Development and Validation of Bioanalytical Method for Simultaneous Estimation of Olmesartan medoximil and Metoprolol succinate by

UHPLC-MS/MS in human plasma

Nirmal Thakker^1,2*, Gajanan Shinde², Abhay Dharamsi², Vishnu Choudhari³, Sarita Pawar⁴

¹Application Support, Spinco Biotech Private Limited, Ahmedabad, Gujarat, India.

²Department of Pharmacy, Parul University, Vadodara, Gujarat, India.

³School of Pharmacy, Dr. Vishwanath Karad MIT World Peace University,

MIT Campus, Kothrud, Pune, MH, India.

⁴Department of Pharmaceutical Chemistry, Sanjivani College of Pharmaceutical Education and Research, Kopargaon, MH, India.

*Corresponding Author E-mail: nirmalthakker117@yahoo.com

ABSTRACT:

A simple, precise bioanalytical UPLC Tandem Mass Spectroscopy gradient elution method was developed and validated for simultaneous estimation of Olmesartan Medoximil (OLM) and Metoprolol Succinate (MET) in human plasma. The quantitation carried out using Shimadzu Shimpack-C18 GIST AQ (50 mm X 2.1 mm, 1.9 µm) column and the mobile phase A comprises of 0.1% formic acid and mobile phase B as acetonitrile used for gradient elution. Analysis completed within run time of 4 min at the mobile phase flow rate of 0.3mL/min. The retention time of metoprolol succinate and olmesartan medoximil were 1.029 min and 2.514min, respectively. Analytes were extracted using acetonitrile as extracting solvent. The method validated in terms of linearity, accuracy, precision, lower limit of quantitation, method sensitivity and various solution stability parameters. Linearity of olmesartan medoximil and metoprolol succinate was in the range of 5-1500ng/mL. All the method validation parameters were determined as per ICH, EMA and FDA guidelines and falls under the stated acceptance criteria. The presented work provides a validated bioanalytical method for simultaneous determination of olmesartan medoximil and metoprolol succinate in human plasma. The method is accurate, simple, precise, fast and suitable for its application for bioequivalence and pharmacokinetic studies.

KEYWORDS: Olmesartan Medoximil, Metoprolol Succinate, Validation, UHPLC-MS/MS, Bioanalytical method, ICH Guidelines, US FDA Guidelines, EMA Guidelines, Human plasma.

1.0 INTRODUCTION:

Olmesartan medoximil (OLM) is chemically (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl5- (2 - hydroxypropan -2-yl)-2-propyl-3-[[4-[2-(2H-tetrazol-5-yl) phenyl]phenyl] methyl] imidazole- 4- carboxylate (Fig. 1a) Olmesartan belongs to a class of angiotensin II receptor blockers. These medicines are closely related to ACE inhibitors which blocks the activity of enzyme responsible for the narrowing of the blood vessels^1,2.

Metoprolol succinate (MET) is chemically butanedioic acid;1-[4-(2-methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol (Fig.1b) is a sensitive cardiac β-blockers used in the treatment of arrhythmia, hypertension, angina pectoris, myocardial infraction^3,4. Both drugs are official in Indian Pharmacopoeia (IP) as well as British Pharmacopoeia (BP). On literature survey, it has been found that there are various chromatography and spectrophotometric methods available for estimation of OLM individually and in combined with various other drugs^2,5-19. Various UV spectrophotometric and HPLC methods are available for evaluation of MET individually in bulk, formulation and in biological samples^20-24 and UV spectrophotometric, TLC and HPLC methods for estimation of MET in combination with other drugs and OLM^25-37. Literature survey also reveals that there is no method reported for estimation of these analytes in combination from human plasma. Therefore, effort were directed towards development and validation of UHPLC-MS/MS TQ method for estimation of the titled analytes form spiked human plasma as a simple yet fast extraction and quantification method; which allow low cost therapeutic monitoring in a reasonable time. By virtue of its short run time the proposed bio-analytical methods would be beneficial in pharmacokinetic or bioequivalence studies where a large no of samples need expedited analysis. Proposed method was developed and validated as per ICH, European Medical Agency (EMA) and US FDA guidelines^38-40.

Fig. 1: Structures of a) Olmesartan and b) Metoprolol succinate.

2.0 MATERIALS AND METHODS:

2.1 Chemicals, reagents and Instrumentation:

The drug sample of Olmesartan medoximil (Assay - 99.81%) and Metoprolol succinate (Assay - 99.77%) were gift by Cadila Healthcare Ltd., Ahmedabad, India. All the reagents and chemical used in presented research work were of HPLC grade quality and purchased from Sisco Research Lab Pvt. Ltd., Mumbai. All the operations were carried out on Shimadzu N Series UHPLC with Shimadzu 8045, LC-MS/MS TQ instrument. Data was integrated using Shimadzu LabSolutions software version 6.89. The column used was Shimadzu Shimpack-C18 GIST AQ (50mm X 2.1 mm, 1.9µm) and the injection volume was 10µL using autosampler mode and mobile phase flow rate was 0.3 mL/min.

2.3 Preparation of Standard and Sample solutions and Calibration Curve:

Analyte standard stock solutions (SSS) were prepared individually, 100mg of each of olmesartan medoximil and metoprolol succinate were accurately weighed and transferred to separate 100mL volumetric flask containing 50ml of acetonitrile: water (1:1 v/v) and dissolved by sonication for 5min. Volume adjusted and suitably diluted to obtain a SSS containing 1000µg/mL of each separately. The above stocks were suitably spiked in Human plasma suitably spiked to get combined standard solutions containing 5ng/mL to 1500ng/mL of each analyte and used for linearity study. Quality control (QC) samples were prepared by spiking QC dilutions in interference free plasma to get lower limit of quantification (LLOQ), lower QC (LQC), middle QC (MQC), higher QC (HQC) and upper limit of quantification (ULOQ) concentrations for both the drugs. Actual concentrations used (level) were 5.00ng/mL (LLOQ), 15.00ng/mL (LQC), 750.00ng/mL (MQC), 1200.00ng/mL (HQC) and 1500ng/mL (ULOQ). All the linearity, spiked, standard and working solutions and standards solutions were stored at -20°C for sample analysis and validation studies.

2.5 Sample Extraction Procedure:

To the 1000µL of plasma spiked sample 5mL of acetonitrile was added and subjected to vortex mixing for 10min at room temperature, then the samples were centrifuged at 10,500rpm for 10minutes. After centrifugation, the upper organic layer was transferred to a glass container and evaporated inside a vacuum oven at 40⁰C. The dried residue was dissolved in 1mL of water: acetonitrile (1:1 v/v). The mixture was properly sonicated for 10 min and 10µL of this solution was injected into liquid chromatography under optimized chromatographic conditions.

2.4 Optimized Chromatographic Conditions:

Table 1: Optimized chromatographic conditions and Optimized MS parameters

Sr. No.	Optimized Chromatographic conditions		Gradient program			Optimized MS parameters
Sr. No.	Parameters	Details	Time (min.)	Mobile Phase-A	Mobile Phase-B	Parameters	Details
1.	Column	Shimadzu Shimpack-C18 GIST AQ (50 mm X 2.1 mm, 1.9 µm)	0.01	80	20	Interference	ESI
2.	Mobile phase A	0.1% Formic acid in water	1.00	80	20	Heat Block temperature	400 ⁰C
3.	Mobile phase B	Acetonitrile	2.00	10	90	DL temperature CVB	250 ⁰C
4.	Column temperature	40 °C	3.00	10	90	Nebulizing gas flow	3.00 L/min
5.	Injection volume	10 µL	3.10	80	20	Heating gas flow	10.00 L/min
6.	Flow rate	0.3 mL/minute	4.00	80	20	Drying gas flow	15.00 L/min
7.	Run time	4 minutes	4.00	80	20	Polarity	Positive

2.5 Sample Extraction Procedure:

To the 1000µL of plasma spiked sample 5mL of acetonitrile was added and subjected to vortex mixing for 10 min at room temperature, then the samples were centrifuged at 10,500rpm for 10minutes. After centrifugation, the upper organic layer was transferred to a glass container and evaporated inside a vacuum oven at 40⁰C. The dried residue was dissolved in 1mL of water: acetonitrile (1:1v/v). The mixture was properly sonicated for 10min and 10µL of this solution was injected into liquid chromatography under optimized chromatographic conditions.

2.6 Method Validation:

Validation of the optimized method was carried out with respect to linearity, precision, LLOQ precision, accuracy, dilution integrity and carryover test Solution stability studies such as solution stability, bench top stability, freeze thaw stability and auto sampler stability were performed. System suitability test (SST) was performed six times injecting standard mixture of drug at MQC before start of the validation experiments and by determining %RSD of the peak areas; which was always < 10% throughout the validation studies.

2.6.1 Selectivity and Carryover:

During the validation, blank plasma samples from 6 different lots were evaluated. During the selectivity run, an LLOQ standards were also used. Responses for the blank plasma to evaluate the selectivity from the six different lots were compared with the response of LLOQ standard. The response in the blank plasma at the retention time of analyte peak should not be more than 20% of response obtained in LLOQ solution. To study the carryover effect, LLOQ and ULOQ followed by a blank preparation was injected. The response in the carryover blank at the analyte RT was compared to the response in LLOQ which should not be more than 20% of response obtained in LLOQ solution.

2.6.3 Calibration Curve (CC), Linearity and LLOQ:

Linearity of the method was carried out by injecting six conc. of the drugs prepared in the plasma matrix in the range 5 to 1500ng/mL for both drugs in six replicates into the UHPLC-MS/MS system by keeping the injection volume constant (10µL). The graph of peak areas were plotted against the corresponding conc. to construct CC equation. In addition, the concentrations of the calibration standards were back calculated; back calculated concentrations of the calibration standards other than LLOQ were evaluated for the limit which should be within ±15% of the nominal value. For the concentrations at LLOQ the limit is ±20%. LLOQ is the lowest amount of analyte in a sample which can be quantified reliably, with specified level of accuracy and precision. The signal to noise ratio obtained in LLOQ should not be less than 10.

2.6.5 Accuracy (Recovery) and Precision:

Accuracy of the method was determined at four levels by applying the method to the both drug samples to which known amount of standard solutions were spiked. Recovery (RE) level explored for the study were LLOQ, LQC, MQC and HQC. Spiked solutions were prepared in 5 replicates and analysed. The recovery samples were analysed against the calibration curve, and the obtained conc. were compared with the nominal value for assay limits as mentioned in previous section. Intra-day recovery and precision was determined by injecting 5 replicates of the said concentration on same day and results were reported. Inter-day recovery and precision was determined by injecting 3 replicates of the said concentration on a different days and results were reported.

2.6.6 Dilution Integrity and Matrix Effect:

To study the effect of sample dilution on accuracy and precision of the method two dilution levels were used. Two dilution levels; 2-fold and 4-fold were used to measure the dilution effect on the developed method. Accuracy and precision is expected to be within ±15%. Dilution samples were prepared by spiking plasma at higher concentration above ULOQ and MQC. Six samples of dilution integrity were prepared by diluting them twice and another six samples by diluting them four times. These samples were analysed against fresh calibration curve standards. Matrix effects was investigated using at least 6 lots of blank matrix. The QC samples were prepared at LLOQ and ULOQ. For both the analytes, the matrix factor (MF) for each lot of matrix was calculated. MF was calculated by determining the ratio of the peak area in the presence of to the peak area in absence of matrix. The % RSD of the matrix factor should not be greater than 15 % when determined for the 6 lots of matrix.

2.6.8 Solution Stability Studies:

All the stability studies were carried out for LLOQ and MQC spiked samples. The %Recovery of calculated concentration should be less than ±15% for the MQC sample, and less than ±20% for the LLOQ compared to initial concentration, when calculated against fresh CC. Solution stability determined for both, olmesartan and metoprolol at room temperature (15 to 20⁰C) after 24 hrs. The bench top stability evaluated by keeping the samples on work bench at ambient temperature for 8 hours. The duration of bench top stability was calculated from the retrieval time of stability samples from the deep freezer to the start time of sample processing. Freeze thaw stability was evaluated by exposing LLOQ and MQC sample to three freeze and thaw cycles. Analyte stability was determined after the third freeze and thaw cycle. Autosampler stability was evaluated by keeping the samples in the autosampler for 72 hours at 10⁰C. The duration of autosampler stability of analyte was calculated from the autosampler loading time of samples to the injection time of the first stability sample. Long-term stability was determined for OLM and MET at 2 - 8 ⁰C for 30 days. The mixed analytes samples at LLOQ and at MQC level were kept at refrigerated conditions for 30 days and analysed against fresh standard.

3.0 RESULT AND DISCUSSION:

3.1 Development and optimization of UHPLC method equipped with MS/MS:

The UHPLC- MS/MS procedure was optimized in order to develop analytical method for simultaneous estimation of olmesartan medoximil and metoprolol succinate with very short chromatography run time. The mixed standard stock solutions were injected into the column. For UHPLC optimization different ratios of different mobile phase were tried in combination with different column including ACN, water, methanol and different columns such as C8, C18 with different dimensions such as (50 mm X 2.1mm, 1.9µm), (75mm X 2.1mm, 1.9µm), (50 mm X 2.1mm, 1.7µm. But it was found that none of the combination gives the appropriate results. Further a trial of 0.1% Formic acid in water: acetonitrile with gradient elution and flow rate of 0.3mL/minute using Shimadzu Shimpack-C18 GIST AQ (50mm X 2.1mm, 1.9µm) column resulted in fast method with MET and OLMrun time of 1.029min and 2.514min, respectively with acceptable system suitability parameters. Optimized chromatographic conditions and gradient program presented in Table 1.

Development and optimization of MS/MS parameters initiated aiming a highly sensitive and selective method. Standard solution mixture was directly infused into the mass spectrometer, and the operating conditions were optimized to monitor the analyte. Positive mode tuning was used to find the parent and daughter ions, thus, to achieve maximum response for both the drugs. For each drug, the MRM channel chosen was the one that gave minimal or no response from the other drug to minimize the cross stalk. First, both drugs were ionized using ESI source prior to detection by multiple reactions monitoring (MRM) mode while monitoring at the following transitions: 559.15/207.00 and 268.20/116.15 for OLM and MET, respectively, Optimized MS parameters listed in Table 1 and full scan of mixture presented in Fig. 2. MRM Identification spectra for metoprolol and olmesartan presented in Fig. 3. For sensitivity adjustment, during the optimization of instrument parameters, a study carried out to evaluate the effect of change of sensitivity of the instrument on the peak response. The sensitivities evaluated at different voltage, peak response of OLM and MET obtained optimum using conditions as per Table 2.

3.2 Method Validation:

Results for various validation parameters are given below:

3.2.3 Calibration Curve, Linearity and Solution Stability Studies:

Linearity of the method was carried out by injecting six concentrations of the stated drugs prepared in the plasma matrix in the range 5 to 1500ng/mL for both olmesartan and metoprolol in six replicates into the UHPLC-MS/MS system. All samples were analysed using optimised LC-MS/MS conditions. The linearity range for both the analyte found to be 5 to 1500ng/mL, linearity data for both the analyte presented in Table 3 and linearity curves presented in Fig. 5. At all the selected concentration levels of the calibration curves back calculated amounts of the calibration standards were always less than ±15% of the OLM and MET nominal values. All the stability studies were carried out for LLOQ and MQC spiked samples. The % Recovery obtained are depicted in below Table 3.

3.2.4 Lower limit of Quantification:

The LLOQ is the lowest calibration standard which is 5 ng/mL for both the analytes. The lower limit of quantification (LLOQ) for both the analyte was 5ng/mL. The accuracy for LLOQ was found to be 97.8% for OLM and 106.7% for MET. The signal to noise ratio obtained in LLOQ were 19.1 and 33.2 for OLM and MET, respectively.

3.2.5 Accuracy (Recovery) and Precision:

Accuracy of the method was determined at LLOQ, LQC, MQC and HQC levels. The recovery samples for accuracy and precision were analysed against the calibration curve. Accuracy and precision data is presented in Table 4, experimental concentrations were compared with the nominal values of both the analytes and the values were always within the limit.

Fig. 5: UHPLC-MS/MS calibration curve for Olmesartan medoximil and metoprolol succinate

Table 3: Linearity data of analyte and Results of Solution Stability Studies (n=6)

Linearity data					Results of Solution Stability Studies
Concentration (ng/mL)	OLM		MET		Results of Solution Stability Studies
Concentration (ng/mL)	Area	% RSD	Area	% RSD	Stability Storage condition	OLM		MET
5	11304	7.51	16820	7.31		Mean % RE (Limit ± 15%)
100	223073	4.84	331713	6.09		LLOQ	MQC	LLOQ	MQC
400	906914	3.69	1340092	2.50	Solution stability (24 hours at RT)	5.63	3.99	4.29	3.29
800	1813849	4.08	2715345	2.73	Solution stability (24 hours at RT)	5.63	3.99	4.29	3.29
1200	2690267	6.36	4034438	3.25	Bench top stability (8 hours)	4.88	4.01	6.19	4.39
1500	3395118	4.39	5052689	3.81	Freeze and thaw stability (3 cycles)	6.23	4.34	5.34	3.94
Slope	2256.9	--	3370.4	--	Auto-sampler stability (72 hours at 10 ºC)	6.11	3.87	7.02	5.12
Correlation Coefficient	0.999	--	0.999	--	Long term stability (30 days at 2-8 ºC)	4.34	3.78	5.76	4.56
y intercept	271.5	--	-1208.9	--	Long term stability (30 days at 2-8 ºC)	4.34	3.78	5.76	4.56

Table 4: Accuracy and Precision data

Analyte	OLM					MET
Intra-day, n=5
Recovery Level	Spiked Conc (ng/mL)	Mean Conc Obtained (ng/mL)	RSD (%)	Mean Recovery (%)	Mean RE (%)	Spiked Conc (ng/mL)	Mean Conc Obtained (ng/mL)	RSD (%)	Mean Recovery (%)	Mean RE (%)
LLOQ	5.003	4.634	6.45	92.62	7.38	5.065	5.431	7.12	107.23	7.23
LQC	15.009	15.831	5.01	105.48	5.48	15.195	15.983	4.87	105.19	5.19
MQC	750.450	789.452	6.12	105.20	5.20	759.750	785.092	5.98	103.34	3.34
HQC	1200.720	1274.34	4.78	106.13	6.13	1215.600	1298.458	5.66	106.82	6.82
Inter-day, n=3
Recovery Level	Spiked Conc (ng/mL)	Mean Conc Obtained (ng/mL)	RSD (%)	Mean Recovery (%)	Mean RE (%)	Spiked Conc (ng/mL)	Mean Conc Obtained (ng/mL)	RSD (%)	Mean Recovery (%)	Mean RE (%)
LLOQ	5.058	5.344	7.89	105.65	5.65	5.276	5.651	5.01	107.11	7.11
LQC	15.174	15.884	4.56	104.68	4.68	15.828	16.801	6.34	106.15	6.15
MQC	758.700	800.345	3.09	105.49	5.49	791.400	835.895	4.78	105.62	5.62
HQC	1213.920	1270.454	5.87	104.66	4.66	1266.240	1312.045	6.23	103.62	3.62

3.2.6 Dilution Integrity and Matrix Effect:

The precision and accuracy for OLM were found to be 4.43% (RSD) and 94.78% (mean % nominal) for all the samples with dilution factor of one half. For its samples with dilution factor of one fourth, precision and accuracy observed were 2.51% and 104.17%, respectively. Precision and accuracy for MET were found to be 2.61% (RSD) and 104.88% (mean % nominal) for all the samples with dilution factor of one half. For its samples with dilution factor of one fourth, precision and accuracy observed were 3.11% and 105.15%, respectively. The % RSD of Mean Matrix Factor (MF) for OLM were found to be 3.81% and 5.34% for LLOQ and ULOQ respectively. Again, the %RSD of Mean Matrix Factor (MF) for MET were found to be 5.64% and 3.78% for LLOQ and ULOQ respectively.

4.0 CONCLUSION:

UHPLC method was developed and validated as per ICH, EMA and FDA guidelines. Study results indicate that the proposed method is suitable for simultaneous estimation of Olmesartan medoximil and Metoprolol succinate in bioanalytical samples. The method has linear response in the developed range and is accurate, precise, sensitive and specific for analytes. As the mobile phase is MS compatible, it is possible to estimate the stated drugs in biological matrices; thus the proposed bioanalytical method is accurate, simple, precise, fast and suitable for its application for bioequivalence and pharmacokinetic studies.

5.0 ACKNOLEDGEMNTS:

The authors are thankful to Spinco Biotech Private Limited, Chennai (India), for providing facility and necessary guidance to do work.

7. CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

8.0 REFERENCES:

1. Sweetman SC. Martindale: The complete drug reference. 35th edn. London, UK: Pharmaceutical Press; 2007. p. 1224.

2. Dash AK, Palo A. Stress induced method development and validation of olmesartan in bulk and pharmaceutical dosage form by UV Spectrophotometric method; https://www.pharmatutor.org/articles/stress-induced-method-development-and-validation-of-olmesartan [Last accessed on April 01, 2021].

3. British Pharmacopoeia. Vol. II. London: The British Pharmacopoeial commission 2010. p. 1419.

4. Indian Pharmacopoeia. Government of India, Ministry of Health and Family Welfare. 6th ed. Vol. II. Ghaziabad: Indian Pharmacopoeia Commission; 2010. p. 1681.

5. Ritesh N. Pancholi S. Validated stability indicating LC-DAD method for determination of olmesartan medoxomil in tablets exposed to stress conditions. Acta Pharm Sci 2009; 51:323-31.

6. Prabhakar V. Vipan K. Bharti S. Development and validation of an RP-HPLC method for estimation of olmesartan medoxomil in tablet formulations and stability studies. J Pharm Res 2010; 3: 1015-1018.

7. Pai N. Sawant S. Development and validation of RP-HPLC method for estimation of olmesartan medoxomil in tablet dosage form. Research J. Pharm. and Tech. 2013; 6(11): 1279-1284.

8. Patel R. Patel M. Raj H. Shah N. Forced degradation studies of olmesartan medoxomil and characterization of its major degradation products by LC-MS/MS, NMR, IR and TLC. Asian J. Pharm. Ana. 2015; 5(3): 119-125. doi:10.5958/2231-5675.2015.00019.8

9. Rao T. Vijayalakshmi A. Apparao K. Krishnarao N. A new analytical method validation and quantification of olmesartan medoxomil and its related impurities in bulk drug product by HPLC. Asian J. Pharm. Tech. 2017; 7(3): 147-152. doi:10.5958/2231-5713.2017.00024.1

10. Babu K. Jayakar B. Vinoth Kumar G. Absorption correction method for estimation of amlodipine besylate and olmesartan medoxomil in combined tablet dosage form. Asian J. Research Chem. 2011; 4(7):1100-1102.

11. Shah S. Asnani A. Kawade D. Dangre S. Spectrophotometric method for simultaneous estimation of olmesartan medoxomil and amlodipine besylate in pharmaceutical preparations. Research J. Pharm. and Tech. 2012; 5(7): 955-957.

12. Kachave R. Bhadane R. Wagh R. Jain D. Simultaneous estimation of olmesartan medoxomil and hydrochlorothiazide by spectrophotometry in tablet formulation. Research J. Pharm. and Tech. 2010; 3(4): 1047-1049.

13. Patel R. Patel M. Raj H. Shah N. Development and validation of stability indicating HPLC method for olmesartan medoxomil and indapamide in tablet. Asian J. Pharm. Res. 2015; 5(1): 15-23. doi:10.5958/2231-5659.2015.00016.8

14. Saravanan G. Bajidbhee S. Sri Krishnanjaneyulu I. Development and validation of RP-HPLC method for simultaneous estimation of hydrochlorothiazide and olmesartan medoxomil in bulk and pharmaceutical dosage form. Asian J. Research Chem. 2015; 8(2): 147-152. doi:10.5958/0974-4150.2015.00026.7

15. Priyadharisini J. Saraswathi D. Ajitha A. Jerad S. RP-HPLC determination of olmesartan medoxomil and amlodipine besylate in tablets. Research J. Pharm. and Tech. 2011; 4(6): 903-904.

16. Raja B. Lakshmana R. Development and validation of a reversed phase HPLC method for simultaneous estimation of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage form. Int J Res Pharm Chem. 2011; 3: 714-717. doi.org/10.13040/IJPSR.0975-8232.1(12).80-84

17. Patil P. More H. Pishwikar S. RP-HPLC method for simultaneous estimation of amlodipine besylate and olmesartan medoxomil from tablet. Int J Pharm Pharm Sci. 2011; 3(3):3-6.

18. Nagavalli D. Venkata S. Aluri B. Development of UV spectrophotometric method for the simultaneous estimation of olmesartan medoxomil and atorvastatin calcium in tablet by simultaneous equation and first order derivative method. Journal of Pharmacy research. 2011; 4(6): 1711-1712.

19. Wankhede S. Wadkar S. Raka K. Chitlange S. Simultaneous estimation of amlodipine besylate and olmesartan medoxomil in pharmaceutical dosage form. Journal of Pharmaceutical Sciences. 2009; (5): 563-567. doi:10.4103/0250-474X.58190

20. Verma N. Ghosh A. Chattopadhyay P. UV- Spectrophotometric determination of metoprolol succinate. Research J. Pharm. and Tech. 4(2); 2011: 271-272.

21. Phale M. Hamrapurkar P. A Validated and Simplified RP-HPLC method for metoprolol succinate from bulk drugs. Asian J. Research Chem. 2009; 2(2): 119-122.

22. Soni S. Verma R. Verma D. Verma A. Analytical method development and validation of metoprolol succinate by HPLC and ultraviolet spectroscopy technique. Research J. Pharm. and Tech. 2021; 14 (2): 931-937. Doi:10.5958/0974-360X.2021.00166.9

23. Mahvash I. Shobha Rani R. Estimation of metoprolol in human plasma by HPLC method. Int. J. Pharm. Pharm. Sci. 2014; 7(1): 442-446.

24. Albers S. Elshoff J. Völker C. Richter A. Läer S. HPLC quantification of metoprolol with solid-phase extraction for the drug monitoring of paediatric patients. Biomed. Chromatogr. 2005; 19: 202-207. doi:10.1002/bmc.436

25. Chabukswar A. Tambe S. Choudhari V. Sharma S. Mohokar M. Chate S. Ratio derivative spectrophotometry method for simultaneous estimation of metoprolol and amlodipine in their combined dosage form. Research J. Pharm. and Tech. 2012; 5(7): 950-954.

26. Vora B. Parmar R. Nayak P. Shah D. Development and validation of the simultaneous UV spectrophotometric method for estimation of metoprolol succinate and olmesartan medoxomil in the tablet dosage form. Pharmaceutical Methods. 2012; 3(1): 44–47. doi:10.4103/2229-4708.9772

27. Chabukswar A. Mohokar M. Choudhari V. Sharma S. Tambe S. Pagare B. Absorption corrected method and isoabsorptive point method for simultaneous estimation of metoprolol and amlodipine in their combined dosage form. International Journal of Pharmaceutical Sciences and Drug Research. 2012; 4(4): 240-244.

28. Venkatachalam T. Kishor K., Kalai S. Srinivasan R. Mariammal R. Lalitha K. New spectrophotometric method applied to the simultaneous determination of metoprolol succinate and hydrochlorthiazide. Asian J. Research Chem. 2010; 3(2): 464-467. DOI: 10.4103/0250-474X.44606

29. Rjanit S. Paras V. Raj H. Absorption correction method for simultaneous estimation of nifedipine and metoprolol succinate in their synthetic mixture using spectrophotometry. Asian J. Pharm. Tech. 2015; 5 (1): 13-16.doi:10.5958/2231-5713.2015.00003.3

30. Garg G. Saraf S. Simultaneous estimation of ramipril and metoprolol tartrate in combined dosage forms. J Indian Chem. Soc. 2007; 84: 609-11.

31. Baldania S. Parmar A. Bhatt K. Shah D. Chhalotiya U. Simultaneous estimation of metoprolol succinate and olmesartan medoxomil in pharmaceutical formulation by TLC-densitometric method. ISRN Analytical Chemistry. 2012; 2012: 1-7. doi.org/10.5402/2012/245429

32. Wankhede S. Dixit N. Zambare S. Chitlange S. Development and validation of RP-HPLC Method for quantitative estimation of atorvastatin calcium and metoprolol succinate in combined dose capsule formulation. Asian J. Research Chem. 2010; 3(3): 663-665.

33. Neelima R. Durga Devi N. Prameela R. Madhavi B. Praveen P. Mrudula B. Determination of metoprolol succinate and atorvastatin calcium in capsules using RP-HPLC. Asian J. Research Chem. 2010; 3(4): 892-894.

34. Rao P. Rahaman S. Prasad R. Reddy G. RP-HPLC method of simultaneous estimation of amlodipine besylate and metoprolol in combined dosage form. Int J Pharm Res Dev. 2010; 9: 69-76.

35. Thakker NM, Choudhari VP, Kuchekar BS, Panchal HB, Rakholiya DR, Murugan R. Development and validation of a stability indicating RP-HPLC method for simultaneous estimation of olmesartan medoxomil and metoprolol succinate in pharmaceutical dosage form. Pharmaceutical Methods. 2012; 3(2): 84–89. doi:10.4103/2229-4708.103880

36. Singh A. Dwivedi J. Gandhi S. Development and validation of stability-indicating RP-HPLC method for determination of metoprolol succinate and olmesartan medoxomil in bulk and in formulation. Research J. Pharm. and Tech. 2014; 7(12): 1368-1373.

37. Mehulkumar P. Ramesh V. Vinay kumar V. Srinivas R. Diwan P. Simultaneous spectroscopic estimation of amlodipine besylate and olmesartan medoximil in tablet dosage form. Asian J Research Chem. 2009; 2(2): 127-130.

38. International Conference on Harmonization (ICH). Guidance for Industry, Q1A (R2), stability testing of new drug substances and products. Geneva: IFPMA; 2003.

39. Food and Drug Administration: USFDA Guidance for Industry: Bioanalytical Method Validation 2001. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf [Last accessed on April 01, 2021].

40. Van Amsterdam P: The EMA Bioanalytical Method Validation Guideline: process, history, discussions and evaluation of its content 2013. https://e-b-f.eu/wp-content/uploads/2018/05/bcn2012-S62.-5_van_amsterdam.pdf [Last accessed on April 01, 2021].

Received on 03.05.2021 Modified on 13.07.2021

Research J. Pharm. and Tech. 2022; 15(7):2909-2916.

DOI: 10.52711/0974-360X.2022.00485

Optimized Sensitivity Conditions						Carryover Data of Analytes
Analyte	Retention time (min)	m/z	Q1 Pre Bias (V)	CE	Q3 Pre Bias (V)	Area (Response) at Level
Analyte	Retention time (min)	m/z	Q1 Pre Bias (V)	CE	Q3 Pre Bias (V)	Blank	LLOQ	ULOQ	% Carryover (w.r.t LLOQ)
OLM	2.514	559.15 > 207.00	-30.0	-35.0	-20.0	1098	12009	3594009	9.1
MET	1.029	268.20 > 116.15	-20.0	-20.0	-20.0	1309	18456	5199345	7.1