Green and sensitive spectrofluorimetric method for the determination of two cephalosporins in dosage forms

Using two green and sensitive spectrofluorimetric methods, we quantified two cephalosporins, cefepime (CFM) and cefazolin (CFZ), in raw and pharmaceutical formulations. The first method is based on the reaction between CFM and fluorescamine (borate buffer, pH 8), which yields a highly fluorescent product. After excitation at 384 nm, the fluorescent product emits light at 484 nm. At concentration ranges from 12.0 to 120.0 ng ml−1, the relative fluorescence intensity/concentration curve was linear with a limit of quantification (LOQ) of 2.46 ng ml−1. The second method relied on measuring the CFZ quenching action on acriflavine fluorescence through formation of an ion-associate complex using Britton–Robinson buffer at pH 8. We measured acriflavine fluorescence at 505 nm after excitation at 265 nm. The decrease in acriflavine fluorescence intensity was CFZ concentration-dependent. Using this method, we quantified CFZ in concentrations ranging from 1 to 10 µg ml−1 with a LOQ of 0.48 µg ml−1. We studied and optimized the factors influencing reaction product formation. Moreover, we adapted our methods to the investigation of the mentioned drugs in raw and pharmaceutical formulations with greatly satisfying results. We statistically validated our methods according to International Council on Harmonisation Guidelines. The obtained results were consistent with those obtained with the official high-performance liquid chromatography methods.


Introduction
Cephalosporins are the most beneficial β-lactam antibiotics after penicillin. They are commonly used to treat bacterial infections [1]. Cephalosporins are semisynthetic antimicrobials derived from natural antibacterial, cephalosporin C, which was produced by the mould, Cephalosporium acremonium. Cephalosporins exert their bactericidal effect by inhibiting bacterial cell wall synthesis. Cephalosporins are usually classified by 'generation'. Cephalosporins from each generation usually have similar antibacterial activity, but it may partly depend on when they were discovered [1].
Cefepime (CFM) hydrochloride (figure 1a) is a broad-spectrum, fourth-generation parenteral cephalosporin used against pneumonia caused by many organisms and for urinary tract infections [1].
The British Pharmacopoeia [2] and the United States Pharmacopeia [3] listed CFM and CFZ as standard medicinal products.
Since both CFM and CFZ have no native fluorescence, quantifying them through spectrofluorometry requires derivatization. In this study, we selected spectrofluorimetry for its high sensitivity, low cost and wide availability in most quality control laboratories. Besides their high sensitivity, our methods have several advantages over the previously published spectrofluorimetric methods. They are simple, rapid, inexpensive and environment-friendly. On the other hand, the previously published spectrofluorimetric methods suffered from using tedious and complex procedure with limited sensitivity as in the case of Hantzsch reaction [15], complexation with terbium which used highly expensive reagents [16] and reactions with safranin required toxic and environmentally harmful organic solvents [17]. Since we used water (a highly green solvent) as a solubilizing and diluting solvent, we considered our proposed quantification methods green. We aimed to create highly sensitive, simple, inexpensive and safe spectrofluorimetric methods to quantify the previously mentioned cephalosporins. In Method I, CFM reacted with fluorescamine in a borate buffer solution (pH 8) to form a product with a high emission at 484 nm after excitation at 384 nm. By contrast, Method II used the quenching impact of CFZ on acriflavine reagent native fluorescence. CFZ formed a non-fluorescent ion-associate complex with acriflavine in a Britton-Robinson buffer solution ( pH 8). Moreover, Method II could assess the stability of CFZ since it depended on the presence of the carboxylic group that was absent in the degradation product of the drug [

Preparation of stock solutions
We freshly prepared the CFM hydrochloride and CFZ sodium aqueous stock solutions by dissolving 10 mg of each cephalosporin separately in a 100 ml volumetric flask using double-distilled water to attain 100 µg ml −1 . We prepared the working solutions by diluting the stock solutions adequately using the same solvent.

Recommended procedures 2.4.1. Procedure for calibration graphs
For method I, we transferred accurately measured volumes of the CFM working solution into 10 ml volumetric flasks to obtain concentrations of 12-120 ng ml −1 . We added 1 ml of borate buffer ( pH 8), mixed, added 1 ml of fluorescamine 0.02% (w/v), mixed and filled the flasks with double-distilled water to the mark. After excitation at 384 nm, we measured the fluorescence of the resulting solutions at 484 nm. We plotted the relative fluorescence intensity against CFM concentration and generated the corresponding regression equation.
For Method II, we transferred accurately measured volumes of the CFZ working solution into 10 ml volumetric flasks to obtain concentrations of 1-10 µg ml −1 . We added 1 ml of Britton-Robinson buffer (pH 8) and 0.9 ml of 2 × 10 −6 M acriflavine solution and then thoroughly mixed and filled the flasks with double-distilled water. After excitation at 265 nm, we measured the decrease in fluorescence intensity at 505 nm. To establish the calibration curve, we plotted the decrease in fluorescence intensity (ΔF) against the CFZ concentration and then derived the regression equation. In both methods, we measured a blank sample (without the drug).

Procedure for assay of vials
We accurately weighed 10 mg of sterile powder from the CFM or zinol vials and transferred it to a 100 ml volumetric flask. We filled the flask with double-distilled water and sonicated it for 5 min. We then royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329 carried out the same procedure as stated under calibration graphs and calculated the concentrations using the derived regression equations.

Results and discussion
-Method I (using fluorescamine) Fluorescamine was previously used to quantify many pharmaceutically important compounds with a primary amino group [28][29][30][31]. Fluorescamine is an extremely poor fluorescent derivatizing agent. However, it reacts swiftly with primary amines yielding derivatives with high fluorescence. Then, excess fluorescamine reacts with water to yield a non-fluorescent compound. It is thus useful to quantify water-soluble compounds with amine groups [32,33]. CFM, non-fluorescent compound with a primary amine, reacts with fluorescamine in a pH 8 borate buffer resulting in a fluorescent product. The formed fluorophore emits light at 484 nm after excitation at 384 nm (figure 2).
-Method II (using acriflavine) Acridine dyes like acriflavine are highly fluorescent natural compounds that can be used as derivatizing agents [34]. Acriflavine was used for estimation of compounds having pharmaceutical interest such as ascorbic acid [35]. CFZ is non-fluorescent and has a free carboxylic group that ionizes (forming an anion) under alkaline conditions ( pH 8). Under the same conditions, the cationic form of acriflavine predominates. The two compounds form a complex via electrostatic interaction between the two oppositely charged ions. The acriflavine reagent fluorescence intensity was measured at emission wavelength 505 nm subsequent to excitation at 265 nm and decreased appreciably upon addition of CFZ drug (figure 3). The quenching effect of CFZ was concentration-dependent (figure 4). We then carefully investigated and optimized the experimental parameters that affected the reaction product for both methods.
Comparison was performed between the two suggested methods and the previously reported ones (table 1).

Experimental condition optimization
The fluorescence of the CFM-fluorescamine derivatization product occurs under alkaline conditions and disappeared under acidic conditions [31]. We thus performed our experiments using borate buffer at pH royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329 7.5-12. We selected the borate buffer as it results in higher fluorescence intensity than other buffers of the same pH [31]. We obtained the highest fluorescence intensity at pH 8 ± 0.2 (figure 5a).

Effect of fluorescamine concentration
We assessed the impact of fluorescamine amount on the fluorescence intensity by recording the fluorescence intensity of solutions containing a fixed CFM concentration and different reagent volumes. We found that 1 ml of fluorescamine 0.02% (w/v) produced maximum fluorescence. At higher volumes, we observed a slight fluorescence intensity decrease (figure 6a).

Effect of reaction time and stability
We noticed that, at room temperature, the fluorescent product formed instantly, and the fluorescence intensity reached a maximum within 5 min. It remained stable for at least 3 h and then slowly decreased.

.3. Effect of reaction time
At room temperature, the effect of reaction time has been studied to detect the time needed for complex formation which is indicated by maximum fluorescence quenching. The derivatization reaction occurred immediately after mixing and remained stable for up to 2 h.

Validation of the developed methods
To assess the validity of the two proposed methods, we determined the linearity, detection and quantification limits, accuracy, precision and specificity, as recommended by International Conference on Harmonization (ICH) Q2(R1) guidelines [36].

Linearity and quantification/detection limits
Under the optimal reaction conditions, the two proposed methods exhibit linearity in the ranges cited in table 2, with r = 0.9999. We calculated the limit of quantification (LOQ) and limit of detection (LOD) following the ICH Q2(R1) [36] using the following equations: where S a is the standard deviation of the calibration graph intercept and b is the calibration graph slope. Table 2 shows the LOD and LOQ values for the investigated drugs. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329

Accuracy
Our methods are suitable to quantify the studied drugs over the concentration ranges shown in table 2.
We compared the results of our methods with findings of the official chromatographic methods [3]. The  The values between parentheses are the tabulated values of t and F at p = 0.05 [37]. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329 official CFM quantification method is based on HPLC using sodium-1-pentane sulfonate solution/ acetonitrile (94 : 6 v/v) as a mobile phase with UV detection at 254 nm. The official CFZ quantification method also uses chromatography with anhydrous dibasic sodium phosphate and citric acid solution/acetonitrile (90 : 10 v/v) as a mobile phase and UV detection at 254 nm. The statistical comparison [37] of our methods and the official methods [3] using Student's t-test and variance ratio F-test revealed no substantial accuracy and precision differences (table 3). Table 4. Precision data for the determination of the the studied drugs using the two proposed spectrofluorimetric methods. The values between parentheses are the tabulated values of t and F at p = 0.05 [37]. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329

Precision
To check precision, we used three concentrations and performed triplicate measurements for each of them. Table 4 shows the results. The % relative standard deviation (RSD) values were inferior to 2, which confirmed the acceptable precision of our methods.

Robustness
To assess the robustness of our methods, we measured fluorescence constancy with little intentional variations in the empirical conditions such as fluorescamine volume (1.0 ± 0.2 ml) for method I, acriflavine volume (0.9 ± 0.2 ml) for method II, and change in pH (8.0 ± 0.2) for both methods. These minute alterations might occur during practical experiments and did not affect the emission intensity of the formed product.

Selectivity
Since the spectrofluorimetric quantification of the two cephalosporins was based on chemical reactions, our methods have high selectivity towards the investigated drugs due to the presence of certain functional groups. In Method II, CFZ can be quantified in the presence of its degradation product.

Application of the proposed methods to pharmaceuticals analysis
Our methods are suitable for the quantification of CFM and CFZ in their dosage forms. Our results showed good agreement with findings obtained using the official methods [3] (table 5). Student's t-test and F-test showed that there was no appreciable difference in their performances [37]. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 8: 210329 11

Assessment of greenness
We assessed the greenness of our methods using the analytical eco-scale method. Methods I (fluorescamine) and II (acriflavine) obtained scores of 92 and 89, respectively, which are both excellent green (table 6).

Conclusion
We applied two different simple derivatization reactions to quantify two important cephalosporins, CFM and CFZ, in their dosage forms. Our methods are rapid, sensitive, effortless and cheap. Moreover, these methods are green (they have little impact on the environment). Another important feature is their high selectively for the studied drugs, thanks to the selectivity of the reactions. Moreover, method II can assess the stability of CFZ because acriflavine does not react with the CFZ degradation product.
Data accessibility. The data of the work were deposited in the Dryad Digital Repository: https://doi.org/10.5061/dryad. 866t1g1q3 [38]. Competing interests. We declare we have no competing interests. Funding. We received no funding for this study.