INTRODUCTION:

Disinfectants and antiseptics both are used to some extend by individuals and hospitals and when put to good use they can become a best choice against microbes^1-3. Cetrimide is a quaternary ammonium compound and polar as cationic surfactant and widely used in the medicine^4-6. It consists t wo parts: the hydrophobic chain which is the large part, and quaternary ammonium ion with a positive charge, which is the part that is responsible for the antiseptic action of the molecule^7,8,9. Due to its polarity Cetrimide remain ionic in the whole range of pH^10-1³.

MATERIALS AND METHOD:

Materials:

Pure Cetrimide (CE) as a standard was supplied by Samara Drug Industries (SDI) in Iraq. Different samples of cream that contain Cetrimide commercially available were used. Methanol and Acetonitrile HPLC grade (BDH), acetic acid and ammonium acetate salt powder (BDH) and deionized water which freshly prepared were used throughout the experiment.

Instruments and chromatographic analysis conditions:

The instruments are from the following countries: Sartorius balance (Germany), shaking water bath (Taiwan), Karl Kolb (German) for sonicating bath, Shimadzu LC-20 A (Japan) for HPLC, and Memmert (German) for oven. For the separation of CE, a Phenomenex C-18 (15 × 4.6mm I.D. and 5m particle size) was employed. Under reversed phase partition conditions, the analysis was done for 6 minutes at room temperature (~25 ^oC). Acetic acid was used to set the pH at 3.0 with a flow rate 1.0mL/min, and the mobile phase was an ammonium acetate solution 0.1M (AA), methanol (MeOH), and methyl cyanide in the ratio of (15:60:25 V/V/V). Using a loop injector with a volume of 10μL, samples were injected, and 280nm was used for detection. Before analysis, the mobile phase was degassed using ultrasonic cleaner equipment (Power Sonic 420), followed by using nylon filter 0.45μm for filtration. By contrasting the compound's retention period in the sample solution with the reference solution, the compound's identity was determined. Chromatography was done in a column with the temperature kept at around 25±2°C.

Cetrimide stock solutions (1000 mg/L) preparation:

In a volumetric flask of 100mL, 0.1g of the standard Cetrimide has dissolved to the mark using (AA: ACN: MOH 15:60:25 V/V). Different diluted concentrations of medicines were prepared using stock solutions.

Diluent:

The second dilution, which employed a stock solution of 1000 μg/mL, was carried out by taking various aliquots (0.1–10mL) from the standard solution and then transferring them to calibrated volumetric flasks having a 10 mL capacity. To prepare standard solutions at various concentrations (10–1000μg/mL), each of these was brought up to the mark with mobile phase.

The Cetrimide assay in pharmaceuticals form:

Two samples of the CE-formulated cream were precisely weighed. In a 100mL volumetric flask, 0.5g was dissolved using the mobile phase after the solvent had been added to the appropriate level. A volume containing the right quantity of each cream in accordance with the calibration curve's range was put into a 25mL bottle. To determine the drug concentration, a straight line equation was used.

RESULT AND DISCUSSION:

Method optimization:

Several attempts were made employing different mobile phases with predetermined percentage of water and the organic solvents in order to obtain good separation resolution of CE¹⁴. The outcome demonstrates that the optimal mobile phase is about 0.1M of ammonium acetate solution, which is composed of acetonitrile (ACN), methanol (MOH), and in the ratio (15:60:25 V/V/V) of methanol. When the mobile phase pH is altered by ±0.2, the retention time of the analyte does not change considerably. The CE retention time in the column was estimated for 10μL injection volume at flow rate 1.0mL/min. The created samples, however, made it possible to determine the retention time accurately for both the CE sample and the reference. The optimization was concentrated on the requirements to create a straightforward, quick, and affordable strategy, through which the mobile phase can produce satisfactory results. In order to determine the chromatographic conditions and provide a better separation some conditions have been changed such as; the type and the strength (volume fraction of organic solvents for mobile phase and the buffer solution pH)¹⁵ of solvents, the rate of flow and wavelength. The mobile phase's parameters have been tuned to eliminate any solvent or excipient influence. See Table 1 lists for ideal chromatographic conditions. The CE chromatogram that obtained under ideal conditions is shown in Figure 1.

Table 1: The optimum parameters for optimized method

Mobile phase 0.1 M AA: MOH: ACN 15:60:25 pH = 3	Column type: ODS: 150mm, 4.6 mm, 5.0µm
Mobile phase 0.1 M AA: MOH: ACN 15:60:25 pH = 3	Sample temperature: ambient
rate of Flow 1.0mL/min.	Temperature of Column: 25±2°C
Volume of Injection 10µL	Run time 5.00min
Wavelength nm 280	Retention time 1.803min

Fig. 1: The chromatogram of Cetrimide using optimized condition

Calibration graphs of HPLC:

Towards the preparation of series of standard solutions, range of drug concentrations (10–1000mg/L) were generated from the standard stock solution. Then the prepared solutions have injected via the injector loop of 10 μL, and the chromatograms were recorded¹⁶. Plotting graphs of the concentration versus the peaks area resulted in a straight line.

System Suitability Test:

The tailing factor, Column efficiency, and the observed values of Resolution are listed in Table 2.

Table 2: Suitability study for CE.

Parameters of Suitability	Results	Acceptance Criteria
Time of Retention	1.803	Acceptance Criteria
The area percentage of RSD of CE for 7 injections	0.415	NMT 2.0
CE peak tailing factor	1.502	NMT 2.0
The CEP theoretical plates	3022	NLT 2000

Analytical validation:

The optimized approach has been validated correspondingly to the guidelines of ICH Q2 R¹⁷. As tests were used for the validation, linearity, precision, specificity, accuracy, ruggedness, robustness, LOD, and LOQ.

Linearity and Range:

For the concentration range of 10 to 1000mg/L of CE, the recommended revers phase HPLC exhibits high linearity; see Figure 2. For CE, the calibration's intercept and slope values were y = 1618.5 x - 19620 (R² = 0.9985), the sizable correlation between the concentration of drug and the response factor within the concentration range are shown in Table 3, and the results appear to be satisfactory.

Fig. 2: The Cetrimide calibration graph using HPLC method.

Precision:

The interday precision has been assessed by the samples analyzing using different concentrations of CE separately in three different days, while the intraday precision has been estimated by analyzing CE samples using different concentrations as a three times daily to calculate the % RSD. Five times during the day, the CE standard solution (80 mg/L) was injected to assess repeatability and determine the value of % RSD 17. Table 3 presents the outcomes that were attained.

LOD and LOQ:

Table 3: Illustrates the obtained results for the calculation of LOD and LOQ using gradual dilution of the lowest concentration.

Table 3: the summary of parameters validation.

Sr. No	Parameters of validation	Results	Standard values
1	Range of Linearity	10 – 1000mg/L	-
2	equation for Straight line	y =1618.5 x - 19620	-
3	Coefficient of Correlation	0.9985	≤ 0.9999
4	Precision (% R.S.D.)		≤ 2.0 % R.S.D.
4	Repeatability Intraday Interday	0.51 0.43 0.68	≤ 2.0 % R.S.D.
5	Mean % Recovery	97.56	95.0 – 105.0 %
6	Specificity	Specific
7	LOD (µg/mL)	5	-
8	LOQ (µg/mL)	16.5	-
9	Ruggedness	Complies	≤ 2.0 % R.S.D.
10	Robustness		≤ 2.0 % R.S.D.
10	Flow rate change Wavelength change Solution pH change	Complies	≤ 2.0 % R.S.D.

The accuracy:

The accuracy of the results for the analytical methods was investigated, and three distinct Cetrimide concentrations of 50, 150, and 300mg/L were selected for the HPLC method¹⁸. Regarding techniques and the computation of parameters, the established approach for the detection of cetrimide was fascinating and quite practical. Table 4 provides an overview of the proposed methodologies' data precision and accuracy.

Table 4: The accuracy of method for drugs determination.

Cetrimide μg/mL		%Recovery		%Error	R.S.D n =3
Taken	Found	%Recovery		%Error	R.S.D n =3
50	48.84	97.68	Mean= 97.56	-2.32	0.32
150	145.13	96.75		-3.25	0.45
300	294.75	98.25		- 1.75	0.33

Specificity:

To study the specificity, the capacity that evaluates the analyte in the presence of elements would be anticipated to be present. By comparing the placebo chromatogram with the one that obtained from the CE standard solution, led to discovered that there were no inferring peaks at the CE's retention period when a solution of placebo was introduced into the mobile phase. The methods of specificity were demonstrated using strong association for tR and area. Figures 3 and 4 show chromatograms demonstrating the CE's specificity.

Fig: 3: Specificity chromatogram of the placebo in 0.1 AA: MOH: ACN 15:60:25.

Fig: 4: Specificity chromatogram of standard CE (1000 mg/ L).

Ruggedness:

By analyzing aliquots of CE solution (700mg/L) with two analysts under identical operational and environmental settings, the robustness of the proposed approach was evaluated¹⁹. The results are shown in Table 5.

Robustness:

The rate of flow has adjusted by ±0.1mL/min (0.9 mL/min and 1.1mL/min), the pH of mobile phase for the buffer solution was adjusted by ±0.2% (2.8 and 3.2%), and the detection wavelength was adjusted at 278nm and 282nm by ±2nm in order to assess the robustness of the method. The outcomes are in Table 5.

Table 5: The results for the both method the Ruggedness and robustness.

Robustness results
Parameters of the Method	Mean n = 3	S.D.	R.S.D. %
Rate of Flow 1.0 ± 0.1 mL/min.	98.96	0.9117	0.9212
pH of Mobile phase 3.0 ± 0.2	97.58	0.7183	0.7361
Wavelength of Detection 280 ± 2 nm	96.91	0.8401	0.8668
Ruggedness results
	1^ST Analyst		2^nd Analyst
Mean % Assay* ± SD	97.76 ± 0.460		97.82 ± 0.570
% R.S.D.	0.4725		0.5827

Analytical assays:

Through the use of UV detection at 280nm and a validated high performance liquid chromatography (HPLC) method, the two samples as cream formula were examined for the presence of CE. The material was injected into the HPLC analysis with 10μL under the ideal separation conditions. Ammonium acetate solution 0.1M (AA), methanol (MOH), and acetonitrile (ACN) were employed as the eluent phase 1.0mL/min for flow of rate at 280nm. The column is a Phenomenex C-18, with 5m-sized particles and 150mm 4.6mm I.D. The analysis took place at a temperature of 25^oC for a total of 4 minutes. The obtained results are listed in Table 6. The chromatograms in Figure 5 shows the drugs separation in their formulas. The drugs recoveries in samples were 95.61 and 96.02%.

Fig. 5: Chromatogram of pharmaceutical preparation (WADI – AL-RAFIDAIN and Modern company).

Table 6: The drugs estimation quantity in different formulas

Cetrimide	Label Claim	Mean amount	% Mean amount	R.S.D
Company	W/W%	found mg/tab.	found	n = 3
WADI – AL-RAFIDAIN	0.5	0.4801	96.02	0.35
Modern	0.5	0.4781	95.61	0.51

CONCLUSION:

The results were obtained using the validated UV-spectroscopy and RP-HPLC procedures, which appear to be straightforward, quick, accurate, precise, and resilient. However, they can be used for the routine analysis of CEP in their formulated and aqueous solutions.

ACKNOWLEDGMENTS:

We would like to thanks the Department of Chemistry/ College of Education for Pure Science/ University of Diyala for the facilities.

AUTHORS' CONTRIBUTIONS:

All the authors have contributed equally.

CONFLICT OF INTEREST:

Declared none.

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Received on 26.05.2023 Modified on 12.08.2023

Research J. Pharm. and Tech 2023; 16(12):5581-5585.

DOI: 10.52711/0974-360X.2023.00902