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Validated stability indicating RP-HPLC method for

determination of paracetamol, methocarbamol and
Cite this: Anal. Methods, 2013, 5, 541
their related substances
Eglal A. Abdelaleem* and Nada S. Abdelwahab

Paracetamol (PAR) and methocarbamol (MET) are co-formulated together in Methorelax
tablets which
are widely used as a muscle relaxant and in the treatment of muscle-skeletal pain. On the other hand,
4-aminophenol (4-AP) and guaifenesine (GU) have been reported to be related substances and
degradation products of PAR and MET, respectively. The target of this work was to develop and validate
a simple, sensitive and selective stability indicating RP-HPLC method for the determination of PAR, MET,
4-AP and GU in their bulk powders and laboratory prepared mixtures. Chromatographic separation was
achieved within 10 minutes with the required asymmetry, accuracy and precision on ODS column using
0.05 M KH2PO4 buffer : acetonitrile (72.5 : 27.5, v/v, pH ¼ 6) as the mobile phase at a flow rate of 1 mL
min1 with UV detection at 225 nm. The developed method has been validated as per ICH guidelines
Received 24th September 2012
Accepted 11th November 2012
and the calibration plots were linear over the concentration ranges of 3–20, 4–25, 0.6–8 and 0.6–8 mg
mL1 for PAR, MET, 4-AP and GU, respectively. The method has been successfully applied in the analysis
DOI: 10.1039/c2ay26085a
of Methorelax
tablets and good results were obtained. Moreover, its results have been compared to a
[Link]/methods previously reported RP-HPLC method and no significant difference was found between the two methods.

1 Introduction their mixtures with other drugs. The binary mixtures of PAR and
MET have been determined by ratio spectra,11 second derivative
Paracetamol (PAR) is N-(4-hydroxyphenyl)acetamide,1,2 it is spectrophotometric12 and RP-HPLC11,13 methods. On the other
widely used as a minor analgesic and is used as an alternative to hand, ternary mixtures of PAR, MET and diclofenac-Na or K
aspirin without the side effects of salicylate on gastric mucosa.3 have been analysed by different RP-HPLC methods.14–16 To the
Methocarbamol (MET) is 2-hydroxy-3-(2-methoxyphenoxy)pro- best of our knowledge, there are no reported methods on the
pylcarbamate,3 it is a centrally acting skeletal muscle relaxant determination of the four studied components. Moreover, aer
and is used as an adjunct in the short term symptomatic treat- testing all the reported RP-HPLC mobile phases, all of them
ment of painful muscle spam.4 MET is sometimes given with failed to separate the components of the studied mixture and so
analgesics for the treatment of skeletal muscle pain.5 4-Amino- the work in this manuscript aims to develop and validate a
phenol (4-AP) is considered to be a PAR impurity and a related sensitive and selective stability indicating RP-HPLC method for
substance1,2 which has nephrotoxic6 and teratogenic7 effects. the determination of PAR, MET, 4-AP and GU. The proposed
Guaifenesine (GUF) is (2RS)-3-(2-methoxyphenoxy)propane-1,2- method offers additional advantages over the reported methods
diol.2 It is used as an expectorant for productive coughs, it acts by in that the former is more selective, has a short analysis time
increasesing the volume and reducing the viscosity of tenacious and good accuracy and precision. Moreover, it can be used for
sputum. It has also been given to patients with altered nasal the quality control determination of 4-AP and GU.
mucociliary clearance associated with HIV infection.4 In the
USP,1 it is reported to be an impurity and related substance of
MET, moreover it is considered to be the starting material in MET 2 Experimental
synthesis.8 Both 4-AP and GU are produced from the hydrolytic
degradation of both PAR and MET, respectively.5,7,9,10 2.1 Samples
A literature survey revealed different methods for the deter- 2.1.1 PURE SAMPLES. Paracetamol was kindly supplied by the
mination of PAR and MET either in their binary mixtures or in Egyptian Co. for Chemicals and Pharmaceuticals, ADWIA, 10th
of Ramadan City, Egypt, with certied purity of 99.84.
Methocarbamol was kindly supplied by October Pharma
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Bani-Suef
University, Alshaheed Shehata Ahmad Hegazy St, 62514, Beni-Suef, Egypt. E-mail:
S.A.E., 6th of October City, Egypt with certied purity of 99.80%.
eglal_bardisi@[Link]; nadasayed2003@[Link]; Fax: +2082 2317950; Tel: Guaiphenesin sample was kindly supplied by NOVARTIS
+201060917535; +201117236884 PHARMA S. A. E Cairo, Egypt with certied purity of 99%.

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Analytical Methods Paper

Pure standard 4AP was purchased from SIGMA-ALDRICH Co., 2.5.2 LINEARITY AND CONSTRUCTION OF CALIBRATION CURVES.
Cairo, Egypt with certied purities of 99.56%. Accurate aliquots of PAR, MET, 4-AP and GU were separately
2.1.2 PHARMACEUTICAL FORMULATIONS. Methorelax
tablets transferred from their respective working standard solutions
(B. no. 12587) labeled to contain 400 and 300 mg MET and PAR, (0.1 mg mL1) into four separate sets of calibrated measuring
respectively per tablet were manufactured by GlaxoSmithkline asks to prepare solutions equivalent to 3–20, 4–25, 0.6–8 and
(GSK), Egypt, S. A. E Elsalam City, Cairo, A. R. E. 0.6–8 mg mL1 of PAR, MET, 4-AP and GU, respectively. Tripli-
cate injections were carried out for each concentration; the
integrated peak area (for PAR, MET and 4-AP) and peak height
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2.2 Chemicals and solvents

(for GU) were used to construct the calibration curve for each
Acetonitrile, methanol and ortho-phosphoric acid were of HPLC component from which its regression equation was
grade (Sigma-Aldrich Chemie GmbH, Germany). NaOH and constructed.
KH2PO4 were of analytical grade (El- NASR Pharmaceutical 2.5.3 APPLICATION TO PHARMACEUTICAL FORMULATION. The
Chemicals Co., Abu- Zabaal, Cairo, Egypt). De-ionized water was procedure under linearity and construction of calibration
from SEDICO Pharmaceuticals Co., Egypt. curves was followed using Methorelax
working solution
(0.1 mg mL1 MET). Concentrations of PAR and MET were then
2.3 Solutions calculated from the corresponding regression equations previ-
ously computed from which the percentage recoveries were
0.05M KH2PO4 was prepared by dissolving 6.8 g KH2PO4 in 1 L
calculated.
de-ionized water.
2.5.4 RECOVERY STUDIES. Recovery studies were carried out
Mobile phase was prepared by mixing 72.5 mL of 0.05 M
to establish the accuracy of the method, it was carried out by
KH2PO4 and 27.5 mL acetonitrile and then adjusting the pH of
spiking pre-analyzed pharmaceutical formulation samples with
the mobile phase to pH ¼ 6 using NaOH and phosphoric acid. It
known amounts of pure PAR and MET at three different
was also used as a diluent for nal dilutions.
concentration levels (80, 100 and 120% of the labeled concen-
Stock standard solutions of PAR, MET, 4AP and GU were
trations). The spiked samples were then analyzed three times at
prepared in methanol at a concentration of 1 mg mL1.
each level and the percentage recoveries of the pure added
Working standard solutions of PAR, MET, 4AP and GU were
drugs were calculated.
prepared in methanol at a concentration of 0.1 mg mL1.
Solutions of pharmaceutical formulation: the content of ten
tablets of Methorelax
was nely powdered, mixed well and 3 Results and discussion
weighed. An accurately weighed portion equivalent to 100 mg
MET and 81.25 mg PAR was accurately transferred to a 100 mL ICH guidelines17 recommend the determination of drug impu-
calibrated measuring ask and then 75 mL methanol was rities, degradation products as well as the active drug for
added. The prepared solution was sonicated for 15 minutes, developing a validated stability indicating assay method (SIAM).
cooled well and then the volume was completed to the mark to No reported method was found in the literature for the simul-
prepare 1 mg mL1 MET (and the corresponding amount of taneous determination of PAR and MET in the presence of 4-AP
PAR) stock solution from which a working solution of 0.1 mg and GU, which have to be determined because of the nephro-
mL1 MET (and the corresponding amount of PAR) was toxic effect of 4-AP (PAR impurity) and the different pharma-
prepared. cological action of GU to the parent drug, MET. Therefore, it was
necessary to develop a sensitive, selective and accurate stability
indicating RP-HPLC method for the simultaneous determina-
2.4 Instruments tion of the four studied components that can be used for quality
The HPLC (Shimadzu) instrument was equipped with a model control and stability studies of PAR and MET.
series LC-10 ADVP pump, SCL-10 AVP controller, DGU-12 A
degasser and SPD-10 AVP UV-VIS detector. Separation and
3.1 Method development and optimization
quantitation were carried out at room temperature using a 250
mm  4.6 mm (i.d.) ODS [RP C18 column (4.6 mm particle size)]. Some important parameters affecting chromatographic sepa-
The detector was set at 225 nm. ration have been studied and optimized such as type of buffer
(acetate and phosphate) used and its concentration (0.02 M and
0.05 M), type of organic modier (methanol and acetonitrile)
2.5 Procedures and its ratio (25–50% of the mobile phase), pH of the mobile
2.5.1 CHROMATOGRAPHIC CONDITIONS. Chromatographic phase (4–8 pH), ow rate (0.8–1.2 mL min1) and scanning
separation was carried out in isocratic mode on an ODS column wavelength (215, 225, 254 and 278 nm). Trials have shown that
with a mobile phase consisting of 0.05 M KH2PO4 buf- 0.05 M KH2PO4 gave better resolution than acetate buffer.
fer : acetonitrile (72.5 : 27.5, v/v, pH ¼ 6) delivered at a ow Moreover, using acetonitrile as an organic modier greatly
rate ¼ 1 mL min1. The injection volume was 20 mL with UV enhanced the resolution and peak asymmetry. The ratio of
scanning at 225 nm at room temperature. The run time was 10 acetonitrile in the mobile phase greatly affected the analysis
min and the total peak area and height were used to quantify time and so it was used in the ratio of 27.5% which gave the best
the studied components. resolution within 10 minutes. On the other hand, the pH of the

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mobile phase has a signicant effect only on the chromato- performing recovery studies at three levels (80, 100 and 120%
graphic separation between PAR and 4-AP where pH ¼ 6 gave addition) and the average percent recovery was then calculated.
the best separation without affecting the resolution of MET and Good percentage recoveries were obtained and are given in
GU. Different scanning wavelengths were tried to obtain Table 1.
maximum sensitivity for all the separated components, scan- 3.2.3 PRECISION. It was studied with respect to both
ning at 225 nm gave the best sensitivity with minimum noise repeatability and intermediate precision. Repeatability was
detected. Also the effect of mobile phase ow rate was tested calculated by analysis of three different concentrations of pure
and a ow rate of 1 mL min1 was found to give good resolution components (10, 15 and 20 mg mL1 for PAR and MET), (2, 4 and
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within a short analysis time. 6 mg mL1 for 4-AP and GU) in triplicate on the same day. The
Aer method optimization, chromatographic separation of experiment was repeated using the same concentrations seven
the four components was achieved using an ODS column with times on four consecutive days to determine the intermediate
0.05 M KH2PO4 buffer : acetonitrile (72.5 : 27.5, v/v pH ¼ 6) as precision. Good results and acceptable RSD%, Table 1, were
the mobile phase at a ow rate ¼ 1 mL min1 and with UV obtained.
scanning at 225 nm, Fig. 1. 3.2.4 SPECIFICITY. The selectivity of the method was
demonstrated by good separation of the four studied compo-
nents, Fig. 1. Also, the absence of any peaks at the retention
3.2 Method validation times of the studied drugs and the good results obtained on
Method validation was carried out according to ICH applying the method to Methorelax
tablets, the results pre-
guidelines.17 sented in Table 2 prove that there was no interference from
3.2.1 LINEARITY. Under optimum chromatographic condi- excepients.
tions, linearity of the method was evaluated by measuring the 3.2.5 LIMITS OF DETECTION AND QUANTITATION (LOD AND
integrated peak area of different concentrations each of PAR, LOQ). ICH recommendations17 using a visual non-instrumental
MET and 4-AP and by measuring the peak height for GU (as method were followed to calculate the values of LOD and LOQ of
peak area gave poor sensitivity and bad correlation coefficient) the four studied components (where LOD is the concentration
and then plotting the calibration graphs relating the peak area at which the signal to noise ratio is equal to 3 : 1 while LOQ is
or peak height against the corresponding concentration from the concentration at which the signal to noise ratio is equal to
which the regression equations were constructed. Linearity 10 : 1). Low values of both LOD and LOQ indicated the high
ranges and regression equation parameters are listed in Table 1. sensitivity of the developed method, Table 1.
3.2.2 ACCURACY. It was calculated as the percentage recov- 3.2.6 ROBUSTNESS. The method was demonstrated to
eries of blind pure components, it was further assured by be robust over an acceptable working range of its HPLC

Fig. 1 RP-HPLC chromatogram of a resolved mixture of standard 6 mg mL1 4-AP (Rt ¼ 3.75 min), 15 mg mL1 PAR (Rt ¼ 4.4 min), 6 mg mL1 GU (Rt ¼ 7.82 min) and 20
mg mL1 MET (Rt ¼ 9.65 min) using 0.05 M KH2PO4 : acetonitrile (72.5 : 27.5, v/v pH ¼ 6).

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Analytical Methods Paper

Table 1 Regression and analytical parameters of the proposed method for the determination of PAR, MET, 4-AP and GU

Parameters PAR MET 4-AP GU

Calibration range 3–20 mg mL1 4–25 mg mL1 0.6–8 mg mL1 0.6–8 mg mL1
Slope 268153.2 71905.4 91343.5 6331.9
Intercept 593247 134161 166168.6 1921.4
Correlation coefficient 0.9998 0.9996 0.9999 0.9998
Accuracy 99.50 99.90 100.36 99.86
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Precision
Repeatability 0.885 0.755 0.632 0.789
Intermediate precision 1.231 1.455 1.025 0.998
LOD 0.30 mg mL1 0.5 mg mL1 0.1 mg mL1 0.1 mg mL1
LOQ 0.5 mg mL1 1.6 mg mL1 1.2 mg mL1 0.5 mg mL1

Table 2 Determination of the studied drugs in Methorelax tablets
by the proposed RP-HPLC method and statistical comparison with the reported method

RP-HPLC method

Parameters PAR MET

Methorelax tabletsa (B. NO. 12587) 99.73  0.881 96.50  1.048

Standard additionb 100.66  1.021 99.66  1.030
Degree of freedom F-test (5.050)c (10) 1.338 (10) 1.492
Degree of freedom Student's t-test (2.228)c (10) 1.872 (10) 0.862
a
Average of 6 determinations. b Average of 3 determinations. c The values in the parenthesis are the corresponding theoretical values at p ¼ 0.05.

co-operational conditions. Any small deliberate variation in the 4 Conclusion

mobile phase pH (0.1 pH unit), organic modier ratio (1%),
ow rate (0.05 mL min1) and scanning wavelength (1 nm) The proposed method provides a sensitive, selective and
showed no dramatic change in Rt value, peak height, area or reproducible means for determination of PAR, MET and their
symmetry of the peaks. impurities. The developed method is the rst stability indi-
3.2.7 SYSTEM SUITABILITY. System suitability testes (SST), cating method for determination of the studied drugs, more-
Table 3, conrmed that the chromatographic system was over it has low LOD and LOQ values and hence it can be used for
adequate for the planned analysis. Also the calculated SST detection of the lowest concentration of 4-AP and GU. On the
parameters were within the acceptable criteria for good HPLC other hand application of the method to a pharmaceutical
practice. preparation showed that excipients do not interfere with the
determination of the studied drugs and hence it can be used for
their determination either in bulk powder or in pharmaceutical
3.3 Application of the method formulation.
Aer method validation, the developed RP-HPLC method was
successfully applied for the determination of PAR and MET in Acknowledgements
Methorelax
tablets, Table 2. Statistical comparison of the
results obtained by applying the proposed method for analysis The authors would like to express their appreciation and thanks
of the two proposed drugs in Methorelax
tablets to those to the Egyptian Co. for Chemicals and Pharmaceuticals, ADWIA,
obtained by applying the reported RP-HPLC method11 showed 10th of Ramadan City, Egypt and October Pharma S.A.E., 6th of
no signicant difference regarding both accuracy and precision, October City, Egypt for the provision of the necessary materials
Table 2. to carry out this work.

References
Table 3 System suitability testing parameters of the developed RP-HPLC
method
1 The United States Pharmacopeia, National Formulary 27,
United States Pharmacopeial convention INC, USA, 32 edn,
Parameters 4-AP PAR GU MET 2009.
2 The British Pharmacopoeia, Her Majesty's, The Stationary
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Office, London, 2007.
Peak a symmetry 1 1.13 1 1
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544 | Anal. Methods, 2013, 5, 541–545 This journal is ª The Royal Society of Chemistry 2013
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Paper Analytical Methods

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