© Wuhan University 2023

0 Introduction

Indapamide is a thiazide-like drug which can achieve diuretic effect by inhibiting the cotransport of sodium chloride in the cortical diluting segment of tubule and the membrane bounding of carbonic anhydrase ^[1]. It has a strong antihypertensive effect and is used in the treatment of adult primary hypertension^[2]. Indapamide preparations include ordinary tablets, capsules and sustained-release tablets. Compared with ordinary tablets and capsules, indapamide sustained-release tablets have advantages of low treatment dose, significant curative effect, high metabolic exclusion rate, long-term medication for hypertension patients, and has same effect as amlodipine, candesartan and enalapril ^[1,3]. The formulation of sustained-release tablets was first developed and produced by Les Laboratories Servier with a trademark of Narilix®. At present, a number of pharmaceutical companies produce generic indapamide sustained-release tablets around the world (Arrotex Pharmaceuticals, Australia:https://apotex.com.au/products/apotex-prescription/; Eurofarma Laboratórios S.A. Brasil:https://www.eurofarma.com.br/produto/indapamida/; Drug International Limited, Bangladesh:https://www.drug-international.com/Web/singleProduct/999; Huangshan Zhonghuang Pharmaceutical Co. Ltd., China:http://www.zhpharm.cn/product.asp).

The type and concentration of impurities in drugs will reduce or change the efficacy of the drug, and even affect the drug safety^[4]. In particular, the genotoxic impurities may directly or indirectly damage cell DNA genetic material, induce genetic gene mutation or cause cancer and tumor. The most obvious feature of these impurities is that even if the concentration is very low, it can still cause structural damage and genetic information changes or deletion to DNA genetic material in the human body, resulting in the mutation of human body gene and the increase of the incidence rate of normal human tumor^{[5, 6]}. Therefore, the contents of impurities in drug play a very important role in drug quality control and quality consistency evaluation of generic drugs, etc. The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has also made clear requirements for drug impurity research^{[7, 8]}.

The monograph of indapamide sustained-release tablets has been listed in British Pharmacopoeia (BP), but not in Chinese Pharmacopoeia (ChP), United States Pharmacopoeia (USP) and other pharmacopoeias^[9-11]. In the tests section of the indapamide sustained-release tablets monograph of BP, two specific impurities are listed, namely 4-chloro-N-(2-methyl-1H-indol-1-yl)-3-sulphamoyl-benzamide (Impurity B, denoted as ImB) and 4-chloro-3-sulfamoylbenzoic acid (denoted as Im1) which are tested at the test high performance liquid chromatography (HPLC) conditions^[9]. In recent years, Kaddah^[12], Gumieniczek^[13], Pilard^[14] and other research groups have studied the stability of indapamide under different conditions, such as heat and humidity, light, oxidation, acid-base, etc., referring to ICH Q1A (R2) and Q1B impurity research guidelines^{[7, 8]}, and obtained the possible degradation products. In order to avoid the potential danger of impurities in indapamide sustained-release tablets on patients with hypertension and guide the consistency evaluation of generic drugs, it is necessary to control the limits of the related impurities of generic indapamide sustained-release tablets, which has been seldom reported as we know.

Referring to the quality standards of indapamide, indapamide tablets and indapamide sustained-release tablets in BP, USP, ChP and other literatures^[15-17], the contents of indapamide and the related impurities in generic indapamide sustained-release tablets, including 2-methyl-1-nitroso-2,3-dihydro-1H-indole (Impurity A, denoted as ImA), ImB, Im1 and other unspecified impurities, were firstly detected simultaneously by a single-run high performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) method in this paper. The results provided references for the quality control, consistency evaluation and quality standards study of generic indapamide sustained-release tablets.

1 Experimental

1.1 Materials and Reagents

Generic indapamide sustained-release tablets (labeled amount: 1.5 mg) were produced by Huangshan Zhonghuang Pharmaceutical Co., Ltd. Indapamide reference substances were purchased from National Institute of Food and Drug Control of China. Im1 was purchased from Shanghai Chemical Reagent Co., Ltd. Reference substances of ImA and ImB were purchased from Shandong Bolode Bio-technology Co., Ltd. The excipients and prescription of indapamide sustained-release tablets were provided by Huangshan Zhonghuang Pharmaceutical Co., Ltd. Acetonitrile (sigma HPLC), ethanol (sigma HPLC), 2-butanol, triethylamine, sodium dodecylsulfate, glacial acetic acid and phosphoric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

1.2 Apparatus and HPLC Conditions

The EssentiaLC-16 HPLC system (Shimadzu) consisted of an LC-20AT pump, a WondaCract ODS-2 column (150 mm×4.6 mm, 5 µm) and an SPD-M20A photodiode array (PDA) UV-visible detector. AL104 electronic analytical balance (Mettler Toledo) and EX125DZH electronic analytical balance (Ohaus Instruments (Shanghai) Co., Ltd.) were used as the weighing instruments. Water for experiments (resistivity minimum 18.2 MΩ) was produced by a Milli-Q advantage ultra-pure water system (Merck Millipore).

The isocratic elution was carried out with a mixed solution as the mobile phase, which was composed of water, acetonitrile, 2-butanol, triethylamine and sodium dodecylsulfate solution (5 g sodium dodecylsulfate, 3 mL glacial acetic acid, 100 mL water, and shaken well) with a volumetric ratio of 690:310:20:10:6. The pH of mobile phase was adjusted to 3.0 with phosphate acid and filtered through 0.22 μm microporous membrane before use. The detection wavelength was 240 nm, the flow rate was 1.6 mL/min, the column temperature was 30 ℃, and the injection volume was 20 μL.

1.3 Preparations

Standard stock solutions of indapamide, Im1, ImA and ImB, were prepared by dissolving standard reference substances accurately weighed in ethanol and diluted to 120.00, 6.00, 12.00 and 4.80 μg/mL with the mobile phase, respectively. The system suitability solution was prepared by mixing the accurately measured standard stock solutions of indapamide, Im1, ImA and ImB. The calibration solutions of these four standard substances with different concentrations were prepared by diluting the stock solutions with mobile phase step by step.

20 tablets of generic indapamide sustained-release tablets were accurately weighed and the total weight was 4.204 0 g. These tablets were ground to powder in a mortar. Test solutions and test reference solutions of generic indapamide sustained-release tablets were prepared as follow. A portion of powdered tablets was accurately weighed and put into a 50.00 mL volumetric flask. 30.00 mL ethanol was added into the volumetric flask which was shaken and ultrasonicated for 10 min for fully dissolving indapamide. Then, it was diluted with ethanol to the volume, well mixed, and filtered with microporous membrane. 4.00 mL of this solution precisely measured was diluted to 10.00 mL as the test solution. The test reference solution was prepared by diluting 1.00 mL of test solution to 100.00 mL with the mobile phase solution and mixing well.

Assay solutions were prepared similarly to the test solution. A portion of powdered tablets was accurately weighed and put into a 50.00 mL volumetric flask. 30.00 mL ethanol was added into the volumetric flask which was shaken and ultrasonicated for 10 min. After being diluted to the volume with ethanol, mixed well, and filtered with microporous membrane, 2.00 mL of this solution precisely measured was diluted to 50.00 mL as the assay solution.

The blank excipient solution was prepared according to the blank prescription (provided by Huangshan Zhonghuang Pharmaceutical Co., Ltd.). 1.000 0 g of blank excipients was accurately weighed and put into a 50.00 mL volumetric flask. The next steps were same with that of the test solution.

All these solutions were kept away from light at 4 ℃ for later use.

2 Results and Discussion

2.1 PDA Detection Wavelength

The UV absorption spectra of indapamide, Im1, ImA and ImB were shown in Fig. 1. It can be seen that the four compounds have strong absorption and medium intensity absorption at 210 and 240 nm respectively, and ImA also has a maximum absorption at 310 nm. The chromatographic noise signal of 210 nm detection wavelength was larger than that of 240 nm. Referring to BP, USP and ChP, 240 nm was selected as the detection wavelength in this experiment.

Fig. 1 UV absorption spectra of indapamide (a), Im1 (b), ImA (c) and ImB (d)

2.2 Three Dimensional Chromatogram and System Suitability Experiment

20.0 μL blank excipient solution, indapamide, Im1, ImA and ImB stock solutions, and system suitability solution were taken and injected into the liquid chromatograph, respectively. The three-dimensional and two-dimensional chromatograms were shown in Fig. 2 and Fig. 3.

Fig. 2 Three-dimensional chromatogram of system suitability experiment (a) Blank excipient solution; (b) Indapamide stock solution; (c) Im1 stock solution; (d) ImA stock solution; (e) ImB stock solution; (f) System suitability solution

Fig. 3 Two-dimensional chromatogram of system suitability experiment (a) Blank excipient solution; (b) Indapamide stock solution; (c) Im1 stock solution; (d) ImA stock solution; (e) ImB stock solution; (f) System suitability solution. Note: 1-Im1; 2-indapamide; 3-ImA; 4-ImB

As shown in Fig. 2 and Fig. 3, it can be seen that the excipients have no influence on the chromatographic peaks of other components. The smallest resolution was 6.677 and there were no interferences between each other. The theoretical plate numbers and resolutions of chromatographic peaks were shown in Table 1. It could be concluded that the chromatographic conditions were appropriate for the experiments.

2.3 Linear Relationship, Limit of Quantitations and Limit of Detections

The stock solutions of indapamide, Im1, ImA and ImB were precisely measured and diluted with mobile phase to prepare solutions with different concentrations. 20.0 μL of each solution was measured and injected into the chromatographic system. Under the same chromatographic conditions, the peak areas of each component at different concentrations were recorded.

The results were shown in Fig. 4 and Table 2. It can be seen that there are good linear relationship (A = a +bC, C (μg/mL) is the concentration, and A is the peak area), low limit of quantitations (LOQ) and low limit of detections (LOD).

Fig. 4 Linear regression curves of indapamide (a), Im1 (b), ImA (c) and ImB (d)

2.4 Simultaneous Analysis of Indapamide and Related Impurities

Test solutions and test reference solutions were precisely injected respectively for tests, and the chromatographic curves were obtained, two of which were shown in Fig. 5. It can be seen from Fig. 5(a) that there are no chromatographic peak signals of Im1 and ImA, namely, both the impurities are not detectable in the sustained-release tablets. But ImB is found in these samples.

Fig. 5 Chromatograms of test solution (a) and test reference solution (b) of generic indapamide sustained-release tablets 1-indapamide; 2-ImB; 3-Im3; 4-Im4; 5-Im5

In addition to ImB, there are other three unspecified impurities denoted as Im3, Im4 and Im5, which represent the peak 3, peak 4 and peak 5, respectively. These impurities in the preparation may be due to the introduction from the indapamide substances or the preparation

processes. Assay solutions were also precisely injected for the determination of indapamide.

The contents of indapamide and the impurities in the generic indapamide sustained-release tablets, and the limits standards in differernt pharmacopoeias were shown in Table 3. It can be seen that Im1 and ImA are not detectable. The contents of indapamide, Im3, Im4, Im5, ImB and the total impurities are 96.7%, 0.035%, 0.074%, 0.032%, 0.106% and 0.247% respectively, which are all compliant with BP and other pharmacopoeia reference contents or limits standards.

2.5 Sample Repeatability Experiments

Six samples of the drug powder were accurately weighed, and the sample solutions were prepared according to the test solution. Under the same chromatographic conditions, 20.0 μL sample solutions were injected in turn.

The mean content of ImB relative to the labeled amounts of indapamide was 0.106% with a relative standard deviation (RSD) of 1.82%. Then, six assay solutions were prepared and injected in turn. The mean content of indapamide was 96.7% of the labeled amount with an RSD of 1.30%. The results were shown in Table 4.

2.6 Sample Recovery Experiments

Six samples of the drug powder were accurately weighed, and a certain amount of ImB reference solution were accurately added in turn. The experimental solutions were prepared according to the method for the test solution, and 20.0 μL of each sample solutions was injected under the same chromatographic conditions. Then, another six sample solutions were prepared according to that of the assay solution and injected in turn. The recovery results were shown in Table 5 and Table 6. The average values of recovery of ImB and indapamide were 99.3% and 98.4% with an RSD of 1.36% and 1.48% respectively, which showed a high accuracy.

3 Conclusion

In this paper, the contents of indapamide and related impurities of generic indapamide sustained-release tablets was simultaneously detected by HPLC-PDA. The results showed that Im1 and ImA were not detectable in the generic indapamide sustained-release tablets, and the contents of indapamide, Im3, Im4, Im5, ImB and the total impurities were all met the reference quality standards of indapamide in BP, USP and ChP. The method was rapid, simple, reproducible, sensitive and accurate. This method and results can provide references for the content analysis of indapamide, the impurity profile analysis and the quality standard study of generic indapamide sustained-release tablets.

References

All Tables

All Figures

	Fig. 1 UV absorption spectra of indapamide (a), Im1 (b), ImA (c) and ImB (d)
In the text

	Fig. 2 Three-dimensional chromatogram of system suitability experiment (a) Blank excipient solution; (b) Indapamide stock solution; (c) Im1 stock solution; (d) ImA stock solution; (e) ImB stock solution; (f) System suitability solution
In the text

	Fig. 3 Two-dimensional chromatogram of system suitability experiment (a) Blank excipient solution; (b) Indapamide stock solution; (c) Im1 stock solution; (d) ImA stock solution; (e) ImB stock solution; (f) System suitability solution. Note: 1-Im1; 2-indapamide; 3-ImA; 4-ImB
In the text

	Fig. 4 Linear regression curves of indapamide (a), Im1 (b), ImA (c) and ImB (d)
In the text

	Fig. 5 Chromatograms of test solution (a) and test reference solution (b) of generic indapamide sustained-release tablets 1-indapamide; 2-ImB; 3-Im3; 4-Im4; 5-Im5
In the text