Introduction
Metolazone is a thiazide-like diuretic commonly marketed under brand names such as Zytanix, Metoz, Zaroxolyn, and Mykrox. It is primarily indicated for the management of congestive heart failure (CHF) and hypertension. The drug exerts its diuretic effect by reducing the reabsorption of water in the kidneys, thereby decreasing blood volume and increasing urine output. This action leads to a reduction in blood pressure and helps prevent fluid accumulation, particularly beneficial in heart failure management.
One of metolazone’s main therapeutic roles is in the treatment of edema associated with CHF. In cases of mild heart failure, it may be administered alone or in combination with other diuretics in more severe conditions. Besides controlling fluid retention, metolazone use may permit a less restrictive sodium intake for patients. Unlike most thiazide diuretics that become ineffective in patients with renal impairment, metolazone retains its activity even when the glomerular filtration rate (GFR) drops below 30–40 mL/min, making it effective in moderate chronic kidney disease.
Pharmacologically, metolazone acts at the renal tubular sites involved in electrolyte reabsorption. It primarily inhibits sodium reabsorption at the cortical diluting segment and, to a lesser extent, at the proximal convoluted tubule. This results in the excretion of sodium and chloride in nearly equal amounts. The enhanced sodium delivery to the distal tubule increases potassium excretion, a common feature of diuretic therapy.
As a cardiovascular agent, metolazone is classified as a quinazoline diuretic, structurally related to thiazides. Its mechanism involves inhibiting sodium transport across the epithelial cells of the renal tubules, predominantly within the distal segments, thereby enhancing the excretion of sodium, chloride, and water.
Fig.1: Chemical structure of Metolazone
Extensive literature review was conducted and an attempt was made to develop an unambiguous, valid method for the estimation of Metolazone. Few of spectroscopic, chromatographic, and other analytical methods [4-19] have been reported for the estimation of Metolazone individually and or along with drug combinations in pharmaceutical preparations. The aim of this study is to develop and validate a new simple, accurate and economic stability- indicating RP-HPLC method with less runtime, which would be able to separate and quantify Metolazone in a single run. The developed method was validated as per ICH guidelines [20-21] and can be applied lucratively to quality control purposes.
Materials and Methods:
Equipment:
The Method development and Validation was carried out using Waters Alliance-HPLC system equipped with waters 1525 binary HPLC pump, 2695- separation module connected to 2996-photo diode array detector, and Waters 2707 auto sampler. The data was acquired by Empower® version 2. The other equipment used were Ascoset Electronic balance, ADWA pH meter, heating mantle. Ultrasonic bath was used for sonication of the samples. Hot air oven was used to carry out thermal degradation studies. UV cross linker, with series of 23400 model UV chamber, equipped with a UV fluorescence lamp with the wavelength range between 200 & 300 nm was used for photo degradation studies.
Chemicals and Reagents:
Metolazone working standard was kindly given as gift sample by Spectrum Pharma Limited, Hyderabad. HPLC grade solvents include acetonitrile, water and methanol. Analytical grade chemicals include sodium hydroxide, hydrochloric acid, 20% hydrogen peroxide, Ortho phosphoric acid, Triethyl amine and potassium dihydrogen phosphate were purchased from E. Merck Limited, Mumbai, India.
Chromatographic conditions:
HPLC analysis was carried out on Waters Alliance-HPLC system equipped with 2695-separation module connected to 2996-photo diode array detector and the data was acquired by Empower® version 2. Separation was achieved using Hypersil BDS C18 column, 150×4.6mm, particle size 5µm as a column with mobile phase of Acetonitrile: HPLC water in the ration of 50:50. The samples were analyzed using 10 µL injection volume, Flow rate was maintained at 0.7 mL/min with runtime of 8 min and the temperature was maintained at 30°C throughout the analysis. Detection and purity establishment of the drugs were achieved using PDA detector at 236 nm wavelength.
Preparation of Working Standard Solution:
Accurately weighed and transferred 100mg of Metolazone into 100mL volumetric flask. 70mL of diluent was added, mixed well to dissolve and diluted up to the mark with diluent to obtain 1000µg/mL solution (Solution –A). 10mL of the above solution was pipetted out into 100mL volumetric flask and diluted up to the mark with diluent to obtain 100µg/mL solution (Solution-B). 6.0mL from Solution-B was pipetted out into 100mL volumetric flask and diluted op to the mark with the diluent (6µg/mL) concentration and sonicated for about 10 minutes with intermediate shaking.
Preparation of Sample Solution:
10mg of drug i.e., equivalent to 10 tablets were weighed and transferred into a 50 mL volumetric flask, 30mL of diluent was added, sonicated for about 30 min with intermediate shaking and volume diluted up to the mark with diluent. The sample solution was filtered with 0.45µ Millipore Nylon filter. Further 3mL of the above solution was pipetted out, transferred into a 100mL volumetric flask and diluted up to the mark with diluent.
METHOD VALIDATION:
The developed and optimized RP-HPLC method was validated according to international conference on harmonization (ICH) guidelines Q2(R1) in order to determine the system suitability, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, ruggedness and robustness.
System suitability:
System suitability parameters were evaluated to verify system performance. 10 µL of standard solution was injected five times into the chromatograph, and the chromatograms were recorded. Parameters such as number of theoretical plates and peak tailing were determined.
Specificity:
The specificity of the analytical method was established by injecting the solutions of diluent (blank), placebo, working standards and sample solution individually to investigate interference from the representative peaks.
Precision:
Repeatability/ method precision was performed by injecting six replicates of same concentrations of Metolazone, calculated % assay and %RSD. Reproducibility/ Ruggedness/ Intermediate precision was performed using different analysts and a different instrument in the same laboratory.
Accuracy:
Accuracy of the proposed method was determined using recovery studies by spiking method. The recovery studies were carried out by adding known amounts (50%, 100% and 150%) of the working standard solutions of Metolazone to the pre-analysed sample. The solutions were prepared in triplicates to determine the accuracy.
Linearity:
Linearity was evaluated by analyzing different concentrations of the standard solutions of Metolazone. Six working standard solutions ranging between 10µg/mL-80µg/mL were prepared and injected. The response was a linear function of concentration over peak area and were subjected to linear least- squares regression analysis to calculate the calibration equation and correlation coefficient.
Limit of detection and Limit of quantification:
Limit of detection (LoD) and limit of quantification (LoQ) of Metolazone were determined by calibration curve method. Solutions of Metolazone were prepared in linearity range and injected (n = 3).
Robustness:
To examine the robustness of the developed method, experimental conditions were deliberately changed, resolution, tailing factor, and theoretical plates of Metolazone peaks were evaluated. To study the outcome of the flow rate on the developed method, it was changed ± 0.1mL/minute, organic phase composition in mobile phase was changed ±5% and the detection wavelength was changed ±10nm. In all the above varied conditions, the composition of aqueous component of the mobile phase was held constant.
Forced Degradation Studies:
Stress studies were performed by considering Metolazone working standard solution to provide the stability-indicating property and specificity of the proposed method. Intended degradation was attempted by the stress conditions of exposure to photolytic stress (1.2 million lux hours followed by 200 Watt hours), heat (exposed at 80°C for 24 hours), acid (0.1N HCl for 24 hours at 60°C), base (0.1N NaOH for 24 hours at 60°C), oxidation (3% peroxide for 24 hours at 60°C), water (refluxed for 12 hours at 60°C), and humidity (exposed to 90% RH for 72 hours). The solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.
RESULTS AND DISCUSSION:
System Suitability:
From the results in table 1, the column efficiency for Metolazone peak was identified from the theoretical plate count which is more than 3000, tailing factor less than 2.0, %RSD was found to be less than 2.0%.
Table 1: System suitability data
| System suitability parameter | Observed value | Acceptance criteria |
| Retention time | 3.548 | — |
| USP Tailing factor | 1.2 | NMT 2.0 |
| USP Theoretical Plate Count | 5765 | NLT 2000 |
| % RSD | 0.5 | NMT 2.0% |
Specificity:
From the obtained chromatograms in figures 2 to 4 it can be inferred that there were no co-eluting peaks at the retention time of Metolazone which shows that peak of analyte was pure and the excipients in the formulation did not interfere with the analyte of interest.
Fig. 2: Blank chromatogram
Fig. 3: Placebo chromatogram
Fig. 4: Chromatogram of Metolazone
Precision:
From the results in table 2, % Assay for Metolazone was found to be in the range of 98 – 102%, and the % RSD for Metolazone to be within 2%. Hence the method is precise, reproducible and rugged for 48 hours’ study.
Table 2: Precision data (system precision and method precision)
| Number of injections | Retention time | Peak area | %Assay |
| 1 | 3.540 | 787571 | 99.7 |
| 2 | 3.547 | 799985 | 99.8 |
| 3 | 3.546 | 787899 | 99.3 |
| 4 | 3.545 | 792132 | 99.6 |
| 5 | 3.546 | 772685 | 99.8 |
| 6 | 3.544 | 768429 | 100 |
| Mean | 782983 | 99.7 | |
| SD | 7369.746 | 0.2162 | |
| %RSD | 0.938238 | 0.9067 | |
Linearity:
Linearity was evaluated by analysing different concentrations of Metolazone. From the results tabulated in table 3, it is inferred that the correlation coefficient was greater than 0.999. The slope and y-intercept values were also provided, which confirmed good linearity between peak areas and concentration.
Table 3: Linearity data
| Concentration (µg/mL) | Peak area |
| 1 | 133264 |
| 2 | 239080 |
| 3 | 399496 |
| 4 | 489978 |
| 6 | 699899 |
| 8 | 952859 |
| 10 | 1191256 |
Fig.5: Calibration curve of Metolazone
Accuracy:
From the results in table 4, the % recovery for Metolazone found to be in the range of 98 –102% and the % RSD for Metolazone is less than 2%. Hence the proposed method was accurate.
Table 4: Accuracy data
| % Concentration (at specific level) | Area | % recovery | % Mean recovery | Overall %Mean recovery | ||
| Sample area | Average Sample area | Standard area | ||||
| 50% | 392057 | 391547.3 | 785488 | 100.2 | 100.6% | 99.89% |
| 391065 | 100.0 | |||||
| 391520 | 100.0 | |||||
| 100% | 782015 | 780680.7 | 99.9 | 99.76% | ||
| 779812 | 99.7 | |||||
| 780215 | 99.7 | |||||
| 150% | 1164589 | 1171920 | 99.2 | 99.86% | ||
| 1172586 | 99.9 | |||||
| 1178586 | 100.5 | |||||
LoD and LoQ:
The Limit of Detection and Limit of Quantification of Metolazone were calculated by using following equations (ICH, Q2 (R1)) and the LoD and LoQ values are reported in table 5. These LOD = 3.3 × σ/S and LOQ = 10 × σ/S
Where σ = the standard deviation of the response and S = slope of the calibration curve.
Table 5: LoD and LoQ data
| Drug | LOD (µg/mL) | LOQ (µg/mL) |
| Metolazone | 0.1 | 0.3 |
Robustness:
From the results in table 6, it is evident that the system suitability parameters such as resolution, RSD, tailing factor, and the theoretical plate count of Metolazone remained unaffected by deliberate changes. The results were presented along with the system suitability parameters of optimized conditions. Thus, the method was found to be robust with respect to variability in applied conditions.
Table 6: Robustness data
| S. No | Conditions | Metolazone | |||
| Retention time (min) | Theoretical plate count | Tailing factor | %RSD | ||
| 1. | Flow rate (+) 0.8mL | 3.110 | 4042 | 1.271 | 0.89% |
| 2. | Flow rate (-) 0.6mL | 4.157 | 5130 | 1.232 | 0.77% |
| 3. | Organic phase (-) 45:55 v/v | 4.091 | 5087 | 1.225 | 0.9% |
| 4. | Organic phase (+) 55:45 v/v | 3.201 | 4153 | 1.276 | 0.94% |
| 5. | Wave length (+) 240nm | 3.110 | 4039 | 1.271 | 0.7% |
| 6. | Wave length (-) 232nm | 4.136 | 5039 | 1.233 | 0.87% |
Forced Degradation Studies:
The samples were analyzed with the above mentioned HPLC conditions using a PDA detector to monitor the homogeneity and purity of the Metolazone. The results which were shown in table 7 indicates that the degradation was not observed in photolytic stress, humidity, acid, base, water hydrolysis, and thermal stress studies. It was interesting to note that all the peaks due to degradation were well resolved from the peaks of Metolazone. Further, the peak purity of Metolazone was found to be homogeneous based on the evaluation parameters such as purity angle and purity threshold. Hence, the method is considered to be “stability-indicating.”
Table 7: Forced degradation studies at different stress conditions
| S. No | Degradation | % Degradation Assay | % Assay | |
| API | Formulation | |||
| 1. | Acid degradation | 89.8% | 89.6% | 100.8% |
| 2. | Alkali degradation | 91.9% | 91.9% | |
| 3. | Oxidative degradation | 90.6% | 90.1% | |
| 4. | Thermal degradation | 93.3% | 94.5% | |
Fig. 6: Chromatogram of acid degradation
Fig. 7: Chromatogram of alkali degradation
Conclusion
A simple, rapid, cost-effective, and accurate RP-HPLC method was successfully developed for the quantification of Metolazone in tablet formulations using isocratic elution. The selected analytical conditions and solvent system enabled efficient separation with a short runtime. The method was validated in accordance with ICH guidelines and showed excellent linearity, precision, accuracy, and specificity. These attributes make the developed RP-HPLC method well-suited for routine quality control and analysis of Metolazone.
Furthermore, the method’s high sensitivity and specificity make it particularly valuable for routine analytical applications requiring the processing of large sample numbers within a short time, without the need for extensive sample preparation. The assay results obtained from both commercial tablet formulations and laboratory-prepared mixtures were in good agreement, supporting the reliability of the method. In conclusion, the proposed RP-HPLC method is a robust and reliable tool for the routine estimation of Metolazone in pharmaceutical dosage forms.
References
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