Viswanadha Institute of Pharmaceutical Sciences
A rapid, sensitive, and high precise reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous quantification of Cefepime and Enmetazobactam using a Waters HPLC system. Chromatographic separation was achieved on a Inertsil ODS C18 column (250 × 4.6 mm, 5 µm particle size) maintained at ambient temperature. The mobile phase consisted of acetonitrile and buffer in the ratio of 80 :20(v/v), which was filtered through a 0.45µm membrane filter prior to use. The flow rate was maintained at 1.0 mL/min, and detection was carried out at 228 nm using PDA detector.
Exblifep is a combination antibiotic used for complicated urinary tract infections (cUTIs) that contains two antibiotics, cefepime and enmetazobactam. Exblifep is used when the bacteria causing the UTI is resistant to other antibiotics, especially if the resistance is due to Extended Spectrum Beta Lactamases (ESBLs).
Exblifep contains cefepime (Maxipime), which is fourth-generation cephalosporin combined with enmetazobactam, which is a beta-lactamase inhibitor. Exblifep is given as an IV infusion that takes about 2 hours and is given every 8 hours for 7 to 14 days.
Exblifep received FDA approval to treat adults (18 years and older) with complicated UTI, including kidney infection (pyelonephritis) caused by specific bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, and Enterobacter cloacae complex, that susceptible to Exblifep.
II. MATERIALS AND METHODS
Preparation of Stock solution: Accurately weigh 125 mg of Enmetazobactam and 120 mg of Cefepime separately in a 100ml volumetric flask sonicated for 20 minutes. Take 10ml of each of each solution and place them in a 100ml volumetric flask. Next, add mobile phase, and after 10 minutes, sonicate the mixture.
Preparation of working standard solution: 10mg of the drugs Cefepime and Enmetazobactam were weighed, and 10 ml of mobile phase was added to each of two separate 10 ml volumetric flasks. The mixture was then sonicated for 20 minutes to obtain 1000 ppm, then taking 1ml of each solution and diluting it to 10ml with mobile phase.
III. RESULTS AND DISCUSSION
Method validation: Validation parameters include specificity, linearity, range, accuracy, precision, limit of detection, limit of quantification, robustness and assay (1-6).
Specificity: Specificity is the ability to assessing equivocally the analyte in the presence of components which may be expected to be present. Typically, these components include impurities, degradants, matrix etc. Blank solution and standard solutions of Cefepime (40μg/ml) and Enmetazobactam. (40μg/ ml) were injected into the HPLC system. The peak purity data of Cefepime and Enmetazobactam. were compared. There should not be any interference at the retention time of the main peaks.
Linearity: Linearity for the drugs Cefepime and Enmetazobactam .(17-27)was determined by preparing the standard solutions at six concentrations levels in six replicates in the range of 20-70μg/ml Cefepime and 20-70μg/ml for and Enmetazobactam hydrochloride from stock solution. The linearity charts of Cefepime and Enmetazo bactam was shown in the figure no 2&3. The correlation coefficient was found to be 0.9996 and 0.9994for Cefepime and Enmetazo bactam respectively. Linearity results were tabulated in table 2.
Accuracy: Accuracy was performed by spiking known amounts of standard solution to sample solution at three different concentrations levels (50%, 100%, 150%) and there by analyzed for %RSD which should not be more than 2.0.The % recovery was calculated and the results was reported in table no. 3 & 4.
Precision: The precision (7-12) of the analytical method was studied by injecting six replicates of standard containing 40μg/ml of Cefepime and 40μg/ml of Enmetazo bactam which were injected into HPLC system. The % RSD was calculated and the results were reported in the table no.5 & 6.
Limit of Detection (LOD) and Limit of Quantification (LOQ): The limit of detection was defined as the concentration which yields a signal - to – noise ratio 3:1 whereas the limit of quantification was calculated to be the lowest concentration that could be measured with signal - to – noise ratio10:1. LOD and LOQ were calculated from slope and standard deviation. The results were tabulated in table no. 7.
Robustness: The smallest deliberate changes in method like change in flow rate are made but there were no predictable changes in the results and are in the range as per ICH guidelines. Conditions like decrease in flow rate (0.8 ml/min), increase in flow rate (1.2 ml/min) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. % RSD was found to be within the limits and results were tabulated in table no. 8.
Assay: Assay was conducted on marketed formulation and mean % assay was found. The results were tabulated in table no. 9.
Table1:OptimisedChromatographicConditions
|
Parameter |
Method |
|
Stationary Phase (column) |
Inertsil -ODS C18 (250 x 4.6 mm, 5 µ) |
|
Mobile Phase |
Methanol and Acetonitrile(80:20) |
|
Flow rate (ml/min) |
1.0 ml/min |
|
Run time (minutes) |
14 min |
|
Temperature in the column (°C) |
Ambient |
|
Injection volume (µl) |
20 |
|
Detection wavelength (nm) |
262nm |
|
Drug RT (min) |
6.120 min for Cefepime and 9.336 for Enmetazobactum. |
Figure 1: OptimisedChromatogram
Table 2: Linearity data of Cefepime and Enmetazobactam
|
Cefepime |
Enmetazobactam |
||
|
Conc (µg/ml) |
Peak area |
Conc (µg/ml) |
Peak area |
|
20 |
442574 |
20 |
1867314 |
|
30 |
663321 |
30 |
2726631 |
|
40 |
863425 |
40 |
3613860 |
|
50 |
1086345 |
50 |
4385621 |
|
60 |
1296547 |
60 |
5263754 |
|
70 |
1535742 |
70 |
6132485 |
Figure 2:Calibration curve of Cefepime
Figure 3:Calibration curve of Enmetazobactam
Table 3: Accuracy Data of Cefepime
|
Concentration % of spiked level |
Amount added (ppm) |
Amount found (ppm) |
% Recovery |
Statistical Analysis of % Recovery |
|
|
50% - 1 |
20 |
20.12 |
100.14 |
MEAN |
99.93 |
|
50% - 2 |
20 |
19.98 |
99.91 |
|
|
|
50% - 3 |
20 |
19.94 |
99.89 |
%RSD |
0.74 |
|
100 % - 1 |
40 |
40.14 |
100.12 |
MEAN |
99.84 |
|
100 % - 2 |
40 |
39.96 |
99.98 |
|
|
|
100% - 3 |
40 |
39.86 |
99.75 |
%RSD |
0.639 |
|
150% - 1 |
60 |
59.94 |
99.99 |
MEAN |
100.04 |
|
150% - 2 |
60 |
60.01 |
100.02 |
|
|
|
150% - 3 |
60 |
60.02 |
100.02 |
%RSD |
0.667 |
Table 4:Accuracy Data for Enmetazobactam
|
Concentration % of spiked level |
Amount added (ppm) |
Amount found (ppm) |
% Recovery |
Statistical Analysis of % Recovery |
|
|
50% - 1 |
20 |
19.93 |
99.97 |
MEAN |
99.98 |
|
50% - 2 |
20 |
19.84 |
99.87 |
|
|
|
50% - 3 |
20 |
20.05 |
100.4 |
%RSD |
0.867 |
|
100 % - 1 |
40 |
39.90 |
99.84 |
MEAN |
99.96 |
|
100 % - 2 |
40 |
40.04 |
100.06 |
|
|
|
100% - 3 |
40 |
40.01 |
100.04 |
%RSD |
0.687 |
|
150% - 1 |
60 |
59.98 |
99.97 |
MEAN |
99.94 |
|
150% - 2 |
60 |
59.95 |
99.93 |
|
|
|
150% - 3 |
60 |
59.92 |
99.91 |
%RSD |
0.687 |
Table 5: System Precision data of Cefepime and Enmetazobactam
|
Sr. No |
Cefepime |
Enmetazobactam |
|
1 |
865021 |
3604637 |
|
2 |
865327 |
3607638 |
|
3 |
865674 |
3609784 |
|
4 |
865780 |
3612341 |
|
5 |
865732 |
3606721 |
|
Mean |
865506.8 |
3608224 |
|
SD |
324.6686 |
2951.79 |
|
% RSD |
0.037512 |
0.081807 |
Table 6: Method Precision data of Cefepime and Enmetazobactam
|
Sr. No |
Cefepime |
Enmetazobactam |
|
1 |
865237 |
3607684 |
|
2 |
865762 |
3604672 |
|
3 |
865760 |
3605241 |
|
4 |
865091 |
3606754 |
|
5 |
865374 |
3607878 |
|
6 |
865767 |
3606752 |
|
Mean |
865498.5 |
3606497 |
|
SD |
303.2641 |
1292.7 |
|
% RSD |
0.035039 |
0.035844 |
Table 7: LOD and LOQ data of Cefepime and Enmetazobactam
|
Drug Name |
LOD (µg/ml) |
LOQ (µg/ml) |
|
Cefepime |
0.11 |
0.33 |
|
Enmetazobactam |
0.09 |
0.27 |
Table 8: Robustness data of Cefepime and Enmetazobactam
|
Sr No |
Drug Name |
Condition |
Peak area |
% RSD |
|
1 |
Cefepime |
Decreased Flow rate of 0.8 ml/min |
864680 |
0.037 |
|
2 |
Increased Flow rate of 1.2 ml/min |
866350 |
0.0038 |
|
|
3 |
Enmetazobactam |
Decreased Flow rate of 0.8 ml/min |
359512 |
0.089 |
|
4 |
Increased Flow rate of 1.2 ml/min |
362053 |
0.066 |
Table 9: Assay data Cefepime and Enmetazobactam
|
Sr. No |
Peak area of Cefepime |
% Assay |
Peak area of Enmetazobactam |
% Assay |
|
1 |
865354 |
99.24 |
3608756 |
101.57 |
|
2 |
865037 |
3605937 |
||
|
3 |
865762 |
3602734 |
||
|
4 |
865972 |
3607961 |
CONCLUSION
The developed RP-HPLC method was validated as per ICH guidelines. All the system suitability parameters were within the range as stated by ICH guidelines. Interference peaks were not observed in blank, standard and sample chromatogram. Hence simple, precise and accurate, sensitive, specific and robust method was developed and validated. This can be used in quality control department with respect to routine analysis.
ACKNOWLEDGEMENTS
Authors are thankful to the management of Viswanadha Institute of Pharmaceutical Sciences (VNIPS) for providing facilities and support to carry out this work.
REFERENCES
V. Vijaya, K Suvarna, Dr. P V Madhavi Latha, Dr. P Uma Devi, Method Development and Validation for the Simultaneous Estimation of Cefepime and Enmetazobactam in Bulk and Pharmaceutical Dosage form by RP-HPLC Method, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 12, 873-879. https://doi.org/10.5281/zenodo.17829859
10.5281/zenodo.17829859
