Development and Validation of RP-HPLC Method for Quantitative Analysis of Sulfamethoxazole and Trimethoprim in Liquid Suspension: A Comparative Study with Compendial Method

Muhammad Aslam*, Shahzad Ali, Mahmood Ahmed, Muhammad Aneeq Javed, Afsah Iftikhar, Yousaf Abbas, Aamir Sohail, Mehvish Abdul-Rehman, and Khansa Habibullah

Department of Chemistry, Division of Science and Technology, University of Education, Lahore, Pakistan

* Corresponding Author: [email protected]

ABSTRACT

Co-trimoxazole is a combination of trimethoprim or sulfamethoxazole. It is used to treat common infectious diseases, including the lung disorders, urinary disorders, and gastrointestinal infections. The current study was performed to develop a new RP-HPLC technique. The main purpose was to analyze SMX (Sulfamethoxazole) and TMP (Trimethoprim) in a liquid medium of 60mL. The analyses of SMX and TMP were performed on RP-HPLC with a C18 column (25 cm × 4.6 mm) packed with 5 µm ODS, L1 stationary phase, while the mobile phase consisted of methanol and water with a ratio of 6:4. The pH of the system was adjusted to 2.6 by using dilute phosphoric acid. The injection volume was 20μL having a flow rate of 1 mL/minute and column temperature of 40ºC. The analysis of all chromatograms was performed at a single wavelength of 254 nm. The validation of the method was determined for range, precision, linearity, specificity, accuracy, and system suitability. This method was found to be more environmentally friendly with respect to the other compendial methods, which are used for the TMP and SMX analysis.

Keywords: co-trimoxazole, chromatogram, RP-HPLC, sulfamethoxazole, trimethoprim

1. INTRODUCTION

Sulfamethoxazole (SMX) is an antibiotic drug that belongs to sulfanilamides [1]. It is utilized to address a range of health conditions that are generated within the body [2, 3]. These antibiotics can be provided to the body either through the mouth or by penetration through the injection [4]. They are quickly absorbed by the body and eliminated through the kidneys [5]. It is 4-amino–N-(5-methel-1,2-oxazol-3-yl)benzene-1-sulfonamide.  It is insoluble in water but easily soluble in acetone and sparingly soluble in alcohol [5, 6]. Trimethoprim (TMP) is a known biological agent, which inhibits bacterial activity. It is used to treat bacterial infections of the respiratory and urinary systems [7]. It is 2,4-diamino-5-(3’4’5’-trimethoxy benzyl)pyrimidine. These are white and cream colour substances [8]. The antibiotic mechanism of TMP is enhanced by sulfonamides [9]. They are used to cure bacterial infections [10]. The phenomenon of supra-additive takes place by sulfonamides addition. This is also the most common addition of multiple combinations for two antibiotic medicines of sulphamides. They have synergic effects and there is a 5:1 ratio of trimethoprim and sulfamethoxazole, respectively [11]. Their combination is known as co-trimoxazole, which is mainly used  to treat lung disorders like pneumonia, Coccidiosis, diarrhea, and gastroenteritis. They are also used for hyper-alimentation in the form of an aqueous solution. Their injections can cure bacterial infections in cattle and horses [12, 13].

Previous research has described that there are many analytical tools to determine SMX and TMP; either in individual form or in combination form [14]. A number of HPLC (high-pressure liquid chromatography) methods have been used for the estimation of TMP and SMX in pharmaceutical or biological samples [15]. Reverse phase high-pressure liquid chromatography (RP-HPLC) is the most used HPLC having 65-90%, respectively [16]. Reasonably, its extensive use is because of its features, which are unity and ease of its use for handling those substances, which have very diverse polarity [17, 18]. Compendial methods are also used to know the quality level of various medicinal products. These methods are not validated [18, 19].

In this research study, the RP-HPLC method was developed and its validity was checked for the identification of SMX and TMP as compared to the compendial methods.

2. MATERIALS AND METHODS

2.1. Reagent and Chemicals

Trimethoprims and sulfamethoxazole were bought from Shandong Rongyuan Pharmaceutical Company Limited, China and Andhra Organics Limited, India, respectively. The distilled water of 0.01 µS/cm conductivity was prepared in a laboratory. The source of methanol and orthophosphoric acid was Merck, Germany.

2.2. Instrumentation

The analyses were done by using the instruments including, Sonicator (Korea 60^oC), aluminumfoil (China 0.2 mm), hot plate (China 400^oC), analytical balance (Sartorius, Germany Min 0.0001g: Max 320 g), column (Merck Germany, C₁₈), nylon filters (Sartorius, Germany 0.45µ),  USA pH 0-14, pH metre (Jenco), vacuum pump (Japan20psi), pH meter (Jenco 6173), Shimadzu LC-20AT Series with a dual pump, Dynamica, HALO DB-20 UV/Visible spectrophotometer, and 254 nm wavelength was used for analysis.

2.3. Collection of Samples

The standard molecules of SMX and TMP were taken from Andhra Organics Ltd. in India and Shandong Rongyuan Pharmaceutical Co., Ltd. in China with high purity levels of 99.44% and 99.66%, respectively, on an anhydrous basis.

2.4. Mobile Phase Preparation

The mobile phase was prepared by adding 600 mL of methanol in 400 mL of distilled water and adjusting the volume to the desired amount. The pH level of the solution was maintained at 2.6 level by adding a small amount of dilute phosphoric acid.

2.5. Standard Preparation

200 mg of sulfamethoxazole and 40 mg of trimethoprim were put into a flask of 100 mL. The mixture was diluted using methanol. A separate 50 mL volumetric flask was used to transfer 10 mL of this solution for further analysis.

2.6. Sample Preparation

To prepare the samples, 200 mg of SMX and 40 mg of TMP were added to a flask of 100 mL. From this solution, 10 mL was transferred to a 50 mL flask and the resulting solution was filtered. The concentration of TMP in the filtered solution was found to be 0.08 mg/mL and the concentration of SMX was found to be 0.4 mg/L.

2.7. Chromatographic System Configuration

The column used for analysis was 25 cm in length and 4.6 mm in diameter, it was also loaded with an ODS (octadecylsilyl) stationary phase with a particle size of 5 µm, specifically the L1 type. The detector used in the analysis had a wavelength of 254 nm. The column was maintained at a temperature of 40°C and the flow rate of the mobile phase was set at 1 mL/minute. A sample injection volume of 20 µL was used for the analysis.

2.8. Method Validation

The developed method was validated through the following characteristics:

2.9. Linearity

Linearity is a measure of the relationship between the concentration of an analyte and the corresponding response of a measurement method. The coefficient of determination (r²) of the regression line is used to determine the linearity of the method. For quantitative analysis, a value of r² greater than 0.99 generally denotes a strong linear connection between the analyte concentration and the observed response, which is considered acceptable. This indicates that the test results obtained by the technique will fall within a predictable range that is directly proportional to the analyte concentration that is being measured.

2.10. Specificity

The ability to dissociate analyte components in the presence of other components like matrix components is known as specificity [17]. If the method remained unaffected in the presence of impurities and exponents, it means that the method has specificity.

2.11. Accuracy

Accuracy is a measure of how closely an analytical method can determine the true value of a sample. An accurate method gave us an accurate value under different measurements. The accuracy of the method was shown because no divergence occurred from the true value. Accuracy reflects the degree to which an analytical method provides reliable and correct results that are free from significant errors or biases.

2.12. Precision

If the procedure is repeated multiple times for the samplings of a homogeneous sample and the results give closed values to each other, the analytical method is said to have precision.

3. RESULTS AND DISCUSSION

It is mandatory to analyze the drugs before their utilization. Analyzing data can provide both qualitative and quantitative information, which is crucially significant. Understanding the therapeutic mechanism of drugs is a vital aspect that cannot be overlooked. A number of methods are being used for simultaneous analysis of trimethoprim and sulfamethoxazole. FT-IR, amperometry, HPTLC, UV spectroscopy, and spectrometry were used for SMX and TMP analysis (see Table 1).

Table 1. Assay Calculations

Figure 1. Chromatogram of TMP-SMZ Standard 1

Pharmaceutical industries specifically use RP-HPLC for conducting their analysis. The results obtained from the analysis of SMX and TMP are as follows (Figures 1 to 4; Tables 1-5).

Table 2. TMP-SMZ Standard 1 at 254 nm

Figure 2. Chromatogram of TMP-SMZ Standard 2

Table 3. TMP-SMZ Standard 2 at 254 nm

Figure 3. Chromatogram of TMP-SMZ Test Sample 1

Table 4. TMP-SMZ Test Sample 1 at 254 nm

Figure 4. Chromatogram of TMP-SMZ Test Sample 2

Table 5. Results of TMP-SMZ Test Sample 2 at 254 nm

3.1. Method validation

3.1.1. Linearity. The method will show linearity if the results of the test are directly proportional to the quantity of the drug [18]. The percent concentrations of 80, 90, 100, 110, and 120 were injected, giving the following linearity results. Table 6 and Figure 5 & 6 show the results for the linearity.

Table 6. Linearity Values

Figure 5. Linearity (Sulfamethoxazole)

Figure 6. Linearity (Trimethoprim)

3.1.2. Specificity. Specificity is the ability of a solution in which components of the matrix dissolve the analyte. In the case of standard and placebo, the following results were obtained (see Table 7).

Table 7. Specificity

3.1.3. Accuracy. Various trials were carried out to confirm the accuracy of this newly developed approach and the results are presented in Tables 8 and 9.

3.1.4. Sulfamethoxazole Results. Absorbance area of 80% reference: 16184214

Absorbance area of 100% reference: 16853525

Absorbance area of 120% reference: 17532744

Table 8. Accuracy Results for Sulfamethoxazole Samples

Table 9. Accuracy Results for Trimethoprim Samples

3.1.5. Precision. If there is a degree of closeness between the individual test findings, the procedure has precision.  The following results were obtained for instrument precision (see Table 10).

Table 10. Instrument precision

3.2. Method Precision

It was expressed as RSD and found out by:

3.2.1. Repeatability. The assay was performed on separate samples in 3 replicate sets (see Tables 11 and 12).

3.2.1.1. Sulfamethoxazole Results. Absorbance area of the reference: 16835314

Concentration of reference: 32 µg/ml

Wavelength: 254 nm

3.2.1.2. Trimethoprim Results. Absorbance area of the reference: 2519356

Concentration of reference: 32 µg/ml

Wavelength: 254 nm

Table 11. Repeatability of Sulfamethoxazole Samples

SD = 0.1849; Average = 100.01%; RSD = 0.1849%

Table 12. Repeatability of Trimethoprim Samples

Average = 100.16%; SD = 0.2280; RSD = 0.2277%

3.2.2. Reproducibility

3.2.2.1. Sulfamethoxazole Results. Absorbance area of the reference: 16813456

Concentration of reference: 32 µg/ml

Wavelength: 254 nm

3.2.2.2. Trimethoprim Results. Absorbance area of the reference: 2516209

Concentration of reference: 32 µg/ml

Wavelength: 254 nm

Table 13. Reproducibility of Sulfamethoxazole Samples

SD = 0.0771; RSD = 0.0770%; Mean = 100.06%

Table 14. Reproducibility of Trimethoprim Samples

Mean = 100.28%; SD = 0.1924; RSD = 0.1919%

The RP-HPLC method developed in this research study was found to be the most effective method among all, which covers all the analytical techniques [15–17]. It was also observed that this technique was better than compendial methods since it is free of pollution, which showed that it is environmentally friendly. The mobile phase is methanol: water (6:4). The pH of the mobile phase was maintained at 2.6 level by using dilute phosphoric acid. The combined determination of Sulfamethoxazole and Trimethoprim requires this pollution-free analysis.

4. CONCLUSION

The current study concluded that the newly developed RP-HPLC method was cost-effective and convenient to use. It also fulfilled all the validation parameters like linearity, specificity, accuracy, and precision. The primary significance of this method lies in its environmentally friendly nature and suitability for our environment.  Further studies can be used for the determination of impurities in samples using the LC-MS technique and future researchers can further establish their research by using this study results.

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