A SIMPLE AND SENSITIVE RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF HYDROXYCHLOROQUINE SULPHATE AND NITAZOXANIDE IN BINARY COMBINATION

A SIMPLE AND SENSITIVE RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF HYDROXYCHLOROQUINE SULPHATE AND NITAZOXANIDE IN BINARY COMBINATION

Jasmina Surati *, Mahyavanshi Aayushi, Ashok Akbari, Sagar P. Patel and Sagar K. Patel

Department of Pharmaceutical Quality Assurance, Shree Naranjibhai Lalbhai Patel College of Pharmacy, Bardoli, Gujarat, India.

ABSTRACT: The purpose of the present research was to develop a suitable, simple, precise, accurate, robust and reproducible RP-HPLC method for reliable simultaneous quantification of Hydroxychloroquine sulphate (HCQ) and Nitazoxanide (NTZ) in a synthetic mixture. The samples were assayed by the cyber lab HPLC instrument using a C18 (250mm, × 4.6mm, D., 5µm) –cyber-sil under isocratic conditions. The mobile phase consisted of Phosphate buffer (pH-3 adjusted with 1% OPA): Methanol: Acetonitrile (30:35:35 v/v/v) with a flow rate of 1.5 ml/min. The wavelength for detection was 266nm (Isoabsorptive point). The Retention time of HCQ and NTZ was 2.8 min and 9. 3min, respectively. The method was validated by determining system suitability, selectivity, sensitivity, linearity, inter-day and intra-day precision, accuracy, robustness and stability in accordance with the ICH guidelines. The calibration curve was linear over the concentration range, 2-10μg/ml for HCQ and 5-25μg/ml for NTZ respectively (n=6). The developed chromatographic method proved to be simple, precise, accurate, robust, and reproducible. Moreover, the samples showed stability at room temperature over a period of 48 h. Thus, this method would be employed for routine simultaneous quantification of Hydroxychloroquine sulphate and Nitazoxanide.

Keywords: Hydroxychloroquine sulphate (HCQ), Nitazoxanide (NTZ), High-Performance Liquid Chromatography, Development and Validation, Simultaneous determination, ICH guidelines

INTRODUCTION: Hydroxychloroquine sulphate is chemically 2-[4-[(7-chloroquinoline-4-yl) amino] pentylethylamino] ethanol; sulfuric acid Fig. 1A with Chemical formula C₁₈H₂₈C_lN₃O₅S and Molecular weight 434g/mol. It is used as antimalarial drug ¹. Hydroxychloroquine sulfate (HCQ) is used in combination with Nitazoxanide for SARS-coronavirus 2 (SARS-CoV-2) ².

The ACE2 undergoes glycosylation for it to convert to an active form. When the SARS-CoV-2 S protein bind to it, the ACE2 receptor undergoes glycosylation and gets activated. HCQ prevents the glycosylation of ACE2 receptors. So, HCQ prevents entry of SARS-CoV-2 into the host organisms ³.

Nitazoxanide is chemically [2-[(5-nitro-1,3-thiazol 2yl) carbamoyl] acetate Fig. 1B with Chemical formula C₁₂H₉N₃O₅S and Molecular weight 307.28g/mol. It is used as antiprotozoal ⁴. The reason behind selecting NTZ for SARS-CoV-2 could be derived from its impact on the immune system in potentiating the production of type 1 interferon and bronchodilation of the airways through inhibition of TMEM16A ion channels. NTZ inhibits the production of pro-inflammatory cytokines TNFα, IL-2, IL-4, IL-5, IL-6, IL-8 and IL-10 in peripheral blood mononuclear cells ⁵. Several HPLC methods have been described for quantifying Hydroxychloroquine sulphate, alone or in combination with other drugs ^6–19.

Moreover, several methods have been described for quantifying Nitazoxanide, alone or in combination with other drugs in various pharmaceutical preparations ^20–28.

However, the simultaneous determination of Hydroxychloroquine sulphate and Nitazoxanide in combination has not been reported previously.

Thus, the present investigation aimed to develop a suitable, simple, precise, accurate, robust and reproducible-HPLC method for simultaneously estimating Hydroxychloroquine sulphate and Nitazoxanide in a synthetic mixture. The method was validated according to ICH guidelines ^{29, 30, 31}.

FIG. 1: CHEMICAL STRUCTURE OF (A) HYDROXYCHLOROQUINE SULPHATE AND (B) NITAZOXANIDE

MATERIALS AND METHODS:

Reagents and Material: Hydroxychloroquine sulphate and Nitazoxanide were kindly provided by Globela Pharma Pvt. Ltd. HPLC grade Methanol (Advent Chembio–Pvt. Ltd), HPLC grade Acetonitrile (Rankem), double distilled water, Potassium Hydroxide (ACS Chemicals) and Orthophosphoric acid (Chem think Lab) were used for development of method. All other reagents and solvents used in this study were of analytical grade.

Instrument and Chromatographic Conditions: Chromatographic analysis was performed using LC 100 HPLC system (CYBER LAB, USA), equipped with Column: C18 (250mm, × 4.6mm D., 5µm) –cyber-sil, Detector: D2 lamps-(UV 100 UV detector), DS-100 data system software.

The mobile phase consisted of phosphate buffer (1.0 M) pH-3 adjusted with 1% OPA: Methanol: Acetonitrile (30:35:35 v/v/v). HPLC with chromatographic conditions as flow rate 1.5ml/min, UV detection at wavelength 266nm, Injection volume 20µl, isocratic mode, and run time 15min were used to get the best separation.

Preparation of Standard Solutions: Accurately weighed HCQ and NTZ (25 mg each) were transferred to two separate 25 ml volumetric flasks. They were dissolved in methanol to get a stock solution having a strength of 1000μg/ml.

Further, Aliquot of 2.5 ml from the above standard stock solutions was pipette out into two separate 25ml of volumetric flasks. It diluted up to the mark with methanol to get a stock solution having a strength of 100μg/ml. Five standards with 2, 4, 6, 8, and 10 μg/ml concentrations for HCQ and 5, 10, 15, 20 and 25 μg/ml concentrations for NTZ were derived from the stock solution of HCQ and NTZ.

Preparation of Test Solution: The combination of HCQ and NTZ is under clinical trials, so we have prepared a synthetic mixture. The synthetic mixture of HCQ and NTZ was prepared in a ratio of 2:5. Common excipients like 400 mg of Microcrystalline cellulose, 300 mg of Hydroxypropyl methylcellulose, 900 mg of Lactose monohydrate, 200 mg of talc and magnesium stearate along with 10,000 mg of NTZ and 4,000 mg of HQC were transferred into motor pestle which is equivalent for 20 tablets.

A synthetic mixture equivalent to 20mg HCQ and 50mg NTZ was taken in a 100ml volumetric flask, dissolved in methanol, and sonicated for 10min. The solution was filtered with Whatman filter paper 42. From the filtrate, 0.5 ml solution was taken and diluted to 10 ml with methanol to get a solution strength 10 μg/ml HCQ and 25 μg/ml NTZ. Then, 20 μl of the resulting solution was analyzed using the HPLC method described above.

Validation of RP-HPLC Method:

System Suitability: The system suitability was assured by determining the peak retention time, peak area, theoretical plates, and tailing or asymmetry factor for HCQ and NTZ. The acceptance criteria of % CV<2% for retention time and peak area, theoretical plates >2000 ³² and tailing factor <2.0.

The standard concentration of 6μg/ml HCQ and 15μg/ml NTZ was used to prepare the samples. The sample preparation was accomplished in accordance with the method described above in Section 2.4. Six replicate samples were assayed to determine the system's suitability.

Specificity: The specificity of the method was determined by analyzing standard and sample solutions of HCQ and NTZ in accordance with the method as described above. The excipients present in the synthetic mixture do not interfere in the measurement of HCQ and NTZ.

Sensitivity: The sensitivity of simultaneous determination of HCQ and NTZ was evaluated from the Limit of detection (LOD) and Limit of Quantification (LOQ) of the developed method. LOD and LOQ were calculated from the six-calibration curves.

The LOD and LOQ were calculated by using the formula.

LOD=3.3 × σ / S

LOQ=10 × σ / S

Where, σ = standard deviation of intercept of six calibration curves

S = mean slope of six calibration curves

Linearity: Five standard concentrations of 2, 4, 6, 8 and 10 μg/ml for HCQ and 5, 10, 15, 20 and 25 μg/ml for NTZ were prepared and analyzed according to the method as described above. The slope, intercept, and correlation coefficient (r2) were calculated for HCQ and NTZ regression analysis.

Accuracy: The accuracy was determined by calculating HCQ and NTZ percentage recovery. The spiking was done at levels 50%, 100%, and 150%. The samples were prepared according to the method described in above Section.

Thus, the final concentrations to be assayed by the described method were 6 μg/ml, 8 μg/ml, and 10 μg/ml for HCQ and 15 μg/ml, 20 μg/ml and 25 μg/ml for NTZ. The actual concentration was determined for each sample, and the mean percent recovery was calculated (n = 3).

Precision: For evaluating precision, repeatability, intra-day and inter-day variances were determined over a short interval of time, 1 day and 3 days, respectively. Standard concentrations of 6μg/ml for HCQ and 15μg/ml for NTZ were analyzed seven times for the repeatability study.

While, three concentrations 2, 6 and 10 μg/ml for HCQ and 5, 15 and 25 μg/ml for NTZ were analyzed for intra-day and inter-day precision study. All the samples were prepared and analyzed per the method described above. The % CV was calculated for each sample analyzed.

Robustness: The effect of slight deliberate variation in chromatographic parameters such as mobile phase flow rate and change in detection wavelength on peak area ratio was observed one by one.

The standard concentration 6µg/ml HCQ and 15µg/ml NTZ strength used in Robustness study. The mean peak area was determined for HCQ and NTZ and the % CV was calculated (n=3).

Stability: The standard solution of 4 μg/ml HCQ and 10 μg/ml NTZ was used to test the stability. The samples were prepared and analyzed according to the method described above.

The stability of HCQ and NTZ in the prepared sample was determined by analyzing concentration at 1, 6, 12, 24 and 48 h. The concentration was determined for HCQ and NTZ at each time point (n=3).

RESULT AND DISCUSSION: Hydroxy-chloroquine Sulphate and Nitazoxanide are soluble in methanol. In the present HPLC method, mobile phases were used as mobile phases for the phosphate buffer (1.0 M) pH-3 adjusted with 1% OPA: Methanol: Acetonitrile (30:35:35 v/v/v). Separation was done using C18 Column (250mml, × 4.6mml, D., 5µm) –cyber-sil, D2 lamps-(UV 100 UV detector), DS-100 data system software.  The described chromatographic conditions resulted in HCQ and NTZ retention at about 2.8 ± 0.29 and 9.3 ± 0.12 minutes, respectively Fig. 2.

FIG. 2: CHROMATOGRAM SHOWING RETENTION TIME OF HCQ (2.7 MIN) AND NTZ (9.3 MIN)

According to the USP, the HPLC method is considered suitable when the CV of peak area < 2%, the tailing factor < 2, and the theoretical plates > 2000 ³². The results of system suitability are shown in Table 1.

All the measured parameters are within the recommended limits. Thus, our results suggested that the described method was suitable for the simultaneous determination of HCQ and NTZ.

TABLE 1: SYSTEM SUITABILITY ANALYSIS

The excipients present in the synthetic mixture during testing specificity do not interfere with the measurement of HCQ and NTZ. No additional peak interfering with the analyte-peaks was seen in the chromatogram Fig. 3A, B, C. Accordingly, our results suggested that the described analytical method demonstrated specificity.

FIG. 3A:  CHROMATOGRAM OF MOBILE PHASE

FIG. 3B: CHROMATOGRAM OF SAMPLE HCQ (10µG/ML) AND NTZ (25µG/ML)

FIG. 3C: CHROMATOGRAM OF STANDARD HCQ (10µG/ML) AND NTZ (25µG/ML)

The Limit of detection (LOD) and Limit of Quantification (LOQ) of the developed method was calculated from the six-calibration curve. LOD and LOQ of HCQ were found to be 0.07 μg/ml and 0.21 μg/ml, respectively. While, LOD and LOQ of NTZ was found to be 0.25 μg/ml and 0.75 μg/ml respectively. Which shows sensitivity of the developed method. The range for constructing the calibration curves of HCQ and NTZwas 2-6 µg/ml and 5-25 µg/ml, respectively. Fig. 4A, B. The response was linear throughout the range for both analytes (r² = 0.9997 and r² = 0.9998, respectively). The Overlay Chromatogram of different concentrations of HCQ (2-10 µg/ml) and NTZ (5-25 µg/ml) is shown in Fig. 5 and the regression analysis is shown in Table 2. According to the ICH guidelines, the calibrators should not deviate by more than 15% of the nominal concentrations ³¹. All the concentrations determined were within 85–115% of the corresponding nominal concentrations; therefore, the method exhibited linearity.

TABLE 2: REGRESSION ANALYSIS DATA OF HCQ AND NTZ

FIG. 4A: CALIBRATION CURVE OF HCQ

FIG. 4B: CALIBRATION CURVE OF NTZ

FIG. 5: OVERLAY CHROMATOGRAM OF DIFFERENT CONCENTRATIONS OF HCQ (2-10 µG/ML) AND NTZ (5-25 µG/ML)

The accuracy was assessed by the percent recovery method. The accuracy data are shown in Table 3. According to the ICH guidelines, the mean value should be within 15% of the nominal value, ³¹.

All the accuracy values were within 98–102% of the recovery range. Thus, our results suggested that the described method was accurate.

TABLE 3: ACCURACY STUDY DATA OF HCQ AND NTZ

The precision was determined by % CV. The inter-day and intra-day precision data are shown in Table 4. According to the ICH guidelines, the precision determined at each concentration should not exceed 2% of the CV ³¹. All the tested samples showed the % CV within the accepted limits, suggesting that the described method was precise.

TABLE 4: PRECISION DATA OF HCQ AND NTZ

The effect of minor intentional changes in the described chromatographic conditions is shown in Table 5. “System suitability” and “robustness” were performed to ensure the reliability of our method. The slight variations in the mobile phase flow rate and change in detection wavelength did not result in considerable differences in the analytes' retention time and peak area ratio. Thus, the described method exhibited robustness.

TABLE 5: ROBUSTNESS DATA OF HCQ AND NTZ

The stability test of HCQ and NTZin the samples was performed at 1, 6, 24 and 48 h Table 6. The test was carried out in triplicate at each time point. The concentration results at various time points were not significantly different from one another.

TABLE 6: STABILITY OF SOLUTION

Application of the proposed method for analysis of HCQ and NTZ in synthetic mixture. The concentrations of HCQ and NTZ in synthetic mixture were determined using the developed HPLC method. The % assay values are given in Table 7.

TABLE 7: ASSAY OF HCQ AND NTZ IN SYNTHETIC MIXTURE

CONCLUSION: All the parameters for HCQ and NTZ met the criteria of the ICH guidelines for the method validation and found to be suitable for routine quantitative analysis. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. So, developed method is accurate, sensitive, specific, robust, and precise.

ACKNOWLEDGMENTS: This work was supported by Department of Pharmaceutical Quality Assurance, Shree Naranjibhai Lalbhai Patel College of Pharmacy, Vidyabharti Campus Bardoli, Gujarat, India. 394345 and Pfizer Inc through Eliassen Group, Somerset, New Jersey, USA. 08873.

CONFLICTS OF INTEREST: The authors declare that they have no competing interests.

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    How to cite this article:

    Surati J, Aayushi M, Akbari A, Patel SP and Patel SK: A simple and sensitive RP-HPLC method for simultaneous determination of hydroxychloroquine sulphate and nitazoxanide in binary combination. Int J Pharm Sci & Res 2023; 14(5): 2586-93. doi: 10.13040/IJPSR.0975-8232.14(5).2586-93.

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