Paracetamol

Metoclopramide

Fig (1): Chemical structure of paracetamol and metoclopramide

Literature survey reveals that gas chromatography⁽²⁾, HPLC⁽³⁾, spectrophotometric⁽⁴⁾ and densitometric⁽⁵⁾methods are available for the determination of paracetamol and spectrphotometric^(6-
8) HPLC⁽⁹⁾and ¹H NMR spectroscopic⁽¹⁰⁾ methods for the determination of metoclopramide.

In modern analytical laboratory, there is always a need for simple, rapid and accurate methods for simultaneous determination of drug combinations that could be used for routine analysis. The present work aimed to develop simple instrumental methods for the quantification of paracetamol and metoclopramide in bulk form or in their pharmaceutical formulations. These methods include chromatographic methods; namely, TLC densitometry and HPLC.

MATERIALS AND METHODS:

Instrument:

· Shimadzu-Dual wavelength lamp flying CS9301 densitometer with PC. With Hamilton syringe 10 µl capacity.

· Florescent TLC plates (20 × 20 cm) with 0.25 mm thickness silica gel F254, (E. Merck).

· The HPLC system comprised an Agilent pump with different flow rates (model 1260 series, Agilent, USA), equipped with a variable wavelength detector and a 20 µl volume injection loop. A Zorbax SB-C18 (4.6 x 100 mm, 3.5µm) column was used as stationary phase. The samples were injected with a 50 µl Hamilton analytical syringe.

Reagents and Chemicals:

All chemicals and solvents used in this investigation were of analytical reagent grade (A.R.) used as such without any further purification.

Paracetamol and metoclopramide were of sufficient purity and passed the British Pharmacopeia (B.P.) requirements. Paracetamol and metoclopramide were supplied by Amoun, Egypt.

Pharmaceutical dosage forms of paracetamol and metoclopramide are supplied by local market company Cid (migracid^®).

Solutions:

Stock standard solution:

For TLC: Solutions were prepared by transferring accurately weighed 100 mg of paracetamol and metoclopramide into 50 ml volumetric flasks. 20 ml of ethanol were added to dissolve by shaking and the volume was completed to the mark with ethanol.

For HPLC: An accurate weight (200 mg) of each of paracetamol and metoclopramide was transferred into 50 ml volumetric flask. Twenty five ml aliquot of the mobile phase was added for each flask and the flasks were shaken for 10 min. The solutions were completed to mark using the same solvent. One ml aliquot of each solution was transferred into 10 ml volumetric flask and the volume was completed with the mobile phase to form 400 µg/ml of the intact standard solutions.

General analytical procedure:

Bulk sample:

For TLC: in 10 ml measuring flasks aliquots containing 0.5-20 mg/ml of paracetamol and metoclopramide working solutions were transferred. The volumes of solutions were completed to the mark with ethanol. 10 µl from drug solutions of paracetamol and metoclopramide having the concentrations 0.05-5 mg/ml were spotted on TLC plates and the specified chromatographic conditions mentioned are adopted.

The calibration curve was constructed relating the area under peak to the corresponding concentration of each drug spot and regression equation was computed.

For HPLC: Aliquot portions (0.25- 8ml) of paracetamol and metoclopramide intact standard solutions equivalent to (0.1- 3.2 mg) were transferred into two series of 10 ml volumetric flasks and the volumes were completed to mark using the mobile phase. Twenty µl of each solution was injected to HPLC system.

The peak area AUP x 10³ was plotted versus the concentration (µg/ml) for constructing the calibration curves.

Laboratory prepared mixtures:

Solutions containing different ratios of paracetamol and metoclopramide were prepared by transferring aliquots from their working solutions into a series of 10 ml volumetric flasks and the volume of each was completed to the mark with ethanol in case of TLC-densitometry. For HPLC, the volume was completed to the mark with the mobile phase.

The peak area of the laboratory prepared mixtures were scanned and processed as described for the calibration curve for each of the proposed TLC and HPLC methods, respectively. The concentrations of paracetamol and metoclopramide in each mixture were calculated using the specified regression equations.

Assay of pharmaceutical dosage forms:

For TLC: Weigh twenty tablets and thoroughly grind well to fine powder, extract an accurate weighed portions of the obtained powder equivalent to 500 mg paracetamol and 5 mg metoclopramide in 50 ml ethanol. Shake for about 15 minutes, filter the solution in 100 ml measuring flask, wash the residue several times and dilute to the mark with ethanol. The procedure of the proposed method was applied under the specified conditions mentioned above. The concentration of each drug was calculated from its corresponding regression equation.

For HPLC: Accurate weight of the powdered Migracid tablets equivalent to 500 mg of paracetamol and 5 mg of metoclopramide was transferred into 150 ml conical flask. Forty ml aliquot of the mobile phase was added to the flask. The flask was shaken for 15 min. The solution was filtered. The filter papers and the residues were washed three times each with 5 ml mobile phase. The combined filtrates and washings were collected into 100 ml volumetric flask and the volume was completed with the same solvent. 2.5 ml aliquot of solution was transferred into 50 ml volumetric flask and the volume was completed to mark with the mobile phase to form a solution of 250 µg/ml of paracetamol and 2.5 µg/ml of metoclopramide.

RESULTS AND DISCUSSION:

TLC-densitometry:

A TLC-densitometric method could be used for the simultaneous determination of paracetamol and metoclopramide without prior separation. Different solvent systems were tried for the separation of paracetamol and metoclopramide. Satisfactory results were obtained by using a mobile phase composed of Ethylacetate/ methanol/ ammonia (85:10:5 v/v/v) where Rf = 0.82 and 0.37 for paracetamol and metoclopramide, respectively. The separation allowed the determination of paracetamol and metoclopramide with no interference [Fig. 2]. The linearity was confirmed by plotting the measured peak area versus the corresponding concentrations at 254 nm over a range of 1-50 μg/spot for paracetamol and over a range of 0.5-10μg/ spot for metoclopramide, where a linear response was obtained [Fig.3,4], regression equations were found to be:

A= 1.044 × C + 0.695 r²= 0.998 for metoclopramide

A= 2.488 × C + 1.044 r²= 0.9998 for paracetamol

Where A is the AUP ×10^-3, C is the concentration in µg/spot and r²is the regression coefficient.

The precision of the proposed method was checked by the analysis of different concentrations of authentic samples in triplicates. The mean percentage recovery was found to be 100.4 for paracetamol and 100.23 for metoclopramide.

Fig (2): The selected developing system give complete separation of the intact drugs (A) paracetamol, (B) metoclopramide and (C) mixture of drugs.

Fig (3): Linearity of the peak area to the corresponding concentrations of metoclopramide (0.5-10 µg/spot) at 254 nm

Fig (4): Linearity of the peak area to the corresponding concentrations of paracetamol (1-50 µg/spot) at 254 nm

High performance liquid chromatography method:

A simple isocratic high-performance liquid chromatography method was developed for the determination of paracetamol and metoclopramide in pure form and in pharmaceutical formulations using a Zorbax SB-C18 (4.6 x 100 mm, 3.5µm). The mobile phase consisted of phosphate buffer pH 4: methanol (75:25 v/v). The mobile phase was chosen after several trials to reach the optimum stationary /mobile –phase matching. The average retention times under the conditions described were 1.3 minutes for paracetamol and 1.7 minutes for metoclopramide. One sample could be chromatographed in 5 minutes. The chromatographic system in this work allowed complete baseline separation of paracetamol from metoclopramide [Fig. 5]. Calibration graphs were obtained by plotting the peak areas versus concentrations of paracetamol and metoclopramide [Fig. 6, 7], Linearity ranges were found to be 10-320 μg/ml for paracetamol and 10-160 μg/ml for metoclopramide using the following regression equations:

Y= 0.01898 C+ 0.06324 r²= 0.9994 for paracetamol

Y= 0.05805 C+ 0.02083 r²= 0.9999 for metoclopramide

Where Y is the peak area AUP x 10³, C is the corresponding concentration in µg/ml, and r²is the regression coefficient. The mean percentage recoveries were found to be 100.216 and 100.69 for paracetamol and metoclopramide respectively.

The robustness of the HPLC method was investigated by analysis of samples under a variety of experimental conditions. It was found that the method was robust when the column and the mobile phase ratio were varied. During these investigations, the retention times were modified, however the areas and peak symmetry were conserved.

Fig (5): HPLC chromatogram of paracetamol at t_R=1.3 min and metoclopramide at t_R=1.7 min, using the specified chromatographic conditions.

Fig (6): Calibration curve for the determination of paracetamol using HPLC.

Fig (7): Calibration curve for the determination of metoclopramide using HPLC

Statistical analysis:

The suggested methods were successfully applied for the determination of paracetamol and metoclopramide in their laboratory prepared mixtures with good precision as shown in table 1. The proposed methods were also used for estimating the concentration of both drugs in their pharmaceutical formulations. The results are shown in table 2. Assay parameters and a validation sheet for determination of the studied drugs are shown in table 3. Statistical comparison for the results obtained by the proposed methods and reported method for the studied drugs are shown in table 4. The calculated t- and F-values were found to be less than the tabulated ones, confirming good accuracy and excellent precision.

Table (1): Determination of paracetamol and metoclopramide in laboratory prepared mixtures by the proposed methods.

Drug determined	TLC-Densitometry	HPLC
paracetamol	100.4 ± 0.336	100.29 ± 0.8
metoclopramide	100.23 ± 0.91	100.45 ± 0.79

Table (2): Determination of paracetamol and metoclopramide in Migracid^® tablets by the proposed methods.

Preparation

TLC-Densitometry

HPLC

Migracid^®tablets

(500mg para., 5mg met.)

Para.

100.26 ± 1.14

100.95 ± 0.73

Met.

100.09 ± 0.71

100.04 ± 0.42

Table (3): Assay parameters and validation sheet for determination of paracetamol and metoclopramide.

Parameter

TLC-densitometry

HPLC

Para.

Met.

Para.

Met.

Range

Slope

Intercept

Correlation coefficient(r²)

1-50µg/spot

2.488

1.044

0.9993

0.5-10µg/spot

1.044

0.695

0.998

10-320µg/ml

0.01898

0.006324

0.9994

10-160µg/ml

0.05805

0.02083

0.9999

R.S.D%^a

R.S.D%^b

0.987

1.49

2.13

2.24

2.7

1.2

1.8

1.08

^a The intraday (n=5) relative standard deviations of (50µg/ml) of paracetamol by HPLC method and (20µg/spot) for the TLC-densitometric method, and of (30µg/ml) of metoclopramide by the HPLC method and (2µg/spot) for the TLC-densitometric method.

^b The interday (n=5) relative standard deviations of (50µg/ml) of paracetamol by HPLC method and (2µg/spot) for the TLC-densitometric method, and of (30µg/ml) of metoclopramide by the HPLC method and (10µg/spot) for the TLC-densitometric method.

Table (4): Statistical comparison for the results obtained by the proposed methods and the reported method for the analysis of paracetamol and metoclopramide.

Parameters

TLC-densitometry

HPLC

Reported method⁽¹⁰⁾

Met.

Para.

Met.

Para.

Met.

Para.

Mean

S.D.

F-test

Student's t-test

100.09

0.71

1.19

(6.39)^*

1.487

(2.78)^**

100.26

1.14

0.126

(6.39)^*

1.84

(2.78)^**

100.04

0.42

2.4

(6.39)^*

1.7

(2.78)^**

100.95

0.73

1.32

(6.39)^*

1.226

(2.78)^**

99.45

0.65

100.34

0.84

^* The tabulated F test.

^**The tabulated student's t-test.