A simple, rapid, and selective HPLC-UV method was developed for the determination of antihypertensive drug substances: amlodipine besilat (AML), olmesartan medoxomil (OLM), valsartan (VAL), and hydrochlorothiazide (HCT) in pharmaceuticals and plasma. These substances are mostly used as combinations. The combinations are found in various forms, especially in current pharmaceuticals as threesome components: OLM, AML, and HCT (
Hypertension currently affects more than 1 billion adults worldwide, and by 2025, the projected estimate is 1.5 billion [
Chemical structures of analyzed drug substances.
The effective treatment of moderate or severe hypertension often requires the use of multiple antihypertensive agents from different drug classes [
The HPLC analyses were performed on a Thermo Separation Products Liquid Chromatograph (TX, USA) which consisted of a P4000 solvent delivery system equipped with a Rheodyne injection valve with a 20
AML, OLM, VAL, HCT, donepezil HCl, acetylcystein, metformin HCl, atorvastatin, naproxen sodium, and cilostazol were kindly supplied by Abdi Ibrahim Ilaç (Istanbul, Turkey). Tizanidine and telmisartan were obtained from Novartis. The pharmaceutical preparations of the investigated substances Sevikar HCT (40 mg olmesartan medoxomil, 10 mg amlodipine besilat, and 25 mg hydrochlorothiazide) and Exforge HCT (10 mg amlodipine besilat, 320 mg valsartan, and 25 mg hydrochlorothiazide) were obtained from a drug store. All chemicals and reagents were of an analytical grade.
Stock solutions of the drug substances (1 mg/mL) were prepared in methanol. Prior to measurements, stock solutions of AML, OLM, VAL, and HCT were diluted with acetonitrile-methanol-water (7 : 13 : 80, v/v/v) so as to prepare the working standard solutions of 100
Five tablets of each preparation (Sevikar HCT which includes
In order to develop a stability-indicating method, forced degradation (stress testing) is undertaken to demonstrate selectivity, particularly when little information is available about potential degradation products [
The selectivity of the proposed method for tablet analyses was determined by checking the peak purities of the related drug substances during the force degradation studies.
The stress conditions were as follows.
Each of the stressed solutions was diluted with the acetonitrile-methanol-water (7 : 13 : 80, v/v/v) to obtain a theoretical concentration of 1
Plasma sample collection and preparation: plasma samples were collected from 8 hypertensive patients after oral administration of the investigated antihipertansive drug substances. The main characteristics of the patients and the drugs they used are summarized in Table
Patients and administered drugs.
Patient |
Age | Gender | Druga and dose | Drugb substance | Time period after last administration | Coadministered drug | Coadministered drug substance and dose |
---|---|---|---|---|---|---|---|
1 | 79 | F | Co-Diovan |
VAL, HCT | 8 | Aricept | 5 mg dorepezil HCl |
2 | 52 | F | Norvasc |
AML | 12 | Mentopin |
200 mg acetylcysteine |
3 | 80 | F | Norvasc |
AML | 6 | Glukofen | 850 mg metformin HCl |
4 | 63 | M | Exforge |
AML, VAL | 8 | Ator | 10 mg atorvastatin |
5 | 71 | F | Diovan |
VAL | 8 | Apranax | 275 mg naproxen sodium |
6 | 65 | M | Hipersar Plus |
OLM, HCT | 7 | Lipitor | 20 mg atorvastatin |
7 | 74 | M | Sevikar HCT |
OLM, AML, HCT | 8.5 | Pletal | 10 mg cilestazol |
8 | 56 | F | Cardopan Plus |
VAL, HCT | 24 | Sirdalud | 2 mg tizanidine |
bThe active substances of the antihypertensive drugs.
Drug-free human plasma samples were obtained from the Blood Bank of Bezmialem Vakif University (Istanbul, TURKEY) and stored in polypropylene tubes at −20°C until analysis. Blood samples were collected into the tubes containing disodium EDTA (ethylenediaminetetraacetic acid) and centrifuged at 4500 rpm for 10 min. 1 mL of the resultant plasma was spiked with various concentrations of working solutions of the drug substances. Each plasma sample was basified with 0.5 mL of aqueous 0.1 M NaOH solution. Then, the analytes were extracted from plasma using 5 mL of n-hexane-ethylacetate-isoamyl alcohol (88 : 10 : 2, v/v/v) and vortex mixing for 2 min. The samples were centrifuged for 1 min at 1500 rpm. For each sample, the organic phase was transferred into another tube for evaporation at 45°C, under nitrogen, and the residue was dissolved in 0.5 mL of mobile phase solution. The samples were filtered through a 0.22 membrane filter before injection into the HPLC column. Plasma samples were quantified using the peak area of the analytes. Each plasma sample was analyzed for three times.
The validation of the method was carried out according to the guidelines given by the FDA [
Calibration curves were prepared by the analysis of 1 mL of human blank plasma samples spiked with various concentrations of working solutions of the drug substances. The samples were then submitted to the processes such as extraction, chromatographic separation, and UV detection described above. Calibration curves were obtained by linear least-squares regression analysis plotting of peak areas versus the concentrations. The calibration curve equation is
LOD was determined as the lowest concentration giving a signal to noise ratio (S/N) of 3 for all of the drug substances. LOQ, the lowest amount of analyte that can be quantified with acceptable precision and accuracy, was determined as S/N of 10.
Precision and accuracy of the method for intraday and interday plasma analyses were determined by studying with the QC (quality control) samples at three different concentration levels (low, medium, and high) for each drug. For intra-day investigation, six replicates of samples for each drug at each QC level were analyzed in the same day. Interday precision and accuracy values were determined by studying the samples every day during five consecutive days. Six replicates at each concentration were assayed per day.
Absolute recoveries of the drugs at three QC levels were measured by comparing the peak areas of each drug obtained from the plasma with peak areas obtained by the direct injection of pure aqueous drug standards. The relative recoveries of the drugs at three QC levels were calculated by comparing the found concentrations obtained from the drugs spiked with plasma to the actually added concentrations.
The stability of the working solution (in acetonitrile-methanol-water (7 : 13 : 80, v/v/v)) of each drug substance was tested at several storage conditions (at room temperature for 2 weeks and 4°C for 1 month). The stabilities of the drug substances in the extraction solvent were also investigated (at room temperature for 1 day and 4°C for 1 week). The freeze-thaw stability of the drug substances in plasma samples was evaluated over five freeze-thaw cycles. Plasma samples in three QC levels were immediately frozen at −20°C and thawed at room temperature for five consecutive times. After that, the samples were processed and assayed. In order to determine the stability of the drug substances in plasma, the spiked plasma samples were stored at room temperature for 24 h and −20°C for 2 weeks, and the evaluations were carried out at intervals. Long-term stability was assessed using the samples stored at −20°C over a period of 8 weeks.
Selectivity of the method was tested by analyzing blank human plasma samples from 8 different sources and by comparing them with the spiked plasma samples under optimized chromatographic conditions.
Reversed-phase HPLC-UV method was preferred for the determination of AML, OLM, VAL, and HCT. Preliminary experiments were carried out to achieve the best chromatographic conditions for the simultaneous determination of the drug substances. Several column types and lengths were trialed considering other chromatographic parameters. 25 cm CN column with a 4.6 mm inner diameter and a 5
(a) The chromatogram of 1
The regression equations were
The recovery values of the extraction procedure with methanol were 97.3%, 93.5%, 95.8%, and 98.9%, the RSD% values were 1.35, 2.42, 1.67, 3.72 for AML, OLM, VAL, and HCT (
For tablet analyses, selectivity was assessed immediately after AML, OLM, VAL, and HCT solutions were exposed to neutral, acidic, and basic hydrolysis and chemical oxidation with
Chromatograms corresponding to drug solutions subjected to (a) neutral hydrolysis, (b) acid hydrolysis, (c) alkaline hydrolysis, (d) chemical oxidation, and (e) exposure to sunlight.
In the initial studies, for protein precipitation some trials were conducted with acetonitrile, methanol, and perchloric acid; however, ion suppression and lower recovery values (<60%) were observed. Therefore, the LLE procedure was preferred to remove the sample matrix and gain the investigated drug substances. The mixture of n-hexane-ethylacetate-isoamyl alcohol (88 : 10 : 2, v/v/v) was selected as an LLE solvent to extract the substances from the spiked plasma.
Parameters corresponding to linear regression obtained from the calibration curves for the plasma samples.
Drug substance | Slope |
Intercept |
Correlation coefficient | Linear range |
---|---|---|---|---|
OLM | 31947 | −5036.2 | 0.9995 | 0.4–25.6 |
HCT | 25436 | −5846.7 | 0.9999 | 0.3–22 |
VAL | 36544 | −1005.4 | 0.9998 | 0.3–15.5 |
AML | 34770 | −10953 | 0.9998 | 0.1–18.5 |
The LOD values were found as 0.3, 0.08, 0.1, and 0.2 ng/mL, which is the concentration that yields a S/N of 3 : 1, and the LOQ values were 0.1, 0.4, 0.3, and 0.3,
Precision and accuracy of intraday and interday analyses at the low (1 µg/mL for all substances), medium (10 µg/mL for all substances), and high (15 µg/mL for AML and VAL, 20 µg/mL for OLM and HCT) concentration levels.
Low ( |
Medium ( |
High ( |
||||
---|---|---|---|---|---|---|
Intraday | Interday | Interday | Intraday | Interday | Intraday | |
RSD% | ||||||
AML | 5.2 | 6.1 | 4.8 | 6.0 | 4.2 | 5.4 |
OLM | 5.4 | 5.7 | 3.9 | 4.3 | 3.5 | 4.1 |
VAL | 6.5 | 6.9 | 6.3 | 6.5 | 5.8 | 6.3 |
HCT | 4.2 | 4.6 | 4.1 | 4.5 | 3.6 | 4.3 |
RME% | ||||||
AML | 9.3 | 10.6 | 9.1 | 9.6 | 7.4 | 8.2 |
OLM | 4.5 | 5.2 | 4.7 | 5.8 | 3.3 | 5.4 |
VAL | 6.5 | 5.4 | 6.2 | 4.3 | 5.7 | 3.9 |
HCT | 5.6 | 6.7 | 5.5 | 5.9 | 6.5 | 6.8 |
Absolute and relative recoveries of plasma analysis.
Concentration of AML |
Concentration of OLM |
Concentration of VAL |
Concentration of HCT |
|||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
0.5 | 10 | 18 | 0.5 | 15 | 25 | 0.5 | 10 | 15 | 0.8 | 10 | 20 | |
Absolute recovery % | 77.3 | 81.6 | 80.2 | 73.5 | 74.2 | 77.6 | 75.8 | 78 | 80.8 | 79.6 | 81.3 | 80 |
RSD% | 4.5 | 3.7 | 3.5 | 2.6 | 2.5 | 1.8 | 4.2 | 3.9 | 2.5 | 2.7 | 1.7 | 1.6 |
Relative recovery % | 95.5 | 97.6 | 97.9 | 92.6 | 95.8 | 96.7 | 100.3 | 99.8 | 98.7 | 92.7 | 100.3 | 100.1 |
RSD% | 3.3 | 3.7 | 2.9 | 2.8 | 3.2 | 3.1 | 1.9 | 2.6 | 2.3 | 4.7 | 3.2 | 3.5 |
The stability of the drug substances in human plasma and in acetonitrile-methanol-water (7 : 13 : 80, v/v/v) was investigated as described in Section
Chromatograms of (a) blank plasma and (b) a plasma sample of 1 mL spiked with 1
The developed method was applied to the plasma samples which were obtained from patients that used antihypertensive drugs, including the investigated compounds. The amounts of the drug substances found in the plasma of the patients are shown in Table
Concentrations of the drug substances in the plasma samples obtained from patients (results given as mean values,
Patient |
Time after administration | Concentration (ng/mL) | |||
---|---|---|---|---|---|
AML | OLM | VAL | HCT | ||
1 | 8 | 1200 | 720 | ||
2 | 12 | 118.7 | |||
3 | 6 | 152.3 | |||
4 | 8 | 103.8 | 1000 | ||
5 | 8 | 1242 | |||
6 | 7 | 602.5 | 460 | ||
7 | 8.5 | 126 | 1023 | 920 | |
8 | 24 | 352 | 412 |
Chromatograms corresponding to plasma extracts of patients 1 (a) and 7 (b).
The proposed HPLC method is specific, accurate, and precise for the simultaneous determination of AML, OLM, VAL, and HCT. An important advantage of the method is the availability for the determination of the drug substances in pharmaceutical preparations and human plasma with a cost effective technique than other techniques including mass detection [
In addition, this method has other advantages over most of the previously published methods, such as its simplicity and less time-consuming procedure. Moreover, RSD and RME values of the method were very low, indicating high precision and accuracy.
The pretreatment procedure is very simple, and it does not require any equipment like solid phase extraction cartridges or any more steps like double LLE. The main difference in the method compared to the previous ones is its ability to determine AML, OLM, VAL, and HCT simultaneously.
In conclusion, the presented HPLC method is simple, selective, cost-effective, and reproducible and can be reliably used by almost every drug laboratory. The method enables simultaneous determination of AML, OLM, VAL, and HCT in pharmaceutical preparations and plasma. Due to the fact that these substances are mainly used as combinations for hypertension therapy, this new procedure is very important. In the process of developing the method, forced degradation and validation studies were carried out. Finally, the method was applied to the analysis for triple drug formulations, including
This research was supported by the Research Fund of Bezmialem Vakif University (Project no. BVU-BAP-19/10). The study protocol was approved by the Clinical Trials Ethics Committee of Bezmialem Vakif University and informed consents of all participating subjects were obtained.