INTRODUCTION:

The UHPLC/Q-TOF-MS (Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry) technique is comparatively new chromatographic technique and has been used worldwide in the identification and quantitative analysis of drug products. This technique has been successfully applied in every area of chromatographic and pharmaceutical analysis. 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. The degradation study, metabolite profiling and pharmacokinetic study has been investigated in various biological samples by applying UHPLC/Q-TOF-MS technique. In the Q-TOF mass spectrometry compounds are converted into smaller particles in the capillary tube. These smaller particles enter in to evacuated flight tube and detected according to their mass values. Q-TOF mass is an ideal mass spectrometer for structural identification and structural elucidation of synthetic organic compounds and biomolecules. This gives the accurate mass, reliable chemical fragmentation of compounds^[1-9]. Losartan, Telmisartan, Valsartan, and Candesartan are widely used antihypertensive drug, belongs to a group of Angiotensin II Receptor Blockers (ARBs). They modulate the Renin-Angiotensin-Aldosterone-System (RAAS) by blocking the activation of Angiotensin II AT₁Receptors resulting in Vasodilation and Vasodilation ultimately cause the fall in blood pressure. These drugs have been used worldwide for the treatment of hypertension^[10-16]. The literature survey revealed that several Liquid chromatography-mass spectrometry methods have been reported for determination of Losartan^[17-20], Telmisartan ^[21-28], Valsartan^[29-32], Candesartan.^[33-35] However in our earlier work an UHPLC/Q-TOF-MS method has been developed for structural identification and quantitative determination of these drugs in their pure samples^[36]. But the study has been extended to explain the entire mass fragmentations of these drugs, establishment of breakdown mechanisms and its application in blood samples. Hence in the presented work a new UHPLC/Q-TOF-MS method is developed, validated and applied for pharmacokinetic study of all ARBs Antihypertensive drugs in human plasma.

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) 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. All other reagents used were of LC-MS grade.

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 3.0 min. 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 3.0 kV, 40 V, 80ºC, 50 L/h, 2.5×10^-4 mbar and 12 V, 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 5 min. 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.

Preparation of Sample Solutions:

500μL of plasma sample was transferred to 10mL glass tube. To this 5mL of extraction solvent (diethyl ether: dichloromethane 70:30, v/v) was added. The sample was mixed by vortexer for 5 min. The organic layer was transferred to another glass tube. The solid residue was evaporated to dryness using evaporator at 40ºC under a stream of nitrogen. The dried extract was reconstituted in 200μL of diluent (methanol: water, 50:50, v/v). This solution was filtered through 0.45μm nylon membrane filter to remove all the particulate materials. 20μL aliquot was injected in to UPLC system.

Validation of Method:

The developed method was validated according to ICH validation guidelines^[37].Different standard concentrations each of the compound in the range of 1-1000 ng/mL (1, 10, 50, 100, 200, 500, and 1000ng/mL) was spiked to 100μL of blank human plasma separately in methanol: water (50:50, v/v). Similarly the low, medium and high concentration QC samples containing different concentrations of the drugs were prepared independently using the same procedure. The solutions were filtered through 0.20μm nylon syringe filter and injected in to the UPLC/QTOF-MS system for analysis. Linearity graph was prepared by average peak area of each concentration. Intraday and interday precision and accuracy was also evaluated by analyzing the samples for three consecutive days. The specificity of the method was examined by analyzing blank plasma extract.

Pharmacokinetic Study:

The method was applied for pharmacokinetic study to determine the plasma concentrations of all the drugs from a clinical trial in which 3 healthy male volunteers received individual tablets containing each of the drugs. Tablets containing single individual drugs were taken from the market and given to 3 healthy male volunteers. The clinical study was performed in three consecutive days taking low dose of drugs. Blood samples were collected before and after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h post-dosing. Plasma was separated by centrifugation and stored in refrigerator until analysis.

RESULTS AND DISCUSSION:

The isocratic mobile phase containing acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.25 mL/min provide peaks with short retention times. The retention time was found to be 0.50 min for Losartan, 0.80 min for Telmisartan, 1.50 min for Valsartan, 2.0 min for Candesartan. The total chromatographic run time was 3.0 min for each compound. The 10ng/mL concentration of each of the compound was prepared separately in methanol: water (50:50, v/v). 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. Therefore, 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 as shown in Figure 1, 2, 3, and 4, 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

On the basis of obtained TOF-MS/MS Spectra of each drug, the mass fragmentation mechanism was established. 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 and Candesartan (M-H^-) at m/z 439.10 is fragmented in to product ion at m/z 309.9 from left side of the structure that is ring containing –OH. Product ions obtained for these drugs under MS/MS conditions is presented in Table 1. The proposed MS/MS fragmentation mechanism of Losartan, Telmisartan, Valsartan, and Candesartan are shown in Figure 5, 6, 7, and 8, respectively.

Table 1: Obtained Product Ions Under MS/MS Conditions in Proposed Method

Drugs	Precursor Ion [M-H]^- (m/z)	Major Product Ion (m/z)	Small Product Ions (m/z)
Losartan	421.20	127.00	294.00, 157.00, 335.00
Telmisartan	513.16	469.13	426.13
Valsartan	434.50	255.50	119.50, 235.50, 350.50
Candesartan	439.10	309.90	380.00, 175.00, 131.80

Figure 5: Proposed MS/MS Fragmentation Mechanism of Losartan

Figure 6: Proposed MS/MS Fragmentation Mechanism of Telmisartan

Figure 7: Proposed MS/MS Fragmentation Mechanism of Valsartan

Figure 8: Proposed MS/MS Fragmentation Mechanism of Candesartan

Table 2: Results Obtained from Pharmacokinetic Studies

Drugs	Brands	Bioavailability (%)^a	t_max (hrs)^b	C_max (ng/mL)^b	AUC (ng.h/mL)^b	Half Life (T_1/2) (hrs)
Losartan	Losakind (50 mg tab, Mankind)	25-35	1.2	350	945	1.5- 2
Telmisartan	Telma (40 mg tab, Glenmark)	42-100	2.15	550	1150	24
Valsartan	Diovan (40 mg tab, Novartis)	25	2.5	352	850	6
Candesartan	Candesar (4 mg tab, Solus)	15	2.0	325	755	9

^aBioavailability in Literature; ^bMean of three replicates (n = 3)

Validation of the method:

The linearity range for all the drugs was 1-1000ng/mL. The LOD and LOQ were 0.1ng/mL and 1.0ng/mL, respectively for all the compounds. 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 and accuracy of the method. No significant interference in the blank plasma traces was seen from endogenous substances at the retention time of both drugs, suggested that method was specific.

Pharmacokinetic Study and Identification of Metabolites:

The method was applied to pharmacokinetic study in human plasma. The tablets containing adult dose of each individual drug was selected for clinical studies. The results of pharmacokinetic parameters obtained from mean plasma concentration time curve after administration of single tablets of each drug are presented in Table 2. By the identification of metabolites, its therapeutic and toxic aspects can be determined. After injecting the plasma samples of each drug, the mass spectra were obtained and possible structures of metabolites were established.

CONCLUSION:

The Q-TOF-MS/MS method was developed and applied for simultaneous determination of ARBs antihypertensive drugs. The mass spectra of drugs were obtained and the fragmentation mechanisms of the drugs were established. The quantification of drugs was carried out by UHPLC using MS/MS transition selecting precursor to major product ion. The proposed fragmentation mechanisms of the drugs are helpful in structural identification of the drugs, their related impurities and metabolites. The method was applied for pharmacokinetic study of these drugs in human plasma.

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 Q-TOF-MS/MS system.

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Received on 09.08.2020 Revised on 04.09.2020

Asian J. Pharm. Ana. 2021; 11(1):22-26.

DOI: 10.5958/2231-5675.2021.00004.1