QBD driven stability indicating RP-HPLC assay method for Flibanserin; development, validation and characterization of major degradants by LC/QTOF-MS/MS

Manish Kumar Sharma

National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, India

Abstract

Abstract :

The characteristic chemical soundness of Flibanserin (FLB) was examined by utilizing Plackett Burman plan for screening of free factors. Box- Behnken plan was utilized for optimization of tall execution fluid chromatography (HPLC) steadiness demonstrating test strategy. Constrained debasement of FLB was carried out beneath hydrolysis (acidic, fundamental, and unbiased), photolysis, oxidation and warm stretch conditions. The major oxidative debasement item was separated by preparative HPLC. The corruption item was recognized as 2-H¬Benzimidazol-2-one, 1, 3-dihydro-1-[2-[4-[3(trifluoro methyl) phenyl]-1-N- oxide-piperazinyl] ethyl] taking after characterization by UV, IR, HRMS and NMR procedures. The parent particle mass of the oxidative degradants was watched mass 407.1710 and major parts (389.1582, 375.1422, 255.1100, 243.1103, 228.0866, 216.1127, and 200.0680) in LC-QTOF-MS when examined in positive ionization mode. Oxidation and hydrolysis were found to be the essential corruption pathways for this particle. The chromatographic partition was accomplished on Cromasil C18 column (4.6mm×250mm, 5μm) employing a versatile stage comprising of a blend of ammonium acetic acid derivation (pH 5.5) and acetonitrile in direct angle elution mode. The strategy was found to be direct within the concentration extend of LOQ (0.5 to 70 µg/mL). The strategy was approved as per ICH rule Q2 (R1). Corruption of FLB taken after first-order energy beneath all test conditions. V-shaped pH-rate profile energy over the pH run 2–10 was watched with greatest steadiness at pH 6.8. In conclusion, a switch stage tall execution fluid chromatographic strategy has been created and approved for quantitation of FLB in nearness of their corruption items. The major corruption item has been distinguished and completely characterized that has not been detailed till date. Usually the primary time to report a steadiness showing measure strategy for FLB.

Background:

In this work, we point to create and approve a quick, straightforward, and delicate strategy for the quantitative assurance of flibanserin and the investigation of its pharmacokinetics.

Materials and Methods:

Ultra-performance fluid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was the strategy of choice for this examination and carbamazepine was chosen as an inner standard (IS). The plasma tests were prepared by one-step protein precipitation utilizing acetonitrile. The exceedingly particular chromatographic partition of flibanserin and carbamazepine (IS) was figured it out utilizing an Agilent RRHD Obscure Furthermore C18 (2.1 × 50 mm, 1.8 µ) column with a slope portable stage comprising of 0.1% formic corrosive in water and acetonitrile. The analytes were recognized utilizing positive-ion electrospray ionization mass spectrometry through different response observing (MRM). The target part particles were m/z 391.3 → 161.3 for flibanserin and m/z 237.1 → 194 for carbamazepine (IS). The strategy was approved by straight calibration plots over the run of 100–120,000 ng/mL for flibanserin (R2 = 0.999) in rodent plasma.

Chemicals and reagents:

We obtained flibanserin (over 98% immaculateness) from perfemiker (Shanghai, China). The carbamazepine (purity > 98%) was procured from Sigma-Aldrich (St. Louis, Moment, USA). As HPLC review, the methanol, formic corrosive and acetonitrile were bought in Merck Company (Darmstadt, Germany). In expansion, the ultrapure water was gotten from the Milli-Q Reagent water framework (Millipore, MA, and USA).

Instrumentation and conditions:

We conducted the tests examination by chromatographic framework of Agilent 1290 of ultra-performance fluid chromatography (UHPLC, Agilent Innovations, Santa Clara, CA, USA) coupled to an Agilent 6490 Triple Quadrupole mass spectrometer (Agilent Advances), which had the triple quadrupole mass spectrometer, a degasser, a HiP sampler, a column compartment and a twofold pump. The column of RRHD Overshadow Additionally C18 (2.1 × 50 mm, 1.8 µ) at the steady temperature of 35 °C was connected to the partition of compounds. The ideal choice of stage of versatile comprised acetonitrile (B) and formic corrosive (A) of 0.1%. Angle elution’s course was utilized within the taking after: direct increment for 0–1.0 min to 90% of B, 1.0–2.6 min kept up at 90%, 2.6–3 min direct diminish to 20% of B. Infusion of analyte volume was 2 µL and stream rate of versatile was 0.4 mL/min.

Result and Discussion:

The fluid chromatography conditions were examined with the objective of isolating interferometer analytes, moving forward the discovery affectability and shortening the runtime. This included optimization of the composition and proportions of the versatile stage, and the column and its temperature. The RRHD Overshadow Also C18 column (2.1 × 50 mm, 1.8 µm) illustrated great symmetry for the analytic top and an appropriate maintenance time.

In arrange to realize effective separation, the crest shape must be symmetrical and the maintenance time ought to be abbreviated. A blend of 0.1% formic corrosive in water and acetonitrile was utilized as the versatile stage composition and angle elution was connected. The stream rate was explored over a extend between 0.2 and 1.0 mL/min and the impact of the column temperature was examined within the extend of 20 to 40 °C. For a portable stage formed with 0.1% of formic corrosive in water (A) and acetonitrile (B), ideal comes about can be gotten employing a stream rate of 0.4 mL/min and a column temperature of 35 °C

We optimized the mass parameters in arrange to attain the next reaction and way better determination. To begin with, the fragment or was set in a unpleasant run from 50 to 240, and the Collision Vitality (CE) extend was between 10 and 50 within the positive mode. After completing the MS/MS optimization strategy, the foremost strongly part was utilized for the evaluation of flibanserin and IS, and the moment most seriously one was utilized for capability of the target analyses.

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