Simultaneous determination of some illegal antihypertensive and diuretic drugs in traditional herbal preparations by HPLC-DAD

Research Article
99Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and 
diuretic drugs in traditional herbal preparations by HPLC-DAD
Pham Van Hung1*, Tran Cao Son2, Nguyen Thi Kieu Anh3
1Yen Bai Drug, Cosmetics and Food Quality Control Center, Yen Bai, Vietnam
2National Institute for Food Control, Hanoi, Vietnam
 3Hanoi University of Pharmacy, Hanoi, Vietnam
(Received: 06/03/2021; Accepted: 27/04/2021)
Abstract
A simple, stable, and specific high-performance liquid chromatography coupled with a 
DAD detector (HPLC-DAD) method has been developed and validated for the simultaneous 
determination of amlodipine, felodipine, furosemide, nifedipine, and spironolactone in 
traditional herbal products. The analytes were extracted in acetonitrile: water (50 : 50, v/v) with 
help of the ultrasonic. The separation of analytes was performed in an Apollo C18 column (250 
× 4.6 mm; 5 µm) and a mobile phase consisting of mixture acetonitrile: 0.1% phosphoric acid in 
gradient elution. The analyzed drugs were detected at 238 nm. The method was validated according 
to the AOAC International guidelines concerning specificity, linearity, precision (repeatability, 
intermediate precision), accuracy, limit of detection (LOD), and limit of quantification (LOQ). 
The method can detect the studied drugs at the concentration of 0.66 to 1.25 µg/g for dry 
samples and 0.10 to 0.24 µg/mL for liquid samples. The method was successfully applied in the 
analysis of 17 samples in the local market. No samples were found positive for the substances to 
be analyzed.
 Keywords: amlodipine, felodipine, nifedipine, furosemide, spironolactone, traditional 
herbal products.
1. INTRODUCTION
Hypertension is a common chronic medical condition, and it is the cause of many 
cardiovascular complications such as cerebrovascular accidents, myocardial infarction, 
arrhythmia, and heart failure. In addition to the use of pharmaceutical drugs in treatment, many 
traditional herbal products have been used by hypertensive patients because they believe that 
their therapeutic effects are more sustainable and safer than chemical drugs. However, to increase 
the effectiveness of treatment, some manufacturers have mixed chemical drugs into herbal 
products. There have been several studies to detect chemical drugs mixed in antihypertensive 
products such as nifedipine, hydrochlorothiazide, atenolol,... [1-4].
Amlodipine, felodipine, nifedipine, furosemide, spironolactone are drugs with 
antihypertensive and diuretic effects commonly used in the treatment of hypertension today 
with many products circulating on the market, at low cost. Therefore, these drugs are likely to 
be illegally mixed into herbal products. When using these products, with uncontrolled doses 
for a long time, users may experience unwanted effects such as heart rhythm disturbances, 
*Corresponding author: Tel: 0967033804 Email: phamhungknyb@gmail.com
100 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and diuretic...
electrolyte imbalance, digestive disorders,... [5]. Therefore, the detection of substances with 
antihypertensive and diuretic effects mixed in herbal products is an urgent matter to protect the 
customer’s health. 
This study was conducted to develop and validate a method to simultaneously detect five 
drugs with antihypertensive and diuretic effects including amlodipine, felodipine, nifedipine, 
furosemide, and spironolactone in herbal products by high-performance liquid chromatography 
coupled with a diode array detector (HPLC-DAD). Our study would contribute to the quality 
control and supervision of health products derived from medicinal herbs to treat or support the 
treatment of hypertension.
2. MATERIALS AND METHODS 
2.1. Samples
The samples of our study consist of two forms: liquid and dry glue samples that were 
prepared from folk remedy for antihypertensive and diuretic treatment. The remedies usually 
include the main drugs that meet the principles of antihypertensive treatment according to 
traditional medicine such as Morinda Citrifolia (lower blood pressure); Ziziphus Mauritiana 
LanK (sedation); Alisma Plantago-Aquatica, Achyranthes Bidentata (diuretics), and 
Styphnolobium Japonicum L Schott (strengthen the vessel walls) [6]. The composition of the 
drugs used to prepare the research sample matrix is presented in Table 1.
Table 1. Compositions of the drug matrix
Drug remedy Compositions
Six components 
remedy
Dioscorea Batatas 16 g, Cortex moutan 12 g, Rehmannia glutinosa 32 
g, Cornus Officinalis Sieb et Zuce 16g, Alisma Plantago-aquatica 12 g, 
Poria cocos (Schw.) Wolf-polyporaceae 12 g.
Lower blood 
pressure remedy
Morinda citrifolia 20 g, Rehmannia glutinosa 20 g, Plantago major 20 
g, Styphnolobium Japonicum L. Schott 10 g, Achyranthes bidentata 10 
g, Ali ... hased from Hanoi University of Pharmacy. 
Acetonitrile, ethanol, methanol (HPLC grade), kali dihydro phosphate, and acid phosphoric 
(analytical grade) were obtained from Merck, German.
101Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Pham Van Hung, Tran Cao Son, Nguyen Thi Kieu Anh
2.3. Method
2.3.1. Optimization of the analytical method
Sample preparation: After being homogenized, 1.0 g for the solid glue sample or 5.0 mL 
for the liquid glue sample was transferred into a 25 mL volumetric flask. A portion of 15 mL 
extraction solvent was added into the volumetric flask, then it is sonicated for 15 minutes at 
room temperature. After that, the extraction solvent was added up to the mark of the volumetric 
flask. The solution was mixed well and centrifuged at the speed of 6,000 rpm for 10 minutes. The 
extraction was filtered through a 0.45 µm filter. In our study, we tested four extraction solvents 
including acetonitrile, methanol, acetonitrile: water (50 : 50, v/v), methanol: water (50 : 50, v/v). 
The most effective solvent giving the best peak shape would be chosen as the extraction solvent.
Chromatography conditions: Based on previous studies [1, 8-9], we investigated five reversed 
chromatography columns including Inertsil ODS-3 (C18, 250 × 4.6 mm; 5 µm), InertSustain - 
AQ1 (C18, 250 × 4.6 mm; 5µm), Eclipse XBD - C18 (250 × 4.6 mm; 5 µm), and Apollo - C18 
(250 mm × 4.6 mm, 5 µm). We also tested different mobile phases and gradients to optimized 
the separation of five analytes.
2.3.2. Method validation
The method was validated according to the AOAC 2016 guidelines [7] for selectivity, 
linear concentration range, accuracy and repeatability, the limit of quantification, and the limit 
of detection.
3. RESULTS AND DISCUSSIONS 
3.1. Chromatography conditions
Based on references [1, 8-9], we selected reversed-phase liquid chromatography with 
column C18 to analyze AML, FEL, NIF, FUR, and SPI in the sample. Using acetonitrile (ACN) 
and 0.1% phosphoric acid solution, analyze according to the gradient mode to be able to elute 
the analytes in the test sample in a suitable time (Figure 1).
Figure 1. Chromatogram of five analytes (AML, FEL, NIF, FUR, SPI) 
 with the mobile phases of acetonitrile and 0.01% acid phosphoric (gradient)
We carried out UV-Vis spectroscopic scanning of each research substance to determine 
the maximum absorption peak wavelength. The results showed that five analytes all had one 
absorption maximum in the range from 236 to 240 nm. At this wavelength, the absorption 
102 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and diuretic...
signal is the largest. Therefore, the wavelength of 238 nm was selected as the wavelength for 
quantitative analysis.
We investigate sample injection volumes from 20 to 60 µL of mixed standard solution at 
a concentration of 10 µg/mL. At different injection volumes, all the peaks had compact shapes 
and good resolution. However, as the injection volume increased, the resolution value between 
the FUR and AML peaks decreased. Therefore, the study selected an injection volume of 50 µL 
to improve the ability to detect substances in herbal products. From the obtained survey results, 
the analysis conditions were selected as follows:
Mobile phase A was ACN and mobile phase B was 0.1% phosphoric acid solution. The 
gradient program was: A - 30% (0.0 - 10.0 min), increase A from 30 to 60% (10.0 - 20.0 min), 
A - 60% (20.0 - 32.0 min), decrease A from 60 to 30% (32.0 - 33.0 min), A - 30% (33.0 - 38.0 
min). The chromatography column was Apolo (4.6 mm × 25 cm; 5µm); column temperature was 
set at 30 oC; flow rate was 1.0 mL/min; the injection volume was 50 µL; the detection wavelength 
was 238 nm.
3.2. Extraction
Based on the solubility of substances, we have investigated the extraction solvents: 
methanol, acetonitrile, acetonitrile: water (50 : 50, v/v), methanol: water (50 : 50, v/v). The survey 
results showed that the two solvents acetonitrile and methanol were not suitable for the method 
because the peaks of the analytes are not balanced. Two extraction solvents acetonitrile: water 
(50 : 50, v/v) and methanol: water (50 : 50, v/v) both gave balanced peaks, the extraction efficiency 
on solid glue sample matrix was from 94.2 to 97.5%; on liquid glue sample background, the 
solvent acetonitrile: water (50 : 50, v/v) gave higher extraction efficiency. Therefore, acetonitrile: 
water (50 : 50, v/v) was chosen as the extraction solvent. The results of the survey were shown 
in Figure 2.
Figure 2. The extraction efficiency of acetonitrile: water (50 : 50, v/v) and methanol: 
 water (50 : 50, v/v) in the solid (A) and liquid (B) glue sample matrix
3.3. Validation
3.3.1. Specificity
We analyzed the matrix sample, the spiked sample, and the standard sample under the 
103Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Pham Van Hung, Tran Cao Son, Nguyen Thi Kieu Anh
selected optimal conditions. The results showed that the chromatograms of the spiked sample 
appear peaks corresponding to the positions of the analytes on the chromatograms of the mixed 
standard solutions. On the chromatograms of the matrix samples, no peaks appeared at positions 
corresponding to the research substances. The spectral superposition coefficient and sample 
purity factor were both greater than 0.99. Thus, the analytical method ensures specificity. Figure 
3 showed the chromatogram to evaluate the specificity of the method for the liquid matrix.
Figure 3. Chromatograms of blank matrix sample, standard sample, and spiked sample
3.3.2. Linearity
We analyzed mixed standard solutions of the studied substances at ten different 
104 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and diuretic...
concentrations and established the correlation between the peak area and the concentration of 
the studied substances, the results were presented in Figure 4.
Figure 4. Calibration curves of the analytes
Figure 4 showed that in the tested concentration AML, FEL, NIF, FUR, SPI have a linear 
correlation between peak area and concentration, the regression coefficients of all the analytes 
were greater than 0.99.
3.3.3. Accuracy and repeatability
The repeatability and precision were obtained by analyzing the spiked sample at three 
concentrations of low, medium, and high with seven replicates. Based on the peak areas of the 
standard addition samples (spiked sample) and the calibration curve constructed on the same 
day, the recovery of substances can be calculated. The repeatability was assessed through the 
relative standard deviation (RSD %). The results were presented in Table 2.
Table 2. Accuracy and repeatability of the method
Concen-
tration 
(µg/mL)
Solid glue sample Liquid glue sample
% Recovery
RSD
% Recovery
RSD
Min Medium Max Min Medium Max
Furosemide
7.13 101.1 101.9 103.5 1.35 101.2 101.9 103.2 1.11
11.14 99.1 102.2 107.9 3.13 97.8 101.2 105.4 3.27
16.04 98.4 99.7 101.6 1.71 100.6 100.9 101.4 0.43
Amlodipine besylate
6.88 101.6 102.3 103.4 0.95 102.1 102.6 103.8 0.95
10.75 99.4 100.9 104.2 2.06 98.4 99.5 103.2 1.72
15.47 98.3 99.8 101.8 1.81 101.9 102.1 102.6 0.40
105Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Pham Van Hung, Tran Cao Son, Nguyen Thi Kieu Anh
Nifedipine
6.95 100.2 100.5 101.0 0.42 98.4 99.3 100.8 1.32
10.87 98.9 99.3 100.8 0.85 96.9 97.9 98.5 0.63
15.65 96.4 97.8 99.8 1.80 99.0 99.3 99.9 0.48
Spironolactone
7.03 103.2 103.6 104.5 0.72 103.5 104.3 105.7 1.15
10.99 99.0 100.4 101.8 0.90 97.9 99.1 100.1 0.71
15.82 99.6 100.9 102.9 1.73 103.4 103.8 104.1 0.40
Felodipine
6.86 101.2 101.5 101.9 0.42 101.5 102.2 103.6 1.21
10.72 98.0 99.4 100.4 0.80 97.8 98.5 99.7 0.63
15.43 97.9 99.2 101.2 1.77 101.3 101.7 102.1 0.38
It could be seen from Table 2 that for both solid and liquid glue matrix, three concentration 
levels, all analytes gave recoveries from 96.4 - 107.9%, and RSD in the range of 0.42 - 3.27% that 
meet the requirement of AOAC 2016 (recovery 80 - 115%, and RSD < 4.0%).
3.3.4. Limit of detection and limit of quantification
We use the method of determining the standard deviation on a standard addition basis to 
evaluate the LOD. LOQ: analyze the test sample ten times according to the established procedure, 
calculate the mean and the standard deviation (SD). From there, calculate LOD as three times of 
SD and LOQ as ten times of SD. The results obtained are as follows:
Limits of detection (LOD) of the investigated substances ranged from 0.03 to 0.05 µg/mL, 
equivalent to 0.66 to 1.25 µg/g of the dry sample and 0.10 to 0.24 µg/mL of the liquid sample. 
The limit of quantification (LOQ) ranged from 0.14 to 0.17 µg/mL equivalent to 2.21 - 4.18 µg/g 
dry sample and 0.48 to 0.80 µg/mL liquid sample. R values were between 5.6 - 9.5 indicate LOD 
and LOQ were reliable.
3.4. Analyze real sample
This procedure was applied to analyze 17 samples of herbal products currently circulating 
in the local market, including four samples of tablets, six samples of capsules, two samples 
of a liquid solution, two samples of nuggets powder, one sample of hard pellets, and two tea 
bags samples. Of which, 12 samples have indications for treatment and support for high blood 
pressure, and five samples have liver-cooling and diuretic effects.
The results of analysis of the real samples showed that 14/17 samples did not detect peaks 
corresponding to the retention time of all five analytes on the chromatogram; two samples 
appeared a peak with the same retention time as spironolactone; one sample showed a peak 
with the same retention time as felodipine. We have performed UV-VIS superimposition of the 
suspect peaks against the standard peak. The results were illustrated in Figure 5.
106 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and diuretic...
Figure 5. Chromatograms of THA11 and TLT5 sample; UV-VIS superimposition 
 of the suspect peaks against the SPI and FEL standard peaks 
The results showed that the test spectral shapes did not coincide with the standard spectral 
shapes. The spectral overlap coefficients were 20.28, 518.37, and 692.31. So the suspect peaks 
were not the active substance under analysis.
107Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Pham Van Hung, Tran Cao Son, Nguyen Thi Kieu Anh
4. CONCLUSION
A method for simultaneous determination of amlodipine, felodipine, furosemide, 
nifedipine, and spironolactone mixed in herbal products for the treatment or support 
of hypertension and diuresis on solid and liquid glue samples by HPLC-DAD has been 
successfully developed. The method has a linear range from 0.20 to 20 µg/mL, correlation 
coefficient R2 > 0.997; high sensitivity with LOD from 0.03 to 0.05 µg/mL, the accuracy and 
precision met the requirements with the recovery from 96.4 to 107.9% and the RSD of 0.42 to 
3.77%. Analysis on 17 real samples showed that there was no sample containing amlodipine, 
felodipine, furosemide. nifedipine and spironolactone.
ACKNOWLEDGMENT  
This study was supported by funding from the Ministry of Health’s project “Development of 
a method for simultaneous analysis of some new drugs of the antihistamine and antihypertensive 
groups illegally mixed in herbal preparations using HPTLC, HPLC, and LC-MS/MS” period 
2021 - 2022.
REFERENCES
[1]. S. H. Geum, J. Y. Ji, Y. Choi, H. J. Park, S-K. Park, and S. Y. Baek, “A rapid method for the 
simultaneous determination of 25 anti-hypertensive compounds in dietary supplements 
using ultra-high-pressure liquid chromatography,” Food Additive Contaminant. Part A 
Chemistry Analysis Control Exposure Risk Assessesment, vol. 33, no.11, pp. 1627-1636, 2016.
[2]. J. R. Kesting, J. Huang, and D. Sørensen, “Identification of adulterants in a Chinese herbal 
medicine by LC-HRMS and LC-MS-SPE/NMR and comparative in vivo study with 
standards in a hypertensive rat model,” Journal of Pharmaceutical Biomedical Analysis, vol. 
51, no. 3, pp. 705-711, 2010.
[3]. Y. L. Lu, N. L. Zhou, S. Y. Liao, N. Su, D. X. He, Q. Q. Tian, B. Chen, and S. Z. Yao, “Detection 
of adulteration of anti-hypertension dietary supplements and traditional Chinese medicines 
with synthetic drugs using LC/MS,” Food Additive Contaminant. Part A Chemistry Analysis 
Control Exposure Risk Assessesment, vol. 27, no. 7, pp. 893-902, 2010.
[4]. A. P. L. Moreira, L. A. Gobo, C. Viana, and  Leandro Machado de Carvalho, “Simultaneous 
analysis of antihypertensive drugs and diuretics as adulterants in herbal-based products by 
ultra-high performance liquid chromatography-electrospray tandem mass spectrometry,” 
Analytical Methods, vol. 8, no. 8, pp. 1881-1888, 2016.
[5]. Ministry of Health, Vietnam National Pharmacopoeia. Hanoi: Medical Publisher, 2018.
[6]. Ministry of Health, Pathology and medical treatment (Combining Eastern - Western 
medicine). Hanoi: Medical Publisher, 2007, pp. 9-33.
[7]. AOAC International, Guidelines for Dietary Supplements and Botanicals: Appendix K, 2016.
[8]. Ministry of Health, Pharmacopoeia of Vietnam. Hanoi: Medical Publisher, 2017.
[9]. The United States Pharmacopoeia 40. 2017. 
108 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Simultaneous determination of some illegal antihypertensive and diuretic...
Xác định đồng thời một số thuốc hạ huyết áp và lợi tiểu trộn 
 trái phép trong các chế phẩm đông dược bằng HPLC-DAD
Phạm Văn Hùng1, Trần Cao Sơn2, Nguyễn Thị Kiều Anh3
1Trung tâm Kiểm nghiệm Thuốc - Mỹ Phẩm - Thực Phẩm tỉnh Yên Bái, Yên Bái, Việt Nam
2Viện Kiểm nghiệm an toàn vệ sinh thực phẩm Quốc gia, Hà Nội, Việt Nam
 3Trường Đại học Dược Hà Nội, Hà Nội, Việt Nam
Tóm tắt 
Nghiên cứu sử dụng phương pháp HPLC kết hợp detector DAD để xác định đồng thời 
amlodipine, felodipine, furosemide, nifedipine và spironolacton trộn trong chế phẩm có nguồn 
gốc thảo dược. Mẫu phân tích được chiết siêu âm trong dung môi acetonitril : nước tỷ lệ (50 : 50, 
v/v) và phân tích trên cột Apollo C18 (250 × 4,6 mm; 5 µm). pha động gồm acetonitril và acid 
phosphoric 0.1% theo chương trình gradient. Bước sóng phân tích là 238 nm. Phương pháp đã 
được thẩm định theo hướng dẫn của AOAC về độ chọn lọc, khoảng tuyến tính, độ đúng, độ lặp 
lại, LOD và LOQ. Phương pháp có thể xác định các chất nghiên cứu ở hàm lượng từ 0,66 - 1,25 
µg/g đối với mẫu rắn và từ 0,10 - 0,24 µg/mL đối với mẫu lỏng. Ứng dụng quy trình đã xây dựng 
để xác định đồng thời amlodipine, felodipine, furosemide, nifedipine và spironolacton trong 17 
mẫu đang lưu hành trên thị trường, không phát hiện mẫu nào dương tính.
Từ khóa: amlodipin, felodipin, nifedipin, furosemid, spironolacton, chế phẩm đông dược, 
HPLC - DAD.