Development of reverse phase chiral liquid chromatographic method for determination of sertraline 1R4R cis enantiomer from sertraline hydrochloride in bulk drug and tablet

 

V. Dighe*, P. Pawaskar, S. Adhyapak, N. Shambhu, D. Mestry

Department of Chemistry, Ramnarain Ruia College, Matunga (East), Mumbai-400019,India.

*Corresponding Author E-mail: dighevidya@yahoo.in

 

Validated for sertraline 1R4R cis enantiomer of sertraline hydrochloride in bulk drug and tablet using UV detector at 215 nm. The developed method was able to separate sertraline 1R4R cis enantiomer of sertraline hydrochloride from its bulk drug and tablet within 25 min. The chromatographic separation was carried out by reverse phase chromatography using column of protein based chiral stationary phase (Chiral AGP 100 mm x 4.0 mm x 5 µm) with mobile phase comprising of ammonium phosphate buffer and isopropanol (80:20, v/v) at a flow rate of 1.0 mL/min at 25°C temperature. The limit of detection (LOD) and limit of quantitation (LOQ) of sertraline 1R4R cis enantiomer were found to be 0.016 µg/mL and 0.05 µg/mL respectively. The linearity of response of sertraline 1R4R cis enantiomer was in the range of 0.05 µg/mL to 0.30 µg/mL with r > 0.9995. The percentage recovery of the sertraline 1R4R cis enantiomer from sertraline hydrochloride bulk drug sample ranged between 97.85 to 100.29 and for sertraline hydrochloride tablet ranged between 96.15 to 100.15. The method was validated and found to suitable for determination of sertraline 1R4R cis enantiomer of sertraline hydrochloride in bulk drug and tablet.

 

KEYWORDS: Sertraline Hydrochloride, Sertraline 1R4R cis enantiomer ,High performance liquid chromatography,Method validation and quantitation.

 

INTRODUCTION:

Sertraline or cis-(1S, 4S)-4-(3, 4-dichlorophenyl)-1, 2, 3, 4-tetrahydro-N-methyl-1-naphthalenamine is administered as its hydrochloride. It is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class ^[1]. The molecule of sertraline contains two stereogenic centers and it is quite likely that its stereoisomers like cis-(1R,4R)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthalenamine hydrochloride, trans-(1S,4R)-4-(3,4-dichloro- phenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthalenamine hydrochloride, trans-(1R,4S)-4-(3,4-dichlorophenyl) -1,2,3,4-tetrahydro-N-methyl-1-naphthalenamine hydrochloride are introduced as impurities during its synthesis.

  Its stereoisomer cis-(1R, 4R)-4-(3, 4-dichlorophenyl)-1, 2, 3, 4-tetrahydro-N-methyl-1-naphthalenamine hydrochloride [Sertraline 1R4R cis enantiomer] which is also present as an impurity in sertraline hydrochloride is quantitated in the present research work. 

 

Sertraline hydrochloride is primarily used to treat major depression in adult outpatients as well as obsessive–compulsive, panic and social anxiety disorders in both adults and children ^[2]. The efficacy of sertraline for depression is similar to that of older tricyclic antidepressants, but its side effects are much less pronounced. Differences with newer antidepressants are subtler and also mostly confined to side effects. Evidence suggests that sertraline may work better than fluoxetine for some subtypes of depression ^[3]. Sertraline is highly effective for the treatment of panic disorders, but cognitive behavioral therapy in combination with sertraline is a better treatment for obsessive-compulsive disorder than sertraline alone. Although approved for social phobia and posttraumatic stress disorder, sertraline leads to only modest improvement in these conditions ^[4]. Sertraline also alleviates the symptoms of premenstrual dysphoric disorder and can be used in sub-therapeutic doses or intermittently for its treatment. Sertraline shares the common side effects and contraindications of other SSRIs, with high rates of nausea, diarrhea, insomnia, and sexual side effects; however, it has relatively mild effects on both cognition and vigilance. The unique effect of sertraline on dopaminergic neurotransmission may be related to these milder effects on cognition and vigilance ^{[5, 6]}. In pregnant women taking sertraline, the drug was found to be present in significant concentrations in fetal blood, and was also associated with a higher rate of various birth defects ^[7].

 

The most potent and selective (+)-isomer was taken into further development and eventually named sertraline. During the synthesis of sertraline, there is a chance of formation of potential sertraline 1R4R cis enantiomer. Chromatographic separation of sertraline 1R4R cis enantiomer from sertraline and followed by its quantitative determination is one of the key quality parameter before sertraline is released to market. Owing to the pharmacological and toxicological differences between these isomers, it is quite important to develop stereo specific assay for separation these of drugs.

 

Literature survey revealed that various methods have been reported for the determination of sertraline hydrochloride in human plasma ^[8], These methods include Analysis of cis-trans isomers and enantiomers of sertraline by cyclodextrin-modified micellar electrokinetic chromatography ^[9], quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients^[10], analytical method for the quantitation of sertraline hydrochloride stereoisomers by electrokinetic chromatography ^[11], chiral HPLC method for separation of enantiomers ^[12-17], However reverse phase LC method for quantitation of sertraline 1R4R cis enantiomer from sertraline hydrochloride using chiral AGP column within 25 min. has not been reported in the literature.

 

In the present research work, a simple, sensitive and accurate reverse phase HPLC method to separate sertraline 1R4R cis enantiomer from sertraline hydrochloride in bulk drug and tablet using chiral AGP column has been reported for first time. The method was also validated to ensure the compliance in accordance with the ICH guidelines ^[18].

 

MATERIAL AND METHOD:

Materials:

Isopropanol (purity 99.0%), methanol (purity 99.0%) used in the present research work were of HPLC Grade and were purchased from Merck (Mumbai, India). Ammonium dihydrogen orthophosphate and sodium hydroxide AR grade were purchased from Merck (Mumbai, India). Sertraline hydrochloride reference standard (no.: LOT F0I030 Purity: 99.7%) was obtained from LGC Promochem India Private Limited. (1R4R)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-tetrahydronaphthalen-1-amine hydrochloride [Sertraline 1R4R cis enantiomer] ( B.no.: RD/00/2755 Purity: 99.8%)  was obtained from RPG Life Sciences Ltd, Navi Mumbai, India. Sample of sertraline hydrochloride (sertraline hydrochloride API B. No. : 33) was obtained from the Process Development Laboratory of active pharmaceutical ingredient plant unit of RPG Life Sciences Ltd, Navi Mumbai, India. Sertra-100 tablet commercial formulations were purchased from the local market. Each tablet contains sertraline hydrochloride equivalent to 100.0 mg of sertraline. Water was obtained from Milli-Q Gradient water purification system. All solutions were filtered through 0.45µm membrane filters.

 

Equipment:

The HPLC system composed of shimadzu 10A VP series with variable wavelength UV-Visible detector, auto injector and Class VP, version 5.03 data processor. Chiral column chiral AGP (100 mm x 4.0 mm) 5 µm, (Chrom Tech Ltd, Japan) were used for separation. The chromatographic and integrated data were recorded using HP computer system.

 

Chromatographic conditions:

Chromatographic separation was achieved on chiral AGP (100 mm x 4.0 mm) 5µm   column with mobile phase consisting of ammonium dihydrogen orthophosphate buffer (pH 5.5) and isopropanol in the volume ratio (80:20) at 25°C. The flow rate was 1.0 mL/min and detector wavelength was kept at 215 nm for monitoring separation. 20 µL volume was injected into the system with total run time of 25 min.

 

Preparation of standard stock solutions of sertraline 1R4R cis enantiomer  (100 µg/mL):

 10.0 mg of sertraline 1R4R cis enantiomer was accurately weighed and dissolved in 5.0 mL methanol first and diluted to the mark in 100 mL standard volumetric flask with mobile phase to get the concentration of 100 µg/mL.  This stock solution was stored in a refrigerator at 5°C. This stock solution was further diluted to get the required concentrations for the method validation.

 

Preparation of working standard solution of sertraline 1R4R cis enantiomer   (0.2 µg/mL)

Working standard solution of Sertraline 1R4R cis enantiomer was prepared by diluting aliquot of 0.2 mL of stock solution of sertraline 1R4R cis enantiomer in 100 mL standard volumetric flask with mobile phase to get the concentration of 0.2 µg/mL.

 

Preparation of sample solution of sertraline hydrochloride (100 µg/mL):

10 mg of sertraline hydrochloride was accurately weighed and dissolved in 5.0 mL methanol first and sonicated for 10 minutes. To this solution, add 30 mL mobile phase sonicate for 5 minutes and  diluted to the mark in 100 mL standard volumetric flask with mobile phase to get the concentration of 100 µg/mL.

 

 Preparation of sample solution from tablet (100 µg/mL):

Twenty tablets of sertraline hydrochloride were weighed accurately and finely powdered. The powder equivalent to 10 mg of sertraline hydrochloride was accurately weighed and dissolved in 5.0 mL methanol first, sonicated for 10 minutes and diluted to the mark in 100 mL standard volumetric flask with mobile phase to get the concentration of 100 µg/mL and filtered through 0.45 µ membrane.

 

 

METHOD VALIDATION:

Validation parameters:

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of its impurity. Enantiomer resolution of sertraline 1R4R cis enantiomer and baseline separation was achieved using chiral AGP column. There were no interfering peaks co-eluted with the compound of interest. This has indicated appropriate specificity of elaborated procedure. The order of elution was determined using UV detector, the retention times of sertraline hydrochloride and sertraline 1R4R cis enantiomer were approximately 12.7 min and 16.8 min, respectively. A typical chromatogram of sertraline 1R4R cis enantiomer, sertraline hydrochloride and sertraline 1R4R cis enantiomer spiked with sertraline hydrochloride bulk drug has been represented in Fig. 1 to Fig 3.

 

Fig.1. A typical chromatogram of  sertraline 1R4R cis enantiomer

 

 

A – Sertraline hydrochloride ,B – Sertraline 1R4R cis enantiomer impurity

Fig.2. A typical chromatogram of  sertraline hydrochloride bulk drug

 

Precision:

The method was validated in terms of system precision and intermediate precision. The system precision was studied by separate, repetitive injections (n = 6) of standard solution of sertraline 1R4R cis enantiomer (0.2 µg/mL), in the chromatographic system under the specified conditions. The percent relative standard was found to be 0.76. The method precision was evaluated by carrying out six replicates of test sample (100 µg/mL). The values of percent relative standard deviation of retention times and peak areas for sertraline 1R4R cis enantiomer was found to be less than 2. The intermediate precision of the method was evaluated using on different days in the same laboratory. The values of percent relative standard deviation of peak area of sertraline 1R4R cis enantiomer for intermediate precision was found to be 1.98. The results indicate that method is precise and reproducible.

 

A – Sertraline hydrochloride ,B – Sertraline 1R4R cis enantiomer impurity

Fig.3. A typical chromatogram of sertraline 1R4R cis enantiomer spiked with sertraline hydrochloride bulk drug

 

Limit of Detection (LOD) And Limit of Quantification (LOQ) of the sertraline 1R4R cis enantiomer:

The limit of detection and limit of Quantitation of the sertraline 1R4R cis enantiomer was estimated at a signal to noise ratio of 3:1and 10:1 respectively, by injecting a series of diluted solution of sertraline 1R4R cis enantiomer with known concentration. The value of LOD and LOQ were found to be 0.016 µg/mL and 0.05 µg/mL respectively.

 

 

Table 1. Recovery results of studies of sertraline 1R4R cis enantiomer from sertraline hydrochloride bulk drug using the proposed HPLC method

*n=3

Table 2. Recovery results of studies of sertraline 1R4R cis enantiomer from sertraline hydrochloride tablet using the proposed HPLC method

*n=3

 

Linearity of the sertraline 1R4R cis enantiomer:

Linearity was evaluated by analyzing working standard solutions of sertraline 1R4R cis enantiomer in the concentration range 0.05 µg/mL to 0.30 µg/mL. The solutions were injected in duplicate in the chromatographic system under optimized conditions described earlier. The calibration plot for sertraline 1R4R cis enantiomer was found to be linear in the concentration range 0.05 µg/mL to 0.30 µg/mL with correlation coefficient r as 0.9995

 

Accuracy:

The accuracy of the method was established by performing recovery experiment using standard addition method. For zero level, only sample solution was analyzed by HPLC in triplicates. To 10 mg of sample, pure standard of sertraline 1R4R cis enantiomer with varying concentrations (0.05 µg/mL, 0.20 µg/mL, 0.30 µg/mL) respectively were added to sample. The solutions were prepared and analyzed by HPLC for each level and mean amounts of sertraline 1R4R cis enantiomer present in each level of sample solution were determined. The average value of percent recovery for sertraline 1R4R cis enantiomer in bulk drug was found to be 99.19 and for percent recovery for sertraline 1R4R cis enantiomer in sertraline hydrochloride tablet was found to be 98.43. As the values are close to 100 %, it indicates a good accuracy of the method.  The results are given in Table 1 and Table 2.

 

Robustness:

Robustness of the method was determined by making small deliberate changes in the chromatographic conditions utilized in present method. The robustness of the method is evaluated by replicate analysis of standard solutions of sertraline 1R4R cis enantiomer (0.2 µg/mL).The chromatographic conditions changed were; mobile phase composition (+/- 10 % of lowest component), flow rate (+/- 10.0 %) and wavelength (+/- 2 nm).

 

The amount of sertraline 1R4R cis enantiomer from sertraline hydrochloride bulk drug obtained by normal method did not affect the system suitability criteria. The resolution between sertraline 1R4R cis enantiomer and sertraline hydrochloride was greater than 3.5; under all separation conditions tested, demonstrating sufficient robustness. As deliberate changes made to the chromatographic method did not affect the results, it can be concluded that method was found to be robust.

 

RESULT AND DISCUSSION:

In the present research work, HPLC method has been developed for the quantitation of sertraline 1R4R cis enantiomer present as impurity from sertraline hydrochloride in bulk drug and tablet. The chromatographic

analysis was carried out on chiral AGP column (100 mm x 4.0 mm, 5 µm), using a mobile phase comprising of 20 mM

ammonium dihydrogen orthophosphate buffer (pH 5.5): isopropanol in volume ratio of 80:20 (v/v). The detection was carried out λ = 215 nm.

 

The limit of detection (LOD) and limit of quantitation (LOQ) concentration were found to 0.016 µg/mL and 0.050 µg/mL for sertraline 1R4R cis enantiomer.  Good linearity was observed for sertraline 1R4R cis enantiomer over the concentration range of 0.05 µg/mL - 0.30 µg/mL, with linear regression equation  y = 86545 x + 164 and correlation coefficient with r > 0.9995. The accuracy of method was evaluated by using freshly prepared solution at four concentration levels of 0.05 µg/mL, 0.2 µg/mL and 0.3 µg/mL of analyte concentration. The percentage recovery values were in the range of 97.85 to 100.29 in bulk drug. The percentage recovery values were between 96.15 to 100.15 in tablet. The resolution between sertraline 1R4R cis enantiomer and sertraline hydrochloride was greater than 3.5; under all separation conditions tested, demonstrating sufficient robustness. As deliberate changes made to the chromatographic methods did not affect the results, it can be concluded that the method is robust.

 

Several chromatographic methods have been reported in the literature for determination of enantiomer from sertaline hydrochloride bulk drug and its formulation likewise ^[12-17].

HPLC method has been reported in the literature for development and validation of a liquid chromatographic method for the determination of sertraline and three of its more closely related synthetical and non-chiral impurities, without using ion-pair reagents [12]. The HPLC analysis was carried out using Zorbax Bonus-RP column (150 mm x 4.6 mm, 5 µm), under isocratic conditions with phosphate buffer (pH 2.8; 10 mM)-methanol (63:37 v/v) at 50°C, at the flow-rate of 1.0 mL/min. UV detection was set at 220 nm. The reported method is used for only separation of non-chiral impurities from sertaline hydrochloride and detection wavelength is different.

 

HPLC method has been reported for separation of stereoisomers of sertraline and its related enantiomeric impurities on a dimethylated β-cyclodextrin stationary phase by HPLC ^[13]. But the selectivity of unmodified Cyclodextrins for stereoisomeric separation of analytes is limited.

A sensitive and stereospecific reverse phase high-performance liquid chromatography method for determination of sertraline hydrochloride in bulk drug, tablets and capsules was developed [14]. The column used was Alltima C18 (250 mm × 4.6 mm, 5 µm) column with hydroxypropyl-β-cyclodextrin (HP-β-CD) as mobile phase additive. The composition of the mobile phase was 68:32 (v/v) aqueous 170 mM phosphate buffer, pH 3.0 (adjusted with 85% phosphoric acid) containing 18 mM HP-β-CD/acetonitrile at a flow rate of 1.0 mL/ min. The UV detector was set at 225 nm.

 

Cyclodextrins are generally added to the above mobile phase but the disadvantages include (i) the cost of chiral additives, (ii) the complex mode of operation and (iii) inconvenience for preparative applications since the chiral additive must be removed from the enantiomeric solutes.

A normal phase HPLC method has been reported in the literature for development and validation of a liquid chromatographic method for the enantiomeric separation of sertraline and its undesired isomer [15]. The column used was chiralpak ADH (250 mm x 4.6 mm) 5µm, using mobile phase containing n-hexane, isopropanol, ethanol, diethylmine (850:100:50:0.1 v/v/v), at the flow-rate of 1.0 mL/min. UV detection was set at 220 nm. In the present research work, the mobile phase is 20 mM ammonium dihydrogen orthophosphate buffer (pH 5.5 with diluted sodium hydroxide solution) and isopropanol in the volume ratio (80:20) which is much simpler as it comprises of only two solvents and shows a better resolution of sertraline 1R4R cis enantiomer present as impurity from sertraline hydrochloride bulk drug and tablet. Isopropanol used in the mobile phase is water miscibile, has low visocity, gives sharp peak shape and is readily available in pure form than the reported methods which has combination of four solvents.

 

A normal phase HPLC method has been reported in the literature for enantiomeric purity test of sertraline and its related enantiomeric impurities [16]. The column used was chiralpak AD  (250 mm x 4.6 mm) 5 µm , using mobile phase containing mixture of 30 volumes of n-hexane and 70 volumes of a mixture of 1 volume of diethylamine, 25 volume of isopropanol and 975 volumes of n-hexane , at the flow-rate of 0.4 mL/min. UV detection was set at 275 nm.

In the present research work, reverse phase HPLC method for quantitation of sertraline 1R4R cis enantiomer from sertraline hydrochloride. Two well separated sharp peaks were detected at wavelength 215 nm by using chiral AGP column (100 mm x 4.0 mm, 5 µm) which is a short.

 

However reverse phase HPLC method for quantitation of sertraline 1R4R cis enantiomer from sertraline hydrochloride using chiral AGP column within 25 min. has not been reported in the literature.

 

In the present research work, a simple, sensitive and accurate reverse phase HPLC method to separate sertraline 1R4R cis enantiomer from sertraline hydrochloride in bulk drug and tablet using chiral AGP column has been reported for first time. The method was also validated to ensure the compliance in accordance with the ICH guidelines ^[18]. 

 

CONCLUSION:

An isocratic, stereo selective and rapid chiral liquid chromatographic method was developed for enantiomeric separation and quantitative determination of sertraline hydrochloride from its bulk drug and tablet. The method was found to be precise, sensitive, accurate and specific in bulk active substances and formulation. The method was completely validated showing satisfactory data for all validation parameters tested. The developed method could be used for quantitation of sertraline 1R4R cis enantiomer in bulk samples and tablets of sertraline hydrochloride. 

 

ACKNOWLEDGEMENTS:

The authors wish to thank the management of RPG Life Sciences Group, Navi Mumbai for all kind of supports for this research work. Authors wish to acknowledge the API group for providing the samples for our research.

 

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Received on 18.12.2012       Modified on 02.01.2013

Accepted on 13.01.2013      © RJPT All right reserved