Condensation Methods for the Determination of Methoxamine Hydro Chloride in Pure and Pharmaceutical Formulations

M.L.N. Acharyulu¹, P.V.S.R. Mohana Rao², I. Siva Ramakoti³,P. Jayarangarao⁴

¹Associate Professor, Centurion University of Technology and Management, Andhra Pradesh- 530017, India

²Research Scholar, Department of Engineering Chemistry, A.U. College of Engineering (A),

Visakhapatnam - 530003, A.P; India

³Assistant Professor, Department of Chemistry, Centurion University of Technology and Management,

Odisha- 761211, India

⁴Assistant Professor, Baba Institute of Technology and Sciences, Visakhapatnam, A.P. India.

*Corresponding Author E-mail: acharyulu@cutmap.ac.in

ABSTRACT:

Two visible spectrophotometric methods were developed Aand B for the determination of Darunavir in pure and pharmaceutical formulations. The methods are based on condensation reaction with PDAB (Method-A) and ONB (Method-B) in presence of acidic medium with the primary amine group in DNV. The coloured products exhibit absorption λ_max at 639 nm and 452nm for methods A and B respectively. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges 10-60μg/ml, 50-300μg/ml, correlation co-efficients are 0.9983, 0.9989; Sandell’s sensitivities are 9.9833 x 10^-3, 3.0456 x 10^-2(1 mole cm^-1); and molar absorptivity values are 5.4857 x 10⁴, 1.7981x 10⁴ (µg cm^-2) for methods-Aand B respectively. The proposed methods are applied to commercial available formulations and the results are statistically compared with those obtained by the UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the DNV in the presence of other ingredients that are usually present in formulations. These methods offer the advantages of rapidity, simplicity and sensitivity and low cost without the need for expensive instrumentation and reagents.

KEYWORDS: Condensation, PDAB, ONB, Regression Analysis.

INTRODUCTION:

Methoxamine hydrochloride (MHC), chemically 2-amino-1-(2,5-dimethoxyphenyl) propan-1-ol (Fig.1), is relatively selective α₁-adrenoreceptor agonist producing an increasing in peripheral vascular resistance1,2. MHC is a potent sympathomimetic that increases both systolic and diastolic blood pressure. It is used in the treatment of some hypotensive patients, particularly those with paroxysmal a trial tachycardia (arrhythmia) andacts almost exclusively on alpha-adrenergic receptors.

Literature survey reveals few analytical methods were reported for the determination of MHC in biological fluids by HPLC with fluorescence detection3,4. Spectrophotometric method analysis 5,6. High performance liquid chromatography using pre-column derivatization of MHC with other cardiovascular drugs7. The analytical useful functional groups in MHC have not been fully exploited for designing suitable visible spectrophotometric methods and so still offer a scope to develop more visible spectrophotometric methods with better sensitivity, precision and accuracy.

No analytical methods that have been reported so far for the estimation of MHC by HPLC method. Hence, the author has made an attempt to develop a new, simple, economical, selective, accurate and precise stability indicating reverse phase high-performance liquid chromatographic method 8-12 with good sensitivity for assay of MHC in pharmaceutical dosage form. Indeed the established method was validated with respect to specificity, linearity, precision, accuracy, robustness, LOD and LOQ according to ICH guidelines13.The analytical applications of the reagents used 14-17 and some methods reported 18-20 of the selected drug from the literature are mentioned.

Fig.1. Structure of MHC

EXPERIMENTAL:

Instruments Used:

A Schimadzu UV-Visible spectrophotometer 1801 with 1 cm matched quartz cells was used for all spectral and absorbance measurements. A Systronics digital pH meter 361 was used for pH measurements.

Preparation of standard Drug solution:

The stock solution (1mg/ml) of Methoxamine Hrdrochloride (MHC) was prepared by dissolving 100 mg of it in 100ml of millipore-distilled water. A portion of this stock solution was diluted stepwise with the distilled water to obtain the working standard MHC solution of concentrations 60µg/ml for proposed methods

Procedure of Assay of MHC in formulations-Reference method:

An accurately weighed amount of formulation (injection powder) equivalent to 100 mg of drug was dissolved in 20 ml of distilled water, shaken well and filtered. The filtrate was further diluted to 100 ml with distilled water to get 1 mg/ml solution of drug in formulations. One ml of this solution was furthered diluted to 25 ml to get 40 µg/ml solution. The absorbance of the solution was determined λ_max270 nm (Fig.2). The quantity of the drug was computed from the Beer’s law plot (Fig.3) of the standard drug in distilled water.

Fig.2 Absorption Spectra of MHC in Methanol (UV reference method)

Fig.3 Beer’s law plot of MHC in methanol (UV reference method)

Recommended Procedures:

After systematic and detailed study of the various parameters involved, as described under results and discussion in this chapter, the following procedures were recommended for the determination of MHC in bulk samples.

Fig.4 Absorption spectra of MHC: PDAB/H₂SO₄

Fig.5 Beer’s plot of MHC: PDAB/H₂SO₄

Method-A:

To each of 10ml calibrated tubes, aliquots of standard MHC solution, (0.1 – 0.6ml, 60µg/ml) 2.0ml of PDAB and 3.0ml of conc. sulphuric acid were added successively and the total volume in each flask was brought to 9ml by the addition of methanol and placed in a heating water bath for 15 min. Then the flasks were cooled and made up to the mark with methanol and absorbance was measured after 5 min. at λ_max 655nm (Fig.4) against a reagent blank prepared in a similar way. The amount of MHC in sample solution was obtained from the Beers-Lambert’s plot (Fig.5)

Fig.6 Absorption spectra of MHC: ONB/H₂SO₄

Method-B:

Into a series of 10ml calibrated tubes, aliquots of standard MHC solution (0.1-0.6ml, 60µg/ml) 1ml of ortho nitro benzaldehyde, 2 ml of concentrated sulphuric acid and 2ml of methanol was added successively and solution allowed to stand for 5 minutes. The solution made up to the volume by adding distilled water. The absorbance of each solution was measured at λ_max 451 nm (Fig.6) against reagent blank prepared in similar manner. The amount of MHC in a sample solution was obtained from the Beers-Lamberts Plot (Fig.7).

Fig.7 Beer’s plot of MHC: ONB/H₂SO₄

Table:1 Optical and Regression characteristics, precision and accuracyof the proposed methods for MHC

Sl. No	Parameter	Method-A	Method-B
1	Wave length λ_max (nm)	655	451
2	Beer’s law limits (µg ml^-1)	10-60	10-60
3	Detection limits (µg ml^‑1)	0.9324	3.4397
4	Molar absorptivity (1 mole cm^-1)	3.6827 x 10⁴	4.6420 x 10⁴
5	Sandell’s sensitivity (µg cm^-2 / 0.001 absorbance unit)	6.7264 x 10^-3	9.9833 x 10^-3
6	Regressionequation (Y = a + bC) Slope (b)	0.0149	0.0101
7	Standard deviation of slope (S_b)	1.1892 x 10^-4	2.9736 x 10^-4
8	Intercept (a)	0.004	0.0077
9	Standard deviation of intercept (S_a)	4.6314 x 10^-3	1.1580 x 10^-2
10	Standard error of estimation (S_e)	4.9749 x 10^-3	1.2439 x 10^-2
11	Correlation coefficient (r²)	0.9998	0.9966
12	Relative standard deviation (%)*	1.0695	1.2909
13	% Range of error (Confidence Limits) 0.05 level*	1.1226	1.3549
14	% Range of error (Confidence Limits)0.01 level	1.7605	2.1249
15	% Error in bulk samples**	0.415	0.545

Average of six determinations considered**: Average of three determinations

Table:2. Assay and recovery of MHC in Pharmaceutical Formulations

Sample	Amount taken (mg)	Amount found by proposed methods		Reference Methods	Percentage recovery by proposed methods
Sample	Amount taken (mg)	Method-A	Method-B	Reference Methods	Method-A	Method-B
Injection I	20	19.96 ± 0.013 F=1.159 t=0.58	19.89 ± 0.016 F=1.306 t=1.33	19.95 ± 0.014	99.90 ± 0.010	99.882 ± 0.019
Injection II	20	19.85 ± 0.013 F=1.331 t=0.69	19.78 ± 0.018 F=1.917 t=0.84	19.92 ± 0.015	99.89 ± 0.020	99.825 ± 0.025

*: Average ± standard deviation of six determinations; the t- and F- values refer to comparison of the proposed method with the reference method. Theoretical values at 95% confidence limit t=2.57, F=5.05.

**: After adding 3 different amounts of the pure labeled to the pharmaceutical formulations, each value is an average of 3 determinations

RESULTS AND DISCUSSION:

Optimum operating conditions used in the procedure were established adopting variation of one variable at a time (OVAT) method. The effect of various parameters such as were studied.Volume of PDAB, Volume of conc. H₂SO₄on colour development,order of addition of reagents on colour development, temperature, shaking time, Solvent for final dilution, Stability period after final dilution were studied for Method-A.Volume of ONB required for condensation,volume of H₂SO_4,effect of CH₃OH,reaction time, Effect of temperature and stability period were studied for method-B.The optical characteristics such as Beer’s law limit, Sandell’s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements, Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (Sa), standard error of estimation (S_e) and % range of error (0.05 and 0.01confidence limits) were calculated and the results are summarized in Table-1. Commercial formulations containing MHC were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-test and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pure analyzed formulations at three different concentration levels. These results are summarized in Table-2.

CONCLUSION:

The results presented above indicate that the proposed methods have good sensitivity, selectivity, precison and accuracy. Results of analysis in bulk form and formulations reveal that the proposed methods are suitable for the estimation of MHC in them, as impurities and excipients present in them cause no interference virtually.

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Received on 14.08.2020 Modified on 28.09.2020

Asian J. Research Chem. 2021; 14(1):23-27.

DOI: 10.5958/0974-4150.2021.00004.3