coefficient (r2) 0.999Slope 15,742STEYX 2078LOD 0.44 µg/mlLOQ 1.32 µg/mlLOQ: Limit of quantification, LOD: Limit of detection, MSC: Methane sulfonyl chlorideTable 6: Repeatability data for MSC at LOQ concentrationNumber of injections MSC areaInjection 1 12,024Injection 2 13,884Injection 3 12,076Injection 4 15,470Injection 5 11,292Injection 6 14,058Average area 13,134Standard deviation 1589Percentage of RSD 12.10RSD: Relative standard deviation, LOQ: Limit of quantification, MSC: Methane sulfonyl chlorideTable 7: LOQ accuracy data for MSCS. No. Sample+LOQ level area1 14,0642 13,6973 14,686Average area 14,149Percentage recovery 13,134Standard average area 107.73%LOQ: Limit of quantification, MSC: Methane sulfonyl chlorideAccuracy (% recovery)Weighed accurately 10.0 g of the ICR API into three different 20 ml of volumetric flasks and spiked with 1.9, 3.75, and 5.6 µg/ml standard solutions of MSC, add 10 ml of diluents mix well then make up with the same diluents. Inject three levels in triplicate. From accuracy data, the % recovery of MSC was found within the limits (100±15%). Results indicate that the method has an acceptable level of accuracy. The results are presented in Table 4.LOD and LOQThe LOD and LOQ were calculated by instrumental and statistical methods. For the instrumental method, LOD is determined as the lowest amount to detect, and LOQ is the lowest amount to quantify by the detector. The LOD and LOQ of MSC in ICR API were determined based on linearity. Prepare the standard MSC solution at LOD (0.44 µg/ml) and LOQ (1.32 µg/ml) concentrations. The area of MSC at LOD concentration is 3985 and LOQ concentration 13134. The linearity also passed at LOQ concentration. The data of LOD and LOQ were as shown in Table 5.Repeatability at LOQ concentrationPrepare the standard MSC solution at LOQ concentration (1.32 ppm) and injected in six replicates. The RSD (n=6) values obtained for the area of MSC is 13134. The acceptance criteria of % RSD for MSC are more than 15%. The LOQ repeatability data and chromatograms of LOD and LOQ were as shown in Table 6 and Fig. 6.Accuracy at LOQ concentrationWeighed accurately 10.0 g of the ICR API into three different 20 ml of volumetric flasks and spiked with LOQ level (1.32 µg/ml) standard solution of MSC, add 10 ml of diluents mix well then make up with the same diluents and inject in triplicate. From accuracy data at LOQ level, the % recovery of MSC was found within the limits (100±15%). The data of LOQ-accuracy were as shown in Table 7.RuggednessRuggedness of the method was evaluated by performing the sample analysis in six replicates using different analyst on different days. The % RSD values of <15.0% for MSC content indicate that the method adopted is rugged. The data of ruggedness were shown in Table 8RobustnessThis study was performed by making small but deliberate variations in the method parameters. The effect of variations in flow rate of carrier gas and column oven temperature was studied. Under all the variations, system suitability requirement is found to be within the acceptance criteria and hence the proposed method is robust. The RSD of area counts for MSC peak obtained from six replicate injections of standard solution should be not more than 15.0%. The data of robustness were shown in Table 9.CONCLUSIONA simple high throughput GC-MS method has been developed and fully validated for the determination of MSC in ICR API. This method is specific, sensitive, and reproducible and has been successfully to 407Asian J Pharm Clin Res, Vol 9, Issue 5, 2016, 403-407 Babu et al. Fig. 6: Typical % relative standard deviation chromatograms at limit of quantification concentrationTable 8: Result of ruggednessDay-1 (% RSD) Day-2 (% RSD) Analyst-1 (% RSD) Analyst-2 (% RSD)Analyst-1 Analyst-2 Analyst-1 and Analyst-2Analyst-1 Analyst-2 Analyst-1 and Analyst-2Day-1 and 2 Day-1 and 26.09 5.01 5.32 4.84 4.91 4.69 5.44 4.77RSD: Relative standard deviationTable 9: Result of robustnessFlow variationParameter 0.5 ml/min (flow minus) 1.0 ml/min (control) 1.5 ml/min (flow plus)Percentage of RSD 5.69 5.28 5.58Column oven temperatureParameter 195°C (temperature minus) 200°C (control) 205°C (temperature plus)Percentage of RSD 4.83 4.71 4.39RSD: Relative standard deviationmonitor and control impurity level. The residue MSC was determined in ppm levels also. The method well suits for the intended purpose.ACNOWLEDGMENTThe author expresses sincere thanks to the Principal and Head of the Chemistry Department, SVRM College, for excellent guidance and encouragement.REFERENCES1. Verenitch SS, Lowe CJ, Mazumder A. Determination of acidic drugs and caffeine in municipal wastewaters and receiving waters by gas chromatography-ion trap tandem mass spectrometry. J Chromatogr A 2006;1116(1-2):193-203.2. Available from: https://www.en.wikipedia.org/wiki/Gas_chromatography%E2%80%93 mass_spectrometry.3. Available from: http://www.sge.com/uploads/55/6a/556ae4e55c042c95832191d30557f017/TA-0061-C.pdf.4. The European Agency for the Evaluation of Medicinal Products, ICH Topic S1B, Note for Guidance on Carcinogenicity: Testing for Carcinogenicity of Pharmaceuticals; 1998.5. European Medicines Agency, Evaluation of Medicines for Human Use, Guideline on the Limits of Genotoxic Impurities; 2007.6. Dileep Kumar M. Analysis of Genotoxic Impurities in API’s in Compliance with ICH Guidelines using Gas Chromatography Mass Spectrometry. Waltham, United States: Centre of Excellence for Analytical Sciences Perkin Elmer (India.) Pvt., Ltd.7. Zhao L, Quimby B. Analysis of Drugs by using the Ultra Inert Inlet. Liners with Wool. Wilmington, DE, USA: Agilent Technologies, Inc.8. Bari SB, Kadam BR, Jaiswal YS, Shirkhedkar AA. Impurity profile: Significance in active pharmaceutical ingredient. Eurasian J Anal Chem 2007;2(1):33-53.9. Veenaeesh P, Manikumar G, Manjusha. P, Padmasri A. Determination of methyl methane sulfonate, ethyl methane sulfonate and isopropyl methane sulfonate impurities in Lopinavir API by GC/MS/MS using electron ionization. Int J Pharm Res Rev 2014; 3(2):11-6.10. Kashif UL, Kumar N. Validation of LC-MS/MS method for the simultaneous estimation of Itraconazole and its metabolite hydroxyl Itraconazole in plasma. Asian J Pharm Clin Res 2014;7 Suppl 1:131-6.11. Geneva .The Impact of Implementation of ICH Guidelines in Non ICH Countries. English Distribution General 2001; 1-29.12. United States Pharmacopoeia (USP). 2006. p. 1577-8.

DEVELOPMENT AND VALIDATION OF A GAS CHROMATOGRAPHY-MASS SPECTROMETRY WITH SELECTED ION MONITORING METHOD FOR THE DETERMINATION OF TRACE  LEVELS OF METHANE SULFONYL CHLORIDE AS AN IMPURITY IN ITRACONAZOLE ACTIVE PHARMACEUTICAL INGREDIENT

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DEVELOPMENT AND VALIDATION OF A GAS CHROMATOGRAPHY-MASS SPECTROMETRY WITH SELECTED ION MONITORING METHOD FOR THE DETERMINATION OF TRACE LEVELS OF METHANE SULFONYL CHLORIDE AS AN IMPURITY IN ITRACONAZOLE ACTIVE PHARMACEUTICAL INGREDIENT

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