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@#Gas chromatography-mass spectrometry (GC-MS) method was established for trace analysis of the potential genotoxic impurity chlorocyclohexane in trihexyphenidyl hydrochloride bulk drug, utilizing an RXI-5SIL MS column at isothermal temperature of 60 °C for the entire 6-minute run time.The inlet temperature was 180 °C and a split ratio of 10∶1 was used with the injection volume of 1.0 μL.The selective ion monitoring mode was set at m/z 82 for chlorocyclohexane with a detector voltage of 0.3 kV and an ion source temperature of 240 °C.The method was verified with respect to specificity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision and robustness.Good linear correlation was achieved with coefficient r of 0.999 9 in the concentration range of 59.72-493 ng/mL.The intra- and inter-day precision was satisfactory (RSD ≤ 5.0%) and robust (RSD ≤ 1.65%).The proposed method in this study can be adequately adopted as a tool for quality assurance of trihexyphenidyl hydrochloride in routine test of potential genotoxic impurity.
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@#In this paper, chemical derivatization-high performance liquid chromatography was used to determine the potential genotoxic impurities chloroacetyl chloride and chloroacetic acid, respectively, in the raw material of azintamide.Derivatization was carried out using 2-nitrophenylhydrazine followed by the determination.Separation was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5 μm), with mobile phase consisting of 0.1% phosphoric acid in water (A) and acetonitrile(B) by gradient elution, at a flow rate of 1 mL/min.The column temperature was 40 °C and the detection wavelength was 226 nm.The blank solvent, derivatization reagent, and azintamide did not interfere with the peak of the test substance, and the target component was well separated from the others.For impurities chloroacetyl chloride and chloroacetic acid, the limits of detection (LOD) were 7.5 ng/mL and 15 ng/mL respectively. There was a good linear relationship between the integral area and the concentration in the range of 30-300 ng/mL.The sample recovery rate was in the range of 87.37% ~ 109.75%.The two methods established in this study have good specificity, good precision, high sensitivity and simple operation, which can be used for the trace determination of potential genotoxic impurities chloroacetyl chloride and chloroacetic acid in the raw material of azintamide.
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@#A UPLC-MS/MS method was established for the determination of the genotoxic impurity (R)-5-(azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinone in linezolid API and its glucose injection. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) with 0.1% formic acid water-0.1% formic acid acetonitrile (60∶40) at a flow rate of 0.3 mL/min. The UPLC-MS/MS was equipped with electrospray ionization in positive ionization mode and multiple reaction monitoring mode. The results showed that the calibration curve was linear in the range of 4-12 ng/mL and the limit of quantification was 0.073 ng/mL.The average recoveries of the low, medium and high concentration (80%,100%,120% limit concentration) loading solutions were 101.14%, 100.59% and 101.47%, respectively (RSDs:0.73%, 1.10% and 0.91%, respectively).The sample solution was stable for 6 d.No genotoxic impurity of (R)-5-(Azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinonewas not detected in the samples of linezolid API and its glucose injection.
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An UPLC-MS/MS method was established for the quantification of the genotoxic impurities bis(2-chloroethyl)amine hydrochloride and 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride in trazodone hydrochloride. The chromatographic separation of the two genotoxic impurities was performed on Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) at 20 ℃. A mixture of 5 mmol·L-1 ammonium hydrogen carbonate aqueous solution and acetonitrile at a flow rate of 0.3 mL·min-1 in gradient elution mode was employed as mobile phase. The UPLC-MS/MS was equipped with electrospray ionization in positive ionization mode and adopted multiple reaction monitoring mode. We found that the calibration curves of the two genotoxic impurities were linear in the range of 0.1-10 ng·mL-1. The limit of detection was 0.10 ng·mL-1 for bis(2-chloroethyl)amine hydrochloride and the average recovery was 101.53% (RSD = 4.06%). The limit of detection was 0.01 ng·mL-1 for 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride and the average recovery was 97.95% (RSD = 1.27%). The sample solution was stable for 24 h. No bis(2-chloroethyl)amine hydrochloride was detected in the samples, and the content of 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride in the samples was within the limit. This research provides a method to improve the quality control standards of trazadone.
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OBJECTIVE：To establish a method for the content determination of potential genotoxic impurity maleic hydrazide in azintamide raw material. METHODS ：HPLC-FLD method was adopted. The determination was performed on Thermo Syncronis C18 column with mobile phase consisted of 0.2 mol/L acetic acid-methanol （gradient elution ）. The column temperature was set at 30 ℃，the excitation wavelength was 315 nm and emission wavelength was 389 nm. The flow rate was 1 mL/min，and the sample size was 20 μL. RESULTS：The blank solvent and azintamide did not interfere with the determination of maleic hydrazide. The linear range of maleic hydrazide was 19.5-300 ng/mL（r＝0.999 9）. The limit of detection was 4.5 ng/mL and the limit of quantification was 19.5 ng/mL. The recovery ranged from 98.79％ to 103.76％（RSDs were lower than 3.00％，n＝9）. RSDs of precision and stability （24 h）tests were no more than 5.63％，and those of durability tests were less than 2.00％（n＝6）. Maleic hydrazide was not detected in 3 batches of azinamide raw material. CONCLUSIONS ：The method is specific ，sensitive and accurate. It can be used for the trace determination of maleic hydrazide in azintamide or other matrix.
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@#To establish a method for the determination of formaldehyde and glyoxal simultaneously in varenicline tartrate active pharmaceutical ingredient (API) and its intermediate, formaldehyde and glyoxal were derivatized by 2, 4-dinitrophenylhydrazine (2,4-DNPH) to improve the HPLC retention and UV detection sensitivity. Separation was performed on a C8 (150 mm × 4.6 mm, 5 μm) column by linear gradient elution using acetonitrile and water as the mobile phase; the detective wavelength was set at 380 nm.Formaldehyde and glyoxal were quantitatively determined by an external reference method.Linear calibration was established for both formaldehyde and glyoxal in the range from 0.094 to 1.88 μg/mL.The detection and the quantification limits were 0.047 μg/mL (19 μg/g) and 0.094 μg/mL (38 μg/g), respectively.The recoveries were (95.0±1.1)% and (99.4 ± 2.6)% for formaldehyde and glyoxal, respectively.This method has been fully validated to be applicable to quantitative analysis of trace amount of formaldehyde and glyoxal in varenicline tartrate API and its intermediate.Test results demonstrated that the contents of both formaldehyde and glyoxal met the permitted daily exposure (PDE) limits for the finished products of varenicline tartrate API as well as its intermediate, though the glyoxal contents in the crude intermediates were likely to exceed the limit.The established method is valuable for the manufacturing process and quality control of varenicline tartrate.
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OBJECTIVE: To establish a method to determine the genotoxic impurity, N-nitroso-N-methyl-4-aminobytyric acid, in losartan potassium using high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-MS/MS). METHODS: The method was developed by using Shimadzu Shim-pack XR-ODS II column (2.0 mm×150 mm, 2.2 μm). Time program was conducted with mobile phase consisting of water (0.1% formic acid, A) and methanol (B). The flow rate was 0.3 mL•min-1, and the column oven temperature was maintained at 40 ℃. The samples were ionized by electrospray ionization (ESI) with multi reaction monitoring (MRM) data acquisition mode. The collision energies were -11, -13, and -13 V, CID gas was argon with pressure of 270 kPa.3 pairs of precursor, and product ions (m/z) of NMBA were 147.15→117.10, 147.15→87.10, and 147.15→44.10, respectively. RESULTS: The genotoxic impurity NMBA showed linearity between 1 and 100 ng•mL-1 with correlation coefficient of 0.999 9. The intra-day and inter-day repeatability was examined by relative standard deviations (RSDs) of retention time and peak area (RSD<1.10%, n=6 for intra-day repeatability and n=18 for inter-day repeatability). The accuracy was examined by percent recovery at three concentration levels, and the average percent recovery was between 94.40% and 98.04%. CONCLUSION: The established LC-MS/MS method is efficient for limit test and quantitation of NMBA in losartan potassium bulk drug.
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Objective:To establish a method for the determination of the potential genotoxic impurities [ methyl chloroacetate and ( R, S) 4-ethyl chloro-3-hydroxybutyrate ] in oxiracetam raw material .Methods:GC-MS was conducted , and the potential genotoxic impurities were extracted by ethyl acetate .The column was a VF-1701 ms capillary column (30 m ×0.25 mm, 0.25 μm) with pro-grammed temperature and the inlet temperature was 220 ℃.The column flow was 1.0 ml· min-1 and the purge flow was 5.0 ml· min-1 .The split injection was used and the split ratio was 5:1 .The carrier gas was high purity helium and the detector was a mass spectrometer detector .The ion source temperature was 230℃and the interface temperature was 230 ℃.The delay time of solvent was 4 min and the ionization mode was electron impact .The scanning ( detection ) method was selective ion monitoring , the electron energy was 70 eV, and the injection volume was 1.0 μl.Results:The separation between the impurities met the requirements .The concen-tration linear range was 50-400 ng· ml-1 (r≥0.9995).The recoveries were 89.7％-96.3％ (RSD=2.3％, n=9) and 91.0％-105.3％(RSD=4.4％, n=9), respectively.Conclusion:The method is simple, accurate, sensitive and rapid, and can be used for the determination of two potential genotoxic impurities in oxiracetam raw material .
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Objective: To establish an LC-MS/MS method for the determination of 4-( 4-amino-3-fluorophenoxy )-N-methylpyri-dine-2-carboxamide ( AFP-PMA) as a genotoxic impurity in regorafenib. Methods: The content of AFP-PMA was determined by an LC-MS/MS method. A Waters XBridge Shield RP18 column was adopted to separate the samples and the column temperature was 50℃. The mobile phase consisted of 5 mmol·L-1ammonium acetate aqueous (A)-acetonitrile (B) with gradient elution (0~9 min, 5%B→90%B) at a flow rate of 1. 0 ml·min-1. An electrospray ionization source (ESI) was used in a positive-ion and multiple reactions monitoring mode. The ion channel was m/z 262. 2→244. 1. Results:The standard curve was linear within the range of 2. 41-980. 90 ng·ml-1(r=0. 9998) and the limit of quantification was 8. 02 ng·ml-1. The limit of detection was 2. 41 ng·ml-1, which was e-quivalent to 0.000241% for the concentration of regorafenib. The average recovery was 100.95% and RSD was 2.37% (n=9). Conclusion:The method has good specificity, promising accuracy and high sensitivity, which can be used for determining the trace genotoxic impurity AFP-PMA in regorafenib.
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Objective:To establish a method for the determination of 4-hydrazinobenzenesulfonamide hydrochloride in celecoxib. Methods:HPLC was used and the chromatographic column was a Hypercarb series C18 column (150 mm × 4. 6 mm, 3 μm) with 100% porous graphite as the filler. The mobile phase consisted of 10 mmol·L-1 ammonium acetate aqueous solution-methanol (50 :50) and methanol. The flow rate was 0. 8 ml·min-1 and the detection wavelength was 220 nm. The column temperature was 35 ℃and the injection volume was 20 μl. Results:The linear range was 99. 897-599. 382 ng·ml-1(r=0. 9982) for 4-hydrazinobenzene-sulfonamide hydrochloride. The average recovery was 97. 4%(RSD=3. 0%,n=9). Conclusion:The method is simple and accurate, and can be used for the determination of 4-hydrazinobenzenesulfonamide hydrochloride in celecoxib.