Stability indicating UPLC-MS/MS method for quantification and identification of cefepime and its degradants in API

An analytical method was developed and validated for determining stability studies of the drug cefepime (CFP) by Ultra Performance Liquid Chromatography - Tandem Mass Spectrometer (UPLC-MS/MS). In this research, Ultra Performance Liquid Chromatography (UPLC) C18 ethylene hybrid column was used. About 0.2% formic acid in the water and acetonitrile (ACN), in 20:80 v/v ratios, was used as the mobile phase with a 0.15 ml/minute flow rate. In multiple reaction monitoring modes, positive electrospray ionization (ESI) was used. The chromatogram of standard Cefepime (CFP) was found to have a retention time of 0.82 ± 0.02 minute. The degradation of CFP was tested under different stress conditions and the method was validated to meet the International Conference on Harmonization of Technical Requirement for Registration of Pharmaceuticals for Human Use guidelines. The retention time of the forced degraded solutions of CFP was found to be 0.76, 0.83, 0.81, and 0.82 minutes for forced acidic, oxidation, thermal, and neutral conditions, respectively. The MS/MS spectra for CFP at 179.15 and 241.33 ± 0.06 in different stress conditions (except basic) with retention times at 0.90 ± 0.02 and 0.84 ± 0.10 minutes indicate Cefepime degradant 1 (CD1) and Cefepime degradant 2 (CD2), respectively. The MS/MS spectra for CFP basic degradant (CD1) in the basic medium were obtained at an m/z ratio of 179.15. The research findings conclude that CFP was unstable with partial degradations in all conditions, and complete degradation in basic medium.


INTRODUCTION
Pharmaceuticals tend to deteriorate to degrade with time. The degradation process is a decrease in distinct characteristics over time. A drug's shelf life is estimated by analyzing two independent properties after it has been stressed to degrade. First, determine how much active ingredient is lost. Second, identify the degradation products that result from the aging process. Degradants need to be predicted and assessed at concentrations greater than 0.1% of the parent molecule in this test, which can be challenging. Trace analysis is required for this part of the assay, which demands the establishment of a stability-indicating assay method (SIAM) for the drug and its dosage forms. Physical, chemical, microbiological, and environmental degradation determine the quality and purity of a drug product. Over time, an active pharmaceutical ingredient (API) or excipients may transit from a metastable state to a more thermodynamically stable state. A drug molecule can undergo chemical degradation due to several reactions such as oxidation, isomerization, hydrolysis, racemization, elimination, and UV degradation. In addition, there may be interactions with excipients and other pharmaceuticals (Connors et al., 1986). When water interacts with parental formulations, it leads to hydrolytic breakdown (Waterman et al., 2002). The most frequent cause of pharmaceutical degradation is oxidative degradation, which can take one of three paths: i) peroxide-accelerated, which involves exposing the drug to dilute peroxide solutions; ii) photochemically stimulated; and iii) radically simulated, which involves reversal loss of electrons (Bajaj et al., 2012;Cosa, 2004;Deidda et al., 2018;Quadri et al., 2014;Sahu et al., 2018).

Chemicals and reagents
CFP standard (99.8%) was acquired as samples from Kshetra Analyticals. The liquid chromatography grade water, formic acid, dimethyl sulfoxide (DMSO), acetonitrile (ACN), methanol, and all other reagents were procured from SD fine chemicals limited (Hyderabad, India).

Instrumentation and chromatographic conditions
The chromatographic separations were performed on a UPLC system (Acquity C18 BEH, 50 × 2.1 mm, 1.7 m) with MS/ MS (UPLC-MS/MS-All Waters, USA). Waters Quattro premier mass spectrophotometer was utilized in tandem MS/MS using a triple quadrupole mass analyzer with an electrospray ionization (+ESI) probe. The system data were collected using the masslynx version 4.1 software. The isocratic elution with a mobile phase composed of 20:80 v/v-0.2% formic acid in water and ACN was used. A flow rate of 0.15 ml/minute, with an injection volume of 5 µl, and a run time of 3 minutes were maintained. The column temperature was maintained at 30°C.

Standard stock solutions
For CFP, a 1,000 µg/ml standard stock solution was prepared using DMSO solvent (Stock Solution A). All of the dilutions of standard stock solution A were performed with 50% methanol (diluent solvent).

Standard working solution
To obtain stock solution B of 200 μg/ml, 2 ml of CFP stock solution A was diluted to 10 ml using a diluent (50% methanol) solvent. To make stock solution C, 1 ml of stock solution B was diluted to 10 ml with a diluent solvent to yield a concentration of 20 µg/ml. The entire standard solution was properly sonicated and filtered using 0.45 μm membrane filters before the analysis. Aluminum foil was used to protect stock solution C from light and it was kept in the refrigerator. For stability testing, the normal stock solution C was employed.

Preparation of the mobile phase
A mixture of 0.2% formic acid in water and ACN 20:80 v/v was used as the mobile phase.

Validation procedure
According to the International Conference on Harmonization of Technical Requirement for Registration of Pharmaceuticals for Human Use (ICH) guidelines, the proposed UPLC-MS/MS methodology for CFP was validated (ICH, 2003).

Standard or control
About 0.5 ml of 20 µg/ml CFP solution was diluted with 4.5 ml of diluent to make a standard working solution (2,000 ng/ ml), which was then injected into the UPLC-MS/MS.

Stress conditions
Forced degradation studies were studied per ICH Q1 guidelines using different stress conditions such as acidic, basic, neutral, peroxide, and thermal degradation. The solutions were analyzed regularly at 6, 12, and 24 hours for a week using UPLC-MS/MS. Hydrolysis of CFP in the presence of water was checked because it is reconstituted using water for injection for intravenous or intramuscular purposes. Degradation of the drug under stress conditions is confirmed by the reduction in area in the chromatogram and drug quantity.

Method development and validation
Using ESI in positive ion mode MS/MS detector optimum settings was investigated to acquire transition for each molecule. Full scan spectra were recorded to find the most prevalent m/z value to optimize the cone voltage with the precursor ions as [M+H] + ions. The most frequent ions were found to have the most sensitive transition using collision energies. MS/MS transition for quantification and confirmation for CFP was observed at 481.3 → 166.85. Typical optimized operating conditions were at capillary voltage: 3.00 and 2.94 kV, cone voltage: 25 and 28 V, desolvation temperature: 400°C and 388°C, source temperature: 120°C, desolvation gas flow: 850 and 848 l/hour, cone gas flow: 100 and 97 l/hour, ion energy: 10.5, multiplier: 550-547 V, syringe pump flow: 100 µl/minutes, and collision gas flow: 0.15 and 0.15 ml/minutes. The chromatogram of standard CFP (for n = 6) was found to have a retention time of 0.82 ± 0.02 minutes. The MS/ MS spectrum of [M+H] + of CFP is at m/z of 481.35 with a peak area of 57,736.916 (Fig. 2). The results for CFP validation are summarized in Table 1.

Forced degradation studies
CFP has degraded in all the stress conditions. A typical chromatogram of CFP and its separated degradants is shown in Figure 3. In acidic, neutral, oxidation, and thermal stress conditions, the presence of m/z 481.42 ± 0.07 [M+H] + peak indicates partial degradation of CFP into CFP degradant 1 (CD1) and CFP degradant 2 (CD2

DISCUSSION
In this research, the retention time of the parent compound of CFP is 0.82 minute with MS/MS spectra of standard CFP obtained at an m/z ratio of 481.42. The retention time of degradant CD1 in different condition was 0.91 ±0.01 minute with MS/MS spectra of CD1 at m/z at 179.15. The retention time of degradant CD2 in different condition was 0.84 ± 0.1 minute with MS/MS spectra of CD2 at m/z at 241.33. The presence of these m/z spectra at 481.42 ± 0.09 in all stress conditions, except basic indicates that the CFP is unstable and has partially deteriorated. The peak in the chromatogram and MS/MS spectra of CFP in forced basic medium has been observed at m/z of 179.15 and Retention time at 0.91 ± 0.01 minute of degradant CD1 indicating CFP is more vulnerable to a basic condition resulting in complete degradation of the drug in basic medium. During the   Figure 6. The molecular weight of degraded products with possible fragments was predicted based on the protonated molecular ions [M+H] + and the fragmentation pathway of CFP was established from mass precision and fragmentation mass spectrum. All degradation products and possible fragmentations were characterized and the proposed structures are presented in Table 3.

CONCLUSION
A novel stability-indicating UPLC-MS/MS method for the determination of stability in different stress conditions for CFP has been performed. Rt of the forced degradation studies solution was reported to be 0.82 minute for CFP, CD1 at 0.91 ± 0.01 minute, and CD2 at 0.84 ± 0.1 minute; this method has less Rt when compared to available assay methods found in the literature. Forced degradation studies were successfully developed as per ICH guidelines.

FUTURE PLAN OF WORK
The developed method can be applied to determine degradants of CFP in blood, cerebrospinal fluid, and urine samples by using this automated UPLC-MS/MS technique.

ACKNOWLEDGEMENT
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.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/ guidelines.

FINANCIAL SUPPORT
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICTS OF INTEREST
The authors report no conflicts of interest.

ETHICAL APPROVAL
This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY
All data generated and analyzed are included in this research article.

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