1. INTRODUCTION:

Methotrexate (Fig 1) and Hydroxychloroquine sulfate (Fig 2) both are widely used potent antirheumatic drugs under the class of (DMARDs). They decrease the pain and swelling of these autoimmune disorders. Methotrexate increases adenosine levels and through cellular cascade produces anti-inflammatory activity. While hydroxychloroquine shows an antirheumatic effect by inhibiting the lysosomal function. Both drugs showed a synergistic effect as per the clinical studies and effects were equivalent to gold compound auranofin in clinical studies. Moreover, they are found to be effective in reducing joint damage and prevent long –term disabilities in patients.

The literature review revealed that only a few RP-HPLC methods were available for the simultaneous estimation of methotrexate and hydroxychloroquine sulfate in combination. Hence, an attempt has been made to developed a simple, rapid, and economic RP-HPLC method for estimation of methotrexate and hydroxychloroquine sulfate in bulk drug.^1-4

Figure 1: Methotrexate

Figure 2: Hydroxychloroquine sulfate

2. EXPERIMENTAL WORK^5-10

2.1 Material and Method:

2.1.1 Instrument:

High-Performance Liquid Chromatography (Shimadzu- prominence LC 20 AD), manual sampler, software Win chrome and detector (UV-visible), Column C-18 (Shim-pack) 250 x 4.6mm, particle size 5µm, ultra-sonicator, vacuum filter, analytical valance.

2.1.2 Selection of wavelength:

The sensitivity of the HPLC method that utilizes UV detection depends upon the proper choice of detection wavelength. An ideal wavelength is the one that gives a good response to the drugs that are to be detected. For the selection of wavelength 50µg/ml concentration of methotrexate and 50µg/ml concentration of hydroxychloroquine sulfate was prepared in 50:40:10 (water: acetonitrile: tetrahydrofuran) pH-3 overlain spectra were obtained on UV spectrophotometer. The results show an iso-absorptive point at 318nm wavelength.

2.1.3 Selection of chromatographic condition:

Chromatographic separation was achieved at ambient temperature on a reversed-phase isocratic high-performance liquid chromatography using a mobile phase consisting of water: acetonitrile: tetrahydrofuran 50:40:10 with pH3. The mobile phase was pumped at a rate of 1.0ml/min. The wavelength was set at 318nm and run time at 10 minutes. To optimize the chromatographic conditions, the effect of chromatographic variables such as mobile phase pH and flow rate were studied. The resulting chromatograms were recorded and the chromatographic responses were measured.

2.1.4 Column:

For the chromatographic separation of drugs, the column of Thermo scientific octadecylsilane (ODS) C18 having a length of 250 ×4.6mm, with a particle size of 5µ was used.

2.2 Preparation of standard stock solution:

2.2.1 Methotrexate standard stock solution:

Standard methotrexate 100mg was weighed and transferred to a 100ml volumetric flask and dissolved in water: acetonitrile: tetrahydrofuran in the ratio of 50: 40:10. The contents were mixed and volume was made up to the mark with HPLC grade mobile phase to give a solution containing 1000µg/ml concentration. Then 10 ml of solution was pipette out and transferred to a 100 ml volumetric flask, made up its volume with solvent to get 100µg/ml concentration solutions.

2.2.2 Hydroxychloroquine sulfate standard stock solution:

3. RESULT AND DISCUSSION:

3.1 Final optimized trial:

3.3.1 Chromatographic conditions:

An isocratic mobile phase containing water: acetonitrile: tetrahydrofuran pH 3 (perchloric acid) at 50:40:10 was prepared

3.3.2 Preparation of mobile phase:

The mobile phase was prepared by mixing HPLC grade water: acetonitrile: tetrahydrofuran Ph 3 (perchloric acid) at a ratio of 50:40:10 and absorbance was measured at 318nm. Degassing is done by sonication of the mobile phase on sonicate for 15 minutes.

Figure 3: Chromatogram of methotrexate at wavelength 318nm

Figure 4: Chromatogram of hydroxychloroquine sulfate at wavelength 318nm

Figure 5: Chromatogram of both drugs at 318nm

3.4 Validation of the developed method:

3.4.1 Linearity curve for methotrexate^11-13

From the standard methotrexate stock solution, the volume of 1, 2, 3, 4, 5ml was pipetted out from 100µg/ml and transferred to different volumetric flasks of 10ml capacity. Then volume was made up to the mark having a concentration of 10, 20, 30, 40, 50µg/ml. The injection was given at a time interval of 15 minutes with a run time of 10 minutes.

The linearity was obtained in selected concentration ranges. The result is shown in figure 6 and table 1

Table: 1. Results of the linearity curve of Methotrexate at wavelength 318 nm.

Sr. No	Conc. (µg/mL)	Area (µ volt sec.)
1	10	1134136
2	20	2595242
3	30	4056275
4	40	5517481
5	50	7078587

Figure 6: Linearity curve of methotrexate at 318nm

3.4.2 Linearity curve for hydroxychloroquine sulfate:

From the standard hydroxychloroquine sulfate stock solution the volume of 1, 2, 3, 4, 5ml was pipetted out from 100µg/ml and transferred to different volumetric

flasks of 10ml capacity. Then volume was made up to the mark having a concentration of 10, 20, 30, 40, 50 µg/ml. The injection was given at a time interval of 15 minutes with a run time of 10 minutes.

The linearity was obtained in selected concentration ranges. The results are showing in figure 7 and table 2.

Table 2. Result of linearity curve of hydroxychloroquine sulfate at 318nm

Sr. No	Conc. (µg/mL)	Area (µ volt sec.)
1	10	633656
2	20	730549
3	30	847499
4	40	963864
5	50	1060221

Figure 7. Linearity curve of hydroxychloroquine sulfate at 318nm

3.4.3 Accuracy^14-18

Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy, 3 determinants of the concentration range of 20, 30, 40 µg/ml solutions were prepared to have 80%, 100%, and 120% of spiked level respectively. 3 replicates of the above concentration were prepared and responses were obtained. Percent recovery was calculated for obtained data and calculated according to ICH guidelines. (Table: 3 and 4).

3.4.4 Drug recovery:

Amount found

% Drug recovery = ------------------------------- × 100

Amount added

Mean test absobance

Amount = --------------------------------× Standard concentration

Found Mean standard avsorc bance

Weghit

Amount added = -------------------

Volume

Table 3: % Drug Recovery of methotrexate at wavelength 318nm

Sr. No.	Unfortified sample			Fortified sample			% Recovery
Sr. No.	Conc.(μg/ml)	Area	Mean	Conc.(μg/ml)	Area	Mean	% Recovery
1	20	2595242 2595243 2595244	2595243	20+30	6651419 6651419 6651420	6651419	99.99
2	30	4056275 4056276 4056277	4056276	30+30	8112350 8112353 8112350	8112351	99.99
3	40	5517481 5517482 5517483	5517482	40+30	9573655 9573657 9573655	9573655	99.99

Table 4: Drug recovery of hydroxychloroquine sulfate at wavelength 318nm

Sr. No.	Unfortified sample			Fortified sample			% Recovery
Sr. No.	Conc. (μg/ml)	Area	Mean	Conc. (μg/ml)	Area	Mean	% Recovery
1	20	730549 730549 730547	730548	20+30	1578038 1578045 1578042	1578041	100.00
2	30	847489 847468 847479	847478	30+30	1694962 1694954 1694952	1694956	100.0
3	40	963854 963855 963853	963854	40+30	1811311 1811313 1811312	1811312	99.99

Table 5: Intraday precision of methotrexate and hydroxychloroquine sulfate at 318nm

Drug	Methotrexate			Hydroxychloroquine sulfate
Conc.	10 µg/ml	20 µg/ml	30 µg/ml	30µg/ml	40µg/ml	50µg/ml
Area (µ volt sec.)	1133136	2595143	4056178	847490	963850	1060210
	1133234	2595229	4056225	847459	963896	1060229
	1133299	2595198	4056199	847475	963844	1060274
Mean	1133223	2595190	4056200	847474.7	963863.3	1060238
S D	82.05486	43.55456	23.544285	15.50269	28.44878	32.86842
% RSD	0.007241	0.001678	0.0005805	0.001829	0.002952	0.0031

Table 6: Interday precision of methotrexate at 318nm

Day	Day 1			Day 2			Day3
Conc.	10µg/ml	20µg/ml	30µg/ml	10µg/ml	20µg/ml	30µg/ml	10µg/ml	20µg/ml	30µg/ml
Area (µ volt sec.)	1133136	2595143	4056178	1133137	2595143	4056178	1133136	2595143	4056178
	1133234	2595229	4056225	1133268	2595227	4056227	1133234	2595227	4056226
	1133299	2595198	4056199	1133299	2595197	4056199	1133296	2595193	4056194
Mean	1133223	2595190	4056200.7	1133235	2595189	4056201	1133222	2595188	4056199
S D	82.05486	43.55456	23.544285	85.99031	42.56759	24.58319	80.67218	42.25321	24.4404
%RSD	0.007241	0.001678	0.0005805	0.007588	0.00164	0.000606	0.007119	0.001628	0.000603

Table 7: Interday precision of hydroxychloroquine sulfate at 318nm

Day	Day 1			Day 2			Day 3
Conc.	30µg/ml	40µg/ml	50µg/ml	30µg/ml	40µg/ml	50µg/ml	30µg/ml	40µg/ml	50µg/ml
Area (µ volt sec.)	847490	963850	1060210	847490	963850	1060210	847489	963859	1060210
	847459	963896	1060229	847459	963896	1060229	847452	963897	1060229
	847475	963844	1060274	847475	963844	1060274	847474	963844	1060272
Mean	847474.7	963863.3	1060238	847474.7	963863.3	1060238	847471.7	963866.7	1060237
S D	15.50269	28.44878	32.86842	15.50269	28.44878	32.86842	18.61003	27.3191	31.76476
%RSD	0.001829	0.002952	0.0031	0.001829	0.002952	0.0031	0.002196	0.002834	0.002996

3.4.5 Precision^19-22

The precision of an analytical method is the degree closeness of agreement between a series of measurements obtained from the multiple sampling of the same sample. Precision includes repeatability, inter and intraday precision, and reproducibility.

3.4.5.1 Interday and intraday precision:

Interday and intraday precision of concentration 10, 20, 30µg/ml was prepared and data were obtained for methotrexate.

Interday and intraday precision of concentration 30, 40, 50µg/ml was prepared and data were obtained for hydroxychloroquine sulfate. 3 replicates were prepared for 3 days.

The results of methotrexate and hydroxychloroquine sulfate were shown in table 5 to 7.

3.4.6 Repeatability^23-27

For repeatability minimum of 6 determinants were prepared of 30µg/ml concentration and the chromatogram responses were obtained by injecting one by one. The results of methotrexate and hydroxychloroquine sulfate were shown in table 8 and 9.

The standard deviation and relative standard deviation was calculated for each type of precision.

Table 8: Repeatability of methotrexate at 318nm

Sr. No	Area (µ volt sec.)
1	4056375
2	4056370
3	4056378
4	4056380
5	4056370
6	4056378
Mean	4056375
S D	4.308906
%RSD	0.000106

Table 9: Repeatability of hydroxychloroquine sulfate at 318nm

Sr. No	Area (µ volt sec.)
1	847490
2	847498
3	847495
4	847499
5	847489
6	847491
Mean	847493.7
S D	4.273952
%RSD	0.000504

3.4.7 Robustness:

Robustness is a measure of its capacity to remain unaffected by small, but deliberate variations in the method parameter. For HPLC robustness was carried out by changing the mobile phase and flow rate. The results were shown in table: 10 for change in the mobile phase and table: 11 for change in flow rate.

Table: 10 Robustness of Methotrexate and Hydroxychloroquine sulfate at wavelength 318±2nm.

Wavelength	Difference	R_t of Methotrexate (min.)	R_t of Hydroxychloroquine sulfate (min.)
316	-2	2.995	3.775
318	0	3.007	3.755
320	+2	3.017	3.780

Change in the flow rate of the mobile phase

Table: 11 Robustness of Methotrexate and Hydroxychloroquine sulfate at wavelength 318 nm.

Flow rate (mL/min.)	Difference	R_t of Methotrexate (min.)	R_t of Hydroxychloroquine sulfate (min.)
0.9	-0.1	3.010	3.910
1	0	3.028	3.923
1.1	+0.1	3.035	3.930

Overall, validation parameters were shown in Table 12 and complied with ICH norms and guidelines.²¹

Table 12: Summary of validation parameter of RP-HPLC at 318nm wavelength

Parameter	Methotrexate	Hydroxychloroquine sulfate
Linearity range (µg/ml)	10-50	10- 50
Regression coefficient (R²)	0.999	0.998
% Recovery	99.99	99.99
Repeatability (n=6)	% RSD NMT 2	% RSD NMT 2
Precision Intraday precision Interday precision	% RSD NMT 2	% RSD NMT 2

4. CONCLUSION:

The estimation of methotrexate and hydroxychloroquine sulfate was done by RP-HPLC. The optimized method included mobile phase composition of water: acetonitrile: tetrahydrofuran the ratio of 50:40:10 % v/ v at pH3 by using perchloric acid, and absorbance was measured at 318nm. Retention time was found to be 3.0 and 3.7 minutes for methotrexate and hydroxychloroquine sulfate respectively. The percentage recovery was found in the range from 99.99-100% of methotrexate and hydroxychloroquine sulfate. All the parameters were validated according to the ICH and USP guidelines and meet all the limits (Table: 12). It can be also inferred that the method was found to be simple, accurate, precise, and linear. It can be utilized for routine laboratory analysis for simultaneous estimation of methotrexate and hydroxychloroquine in bulk drug as well as formulation.

5. REFERENCES:

1. Braun J. Methotrexate: Optimizing the Efficacy in Rheumatoid Arthritis. Ther Adv Musculoskelet Dis; 2011. 151-158.

2. HJ Williams, JR Ward, JC Reading, Comparison of auranofin, methotrexate, and the combination of both in the treatment of rheumatoid arthritis. A controlled clinical trial; a journal of the national library of medicine; 1992; 35(3): 259-69.

3. ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedure: Text and Methodology Q2 (R1), Current Step 4 Version, Parent Guidelines on Methodology; 2005. 1-13

4. Sethi PD, Sethi R. Sethi's, HPLC: High-Performance Liquid Chromatography; Quantitative Analysis of Pharmaceutical Formulations. CBS Publ; 2010.

5. Sartori T, Murakami FS, Development and Validation of a Fast RP-HPLC Method for Determination of Methotrexate Entrapment Efficiency in Polymeric Nanocapsules; Journal of Chromatographic Science, 2008; 46(6).505-509.

6. Mobil A, S Rezaee. High-Performance Liquid Chromatography Determination of Methotrexate in Plasma; Iranian Journal of Pharmaceutical Research, 2003; 2(3). 149-152.

7. Suguna P, Sathyanarayana B, Validation of HPLC Method for the Analysis of Methotrexate in Bulk Drug and Pharmaceutical Dosage Forms. Journal of Chemical and Pharmaceutical Research, 2015; 7(9). 27-35.

8. Montemurro M, Maria MD. Optimized High-Performance Liquid Chromatography–Ultraviolet Detection Method Using Core-Shell Particles for the Therapeutic Monitoring of Methotrexate; Journal of Pharmaceutical Analysis, 2016; 6(2). 103-111.

9. Roy M, Mukesh M. Development and Validation of RP- PLC Method for the Determination of Methotrexate in Bulk and Pharmaceutical Tablet Dosage, European Journal of Pharmaceutical and Medical Research, 2016; 3(5). 355-358

10. Agnihotri J, Singh S. Simultaneous Estimation of Antimalarial Drugs Artesunate and Hydroxychloroquine by Validated HPLC Method in Combined Pharmaceutical Formulation. Indo American Journal of Pharmaceutical Research, 2013; (6). 177-122.

11. Karuppiah SP, Basha AK. Analytical Method Development for Impurities Present in Hydroxychloroquine Sulphate from Antimalarial Tablet Formulation. World Journal of Pharmacy and Pharmaceutical Sciences, 2014; 3(7). 586–591.

12. Singh A, Singh CL, Development, and Validation of Reversed-Phase High-Performance Liquid Chromatography Method for Hydroxychloroquine Sulfate. Indian J Pharm Sci, 2015; 77(5). 586-590.

13. McCrudden EA, Tett SE. Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly (styrene-divinylbenzene) column. J Chromatog B, 1999; (721). 87–92.

14. S.JanetBeula,Dr.R.Suthakaran,PremKumarBichala, CH. Shankar, Syed Ghouse, K. Suneetha. A Prospective Validation of Hydrochlorothiazide and Bisoprolol Fumerate in its Pure and Bulk Dosages Forms by using RP-HPLC Technique .Asian J.Pharma.Ana.2020;10(1): 01-06.

15. B.Bhavya, P.Nagaraju, V.Mounika, G. Indira Priyadarshini. Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Albendazole and lvermectin in Pharmaceutical Dosage form. Asian J. Pharm. Ana.2017; 7(1) 6-14.

16. Sushil D. Patil, Sunil V. Amurutkar, C. D. Upasani. Development and Validation of Stability Indicating RP-HPLC Method for Empagliflozin. Asian J. Pharm. Ana. 2016;6(4): 201-206

17. Kirthi A,Shanmugam R,Mohana LakshmiS,Ashok Kumar CK,Padmini K,Shanti Prathyusha M,Shilpa V.Analytical Method Development of a Stability-indicating RP-HPLC Method for the Analysis of Danazol in Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2016;6(4): 227-234.

18. Kumaraswamy.Gandla, D.Sudheer Kumar, Joru Praveen, Emmadi Suman. RP-HPLC Method Development and Validation for Simultaneous Estimation of Lignocaine Hydrochloride and Clotrimazole Hydrochloride in Ear Drops. Asian J. Pharm. Ana. 2017; 7(3): 163-168

19. A New Analytical method Validation and Quantification of Entacapone and its Related Substance in bulk G. Kumar, T. B. Patrudu, Tentu Nageswara Rao, M. V. Basaveswara Rao. Drug Product by HPLC. Asian J. Pharm. Ana. 2017; 7(1)1-5.

20. Jayshree Pawar, Sandeep Sonawane, Santosh Chhajied, Sanjay Kshirsagar. Development and Validation of RP-HPLC method for simultaneous Estimation of Metformin HCl and Gliclazide.Asian J.Pharm.Ana.2016;6(3): 151-154.

21. Prashanthi. Y, Tentu Nageswara Rao, Yellapu Srinivas. Method Development and Validation of Alectinib Drug by RP-HPLC in Bulk and Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2018;8(4):186-190.

22. B.Thangabalan, M.Salomi, N.Sunitha, S. Manohar Babu. Development of validated RP-HPLC method for the estimation of Itraconazole in pure and pharmaceutical dosage form .Asian J.Pharm.Ana.2013; 3(4): 119-123.

23. B.Premkuma.Antioxident Defense and Disease activity in Rheumatoid Arthritis.Research J.Pharm.and tech 2018; 11(5): 1810-1814.

24. Kumaraswamy. Gandla, D. Sudheer Kumar, Joru Praveen, Emmadi Suman. RP-HPLC Method Development and Validation for Simultaneous Estimation of Lignocane Hydrochloride and Clotrimazole Hydrochloride in Ear Droos. Asian J. Pharm. Ana. 2017; 7(3):163-168.

25. G. Kumaraswamy, M. A. Zeeshan Hamza, R. Suthakaran. Development and validation of RP-HPLC for simultaneous estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium Tablets, Asian J. Pharm.Ana.2014; 4(4).151-155

26. R. Anusha Naik, Ajay Kumar D., M. Venkatesh. Method development and validation of Taurine and Acetyl Cysteine by using RP-HPLC method, Asian J. Pharm. Ana. 2018; 8(4).223-235

27. Indian Pharmacopoeia. 6thed.Indian Pharmacopeia Commission Ghaziabad; 2010. 147.

Received on 25.09.2020 Revised on 10.11.2020

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):73-78.

DOI: 10.52711/2231-5675.2021.00014