Abstract
This invention describes a novel method encompassing preparing a nitrosamine sample. adding in relevant chemicals that reduces or eliminates in situ nitrosamine formation, utilizing full evaporation static headspace gas chromatograph coupled with nitrogen phosphorous detector (FE-SHSGC-NPD), and analysis of rapid results in the gaseous phase for sensitive detection of semi-volatile nitrosamines, including ND.MA.
Claims
1. A method for analyzing nitrosamines in a pharmaceutical sample wherein in situ nitrosamine formation is reduced or eliminated comprising: a) heating a pharmaceutical sample suspected of containing one or more nitrosamines in the presence of one or more nitrosating inhibitors, b) displacing the nitrosamines from the pharmaceutical sample into into a gaseous phase; and c) analyzing the nitrosamines in the gaseous phase.
2. The method according to claim 1 wherein the nitrosating inhibitor is selected from pyrogallol, phloroglucinol, pyrrole, 2,5-dimethylpyrrole, catechol, ascorbic acid, hydrazine, propylgallate, gallic acid, diphenylamine, and/or N-methyl aniline.
3. The method according to claim 2 wherein the nitrosating inhibitor is pyrogallol, phloroglucinol, pyrrole, 2,5-dimethylpyrrole, catechol, ascorbic acid, hydrazine, propylgallate, or gallic acid in combination with diphenylamine, or N-methyl aniline.
4. The method according to claim 3 wherein the nitrosating inhibitor is pyrogallol in combination with diphenylamine.
5. The method according to claim 1 wherein the nitrosating inhibitor is in a diluent comprising an acid and solvent wherein the acid is non-volatile and selected from the group consisting of phosphoric acid, sulfuric acid, methanesulfonic acid.
6. The method according to claim 5 wherein the solvent is selected from the group consisting of methanol, acetonitrile, acetone, ethanol, isopropanol, and 1-propanol or a mixture thereof.
7. The method according to claim 1 wherein the temperature of the pharmaceutical sample is heated to about 60? C. to about 200? C.
8. The method according to claim 1 wherein the nitrosamine analytes being analyzed are semi-volatile.
9. The method according to claim 5 wherein the ratio of diluent to the nitrosamine containing pharmaceutical sample is from 1:5 to 10:1.
10. The method according to claim 1 wherein the nitrosamines in the gaseous phase are separated and analyzed using gas chromatography (GC) coupled with a GC detector selected from the group consisting of mass spectrometer (MS), nitrogen-phosphorus detector (NPD), thermal energy analyzer (TEA), nitrogen chemiluminescence detector (NCD) or flame ionization detector (FID).
11. The method according to claim 1 wherein the method for analyzing nitrosamines is selected from liquid injection GC-NPD, headspace GC-NPD or full evaporation headspace GC with NPD (FE-HSGC-NPD).
12. The method according to claim 1 wherein the method for analyzing nitrosamines is full evaporation headspace GC with NPD.
13. A method for analyzing nitrosamine analytes in pharmaceutical samples wherein in situ nitrosamine formation is reduced or eliminated, comprising: heating a pharmaceutical sample suspected of containing one or more nitrosamine analytes in the presence of one or more nitrosating inhibitors selected from the group consisting of pyrogallol, phloroglucinol, pyrrole, 2,5-dimethylpyrrole, catechol, ascorbic acid, hydrazine, propylgallate, and/or gallic acid a) displacing the nitrosamine(s) analytes inte into a gaseous phase; and b) analyzing the nitrosamine analytes.
14. The method according to claim 13 wherein the nitrosating inhibitor is in a diluent comprising an acid and solvent wherein the acid is non-volatile and selected from the group consisting of phosphoric acid, sulfuric acid, methanesulfonic acid and the solvent is selected from the group consisting of methanol, acetonitrile, acetone, ethanol, isopropanol, and 1-propanol or a mixture thereof.
15. The method according to claim 13 wherein the temperature of the pharmaceutical sample is heated to about 60? C. to about 200? C.
16. The method according to claim 13 wherein the nitrosamine analytes being analyzed are semi-volatile.
17. The method according to claim 13 wherein the nitrosamine(s) in the gaseous phase are separated and analyzed using gas chromatorgraphy (GC) coupled with a GC detector selected from the group consisting of mass spectrometer (MS), nitrogen-phosphorus detector (NPD), thermal energy analyzer (TEA), nitrogen chemiluminescence detector (NCD) or flame ionization detector (FID).
18. The method according to claim 13 wherein the method for analyzing nitrosamines is selected from liquid injection GC-NPD, headspace GC-NPD or full evaporation headspace GC with NPD (FE-HSGC-NPD).
19. The method according to claim 13 wherein the method for analyzing nitrosamines is full evaporation headspace GC with NPD (FE-HSGC-NPD).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present invention and various aspects, features and advantages thereof are explained in detail below with reference to exemplary and therefore non-limiting embodiments, and with the aid of the drawings, which constitute a part of this specification and include depictions of the exemplary embodiments. In these drawings:
[0032] FIG. 1 shows a schematic illustration of FE-SHSGS-NPD method.
[0033] FIG. 2A shows a schematic comparison of traditional static headspace sampling (SHS).
[0034] FIG. 2B shows a full evaporation static headspace sampling (FE-SHS).
[0035] FIG. 3. Comparison of method sensitivity using static headspace sampling and full evaporation static headspace sampling.
[0036] FIG. 4 An analysis of method sensitivity and linearity shown by overlaid chromatograms of blank and NDMA standard solutions obtained using FE-HSGC-NPD.
[0037] FIG. 5 shows the inhibition of in situ formation of NDMA in metformin HCl drug substance with and without pyrogallol (PGL) during FE-SHSGC-NPD analysis.
[0038] FIG. 6. Analysis of common nitrosamines including NDMA, NDEA, NEIPA, NDIPA, NDBA, NMPA, and NMORP using FE-HSGC-NPD.
[0039] FIG. 7: Analysis of NDMA in metformin-sitagliptin instant release tablets
[0040] FIG. 8: Analysis of NDMA in metformin-sitagliptin extended release tablets
[0041] FIG. 9: Analysis of NDMA in metformin-ertugliflozen tablets
[0042] FIG. 10: Analysis of NDMA in valsartan drug substance
[0043] FIG. 11: Analysis of NDMA in ranitidine HCl drug substance
[0044] FIG. 12: Analysis of NDMA in ranitidine HCl drug substance
[0045] FIG. 13: Analysis of Fate and purge study of NDMA and NDEA in Compound A
[0046] FIG. 14: Shows the overlaid chromatograms for the analysis of NDMA and NDEA in Compound A
DETAILED DESCRIPTION OF THE INVENTION
Process Instrument Conditions
[0047] Traditional analytical techniques such as LC-UV or GC-FID do not provide the required sensitivity and/or specificity for analyzing nitrosamine analytes. Most methods utilize mass spectrometry detection (MS) detection to achieve the required sensitivity and selectivity. Although MS coupled with liquid chromatography (LC) or gas chromatography (GC) is capable of detecting nitrosamines at low ppb level, it is difficult to achieve the same sensitivity consistently and the throughput is often limited due to high instrument operating cost, frequent instrument maintenance and relatively complex data processing. Several other methodologies have been reported including thermal energy analyzer (TEA), nitrogen chemiluminescence detection (NCD), and nitrogen phosphorous detection (NPD). However, TEA or NCD is often limited by its sensitivity, while NPD is not as specific as TEA or NCD. Methods based on MS detection still dominates the nitrosamine testing despite the high cost and extensive maintenance.
[0048] This invention describes a method for analyzing nitrosamines in pharmaceutical samples using full evaporation headspace gas chromatography with a GC detector. An aspect of this invention is realized when the method used is full evaporation headspace gas chromatography with NPD as a GC detector. The method offers superior sensitivity and is suitable for the analysis of common nitrosamines, such as NDMA, NDEA, NEIPA, NDIPA, NDBA, NMPA, and NMORP in drug substances and drug products. Thus, an embodiment of this invention is a method for analyzing nitrosamines in pharmaceutical samples using full evaporation static headspace gas chromatograph with nitrogen-phosphorous detection (FE-SHSGC-NPD). A subembodiment of this aspect of the invention is realized when the method is used to analyze semi-volatile nitrosamine containing pharmaceutical samples as exemplified in FIG. 5.
[0049] Although this invention exemplifies a method for analyzing nitrosamines using nitrogen phosphorous detector (NPD) this method can be coupled with any GC detector including MS, TEA, NCD or FID to achieve the desired sensitivity and selectivity. Thus, an aspect of this invention includes GC detectors MS, TEA, NCD, NPD, or FID. The technique is simple, sensitive and fast, and can be easily implemented in a supply analytical laboratory. The simple sample preparation enables this method to be used for nitrosamines in different drug substances or drug products as is or with minor modifications.
Example Instrument Parameters
[0050] The instrument used for the analysis was Agilent 7890B GC system equipped with nitrogen-phosphorous detector, and Agilent 7697A headspace sampler.
[0051] Example GC parameters are shown below:
TABLE-US-00001 GC column: BD-Wax, 30 m ? 0.25 mm, 0.5 ?m film thickness or equivalent Inlet 200? C. Temperature: Split Ratio: 5:1 Carrier Gas: Helium Flow Rate: Constant flow at flow rate 3 mL/min Detector: Nitrogen Phosphorous Detector (NPD). Temperature: 330? C. Fuel flow (Hydrogen): 3 mL/min Oxidizer flow (Air): 60 mL/min Makeup gas: Nitrogen, constant makeup at 5 mL/min Recommended Offset: 20 pA Oven Hold 60? C. for 1.5 min, ramp at 20? C./min to 150? C., Temperature then 40? C./min to 240? C. and hold for 3 min Program: Total run 11.25 min time:
[0052] Example Headspace parameters are shown below:
TABLE-US-00002 Vial size: 10 mL Vial oven 115? C. temperature: Injection volume 1 mL (Sample Loop): Loop 160? C. temperature: Transfer line 170? C. temperature: Vial shaking: high GC cycle time: 14 min Vial equilibration 15 min time: Pressure 0.1 min equilibration time: Injection time: 0.5 min Vial fill mode: Fill to pressure at 30 psi
Example 1
[0053] Shown in FIG. 1 is a schematic illustration of this invention. A solid sample (e.g. tablet) is ground into fine powder. A portion of the powder (e.g. 20-100 mg) is weighed into a headspace vial. A small volume of volatile solvent containing nitrosation inhibitor (e.g. 50 ?L 20 mg/mL pyrogallo+0.1% phosphoric acid in IPA) is added. The headspace vial is sealed, and is subjected to headspace extraction at high temperature for extended period (e.g. 115? C. for 15 min). An aliquot of the headspace is injected into a GC column (e.g. DB-Wax) to separate nitrosamine from other volatile components, followed by a sensitive detection using NPD. The nitrosamine in the solid sample is quantitated using an external standard. As shown in FIG. 1, high sensitivity (e.g. below 1 ppb NDMA) can be achieved as the solvent is fully evaporated and there is no headspace-liquid phase partition. Thus, an aspect of this invention is realized by a method for analyzing nitrosamine analytes in pharmaceutical samples that reduces or eliminates in situ nitrosamine formation with a sensitivity level below 1 part per billion nitrosamines. The in situ formation of nitrosamine is minimized with the addition of a nitrosating inhibitor.
Example 2
[0054] FIG. 2 shows a schematic comparison of traditional static headspace sampling (SHS: Sample A) and full evaporation static headspace sampling (FE-SHS: Sample B). In traditional SHS, the analyte of interest (e.g. nitrosamine) mainly stays in the liquid phase due to its high partition coefficient, resulting in low sensitivity. In FE-SHS, analyte of interest and the solvent is fully evaporated into headspace while the sample matrix is not volatized. Without the undesirable partition, better sensitivity can be achieved even though a smaller amount of sample is used.
Example 3
[0055] The experimental results shown in FIG. 3 illustrates the sensitivity achieved with static headspace sampling and full evaporation static headspace sampling. For static headspace sampling, 1 mL of 2 ng/mL NDMA in DMSO was added to a 10 mL headspace vial, which corresponds to 20 ppb NDMA with respect to 1 mL of 100 mg/ml sample. The vial is heated at 115? C. for 15 min. The injection volume is 1 mL and the split ratio is 5:1. For FE-SHS, 50 ?L of 20 ng/mL NDMA in diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA was added to a 10 mL headspace vial, which corresponds to 20 ppb with respect to 50 mg sample. The vial is heated at 115? C. for 15 min, and 1 mL headspace is injected with a split ratio of 5:1. The sensitivity with FE-SHS is 38 times higher than that with SHS. The improved sensitivity in FE-SHS can be attributed to the elimination of headspace-liquid partition. For the data shown in FIG. 3, DMSO was used as diluent as it provided high solubility for drug substances such as valsartan or metformin HCl, and had a high boiling point, a preferred property for SHS. However, the injection volume for SHS is limited due to phase soaking effect of condensed DMSO solvent. There is no such limitation for FE-HSGC as IPA had a low boiling point and did not affect the chromatographic behavior of NDMA. As a result, the injection volume can be easily increased for FE-SHS (e.g. from 1 mL to 5 mL) to further improve the sensitivity.
Example 4: Method Sensitivity and Linearity
[0056] FIG. 4 shows the overlaid chromatograms of blank and NDMA standard solutions obtained using FE-HSGC-NPD. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at constant flow rate at 3 mL/min. The oven temperature was held at 60? C. for 1.5 min, then ramped up at 20? C./min to 150? C. then 40? C./min to 240? C., and held at 240? C. for 3 min. The diluent contained 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. For each solution, 50 ?l was pipetted to a 10-mL headspace vial, capped and crimped tightly. The vial was heated at 115? C. for 15 min, and 1-mL headspace was injected with a split ratio of 5:1. The NDMA concentrations range from 0.1 to 20 ng/ml, corresponding to 0.1 ppb to 20 ppb relative to 50 mg metformin HCl drug substance. The quantitation limit was estimated to be 0.25 ppb based on a signal to noise ratio of 10:1, and the detection limit was estimated to be 0.10 ppb based on a signal to noise ratio of 3:1. The sensitivity obtained using this method meets the regulatory requirement, e.g. 4.8 ppb NDMA in metformin HCl based on a maximum daily dose of 2 g.
Example 5: Inhibition of In Situ Formation of NDMA in Metformin HCl by FE-HSGC-NPD
[0057] FIG. 5 shows the inhibition of in situ formation of NDMA in metformin HCl drug substance during FE-HSGC-NPD analysis. A DB-Wax column (30 m?0.25 mm, 0.5 um film thickness) was used. The carrier gas was helium at constant flow rate at 3 mL/min. The oven temperature was held at 50? C. for 1.5 min, then ramped up at 20? C./min to 150? C. then 40? C./min to 240? C., and held at 240? C. for 1 min. About 30 mg metformin HCl drug substance was added to a 10-mL headspace vial, along with 50 ?l diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The vial was heated at 115? C. for 15 min, and 1-mL headspace was injected with a split ratio of 5:1. Several nitrosation inhibitors have been evaluated including pyrrole, 2,5-dimethylpyrrole, pyrogallol, phloroglucinol, catechol, ascorbic acid, hydrazine, propylgallate and gallic acid. Exemplified in FIG. 5 is the combination of pyrogallol and phosphoric acid in isopropanol solvent, which provided the best inhibition effect. When IPA diluent is used, about 1000 ppb was detected in a metformin HCl material in which no NDMA was detected using a validated LC/MS method (<10 ppb). The NDMA level decreased to 28 ppb with the addition of 20 mg/mL pyrogallol in IPA, and to 0.7 ppb with the addition of both 20 mg pyrogallol and 0.1% phosphoric acid in IPA. Therefore, the risk of false positive due to in situ NDMA formation is deemed negligible (i.e., at least 99.93% of the in situ nitrosation reaction was inhibited) It was unexpected that pyrogallol inhibited the nitrosation more effectively in the presence of phosphoric acid as nitrosation is believed to be catalyzed by acid. One explanation is that under acidic conditions (pH is ?2.5 for 0.1% phosphoric acid), dimethylamine exists as protonated base, resulting in slow nitrosation while pyrogallol is more effective as a nitrosation inhibitor. One additional benefit of phosphoric acid is that the amine impurities in the sample matrix are protonated and become non-volatile, and do not contribute to the interference in GC separation.
Example 6: Analysis of Common Nitrosamines
[0058] FIG. 6 shows analysis of common nitrosamines including NDMA, NDEA, NEIPA, NDIPA, NDBA, NMPA, and NMORP using FE-HSGC-NPD and further demonstrates the unversality of this method. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at constant flow rate at 2 mL/min. The oven temperature was held at 70? C. for 1 min, then increased at 25? C./min to 240? C., and held at 240? C. for 1 min. The diluent contained 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The standard solution contained 500 ng/ml each of NDMA, NDEA, NEIPA, NDIPA, NDBA, NMPA, and NMORP, and 40 ?l was added to a 20-mL headspace vial, capped and crimped tightly. The vial was heated at 150? C. for 30 min, and 1-mL headspace is injected with a split ratio of 20:1.
Example 7: Analysis of NDMA in Metformin-Sitagliptin Instant Release Tablets
[0059] FIG. 7 shows the analysis of NDMA in metformin-sitagliptin instant release tablets. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at a constant flow rate of 3 mL/min. The oven temperature esd held at 60? C. for 1.5 min, then increased by 20? C./min to 150? C., followed by 40? C./min to 240? C., and finally held at 240? C. for 3 min. Ground tablet powder equivalent to ?21 mg metformin HCl drug substance was added to a 10 mL headspace vial, along with 50 ?L diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The vial was heated at 115? C. for 15 min, and 1 mL headspace was injected with a split ratio of 5:1. The quantitation limit for NDMA is 4.8 ppb, corresponding to 10% of the acceptable intake limit of 96 ng/day and maximum daily dose of 2 g.
Example 8: Analysis of NDMA in Metformin-Sitagliptin Extended Release Tablets
[0060] FIG. 8 shows the analysis of NDMA in metformin-sitagliptin extended release tablets. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at a constant flow rate of 3 mL/min. The oven temperature was held at 60? C. for 1.5 min, then increased by 20? C./min to 150? C., followed by 40? C./min to 240? C., and finally held at 240? C. for 3 min. Ground tablet powder equivalent to ?21 mg metformin HCl drug substance was added to a 10 mL headspace vial, along with 50 ?L diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The vial was heated at 115? C. for 15 min, and 1 mL headspace was injected with a split ratio of 5:1. The quantitation limit for NDMA is 4.8 ppb, corresponding to 10% of the acceptable intake limit of 96 ng/day and maximum daily dose of 2 g.
Example 9: Analysis of NDMA in Metformin-Ertugliflozen Release Tablets
[0061] FIG. 9 shows the analysis of NDMA in metformin-ertugliflozen release tablets. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at aconstant flow rate of 3 mL/min. The oven temperature was held at 60? C. for 1.5 min, then increased 20? C./min to 150? C., followed by 40? C./min to 240? C., and finally held at 240? C. for 3 min. Ground tablet powder equivalent to ?21 mg metformin HCl drug substance was added to a 10 mL headspace vial, along with 50 ?L diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The vial was heated at 115? C. for 15 min, and 1 mL headspace was injected with a split ratio of 5:1. The quantitation limit for NDMA is 4.8 ppb, corresponding to 10% of the acceptable intake limit of 96 ng/day and maximum daily dose of 2 g.
Example 10: Analysis of NDMA in Valsartan Drug Substance
[0062] FIG. 10 shows the analysis of NDMA in valsartan drug substance. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) was used. The carrier gas was helium at a constant flow rate at 3 mL/min. The oven temperature was held at 60? C. for 1.5 min, then increased by 20? C./min to 150? C., followed by 40? C./min to 240? C., and finally held at 240? C. for 3 min. Approximately 50 mg valsartan drug substance was added to a 10 mL headspace vial, along with 50 ?L diluent containing 20 mg/mL pyrogallol and 0.1% phosphoric acid in IPA. The vial was heated at 115? C. for 15 min, and 1 mL headspace was injected with a split ratio of 5:1. The quantitation limit for NDMA is 2 ppb with respect to valsartan, which is below 10% of acceptable intake limit of 30 ppb.
Example 11: Analysis of NDMA in Ranitidine Hydrochloride Drug Substance
[0063] FIG. 11 shows the analysis of NDMA in ranitidine hydrochloride drug substance. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) is used. The carrier gas is helium at constant flow rate at 3 mL/min. The oven temperature is held at 60? C. for 1.5 min, then ramped by 30? C./min to 240? C., and finally held at 240? C. for 2 min. For sample preparation, 50 mg/mL ranitidine HCl was dissolved in a diluent containing 100 mg/mL pyrogallol. 20 mg/mL diphenylamine and 0.1% phosphoric acid in methanol. For GC analysis, 40 ?L sample solution was transferred into a 20-mL head-space vial. The vial is equilibrated at 100? C. for 20 min, and 1 mL headspace is injected with a split ratio of 5:1. The quantitation limit for NDMA is 32 ppb with respect to ranitidine HCl, which is 10% of the acceptable intake limit of 96 ng/day and maximum daily dose of 300 mg.
Example 12: Analysis of NDMA in Ranitidine Hydrochloride Drug Substance
[0064] FIG. 12 shows an analysis of NDMA in ranitidine hydrochloride drug substance. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) is used. The carrier gas is helium at constant flow rate at 3 mL/min. The oven temperature is held at 60? C. for 1.5 min, then ramped by 30? C./min to 240? C., and finally held at 240? C. for 2 min. For sample preparation, 50 mg/mL ranitidine HCl was dissolved in a diluent containing 100 mg/mL pyrogallol, 20 mg/mL diphenylamine and 0.1% phosphoric acid in methanol. For GC analysis, 40 ?L sample solution was transferred into a 20-mL head-space vial. The vial is equilibrated at 100? C. for 20 min, and 1 mL headspace is injected with a split ratio of 5:1. The quantitation limit for NDMA is 32 ppb with respect to ranitidine HCl, which is 10% of the acceptable intake limit of 96 ng/day and maximum daily dose of 300 mg.
Example 13: Fate and Purge Study of NDMA and NDEA in Compound A
[0065] FIG. 13 shows the overlaid chromatograms for the analysis of NDMA and NDEA in Compound A and its intermediate to understand the fate and purge of NDMA and NDEA in the synthesis process. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) is used. The carrier gas is helium at constant flow rate at 2 mL/min. The oven temperature is held at 55? C. for 1 min, then ramped by 25? C./min to 240? C., and finally held at 240? C. for 4 min. For sample preparation, 50 mg/mL Compound A or its intermediate was dissolved in a diluent containing 20 mg/mL pyrogallol in methanol. For GC analysis, 50 ?L sample solution was transferred into a 20-mL head-space vial. The vial is equilibrated at 125? C. for 15 min, and 1 mL headspace is injected with a split ratio of 5:1. The quantitation limit is 6 ppb for NDMA and 2 ppb for NDEA with respect to 50 mg Compound A or its intermediate, which is 10% of the acceptable intake limit of 96 ng/day NDMA and 26.5 ng/day NDEA and maximum daily dose of 1600 mg.
Example 14: Testing for NDMA and NDEA in Compound A Drug Product
[0066] FIG. 14 shows the overlaid chromatograms for the analysis of NDMA and NDEA in drug product with Compound A. A DB-Wax column (30 m?0.25 mm, 0.5 ?m film thickness) is used. The carrier gas is helium at constant flow rate at 2 mL/min. The oven temperature is held at 55? C. for 1 min, then ramped by 25? C./min to 240? C., and finally held at 240? C. for 4 min. For sample preparation, 50 mg/mL Compound A or its intermediate was dissolved in a diluent containing 20 mg/mL pyrogallol in methanol. For GC analysis, 50 ?L sample solution was transferred into a 20-mL head-space vial. The vial is equilibrated at 125? C. for 15 min, and 1 mL headspace is injected with a split ratio of 5:1. The quantitation limit is 6 ppb for NDMA and 2 ppb for NDEA with respect to 50 mg Compound A or its intermediate, which is 10% of the acceptable intake limit of 96 ng/day NDMA and 26.5 ng/day NDEA and maximum daily dose of 1600 mg.
