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VALIDATED UPLC/Q TOF MS METHOD FOR STRUCTURAL IDENTIFICATION AND QUANTITATIVE DETERMINATION OF ANTIHYPERTENSIVE DRUGS

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VALIDATED UPLC/Q-TOF-MS METHOD FOR STRUCTURAL

IDENTIFICATION AND QUANTITATIVE DETERMINATION OF

ANTIHYPERTENSIVE DRUGS

Hamid Khan*

ISBM University School of Pharmacy, Chhattisgarh, India.

ABSTRACT

In the presented work the ultra-performance liquid chromatographic/quadrupole time-of-flight mass spectrometric (UPLC/Q-TOF-MS) method has been developed for Structural Identification and Quantitative Determination of Antihypertensive Drugs such as Losartan, Telmisartan, Valsartan Candesartan and Irbesartan,. 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 421.2 to 127.00 for Losartan, m/z 513.18 to 469.13 for telmisartan, m/z 435.02 to 157.00 for valsartan, m/z 439.1 to 309.9 for Candesartan and m/z 427.2 to 193.08 for Irbesartan. The chromatographic separation was achieved on Acquity UPLCTM 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 calibration curves were linear over the concentration range of 1-1000 ng/mL for all the drugs. The developed method was validated according to ICH guidelines. The method is helpful in Structural Identification and Quantitative Determination of Antihypertensive Drugs.

KEYWORDS: UPLC/Q-TOF-MS, Antihypertensive Drugs, Structural Identification.

INTRODUCTION

The UPLC/Q-TOF-MS technique is the latest amongst all the chromatographic technique and has been used worldwide in drug discovery and development. It has been applied in pharmaceutical development particularly in the identification and quantitative analysis of drug products. The Q-TOF mass spectrometry gives the accurate mass, reliable chemical

Volume 8, Issue 13, 849-856. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Hamid Khan

ISBM University School of

Pharmacy, Chhattisgarh,

India.

Article Received on 27 Sept. 2019,

Revised on 17 Oct. 2019, Accepted on 07 Nov. 2019

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fragmentation of synthetic compounds.[1, 2] Losartan, Telmisartan, Valsartan, Candesartan and Irbesartan are antihypertensive drug, belongs to a group of angiotensin converting enzyme (ACE) inhibitors. It is used for the treatment of hypertension.[3,4] The literature survey revealed that few analytical methods have been reported for determination of Losartan, Telmisartan, Valsartan, Candesartan and Irbesartan as an individual drug in human plasma by LC-MS.[5-9] In the presented work an UPLC/Q-TOF-MS method is developed and validated for structural identification and quantitative determination of five antihypertensive drugs that are, Losartan, Telmisartan, Valsartan, Candesartan and Irbesartan using isocratic elution.

EXPERIMENTAL Chemicals and Reagents

Losartan (C22H23ClN6O, Molecular weight 435.51), Telmisartan (C33H20N4O2, Molecular

weight 514.61) Valsartan (C24H29N5O3, Molecular weight 435.51), Candesartan (C24H20N6O3,

Molecular weight 440.45) and Irbesartan (C25H28N6O, Molecular weight 429.53) 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.

Q-TOF-MS and UPLC Conditions

Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. 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 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. The Acquity UPLC BEH C18 column with dimension of 100.0 × 2.1 mm, 1.7µm, Mobile phase of Acetonitrile–2

mM ammonium acetate (50:50, v/v) with flow rate of 0.25mL/min and isocratic elution with 3 min total run time was selected for optimum UPLC conditions.

Preparation of Standard Solutions

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with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound.

Validation of the Method

The developed method was validated according to ICH validation guidelines.[10] Different standard concentrations each of the compound in the range of 1-1000 ng/mL (1, 10, 50, 100, 200, 500, and 1000 ng/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/QTOF-MS system for analysis. Linearity graph was prepared for each drug by average peak area of each concentration. Intraday and interday precision and accuracy and was also evaluated.

RESULTS AND DISCUSSION

[image:3.595.147.451.449.700.2]

All the compounds have strong responses in the negative ionization mode. Therefore, under the selected MS/MS conditions the precursor ions [M-H]- were fragmented to major product ions at m/z 421.20 to 127.00 for Losartan, m/z 513.18 to 469.13 for Telmisartan, m/z 435.02 to 157.00 for Valsartan, m/z 439.1 to 309.9 for Candesartan and m/z 427.20 to 194.50 for Irbesartan as shown in Figure 1, 2, 3, 4 and 5 respectively.

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[image:4.595.149.450.75.270.2] [image:4.595.148.449.323.515.2]

Figure 2: TOF-MS/MS Spectra of Telmisartan.

[image:4.595.148.450.565.738.2]
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[image:5.595.144.456.78.338.2]

Figure 5: TOF-MS/MS Spectra of Irbesartan.

On the basis of obtained TOF-MS/MS Spectra of each drug the mass fragementation mechanism was established. The proposed MS/MS fragmentation mechanism of Losartan, Telmisartan, Valsartan, Candesartan and Irbesartan are shown in Figure 6, 7, 8, 9 and 10 respectively.

[image:5.595.151.441.471.620.2]
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[image:6.595.163.428.74.346.2] [image:6.595.144.456.387.544.2]

Figure 7: Proposed MS/MS Fragmentation Mechanism of Telmisartan.

[image:6.595.144.449.590.740.2]
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[image:7.595.143.449.69.299.2]

Figure 10: Proposed MS/MS Fragmentation Mechanism of Irbesartan.

Validation of the method

The results of linearity (1-1000 ng/mL), LOD (0.1 ng/mL) and LOQ (1 ng/mL) for all the drugs were 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 and accuracy of the method.

CONCLUSION

The UPLC/Q-TOF-MS method was developed, validated and applied for structural identification and quantification of antihypertensive drugs. The mass spectra of drugs were obtained. From the MS/MS Spectra, the fragmentation mechanisms of the drugs were established. The proposed fragmentation mechanisms of the drugs are helpful in their structural identification. Furthermore the method is also helpful in quantitative determination of these drugs.

ACKNOWLEDGEMENTS

The authors are grateful to Systopic Laboratories Ltd., Delhi, India, for providing gift Samples of various antihypertensive drugs. 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.

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REFERENCES

1. Plumb R, Castro-Pere. Z J, Granger J, Beattie I, Joncour K, Wright A. Ultra performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry. Rapid Communication in Mass Spectrometry, 2004; 18(19): 2331-2337. 2. Khan H, Ali J. UHPLC/Q-TOF-MS: Introduction and Applications. Letters in Organic

Chemistry, 2015; 12(6): 371-378.

3. Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, 14th edition, Merck Research Laboratories, Merck and Co., Inc., White House Station, NJ, USA, 2006; 1569. 4. British Pharmacopoeia. HMSO: London, 2008; 1036-1037.

5. Rao RN, Raju SS, Vali RM, Sankar GG. Liquid chromatography mass spectrometric determination of Losartan and its active metabolite on dried blood spot. J Chrom B., 2012; 902: 47-54.

6. 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. J. Chrom. B., 2005; 828(1-2): 126-129.

7. Koseki N, Kawashita H, Hara H, Nina M, Tanaka M, Kawai R, Nagae Y, Masuda N. Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography tandem mass spectrometry. J Pharm Biomed Anal, 2007; 43: 1769-1774.

8. Lou HG, Ruan ZR, Jiang Bo. Determination of candesartan in human plasma by liquid chromatography-tandem mass spectrometry and its application to bioequivalence study. J Liq Chrom & Related Technol, 2012; 35(8): 1027-1037.

9. Wani TA, Zargar S. New Ultra performance liquid chromatography tandem mass spectrometry method for the determination of irbesartan in human plasma. J Food Drug Analysis, 2015; 23(3): 569-576.

Figure

Figure 1: TOF-MS/MS Spectra of Losartan
Figure 2: TOF-MS/MS Spectra of Telmisartan.
Figure 6: Proposed MS/MS Fragmentation Mechanism of Losartan.
Figure 9: Proposed MS/MS Fragmentation Mechanism of Candesartan.
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References

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