A developed HPLC method for the determination of Alogliptin Benzoate and its potential impurities in bulk drug and tablets

A developed HPLC method for the determination of Alogliptin Benzoate and its potential impurities in bulk drug and tablets

Kun Zhanga,Panqin Mab,Wenna Jingc,Xiangrong Zhanga,＊
aSchool of Pharmacy,Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang,110016,China bKangya of Ningxia Pharmaceuticals CO.LTD,No.57,Fuan West Lane,Yinchuan,750003,China cSchool of Medical Devices,Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang,110016,China

ARTICLEINFO

Article history:

Received 22 April 2014

Received in revised form

10 July 2014

Accepted 15 January 2015

Available online 17 February 2015

Alogliptin benzoate Impurity

A

Alogliptin(AGLT),active ingredient of Alogliptin Benzoate(AGLT-BZ),is a new dipeptidyl peptidase-4(DPP-4)inhibitor for the treatment of type 2 diabetes.This study aimed to build a suitable method to determine the potential related substances in AGLT-BZ bulk drug and tablets.Seven related substances in Alogliptin Benzoate substances were synthetized and identi fi ed by1H-NMR and ESI-MS.In addition,the impurities were detected by a gradient reverse-phase high performance liquid chromatography(RP-HPLC)with UV detection.The chromatographic system consisted of an Angilent Zobax SB-CN column(250×4.6 mm; 5 μm).The mobile phase consisted of water/acetonitrile/tri fl uoroacetic acid 1900:100:1 v/v/ v(solution A)and acetonitrile/water/tri fl uoroacetic acid 1900:100:1 v/v/v(solution B)using a gradient program at a fl ow rate of 1.0 ml/min with 278 nm detection and an injection volume of 20 μl.Additionally,selectivity,the limit of quantitation(LOQ)and limit of detection(LOD),linearity,accuracy,precision and robustness were determined.Linearity was good over the concentration range 50-1000 ng/ml and the coef fi cient of determination (R2)were 0.9991-0.9998.RSD%of the determination of precision were＜2%(n=6).The method of RP-HPLC for the determination of impurities in AGLT-BZ was proved to be precise,accurate,robust and reliable.Three batches of self-made bulk drug and three dosages of commercial tablets were detected with this method.

?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction

Recent years,diabetes has been a serious disease affecting the patients around the world.According to International Diabetes Federation(IDF),there are totally 382 million diabetes patients in 2013.And this number will rise to 592 million by 2035[1].Type 2 diabetes is a chronic disease and progressive disease usually characterized by resistance of insulin and defunction of β-cell[2-4].Dipeptidyl peptidase-4 (DPP-4)is an enzyme,which is widely distributed in living organismsand blood circulation and could inactivate endogenous glucagon-like peptide-1(GLP-1),an insulin tropic hormones playing an important role in promoting insulin secretion,inhibiting glucagon secretion,delaying gastric emptying,enhancing satiety and moderating food intake [5-9].

Alogliptin,active ingredient of 2-[6-[3(R)-Aminopiperidin-1-yl]-3-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-ylmethyl]benzonitrile Benzoate,is a novel,potent and highly selective DPP-4 inhibitor[10].It has been developed into a commodity fi rstlybyTakedaPharmaceuticalCompany Limited and on list in Japan and America in 2010 and 2013,respectively.

Strict controlof impurities is necessary for the manufacturing of drug products.These impurities may be present in the materials,formed as by-products during the process of production or appear as degradation products during storage.Both actual and potential impurities should be studied and detected by a reliable and convenient method.Though a method was referred to in a patent of Takeda,the practicability of this method has not been veri fi ed[11].Few studies about the related substances of AGLT-BZ have been reported.It is necessary not only to fi nd the potential impurities according to the drug characteristic and synthesis route shown in Fig.1,but to fi nd a convenient, accuracy and sensitive method to detect them.The impurities of AGLT-BZ studied in this paper were named as compounds A,B,C,D,E,F and G shown in Table 1, respectively.

2. Materials and methods

2.1. Materials

Alogliptin Benzoate bulk drug were synthesized in our laboratory.Three dosages of commercial tablets(Nesina)were purchased from Takeda(Japan).Impurity standards A,B,C,D, E,F and G were obtained from Chiral-Tech Pharmaceutical Co.,Ltd(Beijing,China,purity＞98%).Table 1 shows the chemical structures of AGLT-BZ and its known impurities. HPLC-grade acetonitrile and tri fl uoroacetic acid were purchased from Concord(Tianjin,China)and Kelmel(Tianjin,China),respectively.Water for HPLC analysis was puri fi ed by the Yarong water puri fi cation system(Yarong Corp,Shanghai, China)and fi ltered with 0.22-μm membranes.All other reagents were of analytical grade.

2.2. Instrumentation and chromatographic method

The HPLC system(Hitachi Chromaster,Japan)consisted of a 5110 quaternary pump,a 5210 auto sampler,a 5310 column compartment,and a 5410 UV detector and 5430 diode arraydetector(DAD)operated at 278 nm.The chromatographic datum were collected and analyzed by the use of Hitachi Chromaster System software.Separation was achieved under gradientelutiononasphericalsilica-basedAngilentZobaxSBCN column,250×4.6 mm,particlesize5.0 μmfrom USAwith a fl ow rate of 1.0 ml/min at 278 nm and the column temperature was 35°C.The injection volume was20 μl.A mixture consisted of water/acetonitrile/tri fl uoroacetic acid 1900:100:1 v/v(solution A)and acetonitrile/water/tri fl uoroacetic acid 1900:100:1 v/v(solution B).The gradient program started at 99%solution A and then solution B was increased linearly from 1%to 25% for a 30-min period,and in the next 20-min,solution B was increased linearly from 25%to 90%.A 9-min re-equilibration at 99%for solution A was followed by a 1-min return ramp to solution A 99%.

The1H-NMR spectrometry experiments of impurities were conducted on a BRUKER AV500-III(Karlsruhe,Germany;1H 500 MHz)and Mass spectrometry experiments were performed on a LC/MS 2020(Shimadzu,Japan).

2.3. Preparation of standard solutions

2.3.1. Preparation of AGLT-BZ standards

AGLT-BZ stock solution of 1 mg/mL was prepared in diluent solution(water/tri fl uoroacetic acid 200:1 v/v)using the AGLTBZ reference standard(self-made).Calibration standard solutions of 0.5 mg/ml were prepared by diluting the stock solution.

2.3.2. Preparation of impurity standards

Impurity sock solution of 200 μg/ml was prepared by in solution using the impurity reference standard.Work solutions using in this study were prepared by serious dilution with ranging from 50 to 1000 ng/ml.

2.3.3. Sample preparation of drug bulk and commercial tablets

Solutions of bulk drug were prepared samely with AGLT-BZ standards.And the solutions of commercial tablets were prepared as follows:add 10 tablets into a suitable volumetric fl ask(250 ml for dosage 34 mg,100 ml for dosage 17 mg and 50 ml for dosage 8.5 mg),then add right amount solvent solution(water/tri fl uoroacetic acid 200:1 v/v),keep them under ultrasound for 30 min and then dilute to corresponding volumes.Then centrifuge for 10 min at 3500 r.p.m.The sample solutions were fi nally obtained by diluting the supernatant to 0.5 mg/ml with a speci fi c dilution ratio according to the dosage.

2.3.4. Sample preparation of forced degradation test

In order to examine the selectivity of the method and study the degradation of the bulk drug:50 mg AGLT-BZ was destroyed with proper amount of HCl(0.1 M)or NaOH(0.1 M) at 75°C for 30 min.Oxidation of AGLT-BZ was achieved by the use of 15%H2O2for 30 min at 75°C and thermodestruction of bulk drug was operated on a electric resistance furnace.The drug in solid state was exposed under 4000 l×light at room temperature for 15 days.Before being detected,the acidstressed and base-stressed samples were neutralized with base and acid,respectively.

All solutions were fi ltered,prior to the use,through 0.22-μm pore size fi lters.

2.4. Method validation

The validation study was consisted of selectivity,accuracy, precision,robustness,limit of detection(LOD)and limit of quantitation(LOQ),as well as linearity.

2.5. Analysis of AGLT-BZ bulk drug and its formulations

Three batches of self-made bulk drug and three dosages of commercial products were analyzed by this procedure.The amount of each impurity was calculated by comparing the peak areas by external standard method.

3. Results and discussion

3.1. Pathway of each impurity

Impurities appear mainly from two ways:brought in during productive process and degraded from the main drug during storage process.During the reaction,starting materials or intermediates remained or their by-products with main drug may become impurities.Additionally,no matter in the form of bulk drug or pharmaceutics,drug may degrade through various pathways such as oxidation,light,heat and acid/base. Fig.2 shows that the pathways of impurities in AGLT-BZ.

3.2. Con fi rmation of unknown impurities

Impurity A:a brown solid;UV λmax271 nm;1H NMR,see Table 2;ESI-MS m/z 225.1[M+H]+.

Impurity B:a yellow oil;UV λmax274 nm;1H NMR,see Table 2;ESI-MS m/z 358.1[M+H]+.

Impurity C:a white solid;UV λmax275 nm;1H NMR,see Table 2;ESI-MS m/z 326.1[M+H]+.

Impurity D:a yellow solid;UV λmax268 nm;1H NMR,see Table 2;ESI-MS m/z 464.2[M+H]+.

Impurity E:a yellow solid;UV λmax275 nm;1H NMR,see Table 2;ESI-MS m/z 455.2[M+H]+.

Impurity F:a white solid;UV λmax274 nm;1H NMR,see Table 2;ESI-MS m/z 444.2[M+H]+.

Impurity G:a yellow solid;UV λmax268 nm;1H NMR,see Table 2;ESI-MS m/z 579.2[M+H]+.

The ESI-MS results and 1H NMR spectrum data agree with the structure data in Table 2.

3.3. Selection of HPLC conditions

Thismethodwasestablished accordingtothe patentofTakeda Pharmaceutical Company Limited[11].In order to achieve the separation of all substances,we fi rstly compared C18 with CN columns and different mobile phases with different pH and proportions.Finally,an AngilentZobaxSB-CN column (250×4.6 mm;5 μm)with water/acetonitrile/tri fl uoroacetic acid was chosen as the method for the future study,because it provided a better separation,column ef fi ciency,acceptable peak shape and a short analysis time.Tri fl uoroacetic acid provided an acidic condition to guarantee the benzoic acid emerging with the form of molecules and having a good separation with AGLT.To have a high sensitivity for each target analyte,a wavelength of 278 nm was used.Giving consideration tothe separationsandrun time,the fl owrateandcolumn temperature were set at1.0 ml/minand 35°C,respectively,and a gradient elution was fi nally chosen.Fig.3 shows the best separation of AGLT-BZ with its potential impurities.

3.4. Validation assay

3.4.1. Method selectivity

Fig.2 shows that resolution between alogliptin,benzoate and impurities can meet the requirement of baseline separation (Resolution＞1.5).

The selectivity of this method was further assessed by a series of forced degradation tests,including acid or base degradation test,high temperature test,oxidant degradation test and light test.Fig.4 indicates that both known impurities and unknown impurities have good separations with alogliptin and benzoate(Resolution＞1.5).

3.4.2. Limit of Detection(LOD)/Limit of Quantitation(LOQ) The LOD and LOQ were determined at a signal-to-noise ratio of 3:1 and 10:1 by injecting a series of diluted solutions with knownconcentrations,respectively.Table3showsthe detailed results of the LODs and LOQs.

3.4.3. Linearity

Linear regression analysis of each target substances was performed using external standard method by injecting a series of solutions with different concentrations from 100 to 1000 ng/ml for impurities B,C,D,F and G,from 150 to 1000 ng/ ml for impurity E,and from 50 to 1000 ng/ml for impurity A. Table 4 shows the linear responses for the peak areas against concentrations obtained for each impurity with correlation coef fi cients(R2)ranging from 0.9991 to 0.9998.

3.4.4. Accuracy

Accuracy was determined by adding known amount of impurities into AGLT-BZ solutions.Then the recovery of the drug was determined.The fi nal concentrations being detected foreach impurity were at three levels(0.4,0.5,0.6 μg/ml each). Table 5 shows that the average recovery for all impurities ranges from 97.04 to 103.78.

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3.4.5. Precision

The precision of the method was obtained by injecting the same preparation of impurities sample solutions six times continuously.Table 6 shows the RSD%of peak areas and retention times for each impurity.

3.4.6. Solution stability

The impurities solution were left at room temperature and injected at 0-,2-,4-,6-,8-,10-,12-h time intervals.The consistency at each interval was calculated by the changes of peak area.The results indicate all the impurities except impurity A are stable in 12 h.

3.4.7. Robustness

To evaluate the robustness of the analytical HPLC method,the changes on fl ow rate of mobile phase(±20%),column temperature(±5°C),detection wavelength(±5 nm)and two different brand columns were made.And when having each little change for the chromatographic condition,it had still good separations between each target analyte and none of the change caused a signi fi cant effect on the system parameters like retention time,peak area,theoretical and tailing factor.

3.5. Batch results

Three batch self-made bulk drug and three dosages commercial tablets were tested using this HPLC method.Table 7 indicates that no impurity was found to be above 0.1%.Only impurity F was not found in all the samples.Impurity A,C and G were found in self-made bulk drug.

4. Conclusion

Seven new impurities of Alogliptin Benzoate were detected by a developed,precise,fast,accurate HPLC method.All the impurities were con fi rmed by ESI-MS and1H-NMR.The method can obtain a good and reliable separation for each target compound by a series of forced degradation tests.The method may be used to assess the quality of Alogliptin tablets.

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*Corresponding author.Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang 110016,China.Tel./fax:+86 24 23986522. E-mail address:zhangxr@vip.sina.com(X.Zhang).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2015.01.001

1818-0876/?2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

HPLC

Bulk drug

Commercial tablets