INTRODUCTION:

The analytical validation method for the determination of drugs can be performed for the substance derived from a living organism and their metabolites along with characteristics. These are objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses towards the therapeutic intervention for clinical and non-clinical studies¹. The safety with effectiveness of drugs and biological products are supported by the data obtained from the validation methods². The purpose of developing a bioanalytical procedure is to design and optimize a method with suitable operating condition, along with limitations and suitability^3,4. The determination of drug’s physicochemical properties for in-vitro and in-vivo metabolism and protein binding are also important for bioanalytical method analysis⁵.

The estimation of half-life, absorption and excretion rates of any drug can be obtained by mathematical determination of plasma level vs time curves^6,7. This endeavors the method is suitable for validating and developing the bioanalytical method by RP-HPLC for drug by human plasma^8,9.

Paracetamol is chemically N-(4-hydroxyphenyl) acetamide categorized under an analgesic and antipyretic agent¹⁰. But this drug shows a poor anti-inflammatory action. Inhibition of cyclooxygenase is the main mechanism (Fig 1a).

8-bromotheophylline or pamabrom restrain aminoisobutanol showing an active diuretic property. It also contains a xanthine derivative drug which increases the renal blood flow by stimulating cardiac and action on vasodilator¹¹. pamabrom interference in tubular reabsorption of sodium and chloride ions is the main mechanism that leads to less absorption of water and involves in excretion¹² (Fig 1b).

Fig.1. Chemical structure of (a) Paracetamol

(n-(4-hydroxyphenyl) acetamide) and (b) Pamabrom (1:1 mixture of 2-amino-2-methyl-1-proponal and 8-bromotheophylline)

Combination drugs of paracetamol and pamabrom are available for back pain and menstrual relief¹³. The reduction of back pain during menstrual relief is performed by paracetamol. Bloating associated with the cycle is relieved by pamabrom¹⁴. Chromatographic methods likes HPLC and HPTLC methods were reported for the paracetamol and pamabrom combination¹⁵.

The purpose of present study was intended for developing and validating a method of bioanalytical study for the determining the amount of the combination drugs of paracetamol and pamabrom by RP-HPLC method. This method is presumed to have accuracy, precision, linearity, stability and selective guidelines as per ICH¹⁶. The review of literature was found that there were analytical method for estimation of paracetmol and pamabrom individually or in combination but no method for bioanalytical was performed and reported in solid dose combination formulation^17-22. In biological media it is important for measuring drugs for bioavailability and bioequivalence, abuse of the drug, development of new drugs, etc. which are highly dependent on accurate drug measurement in the biological fluids^23-24. Hence in this present study, we have developed a new bioanalytical method for the simultaneous estimation of paracetamol and pamabrom oral combinational solid dosage form.

MATERIALS AND METHODS:

Chemical and reagents used:

Ammonium formate, formic acid, methanol, HPLC grade water, standard drugs of Paracetamol, Pamabrom and Furosemide were obtained from Suven Pharmaceutical, Hyderabad; Akshya Laboratories Pvt. Ltd, Hyderabad; Mentena Drugs Pvt Ltd., Hyderabad. Marketed solid dosage form was purchased from local medical store (Mankind Pharma Ltd, Delhi). Human plasma was obtained by the local blood bank, Govt Headquarters hospital, Ooty. All reagents were of analytical reagent grade.

Instruments used:

Shimaduz digital balance, Systronic pH meter, µ Ph meter system 361, waters HPLC system with 1515 solvent delivery pump system, Rheodyne 7725 injector with 20µl loop, 2487 dual wavelength UV-Vis absorbance detector, breeze data station Shimaduz gradient HPLC with LC-10 AT-VP solvent delivery pump system, SPDM-10AVP Photo Diode array detector, Class-VP data station. Shimaduz UV-1700 spectrophotometer, Ultra sonicator, Analytical columns such as Hibar® C₁₈ (250x4.6mm i.d., 5µ), Zorbex C8 (150x4.6mm i.d., 5µ), agilent zorbex C₈ (150x4.6mm i.d.,3.5µm), Jones chromatography Ltd, Silica gel (150x4.6mm i.d.,5µ)

Method development and optimization of chromatographic condition:

Selection of wavelength:

In the range of 320-400nm, the spectrum of paracetamol and pamabrom in the 25µg/ml concentration were recorded separately. To determine the isosbestic point the obtained spectra were superimposed. The wavelength of 268nm was overlaid for analytical purposes. The drug solution was also checked with the help of an HPLC PDC detector in the range of 220-350nm where the separation was observed at 268nm.

Selection of chromatographic method:

The method depends on the nature of the sample, molecular weight and solubility. The drug for the present study is polar in nature so the reverse phase chromatography method was utilized. Because of simplicity and suitability, the initial separation was performed by RP-HPLC.

Condition of chromatographic method:

A 10 µg/ml solution of paracetamol and pamabrom were prepared and chromatographed using C₁₈ column, ammonium formate (25mM): methanol with gradient elution modes at the flow rate of 1ml/min and pH of 4-6. Furosemide was used as an internal standard. The time of retention for both the drugs paracetamol and pamabrom was 10.05 min and 7.05 min which helped us to calculate capacity factor, resolution, asymmetric factor, column efficiency.

Optimized HPLC conditions:

Hibar C18 column was used as a stationary phase along with the mobile phase containing Ammonium Formate: Methanol in the ratio of 80:20. It was filtered by a membrane 0.22µm and degassing was done using an ultrasonicator. The flow rate was adjusted to 1.0ml/min with an injection volume was 20µl.

Estimation of pamabrom and paracetamol in plasma:

Preparation of standard stock solution of pamabrom, paracetamol and internal standard (1 mg/ml):

In 10ml volumetric flask, weight accurately 10mg of pamabrom, paracetamol and furosemide (internal standard) separately and dissolve it with methanol and store at below 8ºC.

Preparation of working standard solution of pamabrom, paracetamol and internal standard:

The 100µg/ml solutions of paracetamol, pamabrom and furosemide (internal standard) were prepared with the help of methanol solution. From this solution, different working standard solutions were prepared.

Preparation of pamabrom and paracetamol standard:

The serial dilution of 0.10, 0.30, 0.60, 1.02, 3.00, 3.60, 4.80, 5.40 and 6.00µg/ml of pamabrom and 0.20, 0.50, 1.00, 2.00, 4.00, 5.00, 6.00, 8.00, 9.00, and 10.00µg/ml of paracetmol were prepared with spiking technique with 2 ml of 25µg/ml of internal standard.

Preparation of plasma samples by protein precipitation technique:

Take 0.2ml of plasma, 0.21ml of drug solution each and 0.25ml of internal standard in ria vials and vortexed for 15 min. Then add 0.2ml of methanol and vortex for 15 min. Centrifuge these solutions at 3500 RMP. Collect the supernatant liquid and make up the volume till 1 ml and inject it for analysis.

RESULT AND DISCUSSION:

Optimization of chromatographic condition:

The executive of the developed method was to contemplate all the heft like run time, symmetry, sensitivity, resolution, etc. All this enhanced condition was intended to estimate the drugs by the prescribed method. The proper selection of UV wavelength was used to find the sensitivity of the method performed with HPLC. The estimation of paracetamol and pamabrom in plasma samples had undergone the required criteria. Separate spectral for UV was determined for both standard drugs individually for obtaining overlay spectra at 268 nm with isosbestic point (Fig 2).

Fig.2. UV spectrum of paracetamol and pamabrom

The chromatogram was recorded by injecting a blank plasma sample (Fig 3a), standard sample and test sample at optimized condition. This method is also used to show a well defined separation between drugs, internal standard and endogenous components.

Approximately 10 µg/ml each solution of the standard drug combination of paracetamol and pamabrom along with 25 µg/ml of internal standard furosemide was injected and shown an in character chromatogram (Fig 3c).

HPLC method for estimation of paracetamol and pamabrom:

The spiked plasma containing standard drugs and internal standards were constructed for the calibration curve. During the process of in-study validation and pre-study validation, the mobile phase used for the estimation showed a well defined separation from the drugs and internal standard. The calculated concentration of paracetamol and pamabrom was identified by the response factor and present in the (Table 1).

Table 1. Linearity and Range of paracetmol and pamabrom

Linearity
Sl. No	Conc. (µg/ml)		Internal standard conc. (µg/ml)	Response factor
Sl. No	Paracetmol	Pamabrom	Internal standard conc. (µg/ml)	Paracetmol	Pamabrom
1	0	0	0	0	0
2	0.20	0.1	25	0.006212	0.002397
3	0.50	0.3	25	0.014287	0.006748
4	1.00	0.6	25	0.028705	0.013824
5	2.00	1.2	25	0.061003	0.028368
6	4.00	2.4	25	0.123135	0.053266
7	5.00	3.00	25	0.14842	0.070206
8	6.00	3.60	25	0.187747	0.081777
9	8.00	4.80	25	0.245416	0.100988
10	9.00	5.40	25	0.257121	0.112505
11	10.00	6.00	25	0.270689	0.125911

Developed method validation:

The dictate optimized method was found to accurate by relative and absolute experimental recovery. The percentage recovery values for paracetamol in methanol were found to be 98% to 99.5% and paracetmol diluted in mobile phase shown a recovery value of 99.74% to 99.92%. Equally a recovery value ranged for pamabrom in methanol shown from 98.50% to 99.50% with a mobile phase value of 99.50% to 99.90%. Paracetamol and pamabrom had shown a relative recovery value from 98.5 to 99.5% and 99.0 to 99.8% respectively. The adopted method was accurate and reliable since the coefficient of variation of these recovery values was less than 5% (Table 2).

Table 2. Accuracy-Recovery studies of paracetamol and pamabrom

Drugs	Conc. of drug (µg/ml)	Amount of drug recovered in plasma sample (µg/ml)	Percentage recovery in plasma (µg/ml)	Amount of drug recovered in mobile phase (µg/ml)	Percentage recovery in mobile phase (µg/ml)	Relative recovery (%)
Level- I
Paracetamol	1.0	0.9673±0.0095	Mean 99.10 CV:0.96% N : 6	0.9944±0.0012	Mean:99.74 CV:0.12% N: 6	99.15
Pamabrom	0.6	0.5951 ± 0.0011	Mean : 99.68 CV :0.20% N : 6	0.5996±0.0008	Mean :99.9 CV :0.02% N : 6	99.78
Level-II
Paracetamol	5.0	4.839± 0.0378	Mean:98.66 CV:0.77% N : 6	4.977 ± 0.0056	Mean :99.82 CV:0.11% N: 6	98.83
Pamabrom	3.00	2.945 ± 0.0008	Mean :99.00 CV :0.34% N : 6	2.995± 0.1337	Mean :99.90 CV :0.03% N: 6	99.09
Level- III
Paracetamol	10.0	9.974 ± 0.0642	Mean:99.43 CV :0.65% N : 6	9.983 ± 0.0036	Mean :99.92 CV:0.04% N : 6	99.5
Pamabrom	6.00	5.823 ± 0.0008	Mean :98.61 CV :0.64% N : 6	5.955± 0.0099	Mean :99.55 CV :0.12% N: 6	99.05

The optimized methods for the estimation of the drugs were found to be precise. This was evident from the coefficient of variation values, which were less than 10 % at all concentrations (Table 3A, 3B and 3C). It also observed that there was no interference of chromatogram at the retention time of selected drugs and internal standards. There was also no additional peak in the chromatogram of the sample. This scrutiny proved the assay method developed was specific and selective. Paracetamol and pamabrom showed a retention time of 7.05 and 10.02 with no interference with the internal standard showing retention time at 11.97 respectively. Since there was no interference between the peaks obtained (Fig 3b).

Fig.3. Typical standard chromatogram of (a) Blank plasma, (b) Pamabrom, paracetamol and furosemide in plasma and (c) Pamabrom, paracetamol and furosemide (without plasma)

Table 3A. Precision studies of paracetamol

Nominal Concentration (µg/ml)
SL.NO	LQC		MQC		HQC
	1 mcg		5 mcg		10 mcg
	1	2	1	2	1	2
1	0.989	0.985	4.900	4.986	9.880	9.779
2	0.988	0.986	4.901	4.987	9.879	9.778
3	0.986	0.986	4.902	4.985	9.882	9.800
4	0.988	0.987	4.901	4.987	9.881	9.779
5	0.987	0.985	4.902	4.986	9.882	9.776
Mean	0.9876	0.9858	4.901	4.986	9.881	9.782
S.D (+/-)	0.00114	0.000837	0.000837	0.000837	0.001304	0.009915
C.V. (%)	0.12%	0.08%	0.02%	0.02%	0.01%	0.10%
% Nominal	98.76	98.58	98.02	99.72	98.81	97.82
N	5	5	5	5	5	5

Table 3B. Precision studies of pamabrom

Nominal Conc. (µg/ml)
SL.NO	LQC		MQC		HQC
	0.6 mcg		3 mcg		6 mcg
	1	2	1	2	1	2
1	0.589	0.587	2.899	2.895	5.915	5.907
2	0.591	0.589	2.901	2.897	5.917	5.908
3	0.587	0.587	2.899	2.895	5.918	5.906
4	0.589	0.589	2.901	2.896	5.917	5.907
5	0.590	0.586	2.898	2.897	5.916	5.909
Mean	0.5892	0.5876	2.900	2.896	5.917	5.907
S.D (+/-)	0.001483	0.001342	0.001342	0.001	0.00114	0.00114
C.V. (%)	0.25%	0.23	0.05%	0.03	0.02%	0.02
% Nominal	98.2	96.83	96.66	99.6	98.61	99.42
N	5	5	5	5	5	5

The specific concentration range for individual drugs was linear when calibration curves were plotted between response factor and standard concentration solutions.

The R² value of paracetamol and pamabrom was linear and found to be 0.9958 and 0.9963 respectively. The variability of the slops and intercepts was determined for the calibration curve for six different day’s samples. The stipulate results showed no significant variability in the slopes and interpretations at the optimized concentration range. A three-level drug spiked human plasma sample was studied for its stability. The stability sample concentration was compared with the concentration noted theoretically. When stored at frozen state the result showed was observed as in (Table 4A, 4B and 4C).

Table 4A. Stability of drugs in plasma during storage and sample handling (Freeze and thaw stability data)

Nominal Concentration (µg/ml)
SL.NO	LQC		MQC		HQC
	Paracetmol 1 mcg	Pamabrom 0.6 mcg	Paracetmol 5 mcg	Pamabrom 3 mcg	Paracetmol 10 mcg	Pamabrom 6 mcg
Cycle 1	0.997	0.597	4.992	2.94	9.81	5.94
Cycle 2	0.992	0.592	4.989	2.91	9.730	5.89
Cycle 3	0.989	0589	4.986	2.89	9.69	5.84
Mean	0.9927	0.5927	4.989	2.913	9.743	5.890
S.D. (+/-)	0.004041	0.004041	0.003000	0.02517	0.06110	0.05000
C.V. (%)	0.41%	0.41%	0.06%	0.06%	0.63%	0.63%
% Nominal	99.27	98.78	99.78	97.10	97.43	98.16
N	3	3	3	3	3	3

Table 4B. Stability of drugs in plasma during storage and sample handling (Short term stability data (at ambient temperature))

Nominal Concentration (µg/ml)
Short time plasma at ambient temperature	LQC		MQC		HQC
	Paracetmol 1 mcg	Pamabrom 0.6 mcg	Paracetmol 5 mcg	Pamabro 3 mcg	Paracetmo 10 mcg	Pamabrom 6 mcg
After 2 h	0.994	0.599	4.997	2.994	9.991	5.994
After 4 h	0.990	0.597	4.994	2.989	9.989	5.989
After 6 h	0.988	0.592	4.992	2.986	9.984	5.984
Mean	0.9907	0.5960	4.994	2.990	9.988	5.989
S.D. (+/-)	0.003055	0.003606	0.002517	0.004041	0.003606	0.005000
C.V. (%)	0.31%	0.60%	0.05%	0.14%	0.04%	0.08%
% Nominal	99.07	99.33	99.28	99.53	99.88	99.81
N	3	3	3	3	3	3

Table 4C. Stability of drugs in plasma during storage and sample handling (Long term stability data (at 70 ⁰C))

Nominal Concentration (µg/ml)
Long time plasma at- 70 ⁰C temperature	LQC			MQC		HQC
		Paracetmol 1 mcg	Pamabrom 0.6 mcg	Paracetmol 5 mcg	Pamabrom 3 mcg	Paracetmol 10 mcg	Pamabrom 6 mcg
After 1 week		0.969	0.569	4.976	2.74	9.970	5.64
After 2 week		0.966	0.562	4.973	2.701	9.969	5.61
Mean		0.9710	0.5655	4.905	2.720	9.305	5.625
S.D. (+/-)		0.004243	0.004950	0.007778	0.02828	0.007071	0.02121
C.V. (%)		0.44%	0.88%	0.16%	1.04%	0.08%	0.38%
% Nominal		97.10	94.25	99.52	90.66	99.70	93.75
N		3	3	3	3	3	3

The values of limit of a detection (LOD) and limit of quantification (LOQ) for paracetamol and pamabrom was found to be 0.03µg/ml and 0.015µg/ml; 0.1µg/ml and 0.05µg/ml respectively. This refection proved that the method was authentically sensitive (Table 5). The values obtained can be influenced by the change in the instrument, conditions at which the separation is performed, along with usage of non-graded solvents where signal to noise ratio can be observed as a result.

The experimental changing condition was studied to see the ruggedness and robustness of the technique. This showed there were no notable changes in the chromatographic parameters. The satisfactory optimized method of system suitability was found for the column efficiency, resolution factor and peak asymmetry (Table 5).

Table 5. System suitability studies

Sl. No	Parameters	Pamabrom	Paracetamol
1	Theoretical Plate	3920	3278
2	Tailing factor	1.0	1.12
3	Asymmetric factor	1.0	1.5
4	LOD (ng/ml)	15	30
5	LOQ (ng/ml)	50	100
6	Resolution Factor	Paracetamol and Furosemide	Pamabrom and Furosemide
6	Resolution Factor	4.02	2.25

CONCLUSION:

The present study was to develop and validate a RP-HPLC method for bioanalytical contemporaneous evaluation of paracetamol and pamabrom in human plasma. The evolved method was validated using parameters like accuracy, precision, robustness, ruggedness and stability. The data were showed prescribed limits along with accurate recoveries for LQC, MQC and HQC. The obtained values were found to system stability for the performed combination drug in biological plasma. The method was rapid, simple, accurate, precise, robust and selective, for these reasons the current method can be used for a bioequivalence study to assess its future applicability.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest in this manuscript.

ACKNOWLEDGEMENT:

The authors are thankful to JSK Nagarajan, JSS College of Pharmacy, Ooty for their support and guidance in this work.

REFERENCES:

1. USFDA. Bioanalytical Method Validation: Guidance for Industry. U.S. Department of Health and Human Services, Food and Drug Administration. 2018. Available on https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry. [Accessed on 12 October 2020]

2. Kanchana SA, Aruna A, Niraimathi V, Suresh AJ. Simultaneous Estimation of Telmisartan and Amlodipine in Tablet Dosage Form by RP-HPLC. Research Journal of Pharmacy and Technology. 2011;4(3):428-429.

3. Skoog DA. Analytial Chemistry. 7^th edition, Saunders College publishers, Philadelphia. 1996.

4. Naykode MD, Bhagwat DA, Jadhav SD, HN More. Analytical and Bioanalytical Method for Quantification of Pure Azilsartan, Not its Salts by RP-HPLC. Research Journal of Pharmacy and Technology. 2017;10(3):708-714. doi: 10.5958/0974-360X.2017.00133.0

5. Tamilselvi N, Sinha H, Visakh D, Vanathi P. Bio-analytical method development and validation for the estimation of Clotrimazole in human plasma by RP-HPLC method. Research Journal of Pharmacy and Technology. 2016;9(6):671-676. doi: 10.5958/0974-360X.2016.00126.8

6. Robinson JW. Undergraduate Instrumental Analysis. 5^th edition, Marcel Dekker, New York. 1977.

7. Lindsay S. HPLC by Open learning. John Wiley and Sons, London. 1991.

8. Paul K, Nagarajan JSK. Comparative pharmacokinetics studies in certizine tablet in healthy human volunteers. International Journal of Research in Pharmacy and Chemistry. 2011;1(4):1212-1235.

9. Lal CLV, Chandan RS, Maruthi R, Tengli AK. Bio-Analytical Method Development and Validation of Dichlorvos Pesticide by RP-UFLC Method. Research Journal of Pharmacy and Technology. 2020;13(8):3725-3728. doi: 10.5958/0974-360X.2020.00659.9

10. Antakli S, Nejem L, Shawa D. Separation and Determination of Paracetamol and Codeine Phosphate in Pharmaceutical Preparation by using High Performance Liquid Chromatography. Research Journal of Pharmacy and Technology. 2019;12(3):1327-1332. doi: 10.5958/0974-360X.2019.00222.1

11. Padaliya H, Patel H. Development and Validation of RP-HPLC method for simultaneous estimation of Paracetamol and Pamabrom in bulk and pharmaceutical dosage form. International Jourmal of University Pharmacy and Bio sciences. 2013;2(5):129-137.

12. Settle F. Handbook of Instrumental Technique for Analytical Chemistry. HPLC-MS, Chhabil Das. 1977.

13. Snyder LR, Joseph J, Kirkland, Glajch JL. Practical HPLC method development, 2^nd edition. 1997; 1-14. doi: 10.1002/9781118592014

14. Harde MT, RK. Chinchole PD, Chaudhari. Development and Validation of HPTLC method for simultaneous estimation of Paracetamol and Pamabrom in bulk and synthetic mixture. International Journal of Pharmaceutical and Chemical sciences. 2013;3(2):1598-1604.

15. Premal PP, Kumari CAD, Dankit PS. Development and Validation of HPTLC method for simultaneous determination of pamabrom and paracetamol in synthetic mixture. International Research Journal of Pharmacy. 2012;3(11):167-171.

16. Paul K, Gowda BHJ, Shankar SJ, Reddy DN. Development and validation of simplified RP-HPLC method for quantification of Darunavir in commercial tablets. Materials Today: Proceedings. 2021; doi: 10.1016/j.matpr.2021.04.444

17. Dhruvi SR, Himani PN, Bhagiranth PK, Ankita BS. Stability indicating thin layer chromatography method for simultaneous estimation of Paracetamol and Pamabrom in bulk and combined pharmaceutical dosage from. Journal of Pharmaceutical and Scientific Innovation. 2017;3(5):173-177. doi: 10.7897/2277-4572.032132

18. Chandrasekhar K, Manikandan A. A New RP-HPLC Method Development and Validation of Paracetamol and Aceclofenac in Tablets by separating Diclofenac. Research Journal of Pharmacy and Technology. 2020; 13(9): 4334-4338. doi: 10.5958/0974-360X.2020.00766.0

19. Ishaq BM, Prakash KV, Mohan GK. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Tapentadol and Paracetamol in Bulk Drug and its Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology. 2014;7(2):208-212.

20. Bambhrolia S, Rajout SJ. Simultaneous estimation of Paracetamol and Pamabrom in bulk drugs and in pharmaceutical formulation by spectrophotometry. International Journal of Chemtech Research. 2013;5(4):1802-1807.

21. Antakli S, Nejem L, Shawa D. Separation and Determination of Paracetamol and Codeine Phosphate in Pharmaceutical Preparation by using High Performance Liquid Chromatography. Research Journal of Pharmacy and Technology. 2019;12(3):1327-1332. doi: 10.5958/0974-360X.2019.00222.1

22. El-Houssini OM. RP-LC and TLC densitometric of Paracetamol and Pamabrom in presence of hazardous impurity of Paracetamol and application to pharmaceutical. Anal Chemical Insight. 2013;2(7):73-81. doi: 10.4137/ACI.S12349

23. Saida R, Arafatb B, Arafatc T. High performance liquid chromatography-Mass spectrometric bioanalytical method for the determination of Dapoxetine in human plasma, Application for Bioequivalence study. Journal of chromatography B. 2020;1142:1221-1254. doi: 10.1016/j.jchromb.2020.122154

24. Satyavert, Gupta S, Nair AB, Satyavert MA, Gupta S, Nair AB, Attimarad M. Development and validation of bioanalytical method for the determination of hydrazinocurcumin in rat plasma and organs by HPLC-UV. Journal of chromatography B. 2020;1156:122310. doi: 10.1016/j.jchromb.2020.122310

Received on 31.03.2021 Modified on 10.05.2021

Research J. Pharm. and Tech. 2022; 15(5):2047-2052.

DOI: 10.52711/0974-360X.2022.00338