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Related Impurities High-performance Liquid Chromatography Method Development and Validation for drug combinations: Olmesartan Medoxomil, Chlorthalidone and Cilnidipine

The liquid chromatography mass spectrometry (LC-MS) compatible, stability-indicating, specific, linear, accurate, sensitive with less run-time related impurities reversed phase high-performance liquid chromatography (RP-HPLC) related impurities method has been developed for olmesartan medoxomil (OLM), chlorthalidone (CHLR), and cilnidipine (CIL) drug combinations, and the method has been validated according to ICH and US-FDA guidelines. The chromatographic separation was performed by using Hypersil-BDS Thermo-Scientific, C18 (12.5 cm, 4.6 mm, 5 microns particle size) column. Mobile phase-A was prepared by mixing 3.85 gm ammonium acetate in HPLC water and adjust pH 5.0 by using diluted acetic acid. Acetonitrile was taken as mobile phase-B. Initial mobile phase ratio (55:45 v/v) was adjusted for mobile phase-A: mobile phase-B followed by gradient program. Other chromatographic conditions such as column temperature 25 degrees, flow rate 1.0 mL/minutes with the detection wavelength at 260 nm. The retention time for CHLR impurity A, olmesartan (OL), OLM impurity A, were found about 2.7, 3.3, and 7.2 minutes respectively, with a total run time of 18.0 minutes. The linearity calibration plot was performed and found linear relationship over the concentration range of 1.25 limit of quantitation (LoQ)–18.75 μg/mL, 3.6 LoQ–60.0 μg/mL, 3.6 LoQ–60.0 μg/mL respectively for CHLR impurity A, OL and OLM impurity A respectively. The limit of detection (LoD) and LoQ were found 0.4 ppm (μg/mL) and 1.2 ppm (μg/mL), 1.2 ppm (μg/mL) and 3.5 ppm (μg/mL), 1.1 ppm (μg/mL) and 3.3 ppm (μg/mL) for CHLR impurity A, OL and OLM impurity A respectively. The accuracy was determined by recovery studies and was found between 90.0–110.0%. The developed analytical method has been validated for LoD-LoQ, specificity, linearity, accuracy, precision, robustness, and ruggedness, which were well within the acceptance limit as per ICH guidelines. All the degradation products generated by stress conditions were found to be well separated from one another (all drug components and impurities). The developed method with shorter runtime was successfully implemented for routine quality control and stability analysis to check the quality of OLM, CHLR, and CIL drug combinations.


Introduction
Olmesartan medoxomil (OLM) is a synthetic imidazole derivative pro-drug with an antihypertensive property (Fig. 1). The OLM prevents angiotensin II induced vasoconstriction and decreases aldosterone production, thereby preventing aldosterone-stimulated sodium retention and potassium excretion. Chlorthalidone (CHLR) is a diuretic medication used to treat high blood pressure, swelling including that due to heart failure, liver failure and nephrotic syndrome, diabetes insipidus, and renal tubular acidosis. (Fig. 2). CIL is a calcium channel blocker. CIL decreases blood pressure and is used to treat hypertension and its comorbidities. OLM, CHLR and CIL combinations are used to treat hypertension when a single medication is not effective (Fig. 3). It also helps to reduce chances of future heart attack and stroke. The most important related compounds for OLM are OL and OLM impurity-A, for CHLR is CHLR impurity-A. A literature survey discloses that few stability-indicating HPLC methods, [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] HPTLC, [19][20] Spectrophotometric methods [21][22] have been reported for the estimation of OLM and or CHLR and or CIL along with drug combinations in pharmaceutical preparations. To the best to our knowledge, no reports were found for stability-indicating LC-MS compatible related impurities method for OLM, CHLR and CIL drug combinations. In the present work, we are concentrated on to develop and validate a stability-indicating, LC-MS compatible method (with less runtime) along with optimum chromatographic conditions for the determination of related impurities (OL, OLM impurity-A, CHLR impurity-A, and un-known impurities) for OLM, CHLR and CIL drug combinations that may be present during stability study. The developed LC-MS compatible method was validated as per ICH guidelines [23][24] and can be applied lucratively to quality control purposes.

Materials
A pharmaceutical-grade gift sample of OLM (established purity 99.2%), CHLR (purity 98.8%), CIL (purity 99.5%) were acquired from Amoli Organics Pvt Ltd. Olkem Trio 40 tablets containing OLM 40 mg, CHLR 12.5 mg and CIL 10 mg were procured from the domestic market. Water HPLC grade, acetonitrile HPLC grade, and methanol HPLC grade were purchased from Merck. HPLC grade of glacial acetic acid and ammonium acetate were procured from Merck.

Instrumentation
The LC-20AT (Shimadzu) system was used for HPLC method development and validation by using Hypersil BDS, C18 (12.5 cm × 0.46 cm) 5 microns column, as well as UV-visible detector, analyzed at 260 nm. Spinchrom software was used for evaluation and data processing.

Chromatographic Conditions
A mobile phase-A was prepared by dissolving 3.85 gram ammonium acetate into 1 liter water. Adjust pH 5.0 with diluted acetic acid and filter through a 0.22 microns membrane filter, sonicated for 10 minutes for degassing. mobile phase-A kept for a line, and acetonitrile kept for B-line with the initial ratio of mobile phase-A 55% and acetonitrile 45%, prepared gradient program in the software ( Table 1).
The analysis was carried out on LC-20AT (Shimadzu) system. The analytes was separated on an analytical column Hypersil BDS C18 (12.5 cm × 0.46 cm) 5 μm column at 260 nm wavelength. The column temperature was kept at 25°C. The volume of injection was 20 μL and the flow was sustained at 1.0 mL/minutes. The runtime was 15 minutes and after that 3 minutes saturation time with initial mobile phase ratio.

Sample Solution Preparation
Weigh, powdered 20 tablets and the average weight was determined. Tablets were crushed by mortar-pastel and mixed well. Accurately weighed tablet powder 40 mg equivalent of OLM into a 10 mL volumetric flask. Add 8 mL diluent, shake for 15 minutes and sonicate the solution for 10 minutes. Make up the volume with diluent and mix well to obtain OLM (4000 μg/mL), CHLR (1250 μg/mL), and CIL (1000 μg/mL). Filter this solution with a 0.45 µm membrane filter.

Method Validation
This method was validated as per USP and ICH guidelines. All validation parameters, eg. specificity, sensitivity (LoQ and LoD) linearity-range, precision, accuracy, and robustness are included in the study.

Specificity
Specificity is one of the substantial features of HPLC, and it denotes the ability of the analytical method to separate analytes from one another in the complex mixture. Specificity of the method was performed by injecting 20 μL solutions of impurity, sample, and blank solutions individually.

Linearity
To assess the linearity-range of the method, different solutions were prepared by diluting stock solutions with the diluent in different concentrations of OL impurity, OLM Impurity-A and CHLR Impurity-A to achieve LoQ, 50, 75, 100, 125 and 150% with respect to sample concentration respectively. One injection from each concentration was analyzed by using the same conditions. Linearity was plotted by using a linear regression method to evaluate r 2 .

Sensitivity
LoD and LoQ of OL impurity, OLM impurity-A, and CHLR impurity-A were performed by preparing different solutions of OL impurity, OLM impurity-A, and CHLR impurity-A and determine the S/N ratio. LoD is the lowest detection concentration with S/N ratio of approximately 3:1, while LoQ is the lowest quantification concentration with S/N ratio of approximately 10:1 along with %RSD (n = 5) of not more than 15%. Accuracy Accuracy of the related impurities method was determined by recovery studies at four levels of concentration (LoQ, 80.0, 100.0, and 120.0%) for OL impurity, OLM impurity-A, and CHLR impurity-A and triplicate samples for individual concentration were injected. The recovery (%) for added OL impurity, OLM impurity-A and CHLR impurity-A and RSD were measured for individual replicate samples.

Precision
The system precision and repeat abilit y (method precision) for proposed methods were performed by multiple measurements of standard and sample solution, individually. A system precision was performed by five injections of the standard on the same day. Method precision was assessed by five injections of the sample on the same day. The RSD of the obtained results was calculated to evaluate repeatability results.

Robustness
Robustness study was performed for deliberate and minor modifications in the instrumental parameters, for example: • Change in flow: ± 0.2 mL/minutes • Variation in organic composition (± 2.0) • pH of buffer: ± 0.2 The alteration was made to evaluate its impact on the method. The %RSD and difference in percentage was verified against original data for each of the modified parameters.

Results and Discussion
The study was aimed to develop a sensitive, accurate, precise, stability-indicating LC-MS compatible related impurit ies method for OLM, CHLR and CIL drug combinations. A Hypersil BDS, C18 (12.5 cm × 0.46 cm) 5 microns column was selected as the stationary phase for the separation and determination of related impurities method for OLM, CHLR and CIL drug combinations. For the optimization of the mobile phase, sequential trials were performed by changing the ratio of methanol with water, acetonitrile with water, and buffer (ammonium acetate) with acetonitrile by isocratic as well as gradient program and monitored at different ratios. Method optimization results are summarized in Table 2.
Based on the above trails, the mobile phase containing ammonium acetate (pH 5.00) for A-line and acetonitrile for B-line with initial ratio 55: 45 v/v and gradient program was finalized as per Table 3.
Method was optimized with flow rate of 1.0 mL/minutes, wavelength 260 nm, 20 μL volume of injection and 25.0°C column temperature as the best chromatographic conditions for the complete study where OLM, CHLR, CIL, OL impurity, OLM Impurity-A and CHLR Impurity-A were eluted forming symmetrical peak shape and good resolution (Fig. 4).

Specificity
Specificity was assessed by comparing the chromatograms of blank, standard solution (OLM, CHLR and CIL), impurity standard (OL and OLM impurity-A, CHLR impurity-A), as such sample and sample spiked with OL, OLM impurity-A and CHLR impurity-A impurities solution. For the same purpose, 20 μL injection of diluent, standard, impurity standard solution, as such sample solution and sample spiked with OL, OLM impurity-A and CHLR impurity-A impurities sample solution were injected into the HPLC system individually, and the chromatogram are shown in Figs. 5-9. It can be observed that there no co-eluting peaks at the retention time of OLM, CHLR, CIL, OL, OLM impurity-A and CHLR Impurity-A. All analyte peaks were pure and hence proved the specificity of the method.

Linearity and Range
Analytical method linearity is demonstrated as the ability of the method to get test results that are directly proportional to the concentration of analyte within a defined range. The peak area achieved from HPLC was plotted against respective concentrations to get the    3) Linear gradient to achieve buffer: Acetonitrile (10:90) at 15 minutes.
All analyte peak shapes are good and well separated from one another. Trials are taken to reduce run time but CHLR imp-A and OLM are very close to each other. were assessed with concentration range from LoQ (1.25 μg/mL-18.75 μg/mL), LoQ (3.6 μg/mL-60 μg/mL) and LoQ (3.6 μg/mL-60 μg/mL) respectively. Based on regression calculation, a linear equation was obtained: y = mx + c, and r 2 was found greater than 0.990, representative a linear relationship for the concentration of analytes and peak area (Figs. 10-12).

Limit of Detection and Limit of Quantification (LoD and LoQ)
The LoD is the lowest analyte level in a sample that could be detected, but not certainly quantitated and LoQ is the lowest analyte level in a sample can be precisely quantified. The results presented an LoD and LoQ for CHLR impurity-A of 0.4 and 1.2 μg/mL, OL of 1.2 μg/mL and 3.5 μg/mL, OLM impurity-A 1.1 μg/mL and 3.3 μg/mL respectively.

Accuracy
The accuracy of an analytical procedure describes the closeness to the accurate value generated by a method.   4-7. The results of recovery (%) were within accepted limits from 90.0 to 110.0% for 80, 100 and 120%, from 70.0 to 130.0% for LoQ level respectively. The results of percentage RSD were within the accepted limits below 10.0% for 80, 100, and 120%, below 15.0% for LoQ level, respectively. This proves its validating of the method for routine drug analysis.

Precision
The precision of the method is derived as "the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions," and it is generally expressed as the RSD. Based on the results of both systems and method precision proved that the method is precise within satisfactory limits. The tailing factor, RSD, and theoretical plats were determined, and all the results are within acceptance criteria. Acceptable precision was less than 2.0 the tailing factor, NMT 10.0% for the RSD and NLT 2000 for a number of plates, as reported in Tables 8-11.

Robustness
Robustness was evaluated for an analytical method by assessing the influence of minor changes in chromatographic conditions on system suitability parameters and % impurity value difference from as such condition of the proposed method. The results of robustness testing proved that minor deliberate changes in method conditions, eg. flow rate, mobile composition, and pH of the buffer is robust within the acceptable criteria. The results are summarized in Tables 12-15. In all modifications, system suitability was achieved and % impurity value was observed well within acceptable limits as well.

Conclusion
In the described research, a simple, fast, accurate, precise, and linear stability-indicating analytical method has been developed and validated for related impurities of OLM, CHLR, and CIL drug combinations. Hence, it can be further employed for quality control routine analysis. The analytical method conditions and mobile phase provided a good resolution for all peaks of an analyte. In addition, the main advantage of the developed method is with less run time. The method was further validated as per ICH guidelines. The method is robust enough to reproduce precise and accurate results under varied chromatographic conditions.