Cephalothin is a first-generation cephalosporin, that shows great activity against Gram-positive microorganisms. Its effect is bactericidal, and due its action, it is the most efficient first-generation cephalosporin against resistant microorganism (β-lactamase producers). Although this drug has been clearly studied and researched about its antimicrobial activity, pharmacokinetics and pharmacodynamics, there are a few studies in literature regarding the development of analytical methodology for this cephalosporin. The aim of this work was to develop and validate a new method of analysis, using high performance liquid chromatography, resulting in an innovative method, quick and using solvents of low toxicity, minimizing, in this way, its toxic actions to the operators and leavings in the environment. The method developed and validated for the quantification of sodium cephalothin in lyophilized powder for injectable solution used high performance liquid chromatography (HPLC). The mobile phase consisted in water with 0.7% of glacial acetic acid and ethanol (70:30 v/v), wave-length of 237 nm, Zorbax Eclipse Plus C18 AgilentTM column and room temperature of 25°C, retention time of 4,20 minutes. The method was linear in the concentrations of 20, 40, 60, 80 e 100 μg/mL, selective, accurate and robust towards these modifications: ethanol brand, water source, mobile phase rate, glacial acetic acid proportion, flow rate, room temperature and wave length. The dosing for CET was of 106.72%.
Keywords: Cephalotin sodium; Green method; HPLC; Method validation; Quality control
With the advance of the infection diseases and the increase in the world mortality rate, because of pathogenic microorganisms, there came the necessity of discovering substances that would be able to fight this. The last decades were dedicated to the search of new drugs, with great importance the period of 1950 a 1970, known as “The golden era” for the discovery of antimicrobials, emerging several classes of them . However, after this period, there was a decrease in the development of new molecules, what brought the worry about resistant microorganisms. In this way, a new approach to fight bacterial infections was through the improvement of this molecules already used .
An important class of antimicrobials is the cephalosporins, originally produced by Cephalosporium acremonium. The cephalosporins are classified as beta-lactam antibiotics, however, they show a broader action spectrum when compared to penicillins, because they are resistant of penicillinases. Changes in its structure gives a higher potency to this substance .
Cephalothin was one of the first modification obtained from the 7-aminocephalosporanic acid, the pharmacological structure of cephalosporins, classifying as a first-generation cephalosporin. This drug shows higher activity against Gram positive and less against Gram negative . Due its instability in acid, it is administered parenterally. Figure 1 shows the chemical structure of the drug.
The development and validation of analytical methods for the determination of the quality of final products is extremely important, mainly when related to pharmaceutical products [4,5]. The evaluation of quality will determine the efficacy of cephalosporin pharmaceutical products and avoid damage in the patient health [6-11]. Some studies relating the quality control of cephalothin were found in literature for its quantification in biological matrices [12-20], and for analysis of CET in pharmaceutical dosage form [21-24]. Its monograph is in pharmacopoeias like Brazilian , United States , United Kingdom , European  and Japanese Pharmacopoeia .
Besides the importance of quality control, a crescent worry about environment and the worker makes necessary the development of conscious methods and less pollutants, in this way, the aim of this work is to improve the already validated technic for HPLC with a green chemistry approach, using less toxic solvents and a decrease in formation of residues .
The CLAE method was performed using a Waters system, model 1525 (Waters Chromatography Systems, California, USA), connected to a UV/VIS detector Waters 2487 and manual injector 7725i with 20 μL loop (Rheodyne BreezeTM, California, USA), The separation was in isocratic form with a reversed phase column Zorbas Eclipse Plus C18 AgilentTM (150 × 4,6 mm; 5 μm) (Santa Clara, California, USA). It was used analytical balance model DV215CD (Discovery, OhausTM, São Paulo, Brazil); ultrasound bath model USC2800A (Unique, São Paulo, Brazil); purified water Milli-QTM (Direct-QTM 3, Merck Millipore, Germany); micropipette model ResearchTM Plus 100-1000 μL (Eppendorf, Hamburg, Germany) UV chamber with mirrors in the interior and UVC lamp (254 nm); UV-VIS spectrophotometer ShimadzuTM (Tokyo, Japan), model UV-1800, using quartz cells of 1 cm of optical path.
Cephalothin reference substance (CET RS), with declared content of 99.6% were kindly provided by the laboratory União Química Farmacêutica Nacional S/A (São Paulo, Brazil).
The samples used was commercial sodium cephalothin (generic) in lyophilized powder for injectable solution in ampoule containing 1000 mg of active substance. The samples have an adjuvant, sodium bicarbonate. The samples were kindly provided by the laboratory ABL Antibióticos do Brasil Ltda (Cosmópolis, Brazil).
All solutions and the mobile phase used in this method were prepared from ultrapure water obtained through Milli-Q (Direct-QTM 3, Merck Millipore, Germany) equipment. HPLC grade ethanol and glacial acetic acid was used for the mobile phase and its brand was Baker JT (Mexico). For selectivity, it was used: 0.1 M hydrochloric acid solution (SynthTM, São Paulo, Brazil), 0.01 M sodium hydroxide solution (Vetec Química FinaTM, São Paulo, Brazil) and 0.3% hydrogen peroxide solution (Vetec Química FinaTM, São Paulo, Brazil).
Preparation of CET SQR and CET sample solutions: The CET SQR solution was prepared weighting 5.02 mg and transferring to a 25 mL volumetric flask and adding purified water that will give 200 μg/mL stock solution. All other solutions used in the tests were prepared by this stock solution, taking the necessary volume to obtain the desired concentration and transferring to a 10 mL volumetric flask.
The content of 5 vials of CET sample in lyophilized powder for injectable solution were mixed and 5.27 mg was weighted and transferred to a 25 mL volumetric flask that was added purified water. It was obtained a 200 μg/mL stock solution. All solutions used in the tests were prepared taking the necessary volume from the stock solution, to obtain the desired concentration, and transferring to a 10 mL volumetric flask.
HPLC method: The method was performed in isocratic mode and at room temperature of 25°C. The mobile phase consisted in ethanol and acidified water with 0.7% glacial acetic acid (30:70, v/v) that was degassed by ultrasonic bath for 30 minutes before use. The injection volume was of 20 μL at flow rate of 1.0 mL/minute, using UV detection at 237 nm. The solutions tested were filtered through 0.45 μm membrane (Pall Corporation, Michigan, USA) before the injection.
The parameters evaluated for the validation of the HPLC method were: system suitability, linearity, selectivity, precision (repeatability and intermediate precision), accuracy, robustness and limits of detection (LOD) and quantification (LOQ). The method was validated according to what is recommended by ICH guidelines literature .
System suitability: A 60 μg/mL CET sample solution was prepared and injected in sextuplicate. All chromatograms were analyzed, and the parameters evaluated such as retention time (tR), peak area, number of plates (N), peak asymmetry (As), retention factor (k) and tailing factor (TF). It was calculated the relative standard deviation (RSD). The results are shown in Table 1.
|Retention factor (>2.0)||Retention time (min)||Peak asymmetry (= 2.0)||Number of plates (>2000)||Area|
aRSD%: Relative Standard Deviation
Table 1: Navigation lock (operation time) vessel movement upstream to downstream.
Linearity: It was prepared a 200 μg/mL CET RS solution from whom was taken aliquots to prepare solutions of 20, 40, 60, 80 e 100 μg/mL and perform injections in triplicate. The equation of the line was determined by linear regression study, by the least squares method, analysis of variance (ANOVA) and residues analysis.
Precision: It was determined by repeatability precision and intermediate. The repeatability precision consisted in six injections of CET RS solution in the concentration of 60 μg/mL. It was done in the same day and carried by the same analyst. The intermediate was done in two ways. The first was performed by the same analyst but in three different days and following the same experimental conditions. In the second way, the analyst was changed, and the six injections were done in the same day and in the same experimental conditions. Statistical analysis was performed for each test through RSD values.
Accuracy: The accuracy of the method was performed by contaminated placebo, in which known amounts of a CET SR solution were added to a solution prepared with sodium bicarbonate. All injections were made in triplicate for each concentration. First, it was injected a 30 μg/mL solution of CET SR in water, and then, three different concentrations of the contaminated placebo that corresponded to 80, 100 and 120% respectively. Aliquots of 4.8; 6.0 e 7.2 mL of CET SR solution were added to an excipient solution to determine the accuracy in the feedstock. The solutions were prepared as shown in Table 2.
|Volume added of CET SR solution (100 μg/mL) (mL)||Volume added of placebo solution (mL)||Nominal concentration (μg/mL)|
Volumetric flask: 10 mL
Table 2: Procedure to determine the accuracy.
Robustness: The robustness was evaluated by small variations in seven parameters organized in eight experiments and followed the Youden and Steiner method. To determine the robustness, it was used CET SR and sample solutions in the concentration of 60 μg/mL and performed in triplicate. Table 3 shows the parameters and the variations for each one, where the capital letter represents the conditions used in the method and the lower case when there was a variation and in Table 4 there is the range of variation.
|C/c||Proportion of mobile phase||C||C||C||c||C||c||C||c|
|D/d||Proportion of glacial acetic acid (%)||D||D||D||d||d||d||D||D|
|E/e||Flow rate (mL/min)||E||E||E||e||e||E||E||E|
|F/f||Room temperature (ºC)||F||F||F||F||F||f||F||F|
bRSD%: Relative Standard Deviation
Table 3: Shear strength prediction models for SFRC beams used in this study.
|S. No||Factors||Unit||Limit||Varied Condition (1)||Normal Condition (0)||Varied Condition (-1)|
|A||Ethanol brand||-||-||Scharlau||J. T. Baker||Scharlau|
|C||Proportion of mobile phase||%||2||68:32 (v/v)||70:30 (v/v)||72:28 (v/v)|
|D||Proportion of glacial acetic acid||%||0.1||0.8%||0.7%||0.6%|
Table 4: Range of variations for the determination of cephalothin.
The difference between the normal values and the ones changed in module should be lower than the value resulted from 2xS in order to infer that the effects achieved with the variations of the parameters were not significant and therefore the method is robust for all selected factors.
Specificity: Specificity can be accessed by different analysis which can be easily found in the literature [31-36]. CET sample solution in the concentration of 60 μg/mL was submitted to forced degradation in acid, alkaline, oxidative, photolytic and neutral conditions. This parameter was performed to evaluate if there was any interference of degradation products in the quantification of CET sample. The solutions used as degradation solvents were: 0.1 M HCl, 0.01 M NaOH, 0.3% H2O2 and purified water, used in acid, basic, oxidative and neutral/photolytic degradation, respectively. The acid, oxidative and neutral conditions were heated to 60°C while basic and photolytic conditions were maintained at 25°C and, the photolytic degradation was induced by exposure to ultraviolet light (UVC, 254 nm). Aliquots were taken from 10 to 10 minutes until degradation above 10%.
Detection (LOD) and Quantification (LOQ): According to the ICH, the LOD and the LOQ are studies based on the standard deviation of intercept and in the slope of the analytical curve. After obtaining three analytical curves, LOD and LOQ were calculated as:
Where σ is the standard deviation and S is the slope of the calibration curve.
Different chromatographic conditions were tested to develop a quantification method for CET SR e CET samples. The choice of the chromatographic column was based in the peaks resolution. The Zorbax Eclipse Plus C18 AgilentTM (150 × 4.6 mm; 5 μm) showed a better peak symmetry and lower system pressure.
For the determination of the mobile phase there was made several tests varying the concentration of the glacial acetic acid in water and the proportion of ethanol. All mobile phases tested showed appropriate peaks, but just one of them covered all parameters settled in system suitability. The mobile phase used was water with 0.7% glacial acetic acid and ethanol (70:30, v/v). The use of water and a less toxic organic solvent reduced the formation of waste and damage for the chromatographic system. The chromatogram obtained in the method conditions is shown in the Figure 2.
Table 5 shows the area values obtained for each concentration used for the determination of linearity. The residue analysis showed that the regression model used is appropriate. The area values were plotted in each concentration and linearity was observed in the range of 20 to 100 μg/mL. The results were analyzed using test of variance (ANOVA) that showed no deviation from linearity and the regression model is appropriate. The ANOVA calculated is in Table 6. The analytical curve and residue analysis are on Figure 3.
|Concentration (μg/mL)||Areasa (AU)||Average area||RSD%c|
a: Average value of three determinations; cRSD%: Relative Standard Deviation
Table 5: Peak area obtained for each concentration of CET SR solution.
|Source of variation||Degree of freedom||Sum of squares||Variability||F calculated||F critical|
|Deviation of linearity||3||4514583352.89||1504861117.63||0.43||3.71|
*Significant at p<0.05%
Table 6: Analysis of variance (ANOVA) for linearity.
The precision of the method was determined by repeatability, when six solutions of CET sample in the concentration of 60 μg/mL were injected by the same analyst, in the same day and under the same experimental conditions, providing a RSD of 1.91%. For the determination of interday precision, analysis was performed on three consecutive days, and RSD% was 4.90%. For precision between analysts it was made six solutions of CET sample in the same concentration as before, under same experimental conditions, in the same day, bust with a different analyst. The value of RSD% was 1.90. All results for precision are shown in Tables 7 and 8.
bRSD%: Relative Standard Deviation
Table 7: Intraday precision for the analytical method developed for HPLC.
aMean of 6values; bRSD%: Relative Standard Deviation
Table 8: Interdays and between analysts precision for the analytical method developed for HPLC.
The results obtained in interday precision was statistically evaluated by analysis of variance and according to ANOVA there was no significant deviation, as shown in Table 9.
|Source of variation||Sum of squares||DF||Average squares||F calculated||F critical||P-value|
Table 9: Analysis of variance (ANOVA) for interday precision.
The accuracy of the method was made by the contaminate placebo method by adding a known quantity of a CET SR solution to the placebo solution. It was determined in three different concentrations predetermined and resulted in 95.38%, lower than what is recommended in literature [37-39]. The average percentage is shown in Table 10.
|Accuracy (days)||Recuperation (%)||Average recuperation (%)||RSD (%)a|
aRSD%: Relative Standard Deviation
Table 10: Results for CET SR method accuracy.
The robustness was evaluated by the Youden and Steiner method that consists in small variations in seven parameters organized in eight experiments, previously shown on Tables 3 and 4. The effects resulting from the changed parameters were evaluated in comparison to the values obtained as reference for the test 1+=5.39 and 1-=7.83. All effects are shown in Table 11.
|Factor||(1)||Content (%)a,b Effects||(-1)||Content (%)a,b Effects|
|A- Ethanol brand||Scharlau||104.12-106.50=-2.38||Scharlau||103.14-98.93=4.21|
|B- Source of water||CFQ||105.53-105.08=0.45||CFQ||101.81-100.26=1.55|
|C- Proportion of mobile phase (v/v)||68:32||105.25-105.36=-0.10||72:28||102.28-99.79=2.49|
|D- glacial acetic acid concentration (%)||0.8||107.12-103.49=3.63||0.6||102.50-99.56=2.94|
|E- Flow rate (mL/min)||1.1||104.85-105.76=-0.91||0.9||102.65-99.42=3.23|
|F- Room temperature (ºC)||27||106.02-104.59=1.43||23||99.65-102.42=-2.77|
|G - Wavelength (nm)||239||104.37-106.24=-1.86||235||100.66-101.41=-0.75|
aAverage contents obtained in normal conditions - Average contents obtained in altered conditions;
bReference criteria calculated: 5.39 to test +1 e 7.83 to test -1
Table 11: Results for sodium cephalothin method robustness.
The selectivity of the method was evaluated by forced degradation observing the chromatograms of CET SR to make clean if there would be any degradation substance. The chromatograms on Figures 4 and 5 shows that the degradation products have negligible interference with CET peak.
Limit of detection and quantification
The sensitivity of the method was determined by chromatographic detection (LOD) and quantitation (LOQ) limits. The value calculated for the lowest concentration detected by analytical procedure was 1.95 μg/mL. In turn, the calculated LQ was 5.90 μg/mL. The calculated values for the LOD and LOQ indicated the ability of the method to detect and quantify reliably CET.
The qualitative analysis of CET sample in lyophilized powder for injection solution was performed by the organoleptic characteristics and high-performance liquid chromatography (HPLC) that demonstrated that these methods are appropriate to identification.
The mainly objective of this work was the development and validation of an analytical method for the quantification of CET sample, with a green chemistry approach. The developed analytical method used ethanol as organic solvent reducing, in this way, the toxicity to the professional and environment. Moreover, it was used a few quantities of organic solvent and no buffering solutions, reducing the waste .
The proposed method can be considered as innovative because it wasn´t found in the literature any approach for the quantification of CET sample with the view of waste and toxic solvents reduction.
So, we can conclude that the developed and validated method can be used in quality control for CET sample in lyophilized powder for injection because it demonstrated to be effective to quantification.
This work was supported by FAPESP and CNPq-Brazil.
The authors report no declarations of interest.