Validation of a Novel and Sensitive RP-HPLC

CFX; is chemically known as (6R,7R)-7-[[(Z)-2-(2Aminothiazol-4-yl)-2-[(carboxymethoxy) imino] acetyl] amino]-3ethenyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid trihydrate. It has a molecular formula of C16H15N5O7S2, 3H2O and a molecular weight of 507.5 [1,2] (Figure 1A). CFX is a white or almost white, slightly hygroscopic powder. Slightly soluble in water, soluble in methanol, sparingly soluble in anhydrous ethanol, practically insoluble in ethyl acetate. A 5% suspension in water has a pH of 2.6 to 4.1. Stored in airtight container to be protected from light [1,2]. CFX is bactericidal and is stable to hydrolysis by many beta-lactamases. Like other Cephalosporins, CFX possesses a mechanism of action similar to penicillins i.e. inhibition of transpeptidation process resulting in the formation of imperfect cell wall; osmotic drive from the outside isotonic environment of the host cell to the inside of the hypertonic bacterial cytoplasm and finally activation of the autolysin enzyme leading to the lysis of bacteria. Only 40 to 50% of an oral dose of CFX is absorbed from the gastrointestinal tract, whether taken before or after meals, although the rate of absorption may Abstract


CFX; is chemically known as (6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2-[(carboxymethoxy) imino] acetyl] amino]-3ethenyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid trihydrate.
It has a molecular formula of C 16 H 15 N 5 O 7 S 2 , 3H 2 O and a molecular weight of 507.5 [1,2] (Figure 1A). CFX is a white or almost white, slightly hygroscopic powder. Slightly soluble in water, soluble in methanol, sparingly soluble in anhydrous ethanol, practically insoluble in ethyl acetate. A 5% suspension in water has a pH of 2.6 to 4.1. Stored in airtight container to be protected from light [1,2]. CFX is bactericidal and is stable to hydrolysis by many beta-lactamases. Like other Cephalosporins, CFX possesses a mechanism of action similar to penicillins i.e. inhibition of transpeptidation process resulting in the formation of imperfect cell wall; osmotic drive from the outside isotonic environment of the host cell to the inside of the hypertonic bacterial cytoplasm and finally activation of the autolysin enzyme leading to the lysis of bacteria. Only 40 to 50% of an oral dose of CFX is absorbed from the gastrointestinal tract, whether taken before or after meals, although the rate of absorption may be decreased in the presence of food [3]. CFX is better absorbed from oral suspension than from tablets. CFX is generally classified as a third -generation Cephalosporin antibacterial and is given orally to treat infection due to susceptible Gram -positive and Gram-negative bacteria, including gonorrhea and infections of the respiratory and urinary tracts [3]. In spite of the significant progress in chemotherapy of bacterial infections, it was needed to reveal the mystery about the deaths of infants and children with known bacterial infections [4]. In order to establish or confirm the cause of death of children in whom bacterial infections cases, it is very often questioned whether the therapy applied or medicine prescribed were correct and, indeed whether the prescribed medicine was administered at all. So the problem of establishing their presence in post-mortem material can form a problem for forensic medical work. Thus, the development of accurate and simple methods for the determination of traces of CFX besides the isolation and cleanup process by using solid phase extraction for fluoroquinolones and cephalosporins antibiotic drugs, contribute much for the analysis of forensic samples [5]. SDM; is chemically known as sodium benzenecarboxylate. It has a molecular formula of C 7 H 5 NaO 2 and a molecular weight of 144.1 [1,2] (Figure 1B). SDM is a white or almost white, crystalline or granular powder or flakes, slightly hygroscopic. Freely soluble in water, sparingly soluble in ethanol (90 per cent V/V) [1,2]. SDM is used primarily as antimicrobial preservative in cosmetics, foods, and pharmaceuticals. It used in concentrations of 0.02-0.5% in oral medicines, 0.5% in parenteral products, and 0.1-0.5% in cosmetics. The inhibitory concentration of sodium benzoate required in emulsion increases with oil content. SDM has also been used as a tablet lubricant at 2-5% w/w concentrations, providing rapid disintegration times [6]. SDM is mandatory in forensic toxicology laboratories to identify drug abuse; quantification in saliva is important additional information as this gives an indication about the level of abuse, where citric acid is used to stimulate the saliva production and sodium benzoate is used as preservative [7].

I. Mobile phase preparation: Buffer: Acetonitrile (50:50)
Sodium dihydrogen buffer is prepared by dissolving 2.76 gm of sodium dihydrogen phosphate monohydrate in 700 mL distilled water and sonicated to dissolve, adjust the pH to 2.8 by the addition of orthophosphoric acid solution. Make up to 1000 mL with distilled water, filter and degase mixtures of buffer and acetonitrile (50:50) through 0.45μ membrane filter under vacuum pump.

HPLC chromatographic conditions
Chromatographic separation is performed on column Agilent Eclipse XDB-C8 (250 X 4.6 mm i.d, 5 µm particle size). Using a mobile phase mixture of sodium dihydrogen phosphate buffer and acetonitrile in the ratio of 50:50 % v/v at ambient temperature, flow rate of 1.0 mL/min, UV detection is performed at 229 nm, injection volume is 10 μL and run time is 7.0 min.

A. Standard solutions preparation of Cefixime and Sodium benzoate:
The stock standard solution (1.6 mg mL -1 of CFX and 0.04 mg mL -1 of SDM) is prepared by dissolving 160 mg of CFX and 4 mg of SDM in 70 mL of diluent and is sonicated to dissolve. Complete to the mark with the same diluent and mix well. Standard solution is prepared by transferring 5 mL of the standard stock solution to 50 mL volumetric flask of diluent to get a concentration of 0.16 mg mL -1 of CFX and 0.004 mg mL -1 of SDM. The obtained chromatogram is shown in Figure 3A.

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B. Authentic prepared mixture: Due to the low concentration of SDM in the dosage form, where the concentration of CFX is Forty-folds greater than SDM, so we prepared an equal concentration (0.2 mg mL -1 ) of each in the mixture to validate their simultaneous determination. The obtained chromatogram is shown in Figure 4.

Application to pharmaceutical formulation (SUPRAX® 100 mg/5mL POS (60mL) & (30mL))
Constitute Suprax as directed in the labeling. Transfer an accurately 2.0 mL of the oral suspension into a 250-mL volumetric flask. Dilute with solvent to achieve a final concentration of 0.16 mg mL -1 for CFX and 0.004 mg mL -1 for SDM. The obtained chromatogram is shown in Figures 3B & 3C. Also, the standard addition technique has been carried out to assess the validity of the method by spiking the pharmaceutical formulation with known amount of standard solution of CFX and SDM. The recovery of the added standards is then calculated after applying the proposed method.

Construction of calibration curves
Different concentrations of CFX and SDM equivalent to (5-200) μg /mL and (2-40) μg /mL for CFX and SDM, respectively, are separately withdrawn from their respective stock standards into separate series of 100 mL volumetric flasks, and the volumes are made up to volume with the diluent. Duplicate 10 µL injections are made for each concentration maintaining the flow rate at 1.0 mL/min and the effluent is UV-scanned at 229 nm. The chromatographic separation is performed following the procedure under chromatographic conditions. The chromatograms are recorded, peak areas of CFX and SDM are determined, the calibration curves relating the obtained integrated peak areas to the corresponding concentrations are constructed and the regression equations are performed.

Results and Discussion
The purpose of this work is to present simple, sensitive, accurate, precise and novel HPLC method for the simultaneous determination of binary mixture of the two drugs, viz., CFX and SDM in their pure form and in pharmaceutical formulation. Also, to achieve the concentration and recovery for laboratory prepared mixture and applying the standard addition technique for each method. Obtained chromatograms have significantly different R t values of 2.203 and 3.970 min for CFX and with correlation coefficients (r) >0.9999, limits of detection of 1.36 and 0.21 µg mL -1 and limits of quantitation of 4.14 and 0.64 µg mL -1 for CFX and SDM, respectively. No occurrence of interfering peaks is reported.  Figure 5). Preliminary studies involved trying C18, C8 reversed-phase columns, finally, the column Agilent Eclipse XDB-C8 (250 mm X 4.6 mm i.d., 5μm) achieved the best resolution between both drugs and sharp peaks with good R t values without tailing of the separated bands and good theoretical plates.

Validation of the analytical method
The method was validated, in accordance with ICH guidelines (ICH Q2R1), for system suitability, precision, accuracy, linearity, specificity, ruggedness, robustness, LOD and LOQ.  Table 1.

II. Repeatability:
Repeatability of the method is evaluated by calculating the RSD of the peak areas of six replicate injections from the standard concentration (160.0 μg/mL) of CFX and (4.0 μg/mL) of SDM. Results are examined as % RSD values of the concentrations of determined drugs. Low values of % RSD (less than 2) indicate high precision of the method as shown in Table  1.

III. Detection and quantitation limits:
These approaches are based on the Standard Deviation of the Response and the Slope. A specific calibration curve should be studied using samples containing an analyte in the range of LOD and LOQ. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. LOD=3.3 × σ /slope and LOQ =10 × σ /slope, where σ = the standard deviation of the response, Table 1.

IV. Accuracy and recovery:
Accuracy of the proposed methods is calculated as the percentage recoveries of pure samples of the studied drugs. Accuracy is assessed using three different concentrations (40,

V. Formulation assay:
The validated method is applied for the determination of CFX and SDM in commercially available SUPRAX® 100 mg/5 mL POS (60 mL) & (30 mL). The results of the assay undertaken yielded recoveries of 103.51% and 99.90%, 99.93% and 97.64% of the label claim for CFX and SDM, respectively. The results of the assay indicate that the method is selective for the analysis of SUPRAX® 100 mg/5 mL POS (60 mL) & (30 mL) without interference from the excipients used to formulate and produce these suspensions. The results are displayed in Tables 5 & 6. The obtained results for the analysis of CFX and SDM by the suggested methods are statistically compared with those obtained by applying the official HPLC [8] and reported HPLC [55] methods. The results showed no significant differences between the proposed and HPLC methods as presented in Table 7.

VI. Intermediate precision (ruggedness):
Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipments, etc. Good results are obtained and presented in Table 8.

VII. Robustness:
The robustness of the proposed methods is evaluated within the development phase where the different factors affecting the method are studied to obtain the optimum parameters for complete separation. Robustness of the method is studied by deliberately varying parameters like flow rate (± 0.1 mL/min), studying the effect of changing the mobile phase pH by (± 0.2), acetonitrile composition (± 5%) and column temperature which is changed between (30° & 25°). The low values of the % RSD, as given in Table 9, indicate the robustness of the proposed methods.

VIII.
System suitability: System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability is checked by calculating tailing factor (T), column efficiency (N), resolution (R s ) factors. All calculated parameters are within the acceptable limits indicating good selectivity of the methods and ensuring system performance, (Table 10).

IX. Stability of analytical solution:
To demonstrate the stability of the standard solution during analysis, the solution is analyzed over a period of 24 h at room temperature and refrigerator. The results showed that for all solutions, the retention times and peak areas of CFX and SDM remained

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almost unchanged (RSD<2.0%) indicating that no significant degradation occurred within this period, i.e. both solutions are stable for at least 24 h, which is sufficient to complete the whole analytical process. These results are displayed in Table 11. X. Specificity: Specificity is tested against standard compounds and against potential interferences in the presence of placebo. No interference is detected at the retention times of CFX and SDM in placebo solution.

Conclusion
The proposed RP-HPLC method for simultaneous estimation of CFX and SDM in their dosage form is novel, precise, specific, highly accurate and less time consuming for analysis, low cost and rapid. The results of stress testing undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective. Based on the above results, the analytical method is valid, fit for use and can be used for regular routine analysis, stability study and forensic sciences & criminal investigation.