Development and Validation of Analytical HPLC-UV Method for simultaneous estimation of Losartan and its active Metabolite EXP-3174

Alka Singh, Neeraj Upmanyu

School of Pharmacy and Research, People’s University, Bhanpur, Bhopal (M.P.) India.

*Corresponding Author E-mail: singhalka7160@gmail.com

ABSTRACT:

Background: The combination therapy of Losartan and active metabolite has proven to be beneficial as compare to either drug monotherapy. Losartan is a highly selective, orally active, non-peptide angiotensin II receptor antagonist indicated for the treatment of hypertension, which is one of the most important causes of mortality and morbidity in the modern world. It has a more potent active carboxylic acid metabolite EXP-3174 (2-butyl-5-chloro-3-[[4-[2-(2H-tetrazol yl)phenyl]phenyl]methyl]imidazole-4-carboxylic acid). Losartan and its active carboxylic acid metabolite EXP-3174 block the vasoconstrictor and aldosterone-secreting effects of angiotensin II by type ATI receptor blockage. Following oral administration, losartan is rapidly absorbed, reaching maximum concentrations 1–2 h post-dose. Result: The selected analytes were effectively separated on thermo β-basic C18 (100×4.6 mm, 5 µm) column using mobile phase consisting of 20mM monobasic potassium phosphate and 0.2% TEA with acetonitrile and iso-propyl alcohol in gradient mode. The eluent was monitored at 228nm at 1.0 ml min^-1 flow rate. The total run time was less than 12 min. Conclusion: The active carboxylic acid metabolite E-3174 is about ten times more potent than losartan. In healthy volunteers, the concentrations of the active carboxylic acid metabolite EXP-3174 were found to be more closely parallel angiotensin II antagonism than those of the parent compound. Thus, the angiotensin II blocking activity of losartan is predominantly based on its major active carboxylic acid metabolite EXP-3174. Given this as background and to our best knowledge, there is no HPLC-UV method available or published simultaneous estimation of losartan with its active metabolite.

KEYWORDS: Losartan, Carboxylic acid metabolite EXP-3174, Angiotensin II antagonism, ATI receptor blocker, Validation.

INTRODUCTION:

Hypertension is the most common primary cause of cardiovascular morbidities¹. Multiple-drug therapy is frequently prescribed in the treatment of hypertension². The combination therapy of Losartan and active metabolite has proven to be beneficial as compare to either drug monotherapy. Cardiovascular diseases and their nutritional risk factors are among the leading causes of mortality and morbidity globally (Figure 1), and have been predicted to rise over the next few decades^3-5. Aging of the world’s population is a key driver of the expected increase, because cardiovascular disease rates tend to increase with age. In addition to this demographic change, an epidemiological change that involves increases in age-specific rates of cardiovascular diseases in developing countries has also been predicted in some analyses⁶

Losartan is a selective, competitive, reversible angiotensin II receptor for the treatment of hypertension. The active metabolite of losartan, carboxylosartan (also known as EXP-3174) is generated via oxidation mainly by CYP2C9⁷. This carboxylic acid metabolite was estimated to contribute approximately 14-times the activity than the parent drug (8). Following oral administration, losartan is well absorbed, undergoes first-pass metabolism, and about 14% of an oral dose is metabolized to carboxylosartan (9). The bioavailability of losartan is approximately 33%, and peak plasma concentrations are reached after 1 hour for losartan and 4 hours for carboxylosartan. Losartan and carboxylosartan are highly plasma protein-bound (10) and have small volumes of distribution at steady state (11). Similar to other acid drugs, losartan is sensitive to transporter uptake (12).

Losartan Carboxylic acid (EXP-3174)

MATERIALS AND METHODOLOGY:

HPLC equipped with pump, injector and UV/PDA Detector, Make: Agilent 1200 series (Chromleon software), UV-Visible Spectrophotometer, Make: Shimadzu UV-1800 (UV-Winlab software), FTIR spectrometer and Mass spectrometer (for characterization purpose), HPLC Columns, drug Sample, working standard, solvents.

Losartan, working standard, was obtained from Zydus Research Centre Ltd, Mumbai, India. Losartan carboxylic acid (LCA) were generous gift of Mylan laboratories (Nasik, India). Analytical/HPLC grade salts and solvents were purchased from Meark chemical limited (Mumbai, India) and used without further purification.

Analytical methods for Losartan:

Authentication of LOS:

The identification of the sample was carried out by UV (λ_max), IR spectroscopy and mass spectrometry. LOS is a crystalline powder with white appearance.

UV-absorbance pattern of LOS:

The UV-absorption pattern of LOS was matched with literature and the calculated UV- extinction coefficient was similar to that reported in literature. The representative UV-spectrum of LOS in 0.1% Phosphoric acid: acetonitrile (1:1) is shown in Fig 1.

Fig1: UV spectra for determination of λ_maxof LOS

The wavelength maxima was found to be 263 nm and absorptivity () was about 487. The values are indicating that LOS shows high-intensity UV-absorption (pavia reference).

IR-spectral behavior of LOS:

The FTIR was recorded and shown in Fig. 2. The peaks were assigned and matched with literature.

Figure 2: The FT-IR spectra of LOS (using ATR for sampling)

The IR Spectrum of LOS had shown prominent peaks for –NH, =C-H, Aromatic –C-H, -C=O and –C-O stretching.

Calibration Curve of LOS:

The CC standards were prepared in the range of 5-25μg/ml. The absorbance was calculated at λ_max of LOS (225nm) in the 0.1% Phosphoric acid: acetonitrile (1:1). The overlain UV- spectrum of LOS is shown in Fig.3

Fig 3: Overlain spectra of calibration curve for LOS in MeOH: Water at 225nm

The absorbances were recorded in duplicate and mean absorbance calculated for each concentration and shown in table 1.

Table 1: Calibration curve LOS in 0.1% Phosphoric acid: acetonitrile using UV-Spectrophotometer at 225 nm

Conc. (μg/ml)	Absorbance			Line equation and correlation coefficient
Conc. (μg/ml)	Abs-1	Abs-2	Mean	Line equation and correlation coefficient
2	0.258	0.241	0.250	y = 0.076x + 0.094 R² = 0.999
4	0.321	0.318	0.320
6	0.398	0.405	0.402
8	0.487	0.469	0.478
10	0.558	0.548	0.553
12	0.624	0.632	0.628
15	0.714	0.701	0.708

The calibration curve was plotted between mean absorbance and concentration as shown below in Fig 4.

Fig 4: Calibration curve of LOS in 0.1% Phosphoric acid: acetonitrile

The Calibration curve was found linear, with line equation y = 0.076x + 0.094 and correlation coefficient r² was 0.999.

HPLC Method development for Losartan:

Analytical method development of LOS: Pre-validation studies:

Proper selection of the method depends upon the nature of the sample (ionic/ionisable/neutral molecule), its molecular weight and solubility. The drug is official in United State Pharmacopoeia (USP 30), therefore trials for HPLC method development were initiate using pharmacopeial method.

The retention time of LOS was 12.5min. The capacity factor was about 13.1. Aside that the method was seems to be complex. Though the chromatographic conditions were passing system suitability parameters but the method fails in justifying the objectives of work. Therefore, it was further modified to justify the need of simultaneous estimation.

Chemically LOS is a weak base having pKa value 3.10. The hydrophobicity of LOS (log P >3.0) was favorable for retention of it on a reversed-phase stationary phase. Therefore, theseparation conditions were decided on the basis of ionization behavior and logp value, available chromatographic methods and previous UV-spectrophotometric experiments for the same. The reverse phase HPLC was selected for the separations because of its simplicity, suitability and ruggedness. Following conditions were taken into consideration before starting the actual HPLC-experiment-

· The diluent for dissolving sample was selected on the basis of UV-experiments. The diluent, 0.1% H3PO4 (pH 3.0): ACN in the ratio of 1: 1 was used for dissolving sample.

· For the method development purposes, pH of the mobile phase was kept in the range of 5.0-6.0 for complete unionization of LOS (>99% in unionized state).

· The drug is strongly hydrophobic in nature (log p >3.0); therefore proportion of organic to aqueous mobile phase was kept higher for the initial experimentation.

The chromatogram recorded in initial RP-HPLC conditions had shown bifurcation in peak with unacceptable peak shape. In the next trial, the pH of HPLC method was changed form 5.5 to 3.0. In addition to change in pH, 0.2%Triethyl amine (TEA) was added to aqueous phase. In the finally optimized method, diluted LOS standard solution (10µg/mL) was prepared and injected in to the chromatograph. The Chromatogram was shown in the Fig. 4

The chromatographic method for Losartan was developed in lieu with simultaneous elution of losartan carboxylic acid. Analytical challenges were involving similarity in UV absorbacne behavior and hydrophobicity except the ionization behavior of LCA. The active metabolite of Losartan, LCA, is having more acidic–COOH moiety, which was chosen as differentiating property in chromatographic separation (Method 1). The chromatographic method in condition 1 was involving co-elution of both the analytes using official HPLC condition with increased amount of organic phase on an ODS column. The results were quite peculiar and co-elution was observed for both the analytes in same elution window. Further serial and systemic changes in the chromatographic conditions were helpful in effective separation of selected analytes as shown in table below. The overlain chromatograms of different method development conditions were showing simultaneous elution of losartan and LCA was shown in figure below.

Table:2 Chromatographic conditions for co-elution of Losartan and Losartan carboxylic acid

Chromatographic parameters	Conditions 1	Conditions 2	Conditions 3	Conditions 4
Column	C18 (L1 USP) short as compare to USP	C8 (150 × 4.6 mm i.d., 5 μm)	C8 (100 × 4.6 mm i.d., 5 μm)	C18 (100 × 4.6 mm i.d., 3.5 μm)
Mobile phase	0.1% H₃PO₄: ACN	10mM phosphate buffer: ACN	10mM phosphate buffer: ACN	10mM phosphate buffer: ACN
Mobile phase ratio	Isocratic (3:2)	Isocratic (1:2)	Gradient 0.00-1.99 min: 20 % (% B) 2.00-4.49 min: 70 % (% B) 4.50-6.99 min: 100 % (% B) 7.00-9.99 min: 20 % (% B)	Gradient 0.00-0.49 min: 20 % (% B) 0.50-1.49 min: 70 % (% B) 1.50-4.99 min: 100 % (% B) 5.00-9.99 min: 20 % (% B)
Flow rate	1.0 ml/min	1.5 ml/min	1.0 ml/min	1.0 ml/min
Detection wavelength	254 nm	264 nm	264 nm	264 nm
Injection volume	20Μl	20μL	20μL	20Μl
Temperature of column oven	35° C	25° C	25° C	40° C
Remarks	Losartan in alone was passing the assay (κ: 7.41). Co-elution was observed for both the analytes	Early elution of Losartan (κ: 3.54). Co-elution was observed for both the analytes	Simultaneous elution of Losartan and LCA observed with resolution factor value 1.79.	Good and effective separation. Though the LCA was eluted near Column void but the quite resolute with void peak.

Fig 6. Chromatograph of LOS and LCA final optimized conditions

RESULT AND DISCUSSION:

The result of system suitability studies is given in the table. 3

Table 3: System suitability studies for diluted LOS and LCA standard solution (n=6) in the optimized RP-HPLC method

System suitability parameter	USP Limit	LOS	LCA
Retention time	-	3.92 min	2.02 min
% RSD	-	1.11	1.98
Mean peak area	-	855785.0	658525.0
% RSD of peak area	≤ 2.0	0.86	1.85
Peak asymmetry factor (at 10% peak height)	≤ 1.5	1.37	1.12
Capacity factor (k’)	0.5 to 20	8.52	4.54
Mean Number of theoretical plates	≥ 2000	5421 8548

Assay method validation:

Based on the finally optimized procedure as in pre-validation section analytical validation of LOS and LCA was performed as per ICH Q2 (R1) guidelines.

Specificity of LOS and LCA:

Specificity measures only the desired component without interference from other species that might be present. To determine specificity during the validation blanks, sample matrix (placebo), and known related impurities are analyzed to determine whether any interferences occur during the chromatographic development.

In the present method validation, content determination of LOS and LCA in bulk was exercised using RP-HPLC. Therefore, it is necessary to determine the LOS and LCA in the presence of diluent (obviously) and if possible related substance. No interference was recorded in the chromatogram of method diluent (blank) at the retention time window of diluted LOS and LCA.

Linearity and Range of LOS and LCA:

For the assay method validation 10 µg/mL was taken as test concentration and range should be 80-120% of test concentration. Therefore, the range for the assay method validation was 8-12µg/mL of LOS and LC.

Linearity of the proposed method was carried out by the preparation of the linearity curve concentration 80%, 90%, 100%, 110% and 120% of LOS and LCA test concentration (100%) for the concentration of 8.0, 9.0, 10.0, 11.0 and 12.0µg/ml, respectively. The linearity standard solutions were prepared in triplicate (table no 4).

The mean peak area was calculated between mean peak area (n=3). The chromatographic data is given in table 4.

Table no 4: Peak area of LOS and LCA Linearity solutions form 8-12 μg/ml

Conc. (μg/ml)	Peak area OF LOS				Line equation and correlation coefficient
Conc. (μg/ml)	1	2	3	Mean	Line equation and correlation coefficient
8	602841	602151	602841	595892	y = 11946x + 36429 R² = 0.999
9	705841	741587	705841	718558
10	829685	825284	829685	898521
11	998547	948585	998547	925584
12	1114558	1002548	1114558	1122448
Conc. (μg/ml)	Peak area OF LCA				Line equation and correlation coefficient
8	544825	518741	501158	521575	y = 10874x + 30209 R² = 0.999
9	621852	635855	618528	625412
10	755852	712585	741852	736763
11	874582	814582	852485	847216
12	952488	985207	925482	954392

Table 5: The results for system, method and intermediated Precision of LOS and LCA

Precision parameters	Conc. (µg/mL)	Peak area of LOS
Precision parameters	Conc. (µg/mL)	1	2	3	4	5	6	Mean ±SD
System	10	812251	825284	829685	825481	832214	823142	824676.2
Method	10	812548	825487	835524	852478	841778	835847	833943.7
Intermediated	10	854758	863258	821478	826895	841785	836585	840793.2
Precision parameters	Conc. (µg/mL)	Peak area of LCA
		1	2	3	4	5	6	Mean ±SD
System	10	741525	745558	736985	742289	742895	759852	744850.7
Method	10	775852	754185	745852	765895	755824	766985	760765.5
Intermediated	10	745589	748528	763998	758548	744855	723585	747517.2

Fig.7: Precision of LOS and LCA chromatogram

Table 6: Recovery studies of LOS and LCA in bulk

Concentration level	Peak area of LOS				% Mean recovery
Concentration level	1	2	3	mean	% Mean recovery
Unfortified samples
80% (8 µg/mL)	602151	602841	595892	600294.7
100 % (10 µg/mL)	825284	829685	898521	851163.3
120% (12 µg/mL)	1002548	1114558	1122448	1079851
Fortified samples			Mean recovery ± SD		98.26 ± 1.37
80+ 100 % (8+10)	1450281	1448526	1411255	1436687
100+ 100 % (10+10)	1665285	1685258	1625852	1658798
120 + 100 % (12+10)	1891482	1909852	1905528	1902287
Concentration level	Peak area of LCA				% Mean recovery
Concentration level	1	2	3	Mean	% Mean recovery
Unfortified samples
80% (8 µg/mL)	544825	518741	501158	521574.7
100 % (10 µg/mL)	755852	712585	741852	736763
120% (12 µg/mL)	952488	985207	925482	954392.3
Fortified samples			Mean recovery ± SD		101.24 ± 1.60
80+ 100 % (8+10)	1300258	1288582	1262558	1283799
100+ 100 % (10+10)	1470248	1498558	1464582	1477796
120 + 100 % (12+10)	1705586	1699821	1661251	1688886

The % mean recovery of accuracy study of LOS was 98.26 and LCA was 101.24 shown in Table 6

Robustness of LOS and LCA:

A measure of an effective analytical method is how well its performance stands up to less than perfect implementation. The robustness of a method is evaluated by varying method parameters such as changing different conditions in the method as change in flow rate of mobile phase, change in mobile phase composition, change in analytical wavelength and determining the effect (if any) on the results of the method. The overlain chromatogram for effect of flow rate of pump and mobile phase composition were shown in Fig. 8

Fig 8: Overlain chromatogram for effect of flow rate of pump

The %RSD for peak area (n=2x3) was calculated as 0.56. The retention time of LOS and LCA was changed as 3.71min 1.81 min for 1.1ml/min and 2.25 min and 4.22 for 0.90ml/min. The retention time of LOS and LCA were altered accordingly with the mobile phase composition 48:52 and 52:48, respectively.

CONCLUSION:

The analytical validation of LOS and its metabolite was performed as per ICH Q2 (R1) guidelines. The validation study shows that the developed method is accurate, rapid, precise, linear, specific and inexpensive with acceptable correlation co-efficient, RSD (%) and standard deviations which make it versatile and valuable for simultaneous determination of Losartan and Losartan Carboxylic Acid (active metabolite) in bulk form. The proposed method is simple and do not involve laborious time-consuming sample preparation. So this RP-HPLC method can be used for the routine analyses of LOS and LCA. This research work also has future application in bioanalytical HPLC method development in combination with other anti hypertensive drugs.

REFERENCES:

1. Ezzati M, Vander Hoorn S, Lawes CM, et al. Rethinking the “diseases of affluence” paradigm: global patterns of nutritional risks in relation to economic development. PLoS Med 2005; 2: 133.

2. Sica D. Rationale for fixed-dose combinations in the treatment of hypertension. Drugs 2002; 62: 443-462.

3. Murray CJL, Lopez AD, editors (1996) The global burden of disease: A comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to 2020. Cambridge (Massachusetts): Harvard School of Public Health. 990 p.

4. Reddy KS, Yusuf S (1998) Emerging epidemic of cardiovascular disease in developing countries. Circulation 97: 596–601.

5. Yusuf S, Reddy S, Ounpuu S, Anand S (2001) Global burden of cardiovascular diseases: Part I: General considerations, the epidemiological transition, risk factors, and impact of urbanization. Circulation 104: 2746– 2753.

6. Bulatao RA (1993) Mortality by cause, 1970 to 2015. In: Gribble JN, Preston SH, editors. The epidemiological transition: Policy and planning implications for developing countries. Washington (D.C.): National Academy Press. pp. 42–68.

7. Stearns RA, Chakravarty PK, Chen R, Chiu S. Biotransformation of losartan to its active carboxylic acid metabolite in human liver microsomes. Role of cytochrome P4502C and 3A subfamily members. Drug Metab Dispos. 1995; 23: 207-215.

8. Obach RS. Pharmacologically active drug metabolites: impact on drug discoveryand pharmacotherapy. Pharmacol Rev. 2013; 65: 578-640.

9. Lo M-W, Goldberg MR, McCrea JB, Lu H, Furtek CI, Bjornsson TD. Pharmacokinetics of losartan, an angiotensin II receptor antagonist, and its active metabolite EXP3174 in humans. Clin Pharmacol Ther. 1995; 58:641-649.

10. Christ DD. Human plasma protein binding of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174. J Clin Pharmacol. 1995; 35: 515-520.

11. Marciniak T. Clinical Review, Supplemental NDA Submission, NDA 20e386, Cozaar Tablets. Silver Spring, MD: Division of Cardiorenal Drug Products. U.S. Food and Drug Administration. Merck and Co; 2002.

12. Gardiner P, Paine SW. The impact of hepatic uptake on the pharmacokinetics of organic anions. Drug Metab Dispos. 2011; 39: 1930-1938.

Received on 12.02.2021 Modified on 15.03.2021

Asian Journal of Research in Chemistry. 2021; 14(4):275-281.

DOI: 10.52711/0974-4150.2021.00047

Stationary phase	:	Thermo β-basic C₁₈ (100 x 4.6 mm i.d., 3.5 mm)
Mobile phase	:	Solvent A: 10mM monobasic potassium phosphate (pH 3.0 with phosphoric acid)
Mobile phase	:	Solvent B: ACN
Solvent ratio	:	Gradient 0.00-0.49 min: 20 % (% B) 0.50-1.49 min: 70 % (% B) 1.50-4.99 min: 100 % (% B) 5.00-9.99 min: 20 % (% B)
Detection wavelength	:	264 nm
Flow rate	:	0.6 mL/min
Injection volume	:	20 µl
Temperature	:	Ambient (around 25° C)
Sample temperature	:	4 ± 2°C (prior to injection sample was stored in refrigerator)