Innovative stability-indicating LC-Corona CAD method for simultaneous determination of assay in Artesunate and Mefloquine hydrochloride fixed-dose combination product

2.5.1 Neutral, acid and basic conditions

Acid decomposition studies were carried under agitation (ultrasonic bath) of the samples in 0.1 M hydrochloric acid for 1 h. The studies under alkaline conditions were carried out under agitation (ultrasonic bath) of the samples in 0.01 M sodium hydroxide for 5 min. The neutral hydrolysis was performed at 70 °C for 90 min. The resultant solutions were neutralized if degration recommended, followed by a final dilution with acetonitrile/water (1:1) to 25.0 mL in a volumetric flask and filtered through a 0.45 µm PTFE membrane before HPLC injection.

2.5.2 Oxidation with hydrogen peroxide

The samples were exposed to 6% hydrogen peroxide at 70 °C for 90 min to study hydrogen peroxide-induced degradation. The resultant solutions were diluted with acetonitrile/water (1: 1) to 25.0 mL in a volumetric flask and filtered through a 0.45 µm PTFE membrane before HPLC injection.

2.5.3 Metal ions

The samples were exposed to 10 mM of Cu(AcO)₂ at 70 °C for 90 min to study oxidation induced by metal ions. After the degradation process, the samples were chelated with EDTA 10 mM. The resultant solutions were diluted with acetonitrile/water (1: 1) to 25.0 mL in a volumetric flask and filtered through a 0.45 µm PTFE membrane before HPLC injection.

2.5.4 Photochemical degradation

The photochemical stability of the samples was studied by exposing samples in solid form to direct sunlight (4.8 million lux hours) for a total period of 10 days. After the stress conditions, the resultant solutions were prepared as described in item 2.5 and filtered through a 0.45 µm PTFE membrane before HPLC injection.

2.5.5 Dry and wet heat degradation

Different combinations of stress conditions were performed to study dry heat degradation of the samples in solid form. Artesunate and its combinations with placebo and Mefloquine Hydrochloride were placed in an oven at 80 °C for 30 h. Mefloquine Hydrochloride and its combinations with placebo and Artesunate were placed in an oven at 90 °C for 30 h. The drug product was placed in an oven at 80 °C for 4 h. Also, the samples were analyzed in solid form in a saturated vapor atmosphere regarding wet heat. Artesunate and its combinations with placebo and Mefloquine Hydrochloride were placed in an oven at 80 ^◦C for 15 h. Mefloquine Hydrochloride and its combinations with placebo and Artesunate were placed in an oven at 90 °C for 15 h. The drug product was placed in an oven at 80 °C for 4 h. After the stress conditions, the resultant solutions were prepared as described in item 2.5 and filtered through a 0.45 µm PTFE membrane before HPLC injection.

2.6.1 Standard solutions

To prepare the three standard solutions, the APIs were weighed (0,50 AS + 1,10 MQ mg/mL) and transferred to a volumetric flask. Half of the volume was completed with diluent (acetonitrile), and the flasks were submitted to an ultrasonic bath for 20 min. After complete solubilization, the volume was completed. From the three different solutions, the three differents linearity curves were performed.

2.6.2 Precision

Precision was computed here by repeatability and intermediate precision. Repeatability studies were carried out by analyzing the primary standard and also the product sample solutions. In both samples, the Artesunate concentration was 100 µg/mL and the Mefloquine concentration was 220 µg/mL. The standard solution was prepared in duplicate, and one replicate was injected 5 five times (ST1) and the other three times (ST2). Six replicates were prepared for the product sample solution, and one injection of each sample was analyzed. The intermediate precision of the method was checked by repeating studies on another day with a different analyst. The acceptance criteria was determined according to the AOAC INTERNATIONAL (AOAC INTERNATIONAL., 2016).

2.6.3 Accuracy

The linearity samples determined the accuracy of the developed method regarding the recovery of each API in the samples 60% (60 µg/mL of Artesunate and 132 µg/mL of Mefloquine), 100% (100 µg/mL of Artesunate and 220 µg/mL of Mefloquine), and 140 % (130 µg/mL of Artesunate and 308 µg/mL of Mefloquine). The acceptance criteria was determined according to the AOAC INTERNATIONAL (AOAC INTERNATIONAL., 2016). The following equation determined the recovery: $$\mathrm{R}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{y}(\%)=\frac{{\mathrm{C}}_{\mathrm{p}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}}}{{\mathrm{C}}_{\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}}}\times 100$$

2.6.4 Robustness of the method

Few parameters like variation of flow rate in the left and right pump, oven temperature, and detection temperature (vaporization) were altered to evaluate the robustness of the HPLC method. This parameter was performed by analyzing the primary standard solution (ST1) and a solution with a mixture of placebo (140 µg/mL) and the primary standards. In both samples, the Artesunate concentration was 100 µg/mL, and the Mefloquine concentration was 220 µg/mL. The standard solution was prepared and injected three times. Three replicates were prepared for the solution with a mixture of placebo and the primary standards solution, and one injection of each sample was analyzed in the altered method conditions. For the statistical analyses, a 5% significance level was used. The acceptance criteria was determined according to the AOAC INTERNATIONAL (AOAC INTERNATIONAL., 2016).

2.6.5 Stability of the analytical solution.

To prepare the sample solutions, the APIs were weighed (25 mg for AS + 55 mg for MQ) and transferred to a volumetric flask (50 mL). Half of the volume was completed with diluent (acetonitrile), and the flasks were submitted to an ultrasonic bath for 20 min. After complete solubilization, the volume was completed. A 4 mL aliquot was transferd to a 20 mL volumetric flask and the volume was completed with acetonitrile (0.1 AS + 0.22 MQ mg/mL). The stability of the sample and the standard solution was performed by analyzing the primary standard solution (ST1) and a sample solution for 72 h at room temperature. In both samples, the Artesunate concentration was 100 µg/mL, and the Mefloquine concentration was 220 µg/mL. According to the acceptance criteria, the RSD for API areas in the standard and sample solution must be less than 1.0% (17).

3.2.1 Hydrolysis

ASMQ was unstable regarding hydrolysis under heating as 49.12% of AS in the product was degraded under neutral conditions. DHA (Dihydroartemisinin) was the mainly related compound formed as described in Figs. 2 and 3. The acidic and basic medium did not induce a significant degradation as only 14.68% and 3.27 of AS, respectively, was decreased in the drug product (Figs. 4 and 5). MQ was also stable (degradation less than 10%) in all the conditions alone or associated.

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Fig. 2

Degradation pathway of Artesunate in hydrolysis, oxidation and heat.

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Fig. 3

Chromatogram of neutral hydrolysis (70 °C/90 min) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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Fig. 4

Chromatogram of acidic hydrolysis (0.1 M HCl/1 h) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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Fig. 5

Chromatogram of basic hydrolysis (0.01 M NaOH/5 min) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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3.2.2 Oxidation

The oxidative condition under heat was a point of attention concerning the ASMQ. ASMQ was unstable at H₂O₂ reaction as 59.51% degradation was identified after exposing the product (Fig. 6), with the formation of one unknown degradation product and an expressive formation of DHA (Fig. 3). AS alone and associated with the placed also had a strong degradation of 54.4%. MQ was stable in all the conditions alone or associated. Regarding the oxidation via metal ions under heat, ASMQ was also unstable and degraded 57.52% with three unknown degradation products and a strong formation of DHA (Fig. 7). AS alone and associated with the placed had an increased degradation of 63.7% and 76.2%, respectively. MQ was also stable (degradation less than 10%) in all the conditions alone or associated.

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Fig. 6

Chromatogram of oxidative degradation (6% H₂O₂/70 °C/90 min) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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Fig. 7

Chromatogram of metal ions degradation (10 mM of Cu(AcO)₂ at 70 °C for 90 min) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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3.2.3 Photochemical degradation

ASMQ was unstable to photochemical degradation as more than 10% of AS (12.9%) degradation was seen after exposing the product (Fig. 8), with the formation of 5 unknown degradation products and DHA. Both APIs alone or associated with each other or placebo showed a degradation profile under 10%.

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Fig. 8

Chromatogram of photochemical degradation (4.8 million lux hours) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin.

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3.2.4 Dry and wet heat degradation

ASMQ in solid state was unstable to humid heat degradation as more than 10% of AS (10.96%) degradation was identified after exposing the product (Fig. 9), with the formation of 4 unknown degradation products, DHA and Artemisinin. Fig. 3 describes the formation of Artemisin from the hydrolysis of AS to DHA, followed by oxidation of the hemiacetal to Artemisinin. AS alone and associated with the placed had an increased degradation of 23.7% and 28.7%, respectively. MQ was stable in all the conditions alone or associated.

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Fig. 9

Chromatogram of dry heat degradation (80 °C for 4 h) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin, AT Artemisinin.

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No degradation above 10% was noticed in ASMQ, for both APIs in the dry heat condition (Fig. 10). However, AS when associated with placebo had a degradation of 24.74%. MQ was stable in all the conditions alone or associated.

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Fig. 10

Chromatogram of humid heat degradation (80 °C for 4 h in a saturated vapor atmosphere) ASMQ sample. Legend: AS Artesunate, MQ: Mefloquine hydrochloride, DHA Dihydroartemisinin, AT Artemisinin.

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3.3.1 Linearity

For fulfilling regulatory requirements, the linearity assessment must provid statistical data that proves the suitability of the linear regression. Data homoscedasticity shall be investigated for the use of a proper model. Besides the graphic representation of the responses concerning analyte concentration, the residue dispersion graphic must be accompanied by its statistical evaluation and an evaluation of the significance of the angular coefficient (Brasil, 2017).

To assess the homoscedasticity, the Cochran Test was used. The calculated C value was 0.558 and 0.615 for AS and MQ, respectively. These results were not higher than the table critical values for the Cochrantest for variance outliers (0.684), indicating a homoscedasticity behavior. Also, the residues graph for both APIs (Figs. 11 and 12) confirmed this result since the residues are randomly distributed around zero. The ANOVA F-test examined the significance of the slope. The calculated F value was 1114.521 and for AS and MQ, respectively. These results were higher than the table critical values of F(4.67), which indicates that the slope is different from zero. The limit of quantification and detection was estimated from the residual standard deviation of the regression line. All the regression details are summarized in Table 2 and are in accordance with the acceptance criteria (Brasil, 2017).

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Fig. 11

Responses (area) in relation to analyte concentration (%) and residues versus adjusted values of Artesunate.

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Fig. 12

Responses (area) in relation to analyte concentration (%) and residues versus adjusted values of Mefloquine hydrochloride.

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3.3.2 Precision

The results of the repeatability and intermediate precision experiments are described in Table 3. It can be seen that RSD values for both repeatability and intermediate precision suitable according to the acceptance criteria of both APIs as recommended by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline Q2 (R1), illustrating that the developed method was found to be precise.

3.3.3 Accuracy

The accuracy of the method was determined by recovery of three levels of linearity solutions (n = 3) and showed high accuracy with the minimal and maximal recovery of 98.3% and 100%, respectively, for AS. The minimal and maximal recovery of 98% and 102.6% was achieved regarding MQ, as described in Table 4.

3.3.4 Robustness of the method and stability of the analytical solution

According to the results described in Table 5, the method was robust for both APIs assay determination in the oven temperature range of 25.0 + 2.0 °C. Regarding the flow of the right and left the pump, the suitable range of work was 0.6 + 0.06 mL/min and 0.3 + 0.03 mL/min, respectively. The variation in the detection temperature (vaporization) compromised the method suitability since this parameter affects the droplet size, significantly influences the detector response (Ilko, 2015).

The sample and standard solutions were considered stable for 72 h when stored and in a well-sealed bottle at 15 °C since the RSD between the areas was less than 1%.