Development of HPLC Method for Estimation of Darunavir Related Substance in Formulation

A simple, specific and validated reverse phase high performance liquid chromatography method was developed for determination of drug related substances (impurity) of Darunavir in formulation. Method development includes optimization of stationary phase (column) and mobile phase flow rate for the resolution of six known impurities and one unknown impurity and Darunavir. Development of HPLC method for estimation of Darunavir related substance in formulation was carried out on Zorbax SB-C8, 250 x 4.6mm, 5µm by using a gradient mode of the mobile phase flow rate at 1ml/min with the same mobile phase as, ACN : Buffer pH 4.0 (Mobile phase B:Mobile Phase A). The stability-indicating capability of the method was proved through the solution state stability, solid state study and it was concluded


Introduction
International conference for harmonization (ICH) provides and quantification of impurities in drug substances and drug products to determining its thresholds [6,7]. Knowing these USFDA regulatory requirements and need of impurity / drug related impurity profiling [8], Darunavir drug had been selected for our current research work. Various new protease inhibitor drugs used to treat HIV infection. Among them, Darunavir is a second-generation protease inhibitor (PIs), designed specifically to overcome problems (Drug resistance problems) with the older agents in this class [9]. Darunavir drug sold under various brand names (dose:100mg/400mg) [10]. It acts on the HIV-Aspartyl protease enzyme which is needed by virus to cleave the HIV polyprotein into its functional fragments. The chemical name of darunavir [11] is (3R, 3aS, 6aR)-hexahydrofuro [2,3-  form is amorphous. When in the formed crystalline forms exist solvent molecules, water, salt, excipient or impurity has a pseudo polymorphic behavior and it can occur during handling, processing and storing. The tendency of a molecule to form solvates is related to the molecular structure, standards of hydrogen bonding and crystal packing. The solvent serves to stabilize the structure and desolvation process results in the formation of an amorphous form [12]. Literature survey reveals that Stability-indicating HPLC [13], RP-HPLC [14][15][16], IR [17], LC-MS [18] and UV Spectrophotometric methods [19] are reported for determination of darunavir as ethanolate form but no methods are reported for determination of darunavir in its amorphous form. Hence the present work describes the development and validation of HPLC method for its assay and related substance determination in amorphous form of the drug.

Apparatus
The analysis was carried out on a HPLC-Jasco LC-Net II/ADC

Selection of λmax by UV Spectroscopy
Standard solution of Darunavir 10 µg/mL was scanned in the range of 400-200nm in 1.0cm cell against solvent (methanol) blank and spectrum were recorded. The spectra of Darunavir standard solution is shown in Figure 2. The λ max of Darunavir was found to be 265.0nm, hence selected as detection wavelength for further experimentation.  Table 2. The chromatogram recorded for trail no 3 is shown in Figure 3.  All the impurity peaks were well resolved with proper peak shape except Dar-I and eluted out within 40 min

Conclusion
From the above chromatographic condition the finalized RP-HPLC parameters are shown in Table 3.

Study of System Suitability Parameters:
The study of chromatogram Figure 3 revels that the API and all the impurities were well resolved except DAR-I Impurity which elutes very close to Darunavir API peak, hence resolution of the same was studied. System suitability parameters were measured to verify the system performance. For this sample solution prepared which contains DAR-I Impurity was 2.4µg/mL and Darunavir 160µg/mL of concentration and five replicate injections of 20µL injected.The chromatogram recorded is shown in Figure 4 and Obsrevations are recorded in Table 4.

Application of Proposed Method to Formulation
The  Table 5. From the figure and observation it was concluded that no additional peaks were seen in the chromatogram of diluents and standard solution. Hence the proposed method was further applied to formulations.  impurity mixture solution and sample solution spiked with known impurities at specification limit were injected into the HPLC system.
There was no interference from the blank at the retention time of Darunavir and impurity peaks. The chromatogram of impurity spiked sample revels that the impurities were well separated and also the peak purity data revels that there was no co-eluting peaks and no interference of impurities at the retention time of analyte peak. The observed chromatograms are shown in Figures 7a-7k.
The observations of spiked sample for specificity are shown in the             LOQ, 3x 50% , 3x 100% and 3x 150% of the limit concentration.
DAR-I Impurity stock solution was prepared of a concentration 60.7µg/mL as a stock solution.
NOTE: Impurity is spiked in 2 nd dilution of sample solution.

Procedure
Weighed 20 tablets and average weight of tablet calculated,

Precision
The precision of an analytical procedure is usually expressed as the variance, standard deviation, relative standard deviation or coefficient of series of measurements (Table 7).

System Precision for Related Substance: System precision
was performed by single injection of Blank (Diluent) and six replicate injections of DAR standard solution were injected on the system. The results obtained from the observation of chromatogram was shown in Table 8.   Table 9.  Table 10.

Limit of Detection and Limit of Quantitation
Based on determination of prediction linearity, six replicate injections were made for LOQ.Six replicates of above predicted LOD and LOQ solution were injected on system and calculated % RSD are summarized in Table 11.

Forced Degradation
In forced degradation study a drug substance or the drug product is exposed to an environmental vigorous than the normal The amount of standard drug undergarded was calculated using the formula

Au expose W unexposed Drug undegradation
Potency As unexposed Wstd exposed Where, Au = Peak area of standard (exposed), As = Peak area of standard (unexposed), Wstd = Weight of Standard Calculation formula for Mass balance:

Assay of degraded sample total impurities generated Mass balance
Assay of control sample total impurities present mass balance was calculated using the formula 1 and 2 is shown in Table 12.

Solid State Analysis
Photolytic degradation (UV Light): According to ICH Guideline sample should be exposed to light to providing an overall illumination of not less than 200 watt hrs. /square meter. The stability chamber was calibrated using appropriate UV meter for UV Light study and the study was carried out for 7 days. The sample solution was prepared following general procedure.
After photolytic degradation sample solution for assay and for related substance solution were injected and chromatographed separately using optimize chromatographic conditions.

Thermal degradation
It was performed by heating the solution in the oven at 50 °C for 24 h, allowed to cool and volume made up to the mark with diluent and mixed. The sample solution was prepared following general procedure. The sample solution for assay and for related substances were injected and chromatographed separately using optimize chromatographic conditions. From the chromatographic observations of Photolytic degradation (UV Light) and thermal degradation chromatograms, AUC were noted, % undegraded drug (Assay) and mass balance was calculated using the formula 1 and 2 is shown in Table 12. Purity Threshold was found to be greater than purity angle. obtained are shown in Table 13.

Filter Compatibility
The Standard and Sample solution was prepared as per described in general procedure for preparation of solutions and filtered through different types of filters (PVDF, Nylon, PTFE & Teflon + Glass) and injected on to the HPLC. The centrifuged sample and filtrates of different filters were analyzed for their equivalency.
The observations are shown in Table 14.

Robustness
The robustness of an analytical procedure is measured by making small but deliberate variations in method parameters and observing the effect of these variations on the system suitability parameters by injecting standard and sample solution.

a.
Change in chromatographic conditions:
The observations are shown in Table 15a & 15b.