Validation and Development of a Method for Identification and Stability Analysis of Delafloxacin Separation Using LC-MS/MS

Introduction

Delafloxacin(DFA) is used for treating adults those who are facing the problem with ABSSSI.¹  This drug is well admitted as ABT-492, RX-3341, and WQ-3034 at the same time it is concealed by the development.²  One of the famous pharmaceuticals named as Rib-X seized this drug.³ Rib-X is renamed as Melinta Therapeutics in the year 2013.⁴ This is later associated by Cempra.⁵  Veronica Hernandis et.al.,⁶measured plasma delafloxacin strengths by RP chromatography.  Wavelength is 405m and 450 nm. Column as Kromasil C18 column 250 × 4.6 mm id, 5 µm. Mobile phase is 0.05% trifluoroacetic acid/acetonitrile as 52/48. Rt values are 5.4 and 11.6min. Calibration curves are 0.1 µg/mL to 2.5 µg/mL. LOD and LOQ are 0.05 µg/mL, 0.1 µg/mL. Accuracy and precision values are less to than to 11%> LOQ is 16%. Recovery is 98.3%.   Iqbal, M et.al.,⁷used UPLC BEH C18 column (50 × 2.1 mm; 1.7μm.  Mobile phase is 0.1% formic acid in both acetonitrile and in water. Iqbal, M et.al.,⁸ information the MRM transitions are arranged as 441.14 > 379.09 as well as 436.89 > 144.87 for internal standard.   Alam P et.al.,⁹usedRP-18 silica gel 60 F254S HPTLC plates. Ethanol 5v/v, water 4v/v and ammonia 2v/v used as mobile phase. Wavelength is 295 nm. % assay is 98.2 and 101.0% and SLNs are 94.4 and 95.0%.  Jorgensen SCJ et.al.,¹⁰ reveals that maximized penetration of intracellular, enhanced bactericidal exercise beneath to acidic circumstances.   D. Chandrapal Reddy et.al.,¹¹ used X-terra RP8 (50 mmx4.6 mm, 5 μm particle size) column.   30 mM ammonium formate (pH-5.0±0.05) and acetonitrile are used as mobile phase.  Rate of flow is 0.40mL/min.   The linearity is 0.100 ng/mL – 100.017 ng/mL. LOD is 0.04 ng/mL and LOQ is 0.100 ng/mL. Intra and Inter precisions are 5.21 to 7.02. Accuracy is 97.14 to 103.50. Recovery is 80.31% and ISTD recovery is 81.09%.   Lou Y et.al., ¹² used Waters X Bridge C18 (2.1 mm × 100 mm, 3.5 µm).  buffer as 0.2 % formic acid in water and acetonitrile. Run time is 4.0 min.  Volume injected is 5μL. Linearity is 2 ng/mL to 500 ng/ml.  By using this literature review authors are performed this validation and observed that the values obtained in this work are more accurate when compared with other values. 

Experimental

Pure delafloxacin contain 99.9% purity of drug was procured from sigma Aldrich. 70%Acetonitrile, NaOH, 0.2% Formic acid as 30%, and HCl were obtained from Merck life sciences, and di-methyl sulphoxide procured from loba company pvt. Ltd., Mumbai. All the solutions are subjected to filtration via Millipore 0.22µm filter paper (India). Merck millipore provided Milli-Q-Water.  The LC-MS/MS experiments are performed using a Waters micro mass Quattro Premier XEseriesliquidchromatography system all weighing processes were settled using an analyticalbalance. At all weighing events, a (Mettler Toledo) scale was used. Photo stability chamber, sonicator and hot air oven were used as well.  The software used for monitoring and integration of chromatographic and spectrumpeakswas executedwithMass Lynxsoftware.

Detection Method

For optimal results, use a Waters column measuring 2.1 mm x 100 mm, 5 m, C18, with a precise mobile phase ratio as 30:70 for acetonitrile and 0.2% formic acid (pH 2.6). The flow rate of 0.12 µL/min produced the best retention of delafloxacin.

Mass parameter

Optimized for this approach is positive ionization mode (ES+) and triple TOF mass spectrometer from the 4600 series with an ESI source.  The capillary voltage was kept at 2.94 KV and pure nitrogen gas was employed as the nebulizer. The source temperature is appropriate at 119 °C, and the mass spectrum collision energy is fixed at 32 eV.The sample of delafloxacin for the injection into mass spectrometry was modify to 10 µL for better ionization of given sample and both parent daughter ion identified.  The desolvation gas flow (L/Hr) 850 and desolvation Temperature (°C) 400. The Cone Gas Flow (L/Hr) 102 was fixed. Cone voltage and Extractor voltage were set to 33 V, 3V respectively. Mass spectrum development was performed using MRM ion mode (ES+) with mass transitions of m/z 441.10 > 379.10 (amu) at retention time of 1.2 minutes for DLF and data acquired with Software Version: 1.40.1248 (MassLynx 4.1 SCN805).  The related information is shown in the figure 1 to 3. 

Figure 1: Optimized Chromatogram Click here to View Figure

Figure 2: Parent ion spectra – Delafloxacin. Click here to View Figure

Figure 3: Daughter ion spectra – Delafloxacin Click here to View Figure

Results And Discussion

StressDecompositionBehaviourofDelafloxacin

To start with, a working standard of delafloxacin containing 100 ng/ml (ppb) was established. This working standard is recycled to establish the purity peak for delafloxacin during the stress inquiry and to determine the synthesis of its DPs using the photo diode array (PDA) detector. The outcome of delafloxacin is shown in the table.1.

Degradation in acidic condition

Degradation was carried out on a wide variety of acidic pH values. 30 mins of individual exposure to 2 ml of 1% Hcl were given to each sample. It was cooled to room temperature after 30 mints. The aforesaid sample was then diluted to a final concentration of 100ngmL-1 (ppb) using diluents.   Stressed samples were strained through a 0.22 m filter. With RT: 1.25 minutes, 11.1% degradation was attained, although 88.9% of the purity was retained.

Degradation in alkaline condition

Alkaline degradation was carried out over a wide range of basic pH values. Each sample was separately exposed for 30 minutes with 2 millilitres of 1M NaOH. A basic condition was used for the alkaline degradation investigation. 98% of the delafloxacin was still present with an Rt of 1.26 mints during the analysis of the DLF, which revealed 2% degradation.

Oxidative degradation         

Degradation studies are carried out using a variety of oxidizing agents, including metal ions and radical initiators, however hydrogen peroxide is among the most common oxidizing agent (for oxidative degradation). Oxidative drug degradation for the product of reactive anions and cations served as a representation of the electron transfer mechanism. For 30 minutes, 3% H2O2 was treated with delafloxacin and kept at room temperature (28°C). 7.2% degradation was recorded during oxidative stressed degradation. The stressed sample wasdiluted to achieve a final concentration of 100ngmL1 (ppb). During oxidation, DLF degradation (Rt 1.24 min) was discovered.

Thermal conditions

To determine how temperature affects a substance’s capacity to withstand heat. The drug component is placed in a Petri dish and is done in a dry oven to 105°C for six hours. Once the required period of time passed, the sample was removed from the dry oven, and the final concentration was adjusted to 100 ng mL-1 using diluents. 85% of the drug was recognized by thermal analysis (RT: 1.02 mints).

Table 1: Degradation Analysis Report for Delafloxacin

Categoryof Stress	PeakArea response	Assay Value	%DegradationofDLF	Interference
Undegraded	26398	100	0	Peak is    homogeneous and there is no co-eluting peaks
Acid	23474	88.9	11.1
Base	25873	98	2
Peroxide (oxidation)	24518	92.8	7.2
Thermal	22538	85	15

 

Analytical Method Validation of delafloxacin

System Suitability

System suitability assessment is important in analytical procedures. At an LLOQ concentration of 100 ng mL-1, standard solution is passed. Both the system’s features as well as % RSD are established in order to evaluate its suitability. A total of six cloned injections were utilized to calculate the area responses’ percent RSD. Predefined requirements were followed for parameters including resolution, tailing factor, and retention time spent. The system was suitable to undertake other validation parameters since the acquired% RSD of area responses for delafloxacin was 3.5%, which agreed with the maximum limit of not more than 5%. The result of the use of delafloxacin is shown in the table.2.

Table 2: System Suitability Evaluation

Sr.No	Area	Retention timein min.
1	22071	1.24
2	21948	1.23
3	22624	1.24
4	23151	1.23
5	23343	1.23
6	24039	1.23
Mean	22862.67
Std.Deviation	802.238
%RSD	3.5

 

Specificity

With a maximum concentration of 150 ng/ml over six repetitions, selectivity ensures that the procedure reliably measures the target analyte without interference from other substances. There was no interference at the analyte’s retention period, and the analyte’s identity was verified by distinctive MS/MS transitions.The selectivity of the suggested methods report was demonstrated below by contrasting the chromatogram of the working standard solution, blank. Six successive concentrations that do not showed any additional peaks proved that the method being used is adequately appropriate for the task at concern.

Accuracy and Precision of delafloxacin

For the purpose of an accuracy, a known concentration of the delafloxacin (100 ngml-1) was divided into three concentration levels were low, medium and high (LOQ-50%, MOQ-100, and HOQ-150%). For LOQ, MOQ, and HOQ, respectively, the average recovery was determined to be 99.3%, and the percentage RSD was computed as 4.5, 7.8, and 2.7 (mean of three replicates). The chromatogram’s result is shown in Figure 4. The accuracy has to range from 85 to 115% of the nominal values, and % RSD should be within ±15%.  Six LLOQ concentrations (10 ngml-1) of delafloxacin were evaluated in order to carry out the precision study. The determined percent RSD was 0.71%, which yielded results that were within tolerances. The acceptable threshold for precision is 15% or less %RSD. The table (3, 4) below contains the delafloxacin outcome.  And the related chromatograms are represented in the figure 4. 

Table 3: Accuracy data of Delafloxacin(n=3).

Spiked conce. as ngml-1	Conce. found as ngml-1	Area	RSD as %	%Recovery
LOQ-50	49.81	n=3=13012	4.5	113.83
MOQ- 100	91.12	n=3=20831	7.8	91.12
HOQ- 150	139.3	n=3=31863	2.7	92.91

 

Table 4: Precision method of Delafloxacin.

SampleNo	LLOQ conce. as ngmL-1	Precision of DLF
		Area	RSD as %
1	10	3306	0.71
2	10	3317
3	10	3369
4	10	3347
5	10	3316
6	10	3339

 

Figure 4: LOQ, MOQ, HOQ. Click here to View Figure

Linearity

Seven distinct concentrations (7-point) of the standard solution of delafloxacin, ranging from 10 to 150 ng/ml, were used to analyze the method’s linearity. Calculate the slope, correlation coefficient, and intercept for concentration versus response of area plot.The results of calibration curve were 232.8, 0.9968, and 1273, respectively, for slope, coefficient of correlation (r2), and intercept.  The linearity curve and results are represented in the table 5 and figure 5.

Table 5: Linearity of delafloxacin.

Conce. In ng/ ml	PeakArea	Slopeas m	Intercept Value asb	Correlation Co-efficient asr2
10	3368	232.8	1273	0.9968
25	7870
50	12305
75	19243
100	25142
125	29653
150	35931

 

Figure 5: Linear curve of delafloxacin (10 – 150ng/ml). Click here to View Figure

Sensitivity of delafloxacin

By using LOD and LOQ, the approach’s sensitivity has been rendered clear. Select a diluted solution (lowest possible known concentration) from the linearity curve to display the LOD and LOQ values. For LLOD (detection limit) and LLOQ (quantitation limit), the S/N ratio was calculated.The LOQ’s %RSD was calculated as 1.6, and the LOD’s %RSD was 2.0. These estimated values were within acceptable.On measuring the concentration (LLOQ-10 ng/ ml, LLOD-3 ng/ ml) of the technique having signal-to-noise ratio LOD should be three times to noise level, also LOQ should be ten times tolevel of noise. ^[18-19]Results showed in table.6.

Table 6: LOD and LOQ- Sensitivity of delafloxacin.

Conce. In ppb	Retentiontime in min.	Area	S/N	Conce. In ppb	Retention time in min.	Area	S/N
10	1.23	3396	447	3	1.24	1134	249
10	1.23	2791	443	3	1.24	1198	248
10	1.23	3305	451	3	1.24	1193	247
10	1.23	3368	458	3	1.24	1108	245
10	1.23	3346	439	3	1.24	1263	259
10	1.23	3316	441	3	1.24	1207	257
Mean	447			251
%RSD	1.6			2.08

 

Conclusion

A linear, accurate, and specific stability indicating LC-MS/MS technique is established also completely validated in consonence with FDA criteria to determine the stability of delafloxacin in the form of its degradation products. It was described as a particularly sensitive UPLC-MS/MS method that could identifyits degradation to the nanogram level. Delafloxacin investigation was found to be less susceptible to circumstances that cause acidic, alkaline, oxidative, basic, and thermal degradation. Delafloxacin and its DP were identified and characterized using the MS/MS technique in ESI mode of positive. In current study, the research could be very helpful in locating other potential DPs and process-related impurities that might be present in delafloxacin in very small amounts. With the effective identification and characterization of delafloxacin’s degradation during stress testing.

Acknowledgements

The authors are thankful for management of Krishna University for conditional necessary facilities to accomplish this research work. 

Conflicts of Interest

There are no conflicts of interest among the authors to perform this work.

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