INTRODUCTION:

Pharmaceutical parental injections such as Celrim, Tazopime, and Magnova use a combination of Cefepime and Tazobactam drugs which are effective in treating bacterial infections especially intra-abdominal, urinary tract infections, skin infections, septicemia, pneumonia, bronchitis, and febrile neutropenic patients^1,2. In this context, the present study focused on developing an analytical method involving simple pretreatment of samples and increased speed of analysis with more accuracy, recovery, and precision using LCMS-MS/MS^{3, 4}. Chemical name of Cefepime is (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-[(1methylpyrrolidinium-1-yl)methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylate and chemical name of Tazobactam is (2S,3S,5R)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4λ6-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid^5,6.

Various assays using chromatography were described to estimate CFP and TZB^7–17, but these methods have disadvantages such as increased matrix effect, complex mobile phase composition, use of buffer solutions, decreased sensitivity, less eco-friendly, and time consumption. The reported two LC-MS/MS does not give details on the retention time of drugs^16,17. The monograph available in the USP 29 –NF 24¹⁸ for analyzing Cefepime had to use a conductivity detector for detection resulting in detection of the drug at a very trace level being difficult. However, there have been no previous descriptions of trace analysis using UPLC-MS/MS for the simultaneous determination of CFP and TZB. The novelty of the work is the usage of low solvent volume as the column used is short and fast column UPLC BEH column¹⁹, increased sensitivity towards detection of an analyte by using triple quadrapole mass analyzer²⁰ and improved peak integration, and decreased retention time of drugs (in seconds). The greenness of the analytical procedure was measured using two methods, first the NEMI method and the other eco-scale method (Supplementary file)^{21, 22}. The aim was to develop an accurate, selective, and sensitive hyphenated analytical procedure for analysis of the drug combination of CFP and TZB by using UPLC-ESI-QQQ-MS^23–26.

MATERIALS AND METHODS:

Materials:

Cefepime standard (CFP) (99.8%) and Tazobactam standard (TZB) (99.8%) were obtained as samples from Kshetra Analyticals (Hyderabad). The LC grade water, DMSO, acetonitrile, formic acid, and methanol were procured from SD Fine Chemicals Ltd. A parental dosage form of Magnova injection (Cefepime 1gm and Tazobactam 125mg) was bought from Apollo pharmacy (Hyderabad, India).

Instrumentation and chromatographic conditions:

The chromatographic separations were performed on a UPLC system (Acquity C18 BEH, 50mm x 2.1mm, 1.7m) with MS/MS (UPLCMS/MS - All Waters, USA). Waters Quattro Premier mass spectrophotometer was utilized as a tandem MS/MS using a QQQ mass analyzer (triple quadrupole) with an +ESI probe. The system data was collected using the Masslynx version 4.1 software. An isocratic elution uses 0.2 percent formic acid in water and acetonitrile (20:80 v/v) as a mobile phase. A constant mobile phase flow of 0.15ml/min was maintained; with a 5μl injection volume. The column temperature was kept at 30°C. The run time was 3 mins. Typical optimized operating conditions were at Capillary voltage: 3.00kV and 2.94kV, Cone voltage: 25 V and 28 V, Desolvation temperature: 400ºC and 388ºC, Source temperature:120ºC, Desolvation gas flow: 850 l/hr and 848 l/hr, Cone gas flow:100 l/hr and 97 l/hr, Ion Energy:1 0.5, Multiplier: 550V - 547V, Syringe Pump Flow: 100µl/min, and Collision Gas Flow: 0.15ml/min.

Preparations of diluent, mobile phase, standard and sample stock solutions:

a) Diluent and blank solution:

The diluent used for preparing the sample and standard solutions of Cefepime and Tazobactam is a 50% methanol solution. Blank mass spectra were obtained using the diluent.

b) Standard Stock solution:

The standard stock solution of 1000µg/ml was prepared using DMSO solvent (Stock Solution A) for Cefepime and Tazobactam. All the dilutions of standard stock solution A were carried out by using 50% methanol (diluent solvent) to obtain standard working solution concentrations. The concentration of Cefepime Standard working solution was 2000ng/ml. The concentration of Tazobactam Standard working solution was 250ng/ml. The standard working solution was sonicated and filtered using 0.45m membrane filters before the analysis and kept in the refrigerator.

c) Preparation of sample solution (Marketed Formulation):

The sample stock solution was prepared by accurately weighing 19.8mg of injectable powder (equal to 10mg CFP and 1.25mg TZB) and dissolving it in 100ml of diluent to yield 100µg/ml of CFP and 12.5µg/ml of TZB solution. 1ml of sample stock solution A was pipette to a 50ml volumetric flask to prepare the working sample solution. The volume was adjusted with diluent (50 percent methanol) up to the mark to obtain 2000ng/ml of Cefepime and 250ng/ml of Tazobactam. The sample was analyzed using the optimum chromatographic conditions.

d) Preparation of the mobile phase:

A mixture of 0.2 percent formic acid in water and acetonitrile (ACN) 20:80 v/v was used as mobile phase.

Validation procedure^27-29:

a) Specificity and system suitability:

Blank samples (50% methanol) and working standards (2000ng/ml for Cefepime and 250ng/ml for Tazobactam) were prepared according to the UPLC-MS/MS developed method and loaded to evaluate the specificity as well as system suitability parameter.

b) Linearity and range:

From the standard stock solutions of CFP and TZB, seven linear concentration solutions ranging from LOQ to 150% were prepared to estimate the linearity of the proposed method.

c) Sensitivity:

With serial diluted solutions of CFP and TZB, the LOD, as well as the LOQ, were measured using 3:1 and 10:1 signal-to-noise ratios.

d) Precision (% repeatability):

By injecting (n = 6) a working solutions of Tazobactam 25ng/ml and Cefepime 200ng/ml, the precision of the developed method was determined. For both drugs, intermediate precision was evaluated using standard working solutions with concentration levels of LOQ, 25%, 50%, and 100%. By analyzing the corresponding concentration (n = 6) on the same day and different days, intraday and interday precision were determined.

e) Accuracy:

Accuracy was measured at 50%, 100%, and 150% concentration levels for CFP and TZB. The standard solutions of both drugs were added to the pre-analyzed solution (2000ng/ml for Cefepime and 250ng/ml for Tazobactam) of the formulation.

f) Robustness:

Column temperature, injection volume, and flow rate were changed to test the robustness of the suggested methodology for CFP and TZB. As a result, the flow rate was changed to 0.14ml/min and 0.16ml/min, resp. To determine how column temperature affects performance, tests were carried out at 27°C and 32°C.In all of these investigations, the mobile phase mix was kept unchanged.

g) Assay Procedure:

Cefepime and Tazobactam in marketed parental formulation (Magnova) were determined using the proposed approach employing UPLC-MS/MS. The sample working solutions and standard working solutions (5µl) containing 2000ng/ml Cefepime and 250 ng/ml Tazobactam were loaded into the chromatographic system, and the respective areas for CFP peak and TZB peak were evaluated and the percentage assay was determined

RESULTS:

Chromatographic Conditions:

The molecular weight of Cefepime and Tazobactam is 480.6 and 300.29 respectively. Using ESI in positive ion mode; MS/MS detector optimum settings were investigated to acquire transition for each molecule. To optimize the cone voltage with the precursor ions as [M+H]⁺ ions, entire scan spectra were assessed to identify the most prevalent m/z value. Using collision energies, the most numerous ions generated were identified with the most sensitive transition ions. MS/MS transition for quantification and confirmation for Cefepime was observed at 481.35à166.85 with a dwell seconds of 0.200, cone voltage of 25 V, Ion Energy:1 0.5, and collision energy of 20. MS/MS transition for quantification and confirmation for Tazobactam was observed at 301.20 à 167.70 with a dwell seconds of 0.100, cone voltage of 25 V, Ion Energy: 1 0.5, and collision energy of 25 eV. The [M+H]⁺MS/MS spectra of parent ion peaks were at m/z 481.35 and 301.2 for Cefepime and Tazobactam respectively. Figure 1A and 1B represents the MS/MS spectra of parent ions of Cefepime and Tazobactam.

The [M+H]⁺ MS/MS spectrum of fragmented daughter ion peaks for Cefepime and Tazobactam were observed at 166.85 and 167.7 for CFP and TZB respectively. Figure 2A and 2B represents the MS/MS spectra of daughter ions of Cefepime and Tazobactam.

Optimizing the chromatographic variables resulted in better peak resolution and shorter retention times for Cefepime and Tazobactam with minimal trace concentrations. The constituents of the mobile phase were studied using a C18 hybrid bridged column as a stationary phase. Various solvents and solvent concentrations were used in a series of trials. In the study, formic acid in water (a solvent that enhances ionization of drugs) and methanol and acetonitrile were used as mobile phases. Using methanol as a diluent and 0.2 % formic acid: acetonitrile (20: 80 v/v) as a mobile phase yielded the best results. Finally, other variables like flow and column temperature were altered to 0.1 ml/min (from 0.3 ml/min) and from 25ºC to 35ºC, respectively, identifying 0.15 ml/min as the optimal flow rate and 30ºC as the optimal column temperature. Cefepime and Tazobactam were eluted simultaneously in two MRM channels mode with retention times of 0.80 min and 0.96 min, respectively. Figure 3A and 3B shows the typical chromatograms of optimized UPLC-MS/MS of Cefepime and Tazobactam.

Figure 3. Chromatogram of optimized UPLC-MS/MS for: (A) cefepime and (B) tazobactam

Validation:

The findings of the system suitability test of the standard solutions of Cefepime and Tazobactam from n = 6 replicate injections were in the vicinity of recommended range under ICH guidelines³⁰. The system suitability was measured by using 2000ng/ml for Cefepime and 250ng/ml for Tazobactam with %R.S.D of peak area equal to 0.90 and 1.23 respectively. Tailing factor for Cefepime and Tazobactam were reported at 1.03 and 0.45 respectively. Other parameters of the proposed method such as peak area, peak symmetry, and retention time are within acceptable limits. To evaluate the specificity, 50% methanol (blank) and working standards were analyzed and it was found that [M+H]⁺ ion of Cefepime and Tazobactam has a retention time of 0.80min and 0.96 min respectively. The m/z ratio of protonated ions was observed at 481.35 and 301.2 for Cefepime and Tazobactam respectively. To validate linearity of the proposed method calibration curves were constructed in the range of 200ng/ml to 3000 ng/ml for Cefepime and 25ng/ml to 375ng/ml for Tazobactam respectively. The analyte shows an excellent linear correlation between its concentration and its peak area in the examined concentration range with Y = 29.60 * x – 373.2 and Y = 8.784 * x + 42.79 as linear regression equation (r²= 0.9955 and r²= 0.9816) for Cefepime and Tazobactam respectively. The calibration curve is represented in Figure 4 and 5 and the results tabulated are in Table 1.

Figure 4. Calibration curve of cefepime

Figure 5: Calibration curve of Tazobactam

Table 1: Linearity parameter for simultaneous determination of Cefepime and Tazobactam

Concentration Level (%)	Cefepime		Tazobactam
Concentration Level (%)	Area *(n =6)*	% R.S.D.	Area *(n =6)*	% R.S.D.
LOQ	5942.606	0.02	276.976	0.72
25	12360.025	0.03	516.254	0.42
50	30301.859	0.01	1282.589	0.29
75	47866.516	0.01	1635.804	0.11
100	56902.129	0.01	2282.704	0.15
125	72868.961	0.01	2398.766	0.02
150	87952.477	0.01	3655.234	0.85

The sensitivity of the method was measured as LOQ and LOD concentrations. LOD was found to be 66ng/ml and 8.3ng/ml, whereas LOQ was found to be 200ng/ml and 25ng/ml, for Cefepime and Tazobactam respectively. At four concentration levels, the method was validated for intraday and interday precision. The concentrations of Cefepime used for evaluating precision are 200ng/ml, 500ng/ml, 1000ng/ml and 2000ng/ml. The concentrations of Tazobactam used for evaluating precision are 25ng/ml, 62.5ng/ml, 187.5ng/ml and 250 ng/ml. The %R.S.D. observed for intraday and interday precision was found to be less than 2. Precision results were tabulated in Table 2.

Table 2 Intraday and Interday Precision for simultaneous determination of Cefepime and Tazobactam

Drug Name	Conc. Of API (ng/mL)		Observed Conc. Of Cefepime and Tazobactam (ng/mL)
Drug Name	Conc. Of API (ng/mL)		Intra-Day			Inter-Day
Cefepime	Level	conc.	Mean (n=6)	SD	% RSD	Mean (n=6)	SD	% RSD
	LOQ	200	199.0	3.0	1.51	200.66	2.08	1.04
	25%	500	485.166	6.04	1.25	495.0	8.18	1.65
	50%	1000	998.0	16.09	1.61	995.83	5.34	0.54
	100%	2000	1954.66	21.54	1.01	1975.0	11.0	0.56
Tazobactam	LOQ	25	25.133	0.40	1.61	24.07	0.24	1.03
	25%	62.5	61.5	1.15	1.88	59.98	1.17	1.6
	50%	187.5	185.42	3.35	1.81	184.8	2.09	1.13
	100%	250	253	4.0	1.58	246.41	4.01	1.63

Table 3 Accuracy and % Recovery of Spiked Samples of Cefepime and Tazobactam

Spiked Level	Theoretical Conc. of pure drug (ng/mL) added		Area		Conc. of pure Found (ng/mL)		Conc. Mean		Standard deviation		% R.S.D		% Recovery
Spiked Level	CPF	TZB	CPF	TZB	CPF	TZB	CPF	TZB	CPF	TZB	CPF	TZB	CPF	TZB
50%	1000	125	31666	1441.1	998	124	1000	126	4.10	2.10	0.41	1.67	100	100.8
50%	1000	125	31886	1448.0	1005	126
50%	1000	125	31481	1448.9	997	128
100%	2000	250	57977	2282.7	1970	257	1990	253	17.4	4.0	0.88	1.58	99.5	101.2
100%	2000	250	58938	2203.9	2003	253
100%	2000	250	58643	2274.4	1998	249
150%	3000	375	87013	4157.7	2951	371	2972	373	26.0	7.3	0.88	1.97	99	99.46
150%	3000	375	88847	4128.1	3013	368
150%	3000	375	87367	4145.2	2963	382

Accuracy was measured at 50%, 100%, and 150% concentration levels, using standard solutions of Cefepime and Tazobactam simultaneously and was expressed in terms of % recovery. % recovery of Cefepime at 50%, 100% and 150% were 100%, 99.5% and 99.06% respectively. % recovery of Tazobactam at 50%, 100% and 150% were 100.8%, 101.2% and 99.46% respectively. The accuracy findings for the proposed method are shown in Table 3. The method robustness was evaluated by changing the column temperature and flow rate. The %R.S.D. of the same was within 2%. (Table-3).

Assay:

The percentage assay of marketed Magnova formulation containing Cefepime and Tazobactam was found to be 98.63% and 97.6% respectively.

CONCLUSION:

The proposed UPLC-MS/MS method achieved accurate, precise, and sensitive results for the simultaneous determination of Cefepime and Tazobactam. The proposed method is fast with a retention time of less than one minute i.e. 48 seconds (0.80 min) for Cefepime and 57.6 seconds (0.96 min) for Tazobactam. The fragmented daughter ion enhances the extent of sensitivity towards detection as well as quantification. ICH guidelines were used to validate all parameters. Due to the increased selectivity of the proposed method towards the selected drugs (CEF & TZB), this proposed approach is suitable to analyze Magnova, Tazopime, and Celrim parental powder in the pharmaceutical field.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors would like to thank Kshetra Analyticals, Hyderabad, Telangana, India, for providing the necessary facilities (chemicals and equipments) to carry out this research work.

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Received on 01.10.2022 Modified on 15.02.2023

Research J. Pharm. and Tech 2024; 17(1):67-73.

DOI: 10.52711/0974-360X.2024.00011