Validated HPLC Method for the Analysis of Clozapine in Rat Plasma and its Application to Pharmacokinetics

 

Aiyalu Rajasekaran*, Tamilarasan Arulsolomon, Joghee Dharuman

Department of Pharmaceutical Analysis, KMCH College of Pharmacy, Tamilnadu, India

*Corresponding Author E-mail: rsekaran2001in@yahoo.co.in

 

 

ABSTRACT:

As a prerequisite to the determination of pharmacokinetic parameters of clozapine in rats, an HPLC method using UV detection was developed and validated. Clozapine and the internal standard, ambroxol were extracted from plasma samples using hexane and isoamyl alcohol (98.5:1.5 v/v) solutions. Chromatographic separation was achieved on a phenomenex C18 column (5 µ; 25 cm x 4.6 mm id) using a mobile phase of ACN/0.5% triethylamine solution in Ammonium acetate buffer of pH 5 in the ratio of 40:60 at a flow rate of 1.5 ml/min. Detection was at 263 nm. The calibration curve was linear from 50-500 ng/ml. The Intra-day accuracy ranged between 99.99% and 100.05% with precision of 0.006% and 0.03%. The Inter-day accuracy ranged between 99.98% and 100.01% with precision of 0.010% and 0.058%. The main pharmacokinetic parameters for rats were determined after a single intravenous administration of 100 mg/kg clozapine are t1/2, 0.0614 h; AUC0-∞, 83.368 ng.hr/ml; Cmax, 36.16 ng/ml; Tmax, 0.5 hrs; KEli, 11.2847 hrs-1.

 

KEYWORDS: Clozapine; Plasma; Bioavailability; HPLC.


 

INTRODUCTION:

Clozapine, a class of dibenzodiazepine is an atypical antipsychotic drug and drug of choice for patients who are at risk of potential extra pyramidal effect1,2 and for patients who are poor responders to traditional neuroleptic drugs such as chlorpromazine or haloperidol3,4. Clozapine acts as an antagonist of dopamine receptors in the mesolimbic system5. The binding ratio of clozapine to serotonine (5-HT2A) receptor and dopamine (D2) receptor is higher than other conventional antipsychotic drugs6,7. This drug also shows higher affinity to dopamine D1 and D4 receptors as well as acts an antagonist at adrenergic, cholinergic, histaminergic and seronergic receptors8,9. Clozapine is rapidly and almost completely absorbed following oral administration with Tmax of 1.5-2 h and the maximum effect of the drug appears approximately 4 h after administration10.  Approximately 95% of the drug binds to plasma proteins and about 50% undergoes first-pass metabolism before being distributed to the systemic circulation11.

 

This drug is extensively metabolized by cytochromes P450 and the major metabolites detected in urine are N-oxide, N-desmethyl and hydroxylated derivatives12. The desmethyl metabolite has only limited pharmacological activity while the hydroxylated and N-oxide derivatives are inactive. An HPLC method is reported for the determination of clozapine and its metabolites, N-desmethylclozapine and clozapine N-Oxide in dog plasma13 and two HPLC methods was reported for determination of clozapine in rat plasm14-15.  Since clozapine is commonly used for treatment of schizophrenia, several generic products are now available. In order to assure the therapeutic equivalence of these generic products, the bioequivalence study needs to be investigated and hence the presence study was undertaken to compare the pharmacokinetic profiles and to evaluate the bioequivalence of the generic and branded clozapine formulations in albino rats.

 

MATERIALS AND METHODS:

Laboratory animals:

Experimental pharmacokinetic determinations were accomplished using Albino white rats, both males and females, weighing 200 ± 20 g. The animals were kept under standard conditions (24 ± 2°C, 60% air humidity) and had free access to water. No food was supplied in the last 18 hours before the experiment. The study adheres to “Principles of Laboratory Animal Care” and is approved by the animal care committee IAEC/CPSEA- Institutional animal ethics committee/ Committee for the purpose of control and supervision of experiments on animals (KMCRET/M.Pharm/14/09).

 

Blood samples:

The experimental animals were divided into two groups of 12 individuals: a control group, treated with clozapine 100 mg/kg of branded formulation, and an experimental group, treated with an equivalent amount of clozapine generic formulation.

 

Doses were established depending on the linearity of pharmacokinetic parameters in rat and the necessary conditions requested by the analytical method (100 mg/kg, oral administration, corresponding to a dose of 20 mg/ animal weighing about 200 g).

 

An additional animal group was used for blood prelevation in order to obtain rat blank plasma, necessary for developing the analytical method and rat plasma standards preparation.

 

After administration, before each blood prelevation, animal anesthesia was induced with ethylic ether, which is not metabolized in the rat organism; it is not soluble in plasma or bound by plasmatic proteins and also does not cause interferences with other metabolic processes.

 

Blood samples were taken from the all groups by retro orbital nerve puncture, each time being taken 1.5-2.0 mL in tubes using EDTA as anticoagulant, at different time periods, as follows: 0;  0.166; 0.33; 0.5; 1; 2; 3; 4; 5 hours. Right after prelevation, blood samples were transferred in anticoagulant pre-treated tubes and centrifuged at 5000 rotations/minute for 10 minutes in a cooling centrifuge at a constant temperature of 4°C. The separated plasma was transferred into micro tubes and kept at -20°C until being analyzed.

 

HPLC clozapine analysis in rat plasma:

Apparatus:

A HPLC system 1100 of Agilent Technologies with quaternary pump, degasser, automatic injector, Phenomenex column C18, 5 µ x 25 cm x 4.6 mm, protected by a pre-column RP18 (Merck)and UV detector was used for the study. HPLC grade water prepared from water purifying device Direct Q5 (Millipore, France) was used for the study. An ultrasonic bath Transsonic T700/H, Elma (Germany), rotational vacuum concentrator RVC2-25 (Martin Christ, Germany), vortex Mix 20 (Falc Instruments, Italy), sample shaker S20 (CAT, Germany), centrifuge 2-15 (Sigma, Germany) were used for preparing the sample.

 

Reagents and reference substances:

Clozapine and Internal standard, Ambroxol were obtained from M/s. Burgeon pharmaceuticals, Chennai. HPLC grade methanol, acetonitrile, hexane, is amyl alcohol, triethylamine, HCl purchased from Merck was used for the study.

 

Mobile phase:

Triethylamine solution (0.5% v/v) in ammonium acetate buffer 20 mM, pH 5 adjusted with glacial acetic acid (A) and acetonitrile (B) in the ratio of 60(A): 40 (B) was used as mobile phase. Flow rate of mobile phase was maintained at 1.5 mL/min. Column temperature was maintained at 45 °C and the sample was detected at 253 nm.

 

Stock and working solutions:

Stock solution of clozapine (1000 mg/mL) and internal standard ambroxol (100 mg/mL) was prepared in methanol. For plasma spiking, 6 working standard solutions of clozapine in the concentrations of 50, 100, 200, 300, 400, 500 ng/mL methanol-water 50:50 (v/v) were prepared.

 

Plasma calibration standard preparation:

Blank plasma (0.5 mL) was treated with 50 ml W solution and 50 ml Internal STD and stirred 10 seconds on the vortex. Plasma standard solution concentrations for the calibration curve were: 50; 100; 200; 300; 400; 500 ng/mL clozapine, internal standard concentration being 1 mg/mL.

 

Plasma samples preparation for HPLC analysis:

Plasma concentrations of clozapine were assayed by high performance liquid chromatography using the modified method as follows: 0.5 ml of plasma sample was transferred in to the screwed capped tube. 40 ml of an internal standard (ambroxol 31.2 mg/ml) and 200 ml of 2 M NaOH were added into the plasma tube. The sample was then thoroughly mixed and left at room temperature for 5 min. After that, 5 ml of hexane: isoamyl alcohol (98.5:1.5 v/v) was added and vortex mixed at full speed for 2 min. The organic and aqueous phases were separated by centrifugation at 3,000 rpm for 5 min. Only the organic phase (approximately 4 ml) was then transferred to a clean tube. The organic phase was then dried under the stream of nitrogen gas at 40°C. The residue was reconstituted by adding 200 ml of 0.1 M HCl and mixed by vortexing at full speed for 1 min. 80 ml of this acid solution is injected for future analysis.

 

Data analysis:

Maximum observed plasma concentration (Cmax), Minimum observed plasma concentration (Cmin) and time of maximum plasma concentration (Tmax) were obtained from drug concentration-time curves. The area under the clozapine concentration-time curves from 0-5 h (AUC0-5h) were calculated using the trapezoidal method and the first order elimination rate constant (KEli) was estimated. The average concentration of drug in plasma during 5h was calculated. The differences in AUC0-5h, Cmax and Tmax between the branded and generic drugs were determined using student T test.

 

RESULTS:

Method specificity:

Under the mentioned HPLC parameters, the separation of clozapine and internal standard is specific as referred to endogenous compounds, without significant interferences in analytes retention times from endogenous compounds. (Figure1). The method implies washing of the column between 18.01 and 25.00 minutes with acetonitrile for eluting retarded endogenous compounds.

 

Figure 1 Typical chromatogram of blank plasma spiked with clozapine and ambroxol

 

Method’s linearity

The method proved to be linear between 50-500 ng/mL clozapine, with a typical calibration curve of: correlation equation y=376x +546.1, correlation coefficient > 0.998 (Figure 2).

 

Figure 2 Clozapine - calibration curve

Limit of detection and Lower limit of Quantification:

The LOD was 12 ng/mL. The LLOQ was 50 ng/mL with coefficient of variation of 0.03% and accuracy of 100%. The ULOQ was 500 ng/mL with coefficient of variation of 0.006 % and accuracy of 99.99 %.

 

Precision and Accuracy:

The Intra-day accuracy ranged between 99.99% and 100.05% with precision of 0.006% and 0.03%. The Inter-day accuracy ranged between 99.98% and 100.01% with precision of 0.010% and 0.058%.

 

Recovery (extraction efficiency) from plasma:

The extraction efficiency of clozapine from rat plasma at the concentrations of 50, 200 and 500 ng/mL was found to be 100.06%, 99.96% and 99.81% with precision of 0.046%, 0.023% and 0.014% respectively. The mean recovery for internal standard was 98.97%.

 

Stability:

The stability of drug spiked rat plasma samples at three levels were studied for three freeze thaw cycles. The mean concentrations of the stability samples were compared to the theoretical considerations. Similarly, short term, long term and standard solution stability were evaluated. The stability of the internal standard was also performed. The results showed that the selected drugs were stable in plasma for about one month when stored at frozen state.

 

Ruggedness and robustness:

The ruggedness and robustness of the methods were studied by changing the experimental conditions. No significant changes in the chromatographic parameters were observed when changing the experimental conditions (operators, instruments, source of reagents and column of similar type) and optimized conditions (pH, mobile phase ratio and flow rate).

 

System suitability:

System suitability parameters such as column efficiency (theoretical plates), resolution factor and peak asymmetry factor of the optimized methods were found satisfactory.

 

Rat plasma samples analysis:

Pharmacokinetic study

The mean clozapine concentration-time profiles after administration of the branded and generic drugs in subjects are depicted in Figure 3.

 

The typical chromatogram of rat plasma obtained after 30 minutes of oral administration of either generic or branded formulation are depicted in the Figure 4 and Figure 5.

 

Figure 3 Mean plasma concentration-time profiles of clozapine in subjects after oral administration of either a 100 mg branded drug or generic drug

 

Figure 4 Typical chromatogram of rat plasma obtained after 30 min administration of 100 mg of branded formulation

 

The pharmacokinetic parameters for rats after a single intravenous administration of 100 mg/kg of branded drug were t1/2, 0.0614 h; AUC 0-∞, 83.368 ng.hr/ml; Cmax, 36.16 ng/ml; Tmax, 0.5 hrs and KEli,11.2847 hrs-1 and that of generic drug were found to be: t1/2, 0.056 h; AUC 0-∞, 84.363 ng.hr/ml; Cmax, 35.62 ng/ml; Tmax, 0.5 hrs and KEli, 12.1598 hrs-1. All of the pharmacokinetic parameters calculated for the branded drug were almost the same to those of the generic drug and there were no statistically significant differences between the two products (Table 1).

 

Figure 5 Typical chromatogram of rat plasma obtained after 30 min administration of 100 mg of generic formulation

 

Table 1 Mean pharmacokinetic parameters of clozapine obtained from rats after administration of either branded or generic formulation.

S.

No

Para

meters

Branded formulation

Generic formulation

1

AUC 0-t (ng.hr/ml)

82.5959

83.6163

2

AUC 0-∞ (ng.hr/ml)

83.3668

84.363

3

Cmax (ng/ml)

36.16

35.62

4

Tmax (hrs)

0.5

0.5

5

KEli (hrs-1)

11.2847

12.1598

6

T1/2 (hrs)

0.0614

0.056

 

DISCUSSION:

Two drugs are considered to be bioequivalent if they are pharmaceutically equivalent and their bioavailabilities are so similar that they are unlikely to produce clinically relevant difference in regard to safety and efficacy.The bioanalytical method was developed and validated and the pharmacokinetic parameters were studied. From the acquired data the branded and generic formulations of clozapine for bioequivalence study were compared. The pharmacokinetic parameters such as Cmax, tmax, AUC (0-t), AUC (0-∞), t1/2, KEli of branded formulation were found to be closer to those of generic formulation. The results indicated that the analytical method is suitable to measure plasma concentrations of the compound in pre-clinical studies.

 

CONCLUSION:

The method designed was found to be rapid, simple, specific, easy to perform and inexpensive. Baseline HPLC separation was obtained for all assayed compounds within 5.5 min. The procedure enabled the determination of both branded and generic drugs using a liquid-liquid extraction method and HPLC with PDA detection.

 

The pharmacokinetic parameters such as Cmax, Tmax, AUC (0-t), AUC (o-∞), t1/2, KEli are com pared for the bioequivalence study by statistical analysis, the t value obtained from student ‘T’ test was found to be 0.0813 which is considered as nonsignificant. Hence the present study shows that generic drug was bioequivalent to branded drug.

 

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Received on 03.06.2013       Modified on 20.06.2013

Accepted on 24.06.2013      © AJRC All right reserved

Asian J. Research Chem. 6(7): July 2013; Page  654-658