INTRODUCTION:

The release the pharmaceutical ingredient from drug product, the dissolution of the drug under physiological condition, and its probability to cross gastro intestinal barrier determines the drug absorption. In vitro dissolution method serves as a tool in assessing the in vivo performance which helps to address lot to lot uniformity and distinguish acceptable lots from unacceptable lots of the manufactured product. It is important to understand the relationship between the in vitro drug release and in vivo performance to evaluate the impact of formulation and process variable on drug product quality during development to predict the performance of commercial batches and to demonstrate the consistency of manufactured batches. In addition, the dissolution method and specification should be able to predict the potential problems with in vivo bioavailability (e.g. bio inequivalence)¹.

Artemether is a part of the fixed dose combination (FDC) of the antimalarial drug product Artemether and lumefantrine tablets. As per WHO, Artemether is a BCS Class II (low soluble, high permeable) compound, whereas Lumefantrine is a BCS Class IV (low soluble and low permeable) compound. Hence, solubility is expected to be a limiting factor for dissolution and drug absorption of Artemether². The drug substance specifications ensure compliance with the Ph. Int monograph. There are two known polymorphs for Artemether (Polymorphs A and B). Lumefantrine has only one polymorphic form and is isolated as a racemic mixture and is stable at room temperature. Artemether and Lumefantrine tablets are official in the International Pharmacopoeia, but dissolution is not included in the monograph. Therefore, dissolution test method needs to be developed in-house for quality control and in vivo assessment of Artemether and Lumefantrine^3-4. For in vivo absorption of Artemether, dissolution is the rate limiting factors. Artemether peak plasma concentrations are reached after about 2 hours after oral administration. Concomitant intake of a high fat meal enhances the absorption of Artemether, resulting an increase in the relative bioavailability by more than two-fold. Hence, there is a need of improving the solubility and bioavailability of Artemether through novel formulation approaches. Amorphous solid dispersion (ASD) and micro emulsifying drug delivery are the available novel techniques to improve the dissolution and bioavailability of Artemether⁵. As the novel solubilization techniques used to give faster dissolution compared to the conventional immediate release dosage form, it is required to develop a suitable analytical method for dissolution with due consideration of discriminatory significance and clinical relevance. Further the developed method need to be validated in line with ICH recommendation⁶. The aim of the current study was to develop and validate an appropriate in-house dissolution method for Artemether and its amorphous solid dispersion (ASD) in a fixed dose combination (FDC) of Artemether and Lumefantrine Tablets.The development of dissolution method involves selection of the dissolution media, apparatus as agitation rate appropriate to the product. The solubility of drug substance, one of the key aspect in selection of possible dissolution media was duly considered for evaluation of sink condition. The resulting final method was validated for linearity, precision, robustness and solution stability.

EXPERIMENTAL:

Materials and Methods

Artemether Ph Int working reference standard (WRS) was obtained from Vital lab pvt ltd , Mumbai India , Sodium Dihydrogen Orthophosphate AR Grade (Thermofisher Scientific, Mumbai, India), Orthophosphoric acid (SD fines chem ltd), Triethylamine AR Grade (SD fines chemical ltd) ,De mineralised water, Milli Q water, C18, Octadecylsilane column 15cm x 4.6mm, 5µm (Zodiac - life sciences, India), Acetonitrile HPLC grade (Merck Chemicals Germany), Sodium lauryl sulfate (Sigma-Aldrich) and Sodium hydroxide pellets (SD fines chem ltd) were used as received. Test product of Artemether and lumefantrine tablets 20+120 and 80+480 strength prepared with conventional and solid dispersion technique were taken for dissolution study.

Discriminatory dissolution method

Evaluation of sink condition

For evaluation of sink condition of Artemether, the saturation solubility data in different media was considered as presented in Table 1. The sink condition was evaluated based on the saturation solubility (Cs) in the proposed medium and concentration of Artemether in the bulk medium (Cd).

Table 1: Saturation solubility and sink condition of Artemether^7-8

Medium	Solubility (µg/ml)	*Sink Cs/Cd^#**
Medium	Solubility (µg/ml)	80 mg
Water	133	1.662
1 % w/v SLS in Water	4107	51.33
pH 6.8 Phosphate buffer	119	1.4875
1 % SLS in 0.05M Sodium phosphate buffer (pH 6.8)	3511	43.88
0.1N HCl	127	1.5875
0.5% SLS + HCl (pH 1.2)	323	4.0375
1% SLS + HCl (pH 1.2)	337	4.2125
pH 4.5 Acetate buffer	130	1.625
1 % SLS in pH 4.5 Acetate buffer	3896	48.70

*Cs is the saturated solubility of the compound in the medium

# CD is concentration of the compound in the bulk medium.

Evaluation of Impact of surfactant

For evaluation of the discriminatory power of the dissolution medium, one comparative dissolution study for Artemether (20 mg label claim) was performed with the USP II paddle apparatus at 100 RPM in 1000 ml of phosphate buffer pH 6.8 with and without surfactant (0.5% w/v of sodium lauryl sulfate)

Finalization of dissolution method and specification

Based on the appropriate pH solubility, sink condition, and dissolution study, pH 6.8 Buffer with and without 0.5% SLS dissolution method was considered for further evaluation.

HPLC Analytical method

The proposed HPLC method is a modified method of previously reported method for the dissolution of Artemether and lumefantrine in tablets. The proposed method was modified in terms of dissolution medium, volume of mobile phase composition and analytical column to evaluate amorphous solid dispersion of Artemether⁸.

A Shimadzu LC-10 AT VP HPLC system (Shimadzu Corporation, Japan) equipped with a PDA detector was used for quantification of Artemether in dissolution samples. Additionally, Agilent 1260 infinity II HPLC was used for carrying out intermediate precision during analytical method validation. The Analytical column was a C18 reverse-phase column (Zodiac, 150 × 4.6 mm, 5µm). The mobile phase was composed of degassed mixture of 0.5 % v/v of triethylamine (pH adjusted to 3.0) and acetonitrile in a ratio of 30:70, v/v). The column temperature was maintained at 25 °C, and the injection volume was 75 µL with a flow rate of 1.0 mL/min. The absorbance of Artemether was set at 216 nm⁸.

Preparation of dissolution medium:

Sodium dihydrogen phosphate (54.40gms) was transferred in to a beaker containing 8 Liters of purified water. Stirred and sonicated to dissolve and pH adjusted to 6.8 ± 0.05 with Ortho phosphoric acid/Sodium hydroxide solution. Based on requirement, specified quantity of Sodium lauryl sulphate was added with mixing and the obtained dissolution medium finally degassed.

Preparation of standard solution:

Artemether working standard (40 mg) was transferred to a 100 mL volumetric flask. To this added 50 mL of mobile phase and sonicated for 10minutes to dissolve completely. Volume makeup to 100 ml was done with mobile phase. Diluted 5mL of above solution to 100 mL with dissolution medium.

Preparation of sample solution:

1000ml of dissolution medium was placed into each six dissolution bowls of dissolution apparatus [Electrolab, Model TDT-08L] and allowed to attain a temperature of 37° ± 10 C. Dropped six tablets individually in to six dissolution bowls and operated the dissolution apparatus as per the set dissolution parameters.10 ml aliquot were withdrawn from each dissolution bowl and filtered through 0.45 μm syringe filters. For Artemether 80 mg strength, 2 ml of the filtrate was diluted to 10ml with dissolution media and injected the test and standard solutions in to HPLC. For 20 mg Artemether strength no dilution was done.

Analytical method validation

The applied HPLC method is a modified method of a previously developed method for the dissolution Artemether and lumefantrine in tablets. The applied method was modified in terms of dissolution medium, volume of mobile phase composition and analytical column hence re-validated with the following parameters.

System Suitability:

System suitability was carried out to demonstrate and verified the adequacy of system suitability parameters of the system for the subjected analysis with the proposed analytical method. The area of the blank solution and 5 standard solutions were measured and evaluated for system suitability acceptance criteria for Injection precision (for retention time, peak area and peak height) resolution, USP tailing factor, Capacity factor and theoretical plates.

Specificity:

To determine the interference due to blank and placebo during analysis, specified quantity of placebo was added to the dissolution vessel during the dissolution test. The responses were measured and the area were recorded for Blank, placebo, standard solution and test solution to rule out the presence of any extra peak and interference.

Linearity and Range:

The linearity of an analytical method is its ability to elicit test results that are directly or by a well defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. The linearity of Artemether standards were evaluated with the proposed methods in the range of 25% to 150% of specification limit. The area response for each level were recorded. A graph was plotted between concentration and area to get slope, intercept and correlation coefficient to evaluate as per the requirement of acceptance criteria. The range of analytical method is the interval between the upper and lower levels of analyte that has been demonstrated with a suitable accuracy and linearity. The specified range were derived from the Linearity and Accuracy studies and evaluated against the acceptance criteria.

Precision:

To establish the effects of random events on the precision of the analytical procedure, the intermediate precision carried out by studying the effect of the variance of analyst and day of analysis. The procedure and Injection sequence schedule was same as described under repeatability test. To demonstrate the capability of the analytical method to yield closeness of data values between a series of measurements obtained from multiple sampling of the same homogeneous sample, The blank, standard solution and test solution (bowl 1 to 6) were injected to evaluate the area and responses as per the acceptance criteria.

Accuracy:

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value (standard value). Known quantity of Artemether standards were spiked into the placebo at 25%, 100%, and 150% of dissolution specification and analyzed in triplicate for each level. The % recovery from the results were then calculated and evaluated against the acceptance criteria.

Robustness

The robustness of the analytical method was carried out by evaluating the impact of some deliberate variation in mobile phase composition and flow rate on % dissolution and acceptance criteria.

Solution Stability:

The solution stability was carried out by injecting the standard preparation and sample preparation at regular interval and evaluated against the acceptance criteria.

RESULTS:

Discriminatory dissolution method

Based on saturation solubility and sink condition, 0.5 % SLS in 0.05M sodium phosphate buffer (pH 6.8) would be suitable dissolution medium for Artemether. However, the presence of surfactant would lead to lower the discriminatory power of the dissolution medium. Further, as the proposed solid dispersion formulation of Artemether is in a highly solubilized amorphous system, the expected dissolution rate would be high and the dissolution media containing surfactant would lead to a low discriminatory power. The impact of surfactant on the dissolution of Artemether is depicted in Figure 1. The comparative dissolution profile of Artemether conventional IR tablet and solid dispersion tablet in phosphate buffer pH 6.8 (without surfactant) with its discriminatory significance depicted in Figure 2. Based on the discriminatory power, phosphate buffer pH 6.8(without surfactant), 1000 ml, paddle, 100 RPM was considered as the discriminatory dissolution method for amorphous solid dispersion formulation of Artemether. Further, based on the dissolution data, the QC release specification for Artemether in the solid dispersion formulation was set as not less than 80(Q) in 30 minutes.

Figure 1: Comparative dissolution profile of conventional immediate release tablet in Phosphate buffer pH 6.8 with and without surfactant (Dissolution data, shown as mean (n = 6) ± SD, where n is the number of observations)

Figure 2: Comparative dissolution profile of conventional immediate release tablet and solid dispersion tablet of Artemether in discriminatory dissolution medium (Phosphate buffer pH 6.8) (Dissolution data, shown as mean (n = 6) ± SD, where n is the number of observations)

Analytical method validation

The data sheet for system suitability, Specificity, Precision/repeatability, Intermediate Precision, Linearity/range, Accuracy, solution stability and robustness for the proposed dissolution method for Artemether solid dispersion are shown in Table 2-10 along with the acceptance criteria.

Table 2: System suitability for Artemether solid dispersion

System suitability data sheet
System Suitability Parameters	Acceptance Criteria	HPLC results	Criteria met/not met
Injection precision for retention time (min)	RSD ≤ 1%	0.07	Criteria met
Injection precision for peak area (n = 5)	RSD ≤ 1%	0.8	Criteria met
Injection precision for peak height	RSD ≤ 1%	1	Criteria met
Resolution (Rs)	Rs = ≥ 2.0	Not Applicable	Not Applicable
USP tailing factor (T)	T = ≤ 2.0	1.165	Criteria met
Capacity factor (k)	K = ≥ 2.0	Not Applicable	Not Applicable
Theoretical plates (N)	N = ≥ 2000	10978	Criteria met

Table 3: Specificity

Name	Retention time	Interference
Blank	Not detected	NA
Placebo	Not detected	NA
Standard	5.94	No interference
Sample	5.60	No interference
Acceptance criteria: There should be no interference due to blank and placebo at the area of Artemether

Table 4: Precision for standard preparation

Injection number	Peak Area
Standard 1	43061
Standard 2	42973
Standard 3	43275
Standard 4	43183
Standard 5	42809
Standard 6	43324
Mean	43104.17
SD	194.8932186
% RSD (< 2 %)	0.45

Table 5: Precision for the dissolution sample

Injection number	Retention time	Peak Area	Peak Height
Replicate 1	5.616	33841	5056
Replicate 2	5.602	34643	5103
Replicate 3	5.482	34121	4977
Replicate 4	5.432	35946	5274
Replicate 5	5.38	34615	5199
Replicate 6	5.361	34298	5382
Mean	5.47	34577	5165.16
SD	0.109	735	149.12
% RSD (< 5 %)	1.99	2.12	2.88

Table 6: Intermediate Precision for the proposed dissolution method

Sample	Peak area / % Dissolution
	Shimadzu, LC-10 A VP (S1)		Agilent 1260 infinity II (S2)
	Analyst 1, Day 1		Analyst 2 , Day 2
	Peak area	% Dissolution	Peak area	% Dissolution
Replicate 1	33841	96.08	34.4467	94.46
Replicate 2	34643	98.36	34.1058	93.53
Replicate 3	34121	96.87	37.4992	102.83
Replicate 4	35946	102.06	35.9862	98.69
Replicate 5	34615	98.28	35.5153	97.39
Replicate 6	34298	97.38	34.5701	94.80
Mean	34577	98.17	35.35	96.95
% RSD	2.13	2.13	3.58	3.58
% RSD (S1+S2)	Acceptance Criteria: RSD (<5)

Figure 3: Chromatograms for Artemether standards and dissolution samples (Intermediate precision study)

Table 7: Linearity

Conc. (mg/ml)	Conc. as % of analyte target	Peak area (n=3)	Peak area % RSD
0.016	80	35841	0.96
0.018	90	40340	1.03
0.020	100	44894	0.53
0.022	110	49580	0.237
0.024	120	53856	0.346
Acceptance criteria: Correlation coefficient should be NLT 0.999.

Figure 4: Linearity curve

Table 8: Accuracy

Accuracy				Recovery (%)	% RSD
Sample	% Nominal (mean of three injections)	Amount of standard (mg)
Sample	% Nominal (mean of three injections)	Spiked	Found
1	80	63.04	58.894	93.42	0.28
2	100	79.056	73.886	93.46	0.09
3	120	95.083	88.779	93.37	0.05
Acceptance criteria: Individual and mean % recovery at each level should be between 90.0% and 110.0%

Table 9: Solution stability

S.No	Initial	12 hour
1	94.54	95.13
2	98.35	96.91
3	97.85	94.45
4	94.07	96.30
5	96.87	98.66
6	92.55	93.55
Average	95.70	95.83
SD	2.32364576	1.843308619
% RSD	2.43	1.92
Acceptance criteria: The average difference in the dissolution should not be more than 1 after 12 hour

DISCUSSION

Discriminatory dissolution method serves as an important tool in quality control and in vivo assessment of formulations containing low soluble and low bioavailable API. Discriminatory dissolution method becomes essential for the formulations where the dissolution is improved to many fold by various novel solubilization techniques such as amorphous solid dispersion. In the current study dissolution method was developed for Artemether solid dispersion considering the anticipated higher dissolution of Artemether as a result of solubilization⁹.

The addition of surfactant in the dissolution medium plays a very important role with respect to discriminatory significance. In many reported research work, use of surfactant recommended for Artemether dissolution to get desired discrimination. However, use of surfactant in the dissolution medium recommended for conventional immediate release formulation for Artemether. In the current study, no use of surfactant recommended in the medium for dissolution testing of Artemether amorphous solid dispersion due to the fact that, Artemether is already in a highly solubilized state in the amorphous solid dispersion and hence any addition of surfactant would lower the discriminatory power of the dissolution medium¹⁰. The dissolution rate of Artemether solid dispersion was significantly improved by 2-3fold compared to conventional immediate release tablet formulation. The dissolution of Artemether solid dispersion was found to be 99.22% in 30 minutes, Hence the tolerance for QC release for Artemether was finalized as not less than 80(Q) in 30 minutes¹¹.

Artemether is a polymorphic API with reported form A and form B differing in their solubility and polymorphic stability. The crystalline polymorph of Artemether was converted to amorphous form by solid dispersion technique. Since the amorphous form of Artemether comparatively unstable and may undergo transformation during dissolution and HPLC analysis, it is important to achieve the solution stability of Artemether solid dispersion. The solution stability data of Artemether solid dispersion during analytical method validation is in good agreement with the Artemether stability. All validation parameters for the proposed HPLC method in terms of accuracy, precision, linearity, specificity, solution stability and robustness found satisfactory.

CONCLUSION:

The result of the current study revealed that the developed and validated HPLC method for dissolution of Artemether amorphous solid dispersion is discriminatory and is capable in identifying small changes in the formulation variables. The proposed method can be applied to QC release and can be explored further in establishing in vitro -in vivo relations.

LIST OF ABBREVIATIONS:

API: Active Pharmaceutical Ingredients, AR: Analytical reagent, ASD: Amorphous solid dispersion, BCS: Biopharmaceutical classification system, EMA: European Medicines Agency, FDC: Fixed dose combination, HPLC: high-performance liquid chromatography, IR: Immediate release, ICH: International Council for Harmonisation , Ph Int: International pharmacopoeia, QC: Quality control, RSD: relative standard deviation, SD: solid dispersion, SLS: Sodium lauryl sulphate, USP: United state pharmacopoeia, UV: Ultra violet WRS: Working reference standard

REFERENCES:

1. Reflection paper on Dissolution specification for generic solid oral immediate release products. European Medicines Agency. [Cited 2023 Oct 23]. https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-dissolution-specification-generic-solid-oral-immediate-release-products-systemic_en.pdf

2. Proposal to waive in vivo bioequivalence requirements for WHO Model List of Essential Medicines immediate-release,solid oral dosage forms; World health Organization. [cited 2023, Oct 25]. https://www.who.int/medicines/services/expertcommittees/pharmprep/QAS04_109Rev1Waive invivo bioequiv.pdf

3. Artemether and Lumefantrine Tablets; Final Text for addition to The International Pharmacopoeia;QAS/07.192/Final.World health Organization. [cited 2023, Oct 25]. http://www.who.int/medicines/publications/pharmacopoeia/mon_mal/en/index.html

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7. Belew S, Suleman S, Duguma M, Teshome H, Wynendaele E, Duchateau L, De Spiegeleer B. Development of a dissolution method for lumefantrine and Artemether in immediate release fixed dose Artemether/lumefantrine tablets. Malar J. 2020; 19(1): 139.

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Received on 13.10.2023 Modified on 04.11.2023

Research J. Pharm. and Tech 2023; 16(11):5303-5308.

DOI: 10.52711/0974-360X.2023.00859

Dissolution Sample	Peak area / % Dissolution
	Change in mobile phase composition ( > 10 % )		Decrease in flow rate (0.8 ml/min)		Increase in flow rate (1.2ml/min)
	Peak Area	% Dissolution	Peak Area	% Dissolution	Peak Area	% Dissolution
Replicate 1	35621	98.88	37699	95.04	26237	99.56
Replicate 2	33442	92.83	39080	98.52	25258	95.85
Replicate 3	35720	99.15	38595	97.30	24368	92.47
Replicate 4	34164	94.83	39000	98.32	25729	97.63
Replicate 5	34536	95.87	38195	96.29	26452	100.38
Replicate 6	37588	104.34	39143	98.68	25019	94.94
Mean	35178.5	97.65	38618.66	97.36	25510.5	96.80
SD	1466.435	4.070	576.022	1.452	783.913	2.974
% RSD	4.168	4.17	1.491	1.49	3.072	3.07
Acceptance criteria: The overall RSD shall be not more than 5 % with the method precision data of individual experiments.

*Corresponding Author E-mail: 121965201519@gitam.in