Journal of Chromatographic Science Advance Access published July 24, 2014 Journal of Chromatographic Science 2014;1– 11 doi:10.1093/chromsci/bmu084
Article
Stability-Indicating Liquid Chromatographic Method for Determination of Saxagliptin and Structure Elucidation of the Major Degradation Products Using LC – MS Maha F. Abdel-Ghany, Omar Abdel-Aziz, Miriam F. Ayad and Mariam M. Tadros* Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt *Author to whom correspondence should be addressed. Email: [email protected] Received 18 December 2013; revised 22 June 2014
A new, simple, selective, reproducible and sensitive stability-indicating liquid chromatographic method was developed and subsequently validated for the determination of saxagliptin (SXG). SXG was subjected to oxidation, thermal, acid hydrolysis, alkali hydrolysis and photodegradation according to ICH guidelines. The major degradation products were separated from the pure drug and the proposed structures’ elucidation was performed, using an LC–MS technique. Isocratic chromatographic elution was achieved on a Symmetryw C18 column (150 3 4.6 mm, 5 mm), using a mobile phase of potassium dihydrogen phosphate buffer (pH 4.6)–acetonitrile–methanol (40 : 30 : 30, v/v/v) at a flow rate of 1 mL min21 with UV detection at 208 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 25 – 400 mg mL21. All the results were statistically compared with the reference method, using one-way analysis of variance. The developed method was validated and proved to be specific and accurate for quality control of SXG in pharmaceutical dosage form.
Introduction Saxagliptin (SXG), (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1adamantyl)acetyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile (Figure 1), is a new oral hypoglycemic drug of new dipeptidyl peptidase-4 (DPP-4) inhibitor (1). SXG is recently approved for treatment of type-II diabetes mellitus (2). DPP-4 inhibitors represent a new therapeutic approach to the treatment of type-II diabetes that functions to stimulate glucose-dependent insulin release and reduce glucagon levels. This is done through inhibition of incretions0 inactivation, particularly glucagon-like peptide-1 and gastric inhibitory polypeptide, thereby improving glycemic control (3). Literature survey reveals that the drug has been estimated by the LC–MS-MS (4), HPLC methods (5 –8) and by the spectrophotometric method, in which SXG was estimated at 208 nm in methanol (9). Another spectrophotometric method based on charge-transfer reaction using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and 7,7,8,8-tetracyanoquinodimethane (10). The aim of our work is to present a new selective and sensitive stability-indicating liquid chromatographic method for the determination of SXG, where it was subjected to oxidation, thermal, acid and alkali hydrolysis and photodegradation according to ICH forced degradation guidelines (11). Optimization of the chromatographic conditions was of great importance to ensure complete separation of the degradation products from SXG, including mobile phase, column type, detection wavelength and flow rate. The major degradation products were separated from the pure drug, and the proposed structures0 elucidation was performed using the LC–MS technique in the case of the acid and alkaline hydrolysis.
Experimental Instrumentation The HPLC system consisted of a Shimadzu LC-20 AT Liquid Chromatograph (Japan), using a Symmetryw C18 column (150 4.6 mm, 5 mm) (Ireland). The system was equipped with a UV– visible detector (SPD-20A, Japan) and an autosampler (SIL-20A, Shimadzu, Japan). An Elma S100 ultrasonic processor model KBK 4200 (Germany) was used for degassing of the mobile phases.
Reagents and reference samples Pure SXG samples certified to contain 99.85% and Onglyzaw tablets (batch number 0J57932) nominally containing 5 mg of SXG per tablet were supplied by Bristol-Myers Squibb/ AstraZeneca EEIG (UK). Methanol (HiPerSolv for HPLC) and acetonitrile (HiPerSolv) were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Potassium dihydrogen phosphate and orthophosphoric acid (85%) were obtained from VWR Chemicals (Pool, UK). Bi-distilled water was produced in-house (A4000D; Aquatron Water Still, UK). Membrane filters 0.45 mm from Teknokroma (Barcelona, Spain) were used. All other chemicals and reagents used were of analytical chromatographic grade.
Chromatographic conditions Isocratic chromatographic elution was achieved at ambient temperature on a Symmetryw C18 column (150 4.6 mm, 5 mm), using potassium dihydrogen phosphate buffer ( pH 4.6) –acetonitrile – methanol (40 : 30 : 30, v/v/v) as a mobile phase, with UV detection at 208 nm. The buffer solution was filtered through a 0.45-mm membrane filter and degassed for 30 min in an ultrasonic bath prior to use. The mobile phase was pumped through the column at a flow rate of 1 mL min21 with the injection volume of 20 mL.
Stock standard solution preparation Stock standard solutions of SXG (1 mg mL21) was prepared by dissolving 100 mg of the drug in methanol, sonicated and completed to volume in a 100-mL volumetric flask. The required concentrations were prepared by serial dilutions.
Samples’ preparation Forty tablets of SXG were weighed, and the coats were removed by carefully rubbing with a clean tissue wetted with methanol.
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
An accurately weighed amount of finely powdered tablets equivalent to 100 mg of SXG was made up to 100 mL with methanol and then sonicated for 15 min.
Procedure Linearity (calibration curve for SXG) Accurately measured aliquots (0.25 –4 mL) of stock standard solution equivalent to 250 – 4,000 mg were separately transferred into a series of 10-mL volumetric flasks and then completed to volume with methanol. A volume of 20 mL of each solution was injected, and the mentioned chromatographic conditions were adapted. The calibration curve was obtained by plotting area under the peaks (AUP) against concentrations (C).
Table I System Suitability Tests for the LC –UV Method for the Determination of SXG in Bulk
Figure 1. SXG structure.
Item
SXG
N T R Resolution between peaks in the case of alkaline degradation Resolution between peaks in the case of acidic degradation RSD% of six injections Peak area Retention time
871 1.03 4.7 5.2 0.21 0.17
N, number of theoretical plates; T, tailing factor; R, resolution; RSD, relative standard deviation.
Table II Results Obtained for the LC –UV Method for the Determination of SXG in Bulk Item
SXG
Retention time (min) Wavelength of detection Range of linearity (mg mL21) Regression equation Regression coefficient (r) LOD (mg mL21) LOQ (mg mL21) Sb (standard deviation of the intercept) Sa (standard deviation of the slope) Confidence limit of the slope Confidence limit of the intercept Standard error of the estimation Precision Intraday, %RSD Interday, %RSD Drug in bulk (mean + standard deviation) Drug in dosage form (mean + standard deviation) Drug added (mean + standard deviation)
2.7 208 nm 25– 400 Area 1026 ¼ 0.1478 C mg mL21 þ 0.3232 0.9999 7.96 24.13 9.67 1024 0.22 0.1478 + 0.033 0.3232 + 3.12 1024 0.32 0.14 –0.23 0.23 –0.38 100.28 + 1.44 100.21 + 1.00 100.20 + 0.86
Table III Standard Addition Method for Determination of SXG in Dosage Form Amount of SXG taken (mg/mL)
Amount of SXG found (mg/mL)
Tablet
Added
Tablet
Tablet and added
Added
Tablet
Added
50 75 100 125 150
350 275 200 125 50
49.48 75.50 99.33 126.45 151.39
404.66 352.10 298.73 250.54 201.36
355.18 276.61 199.40 124.09 49.97
98.96 100.67 99.33 101.16 100.93
101.48 100.59 99.70 99.27 99.94
100.21 1.00 0.45
100.20 0.86 0.39
Mean SD (+) SE (+)
Figure 2. LC chromatogram of SXG (333.3 mg mL21) using UV detection at 208 nm.
2 Abdel-Ghany et al.
SD, standard deviation; SE, standard error.
Recovery %
Assay of drug in bulk (accuracy) The mentioned procedure under ‘Linearity (calibration curve for SXG)’ was repeated using 0.75 – 2.75 mL of stock standard solution equivalent to 75 – 275 mg mL21, and the concentrations of SXG were calculated by the corresponding calibration equation. Assay of drugs in pharmaceutical dosage forms For determination of SXG in tablets, from the sample solution prepared (as discussed in ‘Samples’ preparation’), aliquots (0.5 – 1.5 mL) equivalent to 50 – 150 mg mL21 were injected in triplicates and the concentrations of SXG were calculated by the corresponding calibration equation. Further, to check the validity of the proposed method, the standard addition technique was applied by adding different known concentrations of the pure drug to different known concentrations of the drug product. Forced degradation of SXG Forced degradation study of SXG included solid-state (thermal and photodegradation) and solution-state (acid, alkali and H2O2) stress conditions, which were performed according to ICH guidelines (11). The stock solution was used for forced degradation study, and the total chromatographic run time was about four times the retention of the drug peak to provide an indication of the stability-indicating property of the method. For study in
the neutral condition, the drug was held in methanol (0.5 mg mL21) in dark at room temperature for 24 h as a protected sample from all the stress conditions under investigation to be used to deduce the accurate amount of the degradation products comparable with SXG.
Preparation of the acid- and base-degraded sample for LC –MS investigation SXG (100 mg) was dissolved in 100 mL of chloroform : methanol (1 : 1), using sonication for 30 min. Then, the procedures, mentioned under ‘Acid-induced forced degradation of SXG for HPLC investigation’ and ‘Base-induced forced degradation of SXG’ sections were repeated. The organic layer was separated, and the aqueous layer was washed with chloroform (5 10 mL). The organic layer was investigated by LC–MS and each of (expected degradation pathways and LC – MS charts) as shown in Figures 5 and 7 –12. Acid-induced forced degradation of SXG for HPLC investigation To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 0.5, 2.5 and 5 mL of 2 M HCl were added and each mixture was completed to mark with distilled water to reach molarities of 0.1, 0.5 and 1 M HCl,
Figure 3. LC chromatograms of SXG in the presence of its acid-degradation product. (A). LC chromatogram of SXG (a) and its acid-degradation product (b) after heating with 0.5 M HCl. (B) LC chromatogram of SXG (a) and its acid-degradation product (b) after heating with 1 M HCl.
Determination of SXG and Structure Elucidation of the Major Degradation Products 3
respectively. The mixtures were kept at room temperature for 8 h in dark and then neutralized with NaOH, having the same molarity of the used HCl, using a pH meter to ensure that pH is 7 before investigation. These experiments were repeated at a
higher temperature of 758C for 0.5 h in dark. Twenty microliters of the degraded solutions were injected in triplicates to HPLC, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Figure 4. LC chromatograms of SXG in the presence of its alkaline degradation products. (A) LC chromatogram of SXG (a) and its alkaline degradation product (b) after 8 h of forced degradation with 0.1 M NaOH. (B) LC chromatogram of SXG (a) and its alkaline degradation product (b) after 8 h of forced degradation with 0.5 M NaOH. (C) LC chromatogram of SXG (a) and its alkaline degradation products (b and c) after 8 h of forced degradation with 1 M NaOH. (D) LC chromatogram of SXG (a) and its alkaline degradation product (b) after heating with 0.1 M NaOH. (E) LC chromatogram of SXG (a) and its alkaline degradation products (b –d) after heating with 0.5 M NaOH. (F) LC chromatogram of SXG alkaline degradation products (a– d) after heating with 1 M NaOH.
4 Abdel-Ghany et al.
Base-induced forced degradation of SXG To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 0.5, 2.5 and 5 mL of 2 M NaOH were added and each mixture was completed to mark with distilled water to reach molarities of 0.1, 0.5 and 1 M NaOH, respectively. The mixtures were kept at room temperature for 8 h in dark and then neutralized with HCl, having the same molarity of the used NaOH, using a pH meter to ensure that pH is 7 before investigation. These experiments were repeated at a higher temperature of 758C for 0.5 h in dark. Twenty microliters of the degraded solutions were injected in triplicates to HPLC, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
System suitability tests System suitability tests are an integral part of liquid chromatographic methods in the course of optimizing the conditions of the proposed method. In the proposed LC method, system suitability tests are used to verify that resolution and reproducibility were adequate for analysis performed. Different parameters affecting the chromatographic separation were studied, including column efficiency (number of theoretical plates), tailing of chromatographic peak, peak resolution factor and repeatability as % relative standard deviation (RSD) of peak areas for six injections of SXG 200 mg mL21 and reproducibility of retention times, as shown in Table I.
Method validation Hydrogen peroxide-induced forced degradation of SXG To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 5 mL of 6% H2O2 and 5 mL of 30% H2O2 were added. The mixtures were kept at room temperature for 8 h in dark and then 5 mL of distilled water was added to stop the reaction. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Thermal degradation of SXG The dry powder of SXG was placed in an oven at 558C for 72 h to study thermal degradation and then dissolved in methanol. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Photodegradation of SXG The dry powder of SXG was placed under a UV lamp for 24 h and then dissolved in methanol. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’. The photodegradation process was carried out using a UV-lamp model UVGL-2 (Minerlightw Lamp multiband UV-254/366 nm, 215–250 V, 50/60 Hz, 0.12 A, USA).
Linearity In this study, a linear relationship between AUP and component concentration (C) was obtained for five chosen concentrations and the regression equation was then computed. The linearity of the calibration curve was validated by the high value of correlation coefficient. The analytical data of the calibration curve including standard deviation for the slope and intercept (Sb and Sa) are summarized in Table II.
Table IV Determination of SXG in the Presence of Its Degradation Products to Ensure the Specificity of the Method Degradation method
Recovery %
Degradation %
Reference sample Thermal degradation Photodegradation 6% H2O2 30% H2O2 0.1 M HCl on cold 0.5 M HCl on cold 1.0 M HCl on cold 0.1 M HCl on hot 0.5 M HCl on hot 1.0 M HCl on hot 0.1 M NaOH on cold 0.5 M NaOH on cold 1.0 M NaOH on cold 0.1 M NaOH on hot 0.5 M NaOH on hot 1.0 M NaOH on hot
101.27 97.86 98.16 97.76 97.81 96.72 95.01 93.66 94.25 83.96 75.43 66.12 57.57 42.57 50.98 36.62 17.53
0 2.14 1.84 2.24 2.19 3.28 4.99 6.34 5.75 16.04 24.57 33.88 42.43 57.43 49.02 63.38 82.47
Results Method development Different chromatographic systems, including various columns and different mobile phases at different pH values, were attempted. Isocratic elution based on potassium dihydrogen phosphate buffer ( pH 4.6) –acetonitrile – methanol (40 : 30 : 30, v/v/v) was applied to obtain the best results. It was found that at least 60% of organic solvent was needed to elute all peaks within 10 min. All the mobile phases that are reported in the literature (5 – 8) failed to obtain a good resolution and separation between SXG and its degradation products. Minimum retention time was obtained at a flow rate of 1 mL min21. The UV detector was operated at 208 nm, where good detector sensitivity was achieved for SXG and all the examined degradation products after the forced degradation. The retention time was 2.7 min as presented in Figure 2.
Table V Statistical Comparison Between the Proposed Method and the Reference Method for the Determination of SXG Statistical term
Reference methoda
HPLC method
Mean SD+ SE+ % RSD N V t (2.306b)
100.20 1.10 0.49 1.10 5 1.21
100.28 1.44 0.64 1.44 5 2.07 0.10
No significant difference between groups by using one-way ANOVA with F equals 0.01 and P equals 0.92. a Reference method: aliquots of standard solutions in methanol containing 5 –40 mg/mL SXG were measured using methanol as a blank (9). b Figures in parentheses are the theoretical t value at P ¼ 0.05.
Determination of SXG and Structure Elucidation of the Major Degradation Products 5
Accuracy Accuracy of the results was calculated by % recovery of five different concentrations of SXG also by a standard addition technique for Onglyzaw tablets. The results including the mean of the recovery and standard deviation are shown in Tables II and III.
Precision
Repeatability: Three concentrations of SXG (160, 200 and 240 mg mL21) were analyzed six times, within the same day, using the procedure mentioned under ‘Procedure’ section. The %RSD was calculated and found to be ,1% in the three concentrations, as shown in Table II.
Figure 5. LC– MS results for SXG and its major degradation products. (A) LC– MS for SXG stock solution. (B) LC– MS for the acid-degradation products of SXG after heating with 1 M HCl. (C) LC–MS for the alkaline degradation products of SXG after heating with 1 M NaOH.
Figure 6. The proposed fragmentation pattern for SXG that ensures its (LC–MS) m/z values.
6 Abdel-Ghany et al.
Intermediate precision: The above-mentioned concentrations of SXG were analyzed on three successive days using the procedure mentioned under ‘Procedure’ section. The %RSD was calculated, and the results are shown in Table II. Specificity Specificity is the ability of the analytical method to measure the analyte response in the presence of interferences, including the degradation products and related substances. In the present work, specificity was checked by analyzing SXG, where good resolution and absence of interference between SXG and most of the degradation products as shown in Figures 3 and 4 and Table IV. Besides, the chromatograms of the pharmaceutical dosage form samples were checked for the appearance of any extra peak. No chromatographic interference from any of the excipients was found at the retention time of SXG. In addition, the chromatograms of SXG in the samples0 solutions were found identical to the chromatograms received by the standard solutions at the wavelengths applied. These results confirm the absence of interference from other materials in the pharmaceutical dosage form and consequently confirm the specificity of the proposed method.
Robustness The flow rate of the mobile phase was changed from 1 to 0.8 mL min21 and 1.2 mL min21. The organic strength was changed by % +2. Finally, the value of pH of the phosphate buffer was varied from 4.6 to 4.4 and 4.8. There is no difference in the results, which was obtained for all these variations, indicating good robustness of the proposed LC method. No interference was observed from the degradation products of SXG in all the previously mentioned conditions. Limit of detection and limit of quantification Limit of detection (LOD), which represents the concentration of analyte at an S/N ratio of 3, and limit of quantification (LOQ) at which S/N is 10 were determined experimentally for the proposed methods and the results are given in Table II. Pharmaceutical dosage forms and the standard addition technique The method had been successfully applied to the pharmaceutical dosage form and to check the validity of the proposed method as in Table III, the standard addition technique was applied by
Figure 7. The first proposed mechanism for the acid-degradation products of SXG.
Determination of SXG and Structure Elucidation of the Major Degradation Products 7
adding different known concentrations of the pure drug to different known concentrations of the drug product and the procedure mentioned above were adopted. The concentrations were calculated using the corresponding regression equations as in Table III.
Figure 8. The second proposed mechanism for the acid-degradation products of SXG.
Figure 9. The first proposed mechanism for the alkaline degradation products of SXG.
8 Abdel-Ghany et al.
Statistical analysis Statistical analysis of the results obtained by the proposed method and the reference method (10) for SXG was carried out by ‘SPSS statistical package version 11’. The significant difference between the reference method and the described method was
tested by one-way analysis of variance (ANOVA) (F-test) at P ¼ 0.05 as shown in Table V. The test ascertained that there was no significant difference among the methods.
Discussion of the degradation behavior Chromatographic conditions in case of LC –MS Isocratic chromatographic elution was achieved at ambient temperature on a Symmetryw C18 column (150 4.6 mm, 5 mm), using distilled water—methanol (50:50, v/v) as a mobile phase. The mobile phase was pumped through the column at a flow rate of 1 mL min21 with injection volume 20 mL. Acid-induced forced degradation of SXG The chromatograms of the acid-degraded samples for SXG, using different molarities of HCl on cold and 0.1 M HCl on hot, showed good recovery percent of the drug with minimal or no peaks. The chromatograms of the acid-degraded samples for SXG, using 0.5 and 1 M HCl on hot, showed a marked decrease of the drug peak at 2.7 min and appearance of an additional peak at 7.7 min
(Figure 3). This reflects that SXG is relatively sensitive to acid. LC – MS for the intact drug was investigated (Figure 5), and the main fragmentation product ensures the purity of the investigated drug (Figure 6). Complete acid-degradation of SXG was not valuable, because of the extensive degradation of the main two degradation products that proved to be formed using LC – MS (Figure 5) and the proposed mechanisms for the aciddegradation products are illustrated in Figures 7 and 8. Under the neutral condition, methanolic solution of SXG did not show any additional peaks.
Base-induced forced degradation of SXG The chromatograms of the base-degraded samples for SXG, using different molarities of NaOH, either on cold or on hot, showed a marked decrease of the drug peak at 2.7 min and appearance of additional peaks at 3.7, 5 and 8.5 min (Figure 4). This reflects that SXG is highly sensitive to base. Complete basic-degradation of SXG was not valuable, because of the extensive degradation of the main degradation products that proved to be formed using
Figure 10. The second proposed mechanism for the alkaline degradation products of SXG.
Determination of SXG and Structure Elucidation of the Major Degradation Products 9
Figure 11. The third proposed mechanism for the alkaline degradation products of SXG.
Figure 12. The fourth proposed mechanism for the alkaline degradation products of SXG.
10 Abdel-Ghany et al.
LC – MS (Figure 5) and the proposed mechanisms for the base degradation products are illustrated in (Figures 9 –12). Hydrogen peroxide, thermal and photo-induced degradation of SXG The samples showed no additional peaks at 208 nm with SXG, indicating relative stability of SXG to those types of degradation.
Conclusion The proposed LC method has the advantages of novelty, simplicity, precision, accuracy and convenience for separation and quantization of SXG in the presence of its degradation products, as a stability-indicating assay. The method can be applied for the determination of SXG in pharmaceutical dosage forms. The method was validated showing satisfactory data for all the method validation parameters tested. The developed method can be conveniently used by quality control laboratories.
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inhibitor, in human plasma using liquid – liquid extraction; Biomedical Chromatography, (2008); 22: 214–222. Fura, A., Khanna, A., Vyas, V., Koplowitz, B., Chang, S.Y., Caporuscio, C., et al.; Pharmacokinetics of the dipeptidyl peptidase 4 inhibitor saxagliptin in rats, dogs, and monkeys and clinical projections; Drug Metabolism and Disposition, (2009); 37: 1164– 1171. Inturi, S., Inturi, R., Tagaram, I.; Validated novel LC determination of saxagliptin in pure bulk and pharmaceutical dosage forms; International Journal of Pharmaceutical Research and Development, (2011); 3: 45–52. Xu, X., Demers, R., Gu, H., Christopher, L., Su, H., Cojocaru, L., et al.; Liquid chromatography and tandem mass spectrometry method for the quantitative determination of saxagliptin and its major pharmacologically active 5-monohydroxy metabolite in human plasma: method validation and overcoming specific and non-specific binding at low concentrations; Journal of Chromatography B, (2012); 889: 77 –86. Hess, C., Musshoff, F., Madea, B.; Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography-mass spectrometry; Analytical and Bioanalytical Chemistry, (2011); 400: 33 –41. Mohammad, M., Elkady, E., Fouad, M.; Development and validation of a reversed-phase column liquid chromatographic method for simultaneous determination of two novel gliptins in their binary mixtures with metformin; European Journal of Chemistry, (2012); 3: 152–155. Kalaichelvi, R., Jayachandran, E.; Validated spectroscopic method for the estimation of saxagliptin in pure and from tablet formulation; International Journal of Pharmacy and Pharmaceutical Science, (2011); 3: 179– 180. El-bagary, R., Elkady, E., Ayoub, B.; Spectrophotometric methods based on charge transfer complexation reactions for the determination of saxagliptin in bulk and pharmaceutical preparation; International Journal of Biomedical Science, (2012); 8: 204– 208. Q2 (R1) Validation of Analytical Procedures. In Proceedings of the International Conference on Harmonisation (ICH), Geneva, Commission of the European Communities; 1996.
Determination of SXG and Structure Elucidation of the Major Degradation Products 11
Article
Stability-Indicating Liquid Chromatographic Method for Determination of Saxagliptin and Structure Elucidation of the Major Degradation Products Using LC – MS Maha F. Abdel-Ghany, Omar Abdel-Aziz, Miriam F. Ayad and Mariam M. Tadros* Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt *Author to whom correspondence should be addressed. Email: [email protected] Received 18 December 2013; revised 22 June 2014
A new, simple, selective, reproducible and sensitive stability-indicating liquid chromatographic method was developed and subsequently validated for the determination of saxagliptin (SXG). SXG was subjected to oxidation, thermal, acid hydrolysis, alkali hydrolysis and photodegradation according to ICH guidelines. The major degradation products were separated from the pure drug and the proposed structures’ elucidation was performed, using an LC–MS technique. Isocratic chromatographic elution was achieved on a Symmetryw C18 column (150 3 4.6 mm, 5 mm), using a mobile phase of potassium dihydrogen phosphate buffer (pH 4.6)–acetonitrile–methanol (40 : 30 : 30, v/v/v) at a flow rate of 1 mL min21 with UV detection at 208 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 25 – 400 mg mL21. All the results were statistically compared with the reference method, using one-way analysis of variance. The developed method was validated and proved to be specific and accurate for quality control of SXG in pharmaceutical dosage form.
Introduction Saxagliptin (SXG), (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1adamantyl)acetyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile (Figure 1), is a new oral hypoglycemic drug of new dipeptidyl peptidase-4 (DPP-4) inhibitor (1). SXG is recently approved for treatment of type-II diabetes mellitus (2). DPP-4 inhibitors represent a new therapeutic approach to the treatment of type-II diabetes that functions to stimulate glucose-dependent insulin release and reduce glucagon levels. This is done through inhibition of incretions0 inactivation, particularly glucagon-like peptide-1 and gastric inhibitory polypeptide, thereby improving glycemic control (3). Literature survey reveals that the drug has been estimated by the LC–MS-MS (4), HPLC methods (5 –8) and by the spectrophotometric method, in which SXG was estimated at 208 nm in methanol (9). Another spectrophotometric method based on charge-transfer reaction using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and 7,7,8,8-tetracyanoquinodimethane (10). The aim of our work is to present a new selective and sensitive stability-indicating liquid chromatographic method for the determination of SXG, where it was subjected to oxidation, thermal, acid and alkali hydrolysis and photodegradation according to ICH forced degradation guidelines (11). Optimization of the chromatographic conditions was of great importance to ensure complete separation of the degradation products from SXG, including mobile phase, column type, detection wavelength and flow rate. The major degradation products were separated from the pure drug, and the proposed structures0 elucidation was performed using the LC–MS technique in the case of the acid and alkaline hydrolysis.
Experimental Instrumentation The HPLC system consisted of a Shimadzu LC-20 AT Liquid Chromatograph (Japan), using a Symmetryw C18 column (150 4.6 mm, 5 mm) (Ireland). The system was equipped with a UV– visible detector (SPD-20A, Japan) and an autosampler (SIL-20A, Shimadzu, Japan). An Elma S100 ultrasonic processor model KBK 4200 (Germany) was used for degassing of the mobile phases.
Reagents and reference samples Pure SXG samples certified to contain 99.85% and Onglyzaw tablets (batch number 0J57932) nominally containing 5 mg of SXG per tablet were supplied by Bristol-Myers Squibb/ AstraZeneca EEIG (UK). Methanol (HiPerSolv for HPLC) and acetonitrile (HiPerSolv) were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Potassium dihydrogen phosphate and orthophosphoric acid (85%) were obtained from VWR Chemicals (Pool, UK). Bi-distilled water was produced in-house (A4000D; Aquatron Water Still, UK). Membrane filters 0.45 mm from Teknokroma (Barcelona, Spain) were used. All other chemicals and reagents used were of analytical chromatographic grade.
Chromatographic conditions Isocratic chromatographic elution was achieved at ambient temperature on a Symmetryw C18 column (150 4.6 mm, 5 mm), using potassium dihydrogen phosphate buffer ( pH 4.6) –acetonitrile – methanol (40 : 30 : 30, v/v/v) as a mobile phase, with UV detection at 208 nm. The buffer solution was filtered through a 0.45-mm membrane filter and degassed for 30 min in an ultrasonic bath prior to use. The mobile phase was pumped through the column at a flow rate of 1 mL min21 with the injection volume of 20 mL.
Stock standard solution preparation Stock standard solutions of SXG (1 mg mL21) was prepared by dissolving 100 mg of the drug in methanol, sonicated and completed to volume in a 100-mL volumetric flask. The required concentrations were prepared by serial dilutions.
Samples’ preparation Forty tablets of SXG were weighed, and the coats were removed by carefully rubbing with a clean tissue wetted with methanol.
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
An accurately weighed amount of finely powdered tablets equivalent to 100 mg of SXG was made up to 100 mL with methanol and then sonicated for 15 min.
Procedure Linearity (calibration curve for SXG) Accurately measured aliquots (0.25 –4 mL) of stock standard solution equivalent to 250 – 4,000 mg were separately transferred into a series of 10-mL volumetric flasks and then completed to volume with methanol. A volume of 20 mL of each solution was injected, and the mentioned chromatographic conditions were adapted. The calibration curve was obtained by plotting area under the peaks (AUP) against concentrations (C).
Table I System Suitability Tests for the LC –UV Method for the Determination of SXG in Bulk
Figure 1. SXG structure.
Item
SXG
N T R Resolution between peaks in the case of alkaline degradation Resolution between peaks in the case of acidic degradation RSD% of six injections Peak area Retention time
871 1.03 4.7 5.2 0.21 0.17
N, number of theoretical plates; T, tailing factor; R, resolution; RSD, relative standard deviation.
Table II Results Obtained for the LC –UV Method for the Determination of SXG in Bulk Item
SXG
Retention time (min) Wavelength of detection Range of linearity (mg mL21) Regression equation Regression coefficient (r) LOD (mg mL21) LOQ (mg mL21) Sb (standard deviation of the intercept) Sa (standard deviation of the slope) Confidence limit of the slope Confidence limit of the intercept Standard error of the estimation Precision Intraday, %RSD Interday, %RSD Drug in bulk (mean + standard deviation) Drug in dosage form (mean + standard deviation) Drug added (mean + standard deviation)
2.7 208 nm 25– 400 Area 1026 ¼ 0.1478 C mg mL21 þ 0.3232 0.9999 7.96 24.13 9.67 1024 0.22 0.1478 + 0.033 0.3232 + 3.12 1024 0.32 0.14 –0.23 0.23 –0.38 100.28 + 1.44 100.21 + 1.00 100.20 + 0.86
Table III Standard Addition Method for Determination of SXG in Dosage Form Amount of SXG taken (mg/mL)
Amount of SXG found (mg/mL)
Tablet
Added
Tablet
Tablet and added
Added
Tablet
Added
50 75 100 125 150
350 275 200 125 50
49.48 75.50 99.33 126.45 151.39
404.66 352.10 298.73 250.54 201.36
355.18 276.61 199.40 124.09 49.97
98.96 100.67 99.33 101.16 100.93
101.48 100.59 99.70 99.27 99.94
100.21 1.00 0.45
100.20 0.86 0.39
Mean SD (+) SE (+)
Figure 2. LC chromatogram of SXG (333.3 mg mL21) using UV detection at 208 nm.
2 Abdel-Ghany et al.
SD, standard deviation; SE, standard error.
Recovery %
Assay of drug in bulk (accuracy) The mentioned procedure under ‘Linearity (calibration curve for SXG)’ was repeated using 0.75 – 2.75 mL of stock standard solution equivalent to 75 – 275 mg mL21, and the concentrations of SXG were calculated by the corresponding calibration equation. Assay of drugs in pharmaceutical dosage forms For determination of SXG in tablets, from the sample solution prepared (as discussed in ‘Samples’ preparation’), aliquots (0.5 – 1.5 mL) equivalent to 50 – 150 mg mL21 were injected in triplicates and the concentrations of SXG were calculated by the corresponding calibration equation. Further, to check the validity of the proposed method, the standard addition technique was applied by adding different known concentrations of the pure drug to different known concentrations of the drug product. Forced degradation of SXG Forced degradation study of SXG included solid-state (thermal and photodegradation) and solution-state (acid, alkali and H2O2) stress conditions, which were performed according to ICH guidelines (11). The stock solution was used for forced degradation study, and the total chromatographic run time was about four times the retention of the drug peak to provide an indication of the stability-indicating property of the method. For study in
the neutral condition, the drug was held in methanol (0.5 mg mL21) in dark at room temperature for 24 h as a protected sample from all the stress conditions under investigation to be used to deduce the accurate amount of the degradation products comparable with SXG.
Preparation of the acid- and base-degraded sample for LC –MS investigation SXG (100 mg) was dissolved in 100 mL of chloroform : methanol (1 : 1), using sonication for 30 min. Then, the procedures, mentioned under ‘Acid-induced forced degradation of SXG for HPLC investigation’ and ‘Base-induced forced degradation of SXG’ sections were repeated. The organic layer was separated, and the aqueous layer was washed with chloroform (5 10 mL). The organic layer was investigated by LC–MS and each of (expected degradation pathways and LC – MS charts) as shown in Figures 5 and 7 –12. Acid-induced forced degradation of SXG for HPLC investigation To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 0.5, 2.5 and 5 mL of 2 M HCl were added and each mixture was completed to mark with distilled water to reach molarities of 0.1, 0.5 and 1 M HCl,
Figure 3. LC chromatograms of SXG in the presence of its acid-degradation product. (A). LC chromatogram of SXG (a) and its acid-degradation product (b) after heating with 0.5 M HCl. (B) LC chromatogram of SXG (a) and its acid-degradation product (b) after heating with 1 M HCl.
Determination of SXG and Structure Elucidation of the Major Degradation Products 3
respectively. The mixtures were kept at room temperature for 8 h in dark and then neutralized with NaOH, having the same molarity of the used HCl, using a pH meter to ensure that pH is 7 before investigation. These experiments were repeated at a
higher temperature of 758C for 0.5 h in dark. Twenty microliters of the degraded solutions were injected in triplicates to HPLC, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Figure 4. LC chromatograms of SXG in the presence of its alkaline degradation products. (A) LC chromatogram of SXG (a) and its alkaline degradation product (b) after 8 h of forced degradation with 0.1 M NaOH. (B) LC chromatogram of SXG (a) and its alkaline degradation product (b) after 8 h of forced degradation with 0.5 M NaOH. (C) LC chromatogram of SXG (a) and its alkaline degradation products (b and c) after 8 h of forced degradation with 1 M NaOH. (D) LC chromatogram of SXG (a) and its alkaline degradation product (b) after heating with 0.1 M NaOH. (E) LC chromatogram of SXG (a) and its alkaline degradation products (b –d) after heating with 0.5 M NaOH. (F) LC chromatogram of SXG alkaline degradation products (a– d) after heating with 1 M NaOH.
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Base-induced forced degradation of SXG To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 0.5, 2.5 and 5 mL of 2 M NaOH were added and each mixture was completed to mark with distilled water to reach molarities of 0.1, 0.5 and 1 M NaOH, respectively. The mixtures were kept at room temperature for 8 h in dark and then neutralized with HCl, having the same molarity of the used NaOH, using a pH meter to ensure that pH is 7 before investigation. These experiments were repeated at a higher temperature of 758C for 0.5 h in dark. Twenty microliters of the degraded solutions were injected in triplicates to HPLC, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
System suitability tests System suitability tests are an integral part of liquid chromatographic methods in the course of optimizing the conditions of the proposed method. In the proposed LC method, system suitability tests are used to verify that resolution and reproducibility were adequate for analysis performed. Different parameters affecting the chromatographic separation were studied, including column efficiency (number of theoretical plates), tailing of chromatographic peak, peak resolution factor and repeatability as % relative standard deviation (RSD) of peak areas for six injections of SXG 200 mg mL21 and reproducibility of retention times, as shown in Table I.
Method validation Hydrogen peroxide-induced forced degradation of SXG To a series of 10-mL volumetric flasks, each one contains 5 mL of methanolic stock standard solution of SXG, 5 mL of 6% H2O2 and 5 mL of 30% H2O2 were added. The mixtures were kept at room temperature for 8 h in dark and then 5 mL of distilled water was added to stop the reaction. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Thermal degradation of SXG The dry powder of SXG was placed in an oven at 558C for 72 h to study thermal degradation and then dissolved in methanol. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’.
Photodegradation of SXG The dry powder of SXG was placed under a UV lamp for 24 h and then dissolved in methanol. Twenty microliters of the degraded solutions were injected in triplicates, and the chromatograms were run as mentioned under ‘Chromatographic conditions’. The photodegradation process was carried out using a UV-lamp model UVGL-2 (Minerlightw Lamp multiband UV-254/366 nm, 215–250 V, 50/60 Hz, 0.12 A, USA).
Linearity In this study, a linear relationship between AUP and component concentration (C) was obtained for five chosen concentrations and the regression equation was then computed. The linearity of the calibration curve was validated by the high value of correlation coefficient. The analytical data of the calibration curve including standard deviation for the slope and intercept (Sb and Sa) are summarized in Table II.
Table IV Determination of SXG in the Presence of Its Degradation Products to Ensure the Specificity of the Method Degradation method
Recovery %
Degradation %
Reference sample Thermal degradation Photodegradation 6% H2O2 30% H2O2 0.1 M HCl on cold 0.5 M HCl on cold 1.0 M HCl on cold 0.1 M HCl on hot 0.5 M HCl on hot 1.0 M HCl on hot 0.1 M NaOH on cold 0.5 M NaOH on cold 1.0 M NaOH on cold 0.1 M NaOH on hot 0.5 M NaOH on hot 1.0 M NaOH on hot
101.27 97.86 98.16 97.76 97.81 96.72 95.01 93.66 94.25 83.96 75.43 66.12 57.57 42.57 50.98 36.62 17.53
0 2.14 1.84 2.24 2.19 3.28 4.99 6.34 5.75 16.04 24.57 33.88 42.43 57.43 49.02 63.38 82.47
Results Method development Different chromatographic systems, including various columns and different mobile phases at different pH values, were attempted. Isocratic elution based on potassium dihydrogen phosphate buffer ( pH 4.6) –acetonitrile – methanol (40 : 30 : 30, v/v/v) was applied to obtain the best results. It was found that at least 60% of organic solvent was needed to elute all peaks within 10 min. All the mobile phases that are reported in the literature (5 – 8) failed to obtain a good resolution and separation between SXG and its degradation products. Minimum retention time was obtained at a flow rate of 1 mL min21. The UV detector was operated at 208 nm, where good detector sensitivity was achieved for SXG and all the examined degradation products after the forced degradation. The retention time was 2.7 min as presented in Figure 2.
Table V Statistical Comparison Between the Proposed Method and the Reference Method for the Determination of SXG Statistical term
Reference methoda
HPLC method
Mean SD+ SE+ % RSD N V t (2.306b)
100.20 1.10 0.49 1.10 5 1.21
100.28 1.44 0.64 1.44 5 2.07 0.10
No significant difference between groups by using one-way ANOVA with F equals 0.01 and P equals 0.92. a Reference method: aliquots of standard solutions in methanol containing 5 –40 mg/mL SXG were measured using methanol as a blank (9). b Figures in parentheses are the theoretical t value at P ¼ 0.05.
Determination of SXG and Structure Elucidation of the Major Degradation Products 5
Accuracy Accuracy of the results was calculated by % recovery of five different concentrations of SXG also by a standard addition technique for Onglyzaw tablets. The results including the mean of the recovery and standard deviation are shown in Tables II and III.
Precision
Repeatability: Three concentrations of SXG (160, 200 and 240 mg mL21) were analyzed six times, within the same day, using the procedure mentioned under ‘Procedure’ section. The %RSD was calculated and found to be ,1% in the three concentrations, as shown in Table II.
Figure 5. LC– MS results for SXG and its major degradation products. (A) LC– MS for SXG stock solution. (B) LC– MS for the acid-degradation products of SXG after heating with 1 M HCl. (C) LC–MS for the alkaline degradation products of SXG after heating with 1 M NaOH.
Figure 6. The proposed fragmentation pattern for SXG that ensures its (LC–MS) m/z values.
6 Abdel-Ghany et al.
Intermediate precision: The above-mentioned concentrations of SXG were analyzed on three successive days using the procedure mentioned under ‘Procedure’ section. The %RSD was calculated, and the results are shown in Table II. Specificity Specificity is the ability of the analytical method to measure the analyte response in the presence of interferences, including the degradation products and related substances. In the present work, specificity was checked by analyzing SXG, where good resolution and absence of interference between SXG and most of the degradation products as shown in Figures 3 and 4 and Table IV. Besides, the chromatograms of the pharmaceutical dosage form samples were checked for the appearance of any extra peak. No chromatographic interference from any of the excipients was found at the retention time of SXG. In addition, the chromatograms of SXG in the samples0 solutions were found identical to the chromatograms received by the standard solutions at the wavelengths applied. These results confirm the absence of interference from other materials in the pharmaceutical dosage form and consequently confirm the specificity of the proposed method.
Robustness The flow rate of the mobile phase was changed from 1 to 0.8 mL min21 and 1.2 mL min21. The organic strength was changed by % +2. Finally, the value of pH of the phosphate buffer was varied from 4.6 to 4.4 and 4.8. There is no difference in the results, which was obtained for all these variations, indicating good robustness of the proposed LC method. No interference was observed from the degradation products of SXG in all the previously mentioned conditions. Limit of detection and limit of quantification Limit of detection (LOD), which represents the concentration of analyte at an S/N ratio of 3, and limit of quantification (LOQ) at which S/N is 10 were determined experimentally for the proposed methods and the results are given in Table II. Pharmaceutical dosage forms and the standard addition technique The method had been successfully applied to the pharmaceutical dosage form and to check the validity of the proposed method as in Table III, the standard addition technique was applied by
Figure 7. The first proposed mechanism for the acid-degradation products of SXG.
Determination of SXG and Structure Elucidation of the Major Degradation Products 7
adding different known concentrations of the pure drug to different known concentrations of the drug product and the procedure mentioned above were adopted. The concentrations were calculated using the corresponding regression equations as in Table III.
Figure 8. The second proposed mechanism for the acid-degradation products of SXG.
Figure 9. The first proposed mechanism for the alkaline degradation products of SXG.
8 Abdel-Ghany et al.
Statistical analysis Statistical analysis of the results obtained by the proposed method and the reference method (10) for SXG was carried out by ‘SPSS statistical package version 11’. The significant difference between the reference method and the described method was
tested by one-way analysis of variance (ANOVA) (F-test) at P ¼ 0.05 as shown in Table V. The test ascertained that there was no significant difference among the methods.
Discussion of the degradation behavior Chromatographic conditions in case of LC –MS Isocratic chromatographic elution was achieved at ambient temperature on a Symmetryw C18 column (150 4.6 mm, 5 mm), using distilled water—methanol (50:50, v/v) as a mobile phase. The mobile phase was pumped through the column at a flow rate of 1 mL min21 with injection volume 20 mL. Acid-induced forced degradation of SXG The chromatograms of the acid-degraded samples for SXG, using different molarities of HCl on cold and 0.1 M HCl on hot, showed good recovery percent of the drug with minimal or no peaks. The chromatograms of the acid-degraded samples for SXG, using 0.5 and 1 M HCl on hot, showed a marked decrease of the drug peak at 2.7 min and appearance of an additional peak at 7.7 min
(Figure 3). This reflects that SXG is relatively sensitive to acid. LC – MS for the intact drug was investigated (Figure 5), and the main fragmentation product ensures the purity of the investigated drug (Figure 6). Complete acid-degradation of SXG was not valuable, because of the extensive degradation of the main two degradation products that proved to be formed using LC – MS (Figure 5) and the proposed mechanisms for the aciddegradation products are illustrated in Figures 7 and 8. Under the neutral condition, methanolic solution of SXG did not show any additional peaks.
Base-induced forced degradation of SXG The chromatograms of the base-degraded samples for SXG, using different molarities of NaOH, either on cold or on hot, showed a marked decrease of the drug peak at 2.7 min and appearance of additional peaks at 3.7, 5 and 8.5 min (Figure 4). This reflects that SXG is highly sensitive to base. Complete basic-degradation of SXG was not valuable, because of the extensive degradation of the main degradation products that proved to be formed using
Figure 10. The second proposed mechanism for the alkaline degradation products of SXG.
Determination of SXG and Structure Elucidation of the Major Degradation Products 9
Figure 11. The third proposed mechanism for the alkaline degradation products of SXG.
Figure 12. The fourth proposed mechanism for the alkaline degradation products of SXG.
10 Abdel-Ghany et al.
LC – MS (Figure 5) and the proposed mechanisms for the base degradation products are illustrated in (Figures 9 –12). Hydrogen peroxide, thermal and photo-induced degradation of SXG The samples showed no additional peaks at 208 nm with SXG, indicating relative stability of SXG to those types of degradation.
Conclusion The proposed LC method has the advantages of novelty, simplicity, precision, accuracy and convenience for separation and quantization of SXG in the presence of its degradation products, as a stability-indicating assay. The method can be applied for the determination of SXG in pharmaceutical dosage forms. The method was validated showing satisfactory data for all the method validation parameters tested. The developed method can be conveniently used by quality control laboratories.
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Determination of SXG and Structure Elucidation of the Major Degradation Products 11
