Application of Validated UPLC/Q-TOF-MS Method for Simultaneous Determination of Telmisartan, Hydrochlorothiazide and their Degradation Products in Tablets

 

Hamid Khan¹* Mushir Ali², Alka Ahuja², Javed Ali²

¹JK College of Pharmacy, Bilaspur, CG, India-495001

²Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India-110062

 

ABSTRACT:

In the presented work the ultra-performance liquid chromatography/ quadrupole time-of-flight mass spectrometric (UPLC/Q-TOF-MS) method is developed for simultaneous determination of telmisartan, hydrochlorothiazide and their degradation products in tablets. For identification of drugs, the Q-TOF mass spectrometer was operated in negative ionization mode and quantification was done using the MS/MS transitions at m/z 513.18 to 469.13 for telmisartan, and 268.90 to 204.94 for hydrochlorothiazide. For quantification, 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 0.25 mL/min. The elution of telmisartan and hydrochlorothiazide was occurred at 2.25 and 1.22 min, respectively. The calibration curves were linear over the concentration range of 1-1000 ng/mL for both the drugs. The developed method was validated according to ICH guidelines.

 

 

 

INTRODUCTION:

Telmisartan, is chemically 4'-[[(4-Methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-benzimidazol-1-yl)]methyl] biphenyl-2-carboxylic acid and is an antihypertensive drug, belongs to a group of angiotensin converting enzyme (ACE) inhibitors. It is used for the treatment of hypertension [1]. Hydrochlorothiazide (HCTZ) is chemically 6-chloro-1, 1-dioxo-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide. It is a diuretic drug used worldwide for lowering the blood pressure individually and in the combination of antihypertensive drugs [2]. Fixed dose combination tablets containing telmisartan and hydrochlorothiazide have been approved for the treatment of mild to moderate hypertension and widely available in the Indian market.

 

The literature survey revealed that few analytical methods have been reported for determination of telmisartan as an individual drug in formulations or its degradation products and/or in biological fluids such as HPLC [3-5], HPTLC [6] and LC-MS [7-9]. Determination of hydrochlorothiazide as an individual drug in formulations or its degradation products and/or in biological fluids has been reported by spectrophotometry [10], HPLC [11-13], and LC-MS [14-16]. Simultaneous determination of telmisartan and hydrochlorothiazide in formulations and/or biological fluids has been reported by derivative spectrophotometry, TLC-densitometry and spectrofluorimetry [17], HPLC [18, 19] and HPTLC [20]. However a HPLC-MS/MS method was developed for simultaneous determination of telmisartan and hydrochlorothiazide in human plasma but the developed method was found very complicated due to the use of internal standard along with main analytes and also gradient elution for chromatographic separation [21]. In the presented work UPLC/Q-TOF-MS method is developed and validated for simultaneous determination of telmisartan, hydrochlorothiazide and their degradation products in tablets, using isocratic elution without use of internal standard.

 

EXPERIMENTAL:

Chemicals and Reagents:

Telmisartan (C₃₃H₃₀N₄O_2, Molecular weight 514.63, and purity 99.98%) and Hydrochlorothiazide (C₇H₈ClN₃O₄S_2, Molecular weight 297.72, and purity 99.99%) were kindly supplied as gift sample by Systopic Pharmaceuticals Ltd. (New Delhi, India). 4-Amino-6-Chloro-1,3-Benzene disulfonamide was supplied by Supra Chemicals Ltd. (Mumbai, India). Tablet formulations were obtained commercially with labeled amounts of 40 mg of telmisartan and 12.5 mg of hydrochlorothiazide. 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. The capillary voltage, sampling cone voltage, extraction cone voltage, source temperature, desolvation temperature, cone gas flow, desolvation gas flow, trap gas flow, and source gas flow were set to 3.0 kV, 40 V, 4 V, 80^oC, 350^oC, 50 L/hr, 800 L/hr, 1.50mL/min, and 0.50mL/min, respectively for all the drugs. Argon was employed as the collision gas at a pressure of 2.5 x 10^-4 mbar. Trap collision energy and transfer collision energy were set to 12 and 6 V, respectively for all the drugs. Quantitation was performed using MS/MS transitions, m/z 513.18 to 469.13 for telmisartan, 268.90 to 204.94 for hydrochlorothiazide. 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 Waters Acquity UPLC BEH C₁₈ (100.0 × 2.1 mm, 1.7µm) column was used for separation. The mobile phase for UPLC analysis consisted of acetonitrile–2 mM ammonium acetate (50:50, v/v) which was filtered through 0.45 mm membrane filter and degassed by sonication. For isocratic elution, the flow rate of the mobile phase was kept at 0.25 mL/min and 10 mL of sample solution was injected in each run.

 

Preparation of Standard Solutions

50 mg each of telmisartan and hydrochlorothiazide individually were weighed accurately and transfer to 50 mL volumetric flasks separately. The powders were then dissolved with approximately 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 give a series of standard solutions containing required concentrations for each compound.

 

Preparation of sample solutions

Twenty tablets were weighed accurately and powdered. Powder equivalent to 40 mg of telmisartan and 12.5 mg of hydrochlorothiazide was taken and transferred to a 50 mL volumetric flask. The powder was dissolved with approximately 25 mL of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. This solution was filtered through 0.45 μm nylon membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 500 ng/mL of telmisartan and 100 ng/mL of hydrochlorothiazide.

 

Validation of the Method:

The developed method was validated according to ICH validation guidelines [22]. The validation parameters addressed were linearity and range, limit of detection and quantitation, precision, accuracy, and specificity.

 

Linearity, Range, LOD and LOQ:

Different standard concentrations each of telmisartan and hydrochlorothiazide in the range of 1-1000 ng/mL (1, 10, 50, 100, 200, 500, and 1000 ng/mL) were 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. Average peak area at each concentration level was subjected to linear regression analysis with the least squares method. Linearity was described by slope, intercept and correlation coefficient obtained from regression equations.

 

Accuracy and Precision:

The intraday precision was assessed by performing six analyses using tablet sample solution containing 500 ng/mL of telmisartan and 100 ng/mL of hydrochlorothiazide. Similarly interday precision was assessed by performing replicate analysis using concentration of all the analytes for three consecutive days under the same experimental conditions. The mean of percentage recoveries and the RSD (%) was calculated. 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 of all the compounds at each level and each replicate were determined.

 

 

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of sample components or matrix such as excipients, potential impurities and degradation products. The samples were chromatographed to determine the extent to which mobile phase components and excipients could contribute to the interference with the analytes. This was done by comparing the chromatograms of each analyte with chromatograms obtained from blank solution. Specificity of the developed method was also assessed by performing forced degradation studies. According to ICHQ1A (R2), stress testing 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 [23, 24]. 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 hr whereas for light study it was 24 hr. All stress conditions employed for forced degradation studies were carried out at room temperature (25^ºC).

Analysis of Marketed Tablets:

The procedure for analysis of marketed tablets was similar as described in preparation of sample solution. The amount of telmisartan and hydrochlorothiazide in tablets was determined by calibration equations obtained from the respective calibration plot.

 

RESULTS AND DISCUSSION:

Optimization of Q-TOF-MS Conditions

All the compounds have strong responses in the negative ionization mode and they form protonated molecules in the full scan mass spectra. Therefore, the negative ions, [M-H]^- at m/z 513.08 for telmisartan, and m/z 295.86 for hydrochlorothiazide were selected as the precursor ions. Under the selected MS/MS conditions the precursor ions were fragmented to major product ions at m/z 513.18 to 469.13 for telmisartan, and 295.80 to 204.94 for hydrochlorothiazide, as shown in Figure 1 and Figure 2, respectively. Quantification was done by taking the major product ions.

 

Figure 1: TOF-MS/MS spectra of telmisartan

 

Figure 2: TOF-MS/MS spectra of hydrochlorothiazide

The product ion spectra of telmisartan was suggested that the fragmentation of molecules occurs from carboxylic group and loss of carbon dioxide results in the formation of one major product ion, at m/z 469.13. The proposed MS/MS fragmentation mechanism of telmisartan is shown in Figure 3. The product ion spectra of hydrochlorothiazide was occurred due to the fragmentation of compound via loss of neutral molecule namely HCN and degraded by hydrolysis, resulted in the formation of one intermediate product ion, at m/z 283.95 which was identified as 4-amino-6-chloro-1,3-benzenedisulfonamide (DSA), a major degradation product of hydrochlorothiazide. This is further fragmented in to another product ion with higher intensity at m/z 268.90 by the loss of NH₃ molecule. This product ion is further converted into major product ion with highest intensity at m/z 204.93 by the loss of SO₂. This in turn fragmented into smallest intensity ion at m/z 169.00. The proposed MS/MS fragmentation and degradation mechanism of hydrochlorothiazide is shown in Figure 4.

 

Figure 3: Proposed MS/MS fragmentation mechanism of telmisartan

 

 

Figure 4: Proposed MS/MS fragmentation and degradation mechanism of hydrochlorothiazide

Optimization of UPLC Conditions:

Various solvents such as acetonitrile, methanol, and water along with ammonium acetate were tested to achieve best results for mass spectrometric-chromatographic analysis. 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 2.25 min for telmisartan, and 1.22 min for hydrochlorothiazide with the total chromatographic run time of 3 min for each compound. UPLC-TOF-MS/MS chromatogram obtained from mixed standards (1ng/mL each) of telmisartan and hydrochlorothiazide is shown in Figure 5. 

 

Figure 5: UPLC-TOF-MS/MS chromatogram obtained from mixed standards (1ng/mL each) of telmisartan (R_t  2.25 min) and hydrochlorothiazide (R_t 1.22 min).

Validation of the method:

For quantitative analysis the developed method was validated for various parameters as per the ICH guidelines. The results of linearity, LOD and LOQ are 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 an acceptable precision of the method. The results of accuracy and precision are presented in Table 2.

Table 1: Results Obtained from Linearity, LOD, and LOQ

 

Table 2: Results Obtained from Recovery Studies and Precision

Conc. Added (ng/mL)	Conc. Found (ng/mL)	Recovery (%)a	RSD (%)
			Intraday	Interday
Telmisartan 250 500 750	249.96  499.92  750.01	99.98 99.98 100.13	1.45 1.78 1.95	1.25 1.57 1.15
Hydrochlorothiazide 50 100 150	50.00 100.02 149.92	100.00 100.02 99.94	1.55 1.78 1.74	1.44 1.52      1.67

^aMean of six replicates (n = 6)

 

Specificity:

The specificity was determined by forced degradation studies. After acidic and alkaline hydrolysis, telmisartan was degraded into unknown degradation product with the appearance of peaks at m/z 162.82 and m/z 188.92, respectively. Whereas it was found stable under oxidative and photolytic stress conditions. The percentage of telmisartan under degradation in acidic and alkaline conditions after 1 h was found to be 94.62% and 96.46%, respectively which was calculated by area (%) with respect to the initial area of telmisartan peaks. After acidic and alkaline hydrolysis, hydrochlorothiazide was degraded into DSA. The proposed degradation mechanism of hydrochlorothiazide is presented in Figure 6. The percentage of hydrochlorothiazide under degradation in acidic and alkaline conditions after 1 h was found to be 93.22% and 92.25%, respectively which was calculated by area (%) with respect to the initial area of hydrochlorothizide peaks. The percentage of DSA from degraded sample of hydrochlorothiazide in acidic and alkaline conditions after 1 h was 6.32% and 8.64%, respectively with respect to the initial area of hydrochlorothiazide peaks. This was further confirmed by co-injection of reference standard solution of DSA, the obtained chromatograms were found similar with that of reference standard of each compound, indicating that there was no co-elution of unknown degradation peak at the retention times of respective compounds. No degradation was observed when both the drugs were subjected to oxidative and photolytic stress conditions. The results of forced degradation studies are presented in Table 3. Earlier reports on the stress testing of telmisartan and hydrochlorothiazide also supported the results obtained from this study [5-7, 10-14].

 

Analysis of Marketed Tablets:

The validated method was applied to the determination of telmisartan and hydrochlorothiazide in commercially available tablets containing 40 mg of telmisartan and 12.5 mg of hydrochlorothiazide. The content of both the drugs in tablets was found to be 98-100% with RSD less than 2%, indicated that method was suitable for routine analysis of drugs in tablets without any interference from excipients.

 

Figure 6 : Proposed degradation mechanism of hydrochlorothiazide

 

 

Table 3: Results obtained from forced degradation studies

^aMean of three replicates (n = 3); ^bPeak area (%) against respective standard

 

 

CONCLUSION:

UPLC/Q-TOF-MS method has been developed and validated for rapid qualitative and quantitative analysis of telmisartan and hydrochlorothiazide along with their degradation products in tablets. Forced degradation studies suggested that telmisartan and hydrochlorothiazide were highly sensitive to acidic and alkaline hydrolysis but remain unaffected under oxidative and photolytic stress conditions. The advantages of our developed method are the short chromatographic run time (3 min), high sensitivity (LOQ: 1.0 ng/mL for telmisartan and hydrochlorothiazide), and exact structure elucidation of the compounds and their degradation products. Hence, it is suggested that the method should be applied for routine quality control analysis of telmisartan and hydrochlorothiazide and to monitor the level of their degradation products, in bulk drugs and in pharmaceutical formulations during stability studies. In addition to this, use of isocratic chromatographic separation without any internal standard makes it an advantageous procedure in high-throughput bioanalysis of telmisartan and hydrochlorothiazide in combination in human plasma.

 

ACKNOWLEDGMENTS:

The authors are grateful to Systopic Laboratories Ltd., Delhi, India, for providing pure samples of telmisartan and hydrochlorothiazide. The authors are grateful to Supra Chemicals Ltd., Mumbai, India for providing pure sample of DSA. The authors are 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.

 

REFERENCES:

1.       Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, 14^th edition, Merck Research Laboratories, Merck and Co., Inc., White House Station, NJ, USA, 2006, pp. 1569.

2.       British Pharmacopoeia. HMSO: London, 2008, pp. 1036-1037.

3.       Torrealday N, Gonzalez L, Alonso RM, Jimenez RM, Lastra EO. Experimental design approach for the optimization of a HPLC-fluorimetric method for the quantitation of the angiotensin II receptor antagonist telmisartan in urine. Journal of Pharmaceutical and Biomedical Analysis, 2003; 32(4/5): 847-857.

4.       Palled MS, Rajesh PMN, Chatter M, Bhat AR. RP-HPLC determination of telmisartan in tablet dosage forms. Indian Journal of Pharmaceutical Sciences, 2005; 67(1):108-110.

5.       Londhe SV, Kaul N, Agrawal H, Mahadik K R. Stability-indicating RP-HPLC method for analysis of telmisartan in the bulk drug and in formulations. Acta Chromatographica, 2010; 22(4): 539-548.

6.       Prabhu C, Subramanian GS, Karthik A, Kini S, Rajan MS, Udupa N. Determination of telmisartan by HPTLC- A stability-indicating assay. Journal of Planar Chromatography, 2007; 20(6):477-481.

7.       Shah RP, Singh S. Identification and characterization of a photolytic degradation product of telmisartan using LC–MS/TOF, LC–MSⁿ, LC–NMR and on-line H/D exchange mass studies. Journal of Pharmaceutical and Biomedical Analysis, 2010; 53(3):755-761.

8.       Chen B, Liang Y, Wang Y, Deng F, Zhou P, Guo F, Huang L. Development and validation of liquid chromatography-mass spectrometry method for the determination of telmisartan in human plasma. Analytica Chimica Acta, 2005; 540(2):367-375. 

9.       Li P, Wang Y, Wang Y, Tang Y, Fawcett JP, Cui Y, Gu J. Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry. Journal of Chromatography B, 2005; 828(1-2):126-129.

10.     Tamat SR, Moore DE. Photolytic decomposition of hydrochlorothiazide. Journal of Pharmaceutical Sciences, 1982; 72(2):180-183.

11.     Daniels SL, Vanderwielen AJ. Stability-indicating assay for hydrochlorothiazide. Journal of Pharmaceutical Sciences, 1981; 70(2): 211-215.

12.     Tagliari MP, Stulzer HK, Murakami FS, Kuminek G, Valente B, Oliveira PR, Segatto SMA. Development and validation of a stability-indicating LC method to quantify hydrochlorothiazide in oral suspension for pediatric use. Chromatographia, 2008; 67(7-8):647-652.

13.     Fang X, Bibart R, Mayr S, Yin Y, Harmon PA, Finnegan J, Tyrrell RJ, Reed RA. Purification and identification of an impurity in bulk hydrochlorothiazide. Journal of Pharmaceutical Sciences, 2001; 90(11):1800-1809.

14.     Franolic JD, Lehr GJ, Barry TL, Petzinger G. Isolation of a 2:1 hydrochlorothiazide-formaldehyde adduct impurity in hydrochlorothiazide drug substance by preparative chromatography and characterization by electrospray ionization LC-MS. Journal of Pharmaceutical and Biomedical Analysis, 2001; 26(4):651-663.

15.     Ramakrishna NVS, Vishwottam KN, Manoj S, Koteshwara M, Wishu S, Varma DP. Sensitive liquid chromatography tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma. Biomedical Chromatography, 2005; 19(10):751-760.

16.     Liu F, Xu Y, Gao S, Zhang J, Guo Q. Determination of hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 2007; 44(5):1187-1191.

17.     Bebawy LI, Abbas SS, Fattah LA, Refaat HH. Application of first derivative, ratio derivative spectrophotometry, TLC-densitometry and spectrofluorimetry for the simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical dosage forms and plasma. Farmaco, 2005; 60(10):859-867.  

18.     Bhat LR, Godge RK, Vora AT, Damle MC. Validated RP-HPLC method for simultaneous determination of telmisartan and hydrochlorothiazude in pharmaceutical formulation. Journal of Liquid Chromatography and Related Technologies, 2007; 30(20): 3059-3067.

19.     Rane VP, Sangshetti JN, Shinde DB. Simultaneous high-performance liquid chromatographic determination of telmisartan and hydrochlorothiazide in pharmaceutical preparation. Journal of Chromatographic Sciences, 2008; 46(10):887-891.

20.     Shah NJ, Suhagia BN, Shah RR, Shah PB. Development and validation of a HPTLC method for the simultaneous estimation of telmisartan and hydrochlorothiazide in tablet dosage form. Indian Journal of Pharmaceutical Sciences, 2007; 69(2): 202-205. 

21.     Yan T, Li H, Deng L, Guo Y, Yu W, Fawcett JP, Zhang D, Cui Y, Gu J. Liquid chromatographic-tandem mass spectrometric method for the simultaneous quantitation of telmisartan and hydrochlorothiazide in human plasma. Journal of Pharmaceutical and Biomedical Analysis, 2008; 48(4):1225-1229.

22.     International Conference on Harmonization, ICH Q2 (R1), Validation of Analytical Procedures: Text and methodology, Geneva, 2005.

23.     Khan H, Ali M, Ahuja A, Ali J. Stability testing of pharmaceutical products: Comparison of stability testing guidelines. Current Pharmaceutical Analysis, 2010; 6 (2):140-148.

24.     International Conference on Harmonization, ICH Q1A (R2), Stability Testing of New Drug Substances and Products, Geneva, 2003.

 

 

 

 

 

 

Received on 03.04.2017       Accepted on 10.06.2017     

© Asian Pharma Press All Right Reserved