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Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876
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INDO AMERICAN
JOURNAL OF
PHARMACEUTICAL
RESEARCH
IDENTIFICATION AND DETERMINATION OF RELATED SUBSTANCES OF
PALIPERIDONE IN BULK DRUG AND PHARMACEUTICAL FORMULATIONS BY HPLC
AND HPLC-MS-MS
Mazahar Farooqui
1*, Rana Z. Ahmed
2, Jaiprakash N.Sangshetti
2, Zahid Zaheer
2, Abdul Wahab
2
Mrinmayee Deshpande2, Sachin Bhojane
2, Salim Rashid Baig
2
1 Dr. Rafiq Zakaria College for Women, Navkhanda, Aurangabad-431001, Maharashtra, India. 2 Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Baugh, Aurangabad-431001, Maharashtra, India.
Corresponding author
Mazahar Farooqui
Dr. Rafiq Zakaria College for Women,
Navkhanda, Aurangabad-431001,
Maharashtra, India.
Affiliated to Dr. Babasaheb Ambedkar Marathwada University.
Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ARTICLE INFO ABSTRACT
Article history
Received 02/12/2013
Available online
30/12/2013
Keywords HPLC,
LC-MS-MS,
Paliperidone,
Related substance,
Impurity.
A novel LC-MS-MS method was developed for identification and determination of related
substances in paliperidone bulk drug and dosage form. Six related substances of paliperidone
API and tablets were identified by the proposed method. The chromatographic system
consisted of Zorbax SBCN column (150 × 4.6mm; 5µ).The mobile phase consisted of 0.077
%w/v ammonium acetate buffer and acetonitrile with a nonlinear gradient program at a flow
rate of 1.3 ml/min, injection volume of 20 µL and UV detection was carried out at 277 nm.
Validation of the developed method was performed. Major impurities have been detected,
identified and quantified using two analytical systems HPLC and LC-MS-MS. The newly
developed method for quantitative determination of paliperidone and its related substances
was found to be specific, accurate, precise and robust. It can therefore be successfully employed for the quality evaluation of impurities in raw material and formulations of
paliperidone.This is the first report where one of the impurities marked as keto impurity was
identified.
Please cite this article in press as Mazahar Farooqui,et al. Identification and determination of related substances of paliperidone in
bulk drug and pharmaceutical formulations by hplc and HPLC-MS-MS .Indo American Journal Of Pharm Research.2013:3(12).
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INTRODUCTION
Paliperidone, is a primary active metabolite of former antipsychotic risperidone. Chemically it is 9-hydroxy risperidone, (9
RS)-3-[2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl) piperidin-1-yl] ethyl]-9-hydroxy-2-methyl-6,7,8,9- tetrahydro- 4H- pyrido[1,2-
α]pyrimidin-4-one. It is a second generation antipsychotic agent. It acts by blocking dopamine (D2) and serotonin (5-HT2A)
receptors. It is used for maintenance and treatment of acute schizophrenia. The drug is available in the form of conventional release
and extended release oral tablets. Clinical studies have discovered its efficacy in the treatment of mania, dementia and bipolar
disorders [1-5].
Raw materials, manufactured by-products and degraded products are a source for impending impurities; therefore,
manufacturing of drug substance and drug products requires stringent control of impurities [6]. In the United States Pharmacopoeia,
five impurities of paliperidone have been reported using HPLC. The chemical structure of paliperidone and its known related substances are shown in the Figure 1.
Paliperidone
(±)-3-[2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido [1,2-α]pyrimidin-
4-one
Related substances
A. 3-(2-chloroethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro pyrido[1,2-α]pyrimidin-4-one (CMHTP)
B. Fluoro-3-(4-piperidinyl)1,2-benzisoxazole (FPBI)
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C.3-[2-[4-[6-fluoro-1,2-benzisoxazole-3-yl)piperidin-1-yl]-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-α]pyrimidin-4-one
(risperidone)
D.3-[2-[4-(6-fluoro-1,2-benzisoxazole-3-yl)-4-oxy-piperidin-1-yl]ethyl]-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-
α]pyrimidin-4-one (N-oxide)
E.3-(2-(4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidinyl)ethyl)-2-methyl-7,8-dihydro-6H-pyrido[1,2-a]pyrimidine-4,9-
dione(Ketoimpurity)
Figure 1: Chemical structures of Paliperidone and its related substances
Literature survey revealed a method for quantitative determination of risperidone and 9- hydroxyrisperidone in biological
samples [7, 8]. HPTLC method for the determination of paliperidone in formulations and for in vitro release study has been reported
[9]. A stability-indicating liquid chromatographic method has also been reported for the determination of paliperidone in osmotic-
controlled release tablets [10]. Other methods which also have been reported include force degradation study and identification of
impurities [11, 12, and 13]. Though, a number of methods are available for identification and a number of impurities of paliperidone have been identified, no method has reported the keto impurity using LC-MS analysis, which our study has detected in
concentrations of 0.0870%.
The present study relates to the development and validation of a simple and precise HPLC method for quality control of
paliperidone bulk drug and in products. The objectives of our work are determination of related impurities in bulk drug and formulated
products and identification and quantitation of impurities by LC-MS. Along with known impurities, keto impurity has been identified
and detected which is major outcome of the present work.
MATERIALS AND METHODS
Chemicals
Paliperidone drug substance (API), reference substance and standard keto impurity was obtained from Wockhardt Ltd.,
Aurangabad, Maharashtra, India. Paliperidone tablets RS were obtained from a local supplier in Aurangabad, Maharashtra, India.
HPLC grade isopropyl alcohol, acetonitrile (Merck), ammonium acetate were of analytical grade. Milli Q/HPLC grade water was obtained from our in-house water purification system.
Instrument and chromatographic conditions
Agilent HPLC system equipped with quaternary solvent delivery pump, degasser, auto sampler and
column thermostat was used for the chromatographic separation using a Zorbax column (150 × 4.6 mm ; 5 µm particle size) A
nonlinear gradient program at a flow rate of 1.3 mL min-1
and injection volume of 20 µL was used. The mobile phase consisted of
0.077% w/v ammonium acetate (solvent A) and acetonitrile (solvent B), which were degassed and filtered through 0.45 µm porosity
membrane before use. The mobile phase programming was done in a non-linear gradient mode, at 0, 20, 30, 70 and 80 min. The
percentage of solvent A was decreased up to 30 min and then kept constant until 70 min as 85 and 70 % and that of solvent B was
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increased linearly with a difference of 15% up to 30 min and then kept constant until 70 min as 15, 30 and 60 % respectively. Further,
percentage of solvent A was increased and stabilized to 85% and that of solvent B was decreased and stabilized to 15% at 77 and 80
min. The column thermostat was set at 30 °C. The related substances were detected at 277 nm using a UV detector, as it gave best
intensity peaks with low noise. The chromatograms of blank, placebo, paliperidone and its related substances are shown in Figure 2.
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Figure 2a: Chromatogram of Blank
Figure 2b: Chromatogram of Paliperidone
Figure 2c: Chromatogram of Placebo
Figure 2d: Chromatogram of Paliperidone and its related impurities. FPBI (4.305), CMHTP (10.044), Risperidone (17),
Paliperidone (23), Keto impurity (24.2), N-Oxide (28.756)
Figure 2: The chromatograms of Blank, Paliperidone, Placebo, and its related impurities
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Sample preparations of paliperidone
The standard stock solutions of reference sample and tablets were prepared by sonicating and dissolving 50 mg of
paliperidone in a 30 ml mixture of isopropyl alcohol, ammonium acetate buffer and acetonitrile (Diluent) (50: 10: 40 v/v/v), then the
volume was made up to 50 mL. All the solutions were filtered through 0.45µm membrane filter before use.
Preparation of resolution stock solution
An accurately weighed 3 mg of standard paliperidone keto impurity was transferred to a 100 ml volumetric flask containing
about 40 ml of diluents and the volume was made up to the mark with diluents.
Preparation of resolution solution Accurately weighed 3 mg of paliperidone was transferred to a 100 ml volumetric flask with about 50 ml of diluents. The
contents of flask were sonicated and 5 ml of resolution stock solution was added, mixed and the volume was made up to the mark with
diluents and mixed. Resulting solution was filtered through 0.45 µm membrane filter.
LC- MS (interface) analysis
The accurate molecular weight of the related substances present in paliperidone bulk drug and formulation was determined by
using mass spectrophotometer (Waters). The chromatographic conditions were carried out as mentioned above. The outlet of the
HPLC apparatus was attached to the inlet of the TOF mass spectrometer operated with electrospray ionization (ESI) dual ESI source
in positive and negative mode. Data was collected in full scan MS mode over the m/z range from 100 to 1,500 Da.
LC-MS/MS analysis
Analysis was performed using mass spectrophotometer (Waters) equipped with ESI source in dual ionization mode. The MS spectra of each related substance was collected from 100 to 1,500 Da m/z range, which provided the structural fragmentation
pattern.The ESI conditions were set as follows: capillary voltage (Vcap) 4 kV, nebulizer pressure 40 psi, drying gas (nitrogen)
temperature 350 o C, skimmer voltage 65 V, fragmenter voltage 200 V and drying gas flow 10 L min-1.
Method Validation
A validation study was performed using parameters such as selectivity, accuracy, limit of detection, limit of quantitation,
precision, linearity and robustness. [14, 15].
Accuracy
Accuracy expresses the closeness of agreement between the individual results and accepted true value. The accuracy of the
assay method was determined in replicate at three concentrations. The percentage recoveries were calculated from the slope and y-
intercept of the calibration curve.
Selectivity The selectivity was proposed from the resolution of principle peak from the nearest peak and also among all the other peaks.
All the related substance peaks were well resolved with each other as well as from the main peak. A resolution of greater than two
establishes the selectivity of the method.
Precision
Precision of an analytical method was checked by injecting six individual sample solutions. Relative standard deviations of
peak areas were determined for each impurity.
Limit of detection and limit of quantitation
The LOD and LOQ were determined using S/N ratio method. They were estimated as the minimum concentration of analyte
showing S/N of 10:1 and 3:1, respectively; by injection of serially diluted solutions of known concentrations. Precision study was also
carried out at the quantitation limit by injection of six individual preparations and calculating the percentage relative standard
deviation (RSD) of the areas.
Linearity
Linearity of the analytical procedure is its response, which is directly proportional to the concentration of the analyte in the
sample, within a given range. The test solution for related substance was prepared by diluting the stock solution to the required
concentrations, a total of 6 concentration solutions were prepared. The calibration curve was established by plotting the peak areas
versus corresponding concentrations. The linearity of response for paliperidone was determined by method of least squares and
regression statistics.
Robustness
Robustness of the method was demonstrated by small deviations in chromatographic conditions such as change in flow rates
(± 0.2 min) and change in column temperature (±3oC).
RESULT AND DISCUSSION
Selection of HPLC conditions Out of some commercially available HPLC columns, selection of appropriate column is an important factor to effectively
implement the HPLC method. Several parameters that include are column dimensions, type of stationary phase, bonding
characteristics and particle size. These parameters are to be carefully studied in terms of peak shape, resolution and analysis time. A
column having small particle size, internal diameter and length indicated shorter run time and high separation efficiency but have
limited sample loading capacity. In order to achieve separation a Zorbax SBCN column (150 × 4.6 mm; 5 µm particle size) was used,
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with mobile phase of ammonium acetate and acetonitrile. The signal intensity was improved with the reduction of solvent peaks at the
wavelength of 277 nm. Flow rate of 1.3 mL min-1 showed improved resolution between peaks by altering the retention time. A
gradient elution method i.e. a non-linear gradient mode, at 0, 20, 30, 70 and 80 min was used. The percentage of solvent A was
decreased up to 30 min and then kept constant until 70 min as 85 and 70 % and that of solvent B was increased linearly with a
difference of 15% up to 30 min and then kept constant until 70 min as 15, 30 and 60 % respectively. Further, percentage of solvent A
was increased and stabilized to 85% and that of solvent B was decreased and stabilized to 15% at 77 and 80 min. This program reduce
the run time and give good resolution. The selected conditions resulted in a chromatogram with resolution >2 for all analytes of
interest and gave a total run time of 80 min.
Detection and identification of related substances in paliperidone bulk drug and tablet
HPLC analysis
The UV detector allowed detection of several related substances in paliperidone along with some very small peaks. The
official method reported in USP 37[16] mentions paliperidone related substances. The HPLC procedure we developed allows
detection and quantitation of six related substances. The new impurity named keto impurity was separated and appeared just after the
main peak. This newly detected impurity has not been reported previously.
LC-MS-MS analysis
To further confirm the identities of related substance LC-MS method was developed. The same column, as for HPLC
analysis was employed to get comparable elution profile. All related substances detected by the HPLC method were confirmed by LC-
MS analysis. The identified peaks confirmed the exact mass. MS-MS scan showed the fragmentation pattern of the related substances.
The mass spectrum obtained for paliperidone indicated a molecular ion [M-H] - at m/z 427.The m/z values and molecular formulae of the related compounds are summarized in Table 1 and the MS data of paliperidone and its related substances is shown in Figure 3.
Table 1: Accurate mass analysis of related substances in Paliperidone
Name Theoretical mass Experimental
mass[M+H]-
Proposed Molecular Formula
FPBI 220.10 221.20 C12H13FNO2
CMHTP 242.08
243.2 C11H15ClN2O2
Keto impurity 424.09 425.3 C23H25FN4O3
Risperidone 410.21 411.3 C23H27FN4O2
N-oxide 442.21 443.3 C23H27FN4O4
Figure 3a: MS data for CMHTP
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Figure 3b: MS Data for FPBI
Figure 3c: MS data for Risperidone
Figure 3d: MS data for N-Oxide
Figure 3e: MS data for Paliperidone
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Figure 3f: MS data for Keto-Impurity
Figure 3: MS data of paliperidone and its related substances
Identification of keto impurity
The elemental calculator revealed the molecular formula as- C23H25FN4O3 . MS/MS showed the daughter ion peaks at 325.3 m/z. Considering the above information, the structure of the keto impurity can be proposed as 3-(2-(4-(6-fluro-1,2-benzisoxazol-3-
yl)piperidinyl)ethyl)-2-methyl-7,8-dihydro-6H-pyrido[1,2α]pyrimidine-4,9-dione.
CONCLUSION
This is the first report where one of the impurities marked as keto impurity was identified a new, accurate, precise, robust and
specific LC-MS method. It has been successfully employed for different sources of raw material and formulated product. A total of six
related substances were identified and quantified in paliperidone tablets by HPLC method. The structure of new impurity was
proposed on the basis of MS-MS data. The identification of keto impurity of paliperidone may open a new window to quality
evaluation of paliperidone.
Authors’ Statements
Competing Interests The authors declare no conflict of interest.
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