High Performance Thin Layer Chromatographic Analysis of Paracetamol and Etoricoxib in Spiked Human Plasma

 

Poonam H. Chaube, Santosh V. Gandhi*, Padmanabh B. Deshpande and Veena G. Kulkarni

Dept. of Pharmaceutical Analysis, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Pune - 411 001, MH, India

*Corresponding Author E-mail: santoshvgandhi@rediffmail.com

 

ABSTRACT:

A new simple, accurate, and precise densitometric method for determination of Paracetamol and Etoricoxib in spiked human plasma has been developed and validated. A Simple precipitation method was carried out by using methanol and a known amount of supernatant solution was spotted on precoated silica gel 60 F254 plates using a Camag Linomat V autosampler. The mobile phase selected was Toluene: Dichloromethane: Methanol (6:2.5:1.5 v/v/v) with UV detection at 240 nm. The retention factors for Paracetamol and Etoricoxib were found to be 0.30 ± 0.03 and 0.48 ± 0.05, respectively. The calibration curve was linear in the concentration range 100 to 600 ng per band for both the drugs in human plasma. The limit of quantitation for Paracetamol and Etoricoxib in human plasma was 100 ng for both the drugs and no interference was found from endogenous compounds. The % recovery from human plasma using the precipitation method was found to be 90.46 for Paracetamol and 87.24 for Etoricoxib, respectively. The method provides a direct estimate of the amount of Paracetamol and present in human plasma. The method was validated with respect to linearity, accuracy, precision and stability as per the Guidance for Industry, Bioanlytical Method Validation guidelines.

 

KEYWORDS: Paracetamol, Etoricoxib, High Performance Thin Layer chromatography, human plasma

 

 

INTRODUCTION:

Paracetamol (PARA), chemically, N-(4 – hydroxyphenyl) acetamide is non-opiate, non-salicylate, centrally and peripherally acting analgesic agent1. It is official in IP, BP and USP2–4. Etoricoxib (ETORI), 5-chloro-6’-methyl- 3-[4-methylsulphonyl)phenyl]-2,3’-bipyridine,  is  a non-steroidal   anti-inflammatory   drug which acts as selective inhibitor of cyclooxygenase type 2 (COX-2)5 The chemical structure of both the drugs are shown  in Figure 1.

 

Extensive Literature survey reveals High Performance Liquid Chromatographic (HPLC)6-12, High Performance Thin Layer Chromatographic13-15 methods for determination of PARA in tablet dosage form and in bioanalytical fluids either as single or in combination with other drugs have been reported. Analytical and bioanalytical methods have been reported for the determination of ETORI includes Spectrophotometry16, HPLC17-22 and HPTLC23-25 as single component or in combinations with other drugs.  Spectrophotometric26 and High Performance Liquid Chromatographic (HPLC)27 method has been reported for determination of paracetamol and Etoricoxib in combined tablet dosage form.

 

To the best of our knowledge, no HPTLC method of analysis has been reported for simultaneous analysis of PARA and ETORI in human plasma. This paper describes simple, accurate and precise HPTLC method for simultaneous determination of PARA and ETORI in human plasma. The method was optimized and validated as per the Guidance for Industry guidelines28.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Working standards PARA and ETORI were obtained from Cipla Ltd. (Mumbai) and Burgeon Pharmaceuticals Ltd. (Kancheepuram, India), respectively used as such without further purification. Toluene, Dichloromethane and Methanol, (all AR grade) were obtained from Sisco Research Laboratories (Mumbai, India).

 

Preparation of Standard Stock Solutions:

Standard stock solution for PARA and ETORI was prepared individually by dissolving 5 mg of each drug in methanol to get the concentrations of  500 µg/ ml. From this stock solution, 0.8, 1.2, 1.6, 2, 2.4 and 2.8 ml of solution  (500µg/ml) was pipetted out into 10 ml volumetric flask and made up the volume with   methanol to get final concentration  40, 60, 80, 100, 120 and 140µg/ml.

          

PARA                                                    ETORI

Fig. 1: Structure of PARA and ETORI

 

Preparation of plasma samples:

To 0.5 ml of plasma, 0.5 ml of PARA and ETORI working standard (Intermediate solution concentration: 40, 60, 80, 100, 120 and 140µg/ml) and 1 ml of methanol as a precipitating agent were added to glass tubes. Each sample was vortex mixed for 3 min and centrifuged (2500 rpm for 15min). Blank plasma sample was prepared in an identical manner except for the addition of PARA and ETORI.

 

Instrumentation and chromatographic conditions:

A remi cyclomixer was used for mixing and vortexing the samples. The 10 µl aliquots of the samples were spotted onto TLC plate in the form of bands of width 6 mm, space between bands was 5 mm, with a 100 µl sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminium plate 60 F254 (10 cm ×10 cm) with 250 µm thickness (E. MERCK, Darmstadt, Germany) using a CAMAG Linomat V sample applicator (Switzerland). The slit dimensions were 5 mm × 0.45 mm and scanning speed of 20 mm/sec was employed. The linear ascending development was carried out in 10 cm × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) using mobile phase Toluene: Dichloromethane: Methanol (6: 2.5:1.5,  v/v/v). The optimized chamber saturation time for mobile phase was 15 min. The length of chromatogram run was 90 mm and development time was approximately 20 min. TLC plates were dried in a current of air with the help of dryer. Densitometric scanning was performed on CAMAG thin layer chromatography scanner at 240 nm for all developments operated by WINCATS software version 1.4.2. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm.

 

Selection of detection wavelength:

After chromatographic development bands were scanned over the range of 200-400 nm and the spectra were overlain. It was observed that both drugs showed considerable absorbance at 240 nm. So 240 nm was selected as the wavelength for detection. The overlain spectrum of PARA and ETORI is shown in Figure 2.

 

Method validation:

Linearity of HPTLC method was constructed by analysis of six solutions containing different concentration of PARA and ETORI. The linearity of the detector response was tested by spotting standards in triplicate for each concentration ranging between 100 to 600 ng. The data were best fitted by a linear equation y = mx + c. Three Quality Control levels used are Low Quality Control  200 ng/band, Medium Quality Control 300 ng/band and  High Quality Control  400 ng/band. The recovery of Paracetamol and Etoricoxib from plasma was determined by comparing peak areas obtained from plasma spiked with PARA and ETORI at concentrations of 200, 300 and 400 ng/ band with the peak areas obtained from standards. The intra-day precision was ascertained by analyzing plasma samples in triplicate spiked with PARA and ETORI at 200, 300 and 400 ng/band on the same day. The inter-day precision was determined by analyzing 200,300 and 400 ng/band plasma samples with blank plasma for three consecutive days. Stability studies for PARA and ETORI were performed on spiked plasma samples having concentrations of 200 and 400 ng. Freeze thaw stability of the spiked quality control samples was determined after three freeze thaw cycles stored at -28 °C ± 5 °C.  Short term stability of the spiked quality control samples was determined for a period of 6 hours 30 min stored at room temperature. Long term stability was determined which exceeds the time between the date of first sample collection and the date of last sample analysis. All the stability samples are compared with freshly prepared plasma sample.

 

Fig. 2: Overlain spectra of PARA and ETORI

 

RESULTS AND DISCUSSION:

Different mobile phases containing various ratios of toluene, ethyl acetate, Dichloromethane, methanol and chloroform were examined (Data not shown). Finally the mobile phase containing Toluene: Dichloromethane: Methanol (6: 2.5:1.5,  v/v/v) was selected as optimum for obtaining well defined and resolved peaks. The Rf value of PARA and ETORI was found to be 0.30±0.03 and 0.48±0.05. The solvent system used for development of the plates produced no interference peaks in the area under the curve, and all other compounds were distinctly separated. The representative densitogram of Blank human plasma and PARA and ETORI extracted from human plasma is shown in Figure 3.

 

Table 1:  Recovery study data of HPTLC method for PARA and ETORI in human plasma

Drug

Level

Concentration

[ng spot-1]

Mean amount [ng spot-1]

Mean recovery

[%]

% RSD

plasma

plane

Plasma Solution

(Mean ± S.D.)

Plane Solution

(Mean ± S.D.)

 

PARA

1

200

177.33 ± 12.66

199.7 ± 1.4

86.69

7.14

0.71

2

300

272.89 ± 13.64

298.6 ± 1.24

89.23

5.0

0.42

3

400

388.75 ± 10.97

397.6 ± 1.59

95.47

2.82

0.40

Average mean recovery [%]

90.46

 

ETORI

1

200

173.35 ± 9.6

199.56 ± 1.75

87.62

5.57

0.88

2

300

253.45 ± 11.75

298.39 ± 1.44

85.66

4.63

0.48

3

400

350.39 ± 14.64

399.7 ± 1.34

88.41

4.18

0.33

Average mean recovery [%]

87.24

 

Fig. 3: Representative densitogram of (1) Blank Human Plasma (2) PARA and ETORI extracted from human plasma

 

Table 2: Stability study data of HPTLC method for PARA and ETORI in human plasma

Procedure

Concentration of PARA

Concentration of ETORI

200 [ng spot-1]

400 [ng spot-1]

200 [ng spot-1]

400 [ng spot-1]

Mean

R.S.D. (%)

Mean

R.S.D. %

Mean

R.S.D. %

Mean

R.S.D. %

Freeze and thaw (n=5)

89.85

4.93

92.73

2.83

88.71

4.25

90.79

2.76

Short term(n=3)

91.69

3.25

93.01

2.15

90.59

6.07

95.70

3.5

Long term(n=3)

89.24

3.90

88.24

3.47

86.82

7.19

88.73

2.77

 

 

Results were found to be linear in the concentration range100–600 ng/ band for both the drugs with good correlation coefficient. The LLOQ for PARA and ETOR in human plasma samples was 100 ng/band. The accuracy, precision and reliability of the procedure were ascertained by adding known concentrations of drug to drug-free plasma and analyzing five samples of each concentration by the method described for precipitation. The intra-day and inter- day precisions of both drugs were within the limit as the coefficient of variation was less than 15%. The mean recovery of PARA and ETORI from plasma was found to be 90.46% and 87.24% respectively. The results are given in Table 1.

 

PARA and ETORI was found to be stable through three freeze–thaw cycles, during storage at  -28°C ± 5°C, and in the short term stability for 5hr at room temperature and stock solution stability for 5hr and 30 min  The results obtained were precise and accurate as shown in Table 2.

 

Acceptance Criteria:  The % CV for LQC and HQC should be sample should be ≤ 15.00.

The % mean stability of LQC and HQC sample should be within 85.00-115.00 %.

 

CONCLUSIONS:

The devloped HPTLC method for quantification of PARAand ETORI in human plasma is accurate, precise, rapid, and selective and economical alternative for studies of the bioavailability, bioequivalence, and pharmacokinetics of these drugs in human plasma.

 

ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to Cipla Ltd., Mumbai, and Burgeon Pharmaceuticals Ltd., Kancheepuram, for providing the sample of pure Paracetamol and Etoricoxib. Thanks are also extended to Principal, Dr. Ashwini R. Madgulkar for providing infrastructure facilities and her constant support.

 

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Received on 12.05.2011       Modified on 22.05.2011

Accepted on 31.05.2011      © RJPT All right reserved

Research J. Pharm. and Tech. 4(8): August 2011; Page 1303-1306