INTRODUCTION:

Valsartan is an antihypertensive drug, belongs to a group of Angiotensin Receptor Blockers (ARBs). It is used for the treatment of hypertension.^[1] The UPLC/Q-TOF-MS technique has been applied in pharmaceutical analysis particularly in the identification and quantitative analysis of drug products. The Q-TOF mass spectrometry gives the accurate mass, reliable chemical fragmentation of synthetic compounds. Liquid Chromatography Coupled to Mass Spectrometry (LC-MS) has become the standard technique for monitoring the degradation products and their detailed structural information.^[2-12]Stability of drug product is directly related with its quality, safety and efficacy. The stability of drug substances is mainly depends on factors such as temperature, moisture, pH, oxygen, and light.

The stability of drugs is determined by stress testing in which drug substances are treated with above conditions as per ICH guidelines. Degradation studies via stress testing simulate the most likely environments that the drug may be subjected to from production to storage. These stress tests are important for inference of the degradation routes of pharmaceutical compounds and inform molecule stability across different stress conditions.^[13-21] The literature survey revealed that few analytical methods have been reported for determination of valsartan as an individual drug and/or its degradation products by UV spectrophotometry,^[22] Stability-indicating UPLC.^[23] Degradation studies by HPLC^[24-26] and both by LC and LC-MS.^[27,28] Determination of valsartan alone and/or with hydrochlorothiazide has been reported by LC-MS.^[29-32] The mass spectral studies reported in the previously published methods not giving full information regarding various mass fragments of Valsartan. Moreover the degradation products of Valsartan and their degradation mechanisms in the earlier reported methods were not explained completely.^[33] Hence in the presented work an attempt was made to apply highly sophisticated UPLC/Q-TOF-MS technique for identification and characterization of degradation products and metabolites of Valsartan.

MATERIAL AND METHODS:

Valsartan (C₂₄H₂₉N₅O_3, Molecular weight 435.50) was 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.

Q-TOF-MS and UPLC Conditions:

Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. Quantification was done by using MS/MS transitions, m/z 434.50 to 255.50. 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. UPLC was performed with Waters Acquity UPLC system (Waters Corporation, MA, USA) equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector. The chromatographic separation was achieved on Acquity UPLC^TM BEH C₁₈(100.0 × 2.1 mm, 1.7µm) column using isocratic mobile phase consisting of acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.25mL/min.

Preparation of Standard Solutions:

100mg of Valsartan pure sample was weighed accurately and transfer to 50mL volumetric flasks separately. The powder was then dissolved with 25 mL 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 get series of standard solutions containing required concentrations.

Validation of the Method:

The developed method was validated according to ICH validation guidelines.^[34]Different standard concentrations of drug in the range of 1-1000 ng/mL (1, 10, 50, 100, 200, 500, and 1000ng/mL) was prepared separately in methanol: water (50:50, v/v). The solutions were filtered through 0.20 μm nylon syringe filter and injected in to the UPLC/Q-TOF-MS system for analysis. Linearity graph was prepared by average peak area of each concentration. The limit of detection (LOD) was defined as the lowest absolute concentration of analyte in a sample that can be detected but not necessarily quantified; whereas, limit of quantitation (LOQ) was defined as the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. Standard stock solutions were diluted to obtain concentrations for the estimation of the LOD and LOQ. Precision was assessed by performing six analyses using standard solution containing 100ng/mL of Valsartan single day (Intraday Precision) and three consecutive days (Interday Precision). The accuracy of the method was determined by standard addition technique. Three different levels (50, 100, and 150%) of standards were added to pre-analyzed tablet sample in six replicates and the mixtures were re-analyzed by the proposed method. The percentage recoveries at each level and each replicate were determined. The mean of percentage recoveries and the RSD (%) was calculated. Specificity is determined by forced degradation/stress testing as per ICH guidelines.

Forced Degradation Studies/Stress Testing:

The specificity is the ability of the method to measure the analyte response in presence of sample components such as excipients, impurities, degradation products or metabolites. Specificity is determined by performing stress testing. According to ICHQ1A (R2), forced degradation studies of the drug substance can help in the identification of degradation products and the intrinsic stability of the molecule and to investigate the stability-indicating power of the analytical methods. ^[3-21] The stress conditions employed for the degradation study includes acid hydrolysis (1 N HCl), alkali hydrolysis (1 N NaOH), oxidation (3% H₂O₂), and light (carried out as per ICH Q1B). For acid, alkali hydrolysis and oxidation, the study period was 1 h whereas for photolytic stress study it was 24 h. Initially all stress conditions employed for forced degradation studies are carried out at room temperature (25ºC). The temperature was subsequently increased to obtain the sufficient degradation for development of stability-indicating analytical method. The UPLC/Q-TOF-MS provide the mass information with higher accuracy and precision, which is ultimately helpful in structure elucidation, identification of fragmentation patterns of the drugs, identification of degradation products and metabolites.

Pharmacokinetic and Metabolite Study:

The method was applied to pharmacokinetic study in human plasma. Study was carried out by determination of the plasma concentrations of Valsartan from a clinical trial in which 3 healthy male volunteers received a single tablet containing 80 mg Valsartan. Blood samples were collected before and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h post-dosing. Plasma was separated by centrifugation for 10 min and was stored in refrigerator until analysis. 20 μL aliquot was injected in to UPLC/Q-TOF-MS system. Pharmacokinetic parameters such as Concentration maxima (C_max)_,Time maxima (T_max) Area under curve (AUC), Plasma half life (T_1/2)and Bioavailability were evaluated from mean plasma concentration-time curve. The mass spectra were obtained to get the mass fragments of possible metabolites in the blood plasma.

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 of Valsartan was found to be 1.50 with total chromatographic run time was 3.0 min. The linearity was found to be 1-1000ng/ml. The LOD and LOQ were 0.1 and 1.0ng/ml, respectively. The RSD less than 2% were obtained for all the parameters such as intraday and interday precision, accuracy suggested an acceptable precision and accuracy of the method. The 10 ng/mL concentration of drug was prepared in methanol: water (50:50, v/v). The solution was injected in to the Q-TOF-MS/MS system to obtain mass spectra. Valsartan showed the strong response in negative ionization mode. Hence the mass spectrometer was operated via negative ionization mode. Under the selected MS conditions it was converted in to precursor ions at m/z 434.50. Therefore, the negative ions, [M-H]^- at m/z 434.50 was selected as the precursor ions. Under the selected MS/MS conditions the precursor ions were fragmented to major product ions at m/z 434.50 to 255.50 as shown in Figure. 1. The proposed MS/MS fragmentation mechanism of Valsartan is shown in Figure. 2.

Forced Degradation Study/Stress Testing:

The specificity of the developed method was determined by forced degradation studies. Degradation was observed when the standard solutions of Valsartan were subjected to acidic and alkaline hydrolysis as seen from the significant drop in assay values and appearance of degradation peaks in the chromatograms. After acidic and alkaline hydrolysis, drug was degraded into unknown major degradation products with the appearance of peaks at m/z 350.50 and at m/z 304.50, respectively in their mass spectrum. By studying the chemical structure and fragmentation mechanism of Valsartan the unknown degradation product at m/z 350.50, was identified and named as 2-methyl-N-{[2'-(1H-tetrazol-5-yl) biphenyl-3-yl] methyl} propan-1-amine. After oxidation with 3% H₂O₂ solution, drug was degraded into unknown degradation products with the appearance of peaks at m/z 334.50 in their mass spectrum. This degradation product from oxidation was identified with the help of fragmentation mechanism of Valsartan and chemically named as N-methyl-N-{[2'-(1H-tetrazol-5-yl) biphenyl-3-yl] methyl} pentanamide. No degradation was observed when the drug was treated with photolytic stress conditions. The results of forced degradation studies are presented in Table 1. The proposed degradation mechanism of Valsartan when treated with acid hydrolysis, alkali hydrolysis, and oxidation, Figure 3, Figure 4 and Figure 5, respectively.

Table 1: Results Obtained from forced Degradation Studies

Stress Conditions	Assay Value (%)^a	Major Degradation Product (m/z value)	Small Degradation Product (m/z value)
Acid hydrolysis (1N HCl, 25 ͦ C,1 h)	92.22	350.50	265.50
Alkali hydrolysis (1N NaOH, 25 ͦ C,1 h)	94.45	304.50	255.15
Oxidation (3% H₂O₂, 25 ͦ C, 1 h)	95.25	335.50	119.50
Photolytic (UV light at 254 nm, 24 h)	99.98	No Degradation	No Degradation

^aMean of three replicates (n = 3)

Figure: 3 Proposed Degradation Mechanism of Valsartan after Acid Hydrolysis

Figure: 4 Proposed Degradation Mechanism of Valsartan after Alkali Hydrolysis

Figure: 5 Proposed Degradation Mechanism of Valsartan after Oxidation

Pharmacokinetic Study and Identification of Metabolites:

The results of pharmacokinetic parameters obtained from mean plasma concentration-time curve after administration of single tablet containing 80 mg Valsartan are presented in Table 2. By the identification of metabolites, its therapeutic and toxic aspects can be determined. The developed method was successfully applied in the metabolite study using selected MS/MS transitions of molecular ion and product ions. After injecting the plasma samples of Valsartan, the obtained mass spectra showed that the appearance of intense peaks at m/z value 450.50 and 435.50. These were peaks were structurally identified as 4-Hydroxy Valsartan and 5-Hydroxy Valsartan, respectively two metabolites of Valsartan.

Table 2: Results of Pharmacokinetic Studies

Pharmacokinetic Parameters	Obtained Values
T_max (h)	2.5 ± 0.50
C_max (ng/mL)	455 ± 125
AUC (ng.h/mL)	1025 ± 225
T_1/2(h)	6.0 ± 1.0
Bioavailability (%)	25 ± 2

CONCLUSION:

The UPLC/Q-TOF-MS method was developed, validated and applied for identification and characterization of degradation products of Valsartan. By obtaining the mass spectra of drug, the fragmentation mechanism was established. Drug was subjected to forced degradation studies, it was suggested that Valsartan was degraded when treated with acid, alkali and oxidative stress conditions but found stable in photolytic condition. Degradation products were identified on the basis of obtained mass spectra. Various degradation pathways were established according to obtained values of mass spectra. Similarly the chemical structures of Valsartan metabolites present in human plasma were also established using mass values of spectrum. Hence the proposed method by applying UPLC/Q-TOF-MS technique should be applied for Valsartan degradation studies during stability testing, pharmacokinetic studies and metabolite studies in metabonomics.

ACKNOWLEDGEMENTS:

The author is grateful to Systopic Laboratories Ltd., Delhi, India, for providing pure sample of Valsartan. 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 system.

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Received on 05.09.2020 Revised on 03.10.2020

Asian J. Pharm. Res. 2021; 11(1):1-5.

DOI: 10.5958/2231-5691.2021.00001.0