INTRODUCTION:

Quinine or (R)-(6-Methoxyquinolin-4-yl)[(1S,2S,4S,5R)-5-vinylquinuclidin-2-yl]methanol is a cinchona alkaloid. It has antipyretic, analgesic, anti-inflammatory and anti-malarial properties¹. It is used as an alternative medication for the treatment of uncomplicated malaria caused by Plasmodium falciparum^2,3. The mechanism of action of the drug is not fully known but it involves in the inhibition of hemozin biocrystallization, resulting in the aggregation of cytotoxic heme. Free cytotoxic heme that accumulates in the parasites will cause their death⁴.

Quinine is a basic amine drug that mostly forms salt with sulfate. Quinine sulfate (Fig. 1) has molecular weight of 746.92 for an anhydrous form. The drug is odourless and has the melting point of 225°C. It is slightly soluble in water with two pKa values at 4.1 and 8.5⁵. The orally effective dose is 300 mg and 10 mg/kg three times a day in adults and children, respectively.

Fig. 1: Chemical Structure of Quinine Sulfate

However, the drug is only available in tablet dosage form in Thailand. Extemporaneous preparation in an oral liquid formulation is useful and suitable as it is a patient-specific weight-based dosage in a friendly preparation for children who are unable to swallow tablets. Several HPLC-based procedures were used for determination of quinine sulfate in pharmaceutical formulations^6-12. A previous study employed a reversed phase C18 column and mobility phase that consisted of acetonitrile, methanol and 0.001 M sodium pentanesulphonate (40:40:20)⁶. This method used the flow rate of 1 mL/min, and the wavelength was set at 254 nm. Diazepam was used as an internal standard. The result showed that quinine was eluted at 4.58 min and well resolved from its thermal degradation product (heated by direct flame for 15 min). Precision of the method was less than 2.5%, and it was linear over the concentration range of 48-240 mg/mL. It was a stability-indicating chromatographic method to study the stability of quinine in the intravenous preparation. Another study reported an isocratic HPLC method for the simultaneous determination of quinine and chloroquine in the pharmaceuticals⁷. The analytical C18 column was operated at ambient temperature. Mobile phase consisted of methanol, acetonitrile and 0.1 M ammonium acetate (45:15:40) at a flow rate of 1.0 mL/min. Salicylic acid was used as internal standard. Fluorescence detection was performed at excitation 325 nm and emission 375 nm, respectively. The drug was eluted at 5.09 min. The method was linear in a concentration range from 0.001 to 0.700 mg/mL. LOD and LOQ values were found to be 0.3 and 0.5 ng, respectively. Accuracy was satisfactory with the mean recovery of 99.0%, and precision was less than 10%. However, forced degradation test was not presented in this study. The method was useful for analysis of quinine in commercial tablets and biological fluids. A C18 column with mobile phase of acetonitrile, water, triethylamine and acetic acid (9:90:0.25:0.75) was also used for separation and analysis of quinine⁹. Flow rate was 1.0 mL/min, and UV detection was performed at 254 nm. The method was selective by which quinine was eluted at 4.64 min. Linearity was found over the range of 1-100 µg/mL. Addition of amine (triethylamine) into the mobile phase was useful for simultaneous separation of quinine and quinidine. However, the result of accuracy and precision did not included in this experiment. These methods were solely developed and validated for analysis of quinine in bulk, tablet, intravenous solution and biological fluids. There were a limited number of methods applied for other pharmaceutical dosage forms such as suspension. Furthermore, stress testing is the best way for obtaining any degradation products that may emerge during storage under different destructive conditions. It is important to perform forced degradation study to develop a stability-indicating analytical method for bulk and dosage form¹³.

The present study was aimed to develop and validate a HPLC method to determine quinine sulfate in extemporaneous preparation. It was followed on ASEAN guideline for validation of the analytical procedure, including specificity, range, linearity, accuracy, precision, LOD and LOQ¹⁴. Forced degradation studies such as acid and basic hydrolysis, oxidation and thermal degradation were also performed to determine the stability-indicating capability of the method.

MATERIAL AND METHODS:

Chemicals and Reagents:

Quinine sulfate tablets were purchased from Government Pharmaceutical Organization (GPO, Thailand). Methylcellulose-4000, methylparaben, propylparaben, saccharine and sucrose were procured from P.C. drug center Co. Ltd, Thailand. Quinine sulfate standard was obtained from Sigma-Aldrich, USA. Other chemicals were analytical grade from Merck, Germany. All solvents used were HPLC grade from RCI Labscan, Thailand.

Extemporaneous Preparation:

Fourty 300 mg quinine sulfate tablets were reduced to a fine powder. A small aliquot of the vehicle, containing 0.5% methylcellulose-4000, 0.25% saccharine, 1% paraben concentrate and chocolate syrup was added to the fine powder and mixed into a uniform paste. The remaining vehicle was mixed and adjusted using a graduated cylinder to obtain an extemporaneous suspension with the nominal strength of 20 mg/mL. The preparation was then placed in the amber glass bottles.

Instrumentation:

UV-visible spectrum of quinine sulfate was carried out on a V-630 double beam spectrophotometer (Jasco, Japan). Quinine sulfate standard (100 mg/mL) was dissolved in methanol, and a matched pair of 1 cm quartz cells was used. Spectrum was measured from 200-800 nm and analyzed by Spectra Manager II software. Ultimate 3000 instrument system (Dionex Corporation, USA) was used to perform liquid chromatography. A C18 reversed phase column (Inertsil^® ODS-3; 4.6 x 250 mm; GL Sciences, Japan) was maintained at 25ºC. The mobile phase was a degassed mixture of 0.1 M ammonium acetate pH 7.0, acetronitrile and methanol (40:25:35) with the flow rate at 1.0 mL/min. Injection volume was set at 50 µL, and UV detection was monitored at a wavelength of 330 nm. Each replicate was analyzed in duplicate. The data were recorded and interpreted using Chromeleon 7 software.

Preparation of Standard Solution:

Fifty mg of quinine sulfate standard was accurately weighed and added to a 50 mL volumetric flask. The standard was dissolved with methanol and used as a stock solution of quinine sulfate (1 mg/mL). It was diluted quantitatively with methanol to obtain standard solutions of known concentration at a range of 0.008-600.00 mg/mL. The standard solution was filtered through a 0.45 mm nylon-66 membrane filter before analysis.

Preparation of Sample Solutions:

Extemporaneous quinine sulfate suspension (20 mg/mL) was shaken well for 1 min. Five mL of the preparation was taken from the center of the container and delivered into a 50mL volumetric flask. An aliquot (30 mL) of methanol was added, and the mixture was sonicated for 10 min before making up the final volume with methanol. Five mL of sample aliquot was then transferred into a 50mL volumetric flask and diluted with methanol. The sample solution (200 mg/mL) was finally filtered through a 0.45 mm membrane filter into a vial.

Preparation of Vehicle Solutions:

Mixture that consists of 0.5% methylcellulose-4000, 0.25% saccharine, 1% paraben concentrate and chocolate syrup was used to prepare vehicle solution. Five mL of the mixture was transferred into a 50 mL flask. Methanol was added, and the mixture was sonicated for 10 min. A 10-fold dilution with methanol was prepared to make the vehicle solution. It was filtered through a 0.45 mm filter before analysis.

Method Validation:

According to ASEAN guideline for validation of the analytical procedure, the method was validated by determining the parameters: system suitability, specificity, linearity, limit of detection (LOD) and limit of quantification (LOQ), accuracy and precision¹⁴.

System Suitability:

Measurement of system suitability was performed by collecting data from 5 replicate injections of quinine sulfate standard (200 mg/mL) to verify an adequate performance of the chromatographic system. Parameters such as peak area, tailing factor and numbers of theoretical plates were measured^15-17.

Specificity:

The specificity of the method was performed by evaluating the interference of the common excipients in the pharmaceutical formulation. Chromatograms of standard solution (200 mg/mL), sample solution and vehicle solution were compared. In addition, the result of standard-spiked sample solution was also assessed.

Linearity, LOD and LOQ:

Linearity was determined by a set of 8 different concentrations (0.008, 0.080, 0.800, 8.00, 80.0, 200, 400 and 600 mg/mL). It was done by accurately weighing quinine sulfate standard in triplicate to prepare stock solutions. Peak area of three replicates was analyzed by linear regression as a function of drug concentration. The parameters such as slope, intercept and regression coefficient were collected. LOD and LOQ values were obtained from calculation based on calibration curves by which factors of 3.3 and 10 were multiplied by a ratio of standard deviation of the response and slope of the curve, respectively.

Accuracy:

The accuracy of the method was determined by standard addition procedure. Quinine sulfate standard was spiked into the sample solution in triplicate at the level of 80%, 100% and 120% of test concentration (160, 200 and 240 mg/mL, respectively). Methanol was used as a diluents, and the standard-spiked sample solutions were filtered through a 0.45 mm membrane filter. Percent recovery and its standard deviation (SD) were calculated to represent the accuracy.

Precision:

Repeatability (intra-day) and intermediate precision (inter-day) were studied to determine the precision of the method. Repeatability was done by injecting 6 replicates of the sample solutions (200 mg/mL) in a same day whereas the intermediate precision was evaluated by analyzing a set of sample solutions on different days. Peak area and percent relative standard deviation (% RSD) were assessed.

Forced Degradation Studies:

Degradation of quinine sulfate in the extemporaneous suspension was conducted to determine the stability-indicating capability of the method. Various stress conditions such as acid and basic hydrolysis, oxidation and thermal stresses were performed in triplicate.

Hydrolytic Studies:

Acid and basic hydrolysis was performed by individually transferring 5 mL of the extemporaneous suspension into a 50 mL flask. About 30 mL of methanol was added, and the samples were sonicated for 10 min. Samples were refluxed at 80°C for 1 h with 5 mL of 1.0 N HCl as acid hydrolysis or 1.0 N NaOH as basic hydrolysis. After completion of the stress, they were allowed to cool and neutralized with the same volume of 1.0 N NaOH or 1.0 N HCl, respectively. Samples with distilled water were used as neutral hydrolysis. Five mL of the samples were transferred to a 50 mL flask. Methanol was filled up to the mark, and the samples were filtered through a 0.45 mm membrane filter.

Oxidation Studies:

Oxidative degradation studies were done by taking 5 mL of the extemporaneous suspension to a 50 mL flask. A portion of methanol was added, and the samples were sonicated for 10 min. An aliquot of 30% H₂O₂ solution was added to make a final concentration of 0.1% H₂O₂. The samples were adjusted the volume with methanol and incubated at room temperature for 1 h in the dark. A 10-fold dilution of the samples was prepared before analysis.

Thermal Studies:

Thermal degradation studies were performed by placing the drug preparations in a hot air oven at 80°C for 1 week. After specified time, 5 mL of the drug was transferred to a 50 mL flask and mixed with methanol. After sonication for 10 min, 5 mL of the samples was taken into a 50 mL flask, and methanol was filled up to the mark.

RESULTS AND DICUSSION:

UV Spectrum of Quinine Sulfate:

Standard solution of quinine sulfate (100mg/mL) was used to determine the lmax. Spectrum of the drug from 200-800 nm was recorded by using methanol as a blank (Fig. 2). The result showed that quinine sulfate had the lmax at the wavelength of 330 nm that was selected for further studies. The heterocyclic quinoline ring was responsible for this unique characteristic¹⁸.

System Suitability Testing:

System suitability testing is an integral part of many analytical methods. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system. They are used to verify the resolution and reproducibility of the chromatographic system that are adequate for the analysis^19,20. Five replicate injections of standard solutions showed that quinine was eluted as a sharp peak at 7.73 min with tailing factor of 1.75. Variation of peak area (% RSD) was 0.08, and theoretical plate was 4424. Therefore, the used method met the general requirements.

Specificity Study:

The specificity of the proposed method was defined by comparing the retention times of quinine sulfate in the extemporaneous samples, standard solutions and vehicle solutions (Fig. 3A, B and C). Retention time of the drug was 7.73 min, and there were no interferences of the pharmaceutical excipients in the formulation. Chromatogram of the standard-spiked sample solution also showed a sharp and large peak at the same time with about double peak area (Fig.3D). Reduction of acetonitrile portion in the mobile phase caused slower elution of the drug from the column (data not shown). Therefore, the proposed method was found to be selective and suitable for determination of quinine sulfate in the extemporaneous suspension.

Linearity, LOD and LOQ Studies:

A linear relationship was constructed from calibration curves of quinine sulfate standard over a concentration range of 0.08-600.00 mg/mL. Regression analysis showed that the method had a correlation coefficient (r²) of 0.9999 with a linear equation Y = 0.6714X + 1.3271; where Y was the peak area, and X was the drug concentration in mg/mL (Fig. 4). LOD and LOQ of the method were calculated based on the standard deviation of the response and the slope of calibration curves. They were found to be 4.32 and 13.09 µg/mL, respectively. These low values indicated that the proposed method was more sensitive, demonstrating its suitability for drug analysis in the extemporaneous suspension⁷.

Accuracy Study:

The accuracy of the method was assessed by standard addition. Known quantity of quinine sulfate standard was spiked into the sample solutions in triplicate at three different levels of test concentration (80%, 100%, and 120%). Percent recovery values for accuracy were in the range of 98.85-100.67 (Table 1). Mean recovery and its SD was 99.83±0.92%. They were in the acceptable criteria in the range between 97-103%²¹. These studies revealed that the proposed method was accurate for routine application. In addition, stability of the sample solution was determined by analyzing the freshly prepared samples at different time intervals (0, 6, 12 and 24 h). The results showed the percent recovery were 100.67, 99.78, 99.82 and 100.13, respectively. They exhibited a complete recovery without any degradations observed in the chromatogram. Therefore, the sample solution of quinine sulfate can be used within 24 h after its preparation.

Table 1: Accuracy of the Proposed Method for Determination of Quinine Sulfate in Drug Product (Mean ± SD, n = 3).

Accuracy	Concentration added (mg/mL)	Concentration found (mg/mL)	% Recovery
80%	160.03	160.00 ± 3.34	99.98 ± 2.09
100%	200.04	201.37 ± 4.23	100.67 ± 2.10
120%	240.05	237.00 ± 3.80	98.85 ± 1.60
Mean recovery			99.83 ± 0.92

Precision Study:

Precision can be represented in the term of repeatability and intermediate precision. Six replicates of the sample solutions were used to describe the analytical variation of the proposed condition on a single day and various days. The studies of repeatability showed that the percentage RSD of the labelled amount were 0.78 and 1.09 in the first and second day, respectively (Table 2). Intermediate precision could be obtained from sample examinations on two different days. The result showed the overall % RSD was 0.99. It was in the acceptable criteria of not more than 2.7²¹. Therefore, the proposed method was precise within the same and between days of analysis.

Table 2: Precision of the HPLC Method for Determination of Quinine Sulfate in Drug Product.

Sample number	% LA
Sample number	1^st Day	2^nd Day
1	99.03	100.64
2	99.31	98.18
3	99.17	100.64
4	98.19	101.33
5	100.55	99.95
6	99.65	99.77
Average	99.32	100.08
Repeatability (%RSD)	0.78	1.09
Overall average	99.70
Intermediate precision (%RSD)	0.99

Stress Testing:

In order to develop a stability-indicating method for determination of the drug, it is important to perform the stress testing under suitable harmful conditions^22-25. Forced degradation studies such as acid and basic hydrolysis, oxidative and thermal conditions were used to produce the degradation products that may be produced during the storage of drug product. Treatments of quinine sulfate samples by refluxing with 0.1 N HCl or 0.1 N NaOH caused a partial degradation of the drug (Fig. 5A and B). The drug content was remained at 80.06±0.94% and 77.27±0.82%, respectively (Table 3). Elution of hydrolytic degradation products was detected at less than 5.00 min, suggesting that they became more polarity. Meroquinene and 6-methoxyepidine might be the acidic-catalyzed degradation products whereas 6-methoxyquinoline was occurred as a basic degradant²⁶. These implied that quinine sulfate was sensitive to acid and basic decomposition. However, the drug was stable under neutral condition with the content of 98.08±0.93%. The significant decrease of quinine sulfate was occurred in the studies with chemical oxidation. There were 49.16±0.61% of the drug content remained from a condition with 0.1% H₂O₂. Drug degradation was proportional to the incubation time and concentration of the oxidizing agent after exposure to H₂O₂²⁷. Oxidative degradation of quinine may cause its carboxylic acid product known as quintenine with the release of formic acid²⁶. Quininone and quininic acid might also be the oxidative by-products.

Furthermore, incubation of the extemporaneous preparation in the dry heat at 80°C for 1 week decreased the drug content (58.19±0.42%). Thermal degradation products were rapidly eluted within 7.00 min. Therefore, these results exhibited the selectivity of the method, allowing the separation of quinine sulfate from other excipients and their degradation products. The proposed method can serve as a stability-indicating procedure that is useful for quantitative determination of quinine sulfate in the preparation during pharmaceutical development and stability studies.

Table 3: Forced Degradation Studies of Extemporaneous Quinine Sulfate Suspension on Various Condition (Mean ± SD, n = 3).

Condition	% Drug content
Acid hydrolysis / 0.1 N HCl, 80°C, 1 h	80.06 ± 0.94
Basic hydrolysis / 0.1 N NaOH, 80°C, 1 h	77.27 ± 0.82
Neutral hydrolysis / Water, 80°C, 1 h	98.08 ± 0.93
Oxidation / 0.1% H₂O₂, 1 h, in the dark	49.16 ± 0.61
Dry heat / 80°C, 1 week	58.19 ± 0.42

Fig. 5: Forced Degradation Studies of Extemporaneous Quinine Sulfate Suspension. (A) Acid Hydrolysis with 0.1 N HCl,

(B) Basic Hydrolysis with 0.1 N NaOH, (C) Neutral Hydrolysis, (D) Oxidation with 0.1% H₂O₂, and

(E) Dry Heat at 80°C. * indicated quinine peak.

Application of the Method for Determination of Quinine Sulfate in the Extemporaneous Suspension:

The proposed method was used to determine of the drug content in the extemporaneous preparation. The result showed that the percent contents of quinine sulfate from 9 different samples were between 97.50 and 101.76 with %RSD of 1.30. Statistics analyses did not presented a significant difference between the data (p-value > 0.05). It indicated that the proposed method was sensitive, accurate and reliable for quantification of quinine sulfate and could be served as a reference method for drug analysis in the suspension preparations.

CONCLUSION:

The proposed HPLC method was validated, according to ASEAN guideline for the validation of analytical procedure, to determine quinine sulfate in an extemporaneous suspension. It exhibited the satisfactory performance with high accuracy, precision and sensitivity. It could separate any degradation products from the main quinine peak that emphasized its selectivity. This indicated that the method was served as a stability-indicating procedure. Therefore, it can be used for routine application on quality control for determination of quinine sulfate in the suspension and its stability study.

ABBREVIATIONS:

HPLC, High Performance Liquid Chromatography; LOD, Limit of Detection; LOQ, Limit of Quantification; SD, Standard Deviation; %RSD, Percent Relative Deviation

ACKNOWLEDGEMENT:

This work was supported by a grant from Walailak University. In addition, we would like to thank Centre of Scientific and Technological Equipments, Walailak University for research facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.02.2017 Modified on 03.03.2017

Research J. Pharm. and Tech. 2017; 10(6): 1603-1610.

DOI: 10.5958/0974-360X.2017.00282.7