New validated HPLC methodology for the determination of (−)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase

A new chromatographic method for the enantioseparation and the determination of (−)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157–164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (−)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets. Figure ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7565-y) contains supplementary material, which is available to authorized users.


Introduction
In case of chiral drugs, substantial differences in transport, distribution, metabolism, and excretion of particular enantiomers may be observed. Hence, for medicinal products marketed as formulations containing only one enantiomer, a very important issue is the content of the second enantiomer, which in the best case may appear as an inactive impurity but may also be responsible for undesired effects. The most popular techniques used for drugs enantiomeric purity estimation are high-performance liquid chromatography (HPLC) [1][2][3] and capillary electrophoresis [4,5], enabling both enantiodifferentiation and enantiomers quantification.
For the determination of paroxetine enantiomers, few HPLC stationary phases (including optically active metal complexes, cyclodextrin, penicillamine, and Pirkle type comp l e x e s ) [ 3 , 8 ] w e r e e x a m i n e d , b u t r e a s o n a b l e enantioseparation was obtained only with the aid of amylose Published in the topical collection Euroanalysis XVII (The European Conference on Analytical Chemistry) with guest editor Ewa Bulska.
In the present paper, we describe new high-performance liquid chromatographic method for paroxetine enantiomers separation and determination with use of silica-based chiral stationary phase containing glycoprotein (ovomucoid [12,13]) as chiral selector, more sensitive and offering better resolution than those recommended before. The method was validated and applied to paroxetine enantiomers quantification in two pharmaceutical formulations: ParoGen (Mc Dermott Laboratories Ltd.)-and Xetanor (Actavis)-coated tablets containing 20 mg paroxetine (as paroxetine hydrochloride, 22.22 mg). Validated HPLC method with amylose tris(3,5dimethylphenylcarbamate) chiral stationary phase was used a reference procedure. It should be noted that the present method was developed as a result of extensive studies with such chiral selectors as hydrocarbons (cellulose and amylose), proteins, and cyklodextins. More than 20 aqueous and non-aqueous mobile phases were examined.

Analysis of medicinal preparations ParoGen 20 mg-coated tablets and Xetanor 20 mg-coated tablets
Paroxetine standard solution preparation: 2 mg of (−)-transparoxetine standard was weighed and transferred into 100 mL volumetric flask. Then, 10 mL of methanol was added and the flask was filled with: ethanol for the analysis with use Chiralpak AD-H column, 10 mM pH 3.5 phosphate buffer for the analysis with use of Ultron ES-OVM column. Placebo extract preparation: accurate weight amount of placebo corresponding to excipients content in a tablet (280 mg) was placed into 100 mL volumetric flask and 10 mL methanol was added. The sample was sonicated for ∼10 min. The volumetric flask was filled with methanol and the resulted suspension was filtered. 5.0 mL of the filtrate was placed into 50 mL volumetric flask and filled with appropriate solvent or solution (depending on the column used, see above).
Extraction of paroxetine from ParGen and Xetanor pharmaceutical formulations: tablets of the formulations were carefully ground. Accurate weight amount of each preparation corresponding to one dose (300 mg) was placed into 100 mL volumetric flask and extracts were prepared as described in above para.
Determination of analyte recovery within the content range of 80-120 % (according to ICH Validation of Analytical Procedures [14]): accurate weight amounts of placebo corresponding to excipients contained in one tablet (280 mg) were transferred into nine volumetric flasks (100 mL). Accurately weighed amounts of (−)-trans-paroxetine hydrochloride were poured into each flask: ca. 16 mg (80 % dose) to first three flasks, ca. 20 mg (100 % dose) to subsequent three flasks and ca. 24 mg (120 % dose) into the last three flasks. Ten milliliter methanol was added to each flask. The flasks were sonicated for ∼10 min, filled with methanol and the resulted suspensions were filtered. Of each filtrate, 5.0 mL was placed into 50 mL volumetric flask and filled with ethanol (Chiralpak) or phosphate buffer (Ultron).

Robustness of the method
Influence of mobile phase composition and flow rate as well as column temperature on the precision of retention times and peak areas measurements and selectivity was examined. Twenty-microliter portions of (−)-trans-paroxetine hydrochloride standard solution (in phosphate buffer) were injected onto a column and chromatograms were registered (n=6 for each measurement). Precision of retention times (t R ), peak areas measurements and resolution (R s ) were calculated. The obtained results prove good repeatability of the developed method with relative standard deviation (RSD) for retention times and peak areas between 0.11 and 3.09 %. It was also found, that changing acetonitrile content (±0.2 %), column temperature (±3°C) and flow rate (±0.5 mL/min) did not much influence resolution factor (|ΔR s |<0.3) although with the flow rate elevation decrease in the resolution factor could be observed (Fig. 1).
Determination of (−)-trans-paroxetine in medicinal products (ParoGen-and Xetanor-coated tablets) (−)-trans-Paroxetine contents were determined in ParoGenand Xetanor-coated tablets with the aid of ovomucoid as well as amylose carbamate stationary phases (Fig. 3, Table 1). They were found to be between 21.13 and 22.27 mg, with RSD between 0.67 and 1.14 %. Recovery of the analyte was in the range of 98.35-101.87 %. Thanks to suitably low LOQ (16 ng/ml), the method possibly could be recommended for the determination of paroxetine enantiomers in body fluids.
Purity of the examined formulations was also checked. According to European Pharmacopeia 7.7 monograph for active substance "Paroxetine hydrochloride anhydrous", an amount of D contamination, (+)-trans-paroxetine hydrochloride, should not exceed 0.2 % with respect to (−)-trans-paroxetine hydrochloride. In the examined tablets, no (+)-transparoxetine enantiomer in the amount ≥0.01 % was found under available analysis conditions.

Conclusion
New chromatographic method for the enantiodifferentiation of (±)-trans-paroxetine and the determination of (−)-and (+)-trans-paroxetine contents was developed with the aid of ovomucoid stationary phase. The new method is selective, precise, accurate, and offers good recovery (>98 %). Resolution, R s =2.8 is similar to the previously reported [10], 2.78. However, it is faster and few times more sensitive (LOD and LOQ, 5 and 16 ng/mL, respectively, comparing to 20 and 60 ng/mL, obtained for modified procedure [9]). The obtained results (elution order) shows that (+)-trans-paroxetine (as compared to (−)-enantiomer) forms more stable solutestationary phase complex on ovomucoid than on amylose tris(3,5-dimethylphenyl)carbamate stationary phase. The developed method allows for the determination of active substance paroxetine hydrochloride in bulk and in pharmaceutical formulations according to European Pharmacopeia 7.7 requirements [14]. In the examined pharmaceutical formulations, ParoGen-and Xetanor-coated tablets, no (+)-trans-paroxetine enantiomer in the amount ≥0.01 % was found under analysis conditions.
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