Using HPLC to analyze (S)-oxiracetam and four related substances in the bulk drug of (S)-oxiracetam

Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113072

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Using HPLC to analyze (S)-oxiracetam and four related substances in the bulk drug of (S)-oxiracetam Chao Wang a,b,1 , Hong Dong a,b,c,1 , Honghui Liu a,b , Zhaoyi Sun d , Ahu Yuan a,b,c , Jinhui Wu a,b,c , Yiqiao Hu a,b,c,∗ a State Key Laboratory of Pharmaceutical Biotechnology, Medical School of Nanjing University & School of Life Sciences, Nanjing University, Nanjing 210093, PR China b Institute of Drug R&D, Nanjing University, Nanjing 210093, PR China c Jiangsu Provincial Key Laboratory for Nano Technology, Nanjing University, Nanjing, 210093, PR China d School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, PR China

a r t i c l e

i n f o

Article history: Received 28 October 2019 Accepted 21 December 2019 Available online 23 December 2019 Keywords: (S)-oxiracetam Analytical methodology HPLC Related substances analysis

a b s t r a c t (S)-oxiracetam is undergoing clinical trials as an active ingredient in the racemic oxiracetam. Here, we report a specific analytical method for analyzing (S)-oxiracetam and four related impurities in the bulk drug of (S)-oxiracetam by using high-performance liquid chromatography (HPLC) system. The chromatographic system included a Capcell pak NH2 analytical column, a mobile phase containing acetonitrile-water (95:5, v/v; pH adjusted to 2.0 with trifluoroacetic acid) at a flow rate of 1.0 mL/min, column temperature at 35 ◦ and the UV detection wavelength is set at 210 nm. This analytical method has shown effective and specific analysis for (S)-oxiracetam and four related substances. Moreover, the molecular weight and chemical structure preliminarily speculated of related substances were characterized by mass spectrometry. The methodology was verified by HPLC and results collected of the method validation included the system suitability, specificity sensitivity, linearity and accuracy, good linear correlation coefficient R2 was more than 0.9991. The analytical method developed and verified in the study, as far as we know, is the most exhaustive HPLC determination report which could be applied for the quality control and stability monitor purposes of the bulk drug of (S)-oxiracetam in the routine pharmaceutical analysis. © 2019 Published by Elsevier B.V.

1. Introduction Racemic oxiracetam, a most generally used nootropic drug contained both (S)-oxiracetam and (R)-oxiracetam (Fig. 1b, c), is a derivative of ␥-aminobutyric acid which belongs to racetam group (Fig. 1a), and it has been using in clinical for treatment of various cognition disorders and has beneficial effects on cerebrovascular trauma and multi-infarct dementia [1–4]. Researches have shown that (S)-oxiracetam has a stronger absorption rate and a weaker elimination rate than (R)-oxiracetam, and (S)-oxiracetam is the effective components in racemic oxiracetam that alleviates the cognitive impairment induced by chronic cerebral hypoperfusion in

∗ Corresponding author at: Nanjing University, No. 22 Hankou Road, Gulou District, Nanjing 210093, PR China. E-mail address: [email protected] (Y. Hu). 1 These authors contributed equally. https://doi.org/10.1016/j.jpba.2019.113072 0731-7085/© 2019 Published by Elsevier B.V.

rats [5]. Moreover, a phase II clinical trial is being conducted to explore the effectiveness and safety of (S)-oxiracetam in China [6]. Various analytical methods were developed for the determination of oxiracetam and its relative substances, including normal phase liquid chromatography with UV detection [7], reversedphase liquid chromatography with fluorescence detection followed by derivatization [8], column-switching high-performance liquid chromatography [9,10], stereoselective HPLC [11] and reversed phase liquid chromatography-tandem mass spectrometry (LC–MS/MS) [12]. The determination of impurities is especially important for drug quality control [13]. However, all these researches are based on racemic oxiracetam. When the synthesis of the active pharmaceutical ingredient (API) is changed from racemic oxiracetam to (S)-oxiracetam, some changes will occur in the known impurities to be detected, such as a new known impurity (S)-4-hydroxy-2- pyrrolidone. To the best of our knowledge, there is no literature or official pharmacopoeial method report about determination of (S)-oxiracetam and relative impurities in the bulk drug of (S)-oxiracetam.

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Fig. 1. Structural formula of oxiracetam, (a) indicates racemic oxiracetam, (b) indicates(S)-oxiracetam and (c) indicates (R)-oxiracetam.

Table 1 Chemical structures of (S)-oxiracetam and its impurities. Name

Structural formula

Molecular Formula (Molecular Weight)

(S)-oxiracetam

C6 H10 N2 O (158.09)

Imp-1

C4 H6 N2 O2 (114.10)

Imp-2

C4 H7 NO2 (101.10)

Imp-3

C8 H13 N3 O4 (215.21)

Imp-4

C6 H9 NO4 (159.14)

In the present study, referring to the import standard of South Korea which is applied for analysis racemic oxiracetam and relative known impurities, a simple, effective and accurate HPLC analytical method for quantitative analysis of (S)-oxiracetam and four relative impurities in the bulk drug of (S)-oxiracetam was developed and validated (Table 1). The four impurities derived from (S)-oxiracetam synthetic process respectively are glycine anhydride (imp-1), (S)-4-hydroxy-2- pyrrolidone (imp2), 2-(4-hydroxyl-2-oxygen-1-acetamide-pyrrolidine)-acetamide (imp-3), and (S)-4-hydroxy-2-oxy-1-pyrrolidine acetic acid (imp4) (Table 1).

dard HPLC-grade acetonitrile and trifluoroacetate were purchased from J&K Chemical Ltd. (Shanghai, China). 2.2. Chromatographic conditions

2. Materials and methods

The HPLC was performed by using LC-20 binary pump system, a thermostated-column device and a UV detector. (Shimadzu, Kyoto, Japan). The analytical column was a Capcell pak NH2 column (4.6 × 250 mm, 5 ␮m) maintained at 35 ◦ C. The mobile phase of HPLC was acetonitrile-water-trifluoroacetate (95:5:0.042, v/v/v, pH = 2.0). The injection volume of sample was 20 ␮L. The column elute was determined with UV detector at 210 nm at a flow rate of 0.9–1.1 mL/min.

2.1. Chemicals and reagents

2.3. Preparation of stock and standard solutions

The bulk drug of (S)-oxiracetam was afforded by Chongqing Dongze Pharmaceutical Technology Development Co., Ltd. (Chongqing, China). Imp-1 reference standard, (S)- oxiracetam reference standard and imp-2 reference standard were afforded by Sichuan Antibiotic Industry Research Institute, China National Pharmaceutical Group Corporation (Sichuan, China). Imp-3 reference standard was purchased from Shenzhen Feisi Biotechnology Co., Ltd. (Shenzhen, China). Imp-4 reference stan-

The test solution containing 1.2 mg/mL of (S)-oxiracetam sample was prepared in mobile phase. The standard solution containing 120 ␮g/mL of (S)-oxiracetam standard was prepared in mobile phase. The stock solutions containing 1.2 mg/mL of imp-1, imp-2, imp-3 and imp-4 were prepared in mobile phase respectively. The four impurities standard working solutions were prepared by dilution of the stock solution with mobile phase respectively to 12 ␮g /mL.

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Table 2 The chromatographic conditions of the import and optimized method. Conditions

Import method

Capcell pak NH2 Water -Acetonitrile (3:97, v/v) pH 2.2 1.2 mL/min Flow rate Column temperature 35◦ Column Mobile phase

Optimized method Capcell pak NH2 Water -Acetonitrile (5:95, v/v) 2.0 1.0 mL/min 35◦

Table 3 Selection of mobile phase for HPLC.

Fig. 2. The system suitability results of the two analytical methods. (a) depicts the analysis results under the import standard method. (b) depicts the analysis results under the optimum analytical method.

2.4. Preparation of system suitability solution The system suitability solution was prepared by mixing the standard of (S)-oxiracetam with its four impurity standards and diluting to 120 ␮g/mL of (S)-oxiracetam and 12 ␮g/mL of four impurities respectively. 2.5. LC–MS conditions The liquid chromatography-mass spectrometry (LC–MS) was conducted on Shimadzu LC–MS 2020 system (Shimadzu, Kyoto, Japan) as described in our previous study [14]. Briefly, in order to avoid interference of trifluoroacetic acid with mass spectrometry, the chromatographic conditions described above was used to identify the four impurities and (S)-oxiracetam by LC–MS, except for altering the trifluoroacetate in mobile phase to ammonium acetate (0.15 %). For mass spectra analysis, test samples were injected into ESI+ mode. The data of the m/z range from 10 to 1000 were collected. The heat block temperature was 200 ◦ , and the desolvation temperature was 250 ◦ with desolvation gas flow 15 L /min. The detector voltage was 1400 V. 3. Results and discussion 3.1. Method development Since there is no literature or official Pharmacopoeia method report on the determination of (S)-oxiracetam and its related impurities. We analyzed (S)-oxiracetam and its four related substances according to the import standard of oxiracetam injection in South Korea. However, the analytical method of imported standards cannot effectively separate all impurities. Imp-1 and imp-2 could not be separated completely, and their resolution was only 0.615. In addition, the peaks of (S)-oxiracetam and imp-4 coincided completely, resulting in a higher tailing factor of the main peak (Fig. 2a).Therefore, it is necessary to optimize the import analytical method to get a specific analytical method for analyzing (S)-oxiracetam and four related impurities in the bulk drug of (S)-oxiracetam.

Composition of mobile phase

Chromatographic peak shape

Water : acetonitrile = 50:50 100 % water Water : acetonitrile-trifluoroacetate (pH1.8) = 3:97 Water : acetonitrile-trifluoroacetate (pH2.2) = 3:97 Water : acetonitrile-trifluoroacetate (pH2.0) = 3:97 Water : acetonitrile-trifluoroacetate (pH1.8) = 2:98 Water : acetonitrile-trifluoroacetate (pH2.2) = 5:95 Water : acetonitrile-trifluoroacetate (pH2.0) = 5:95

C C B B B B B A

Notes: A: Moderate retention time, good separation of impurities; B: Moderate retention time, impurities are not completely separated; C: The impurities are not completely separated and coincide with the main peak.

The end absorption wavelength of (S)-oxiracetam in mobile phase is 200 nm. The UV–vis full-wavelength scanning of (S)oxiracetam and its impurities revealed that the impurities were also absorbed at 210 nm (Fig. 3a). Therefore, a wavelength of 210 nm is selected as the detection wavelength. According to the researches and import standard about the detection method of oxiracetam and its related substances [11,15,16], a series of analytical columns were tested to choose a suitable chromatographic column. It has obviously shown that the Capcell pak NH2 column in all evaluated columns has got better tailing factor, retention time and reproducibility (Fig. 3b). Subsequently, the following mobile phases were screened during the test and the mobile phase was optimized according to the actual conditions of the experiment (Table 3). At the same time, we also evaluated the effect of flow rate, column temperature and mobile phase pH on the peak of (S)-oxiracetam. Briefly, when the test flow rate was 0.9–1.1 mL/min, the retention time and peak shape of (S)-oxiracetam and its impurities were good (Fig. 3c); the column temperature range of 32.5 ◦ C–37.5 ◦ C is suitable for (S)oxiracetam detection (Fig. 3d); but the pH of the mobile phase has a significant effect on the peak of (S)-oxiracetam, it is suitable for detecting (S)-oxiracetam when the pH of mobile phase is 2.0 (Fig. 3e). The comparison of the import method and optimum method was shown in Table 2. The results showed that when the chromatogram was recorded to 3 times the retention time of the main drug peak, the sample impurities were all detected; in addition, all the known impurities and (S)-oxiracetam were well separated (Fig. 2b). In summary, we report that a specific analytical method for analyzing (S)-oxiracetam and four related impurities in the bulk drug of (S)-oxiracetam by using HPLC. 3.2. Method validation Identification and characterization of the four related substances of (S)-oxiracetam were performed by using LC–MS. Preliminary structural elucidations of the related substances were tabulated in Table 1. This is very important supplementary for verifying the potential impurities in (S)-oxiracetam synthesis process and then obtaining the purer compound. It should be noted that all

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Fig. 3. The development of chromatographic conditions. (a) The UV–vis full-wavelength scanning of (S)-oxiracetam, related impurities and mobile phase baseline (b) Effects of the three different stationary phases. (b) Results of different flow rates between 0.9 mL/min and 1.1 mL/min. (c) Effects of the different column oven temperature values (i.e. 32.5, 35, 37.5 ◦ ). (d) Results of three pH values (i.e., 1.8, 2.0, 2.2) of the mobile phases.

Table 4 The study of system precision. Repeated test

1

2

3

4

Main peak area Average of Main peak area RSD (%)

352379 351905 1.38

353101

354244

344414

5

6 348756

358535

Table 5 The result of system suitability.

Tailing factor Resolution

(S)-oxiracetam Imp-1 with (S)-oxiracetam (S)-oxiracetam with Imp-4

Measured value

Acceptability Criteria

1.174 1.8 5.8

Should be less than 2.0 Should be more than 1.5 Should be more than 1.5

the impurities mentioned above were purchased as standards on the method validation studies.

indicated that this optimum analytical method is more suitable than the import.

3.2.1. System suitability For determination of the system precision, briefly, 12 ␮g/mL (S)-oxiracetam solution, which was prepared by transferring 1 mL standard solution to a 100 ml volumetric flask and the volume was fixed to 100 ml with diluent, was injected six times into the HPLC system to calculate the relative standard deviation(RSD) of the peak area. The average of the peak areas was 351,905 and the RSD was calculated as 1.38 % (Table 4). The associated parameters of system suitability were calculated and shown in Table 5 and the chromatograms of system suitability solution shown in Fig. 2(b)

3.2.2. Specificity The specificity study of the liquid phase HPLC method was designed to investigate peak identification and selectivity. The blank solution and the localization solution were separately injected to determine all potential impurities. The resolution between the main peak and the adjacent peak should not be less than 1.0. The results show that the order of the peaks is Imp-2, imp-1, (S)-oxiracetam, imp-4 and imp-3. The resolution of (S)oxiracetam and imp-1 is not less than 1.0. It can be seen from the Table 6 that the minimum resolution between the main peak and the adjacent peak of the known impurity is in accordance with the

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Table 6 The result of the selectivity of method. Solution

Composition

Concentration (␮g/mL)

Retention time (min)

Resolution

Imp-2 standard solution Imp-1 standard solution (S)-oxiracetam standard solution Imp-4 standard solution Imp-3 standard solution

Imp-2 standard Imp-1 standard (S)-oxiracetam standard Imp-4 standard Imp-3 standard Imp-2 standard Imp-1 standard (S)-oxiracetam standard Imp-4 standard Imp-3 standard

12 12 120 12 12 12 12 120 12 12

6.90 7.20 8.10 12.25 13.46 6.79 7.27 7.89 10.85 12.17

2.38 2.16 2.94 9.45 2.98

System suitability solution

Fig. 4. The results of forced degradation study. (a) -(f) described the degradation of (S)-oxiracetam under unstress, acid (1 M HCl for 30 min at room temperature), base (1 M NaOH for 4 min at room temperature), oxidation (30 % H2 O2 for 30 min at room temperature), heat (100 ◦ for 30 min) and light (Illumination of 2500 lx for 30 days) stress condition respectively.

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Table 7 The degradation study of (S)-oxiracetam. Tests

Conditions

Assay of (S)-oxiracetam (%)

Unstressed Acid Base Oxidation Heat Light

—— 1 M HCl for 30 min at room temperature 1 M NaOH for 4 min at room temperature 30 % H2 O2 for 30 min at room temperature 100 ◦ for 30 min Illumination of 2500 lx for 30 days

99.84 98.01 94.44 96.33 99.45 99.12

Table 8 The results of sensitivity, linearity, accuracy and precision. Parameter

(S)-oxiracetam

Imp-1

Imp-2

Imp-3

Imp-4

LOD (␮g/mL) LOQ (␮g/mL) Regression equation(y) Slope(b) Intercept(a) Correlation coefficient Accuracy 80 % 100 % 120 % Precision(%RSD)

0.02 0.01

0.003 0.001

0.02 0.006

0.025 0.0075

0.05 0.015

25640 79.4 0.9997

40014 313.0 0.9996

12303 −527.2 0.9998

19068 −2624.5 0.9994

17787 −2441 0.9991

91.73 % 102.94 % 91.37 % 1.20

86.42 % 94.12 % 88.72 % 1.25

88.02 % 98.29 % 90.15 % 1.19

85.04 % 89.30 % 94.91 % 1.29

Table 9 The robustness of method.

Retention time

Resolution

Number of theoretical plates

Recovery rate

Parameter 33

Column oven temperature (◦ )

Flow rates (mL min-1)

35

37

0.9

1.0

1.1

1

2

(S)-oxiracetam Imp-1 Imp-2 Imp-3 Imp-4 Imp-1 Imp-2 Imp-3 Imp-4 Imp-1 Imp-2 Imp-3 Imp-4 Imp-1 Imp-2 Imp-3 Imp-4

6.950 6.493 6.101 10.452 9.478 1.284 3.831 1.668 4.42 9507 9364 11265 4177 101.39 115.44 94.86 123.96

6.858 6.412 6.028 10.297 9.391 1.285 3.939 1.583 4.468 9470 9789 11255 4141 96.27 90.47 99.85 103.99

6.850 6.418 6.028 10.213 9.468 1.286 3.928 1.367 4.673 9140 9140 13091 4421 93.56 107.90 75.87 111.37

7.834 7.290 6.857 11.889 10.569 1.275 4.066 1.895 4.100 9425 9754 10221 3504 94.52 99.20 96.71 123.20

7.062 6.587 6.198 10.722 9.543 1.259 4.012 1.958 4.251 9379 9663 10745 3925 96.14 99.22 97.55 105.11

6.375 5.948 5.594 9.677 8.609 1.240 3.909 1.910 4.161 8899 9274 9796 3805 101.28 105.96 100.57 99.27

7.062 6.587 6.198 10.722 9.543 1.259 4.012 1.958 4.251 9379 9663 10745 3925 96.14 99.22 97.55 105.11

requirements of the verification scheme (not less than 1.5). Therefore, the optimized analytical method described above is specific for (S)-oxiracetam and four related substances, which may produce as impurities during the synthesis process. In addition, the forced degradation study was performed on the bulk drug of (S)-oxiracetam to demonstrate the stability properties and specificity of the proposed method. Deliberate degradation of bulk drugs under different conditions was evaluated to determine the specificity of the optimized method to detect (S)oxiracetam from its degradation productions (Fig. 4 and Table 7), including acid, base, oxidant, thermal and photodegradation tests. All the results shown above indicate that even if oxiracetam is present or separated from the relevant substances, the method developed in this study can also quantitatively determine (S)oxiracetam, which shows the specificity of the determination of (S)-oxiracetam.

3.2.3. Sensitivity For known impurities, the limits of detection (LOD) and the limits of quantitation (LOQ) are determined by the signal to noise ratio

Column

6.533 6.140 5.816 9.561 8.728 0.951 0.951 1.355 3.715 6684 7018 6531 3719 90.19 91.24 86.15 96.39

method. The LOD and LOQ were examined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with decreasing concentrations. The known concentration of the impurity control solution is diluted to a low concentration, and the measured signal is compared with the signal at the blank (baseline noise) to calculate the lowest concentration or percentage that can be reliably detected. The results of the limits of detection and the limits of quantitation of such related substances were shown in Table 8.

3.2.4. Linearity and range To determine the linearity of the optimized method, a series of (S)-oxiracetam and such related substances standard solutions with concentrations from about LOQ level to 150 % of the specification level were injected into the HPLC system, respectively. The linear relationship is plotted by the measured corresponding signal (peak area) as a function of analyte concentration. Linear regression is performed by least-squares linear regression analysis, and the correlation coefficient R2 is used to confirm a good linearity. The value of the linear regression coefficient R2 is required to be greater than 0.990. The results show that there is a good linear rela-

C. Wang, H. Dong, H. Liu et al. / Journal of Pharmaceutical and Biomedical Analysis 180 (2020) 113072

tionship between peak area and concentration, and the correlation coefficient (R2 ) is between 0.9991 and 0.9998 (Table 8). 3.2.5. Accuracy Accuracy refers to the recovery measured by adding 80 %, 100 %, and 120 % of three different concentrations of each impurity to the test sample respectively. The accuracy of the known impurities is obtained by adding a known amount of impurities, and then calculating the ratio between the measured result and the theoretical value of the known impurity in the sample. The recovery of unknown impurities was determined by replacing (S)-oxiracetam with unknown impurities. The recovery rate is required to be between 70.0 % and 130.0 % to confirm that the method has good accuracy. The test results are shown in the table below (Table 8). 3.2.6. Precision Precision, i.e. repeatability, was verified by preparing 6 sample solutions and testing each solution with 1 injection. It is required that the number of impurity peaks is the same in the six measurement results to confirm that the method has a good precision. Test results are shown in the Table 8, indicating that the method we developed has highly precision. 3.2.7. Robustness The robustness of the optimized method was investigated by changing the flow rate of mobile phase of 10 % (1.0 ± 0.1 ml / min), the column temperature of 2 ◦ (35 ± 2 ◦ ) and different batches of Capcell Pak NH2 . There was no significant difference between normal and changed conditions in all minor but design changes in chromatographic conditions (Table 9). Therefore, the robustness of the developed method can be confirmed and also its reliability in normal use can be demonstrated. 4. Conclusion In this study, a specific analytical method for analyzing (S)oxiracetam and four related impurities in the bulk drug of (S)-oxiracetam by using HPLC was developed. This method has been validated in system suitability, specificity, sensitivity, linearity, accuracy, precision and robustness. As a supplement of this study, the molecular weight and chemical structure preliminarily speculated of the four related substances were characterized by LC–MS and forced degradation studies of (S)-oxiracetam were conducted to further confirm the specificity of the optimized method. In conclusion, this study reports an effective HPLC determination method applied for the quality control and stability monitor purposes of the bulk drug of (S)-oxiracetam in the routine pharmaceutical analysis. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This work was supported by National Key R&D Program of China (2017YFA0205400), National Natural Science Foundation

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of China (No. 31872755, 81872811) and the Central Fundamental Research Funds for the Central Universities (021414380447, 02141480608201). References [1] M. Kometani, M. Okada, E. Takemori, Y. Hasegawa, N. Nakao, T. Inukai, Effect of oxiracetam on cerebrovascular impairment in rats, Arzneimittelforschung 41 (7) (1991) 684–689. [2] G. Bottini, G. Vallar, S. Cappa, G.C. Monza, E. Scarpini, P. Baron, A. Cheldi, G. Scarlato, Oxiracetam in dementia: a double-blind, placebo-controlled study, Acta Neurol. Scand. 86 (3) (1992) 237–241, http://dx.doi.org/10.1111/j.16000404.1992.tb05077.x. [3] B. Baumel, L. Eisner, M. Karukin, R. MacNamara, R.J. Katz, J. Deveaugh-Geiss, Oxiracetam in the treatment of multi-infarct dementia, Prog. Neuropsychopharmacol. Biol. Psychiatry 13 (5) (1989) 673–682, http://dx.doi. org/10.1016/0278-5846(89)90054-7. [4] Z. Hlinak, I. Krejci, Oxiracetam pre- but not post-treatment prevented social recognition deficits produced with trimethyltin in rats, Behav. Brain Res. 161 (2) (2005) 213–219, http://dx.doi.org/10.1016/j.bbr.2005.02.030. [5] W. Li, H. Liu, H. Jiang, C. Wang, Y. Guo, Y. Sun, X. Zhao, X. Xiong, X. Zhang, K. Zhang, Z. Nie, X. Pu, (S)-Oxiracetam is the active ingredient in Oxiracetam that alleviates the cognitive impairment induced by chronic cerebral hypoperfusion in rats, Sci. Rep. 7 (1) (2017) 10052, http://dx.doi.org/10.1038/ s41598-017-10283-4. [6] http://www.chinadrugtrials.org.cn/eap/clinicaltrials. searchlist?keywords=CTR20170517, Registrati-on number CTR20170517, China Drug Clinical Trial Registration and Information Publicity Platform (Accessed 28 August 2017). [7] M. Visconti, R. Spalluto, T. Crolla, G. Pifferi, M. Pinza, Determination of oxiracetam in human serum and urine by high-performance liquid chromatography, J. Chromatogr. 416 (2) (1987) 433–438, http://dx.doi.org/10. 1016/0378-4347(87)80532-7. [8] R.C. Simpson, V.K. Boppana, B.Y. Hwang, G.R. Rhodes, Determination of oxiracetam in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection, J. Chromatogr. 631 (1-2) (1993) 227–232, http://dx.doi.org/10.1016/0021-9673(93)80526-E. [9] J.B. Lecaillon, C. Souppart, F. Le Duigou, J.P. Dubois, Determination of oxiracetam in plasma and urine by column-switching high-performance liquid chromatography, J. Chromatogr. 497 (1989) 223–230, http://dx.doi.org/ 10.1016/0378-4347(89)80021-0. [10] Q. Zhang, W. Yang, Q. Zhang, Y. Yang, J. Li, Y. Lu, Y. Zheng, J. He, D. Zhao, X. Chen, Enantioselective HPLC determination of oxiracetam enantiomers and application to a pharmacokinetic study in beagle dogs, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 993-994 (2015) 9–13, http://dx.doi.org/10. 1016/j.jchromb.2015.04.033. [11] Q. Zhang, W. Yang, Y. Yang, H. Xing, Q. Zhang, J. Li, Y. Lu, J. He, S. Yang, D. Zhao, X. Chen, Comparative pharmacokinetic studies of racemic oxiracetam and its pure enantiomers after oral administration in rats by a stereoselective HPLC method, J. Pharm. Biomed. Anal. 111 (2015) 153–158, http://dx.doi.org/10. 1016/j.jpba.2015.03.039. [12] J. Son, J. Lee, M. Lee, E. Lee, K.T. Lee, S. La, D.H. Kim, Rapid quantitative analysis of oxiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry, J. Pharm. Biomed. Anal. 36 (1) (2004) 183–187, http://dx.doi.org/10.1016/j.jpba.2004.07.031. [13] S. Gorog, Drug safety, drug quality, drug analysis, J. Pharm. Biomed. Anal. 48 (2) (2008) 247–253, http://dx.doi.org/10.1016/j.jpba.2007.10.038. [14] Q. Li, Y. Huang, Y. Zhang, H. Cui, L. Yin, Y. Li, A. Yuan, Y. Hu, J. Wu, A novel HPLC method for analysis of atosiban and its five related substances in atosiban acetate injection, J. Pharm. Biomed. Anal. 177 (2019), 112808, http:// dx.doi.org/10.1016/j.jpba.2019.112808. [15] D. Wang, S. Wu, Development and validation of a RP-HPLC method for the simultaneous determination of aceglutamide and oxiracetam in an injection formulation, J. Chromatogr. Sci. 53 (5) (2015) 767–770, http://dx.doi.org/10. 1093/chromsci/bmu123. [16] L. Gagliardi, D.d. Orsi, G. Cavazzutti, D. Tonelli, S. Zappoli, HPLC determination of oxiracetam, its impurities, and piracetam in pharmaceutical formulations, Anal. Lett. 27 (5) (1994) 879–885.