A green analytical method for estimation of letermovir in degradation studies

The present established analytical method depicts identi ication of degradation products of letermovir with the aid of U.P.L.C-M.S/M.S. Letermovir was subjected to hydrolysis, oxidation, photolysis and thermal pressure, according to with I.C.H conditions. The analyte shows degradation in oxidative and photolysis stress conditions. The analyte elution was inished on an intersil ODS column (150×4.6m.m; 5μ.m)withpolar eluent containswater andmethanol (20:80%, v/v) in an isocratic elution mode at a low rate of 0.6m.L/min. The eluents have been monitored through the use of a U.P.L.C-M.S/M.S and linearity range was obtained from 50-500 ng./m.L. The method was found to be stable and precision study was expressed in %R.S.D, ranged from 0.43 to 1.69. The developed method was validated as per I.C.H guidelines. No prior approach became made concerning degradation behavior of letermovir. The multiple reaction monitoring transitions were set at 573.31 → 381.12(m/z) and 288.10 → 176.10(m/z)for Letermovir and Tenofovir disoproxil respectively. The calibration curvewas linear over the range of 50-550.00ng/mlwith lower limit of quanti ication validated at 50ng/mL. The inter and intra-day accuracy values were below 15% at all quality control levels. The within run and between run precisions were within 15%, while accuracy ranged from 97.07 to 101.98. The degradation products were characterized by comparing their collision induced dissociationmass spectral datawith that of Letermovir and the most possible degradation and fragmentation pathways were identiied.

From our expertise, none of the methods has been stated for determination of letermovir and its degeneration of products by means of L.C.-M.S-M.S. The literature reveled very few analytical methods available methods for the estimation of letermovir, which include letermovir enantiomeric purity by HPLC (Zhang et al., 2016) and estimation in biological luids (Rajanikanth et al., 2019). Based on literature, it is important to research the analytical method to analyze the degradation behavior of letermovir upon exposure to I.C.H., recommended stress parameters making use of an optimized L.C-M.S-M.S (ICH, 2003).

Selectivity & Speci icity
Speci icity studies indicating that the complex matrices did not interfere with the analysis. In order to test the interference at the retention time of Letermovir and Tenofovir disoproxil& blank samples (Eluent) were injected in to U.P.L.C-M.S/M.S. Hence, the chromatographic system used for the estimation of Letermovir and Tenofovir disoproxil were very elective and speci ic.

Linearity and Range
The Letermovir was assessed at 50 % to 150 % of the target concentration at different levels in the range of 50.0 ng./m.L to 550.0 ng./m.L for linearity study.

Precision study
The intra and Inter day precision study was assessed by analyzing six replicates of three different standard samples (200, 300 and 400 ng./m.L) and %CV was calculated.

Detection and quanti ication Limit
For Letermovir, the LOD and LOQ of was determined by calibration curve method. Solutions of Letermovir (LV) were prepared in linearity range and injected in triplicate. Average peak area ratio of three analyses was plotted against concentration.

Solution Stability
A

Preparation of stressed or degradation sample solutions
Weighed about 10 m.g of Letermovir was taken in a 10 m.L volumetric lask individually and dissolved 25 m.L methanol and diluted with 0.1 N HCl, 0.1 N NaOH and 3% v/v H2O2 and water and set aside for 72 h at ambient temperature. For Thermal (Dry heat) and Photolytic Degradation 10m.g Letermovir was taken on Petri dishe and exposed to heat (80ºC for 72 hrs) and UV radiation ((254 n.m) at 1.2 million lux-hours for 72 h). After degeneration, samples were diluted with eluent to get the inal concentration 100 ng./m.L and injected into U.P.L.C-M.S/M.S system.

LC-MS/MS Conditions
For better separation and ionization, a mixture of methanol and water (80:20, v/v) and an "Inertsil ODS-3 C18 (250 mm × 4.6 mm, i.d., 5 µm)" column at low of 0.6m.L/min was used. Due to compatibility with analyze chromatographic conditions Tenofovir disoproxil was selected as IS (internal standard). The chromatographic peaks were eluted at 5.68 for Letermovir and Tenofovir disoproxil for 5.88 min. Mass parameters were optimized positive ion mode with ion transitions of m/z 573.31 → 381.12 and 288.10 → 176.10 for LV and TVIS (Figure 2). In the proposed method, nontoxic, nor hazardous. The pH of the samples and the eluent is about neutral i.e., not corrosive. So according to these criteria, the proposed method passes the greenness pro ile.

Method validation
Precision of Instrument parameter can be de ined as test to ensure that the instrument can generate precise results. In this method %RSD value obtained was less than 2%.
From Figure 3, it was observed at retention times of Letermovir and Tenofovir disoproxil in eluent no signi icant response. It can be concluded that the method is speci ic for estimation of Letermovir in presence of solvent. Linearity was found to be satisfactory with correlation coef icient 0.9960 over the concentration range of 50.0 to 550.0 ng./m.L (Table 1 and Figure 4).
The precision study of the proposed method was evaluated at three different concentration levels. The %RSD and accuracy was found to be 0.43 to 1.27 and 99.36 to 99.73% for intraday precision study. Whereas, for interday precision study %RSD and accuracy was found to be 0.57 to 1.64 and 97.07 to 101.98% (Table 2). The detection and quanti ication limits were evaluated from calibration curve plotted in concentration range of 50.0 -550.0 n.g./m.L. L.O.D and L.O.Q for this method were found to be 45.49 and 137.85 p.g./m.L respectively. These values indicated that the method was sensitive to quantify the drug.
The %RSD of Letermovir was good under most conditions and didn't show any signi icant change when the critical parameters were modi ied and the components (Analyte and IS) were well separated under all the changes carried out. Thus, the method conditions were robust and Stable (Table 3). The % difference values for Letermovir and Tenofovir disoproxil of different ilter materials was found to be 0.17 to 1.39% and 0.45 to 0.94 and no signi icant interference was observed.

Identi ication of major degeneration of product formed under stress conditions by U.P.L.C-M.S/M.S
The fragmentation for the degradants was also carried out for Letermovir using product ion scan by U.P.L.C-M.S/M.S. In these stress studies, a total of two degradation products (D1 and D2) were observed for Letermovir in D1 (Peoxide) and D2 (Photolyic). The degeneration products of oxidative and photolytic were analyzed by U.P.L.C-M.S/M.S, the total ion chromatograms of both degrades was showing molecular ions at m/z 554 and 471 indicating the presence of the degeneration product in both cases. The oxidation stress conditions were studied using 3% v/v H2O2 up to 72 h at room temperature. It was found that, 3% v/v H2O2 was effective in oxidizing the drug even after 72 h. The degradant products of molecular ion at m/z 554 and 471 were shown in Figures 5 and 6. No Acidic, Basic, neutral and thermal degeneration was observed for the solution and solid form of drug after exposure to the water, 80ºC, up to 72 h, respectively. It was con irm that drug was found to be stable solid as well as in solution forms under Acidic, Basic, neutral and thermal stress conditions. The respective degeneration of m/z values of all degradate and their fragmentation ions was represent in Tables 4 and 5 and Figures 5 and 6.

CONCLUSIONS
In this study, the forced degeneration study on letermovir has been performed under I.C.H prescribed situations as to take a look at its degradation proile. An Eco friendly, sensitive, accurate, speci ic and reproducible isocratic stability-indicating U.P.L.C-M.S/M.S technique became evolved. The validation experiment proved that the developed method was linear, unique, speci ic and selective and further identi ied, the drug was sensitive to oxidative and photolytic conditions. This approach may be used for ordinary analysis of letermovir in Q.C laboratories without harming the environmental conditions.