- Corresponding Author:
- K. K. Srinivasan]

Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, MAHE, Manipal - 576104, India.**E-mail:**[email protected]

Date of Submission | 18 November 2005 |

Date of Revision | 7 June 2007 |

Date of Acceptance | 19 July 2007 |

Indian J Pharm Sci, 2007, 69 (4): 540-545 |

## Abstract

A derivative spectrophotometric procedure has been developed for the simultaneous determination of individual combination of aceclofenac and tramadol with paracetamol in combined tablet preparation. Tablet extracts of the drugs were prepared in distilled water. The zero crossing point technique and the compensation technique were used to estimate the amount of each drug in the combined formulations, and were compared. The results were found to be accurate and free from interferences. The procedure is rapid, simple, nondestructive, and does not require solutions of equations. Calibration graphs are linear (r=0.9999), with a zero intercept up to 24 mg/ml of each drug in combination with paracetamol. Detection limits at the p = 0.05 level of significance were calculated to be 0.5 mg/ml of aceclofenac, tramadol and paracetamol respectively.

## Keywords

Derivative spectrophotometry, aceclofenac, tramadol, paracetamol

Derivative spectrophotometry has only recently become a practical analytical method in the general laboratory because of the rapid progress in microcomputers technology. This technique, if properly understood and applied, it will be a valuable tool for problem solving in several areas of analytical chemistry. It can lead to quicker and more accurate quantitation of multicomponent mixtures that previously would have required, e.g., separation by HPLC. In recent years; derivative spectrophotometry has received increasing attention with regard to the assay of drugs in their formulation and in systems of clinical and biological interest. The fundamental principles and convention of derivative spectrophotometry have been described in the works of O’Haver et al. and Fell and Smith [1,2]. The zero crossing technique has found practical application more recently and has become the most often used procedure to resolve binary mixtures by spectrophotometry. Several papers have been published on zero-crossing technique using various orders of derivative spectrophotometry [3-6].

Aceclofenac is an orally administered non-steroidal antiinflammatory drug [7,8], which possesses good analgesic properties and good tolerability profile in a variety of painful conditions. Chemically aceclofenac is 2-[[2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl] oxy]acetic acid. Several methods have been reported for the assay of aceclofenac [9-11].

Tramadol is an orally administered non-steroidal antiinflammatory drug [12,13], which possesses good analgesic properties and good tolerability profile in a variety of painful conditions. The chemical name for tramadol hydrochloride is (±) cis- 2-[(dimethylamino)methyl]-1-(3-methoxyphenyl cyclohexanol hydrochloride. Several methods have been reported for the assay of tramadol [14-16].

Chemically paracetamol is N-(4- hydroxyphenyl)acetamide. Paracetamol exhibits antiinflammatory, analgesic and antipyretic activities, which are due to the inhibition of cyclooxygenase–2 (COX-2). Literature survey revealed that there are UV and HPLC methods reported for the estimation of paracetamol in pharmaceutical formulations [17-20].

The review of the literature revealed that no method is yet reported for the simultaneous estimation of aceclofenac and tramadol in individual combination with paracetamol in combined dosage forms. This paper describes simple, rapid, accurate, reproducible and economical methods for the simultaneous estimation of individual combination of aceclofenac and tramadol with paracetamol in tablet formulations using second derivative zero crossing point technique.

## Materials and Methods

All spectrophotometric measurements were made using a Shimadzu UV/Vis spectrophotometer 1601 model with a spectral bandwidth (resolution) of 0.1 nm and wavelength accuracy (with automatic wavelength correction) of 0.5 nm. An ultrasonicator was used for proper dissolution of the samples.

**Analytical procedure**

Suitable volumes of stock solution, containing up to 24 μg/ml of paracetamol and aceclofenac, were placed in a 10 ml calibrated flask and brought to volume with distilled water. Then the second derivative spectrum of the mixture against water was recorded and values of the derivatives were measured at 279.4 and 289.9 nm for aceclofenac and 259.7 and 295.8 nm for paracetamol, respectively. Similarly suitable volumes of stock solution containing up to 24 μg/ml of paracetamol and tramadol, were placed in a 10 ml calibrated flask and made up to volume with distilled water. Then the second derivative spectrum of the mixture against water was recorded and values of the derivatives were measured at 265.1 nm for paracetamol and 279.1 nm for tramadol, respectively.

**Analysis of tablet formulation**

Twenty tablets were weighed accurately, average weight was determined and then all the 20 tablets were ground to a fine powder. A quantity equivalent to 100 mg of aceclofenac and 500 mg of paracetamol were transferred to a 100 ml volumetric flask. The contents were ultrasonicated for 10 min with distilled water, made to volume and filtered through Whatmann filter paper. The solution was further diluted with distilled water, to give concentrations of 4 and 20 μg/ml of aceclofenac and paracetamol, respectively. Second derivative absorbances of these solutions were measured at 279.4 and 289.4 nm for aceclofenac (A) and 259.7 and 295.8 nm for paracetamol (B). Similarly, quantity equivalent to 100 mg of tramadol and 500 mg of paracetamol were transferred to a 100 ml volumetric flask, ultrasonicated for 10 min with distilled water, made to volume and filtered through Whatmann filter paper. The solution was further diluted with distilled water to give concentrations of 2 and 10 μg/ml of tramadol and paracetamol, respectively. Second derivative absorbances of these solutions were measured at 279.1 nm for tramadol (B) and 265.1 nm for paracetamol (A).

## Results and Discussion

The zero absorption spectra of aceclofenac and
paracetamol (10 μg/ml each) show significant
differences in the absorption values at similar
concentration, hence the traditional Vierordt’s and
modified Vierordt’s methods for the assay of binary
mixtures gave erroneous results. Several tests
were made to select the more suitable order of the
derivative, the type of measurement, i.e., graphical
or zero-crossing measurements, and the working
wavelength exhibiting the best linear response to
analyte concentration and higher sensitivity, and while
not being affected by any other components. The
first derivative spectra of both showed considerable
differences in certain areas; which prevents, in the
present instance, suitable use of this technique.
The corresponding second derivative spectra of
aceclofenac and paracetamol are represented in **fig. 1**. On the contrary zero-crossing second derivative
spectrophotometry offers an extremely valuable means
of simultaneously determining both the drugs in a
mixture.

The second derivative spectra of a mixture of
aceclofenac and paracetamol were recorded against
water and the values of derivative were measured at
279.4 and 289.4 nm for aceclofenac (zero-crossing
wavelength of second derivative of paracetamol) and
259.7 and 295.8 nm (zero-crossing wavelength of
second derivative of aceclofenac) for paracetamol
and the concentration of aceclofenac and paracetamol
was calculated from the calibration graphs. **Fig. 2**.
depicts a typical set of second derivative spectra of a
laboratory mixture containing 4 μg/ml of aceclofenac
and increasing concentrations of paracetamol ranging
from 10-24 μg/ml. **Fig. 3**. exhibits a typical set of
second derivative spectra of a laboratory mixture
containing 20 μg/ml of paracetamol and increasing concentrations of aceclofenac ranging from 4-10
μg/ml.

The height of peaks at 279.4 and 289.4 nm, the zero
crossing wavelengths of paracetamol, was denoted as
h2 **(fig. 2)**, and the height of peaks at 259.7 and 295.8
nm, the zero crossing wavelengths of aceclofenac,
was denoted h1 **(fig. 3)**. These heights, h1 and h2
were proportional to aceclofenac and paracetamol
concentrations, respectively. Moreover, the values
of h1 and h2 were not affected by the presence of
other excipients present in the tablet formulation.
An interaction study was performed, and the results
indicated that when one component is kept constant
(2 μg/ml) and the concentration of the other is varied,
the H1 (at 279.4 nm) and H2 (at 259.7 nm) values are
unaltered up to 30 μg/ml of the second component.
Hence, accurate quantitation of the two drugs was
achieved even when the ratio of concentration was
1:15.

It is also interesting to note there are two distinct
isobestic points each in **fig. 2**. (at 279.4 and 289.4
nm zero crossing wavelengths of paracetamol)
as well as in **fig. 3** (at 259.7 and 295.8 nm zerocrossing
wavelength of aceclofenac), irrespective of
the concentration of paracetamol and aceclofenac,
respectively.

The second derivative spectra of tramadol and
paracetamol are represented in **fig. 4**. The values of
derivative were measured at 279.1 nm for tramadol
(zero-crossing wavelength of second derivative of
paracetamol) and 265.1 nm (zero-crossing wavelength of second derivative of tramadol) for paracetamol.
Then, the concentrations of tramadol and paracetamol
were calculated from the calibration graphs. **Fig. 5**.
shows a typical set of second derivative spectra of
a laboratory mixture of 5 μg/ml of tramadol and
increasing concentrations of paracetamol ranging from
10-24 μg/ml. Similarly **fig. 6**. shows a typical set of
second derivative spectra of a laboratory mixture of
20 μg/ml of paracetamol and increasing concentrations
of tramadol ranging from 4-24 μg/ml.

The height of peak at 279.1 nm, the zero crossing
wavelength of paracetamol, was denoted as h2 (**fig. 6)**, and the height at 265.1 nm, the zero crossing
wavelength of tramadol, was denoted h1 **(fig. 5)**.
These heights, h2 and h1 were proportional to
tramadol and paracetamol concentrations, respectively.
Moreover, the values of h1 and h2 were not affected
by the presence of other excipients present in tablet
formulation. An interaction study was performed,
and the results indicate that when one component
is kept constant (2 μg/ml) and the concentration of the other is varied, the H1 (at 265.1 nm) and H2 (at
279.1 nm) values are unaltered up to 30 μg/ml of the
second component. Hence, accurate quantitation of
the two drugs was achieved even when the ratio of
concentration was 1:15.

It is also interesting to note distinct isobestic points
in **fig. 5**. at 279.1 (zero crossing wavelengths of
paracetamol) and in **fig. 6**. at 265.1 nm (zero
crossing wavelength of tramadol) irrespective of
the concentration of paracetamol and tramadol,
respectively.

The linear regression equations calculated for
individual mixtures of aceclofenac and tramadol with
paracetamol are assembled in **Tables 1 and 2** together
with the correlation coefficients, the variances, and the
detection limits at a level of significance of p=0.05
for 11 standard samples. Beer’s law is followed
for concentrations up to 24 μg/ml of each drug.
Two different tests of significance of the intercepts
of line of regression (H= a+bc) were performed to establish whether the experimental intercept ‘a’ differed significantly from the theoretical value, zero.
These tests are very useful in the case of mixtures to
verify if the analytical method is free from procedural
errors depending on the concentrations of one of
the two components. The first procedure to estimate
the difference ‘a–0’ follows from the determination
of the quantities ‘t= a/Sa’ (Sa’ is an estimate of
the accuracy of the determination of ‘a’) and their
comparison with the tabular data for t-distribution.
The values calculated for ‘t’ are for aceclofenac
and for paracetamol (i.e., they do not exceed the
95% criterion of tp= 2.26 for n= 11 samples); this
indicates that the intercepts of line of regression are
not significantly different from zero.

Regression equations | λ (nm) | r | SD, Intercept, Slope, Sb |
LOD µg/ml | LOQ µg/ml | t=a/Sa | |
---|---|---|---|---|---|---|---|

A | D2'_{A}=4.25x10^{-4}+5.34x10^{-4} CA |
259.7 | 0.9997 | 1.13x10^{-5}, 3.43x10^{-7} |
0.32 | 1.06 | 1.03 |

A | D2'_{A}=1.00x10^{-4}+1.46x10^{-4} CA |
295.8 | 0.9994 | 1.28x10^{-5}, 4.12x10^{-7} |
0.37 | 1.23 | 0.96 |

B | D2'_{B}=7.99x10^{-5}-6.41x10^{-4} CB |
279.4 | 0.9989 | 5.95x10^{-5}, 2.83x10^{-6} |
0.47 | 1.56 | 0.88 |

B | D2'_{B}=-1.43x10^{-4}-3.22x10^{-4} CB |
289.9 | 0.9999 | 5.37x10^{-5}, 3.36x10^{-6} |
0.52 | 1.73 | 0.85 |

A is paracetamol, B represents aceclofenac, CA and CB denote concentrations of drugs (μg/ml). Number of samples n=10, aTheoretical value of t at P= 0.05 level of significance is 2.31

**Table 1:** Statistical data for the calibration graphs of paracetamol and aceclofenac zero crossing derivative spectrophotometry.

Regression equations | λ (nm) | r | SD Intercept, Sa Slope, Sb | LOD µg/ml | LOQ µg/ml | t=a/Sa | |
---|---|---|---|---|---|---|---|

A | D2'_{A}=-0.12x10^{-1}+0.132x10^{-1}CA |
265.1 | 0.9997 | 1.14x10^{-4}, 2.43x10^{-6} |
0.29 | 1.16 | 1.02 |

B | D2'_{B}= 6.32x10^{-3}-7.1x10^{-3}CB |
279.1 | 0.9999 | 4.27x10^{-3}, 2.36x10^{-5} |
0.42 | 1.63 | 0.82 |

A is paracetamol, B represents tramadol. CA and CB denote concentrations of drugs (μg/ml). Number of samples, n= 10. aTheoretical value of t at P= 0.05 level of significance is 2.31

**Table 2:** Statistical data for the calibration graphs of tramadol and paracetamol by zero crossing derivative spectrophotometry

To study accuracy, reproducibility, and precision of
the proposed methods, five successive determinations
on synthetic mixtures of paracetamol and aceclofenac
were carried out. The results reported in **Tables 3
and 4** show that accuracy and precision are very
satisfactory. The complex problem of quantitating
components of above mixture with widely differing
absorption values could be solved. The conceptual
and experimental straightforwardness of the proposed
second derivative method vouches for its suitability
for the routine analysis of pharmaceutical dosage
forms. The method described is simple and confirms
that the technique of derivative spectrophotometry,
if properly used, can give precise, sensitive, and
rapid analysis of mixtures of drugs. In the proposed
method, the values of coefficient of variation were satisfactorily low and recovery was close to 100% for
both the drugs. Hence, it can be employed for routine
analysis in quality control laboratories

Mixture | Nominal value µg/ml |
Mean value±SD µg/mla |
RSD % |
---|---|---|---|

Paracetamol and aceclofenac | 20.0 | 19.89±0.034 | 0.18 |

4.5 | 4.46±0.052 | 0.16 | |

Paracetamol and aceclofenac | 10.0 | 9.94±0.024 | 0.13 |

18.0 | 18.02±0.031 | 0.12 | |

Paracetamol and tramadol | 20.0 | 19.79±0.024 | 0.16 |

4.5 | 4.49±0.032 | 0.15 | |

Paracetamol and tramadol | 10.0 | 9.98±0.025 | 0.12 |

18.0 | 18.03±0.031 | 0.13 |

aMean of five determinations

**Table 3:** Replicate determinations on individual synthetic mixtures of aceclofenac. and tramadol with paracetamol

Combined tablet dosage form | Label claim, mg | Mean recovery ±SD, %b |
---|---|---|

Paracetamol and aceclofenac | 500.0 | 99.96±0.042 |

100.0 | 99.86±0.067 | |

Paracetamol and aceclofenac | 500.0 | 101.04±0.012 |

100.0 | 100.12±0.061 | |

Paracetamol and tramadol | 500.0 | 99.98±0.032 |

100.0 | 99.96±0.077 | |

Paracetamol and acelofenac | 500.0 | 100.04±0.016 |

100.0 | 100.15±0.051 |

bMean of five determinations, assay as percentage of label claim.

**Table 4:** assay of aceclofenac and tramadol with paracetamol in combined tablet dosage form.

## Acknowledgements

Authors are thankful to Indoco Remedies, Mumbai for the gift samples of drugs.

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