A new, simple, rapid, and stability-indicating reversed phase liquid chromatographic (RP-LC) method for the determination of both assay and related substances in paliperidone has been developed and validated. During the forced degradation at hydrolysis, oxidative, photolytic, and thermal stressed conditions, the degradation was observed in the oxidative and acid stress conditions. Five process-related impurities (Imp-A to Imp-E) in test sample of paliperidone have been detected using newly developed RP-LC method. Among the five, Imp-C and Imp-D were found to be degrdants. Good resolution between the peaks corresponding to degradation and process-related impurities from the analyte was achieved on a Hypersil BDS C18 (
Paliperidone, a major metabolite of risperidone (9-hydroxy-risperidone), has been approved by the FDA for the treatment of schizophrenia since 2006. Paliperidone is a centrally active dopamine D2 and serotinergic 5-HT
Synthetic scheme of paliperidone: generation of process-related impurities and degradation-related impurities.
To the best of our knowledge no method has been reported for the determination of paliperidone and its potential process related impurities in the bulk drug using HPLC for the regular analysis and stability studies in quality control laboratory. The core-objective of this research work is to develop a simple and rapid stability-indicating RP-LC method for the determination of process and degradation related substances in paliperidone bulk drugs and validation as per USP and ICH guidance documents [
HPLC-grade acetonitrile and methanol procured from Merck (India) were used. Ammonium dihydrogen orthophosphate, hydrochloric acid, sodium hydroxide and hydrogen peroxide were all of AR grade, procured from Merck (India). HPLC-grade water obtained from Millipore system (Millipore Inc., USA) was used throughout the analysis. The investigated sample of paliperidone and its potential process and degradation-related impurities (Figure
A Waters HPLC (Milford, MA, USA) equipped with Alliance 2695 separations module and 2996 photodiode array detector was used for method development, forced degradation studies, and method validation. The column Hypersil BDS C18 (
A mixture of water and acetonitrile in the ratio of 50 : 50 (v/v) was used as diluent in the preparation of analytical solutions. A solution of paliperidone was prepared at a concentration of 1000
Formulation drug sample prepared as; ten weighed tablets of paliperidone (equivalent to 5 mg each of paliperidone) were ground to powder and an equivalent of 25 mg of active ingredient dissolved in diluent in a 25 mL volumetric flask, ultra sonicated for about 10 min. and its volume made up to the mark with the diluent, filtered through Merck Nylon syringe filter having pore size 0.45
Paliperidone working reference standard solution (1000
The forced degradation studies were conducted on bulk drug substance in order to prove the stability-indicating property and selectivity of the established method [
LC
The 1H NMR spectra were recorded on Bruker 300 MHz spectrometer using deuterated chloroform as solvent and tetramethylsilane (TMS) as internal standard.
Mass spectra were recorded on Waters Micro mass-Quattro micro API mass spectrometer equipped with a quadrupole mass analyzer. Detection of the ions was performed in electron spray ionization with positive ion mode. Spectra were acquired from m/z 60 to 800 in 0.1 amu steps with 10 numbers of scans.
FT-IR spectra were recorded for all the five degradation and process-related impurities (Table
Mass, FTIR spectral data, and 1H NMR chemical shift values.
Name of impurity | Mass value (m/z) (MH)+ | FT-IR (KBr) absorption bands (cm−1) | 1H NMR chemical shift values, |
---|---|---|---|
(1) Imp-A | 210.6 | 3145.80, 2965.68, 1664.21, 1531.82, 1486.08, 1326.72, 1183.89 | 3.64–3.72 (t, 4H), 1.792–2.07 (m, 4H) |
(2) Imp-B | 221.5 | 3057.86, 2934.75, 2500.67, 1612.45, 1417.35, 822.24 | 7.69–7.73 (dd, 1H), 8.12–8.17 (dd, 1H), |
(3) Imp-C | 425.2 | 1721.52, 1655.23, 1608.81, 1519.39, 1276.03, 959.64 | 2.20–2.27 (m, 2H), 2.32–2.38 (m, 4H), |
(4) Imp-D | 443.00 | 3399.9, 3054.70, 1638.38, 1528.97, 1474.56, 1268.70, 1117.65, 959.45 | 7.30–7.36 (td, 1H), 8.01–8.04 (dd, 1H), |
(5) Imp-E | 411.00 | 3059.07, 2942.22, 2757.28, 1651.47, 1610.70, 1591.93, 1536.55, 1270.52, 958.74 | 7.00–7.05 (td, 1H), 7.67–7.70 (dd, 1H), |
s, singlet; m, multiplet; t, triplet; d, doublet; td, triplet of doublet; q, quartet; bs, broad singlet.
The reaction scheme for the synthesis of paliperidone is shown in Figure
Laboratory batches of crude paliperidone were analyzed for their related substances identification using the developed RP-HPLC method. These samples were subjected to LC-MS analysis. Based on the observed mass, starting material and reactants used in the synthetic scheme (Figure
Typical RP-HPLC chromatograms of (a) paliperidone unspiked test preparation and (b) paliperidone spiked test preparation with known impurities (Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E).
Unspiked test sample
Spiked test sample
All impurities (Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E) were characterized with the help of MS, FT-IR, and NMR spectroscopic techniques. The mass, FT-IR spectral data, and 1H NMR chemical shift values of these impurities are presented in Table
The present study aims to develop the chromatographic system capable of eluting and resolving paliperidone, its process related and degradation related impurities (Imp-A, Imp-B, Imp-C, Imp-D, and Imp-E) within the short run time and that complies with the general requirement of system suitability. The Imp-C and Imp-D were the degradation impurities as well as process related impurities present in bulk samples produced in the process of paliperidone. The core objective of the chromatographic method is to get the better peak shape of the paliperidone and its impurities along with the separation of closely eluting peak pairs of paliperidone and Imp-C.
The preliminary investigations were conducted for the effect of various parameters on system suitability of the method. The parameters assessed include the detection wavelength, the type and quantity of organic modifier, the column, the salt concentration, the pH of mobile phase and column temperature. Paliperidone has UV-absorption maxima at about 237 nm wavelengths. Hence detection at 237 nm was selected for method development purpose. Considering the fact that the dissociation constants of paliperidone are pKa1 = 8.2 (piperidine moiety) and pKa2 = 2.6 (pyrimidine moiety) [
System suitability test results.
Compound | Resolution ( | USP tailing factor ( | RRT | RF |
---|---|---|---|---|
Imp-B | — | 1.13 | 0.63 | 0.96 |
Imp-A | 4.04 | 1.06 | 0.76 | 1.50 |
Imp-C | 2.39 | 1.11 | 0.87 | 1.76 |
Paliperidone | 2.06 | 1.17 | 1.00 | 1.00 |
Imp-D | 7.19 | 1.06 | 1.37 | 1.12 |
Imp-E | 2.15 | 1.17 | 1.43 | 0.96 |
RRT, relative retention time; RF, response factor.
Typical RP-HPLC chromatograms of forced degradation; (a) peroxide treated test sample and (b) acid treated test sample, (c) base treated test sample.
Peroxide-treated test sample
Base-treated test sample
Acid-treated test sample
The data on degradation studies revealed that the degradation products were well separated from the paliperidone and the peak purity data (purity angle is less than purity threshold) of paliperidone indicated that itwas spectrally pure. The mass balance is a process of adding together the assay value and the levels of degradation products to see how closely these add up to 100% of initial value with due consideration of the margin of analytical error [
Forced degradation results.
Stress condition | % assay of paliperidone | % of degradant | Observation and Mass balance | Peak purity | |
PA | PT | ||||
Undegraded | 99.70 | — | 0.080 | 0.258 | |
Acid hydrolysis (conc. HCl, 4 h refluxed) | 74.83 | 25.13 | Increase in levels of Imp-A (1.25%) and Imp-C (13.01%) and other 10.88 % of unknown degradation product formed (mass balance: 99.96%) | 0.226 | 0.251 |
Base hydrolysis (5 M NaOH, 48 h refluxed) | 88.59 | 11.05 | Increase in levels of Imp-A (0.31%), Imp-B (1.69%) and other 7.82 % of unknown degradation product formed (mass balance: 99.00%) | 0.195 | 0.248 |
Oxidation (6% H2O2 at RT) | 72.11 | 27.77 | Increase in levels of Imp-A (0.14%) and major degradation product, that is, Imp-D (24.51%) and other 3.12 % of unknown degradation product formed (mass balance: 99.64%) | 0.079 | 0.250 |
Thermal (60°C, 8-days) | 99.85 | Nil | No any known and unknown degradation product formed (mass balance: 100.3%) | 0.078 | 0.258 |
Photolytic as per ICH | 99.63 | Nil | No any known and unknown degradation product formed (mass balance: 99.2%) | 0.080 | 0.258 |
Mass balance = % assay + % sum of all impurities + % sum of all degradants. PA, purity angle; PT, purity threshold.
The linearity of method was established at two different levels. The assay linearity was studied by preparing five different solid weightings of paliperidone from 50 to 150% w/w (50, 75, 100, 125, and 150% w/w) with respect to target analytical concentration (100
Validation data results of related substances and assay of paliperidone.
Compound | Related substances results | Assay results | ||||
IMP-A | IMP-B | IMP-C | IMP-D | IMP-E | paliperidone | |
Precision (mean results ± %RSD) | ||||||
Method precision ( | ||||||
Intermediate Precision ( | ||||||
Overall results ( | ||||||
Limit of detection (LOD) | ||||||
LOD ( | 0.030 | 0.039 | 0.155 | 0.071 | 0.046 | 0.065 |
LOD (% w.r.t. test) | 0.003 | 0.004 | 0.016 | 0.007 | 0.005 | 0.007 |
Limit of quntitations (LOQ) | ||||||
LOQ ( | 0.090 | 0.119 | 0.470 | 0.214 | 0.141 | 0.197 |
LOQ (% w.r.t. test) | 0.009 | 0.012 | 0.047 | 0.021 | 0.014 | 0.020 |
Linearity: for related substances LOQ to 250% of specification level and for assay 50 to 150% of TAC | ||||||
Correlation coefficient (r) | 0.99966 | 0.99978 | 0.99973 | 0.99936 | 0.99925 | 0.99996 |
Slope | 22910.49 | 35819.23 | 19598.94 | 30803.29 | 36086.78 | 31580.49 |
Intercept | −100.38 | −653.37 | 1214.32 | −172.37 | −631.62 | −1131.83 |
Accuracy (mean recovery ± %RSD): LOQ to 150 % of specification level | ||||||
LOQ | — | |||||
50% | — | |||||
100% | — | |||||
150% | — |
% w.r.t. test LOD LOQ values are in % with respect to test concentration of 1000
TAC, Target analytical concentration that is, 100
The RF of each impurity was determined using the slope of the paliperidone plot against each impurity plot. The Y-intercept of each plot was within the 2.5% of the response at 0.15% w/w level of each impurity, describing that the plot is going almost through the origin.
The limit of detection and limit of quantitation were determined for paliperidone and for each of the related substances as per ICH Q2R1 guideline from the standard deviation of the peak areas and slope of linearity data. The values of LOD and LOQ for paliperidone were 0.065
System precision for assay method was verified by injecting the six replicate injections of standard concentration (50
Precision of the method was studied for method precision and intermediate precision. The assay method precision was examined by analysing six determinations of the sample solution at working concentration versus a standard concentration, and RSD of obtained results was evaluated and found to be 0.21%. Related substances method precision was demonstrated by analyzing six separate paliperidone sample solutions that were prepared by spiking the related substances namely, Imp-A, Imp-B, Imp-C, and Imp-D at specification level. The RSD (0.61–3.85%,
Accuracy of the method for all the related substances was determined by analyzing paliperidone sample solutions spiked with all the related substances at four different concentration levels of LOQ, 50, 100, and 150% of each in triplicate at the specified limit. The recovery of all these related substances were found to be in between the predefined acceptance criterion of 80.0–120.0%, and the data is given in Table
To determine the stability of sample solution, the sample solutions of paliperidone spiked with related substances at specified level were prepared and analyzed immediately after preparation and after different time intervals up to 8 days, while maintaining the sample cooler temperature at about 25°C and at about refrigerator temperature (5–8°C). The results from these studies indicated the sample solution was unstable at room temperature and stable for 8 days at 8°C temperature.
To evaluate the mobile phase stability of the method, the paliperidone test sample spiked with related substances at specification level was used. The paliperidone test sample was analyzed after 24 hrs and after 48 hrs by using same mobile phase. The content of each impurity was evaluated and compared to the mean results of method precision. The difference between the mean values (after 48 hrs.) from the method precision mean results is found to be below 10.0%. The studies indicated no effect on the determination of related substances and the selectivity after 48 hrs. Therefore the mobile phase is stable for 48 hrs.
To evaluate the robustness of the developed method, the chromatographic conditions were deliberately altered and the resolution between closely eluting peak pair that is, Imp-C and paliperidone was evaluated. The flow rate of the mobile phase was 1.0 mL min−1. To study the effect of flow rate on the resolution, the same was altered by 0.1 units that is, from 0.9 to 1.1 mL min−1. The effect of column temperature on resolution was studied at 42 and 48°C instead of 45°. All the other mobile phase components were held constant as described above. In all the deliberate varied chromatographic conditions (flow rate and column temperature), the tailing factor of paliperidone was less than 1.20 and the resolution between any two peaks was greater than 2.0. There was a very minor variation in the resolution and tailing factor results observed in all the robustness conditions, illustrating the robustness of the method.
The analysis of commercial formulation sample and bulk drug sample indicates that the method is specific and selective for determination of related substances in the formulation and bulk drug samples (Table
Results (in %) of formulation tablet and bulk drug batches sample analysis.
Sample source | Imp-A | Imp-B | Imp-C | Imp-D | Imp-E | SMUI |
---|---|---|---|---|---|---|
Formulation product analysis results | ||||||
Formulation-1 | ND | ND | 0.19 | 0.01 | ND | 0.02 |
Formulation-2 | ND | ND | 0.19 | 0.01 | ND | 0.02 |
Batch analysis results (%) | ||||||
B.NO.1 | 0.02 | ND | 0.06 | ND | 0.05 | ND |
B.NO.2 | 0.02 | 0.01 | 0.06 | ND | 0.06 | ND |
B.NO.3 | 0.02 | ND | 0.06 | ND | 0.06 | ND |
ND, not detected; SMUI, single maximum unknown impurity.
The method presented in this communication describes the development of a rapid, simple, accurate, and selective gradient RP-HPLC method that separates the all related substances with good resolution. The process, and degradation related impurities, which were present in paliperidone sample were identified by LC-MS and characterized using MS, FT-IR, and 1HNMR spectral data. The developed method was validated to ensure the compliance in accordance with ICH guidelines. The method was found to be simple, selective, precise, accurate, and robust. Therefore, this method can be used for routine testing as well as stability analysis of paliperidone drug substance. All statistical results (percentage, mean, R.S.D., percentage difference, and recovery %) were within the acceptance criteria. The method could be of use not only for routine evaluation of the quality of paliperidone in bulk drug manufacturing unit but also for detection of impurities in pharmaceutical formulations.
The authors wish to thank the management of Megafine group for supporting this paper. The authors would also like to thank colleagues in the division of Research and Development of Megafine Pharma (P) Ltd. for their cooperation in carrying out this paper.