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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (4): 529-536    DOI: 10.3724/zdxbyxb-2021-0247
    
Simultaneous determination of related substances in entecavir film-coated tablet by high performance liquid chromatography
ZHANG Yi(),YUAN Hong()
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
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Abstract  

Objective: To establish a high performance liquid chromatography method to simultaneously quantify eight related substances in entecavir film-coated tablets.Methods:According to USP40 and YBH33292005 standards, a high performance liquid chromatography (HPLC) method for simultaneous determination of 8 related substance in entecavir film-coated tablets was established and validated. The column was WATERS C18 (250 mm×4.6?mm, 5?μm), the mobile phase A was water-acetonitrile-trifluoroacetic acid (990∶10∶1), the mobile phase B was water-acetonitrile-trifluoroacetic acid (700∶300∶1) with gradient elution and ultraviolet-visible light detector, detection wavelength at 254 nm and column temperature of 30?℃. Results: The resolution between entecavir and each impurity peak was more than 1.5, and each impurity had a good linear relationship with the peak area in the linear range. The limits of detection and quantification, precision, stability, durability, specificity, met the verification requirements. Conclusion:The HPLC method established in this study can be used for simultaneous determination of 8 related substance in entecavir film-coated tablets.

Key words： Chromatography, liquid      Entecavir      Film-coated tablet      Ultraviolet-visible light detector      Related substance     
Received: 10 April 2021      Published: 01 November 2021
CLC:  R284.1  
  R917.101  
Corresponding Authors: YUAN Hong     E-mail: zyi0571@sina.com;yuanhong70@zju.edu.cn
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ZHANG Yi
YUAN Hong
Cite this article:

ZHANG Yi,YUAN Hong. Simultaneous determination of related substances in entecavir film-coated tablet by high performance liquid chromatography. J Zhejiang Univ (Med Sci), 2021, 50(4): 529-536.

URL:

http://www.zjujournals.com/med/10.3724/zdxbyxb-2021-0247     OR     http://www.zjujournals.com/med/Y2021/V50/I4/529


利用高效液相色谱法同时检测恩替卡韦薄膜衣片中的有关物质

目的：建立能够同时测定恩替卡韦薄膜衣片8种有关物质的高效液相色谱法。方法：参考美国药典40和中美上海施贵宝制药有限公司的恩替卡韦薄膜衣片（博路定）标准（YBH33292005）等，采用WATERS C18色谱柱（250 mm×4.6?mm，5?μm），流动相A为水-乙腈-三氟乙酸（990∶10∶1），流动相B为水-乙腈-三氟乙酸（700∶300∶1），梯度洗脱，采用紫外-可见光检测器，检测波长为254?nm，柱温30?℃，建立同时测定恩替卡韦薄膜衣片8种有关物质的高效液相色谱法，并对该检测方法进行方法学验证。结果：恩替卡韦与各杂质峰之间的分离度均大于1.5，8种有关物质在线性范围内均与峰面积成良好的线性关系，检测限、定量限、精密度、稳定性、耐用性、专属性等均符合要求。结论：本研究建立的高效液相色谱法可同时检测恩替卡韦薄膜衣片常见的8种有关物质。


关键词： 色谱法，液相,  恩替卡韦,  薄膜衣片,  紫外-可见光检测器,  有关物质 

峰归属

USP40标准方法

本研究建立的方法

保留时间（min）

分离度

拖尾因子

理论板数

保留时间（min）

分离度

拖尾因子

理论板数

鸟嘌呤

3.732

24.65

1.14

16?981

4.955

33.01

1.04

9436

恩替卡韦呋喃杂质

8.703

18.06

1.15

14?417

11.272

17.44

0.99

62?917

非对映异构体1

14.277

1.61

1.04

30?164

14.335

2.47

1.00

110?524

非对映异构体3

14.780

4.74

1.06

39?147

14.768

2.82

0.98

108?564

恩替卡韦

16.098

2.71

1.09

61?615

15.330

5.37

1.36

77?992

非对映异构体2

16.768

5.65

—

80?987

16.428

6.46

0.97

119?169

8-羟基杂质

18.082

18.31

1.11

98?635

17.618

20.41

0.99

154?875

8-甲氧基杂质

22.085

—

1.05

180?106

21.348

—

0.98

207?693
Table 1 Test results of USP40 standard method and the method established in this study for the mixed solution
Figure 1 Chromatograms of system applicability

成分

最低检测浓度（ng/mL）

信噪比

与供试品浓度比（%）

鸟嘌呤

0.5

3.1~4.2

0.001

恩替卡韦呋喃杂质

1.3

4.4~4.7

0.003

非对映异构体1

2.4

4.1~4.8

0.005

非对映异构体3

2.5

6.2~6.6

0.005

恩替卡韦

1.2

4.5~4.9

0.002

8-羟基杂质

2.6

4.5~4.9

0.005

非对映异构体2

1.0

3.6~3.8

0.002

8-甲氧基杂质

1.0

3.8~4.4

0.002

4-羟基二甲基甲硅烷基杂质

1.2

3.3~3.7

0.002
Table 2 Limits of detection of entecavir and related substances

成分

定量限浓度（ng/mL）

信噪比

6针峰面积

与供试品浓度比（%）

鸟嘌呤

1.3

12.3~14.9

0.011~0.012

0.010

恩替卡韦呋喃杂质

5.1

11.9~15.1

0.016~0.018

0.010

非对映异构体1

5.4

12.5~15.6

0.019~0.020

0.011

非对映异构体3

5.5

10.0~14.6

0.022~0.025

0.011

恩替卡韦

2.5

11.5~19.9

0.009~0.010

0.005

8-羟基杂质

9.1

15.9~19.7

0.012~0.013

0.018

非对映异构体2

2.7

21.3~24.5

0.010~0.011

0.005

8-甲氧基杂质

6.4

13.0~14.5

0.023~0.023

0.013

4-羟基二甲基甲硅烷基杂质

3.5

10.1~15.8

0.005~0.006

0.005
Table 3 Limits of quantitation of entecavir and related substances

成分

线性回归方程

相关系数

响应因子RSD（%）

浓度范围（μg/mL）

鸟嘌呤

y=8.3479 x＋0.0026

0.9998

1.85

0.0013~0.3157

恩替卡韦呋喃杂质

y=3.4239 x＋0.0006

1.0000

1.42

0.0051~0.3066

非对映异构体1

y=3.4324 x – 0.0006

0.9998

2.39

0.0051~0.3078

非对映异构体3

y= 3.8999 x ＋0.0035

0.9999

1.29

0.0055~0.3279

恩替卡韦

y=0.0414 x – 0.0016

0.9900

1.75

0.0024~0.5735

8-羟基杂质

y=4.2834 x＋0.0006

0.9999

1.81

0.0027~0.3268

非对映异构体2

y=3.6037 x ＋0.0002

0.9999

1.82

0.0027~0.3248

8-甲氧基杂质

y=3.8217 x – 0.0010

1.0000

0.77

0.0061~0.2942

4-羟基二甲基甲硅烷基杂质

y=1.4790 x – 0.0025

0.9999

4.39

0.0034~0.3069
Table 4 Linear regression analysis of entecavir with related substances

浓度（%）

鸟嘌呤

恩替卡韦呋喃杂质

非对映异构体1

非对映异构体3

8-羟基杂质

非对映异构体2

8-甲氧基杂质

4-羟基二甲基甲硅烷基杂质

50

102.7

94.6

95.6

104.1

101.8

99.4

101.2

96.4

103.4

94.8

96.3

103.9

102.3

100.2

101.6

96.4

103.0

95.1

96.0

104.3

102.8

100.6

100.9

96.9

80

101.2

95.2

98.2

101.4

101.0

99.3

100.5

103.3

101.1

95.1

99.2

101.4

100.4

99.2

100.5

102.6

100.7

94.9

98.3

101.1

99.4

98.8

100.1

103.0

100

101.2

96.1

98.9

101.6

99.2

99.8

100.8

96.7

101.9

96.8

98.9

102.1

100.7

100.5

101.4

96.9

101.3

96.1

98.0

101.8

99.9

100.2

100.6

96.7

120

101.4

97.0

98.6

101.9

98.7

100.6

100.6

98.4

101.3

96.8

98.1

101.7

97.9

100.8

100.6

98.4

101.4

97.0

98.4

101.6

97.7

100.1

100.9

98.4

RSD

0.84

0.97

1.24

1.11

1.89

0.64

0.41

2.73
Table 5 Recovery rates of entecavir related substances
Figure 2 Chromatogram of forced degradation experiment在高温、光照、碱、酸和氧化处理条件下，降解产物与恩替卡韦的峰均可以分离.
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