Methods for the Analysis of Lovastatin Extended-Release Tablets

To start the standard development process and have earlier stakeholder engagement, USP is piloting a new approach for developing and sharing information with our stakeholders. Through a collaboration between USP’s Small Molecules Department and Global Analytical Development Laboratory, methods are being developed and validated for drug substances and drug products for which no monographs are currently available. The Emerging Standards are intended to improve USP’s official standards elaboration process by increasing transparency and allowing for broader stakeholder participation by publishing on the USP website prior to formal notice and comment through publication in the Pharmacopeial Forum.

Lovastatin extended release (ER) tablets has been evaluated and shown to be a suitable candidate for development under this new program. The methods in this document are being published to help analyze lovastatin ER tablets. Additional method development and validation information is provided to justify the use of method parameters.

Certain commercial software, instruments, or material may be identified in this document to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the software, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate.

This document is not a USP compendial standard and is intended to serve as a resource for informational purposes only. It does not reflect USP or USP’s Expert Body opinions of future revisions to the official text of the USP-NF. Parties relying on the information in this document bear independent responsibility for awareness of, and compliance with, any applicable federal, state, or local laws and requirements

Lovastatin is classified as an anticholesteremic agent. This medication belongs to a class of drugs called ”statins,” which reduce the production of cholesterol in the liver, leading to lower blood cholesterol levels. Statins are commonly prescribed to manage hypercholesterolemia and reduce the risk of cardiovascular diseases.¹

No pharmacopeia currently provides a monograph for lovastatin ER tablets. Procedures for identification (ID), assay and organic impurities (OI) were developed and validated for lovastatin ER tablets. The impurities incorporated are mevastatin, lovastatin related compound -A (RC-A), lovastatin acid, dehydrolovastatin, lovastatin angelate, lovastatin dimer and epilovastatin.

This document summarizes method development, validation, robustness, and forced degradation study results. It also describes the procedures for the ID, assay, and OI determination of lovastatin in the presence of various impurities and excipients in lovastatin ER tablets. Summary of validation data and representative chromatographic results are included.

3.1 Lovastatin and Impurities Standards

USP Reference Standards (RS) for lovastatin, mevastatin, lovastatin RC-A, lovastatin impurity identification mixture, lovastatin epimer mixture (1:1 mixture of epilovastatin and lovastatin) were used. USP Lovastatin Impurity Identification Mixture RS contains lovastatin and approximately 0.2% lovastatin acid, 0.2% mevastatin, 0.4% dehydrolovastatin and 0.3% lovastatin dimer. Dehydrolovastatin was obtained from Anant Pharmaceuticals Pvt Ltd, and lovastatin angelate was sourced from TLC Pharmaceuticals Standards. Chemical structures of lovastatin and related impurities are shown in Figures 1 and 2.

Figure 1. Lovastatin

Figure 2a. Mevastatin

Figure 2b. Lovastatin RC-A (4,4a-dihydrolovastatin)

Figure 2c. Lovastatin acid (or) hydroxy acid lovastatin

Figure 2d. Dehydrolovastatin

Figure 2e. Lovastatin angelate

Figure 2f. Lovastatin dimer

Figure 2g. Epilovastatin

Figure 2. Lovastatin related impurities

3.2 Samples

Lovastatin ER tablets, (20 mg) from one commercial source was used to evaluate methods described in this document.

3.3 Reagents

Ortho-phosphoric acid 88% (EMPARTA® ACS grade) was obtained from Merck. sodium hydroxide (NaOH) pellets (Special quality [SQ] grade), acetic acid glacial (HPLC grade), and acetonitrile (HPLC grade) were obtained from Qualigens. Water was obtained from the Sartorius water purification system.

The initial goal of this work was to develop a stability-indicating OI method for all known impurities and potential degradants for lovastatin ER tablets, using the HPLC conditions from the OI procedures of the current European Pharmacopeia (EP) Simvastatin monograph and USP Lovastatin family monographs. However, the separation of lovastatin and epilovastatin was not satisfactory using any of these procedures.

A new OI method was developed using the analytical quality by design approach. This approach optimized the chromatographic conditions to achieve the desired resolution of the target analytes.

OI procedure validation was performed for all the impurities except lovastatin dimer and epilovastatin due to poor quality of the respective impurity materials. Specificity, retention time (RT) and relative retention time (RRT) were reported for these two impurities. The same method was used for assay and identification tests.

4.1 Chromatography Optimization

ACD software was used for the screening of column chemistry and mobile phase composition. The gradient program was optimized to achieve the desired resolution of the target analytes. See the Assay section 6 and Organic Impurity section 7 for the method conditions.

4.2 Forced Degradation Study

Forced degradation study was performed by exposing USP Lovastatin RS to acid, base, oxidation, heat, humidity, and light conditions. The stressed samples and a control sample (unstressed) were analyzed and compared. The chromatograms were processed at 238 nm for lovastatin and all other impurities and at 200 nm for lovastatin RC-A only. The photo diode array (PDA) data from 190-400 nm showed homogeneity of ultraviolet (UV) spectrum for the lovastatin indicating the peak is free from coelution under all the stress conditions. The results for each of the forced degradation conditions are presented in Table 1.

Table 1. Forced degradation study results

In summary, no major degradation was observed under conditions like heat, heat/humidity, light exposure, and oxidation with AIBN.

^a HCl = hydrochloric acid

^b Hydrogen peroxide

^c AIBN = azobisisobutyronitrile

Lovastatin acid impurity was the major degradant formed in conditions like acid (22.7%), oxidation (peroxide, 3.7%), and base (99.9%).

Complete degradation was observed under base condition, even with lower concentrations like 0.05N NaOH and 0.02N NaOH.

All known and unknown impurity peaks were separated from each other and no coelution was observed with the lovastatin peak.

4.3 Robustness Study

Robustness solution: A solution consisting of lovastatin angelate, lovastatin RC-A, epilovastatin, butylated hydroxyanisole (BHA; an excipient), and lovastatin impurity identification mixture was prepared. USP Lovastatin Impurity Identification Mixture RS contains lovastatin, lovastatin acid, mevastatin, dehydrolovastatin and lovastatin dimer.

The Robustness solution was used for robustness study. A multifactorial robustness study was conducted using design of experiment (DOE) software to evaluate the method robustness with respect to the peak separation. The original chromatographic conditions are specified in Assay, section 6. Using DOE software, 25 chromatographic conditions were designed and tested to evaluate the effects of small but concomitant changes in the following parameters:

1. Flow rate (± 0.1 mL/min)
2. Column temperature (-2° and +3°)
3. Initial isocratic hold time (± 0.5 min)
4. Gradient composition at 30 min, ± 5% of Solution B

See Table 2 for the details of the experimental design.

Robustness of the method was determined by monitoring USP resolutions, peak tailing, and relative retention time (RRT). The data demonstrated that the method was robust under the deliberate minor but concomitant changes to chromatographic parameters.

Table 2. Multifactorial robustness study conducted by using DOE software

In summary, the result of the multivariate study indicated that the method is robust for concomitant but small changes in flow rate, column temperature, initial isocratic hold time and gradient composition at 30 min. The resolution criteria of not less than (NLT) 2.0 between lovastatin and adjacent peaks, and NLT 1.5 between other peaks were met in all conditions studied.

Identification of lovastatin in lovastatin ER tablets was evaluated using the HPLC method from the Assay procedure (section 6) with PDA spectral match and chromatographic retention time (RT) match. See the Assay procedure section (6.1) for instruments and method and section (6.2) for solutions.

5.1 PDA Spectral Match

The validation parameters and results are summarized in Table 3, and representative UV spectra of lovastatin from the Standard solution and Sample solution are shown in Figures 3 and 4 respectively.

Table 3. Summary of validation parameter, solutions, and results for the Identification procedure by PDA spectral match

Figure 3. UV spectrum of lovastatin from the Standard solution

Figure 4. UV spectrum of lovastatin from the Sample solution

5.2 Retention Time Match

The validation parameter and results are summarized in Table 4, and representative chromatograms of the System suitability solution (0.4 mg/mL of lovastatin epimer mixture), Standard solution and Sample solutions are shown in Figures 5, 6 and 7, respectively.

Table 4. Summary of validation parameters, solutions, and results for the Identification test by RT match

Figure 5. Chromatogram of System suitability solution

Figure 6. Chromatogram of Standard solution

Figure 7. Chromatogram of Sample solution

Validation of the assay procedure for lovastatin ER tablets was conducted using the criteria described in USP General Chapter 〈1225〉, Validation of Compendial Procedures,² and found to be specific, linear, accurate, and precise for the sample evaluated.

6.1 Instruments and Method

The analysis of lovastatin ER tablets was performed using Waters Alliance 2695 and Agilent 1260 Infinity series instruments equipped with a PDA detector. CORTECS C18, 90Å, (4.6 x 150) mm, 2.7 µm (L1) column from Waters was used. The analysis was performed at 25° column temperature, with a flow rate of 1.0 mL/min and 10 µL as the injection volume. The autosampler temperature was maintained at 8°. The PDA detector was set at 190-400 nm wavelength, and the detection wavelength was at 238 nm for all impurities and at 200 nm for RC A with run time of 60 minutes. The separation was achieved by a gradient program as shown below. The results were processed using Empower (Waters software).

Gradient program

6.2 Solutions

Solution A (0.1% v/v of ortho-phosphoric acid in water): Transferred 5.0 mL of ortho-phosphoric acid in 5000 mL of water and mixed well.

Solution B (0.1% v/v of ortho-phosphoric acid in acetonitrile): Transferred 3.0 mL of ortho-phosphoric acid in 3000 mL of acetonitrile and mixed well.

Solution C (1 N sodium hydroxide solution): Weighed and transferred about 4.0 g of sodium hydroxide pellets into a 100 mL volumetric flask, to this added 50% of the flask volume of water, sonicated to dissolve, diluted with water to volume, and mixed well.

Solution D: Mixed 3.0 mL of glacial acetic acid in 1000 mL of water and adjusted with Solution C to a pH of 4.0

Diluent (acetonitrile: Solution D; 80:20 v/v): Mixed 1600 mL of acetonitrile and 400 mL of Solution D. (Diluent was used as blank).

System suitability solution (0.4 mg/mL of lovastatin epimer mixture): Weighed and transferred about 2.0 mg of USP Lovastatin Epimer Mixture RS into a 5-mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Standard stock solution (1.0 mg/mL of lovastatin): Weighed and transferred about 50.0 mg of USP Lovastatin RS into a 50-mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Standard solution (0.2 mg/mL of lovastatin): Transferred accurately 4.0 mL of Standard stock solution into a 20-mL volumetric flask and diluted with Diluent to volume.

Sample stock solution (1.0 mg/mL of lovastatin): Transfer 10 lovastatin ER tablets (equivalent to 200 mg of lovastatin) into a 200 mL volumetric flask, add about 140 mL of Diluent, sonicate for 10 min, and shake intermittently. The solution should then be shaken on a mechanical shaker at 400 rpm for 75 min and diluted with Diluent to volume. Transfer the solution into a centrifuge tube and centrifuge at 4000 rpm for 5 min. Pass the supernatant solution through nylon syringe filter of 0.45 µm pore size. Collect the solution by discarding the initial 2 mL of the filtrate.

Note-1: PVDF, nylon & PTFE syringe filters were evaluated. Nylon filter was found to be suitable for the assay and OI tests.

Note-2: Initially one tablet was taken for the sample preparation due to a lack of sufficient samples, one tablet was taken, and the volume was accordingly adjusted to achieve the 1.0 mg/mL sample concentration. The results obtained using 1 tablet for sample preparation were compared with the results obtained using 10 tablets to prove the equivalency of the data. The findings of the equivalency study suggested no impact on the assay and OI results. Accordingly, the assay and OI validations were performed by preparing the samples using 1 tablet.

Sample solution (0.2 mg/mL of lovastatin): Transferred accurately 2.0 mL of Sample stock solution into 10-mL volumetric flask, diluted with Diluent to volume and mixed well.

6.3 Validation Parameters and Results

The system suitability requirements and results are summarized in Table 5. The validation parameters, solutions, and results for lovastatin ER tablets are summarized in Table 6. Representative chromatograms of the System suitability solution, Standard solution and Sample solutions are shown in Figures 5, 6 and 7, respectively.

Table 5. Summary of system suitability parameters, solutions, and results for the Assay

Table 6. Summary of validation parameters, solutions, and results for the Assay

The developed HPLC method was validated for the OI procedure, using the criteria described in USP General Chapter 〈1225〉, Validation of Compendial Procedures², and found to be specific, accurate, precise, robust, linear, and free from interference for the samples evaluated.

7.1 Instruments and Method

The analysis for OI procedure of lovastatin ER tablets was performed using the same instruments and method as described in the Assay procedure section 6.

7.2 Solutions

Prepared Solution A, Solution B, Solution C, Solution D and Diluent as per the Assay procedure.

Resolution solution (0.5 mg/mL of lovastatin epimer mixture): Weighed and transferred about 2.5 mg of USP Lovastatin Epimer Mixture RS into a 5-mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Lovastatin stock solution (0.1 mg/mL): Weighed and transferred about 2.5 mg of USP Lovastatin RS into a 25 mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Impurity mixture stock solution-1 (0.1 mg/mL of each mevastatin, lovastatin angelate, lovastatin acid and dehydrolovastatin): Weighed and transferred about 2.5 mg of each USP Mevastatin RS, lovastatin angelate, lovastatin acid and dehydrolovastatin into a 25 mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Lovastatin RC-A stock solution (0.1 mg/mL): Weighed and transferred about 2.5 mg of USP Lovastatin RC-A RS into a 25 mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Lovastatin angelate stock solution (0.1 mg/mL): Weighed and transferred about 2.5 mg of lovastatin angelate into a 25 mL volumetric flask, to this added 50% of the flask volume of Diluent, sonicated to dissolve and diluted with Diluent to volume.

System suitability solution (0.002 mg/mL of lovastatin angelate (0.2%), 0.004 mg/mL of lovastatin RC-A (0.4%), and 0.002 mg/mL of Epilovastatin (0.2%) (from Resolution solution) in 1.0 mg/mL of lovastatin impurity identification mixture)): Weighed and transferred about 5.0 mg of USP Lovastatin Impurity Identification Mixture RS into a 5 mL volumetric flask, added accurately 0.1 mL of Lovastatin angelate stock solution, 0.2 mL of Lovastatin RC-A stock solution and 40 μL of Resolution solution and added 2.5 mL of Diluent, sonicated to dissolve and diluted with Diluent to volume.

Sensitivity solution (0.001 mg/mL (0.1%) of lovastatin and all specified impurities): Transferred accurately 0.1 mL of each Impurity mixture stock solution, Lovastatin RC-A stock solution and Lovastatin stock solution into a 10 mL volumetric flask and diluted with Diluent to volume.

Standard solution (0.002 mg/mL (0.2%) of lovastatin and all specified impurities): Transferred accurately 0.2 mL of each Impurity mixture stock solution, Lovastatin RC-A stock solution and Lovastatin stock solution into a 10 mL volumetric flask and diluted with Diluent to volume.

Sample solution (1.0 mg/mL): Transfer 10 lovastatin ER tablets (equivalent to 200 mg of lovastatin) into a 200 mL volumetric flask, add about 140 mL of Diluent, sonicate for 10 min, and shake intermittently. The solution should then be shaken on a mechanical shaker at 400 rpm for 75 min and diluted with Diluent to volume. Transfer the solution into a centrifuge tube and centrifuge at 4000 rpm for 5 min. Pass the supernatant solution through nylon syringe filter of 0.45-µm pore size. Collect the Sample solution by discarding the initial 2 mL of the filtrate.

Spiked sample solution: (0.1% (or) 0.001 mg/mL of specified impurities and 1.0 mg/mL of lovastatin): Transfer 10 lovastatin ER tablets (equivalent to 200 mg of lovastatin) into a 200 mL volumetric flask, add 2 mL of each Impurity mixture stock solution, Lovastatin RC A stock solution and about 140 mL of Diluent and sonicate for 10 min while shaking intermittently. The solution should then be shaken on a mechanical shaker at 400 rpm for 75 min and diluted with Diluent to volume. Transfer the solution into a centrifuge tube and centrifuge at 4000 rpm for 5 min. Pass the supernatant solution through nylon syringe filter of 0.45-µm pore size. Collect the Spiked sample solution by discarding the initial 2 mL of filtrate.

7.3 Validation Parameters and Results

The system suitability parameters and results are summarized in Table 7. The validation parameters and results are summarized in Table 8. Representative chromatograms of Diluent (Blank), Resolution solution, System suitability solution, Sensitivity solution, Sample solution and Spiked sample solutions are presented in Figures 8-19. Linearity was established for lovastatin and related impurities, whereas accuracy and repeatability were established for lovastatin related impurities. The relative response factors (RRF) are presented in Table 9.

The Sensitivity solution concentration was established for each analyte at 0.001 mg/mL (0.1%) with respect to the sample concentration of 1.0 mg/mL.

Table 7. Summary of system suitability parameters, solutions, and results for Organic Impurities

Table 8. Summary of validation parameters, solutions, and results for Organic Impurities

Table 9. Relative response factors (RRF)