A simple RP-HPLC-PDA method for determination of atenolol (ATN) and trimetazidine (TMZ) in human urine and tablets has been developed. Analytes were separated on a Caltrex BI column (125× 4.0 mm, 5
Atenolol (ATN) (4-(2-hydroxy-3-isopropylamino propoxy) phenyl acetamide) (Figure
Chemical structure of atenolol
Chemical structure of trimetazidine dihydrochloride
Trimetazidine dihydrochloride (TMZ), (1-[(2,3,4-trimethoxyphenyl)methyl]-piperazine dihydrochloride) (Figure
TMZ has no negative inotropic effects or vasodilatory characteristics and can be combined with other antianginal drugs as a complementary therapy mostly for patients with companion diseases such as left ventricular dysfunction and diabetes mellitus [
Coadministration of different medications for controlling chronic diseases especially in geriatric patients may cause polypharmacy problems such as adverse drug effects and toxicity. Changes in kidney and liver functions may affect drug pharmacokinetic and pharmacodynamics and may increase the mortality rates. Monitoring concomitant drugs in blood, plasma, and urine samples is mandatory in some cases [
Atenolol experiences low or no hepatic metabolism and is excreted unchanged mainly in urine [
Different analytical methods have been applied for the determination of atenolol in biological fluids and pharmaceutical products such as HPLC [
Several techniques have been also reported for the determination of trimetazidine including HPLC [
To our knowledge, there are no published methods for determination of TMZ and ATN in human urine simultaneously. This paper describes simple HPLC-PDA methods for simultaneous determination of TMZ and ATN in human urine and also for determination of TMZ and ATN in their corresponding tablets.
All chemicals were of analytical grade and all solvents were of HPLC grade. Trimetazidine was kindly supplied from SIGMA Pharmaceutical Industries (Cairo, Egypt). Atenolol was kindly supplied from Egyptian Int. Pharmaceutical Industries Co. (10th of Ramadan city, Egypt). Metacardia® film-coated tablets labeled to contain 20 mg trimetazidine per tablet and Blokium® film-coated tablets labeled to contain 100 mg atenolol per tablet were obtained from local pharmacy. Acetonitrile, HPLC grade, orthophosphoric acid, and potassium orthophosphate monobasic were obtained from Fischer Scientific UK (Bishop Meadow Road, UK). Methanol and water were obtained from TEDIA Chemicals (USA). Sodium hydroxide, chloroform, and n-butanol were purchased from El-Nasr Pharmaceutical Chemicals Co. (Abu-Zaabal, Cairo, Egypt). Human blank urine was collected from healthy nonsmoker adult volunteers. Urine samples were stored at -20°C.
The HPLC system consists of Agilent Technologies 1200 series chromatographic apparatus (Agilent Technologies, Palo Alto, CA, USA) equipped with an autosampler injector, 100
Stock solutions of either trimetazidine or atenolol were prepared individually at 1.0 mg/mL in methanol. Working solutions were prepared by further dilution of the stock solutions with water for spiking calibration standards and quality control samples. All stock solutions were stored in amber glass containers at 2-8°C.
Calibration standards were prepared in HPLC grade water at concentrations of 1.0, 3.0, 5.0, 10, 25, 50, 75, and 100
Calibration standards were prepared in blank human urine at concentrations of 0.25, 0.5, 1.0, 10, 20, and 25
Five tablets were weighed, ground, and mixed well. An accurately weighed amount of powdered tablets equivalent to 50 mg of trimetazidine was extracted with 50 ml methanol and sonicated for 30 minutes. The solution was filtered and subsequent dilutions were made in water for HPLC-PDA analysis.
Five tablets were weighed, ground, and mixed together. An accurately weighed amount of powdered tablets equivalent to 50 mg of atenolol was extracted with 50 ml methanol and sonicated for 30 minutes. The solution was filtered and the filtrate was subsequently diluted with water for HPLC analysis.
Human urine samples were thawed at room temperature and mixed well. A 2.0 mL aliquot of each sample was transferred into screw cap culture tube, 0.5 mL of 1.0 N NaOH was added, and the tubes were vortex mixed for 30 seconds. Samples were extracted by adding 6.0 ml of (4:1) chloroform: n-butanol, v/v; samples were vortex mixed for 10 minutes. After centrifugation at 3000 rpm for 10 min, the organic layer was transferred into clean tube and evaporated to dryness under a nitrogen stream at approximately 40°C. The residue was reconstituted with 0.5 mL methanol, vortex mixed briefly. A 20
Chromatographic separation was carried out on a Caltrex BI (125 × 4.0 mm, 5
Same conditions as described above were applied for the analysis of target analytes in urine samples except the following: the gradient was 0-2.0 min 10 %B and 0 %C, 2.1 min 20 %B and 0 %C, 5.0 min 15% B and 15 %C, 5.1 min 20 %B and 20%C, 5.1-8.0 min 80%B and 0%C, 8.0-11.0 min 90% B and 0% C, and 11.1-13.0 min 10% B and 0% C. Run time was 13.0 min.
Target analytes exhibit different absorption characteristics, so 210 and 225 nm were selected for detection of TMZ and ATN, respectively. All chromatographic parameters were fully optimized to achieve the best chromatographic separation. Both methanol and acetonitrile were tested as organic modifiers in different proportions using both isocratic and gradient elution; methanol showed higher sensitivity but with asymmetric peak shape for both analytes. A gradient elution using both methanol and acetonitrile (as described above) showed excellent symmetric peak shape for both analytes. Addition of buffer modifiers (using different mobile phase buffers) and effect of mobile phase pH (pH 3-5) have been studied in terms of peak shape, elution time, and sensitivity. Lower pH mobile phase showed rapid elution; however, pH 5 showed late elution for both analytes. 25 mM phosphate buffer, pH 3.3, was found to be the most appropriate buffer modifier, providing well-resolved peaks in reasonable elution time. The flow rate range from 0.5 to 1.0 mL/min was tested. It was found that 1.0 mL/min was optimum for good chromatographic separation in a reasonable time without interference from either pharmaceutical preparation placebo components or biological fluid endogenous peaks. Different gradient programs have been applied; peak symmetry, resolution, selectivity, and number of theoretical plates were calculated in each case and summarized in Table
Analytical performance data for HPLC determination of atenolol and trimetazidine in pure form and urine samples.
Parameter | Atenolol | Trimetazidine | ||
---|---|---|---|---|
Pure form | Urine | Pure form | Urine | |
Linearity range ( | 1.0–100 | 0.5-25 | 1.0– 100 | 0.25-25 |
Correlation coefficient (r2) | 0.9998 | 0.9999 | 0.9999 | 0.9995 |
Regression equation | Y=39.15X-5.7394 | Y=0.037X-8.0947 | Y=118.06X-58.05 | Y=0.3464X+31.83 |
LOD ( | 0.30 | 0.11 | 0.18 | 0.05 |
LOQ ( | 0.99 | 0.38 | 0.60 | 0.16 |
HPLC-PDA chromatogram of standard neat solution of (A) atenolol 10
HPLC-PDA chromatogram of (A) atenolol 25
HPLC-PDA chromatogram of blank urine.
Liquid-liquid extraction using organic solvents has been shown to provide clean extracts of biological fluid samples; it is also considered a cheaper sample treatment approach. Different organic solvents (dichloromethane, chloroform, n-hexane: ethyl acetate (1:1, v/v), and chloroform: n-butanol (4:1, v/v)) have been evaluated for extraction recoveries. It was found that 4:1 chloroform: n-butanol, v/v, not only provided the highest extraction recoveries for both analytes but also eliminated the interference of endogenous matrix components. pH of the sample is known to strongly affect the ionization of different compounds and subsequently affect the extracted portion of the analyte into the organic layer; acidic and basic conditions using 1.0 N HCl and 1.0 N NaOH, respectively, and matrix samples without any pH optimization (using water) have been compared in terms of extraction recoveries and cleanness of the final extracts. Both target drugs are basic compounds (of PKa 9.6 and 9.14 for ATN [
System suitability parameters such as resolution (Rs), tailing factor (T), capacity factor (K'), and selectivity factor (
System suitability parameters for the determination of the atenolol and trimetazidine in pure form/human urine.
Parameters | Atenolol | Trimetazidine | Reference value [ |
---|---|---|---|
Retention time ( | 3.72/4.32 | 4.31/5.79 | ----- |
Number of theoretical plates (N) | 13211/2896 | 13935/13668 | >2000 |
increase with efficiency of separation | |||
Tailing factor (T) | 0.97/1.85 | 0.71/1.11 | ≤2 |
Capacity factor “Mass distribution ratio” (K′) | 2.44/2.32 | 2.98/3.46 | 1-10 acceptable |
Height equivalent to one theoretical plate (HETP) | 0.01/0.04 | 0.01/0.01 | The smaller the value, the higher the column efficacy |
Resolution | 1.62/4.89 | >1.5 | |
Good separation between peaks of interest. | |||
Selectivity factor ( | 1.11/1.49 | > 1 |
Statistical analysis of the proposed method in standard solution and the official/reported methods [
Atenolol | Trimetazidine | |||
---|---|---|---|---|
Proposed Method | Reported method [ | Proposed Method | Official method [ | |
Mean | 100.34 | 99.66 | 100.35 | 98.39 |
SD | 1.17 | 0.87 | 1.06 | 0.77 |
RSD% | 1.16 | 0.88 | 1.06 | 1.12 |
Variance | 1.36 | 0.76 | 1.13 | 0.59 |
N | 8 | 5 | 8 | 3 |
F – test | 1.8 (4.12) | - | 1.09 (4.74) | - |
Student’s | 1.11 (2.201) | - | 1.93 (2.262) | - |
Intra- and interday precision and accuracy calculated from quality control (QC) samples in pure form.
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3 | 2.97 | 0.02 | 0.77 | -1.12 | 3.01 | 0.05 | 1.7 | 0.25 |
50 | 50 .95 | 0.45 | 0.88 | 1.89 | 50.17 | 0.41 | 0.82 | 0.34 |
75 | 75.004 | 0.12 | 0.17 | 0.005 | 76.36 | 0.29 | 0.39 | 1.82 |
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3 | 2.89 | 0.09 | 3.45 | -3.67 | 2.88 | 0.09 | 3.02 | -3.99 |
50 | 50.09 | 0.23 | 0.45 | 0.18 | 50.07 | 0.26 | 0.52 | 0.15 |
75 | 77.14 | 1.31 | 1.7 | 2.85 | 76.34 | 2.05 | 2.68 | 1.78 |
Intra- and interday precision and accuracy calculated from quality control (QC) samples in human urine.
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| 1.00 | 0.02 | 1.76 | 0.08 | 0.99 | 0.03 | 2.85 | -0.01 |
| 9.92 | 0.07 | 0.73 | -0.81 | 9.85 | 0.11 | 1.10 | -1.50 |
| 20.02 | 0.25 | 1.26 | 0.11 | 20.08 | 0.17 | 0.82 | 0.41 |
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| 0.50 | 0.004 | 0.75 | 0.98 | 0.50 | 0.004 | 0.69 | 0.90 |
| 10.19 | 0.01 | 0.98 | 1.9 | 10.14 | 0.12 | 1.18 | 1.37 |
| 19.36 | 0.08 | 0.42 | -3.19 | 19.41 | 0.08 | 0.42 | -2.94 |
Application of the proposed method in standard solution on pharmaceutical tablets.
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| 4.87 | 97.44 | 5.00 | 4.73 | 94.54 | |
| 9.60 | 95.97 | 10.00 | 9.74 | 97.40 | |
| 28.63 | 95.42 | 30.00 | 28.50 | 95.05 | |
| 47.50 | 95.01 | 50.00 | 49.15 | 98.30 | |
| 71.67 | 95.56 | 75.00 | 71.24 | 94.99 | |
| 95.27 | 95.27 | 100.00 | 95.36 | 95.36 | |
| 95.78 | 95.94 | ||||
| 0.88 | 1.53 | ||||
| 0.36 | 0.63 | ||||
| 0.91 | 1.60 | ||||
| 0.77 | 2.34 |
RP-HPLC method with PDA detection was developed for the determination of atenolol and trimetazidine in pure form, pharmaceutical tablets, and human urine. The proposed method was found to be simple, sensitive, and accurate. The optimized chromatographic conditions allowed separation of the studied drugs in reasonable time without any interference from excipients and/or extracted matrix components. This method can be used for therapeutic drug monitoring of patients treated with ATN and TMZ concomitantly and also can be applied for quality control analysis of the studied drugs in their pharmaceutical tablets.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.