TY - JOUR
AU1 - Chawla, Rajesh Kumar
AU2 - Rao, G S N Koteswara
AU3 - Kulandaivelu, Umasankar
AU4 - Panda, Siva Prasad
AU5 - Alavala, Rajasekhar Reddy
AB - Abstract Objective A selective and sensitive liquid chromatography–tandem mass spectrometer (LC–MS/MS) method has been developed for the quantification of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate oral solution. Materials and method The LC–MS/MS analysis was done on X Bridge HILIC (100 × 4.6 mm, 5 μm) analytical column, and the mobile phase used was10 mM ammonium formate with 0.2% formic acid as mobile phase-A and acetonitrile as mobile phase-B with a gradient programme of 5.0 min. The flow rate used was 1.2 mL/min. Triple quadrupole mass detector coupled to positive electrospray ionization operated in multiple reactions monitoring mode was used for the quantification at m/z 116.10 ± 0.5. Results Retention time of impurity was found ~3.2 min. The method was validated in terms of specificity, linearity, accuracy, precision, range, limit of detection, limit of quantitation (LOQ) and robustness. Relative standard deviation (RSD) for system suitability was found 1.3%. Calibration plot was linear over the range of 0.050–2.000 μg/mL. Limit of detection and limit of quantification were found 0.017 and 0.051 μg/mL, respectively. The intra- and inter-day precision RSD was 2.3% and the obtained recovery at LOQ to 200% was in between 86.7 and 107.4%. Conclusion The low RSD values and high recoveries of the method confirm the suitability of the method. Introduction Glycopyrrolate is a white crystalline powder with a melting point of 193°C, which has molecular weight of 393.3 and is soluble in water (1). Glycopyrrolate is a synthetic quaternary ammonium that is an anticholinergic agent with antispasmodic activity. Glycopyrrolate competitively binds to peripheral muscarinic receptors in the autonomic effector cells of, and inhibits cholinergic transmission in smooth muscle, cardiac muscle, the sinoatrial (SA) node, the atrioventricular (AV) node, exocrine glands and in the autonomic ganglia. Blockage of cholinergic transmission, in smooth muscle cells located in the gastrointestinal tract and the bladder, causes smooth muscle relaxation and prevents the occurrence of painful spasms. In addition, glycopyrrolate inhibits the release of gastric, pharyngeal, tracheal and bronchial secretions, it has no rare central effects as it does not cross the blood–brain barrier (2, 3). 1,1-dimethyl-3-hydroxy-pyrrolidinium is a metabolite found in the kidney and liver in response to oral administration of glycopyrrolate (4). Impurity profiling is now receiving critical attention from regulatory authorities. For trace level quantification of potential degradation impurities, conventional analytical techniques like high-performance liquid chromatography (HPLC) with connected detectors like ultraviolet–visible, refractive index, fluorescence, conductivity & evaporative light scattering and gas chromatography with flame ionization, electron capture & thermal conductivity detectors are inadequate; consequently, there is a great need to apply hyphenated analytical techniques to develop sensitive analytical methods for the analysis of pharmaceuticals. Glycopyrrolate active pharmaceutical ingredient and its formulations like glycopyrrolate tablets and glycopyrrolate injections have official monographs in US Pharmacopoeia (5), British Pharmacopoeia (6) and European Pharmacopoeia (7), but 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity is not listed or included in the monographs. Being a degradant impurity/metabolite it is mandatory to monitor and control in drug substance and drug products. A one-pot process for the synthesis of glycopyrrolate using cyclopentyl mandelic acid and 1-methyl-pyrrolidin-3-olwas has been described and investigated the related impurities and byproducts by liquid chromatography–mass spectrometry (LC–MS) (8). Determination of glycopyrrolate and its related impurities has been reported by ion-pair HPLC with ultraviolet detector (9). Impurity profile of glycopyrrolate has been reviewed and compared for the various analytical methods (10, 11). Determination of glycopyrrolate in human by ESI-LC–MS using volatile ion-pair reagent heptafluorobutyric acid has been reported (12). The diastereomeric purity of glycopyrrolate was determined by HPLC on B-cyclodextrin-bonded phase column has been reported (13). A high performance thin layer chromatography (HPTLC) method using densitometry scanning for determination of glycopyrrolate and its related impurities in drug substance and drug product has been reported in (14). Simultaneous estimation of glycopyrrolate and formoterol & indacaterol and glycopyrronium has been reported by RP-HPLC using ultraviolet detector (15, 16). Development and validation for assay of glycopyrronium by RP-HPLC has been also reported in (17). The literature survey revealed that no methods have been reported for the estimation of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate and its drug products. Glycopyrrolate degradates to form glycopyrrolate related compound—C and 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurities (Figure 1). A degradant impurity 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide is lack of chromophore in its structure, hence it does not show any absorbance in UV–visible range. The purpose of this work is to develop and validate a simple, accurate, selective and sensitive method for estimation of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate oral solution (1 mg/5 mL) by liquid chromatography–tandem mass spectroscopy (LC–MS/MS). Figure 1 Open in new tabDownload slide Structures of glycopyrrolate bromide degradates to form glycopyrrolate related compound—C and 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurities. Figure 1 Open in new tabDownload slide Structures of glycopyrrolate bromide degradates to form glycopyrrolate related compound—C and 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurities. Materials Acetonitrile (HPLC grade) and formic acid (LC–MS LiChropur grade) were procured from Merck Millipore, India. Ammonium formate (Optima LC–MS grade) was procured from Fisher Scientific, India. 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity working standard (CAS No. 51052-74-5) was procured from Pharmaffiliates, Panchkula, India. HPLC grade water was used from Milli-Q water purifier system. The mobile phase solution was filtered through a 0.45 μm Ultipor® N66® membrane filter (Pall Life Sciences, USA). The marketed glycopyrrolate oral solution 1 mg/5 mL formulation (Cuvposa, Make: Merz Pharmaceuticals, LLC. North America) was used for analysis of drug product. Method The method was developed and validated on Agilent’s 1200 series automated HPLC system equipped with degasser, quaternary pump with gradient mixing, autosampler with temperature control, column compartment with thermostat connected to SCIEX API 4000—LC–MS/MS. The chromatographic parameters were optimized and optimized chromatographic conditions are shown in Table I. Table I Optimized Chromatographic Conditions of the Developed Method Chromatographic conditions:  Instrument name: Liquid chromatography tandem Mass spectrometry (LC–MS/MS)  Make & model: Agilent 1200 Series & ABSCIEX API 4000  Column: X Bridge HILIC, 100 × 4.6 mm, 5 μm  Flow rate (μL/min): 1,200  Split ratio: 90:10  Injection volume: 5 μL  Elution mode: Gradient program  Column oven temperature: 45.0 ± 1.0°C Gradient programme: Step Time (min) Flow rate (μL/min) %A %B 0 0.5 1,200 2 98 1 1.0 1,200 30 70 2 2.2 1,200 30 70 3 2.5 1,200 2 98 4 5.0 1,200 2 98 Auto sampler temperature: 5.0 ± 0.5°C Run time: 5.00 min Retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium is ~3.2 ± 0.3 min. MRM conditions  Ion source: Turbo spray  Polarity: Positive MRM transitions  1,1-dimethyl-3-hydroxy-pyrrolidinium: 116.10 ± 0.5 (parent), 88.10 ± 0.5 (product)  Collision gas (CAD): 9.0  Curtain gas (CUR): 20.0  Ion source gas 1 (GS1): 30.0  Ion source gas 1 (GS2): 30.0  Ion spray voltage (IS): 5,500  Temperature (TEM): 400  Entrance potential (EP): 10.0  Resolution: Unit  Declustering potential (DP): 46.00  Collision energy (CE): 27.00  Collision cell exit potential (CXP): 6.00 Chromatographic conditions:  Instrument name: Liquid chromatography tandem Mass spectrometry (LC–MS/MS)  Make & model: Agilent 1200 Series & ABSCIEX API 4000  Column: X Bridge HILIC, 100 × 4.6 mm, 5 μm  Flow rate (μL/min): 1,200  Split ratio: 90:10  Injection volume: 5 μL  Elution mode: Gradient program  Column oven temperature: 45.0 ± 1.0°C Gradient programme: Step Time (min) Flow rate (μL/min) %A %B 0 0.5 1,200 2 98 1 1.0 1,200 30 70 2 2.2 1,200 30 70 3 2.5 1,200 2 98 4 5.0 1,200 2 98 Auto sampler temperature: 5.0 ± 0.5°C Run time: 5.00 min Retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium is ~3.2 ± 0.3 min. MRM conditions  Ion source: Turbo spray  Polarity: Positive MRM transitions  1,1-dimethyl-3-hydroxy-pyrrolidinium: 116.10 ± 0.5 (parent), 88.10 ± 0.5 (product)  Collision gas (CAD): 9.0  Curtain gas (CUR): 20.0  Ion source gas 1 (GS1): 30.0  Ion source gas 1 (GS2): 30.0  Ion spray voltage (IS): 5,500  Temperature (TEM): 400  Entrance potential (EP): 10.0  Resolution: Unit  Declustering potential (DP): 46.00  Collision energy (CE): 27.00  Collision cell exit potential (CXP): 6.00 Open in new tab Table I Optimized Chromatographic Conditions of the Developed Method Chromatographic conditions:  Instrument name: Liquid chromatography tandem Mass spectrometry (LC–MS/MS)  Make & model: Agilent 1200 Series & ABSCIEX API 4000  Column: X Bridge HILIC, 100 × 4.6 mm, 5 μm  Flow rate (μL/min): 1,200  Split ratio: 90:10  Injection volume: 5 μL  Elution mode: Gradient program  Column oven temperature: 45.0 ± 1.0°C Gradient programme: Step Time (min) Flow rate (μL/min) %A %B 0 0.5 1,200 2 98 1 1.0 1,200 30 70 2 2.2 1,200 30 70 3 2.5 1,200 2 98 4 5.0 1,200 2 98 Auto sampler temperature: 5.0 ± 0.5°C Run time: 5.00 min Retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium is ~3.2 ± 0.3 min. MRM conditions  Ion source: Turbo spray  Polarity: Positive MRM transitions  1,1-dimethyl-3-hydroxy-pyrrolidinium: 116.10 ± 0.5 (parent), 88.10 ± 0.5 (product)  Collision gas (CAD): 9.0  Curtain gas (CUR): 20.0  Ion source gas 1 (GS1): 30.0  Ion source gas 1 (GS2): 30.0  Ion spray voltage (IS): 5,500  Temperature (TEM): 400  Entrance potential (EP): 10.0  Resolution: Unit  Declustering potential (DP): 46.00  Collision energy (CE): 27.00  Collision cell exit potential (CXP): 6.00 Chromatographic conditions:  Instrument name: Liquid chromatography tandem Mass spectrometry (LC–MS/MS)  Make & model: Agilent 1200 Series & ABSCIEX API 4000  Column: X Bridge HILIC, 100 × 4.6 mm, 5 μm  Flow rate (μL/min): 1,200  Split ratio: 90:10  Injection volume: 5 μL  Elution mode: Gradient program  Column oven temperature: 45.0 ± 1.0°C Gradient programme: Step Time (min) Flow rate (μL/min) %A %B 0 0.5 1,200 2 98 1 1.0 1,200 30 70 2 2.2 1,200 30 70 3 2.5 1,200 2 98 4 5.0 1,200 2 98 Auto sampler temperature: 5.0 ± 0.5°C Run time: 5.00 min Retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium is ~3.2 ± 0.3 min. MRM conditions  Ion source: Turbo spray  Polarity: Positive MRM transitions  1,1-dimethyl-3-hydroxy-pyrrolidinium: 116.10 ± 0.5 (parent), 88.10 ± 0.5 (product)  Collision gas (CAD): 9.0  Curtain gas (CUR): 20.0  Ion source gas 1 (GS1): 30.0  Ion source gas 1 (GS2): 30.0  Ion spray voltage (IS): 5,500  Temperature (TEM): 400  Entrance potential (EP): 10.0  Resolution: Unit  Declustering potential (DP): 46.00  Collision energy (CE): 27.00  Collision cell exit potential (CXP): 6.00 Open in new tab Prepared mobile phase for chromatography, diluents used for solution preparation, blank solution, standard solution and test sample solution Mobile phase A was prepared by weighing and transferring ~0.6306 g of ammonium formate into 1,000-mL of Milli-Q water, to this added 2 mL of formic acid. Mobile phase B was used as 100% acetonitrile. About 0.2% formic acid and acetonitrile in the ratio of 70:30 v/v was used as diluent. Standard solution (1 μg/mL) was prepared by weighting and transferring ~2.5 mg of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity standard into a 100-mL volumetric flask, added 50 mL of diluent, sonicated to dissolve and made up to volume with diluent. Transferred 1.0 mL of above solution into 25 mL volumetric flask and diluted to volume with diluent and mixed. Prepared standard check solution (1 μg/mL) was exactly same as standard solution. Glycopyrrolate oral solution was taken as such for test sample. Diluted the above blank solution, standard solution, standard check solution and test sample solution to 50 folds with diluent and injected into LC–MS/MS. Procedure for sequence of solution injection and recording of chromatographic data Injected blank, standard solution (six times), check standard solution (two times) and test sample preparation into LC–MS/MS. Recorded the chromatograms and measured the peak responses. Injected standard solution after every six sample solution as bracketing standard. Specification limit for the control of impurity in the drug product The specification of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity calculated as per International Conference on Harmonization (ICH) Q3B (R2) (18) guidelines and found not >0.5% in the drug product. Method validation The developed method was validated as per ICH Q2 (R1) (19) guidelines and US Pharmacopoeia general chapter < 1225> (20). System suitability System suitability was checked in accordance with US Pharmacopoeia general chapter < 621> (21). Peak retention time, % relative standard deviation for six replicate standard and similarity factor for check standard of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide peak were evaluated. The acceptance criterion for % relative standard deviation for six replicate standards was not >10.0 and similarity factor for check standard was 0.90–1.10. Specificity The developed method was validated for specificity by injecting blank and placebo solution in triplicate. The placebo solution was prepared by mixing the inactive ingredients listed in the information leaflet of Cuvposa solution. The inactive ingredients used were citric acid, glycerin, natural and artificial cherry flavor, methylparaben, propylene glycol, propylparaben, saccharin sodium, sodium citrate, sorbitol solution and purified water. The chromatograms were evaluated for any interference at the retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide peak. Forced degradation of glycopyrrolate oral solution was carried out, to confirm that during stability study or throughout the shelf life, 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity peak degrading or not and also the forced degradation study will help to identify the type of degradation pathway (oxidative, alkali hydrolysis, acid hydrolysis, photolytic, dry heat and humidity) for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity peak. Precision Precision of the development method was evaluated by injecting six test sample preparations as such and six test sample preparations spiked with 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity at 100% level. The % 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity and % relative standard deviation of test sample preparations as such and six test sample preparations spiked were calculated. Intermediate precision of the method was also evaluated using different analyst, different day and different column by injecting six test sample preparations as such and six test sample preparations spiked prepared as same for precision. The acceptance criteria for individual precision % relative standard deviation (RSD) was not >15.0 and for 12 preparation results was not >20.0. Accuracy (Recovery) Recovery study was performed to evaluate the accuracy of the method by spiking method. Recovery study was done by spiking 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in to placebo in the concentration of limit of quantitation (LOQ), 50, 100 and 200% level of the proposed concentration. The test samples were prepared in triplicate for 50 and 100% level and six preparations for LOQ and 200%. Injected the prepared spiked samples in the proposed experimental conditions. The % recovery of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity was calculated for all the levels. The acceptance criterion for recovery of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity was 80.0–120.0% and % RSD for six recovery results at LOQ and 200% was not >15.0. Limit of detection and limit of quantitation Limit of detection is the lowest amount of analyte in sample that can be detected, but not necessarily quantifiable and limit of quantification is the lowest amount of analyte that can be quantitated with acceptable accuracy, precision, under the stated experimental conditions. The limit of detection (LOD) and LOQ were determined by injecting a known concentration of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity under the stated experimental conditions and determined the signal-to-noise ratio, usually for LOQ 10:1 and for LOD 3:1 can be acceptable. Linearity Linearity of the method was established for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity from LOQ to 200% of the proposed concentration using six calibration levels at LOQ, 25, 50, 100, 150 and 200%. The linearity was established from the 0.050 to 2.000 μg/mL impurity range. The working standard was used to prepare calibration levels. The calibration curve was plotted for each level as concentration of level verses peak response. The result of linearity was evaluated by simple linear regression analysis. Robustness The robustness of the method was evaluated by deliberately altering the method conditions from the original method parameters. For robustness study, two chromatographic parameters were considered such as (i) flow rate 1,200 μL/min ±10% (1,080 and 1,320 μL/min) and (ii) column temperature 45 ± 5°C (40 and 50°C). Robustness of the method was evaluated by system suitability parameters. Solution stability Solution stability was established for standard and test sample preparations. Bench-top (ambient temperature) stability was established by injecting standard and test sample at regular interval for 2 days. Solution stability was established by calculating similarity factor for standard against fresh standard and % impurity difference for test sample from initial value. Results Method development and optimization Initially the method development for estimation of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate oral solution was initiated on HPLC with UV–visible and refractive index detectors. The trials were taken to separate the peaks on ACE C18, 250 × 4.6 mm, 5 μm column using 50 mM phosphate buffer adjusted to pH 3.0 at lower wavelength of 200 nm. Because of lack of chromophore in 1,1-dimethyl-3-hydroxy-pyrrolidinium, no response was observed in UV–visible detector. In refractive index detector, the peak was eluted in void and has interference with other inactive components of test solution. As the conventional HPLC method was not possible, tried to develop method on LC–MS/MS. A gradient program using ammonium formate buffer as mobile phase A and acetonitrile as mobile phase B was tried on ACE C18, 250 × 4.6 mm, 5 μm column but 1,1-dimethyl-3-hydroxy-pyrrolidinium and glycopyrrolate peaks were merged and no separation was observed. To get retained 1,1-dimethyl-3-hydroxy-pyrrolidinium peak, X Bridge HILIC, 100 × 4.6 mm, 5 μm column was used and observed the peaks were eluted at 2.6 and 3.2 min for glycopyrrolate and 1,1-dimethyl-3-hydroxy-pyrrolidinium respectively. By using HILIC column a good selective method was achieved and further optimized the test solution and standard solution to achieve sensitive method. System suitability System suitability of the method was evaluated by means of peak retention time, % relative standard deviation for six replicate standard and similarity factor for check standard of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide peak were evaluated and found within the acceptance criteria. The results are presented in Table II. Table II System suitability results Parameter . Result . Retention time (min) 3.27 Six replicate standard Pear area  Standard solution injection—1 84,331  Standard solution injection—2 82,720  Standard solution injection—3 81,840  Standard solution injection—4 82,992  Standard solution injection—5 84,789  Standard solution injection—6 82,817 Average 83,248 % RSD 1.3 Check standard (similarity factor) 1.00 Parameter . Result . Retention time (min) 3.27 Six replicate standard Pear area  Standard solution injection—1 84,331  Standard solution injection—2 82,720  Standard solution injection—3 81,840  Standard solution injection—4 82,992  Standard solution injection—5 84,789  Standard solution injection—6 82,817 Average 83,248 % RSD 1.3 Check standard (similarity factor) 1.00 Open in new tab Table II System suitability results Parameter . Result . Retention time (min) 3.27 Six replicate standard Pear area  Standard solution injection—1 84,331  Standard solution injection—2 82,720  Standard solution injection—3 81,840  Standard solution injection—4 82,992  Standard solution injection—5 84,789  Standard solution injection—6 82,817 Average 83,248 % RSD 1.3 Check standard (similarity factor) 1.00 Parameter . Result . Retention time (min) 3.27 Six replicate standard Pear area  Standard solution injection—1 84,331  Standard solution injection—2 82,720  Standard solution injection—3 81,840  Standard solution injection—4 82,992  Standard solution injection—5 84,789  Standard solution injection—6 82,817 Average 83,248 % RSD 1.3 Check standard (similarity factor) 1.00 Open in new tab Specificity The specificity was performed to check blank and placebo interference. The chromatograms show (Figures 2–5) no interference at the retention time of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity peak due to blank and placebo. Figure 2 Open in new tabDownload slide LC–MS/MS chromatogram of blank solution. No peak observed for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 2 Open in new tabDownload slide LC–MS/MS chromatogram of blank solution. No peak observed for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 3 Open in new tabDownload slide LC–MS/MS chromatogram of placebo solution. No peak observed for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 3 Open in new tabDownload slide LC–MS/MS chromatogram of placebo solution. No peak observed for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 4 Open in new tabDownload slide LC–MS/MS chromatogram of standard solution. Peak at 3.27 min is due to 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 4 Open in new tabDownload slide LC–MS/MS chromatogram of standard solution. Peak at 3.27 min is due to 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 5 Open in new tabDownload slide LC–MS/MS chromatogram of test sample solution. Peak at 3.27 min is due to 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 5 Open in new tabDownload slide LC–MS/MS chromatogram of test sample solution. Peak at 3.27 min is due to 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Forced degradation of glycopyrrolate oral solution was carried out for acid hydrolysis, alkali hydrolysis, oxidation, photolytic, dry heat and humidity conditions. It was observed that glycopyrrolate is sensitive to acid and alkali hydrolysis results into generation 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. The results are presented in Table III. Table III Specificity—Forced Degradation Results Forced degradation conditions . % 1,1-Dimethyl-3-hydroxy-pyrrolidinium bromide impurity . Control sample 0.142 Acid hydrolysis (5N HCL at 60°C for 2 h) 4.744 Alkali hydrolysis (5N NaOH at 60°C for 2 h) 26.365 Peroxide oxidation (30% Peroxide at bench-top for 2 h) 0.148 Water hydrolysis (Water at 60°C for 2 h) 0.149 Humidity degradation (90%RH for 44 h) 0.144 Thermal degradation (80°C for 43 h) 0.290 UV light degradation (200 watts-hours/sq. meter) 0.147 Photolytic light degradation (1.2 million lux hours) 0.136 Forced degradation conditions . % 1,1-Dimethyl-3-hydroxy-pyrrolidinium bromide impurity . Control sample 0.142 Acid hydrolysis (5N HCL at 60°C for 2 h) 4.744 Alkali hydrolysis (5N NaOH at 60°C for 2 h) 26.365 Peroxide oxidation (30% Peroxide at bench-top for 2 h) 0.148 Water hydrolysis (Water at 60°C for 2 h) 0.149 Humidity degradation (90%RH for 44 h) 0.144 Thermal degradation (80°C for 43 h) 0.290 UV light degradation (200 watts-hours/sq. meter) 0.147 Photolytic light degradation (1.2 million lux hours) 0.136 Open in new tab Table III Specificity—Forced Degradation Results Forced degradation conditions . % 1,1-Dimethyl-3-hydroxy-pyrrolidinium bromide impurity . Control sample 0.142 Acid hydrolysis (5N HCL at 60°C for 2 h) 4.744 Alkali hydrolysis (5N NaOH at 60°C for 2 h) 26.365 Peroxide oxidation (30% Peroxide at bench-top for 2 h) 0.148 Water hydrolysis (Water at 60°C for 2 h) 0.149 Humidity degradation (90%RH for 44 h) 0.144 Thermal degradation (80°C for 43 h) 0.290 UV light degradation (200 watts-hours/sq. meter) 0.147 Photolytic light degradation (1.2 million lux hours) 0.136 Forced degradation conditions . % 1,1-Dimethyl-3-hydroxy-pyrrolidinium bromide impurity . Control sample 0.142 Acid hydrolysis (5N HCL at 60°C for 2 h) 4.744 Alkali hydrolysis (5N NaOH at 60°C for 2 h) 26.365 Peroxide oxidation (30% Peroxide at bench-top for 2 h) 0.148 Water hydrolysis (Water at 60°C for 2 h) 0.149 Humidity degradation (90%RH for 44 h) 0.144 Thermal degradation (80°C for 43 h) 0.290 UV light degradation (200 watts-hours/sq. meter) 0.147 Photolytic light degradation (1.2 million lux hours) 0.136 Open in new tab Precision Precision was evaluated by injecting six test sample preparations as such and six test sample preparations spiked with 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity at 100% level and % RSD of impurity (n = 6) was found to be 2.6 and 1.2, respectively, whereas for intermediate precision it was found to be 2.5 and 1.2, respectively. % RSD of precision and intermediate precision (n = 12) was found to be 2.5 and 2.3, respectively for as such test and test sample spiked with 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. The results were found within acceptance criteria for precision of the developed method. The results of precision are compiled in Table IV. Table IV Precision Results Test sample no. . Test unspike (as such) . Test spike with impurity . Precision (% impurity) . Intermediate precision (% impurity) . Precision (% impurity) . Intermediate precision (% impurity) . 01 0.159 0.162 0.646 0.691 02 0.155 0.157 0.655 0.686 03 0.163 0.167 0.661 0.671 04 0.153 0.161 0.663 0.680 05 0.161 0.156 0.663 0.693 06 0.163 0.159 0.669 0.686 Mean (n = 6) 0.159 0.160 0.660 0.685 % RSD (n = 6) 2.6 2.5 1.2 1.2 Mean (n = 12) 0.160 0.672 % RSD (n = 12) 2.5 2.3 Test sample no. . Test unspike (as such) . Test spike with impurity . Precision (% impurity) . Intermediate precision (% impurity) . Precision (% impurity) . Intermediate precision (% impurity) . 01 0.159 0.162 0.646 0.691 02 0.155 0.157 0.655 0.686 03 0.163 0.167 0.661 0.671 04 0.153 0.161 0.663 0.680 05 0.161 0.156 0.663 0.693 06 0.163 0.159 0.669 0.686 Mean (n = 6) 0.159 0.160 0.660 0.685 % RSD (n = 6) 2.6 2.5 1.2 1.2 Mean (n = 12) 0.160 0.672 % RSD (n = 12) 2.5 2.3 Open in new tab Table IV Precision Results Test sample no. . Test unspike (as such) . Test spike with impurity . Precision (% impurity) . Intermediate precision (% impurity) . Precision (% impurity) . Intermediate precision (% impurity) . 01 0.159 0.162 0.646 0.691 02 0.155 0.157 0.655 0.686 03 0.163 0.167 0.661 0.671 04 0.153 0.161 0.663 0.680 05 0.161 0.156 0.663 0.693 06 0.163 0.159 0.669 0.686 Mean (n = 6) 0.159 0.160 0.660 0.685 % RSD (n = 6) 2.6 2.5 1.2 1.2 Mean (n = 12) 0.160 0.672 % RSD (n = 12) 2.5 2.3 Test sample no. . Test unspike (as such) . Test spike with impurity . Precision (% impurity) . Intermediate precision (% impurity) . Precision (% impurity) . Intermediate precision (% impurity) . 01 0.159 0.162 0.646 0.691 02 0.155 0.157 0.655 0.686 03 0.163 0.167 0.661 0.671 04 0.153 0.161 0.663 0.680 05 0.161 0.156 0.663 0.693 06 0.163 0.159 0.669 0.686 Mean (n = 6) 0.159 0.160 0.660 0.685 % RSD (n = 6) 2.6 2.5 1.2 1.2 Mean (n = 12) 0.160 0.672 % RSD (n = 12) 2.5 2.3 Open in new tab Accuracy (Recovery) The accuracy was evaluated by calculating the recoveries at LOQ, 50, 100 and 200% level of the proposed concentration. The mean % recoveries at LOQ (n = 6) was found 103.2, at 50% (n = 3) was 98.9, at 100% (n = 3) was 89.7 and at 200% (n = 6) was 90.3. The % RSD at LOQ and 200% were found 2.8 and 2.7, respectively. The recoveries were found within the acceptance criteria and method found to be accurate. The results of accuracy are presented in Table V. Table V Accuracy Results Recovery level . Recovery (%) . Mean (%) . % RSD . LOQ sample—1 102.0 LOQ sample—2 99.8 LOQ sample—3 107.4 103.2 2.8 LOQ sample—4 102.6 LOQ sample—5 101.4 LOQ sample—6 106.0 50% sample—1 98.3 50% sample—2 98.6 98.9 NA 50% sample—3 99.7 100% sample—1 89.7 100% sample—2 90.2 89.7 NA 100% sample—3 89.1 200% sample—1 88.6 200% sample—2 91.6 200% sample—3 91.9 90.3 2.7 200% sample—4 89.6 200% sample—5 93.3 200% sample—6 86.7 Recovery level . Recovery (%) . Mean (%) . % RSD . LOQ sample—1 102.0 LOQ sample—2 99.8 LOQ sample—3 107.4 103.2 2.8 LOQ sample—4 102.6 LOQ sample—5 101.4 LOQ sample—6 106.0 50% sample—1 98.3 50% sample—2 98.6 98.9 NA 50% sample—3 99.7 100% sample—1 89.7 100% sample—2 90.2 89.7 NA 100% sample—3 89.1 200% sample—1 88.6 200% sample—2 91.6 200% sample—3 91.9 90.3 2.7 200% sample—4 89.6 200% sample—5 93.3 200% sample—6 86.7 Open in new tab Table V Accuracy Results Recovery level . Recovery (%) . Mean (%) . % RSD . LOQ sample—1 102.0 LOQ sample—2 99.8 LOQ sample—3 107.4 103.2 2.8 LOQ sample—4 102.6 LOQ sample—5 101.4 LOQ sample—6 106.0 50% sample—1 98.3 50% sample—2 98.6 98.9 NA 50% sample—3 99.7 100% sample—1 89.7 100% sample—2 90.2 89.7 NA 100% sample—3 89.1 200% sample—1 88.6 200% sample—2 91.6 200% sample—3 91.9 90.3 2.7 200% sample—4 89.6 200% sample—5 93.3 200% sample—6 86.7 Recovery level . Recovery (%) . Mean (%) . % RSD . LOQ sample—1 102.0 LOQ sample—2 99.8 LOQ sample—3 107.4 103.2 2.8 LOQ sample—4 102.6 LOQ sample—5 101.4 LOQ sample—6 106.0 50% sample—1 98.3 50% sample—2 98.6 98.9 NA 50% sample—3 99.7 100% sample—1 89.7 100% sample—2 90.2 89.7 NA 100% sample—3 89.1 200% sample—1 88.6 200% sample—2 91.6 200% sample—3 91.9 90.3 2.7 200% sample—4 89.6 200% sample—5 93.3 200% sample—6 86.7 Open in new tab LOD and LOQ The LOD and LOQ were determined by injecting a known concentration of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. The LOD and LOQ of the method were found to be 0.017 and 0.051 μg/mL, respectively. Linearity Linearity of the method was established for 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity from LOQ to 200% of the proposed concentration using six calibration levels (LOQ, 25, 50, 100, 150 and 200%) from the range 0.050 to 2.000 μg/mL. The working standard was used to prepare calibration levels. The calibration curve was plotted for each level as concentration of level verses peak response. The result of linearity was evaluated by simple linear regression analysis. The developed method was found to be linear from LOQ to 200% of the proposed concentration over six calibration levels ranging from 0.050 μg/mL (LOQ) to 2.000 μg/mL. The value of coefficient of correlation (R (2)) was found to be 1.000. Linearity graph of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity is shown in Figure 6. Chromatograms at LOQ and 200% are shown in Figures 7 and 8. Figure 6 Open in new tabDownload slide Linearity graph of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity from LOQ to 200% level. Figure 6 Open in new tabDownload slide Linearity graph of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity from LOQ to 200% level. Figure 7 Open in new tabDownload slide LC–MS/MS chromatogram at LOQ level of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 7 Open in new tabDownload slide LC–MS/MS chromatogram at LOQ level of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 8 Open in new tabDownload slide LC–MS/MS Chromatogram at 200% level of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Figure 8 Open in new tabDownload slide LC–MS/MS Chromatogram at 200% level of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity. Robustness The robustness of the method was evaluated by deliberately altering the flow rate and column temperature. Robustness results were found within the acceptance criteria and presented in Table VI. Table VI Robustness Results Chromatographic parameters . Retention time (min) . % RSD of standard . Check standard (similarity factor) . As such method 3.27 1.3 1.00 Flow rate 1,080 μL/min 3.51 1.6 1.01 Flow rate 1,320 μL/min 3.03 2.1 0.99 Column temp. 40°C 3.39 1.1 0.98 Column temp. 50°C 3.13 1.9 1.00 Chromatographic parameters . Retention time (min) . % RSD of standard . Check standard (similarity factor) . As such method 3.27 1.3 1.00 Flow rate 1,080 μL/min 3.51 1.6 1.01 Flow rate 1,320 μL/min 3.03 2.1 0.99 Column temp. 40°C 3.39 1.1 0.98 Column temp. 50°C 3.13 1.9 1.00 Open in new tab Table VI Robustness Results Chromatographic parameters . Retention time (min) . % RSD of standard . Check standard (similarity factor) . As such method 3.27 1.3 1.00 Flow rate 1,080 μL/min 3.51 1.6 1.01 Flow rate 1,320 μL/min 3.03 2.1 0.99 Column temp. 40°C 3.39 1.1 0.98 Column temp. 50°C 3.13 1.9 1.00 Chromatographic parameters . Retention time (min) . % RSD of standard . Check standard (similarity factor) . As such method 3.27 1.3 1.00 Flow rate 1,080 μL/min 3.51 1.6 1.01 Flow rate 1,320 μL/min 3.03 2.1 0.99 Column temp. 40°C 3.39 1.1 0.98 Column temp. 50°C 3.13 1.9 1.00 Open in new tab Solution stability Solution stability of standard and test sample solution was established and found to be stable for 2 days on bench-top (ambient temperature). Similarity factor for standard after 2 days was found to be 0.94 and % impurity difference for test sample from initial value was found to be 0.02. Discussion Literature survey shows that many methods have been reported for the determination of glycopyrrolate and its related impurities in drug substances and drug products by HPLC, HPTLC and LC–MS. In these methods assay, degradation path, diastereomeric purity and simultaneous estimation with other drugs has been discussed but determination of major degradation impurity of glycopyrrolate i.e., 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide has been not reported before. This study is based on the development and validation of method for quantitative estimation of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate oral solution by LC–MS/MS. 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide is lack of chromophore in its structure, hence it does not show any absorbance in UV–visible range. Therefore, the LC–MS/MS method is the most suitable method for estimation of this impurity at very low concentration level. Several trials were taken to optimize the mobile phase, flow rate, gradient programme, injection volume, column oven temperature and standard & test concentrations to achieve good peak shape and better retention and resolution of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity peak. The method was developed on X Bridge HILIC, 100 × 4.6 mm, 5 μm column with very short gradient programme of 5 min. The developed method was validated as per the current guidelines and found suitable. Conclusion The developed method was validated as per ICH and USP guidelines for specificity, precision, accuracy, LOD & LOQ, linearity and robustness over the range of 0.050–2.000 μg/mL and found meeting the acceptance criteria for all parameters. 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TI - A Selective and Sensitive Method Development and Validation of 1,1-Dimethyl-3-Hydroxy-Pyrrolidinium Bromide Impurity in Glycopyrrolate Oral Solution by Liquid Chromatography–Tandem Mass Spectroscopy
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmab003
DA - 2021-02-23
UR - https://www.deepdyve.com/lp/oxford-university-press/a-selective-and-sensitive-method-development-and-validation-of-1-1-SMnVpUFRmg
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -