INTRODUCTION:

The UHPLC/Q-TOF-MS/MS (Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry) technique has been used worldwide in the identification and quantitative analysis of drug products. UHPLC provides the fast, better chromatographic separation with shorter chromatographic run time. Q-TOF-MS provides higher sensitivity, selectivity and accurate mass measurement and reliable chemical fragmentations, which are ultimately helpful in structure elucidations of the compounds.^1-3Losartan, Telmisartan, Valsartan, Candesartan, Irbesartan and Olmesartan are antihypertensive drug, belongs to a group of Angiotensin II Receptor Blockers (ARBs). These drugs have been used worldwide for the treatment of hypertension.^4,5

The literature survey revealed that several liquid chromatography-mass spectrometry methods have been reported for determination of Losartan^6-9, Telmisartan^10-17, Valsartan^18-21, Candesartan^22-24, Irbesartan²⁵ and Olmesartan.^26-28 However in our earlier research work an UHPLC/Q-TOF-MS method has been developed for structural identification and quantitative determination of these drugs in their pure samples. But the reported method does not explain the entire mass fragmentations of these drugs.²⁹ Hence in the presented work, the UHPLC/Q-TOF-MS/MS technique was applied using respective MS and MS/MS conditions and a new method was developed, validated and applied for mass spectrometric identification and quantitative determination of SARTANS antihypertensive drugs.

EXPERIMENTAL:

Chemicals and Reagents:

Losartan (C₂₂H₂₃ClN₆O, Molecular weight 422.91), Telmisartan (C₃₃H₂₀N₄O₂, Molecular weight 514.61) Valsartan (C₂₄H₂₉N₅O_3, Molecular weight 435.50), Candesartan (C₂₄H₂₀N₆O₃, Molecular weight 440.45), Irbesartan (C₂₅H₂₈N₆O, Molecular weight 428.50) and Olmesartan (C₂₄H₂₆N₆O₃ Molecular weight 446.50) were kindly supplied as gift sample by Systopic Pharmaceuticals Ltd. (New Delhi, India). LC-MS grade water; acetonitrile, methanol, and ammonium acetate were purchased from Fluka analytical, Sigma-Aldrich Corporation, St. Louis, MO, USA.

UHPLC and Q-TOF-MS Conditions:

UHPLC was performed with a Waters Acquity UPLC system equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector. Chromatographic separation was performed on a Waters Acquity UPLC BEH C₁₈(100.0 × 2.1mm, 1.7µm) column. The mobile phase for UPLC analysis consisted of acetonitrile–2mM ammonium acetate (50:50, v/v) which was filtered through 0.45mm membrane filter and degassed by sonication. For isocratic elution, the flow rate of the mobile phase was kept at 0.25mL/min and 10 mL of sample solution was injected in each run. The total chromatographic run time was 3.0min. Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. The various parameters for Q-TOF-MS such as Capillary voltage, Sampling cone voltage, Source temperature, Cone gas flow, Source gas flow, Collision gas (Argon) and Collision energy were 3.0kV, 40 V, 80ºC, 50L/h, 2.5×10^-4 mbar and 12V, respectively.

Preparation of Standard Solutions:

Each of the drugs was weighed accurately and transfer to 50mL volumetric flasks separately. The powders were then dissolved with approximately 25mL of methanol and ultrasonicated for 5min. The final volume was made up with methanol. The solutions were further diluted with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound and injected in to UPLC system.

Validation of Method:

The developed method was validated according to ICH validation guidelines.^30-35Different standard concentrations each of the compound in the range of 1-1000ng/mL (1, 10, 50, 100, 200, 500, and 1000ng/mL) were prepared in methanol: water (50:50, v/v). The solutions were injected in to the UPLC/QTOF-MS system for analysis. Linearity, Intraday and interday precision and accuracy were also evaluated.

RESULTS AND DISCUSSION:

Time-of-Flight mass spectrometry is based on bombardment of sample by electron beam which causes fragmentation to form smaller groups of atoms or ions. The fragmented ions travel through evacuated flight tube. The generated ions then measures the time each ion takes to reach the detector. The TOF mass spectrometry provides accurate and reliable chemical fragmentations, which are ultimately helpful in structure elucidations of compounds. The solutions were injected in to the Q-TOF-MS/MS system to obtain mass spectra. The mass spectrometer was operated via negative ionization mode. Under the selected MS conditions the compounds are converted in to their respective precursor ions at m/z 421.20 for Losartan, m/z 513.16 for Telmisartan, m/z 434.50 for Valsartan, m/z 439.10 for Candesartan, m/z 427.20 for Irbesartan and m/z 445.50 for Olmesartan. Under the selected MS/MS conditions the precursor ions [M-H]^- fragmented in to major product ions at m/z 421.20 to 127.00 for Losartan, m/z 513.16 to 469.13 for Telmisartan, m/z 434.50 to 255.50 for Valsartan, m/z 439.10 to 309.90 for Candesartan, m/z 427.20 to 193.10 for Irbesartan and 445.50 to 235.50 for Olmesartan as shown in Figure 1, 2, 3, 4, 5 and 6, respectively.

Figure 1: TOF-MS/MS Spectra of Losartan

Figure 2: TOF-MS/MS Spectra of Telmisartan

Figure 3: TOF-MS/MS Spectra of Valsartan

Figure 4: TOF-MS/MS Spectra of Candesartan

Figure 5: TOF-MS/MS Spectra of Irbesartan

Figure 6: TOF-MS/MS Spectra of Olmesartan

The proposed MS/MS fragmentation mechanism of Losartan, Telmisartan, Valsartan, Candesartan, Irbesartan and Olmesartan are shown in Figure 7, 8, 9, 10, 11 and 12, respectively. Losartan parent ion (M-H^-) at m/z 421.20 is fragmented via –OH and –Cl containing ring and gives product ion at m/z 127.0. Telmisartan precursor ion (M-H^-) at m/z 513.16 converted in to product ion at m/z 469.13 by loss of CO₂ molecule. Valsartan precursor ion (M-H^-) at m/z 434.50 is fragmented in to product ion at m/z 255.50. Candesartan (M-H^-) at m/z 439.10 is fragmented in to product ion at m/z 309.9. Irbesartan precursor ion (M-H^-) at m/z 427.20 is converted in to two products ions one at m/z 205.50.

Figure 7: Proposed MS/MS Fragmentation Mechanism of Losartan

Figure 8: Proposed MS/MS Fragmentation Mechanism of Telmisartan

Figure 9: Proposed MS/MS Fragmentation Mechanism of Valsartan

Figure 10: Proposed MS/MS Fragmentation Mechanism of Candesartan

Figure 11: Proposed MS/MS Fragmentation Mechanism of Irbesartan

Figure 12: Proposed MS/MS Fragmentation Mechanism of Olmesartan

Validation of the method:

The Results Obtained from Linearity, LOD, and LOQ is presented in Table 1. The obtained results indicated that higher sensitivity of the method. The RSD less than 2% were obtained for all the compounds by evaluation of intraday, interday, and different analysts precision suggested that an acceptable precision, accuracy and specificity of the method.

CONCLUSION:

The UHPLC-Q-TOF-MS/MS method was developed, validated and applied for identification and quantitative determination of SARTANS antihypertensive drugs such as Losartan, Telmisartan, Valsartan, Candesartan, Irbesartan, and Olmesartan. The quantification of drugs was carried out by UHPLC and using MS/MS transition by selecting precursor to major product ion. The mass spectra of drugs were obtained and the fragmentation mechanisms were established. The proposed fragmentation mechanisms of the drugs are helpful in mass spectral studies. Hence the method must be adopted for structural identification and quantitative determination of SARTAN antihypertensive drugs.

ACKNOWLEDGEMENTS:

The author is grateful to Systopic Laboratories Ltd., Delhi, India, for providing pure samples of antihypertensive drugs. The author is also thankful to Dean and In-charge of Instrumentation Facilities, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi, India, for providing opportunities to work on UPLC/Q-TOF-MS/MS system.

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Received on 06.08.2023 Modified on 15.01.2024

Research J. Pharm. and Tech 2024; 17(7):3004-3008.

DOI: 10.52711/0974-360X.2024.00469

Parameters	Losartan	Telmisartan	Valsartan	Candesartan	Irbesartan	Olmesartan
Linear range (ng/mL)	1-1000	1-1000	1-1000	1-1000	1-1000	1-1000
Correlation coefficient	0.9997	0.9998	0.9998	0.9997	0.9998	0.9998
LOD (ng/mL)	0.1	0.05	0.05	0.1	0.1	0.1
LOQ (ng/mL)	1	0.1	0.1	1	1	1