Analytical Applications of Isatin in the Determination of Darunavir and Methoxamine Hydrochloride

 

M.L.N. Acharyulu¹*, P.V.S.R. Mohana Rao², P. Jaya Ranga Rao³

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.

³Assistant Professor, Department of Chemistry, Basic Sciences and Humanities, Baba Institute of Technology and Science, Visakhapatnam, Andhra Pradesh- 530017, India.

 

ABSTRACT:

Two simple and sensitive extractive visible spectrophotometric methods (A and B) for the assay of Darunavir (DNV)and Methoxamine hydrochloride (MHC), in pure and pharmaceutical formulations based on under acidic conditions the primary amine group in DNV and MHC forming coloured condensation product with aromatic aldehyde Isatin forms the basis for Method-A and Method-B. The coloured products exhibit absorption λ_max at 412nm and 450nm for methods A and B respectively. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (100-600)μg/ml for method A, (50-300)μg/ml for method B and correlation co-efficients are 0.994(A), 0.9967(B) respectively. The Sandell’s sensitivities are2.0044x 10^4, 1.8991 x 10⁴ (1 mole cm^-1) and molar absorptivity values are 2.7322x 10^-2, 1.3043 x 10^-2(µgcm^­-2). 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 theDarunavir and Methoxamine hydrochloride 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.

 

 

 

INTRODUCTION:

Darunavir:

Darunavir (DNV, Fig-1) is an oral anti-retroviral agent which selectively inhibits the cleavage of Human immunodeficiency virus (HIV-1) encoded Gas-polyproteins in infected cell, thereby the formation of mature virus.

 

Darunavir ethanolate is chemically [(1S, 2R)-3-[[(4-amino phenol) sulfonyl](2-methyl propyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-carbamic acid (3R, 3aS, 6aR)-hexahydrofuro[2, 3-b]furan-3-yl ester monoethanolate(Fig-1)

 

Fig.1 Methoxamine Hydrochloride

 

Methoxamine hydrochloride (MHC, Fig-2), 2-amino-1-(2, 5-dimethoxyphenyl) propan-1-ol, is relatively selective α₁-adrenoreceptor agonist producing an increasing in peripheral vascular resistance. 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 atrial tachycardia (arrhythmia) and acts almost exclusively on alpha-adrenergic receptors.

 

Fig-2. Chemical Structure of Darunavir

 

Literature Survey on the analytical methods for DNV and MHC:

 Darunavir was designed to form robust interactions with the protease enzyme from many strains of HIV, including strains from the treatment-experienced patients with multiple resistance mutations to Protease inhibitors^[1]. It blocks HIV protease, an enzyme which is needed for HIV to multiply. According to in vitro experiments, DNV was active against HIV-1 with PI-resistance mutations and against PI-resistance clinical isolates^[2,3]. This drug is effective in patients experienced in anti-retroviral treatment, such as those carrying HIV-1 strains which are resistance to more than one PI^[4]. The use of advanced instrumentation techniques for the analysis of drugs has been discussed elsewhere^[5]. Literature survey revealed that different analytical methods have been reported for the determination of DNV in plasma using liquid chromatography coupled with tandem mass spectroscopy^[6] simultaneous determination of DNV with other anti-retroviral agents in plasma^[7,8]. Few HPTLC methods for determination of DNV in rat plasma and in tablet dosage form its application to pharmacokinetics studies^[9]. Infrared Spectroscopy method for determination of Darunavir in tablets^[10]. Few methods had been developed for determination of DNV Spectrophotometric method^[11] and electrophoretic method for the separation of DNV^[12]. Literature survey reveals few analytical methods were reported for the determination of MHC in biological fluids by HPLC with fluorescence detection^{[13, 14]}. Spectrophotometric method analysis^[15,16]. High performance liquid chromatography using pre-column derivatization of MHC with other cardiovascular drugs^[17]. Isatin (1H-indole-2, 3-Dione)^[18,19] was first obtained by Erdman and Laurnet in 1841 as a product from the oxidation of indigo by nitric acid and chromic acids. Under acidic conditions isatin reacts with proline and pyrrole to give colored condensation product. Isatin also produces a fluorogenic derivative when reacted with tryptophan which has been used for its detection by TLC^[20,21]. A further application of isatin in steriod analysis is its use as a colored marker in sephadex LH-20 chromatographic separation of steroidal blood components^[22,23].

 

The analytical useful functional groups in DNV and MHC have not been fully exploited for designing suitable visible So, the author made some attempt in developing visible spectrophotometric methods and succeeded in developing two methods. Good number of methods were reported in literature using Isatin^[24-30] as a simpler reagent based on the reaction between drug and isatin other than the drugs chosen by author. The present paper describes two simple and sensitive extraction visible spectrophotometric methods for the determination of assay of DNV and MHC based on condensation reaction of primary amine of the drug with Isatin in presence of acidic medium. The applications of spectrophotometry in other substances were also referred^[31-40]

 

MATERIALS AND METHODS:

Apparatus and chemicals: A Shimadzu UV-Visible spectrophotometer 1601 with1cm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade.

 

Isatin Solution 0.004%:

Prepared by dissolving 4mg of Isatin in 100ml of acetic acid

 

Sulphuric acid:

A.R. Grade sulphuric acid is used as it is.

 

Preparation of standard Drug solution:

The stock solution (1mg/ml) of Darunavir (DNV) was prepared by dissolving 100mg 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 DNV solution of concentrations 4-600µg/ml.

 

Procedure of Assay of DNV in formulations:

An accurately weighed amount of formulation (tablet) equivalent to 100mg of drug was dissolved in 20ml of distilled water, shaken well and filtered. The filtrate was further diluted to 100ml with distilled water to get 1 mg/ml solution of drug in formulations. One ml of this solution was furthered diluted to 25ml to get 40µg/ml solution. The absorbance of the solution was determined λ_max 223nm (Fig-4). The quantity of the drug was computed from the Beer’s law plot (Fig-5) of the standard drug in distilled water.

 

 

Fig-4. Absorption spectra of DNV in Methanol (UV reference method)

 

 

Fig-5. Beer’s law Plot of DNV in(UV reference method)

 

 

Fig-6. Absorption Spectra of MHC in Methanol (UV reference method)

 

 

Fig-7. Beer’s law plot of MHC in Methanol (UV reference method)

 

EXPERIMENTAL:

Instruments Used:

A Schimadzu UV-Visible spectrophotometer 1801 with 1cm 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 4-500µg/ml.

 

Procedure of Assay of MHC in formulations:

An accurately weighed amount of formulation (injection powder) equivalent to 100mg of drug was dissolved in 20ml of distilled water, shaken well and filtered. The filtrate was further diluted to 100ml with distilled water to get 1mg/ml solution of drug in formulations. One ml of this solution was furthered diluted to 25ml to get 40 µg/ml solution. The absorbance of the solution was determined λ_max270nm (Fig-6). The quantity of the drug was computed from the Beer’s law plot (Fig-7) of the standard drug in distilled water.

 

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 DNV in bulk samples.

 

Method-A:

Into series of 20ml calibrated tubes, aliquots of standard DNV solution (0.1-0.6ml, 600µg/ml) were transferred and volume adjusted to 3ml by adding methanol. To this solution 2.5ml of Isatin and 1ml of concentrated sulphuric acid were added successively added and cooling the solution under a tap with constant shaking and the final volume was made up to the mark with methanol. The absorbance was measured at 413nm (Fig-8) against reagent blank prepared in similar manner. The amount of DNV was calculated from its calibration graph (Fig-9)

 

Method -B:

Into series of 20ml calibrated tubes, aliquots of standard MHC solution (0.1-0.6ml, 300µg/ml) were transferred and volume adjusted to 3ml by adding methanol. To this solution 2.5ml of Isatin and 1ml of concentrated sulphuric acid were added successively and cooling the solution under a tap with constant shaking and the final volume was made up to the mark with methanol. The absorbance of the colored species was measured at λ_max 450nm (Fig.10) against reagent blank prepared in similar manner. The amount of MHC was calculated from its calibration graph. (Fig.11)

 

 

Fig-8. Absorption spectra of DNV: ISATIN

 

 

Fig-9. Beer’s plot of DNV: ISATIN/H₂SO₄

 

 

Fig-10. Absorption spectra of MHC: ISATIN

 

 

Fig-11. Beer’s plot of MHC: ISATIN/H₂SO₄

 

Table:1. Optical and Regression characteristics, precision and accuracy of the proposed methods For DNV and MHC

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

 

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

*: 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.

 

Chemistry of coloured species in the present investigation:

Method-A:

DNV possesses different functional moieties such as primary amine, tertiary amine and sulphonyl groups of varied reactivity. The methods proposed are based on condensation reactions with Isatin in presence of acidic medium with primary amine in nonaqueous medium. The nature of coloured species obtained with Isatin, is represented in the following scheme-1.

 

 

Method-B

MHC possesses different functional moieties such as primary amino group and hydroxyl groups. The proposed method is based on reactivity of primary amine of MHC forming coloured condensation product with aromatic aldehyde Isatin in the presence of H₂SO₄ in non aqueous medium. The nature of coloured species obtained with Isatin is represented in the following Scheme-2

 

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 volume of H₂SO₄ time, volume of CH₃OH(ml), Reaction time(nm), temperature( C), Stability period after final dilution (min.) of the colored species were studied. 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 DNV and 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 proposed methods for DNV and MHC determination have many advantages over other analytical methods due to its rapidity, lower cost and environmental safety. Unlike HPLC, LC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are in expensive and available in any analytical laboratory. The proposed methods report new ways for the determination of DNV and MHC in pharmaceuticals.

 

REFERENCES:

1.      Ghosh AK, Dawson ZL, Mitsuya H "Darunavir, “A conceptually new HIV-1protease inhibitor for the treatment of drug-resistant HIV". Bioorg. Med. Chem; 2007, 15(24), 7576–80.

2.      S. Meyer, H. Azojin, D. Surleraux, TMC 114, a novel human immunodeficiency virus type 1 protease inhibitor-resistance viruses, including a board range of clinical isolates. Antimicrob Agents Chemother, 49 (2005), 2314-2321.

3.      N. King, M. Jeyabaran, E. Naliyaika, P. Wifgerinck, M. Bethune, C. Schiffer, Sturctural and Thermodynamics Basis for the Binding of TMC 114, a Next-Generation Human Immunodeficiency Virus Type 1 Protease Inhibitor. J. Virol, 78(2004), 12012-12021.

4.      Clotet, N. Bellos, J.M. Molina, D. Cooper, J.C. Goffard, A. Lazzarin, A. Wohtmann, C. Katlama, T. Wilkin. Efficacy and safety of darunavir-ritonavir at week 48 in treatment-experienced patients with HIV-1 infection in POWER 1 and 2: a pooled subgroup analysis of data from two randomised trials Lancet, 2007, 369, 1169-1178.

5.      "Instrumental Methods of Analysis" by Galen W. Ewing, Mc Graw Hill International Edition, 48, 378.

6.      Amit Patel, Ami Patel and Ashlesha Makwana. An ESI-LC-MS/MS method for simultaneous Estimation of Darunavir and Ritonavir in human plasma. International Journal of Research in Pharmaceutical and Biomedical science. 2013, 4(4)1138-47.

7.      Nageshwar Rao, Ram chandra, Santosh kumar. RP-HPLC speration and characterization of unknown impurties of a novel HIV-1 protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy. J Pharm Biomed Anal 2013; 75: 186-191.

8.      Avolio, M. Siccardi, M. Sciandra, L. Baietto. HPLC-MS method for the simultaneous quantification of the new HIV protease inhibitor darunavir and other anti-retroviral agents in plasma of HIV-infected patients. J Chromatogr B. 859 (2007) 234-240.

9.      Hari Babu k, Sisla Ramakrishna, Kiran kumar, Ramesh, Sita Devi. HPTLC method for determination of Darunavir in rat plasma and its application to pharmaceutics studies. J liq Chromatogra Releated Technol 2013; 36:169-179.

10.   Ana Carolina Kongana, Herida Regina. Development and validation of infra-red spectroscopy method for determination of Darunavir in tablet. Phy Chem 2013; 3, 1-6.

11.   Gholve Sachin B, Asware Baburao S., Kadam Shrihari C., Bhusnure Omprakash G. and Thonte Sanjay S, Development and validation of a simple UV spectrophotometric method for the determination of Darunavir ethanolate both in bulk and marketed dosage formulations. World Journal of pharmaceutical research. 2015, 4(3), 1276-83.

12.   S. Leonard, A. Schepdael, T. Lvanyi, I. Lazar, J. Rosier, M. Vanstockem. Development of a capillary electrophoretic method for the separation of diasteroisomers of a new human immunodeficiency virus protease inhibitor. Electrophoresis. 2005, 26; 627

13.   Ulu, Sevgi Tatar. Determination of Methoxaminein human plasma and urine by a validated HPLC-Fluorescence detection method. J of AOAC Int. 2013, 5(96), 987-990

14.   Al-mesdhal A, El-domiaty M.M, Al-obaid. HPLC analysis of methoxamine in rabbit plasma and pharmaceutical formulations. J of liq. Chrom. 1989, 12(9), 1589-1600.

15.   Abdulrahman A. Alwarthan, Abdulrahman M. Al-Obaid. Spectrophotometric determination of methoxamineusing cerium (IV) in presence of sodium lauryl sulphate and rhodamine-B. J of Pharm & Biom. Anal. 1997, 15(7), 911-916.

16.   Ekram M. Hassan, Heba Abdine, Abdul-Rahman M.A. Ai-Obaid. Spectrophotometric and flurometric methods for the determination of methoxamine Hcl in bulk drugs and in Ampoules. Spectroscopy letters. 2000, 33(1), 9-21.

17.   Han Liu, Fang Feng, Ming Ma, Shuangiin Cui, Darong Xie, Sizhi Xu. Pharmacokinetic study of three cardiovascular drugs by high performance liquid chromatography using pre-column derivatization with 9, 10-anthraquinone-2-sulfonyl chloride. J Chromatogr B. 2007, 858(1), 42-48.

18.   Tomos G.V., Belmore.K.A., Cava. M.P., J.Am. Chem.Soc, 1993, 115, 12.

19.   Trigoso.C.I., Ibebez.N., Stockert, J.C.J. Histochem. Cytochem, 1993, 41, 1157.

20.   Datta.S., Datta.S.C., Spray reagents for the detection of p-phenylazophenylthiohydantoins of amino acids on silica gel plates, J.Chromatogr.1979, 170, 228.

21.   Sybulski.S., Maughan.G.B., Cortisol levels in umbilical cord plasma in relation to labor and delivery. Am J Obstet Gynecol 1976;236–238, Am.J.best.gynecol.1975, 121, 32.

22.   Kachel.C.D., Mendelsohn.F.A., J. Seroidbiochem, 1979, 5, 563.

23.   Pesez.M., Petit, A., Bull. Soc. Chem. Fr., 1947, 122.

24.   Fouad N. Boctor, An improved method for colorimetric determination of proline with isatin, Analytical Biochemistry, Volume 43, Issue 1, 1971, 66-70

25.   Zeida.Alothman, Masoomraza Siddiqui, Saikhmohammadwabaidur, Hamada. Al-lohedan, Mohd. Sajidaliand M.Z.A. Rafiquee, Kinetic Spectrophotometric Methods for the Determination of IsaTin, Asian Journal of Chemistry; 2013, 25(8), 4563-4568

26.   Ken-ichi Mawatari, Makiko Segawa, Rumiko Masatsuka, Yasuko Hanawa, Fumio Iinuma, Mitsuo Watanabe, Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photo irradiation, Analyst, 2001, 126, 33-36

27.   Fouad N. Boctor, An improved method for colorimetric determination of proline with isatin,  Analytical Biochemistry, 1971, 43(1), 66-70

28.   Markus Rienth, Charles Romieu, Rebecca Gregan, Caroline Walsh, Laurent Torregrosa, Mary T. Kelly, Validation and Application of an Improved Method for the Rapid Determination of Proline in Grape Berries, J. Agric. Food Chem, 2014, 62(15), 3384–3389

29.   Nur Nadia Dzulkifli, Yang Farina, Bohari M Yamin, Malaysian Journal of Analytical Sciences, Synthesis, structural, and chemical properties of Nd(III) Isatin 2-Methyl-3-Thiosemicarbazone,  2015, 19(5), 935 -941

30.   Muhammad Aqeel Ashraf, Abdul Wajid, Karamat Mahmood, Mohd.Jamil Maah and Ismail Yusoff, Spectral Investigation of the Activities of Amino Substituted Bases, Oriental Journal of Chemistry, 2011, 2(2), 363-372

31.    Yogesh Vaishnav, Aloksingh Thakur, Chanchal Deep Kaur, Shekhar Verma, Achal Mishra, Sanmati Kumar Jain, Piyush Ghode. QSAR, Analysis of some N, N-diphenyl urea derivatives as CCR5 Receptor Antagonist. Research J. Pharm. and Tech 2018; 11(9): 3802-3810

32.   G. Karthiga Devi, Ar. Alamu. Production of Biopolymer Levan by Bacillus subtilis using Non-Ionic Surfactants. Asian J. Pharm. Tech2013; 3(4), 149-154.

33.   S. Khatrivel, R. Raju, B. Seethadevi, A. Suneetha, J. Pavani. Stability Indicating RP-HPLC Method for the Determination of Process Related Impurities in Asian Journal of Pharmacy and Technology; 2014, 4(4), 167-178

34.   Amol S. Sherikar. Quantitative Structure Activity Relationship of Some Substituted 2-Aminopyridines and Fused Bicyclic Rings as inhibitors of Nitric Oxide Synthases. Asian J. Research Chem.2011, 4(10), 1625-1629.

35.   Rakesh Jasrai, Karan Singh Bhau, Nayana Bhrambhatt. Corrosion Inhibition of Zinc in Hydrochloric Acid by Vanillin, Phenyl Thiourea and N-Allyl Thiourea. Asian J. Research Chem. 2014, 7(12), 1062-1064.

36.   Jonardan Koner, Avinash Purandare. Chennai Port: An ISO 14001: 2004 Certified Port. Int. J. Tech.2015, 5(2), 263-268

37.    Praful Daharwal, Prashant Awchat. Quality Improvement in Submerged Arc Welding Process Using Taguchi Method and Regression    Analysis. Int. J. Tech. 2018; 8(1): 41-46 

38.   Manu K S, Harshita Agarwal. Dynamic Relationship between Crude Oil, Gold and Exchange Rates: The Case of India. Asian Journal of Management. 2018; 9(2):933-938.

39.   Namdeo R. Jadhav, Anant R. Paradkar, Gourav N. Shah. Adsorption Studies of Bromhexine Hydrochloride on Talc. Research J. Pharm. and Tech. 6(11): November 2013; Page 1247-1250.

40.   Naveen Kumar R, Manikandan K, Kathiravan M K. Spectroscopic Estimation of Tramadol using Multilinear Regression Analysis. Research J. Pharm. and Tech 2020; 13(5): 2282-2286.

 

 

 

Received on 04.07.2020                    Modified on 01.08.2020

Accepted on 29.08.2020                   ©AJRC All right reserved