1. INTRODUCTION:

Artesunate (AS) is a medication used to treat malaria. The intravenous form is preferred to quinidine for severe malaria. Often it is used as part of combination therapy. It is not used for the prevention of malaria .Artesunate can be given by injection into a vein, injection into a muscle, or taken by mouth. Artesunate is generally well tolerated. Side effects may include a slow heartbeat, allergic reaction, dizziness, and low white blood cell. [1]

It is in the artemisinin class of medication. Artesunate is on the WHO's List of Essential Medicines, the most important medications needed in a basic health system. Artesunate (active metabolite dihydro artemisinin) and artesunate based combination therapy (ACT) is recommened by WHO for multi drug resistant malaria.[2]

The mechanisms of action of artesunate remains unclear and debatable. Artesunate is a prodrug that is rapidly converted to its active form dihydroartemisinin (DHA). This process involves hydrolysis of the 4-carbon ester group via plasma esterase enzyme. It is hypothesized that the cleavage of endoperoxide bridge in the pharmacophore of DHA generates reactive oxygen species (ROS), which increases oxidative stress and causes malarial protein damage via alkylation.[3] In addition, Artesunate potently inhibits the essential Plasmodium falciparum exported protein 1, a membrane glutathione S-transferase. As a result, the amount of glutathione in the parasite is reduced. In 2016, artemisinin has been shown to bind to a large number targets, suggesting that it acts in a promiscuous manner. There is evidence suggesting DHA inhibition of calcium-dependent ATPase on endoplasmic membrane, which disrupts protein folding of parasite.[4]

Artesunate chemical structure:

Chemical Name: (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-3,6,9-trimetyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-ol, hydrogen succinate .

2: MATERIALS AND METHOD:

2.1. Drug and reagents:

The drug was obtained as a gift sample from Mylan Laboratories Limited (R and D Centre), Bollaram, Hydrabad,Telangana,India. All chemicals and reagents were of analytical reagent grade. Sodium hydroxide was procured from Ranbaxy Laboratories (S.A.S. Nagar, India). Hydrochloric acid was procured from AVRA Synthesis Pvt. Ltd. (Hyderabad, India) . HPLC grade water procured from Martinsynge Pharma sciences PVt. Ltd, Aleap Industrial Estate, Hydrabad. HPLC grade Acetonitrile, potassium Dihydrogen phosphate and ortho phosphoric were Procured from Finar Limited, Gujarat. HPLC grade Methonol was Procured from Standard Reagents Pvt. Ltd, Hydrabad.

2.2. Apparatus and equipment:

Precision water bath equipped with MV controller (Julabo,Seelbach, Germany) was used for solution degradation studies. Adry-bath (Thermolyne, IA, USA) was used for solid state thermal stress studies. pH/Ion analyzer (MA 235, Mettler Toledo,Schwerzenbach, Switzerland) was used to check and adjust the pH of buffer solutions. Other smaller equipments used were sonicator(3210, Branson Ultrasoincs Corporation, Danbury, CT, USA), precision analytical balance (AG 135, Mettler Toledo, Schwerzenbach,Switzerland).

HPLC:

The separation of degradation products was achieved using a Prominence series liquid chromatograph (LC) equipped with a photodiode array detector (Shimadzu, Kyoto, Japan) and a Pursuit column C18 Hypersyl BDS

( 250×4.6) mm,5µ, Varian, North America). Aligent 1260 model HPLC system and software used was open labs.

Preparative HPLC:

Aligent 1200 infinity series

2.3 Preparation of reagents.:

Preparation of buffer:

Potassium dihydrogen phosphate buffer was prepared by taking 0.272gms in 200ml water and pH was adjusted to 3 with ortho phosphoric acid.

Preparation of 0.5 N HCl:

4.12 ml of Conc.HCl was taken and volume was made up to 100 ml with distill water.

Prepartion of 0.5 N NaOH:

2gms of NaOH was taken and volume was made up to 100ml with distilled water.

2.4 Preliminary analytical chromatographic method:

Method was obtained from USP monograph for related compound analysis for the initial identification of degradant impurities which are formed due to forced degradation. Many trials using various mobile phase composition were tried and best composition for identification of degradants are mentioned below.[5]

Chromatographic parameters:

Mobile phase A: 1.36gm KH2PO4 in 1 L water

Mobile phase B: Acetonitrile

Method composition: 58: 42 (A: B)

Column: C₁₈ Hypersyl BDS ( 250* 4.6) mm,5µ

Column temperature: 30^oC

Flow: 1ml/ min

ƛmax: 216 nm

Sample preparation : 4 mg/ml

Injection volume : 20µl

Diluent : Acetonitrile

2.5 Stress studies:

Stress studies were carried out under hydrolytic (acid and alkali), Aqueous, and thermal conditions as per ICH Q1A (R2) recommendations . The International Conference on Harmonization (ICH) guideline entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance.[6] The aim of this work was to perform the stress degradation studies on artesunate. Acidic and alkaline hydrolysis was carried out in different molarity HCl and NaOH solutions, respectively, and the temperature of hydrolytic studies were 50 ^◦C. Aqueous degradation was carried out by keeping the dug on a water bath 50˚C for about 30 minute . Thermal stress testing was carried out by keeping about 200 mg API in the oven for about 4 hours at 100 ˚C .

Procedure for acid degradation:

100 mg Artesunate drug was taken in a 25 ml volumetric flask to which 5 ml of acetonitrile was added to dissolve drug and then 4 ml of 0.5 N HCl and it was kept on water bath 50˚C for about 30 minute , cooled to room temperature and then diluted to 25 ml with acetonitrile and the solution was thoroughly mixed .

Procedure for Alkali degradation:

100 mg Artesunate drug was taken in 25 ml volumetric flask to which 5 ml of acetonitrile was added to dissolve the drug and then 4 ml of 0.5 N NaOH and it was kept on water bath 50˚C for about 30 minute , cooled to room temperature and then diluted to 25 ml with acetonitrile and the solution was thoroughly mixed

Procedure for Aqueous Degradation:

100 mg Artesunate drug was taken in a 25 ml volumetric flask to which 5 ml of acetonitrile was added to dissolve drug and then 4 ml of water and it was kept on water bath 50˚C for about 30 minute, cooled to room temperature and then diluted to 25 ml with acetonitrile and the solution was thoroughly mixed .

Procedure for Thermal Degradation:

About 200 mg API was placed in the oven for about 4 hours at 100 ˚C. Then 100 mg of thermally degraded compounded was taken into a 25 ml volumetric flask to which 5 ml of acetonitrile was added to dissolve and then diluted to 25 ml with acetonitrile and the solution was thoroughly mixed .

Table 1 : Artesunate in acid degradation

RT	%	RRT
2.8	8	0 .24
6.16	53	0.54
7.22	3.9	0.63
7.71	20	0.68
10.9	1.05	0.96
18.53	1.1	1.63
26.06	5.2	2.3

RT - Retention time , RRT -Relative retention time

Table 2 : Artesunate in alkali degradation

RT	%	RRT
2.8	13	0.24
3.3	7.2	0.29
3.9	5.6	0.34
4.5	2.3	0.39
5.4	0.61	0.48
6.1	10.49	0.54
7.1	0.06	0.63
10.9	1.2	0.96

Table 3 : Artesunate in Aqueous degradation

RT	%	RRT
7.8	2.1	0.68

Table 4: Artesunate in Thermal degradation

RT	%	RRT
6.1	0.24	0.53
9.1	0.25	0.80
26.07	1.7	0.45

Table 5 :As per USP monograph impurity profile of Artesunate is given in the following [2]

Name	Relative Retention time	Acceptance criteria (NMT %)
α- dihydro artemisinin, β- dihydro artemisinin.	0.6 0.9	0.5
Artesunate	1.0	-
Anhydro dihydro artemisinin	2.7	0.5
Individual unspecified impurity	-	0.5

3. RESULTS AND DISCUSSION:

The drug Artesunate was found to show significant degradation under acidic (0.1 N HCL ) hydrolytic conditions. While the drug Artesunate remained unaffected to other degradation conditions such as basic, aqueous, and thermal stress. Following are the unknown degradants selected for further study and their Retention time, Relative retention time and % impurity is given in the table below.

Table 6:

Degradation condition	Retention time	% impurity	Relative retention time
Acid degradation	7.22	3.9%	0.63
Acid degradation	6.16	53%	0.54

From the above table and chromatogram of Artesunate in acid degradation shows 6.16 min RT with 0.54 RRT, of which the degradant showing 53% impurity was isolated. Another peak was seen at 7.22 RT with RRT of 0.63. It showed 3.9 % impurity in acid degradation. Since the percentage is very less and within the permissible limits it was not isolated. The peak RT at 6.16 min and RRT 0.54 is found to be major degradant under acid degradation condition it was isolated for further structural identification of characteristics.

Impurity chromatogram after distillation.

ISOLATED IMPURITY PEAK AFTER DISTILATION:

Preparative chromatography:

Preparative HPLC is used to separate and refine high purity target compounds from a mixed solution after the trials or synthesis. Preparative HPLC associated large columns and high flow rates. The objectives of analytical HPLC is qualitative and quantitative determination of compound. But preparative hplc is the isolation and purification of the product. The goal of preparative HPLC is to produce a quantity of pure compound or to purify a material for manufacturing or research purpose.[7] Principle of preparative hplc is adsorption.The only difference is sample goes from detector into fraction collector .The main purpose of preparative HPLC is not quantitation but purification of sample. Therefore, good peak shape is not necessarily. Columns loads were increased for preparatory purposes by increasing the dimensions of the column. [8] In preparative HPLC column inner diameter more. Buffer is changed. Method also changed. Flow rate is increased. In preparative HPLC RT collected at 8.6min due to this reasons RT is changed from 6.1min to 8.6min.

Purification and Isolation of acid impurity by preparative chromatography:

Sample preparation:

100 mg of acid degraded sample is taken in a 25 ml volumetric flask and made up the volume with diluent acetonitrile and kept in a water bath for 30 min about 50- 60 ^o C and injected 5ml / inj . Required fraction collected at RT 8.6 . Organic part is (acetonitrile and tri fluro accetic acid ) removed by rotary evaporator. Aqueous layer is extracted with ethyl acetate and it is distilled. Compound left in round bottom flak 30 mg showed 75% purity. From this 2 mg is submitted for LCMS. Aqueous layer is checked. It has no required impurity.

Fraction collection :

Collected all peaks at RT 2.5, 3.6, 7.6, 8.6,(required) 10.0, 13.8, 16.0 min respectively. Required fraction distilled under rotary evaporator for removing the organic part and the aqueous layer extracted with ethyl acetate. Separated organic and aqueous layer . Required impurity was 30 mg and the HPLC purity of the isolated impuritywas found to be above 73%. This isolated solid impurity was used for spectral characterization without any further purification.

Mobile phase A : 0.1% Trifluro acetic acid (TFA) in water . (1L water and 1ml tri fluro accetic acid)

Mobile phase B : Acetonitrile

Method : Gradient method (0/30, 15/90, 20/90, 21/30, 25/30 )

Column : Symmetry C ₁₈( 300×19 mm×7µ)

Column temperature : 30^oC

Flow : 18 ml/ min

ƛ : 210 nm

Injection volume : 5000µl (5ml)

Concentration : 20mg/inj

Diluent : acetonitrile

Run time: 25 min

Preparative chromatogram of isolated acid hydrolytic impurity:

4. CONCLUSION:

Anti-malarial drug Artesunate was subjected to stress conditions of hydrolysis (acid and alkali), thermal and aqueous degradations. The degradation products were observed for Artesunate only under hydrolytic acid degradation, while it was found stable to remaining other stress conditions. Artesunate in acid degradation shows 6.16 min RT with 0.54 RRT, of which the degradant showing 53% impurity was isolated. Another peak was seen at 7.22 min RT with RRT of 0.63. It showed 3.9 % impurity in acid degradation. Since the percentage is very less and within the permissible limits it was not isolated. The peak RT at 6.16min and RRT 0.54 was found to be major degradant under acid degradation condition and it was isolated for further structural identification of characteristics.

ACKNOWLEDGEMENT:

The authors acknowledge the support and necessary facilities provided by Mylan Laboratories Limited (R and D Centre), Bollaram, Hydrabad, Telangana, India, NGSMIPS, Mangalore, Nitte (deemed to be university), Mangalore, Vidgas Labs,Hydrabad to carry out this work.

REFERENCES:

1. www.drug bank.ca.Available from http://www.drug bank.ca/drugs/DB00709

2. www.wikipedia.org.availble from https://en.wikipedia.org/wiki/ Artesunate

3. www.drug bank.ca.Available from http://www.drug bank.ca/drugs/DB00709

4. http://pubchem.ncbi.nlm.nih.gov/compound/3555370

5. www.usp.org available from http://www.usp.org/sites/default/files/usp-pdf/ EN/USPNF/ pending standards/m3 3429 authorized .pdf

6. ICH Harmonized Tripartite Guideline, Impurities in New Drug Products:Q3 B(R2) 2006.

7. Separation Science and Technology, volume22,1987-Issue 8-10: preparative Scale chromatography.

8. M.U. Phadke, V. K. Jadhav , D. Patil , S. Narayankar, R. K. Jadhav, Y. K. Bansal Development and validation of stability indicating UPLC method for determination of related impurities in Artesunate and Amodiaquine fixed dose tablets Der Pharmacia Lettre, 2010; 2(6):33

9. 9.Nitin g. rathod and manohar v. lokhande identification of impurities related to amodiaquine hydrochloride by using some analytical techniques www.wjpr.net Vol 3, Issue 6, 2014.

10. 10.Santosh Gandhi, Padmanabh Deshpande, Pankaj Jagdale, Varun Godbole A simple and sensitive RP-HPLC method for simultaneous estimation of Artesunate and Amodiaquine in combined tablet dosage form J. Chem. Pharm. Res., 2010; 2(6):429-434.

11. 11.Nelson A. ochekpe, Ayodeji A. Agbowuro and Ezekiel o. Afolabi Characterization of some impurities in two brands of Artesunate tablets International Journal of Drug Development and Research April-June 2010 ; Vol. 2 Issue 2 ISSN 0975-9344.

12. K. Neelima, Y. Rajendra Prasad Simultaneous estimation of Pyronaridone Tetraphosphate and Artesunate in Bulk and Combined Tablet Dosage form by RP-hplc international journal of chemical and analytical science 2014;5(2):93-98.

13. P. Jyothi, K. Geetha, A. Ajitha, V. Uma Maheshwara Rao, Nadendla Ramarao Stability Indicating Method Development and Validation for Simultaneous Estimation of Mefloquine and Artesunate in Tablet Dosage Form Sch. Acad. J. Pharm., 2014; 3(5): 411-417.

14. Panchal Archana H., Prajapati Laxman M., Joshi Amit K., Kharodiya Mahammadali L. stability indicating RP-HPLC method for simultaneous estimation of artesunate and amodiaquine hcl in their combined dosage form International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014; 2319-8141.

15. Odedara M.H Faldu S.D, Dadhania K.P, RP-HPLC Method for Simultaneous Estimation of Artesunate and Amodiaquine HCL in their Combined Pharmaceutical Dosage Form JPSBR: Volume 2, Issue 3: May-Jun 2012; 114-117.

Received on 16.08.2018 Modified on 28.09.2018

Research J. Pharm. and Tech 2018; 11(12): 5587-5592.

DOI: 10.5958/0974-360X.2018.01016.8