Microchemical Journal 148 (2019) 262–269

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

A micellar HPLC method for simultaneous determination of methocarbamol T

in three different tablets using single run; application to human plasma and
evaluation of the method greenness
Samah Abo El Abass Mohamed , Fathalla Fathalla Belal
⁎

Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

ARTICLE INFO ABSTRACT

Keywords: Methocarbamol is a well-known centrally acting skeletal muscle relaxant and is usually combined with an-
Methocarbamol algesics like paracetamol, diclofenac and ibuprofen for treatment of musculoskeletal pain. A new approach based
Micellar mobile phase on applying green analytical procedures was proposed using micellar chromatographic method for the quanti-
Spiked human plasma and the analytical eco- fication of methocarbamol in three different combined dosage forms with isocratic mobile phase system in a
scale
single run in about 11 min. The separation was performed on C8 column (250 mm × 4.6 mm i.d., 5-μm particle
size) with micellar mobile phase consisting of (0.12 M) sodium dodecyl sulfate, (15%) n-propanol, 0.02 M or-
thophosphoric acid, 0.3% triethylamine, adjusted to pH 6. The eluents were monitored by UV detection at
220 nm. The linearity of the method was acceptable over the concentration ranges of 5–200 μg/mL for meth-
ocarbamol, 5–80 μg/mL for paracetamol and 5–100 μg/mL for both diclofenac and ibuprofen. Also, the method
was extended to determine methocarbamol in spiked human plasma. The validation of the developed method
was evaluated based on ICH guidelines acceptance criteria for linearity, accuracy, precision and robustness.
Moreover; the greenness profile of the proposed method was assessed and compared with the published HPLC
methods using the analytical Eco-scale as an assessment tool.

1. Introduction including; spectrophotometry [4–7], spectrofluorimetry [8,9], TLC [10]

Most of analytical methods are far from safety due to volatile or-
ganic solvents, flammable, toxic, harmful and carcinogenic chemicals
used. These reasons made the green chemistry concept development
very fast. Most of analytical methods have harmful risk to human health
and environment. Liquid chromatography is one of harmful techniques
due to toxic and hazardous wastes [1]. For these reasons; an eco-
friendly green analytical method was investigated using micellar mo-
bile phase. It consists of aqueous solution of surfactant with small
proportion of organic solvent, thus it is environmentally friendly [2].
Methocarbamol (MET) or chemically 2-Hydroxy-3- (2-methox-
yphenoxy) propyl carbamate is one of the mostly used drugs for treat-
ment of muscle spasm. It acts as skeletal muscle relaxant through its
depressant effect on CNS. MET is given with analgesics as combined
preparations for treatment of musculoskeletal pain [3].
MET has three famous combined tablets with common non-steroidal
anti-inflammatory drugs including paracetamol (PAR), diclofenac
(DCF) and ibuprofen (IBU) [3]. Such binary combinations of MET with
these analgesics have been determined previously by several methods
and HPLC [11–18]. The studied four drugs are officially reported in
United States Pharmacopeia, USP [19]. The USP recommended a
spectrophotometric method for assay of MET in its pure form and HPLC
method for assay in its tablets.
After an overview for the published methods, the drawbacks may be
due to the low sensitivity of the method or a gradient or isocratic elu-
tion systems but at different chromatographic conditions. Moreover,
few of them determined MET in human plasma and if present, several
extraction steps were followed. Also, there is no micellar HPLC method
have been developed previously for assay of these four drugs. So, we
proposed the first micellar chromatographic method for simultaneous
estimation of MET in its three combined tablets with PAR, DCF or IBU
using single chromatographic conditions in about 11 min with low
percent of organic modifier to be the first green chromatographic assay
method. The greenness of the method was evaluated using eco-scale
profile. Additionally, the micellar system made the assay procedures of
MET in human plasma so simple by direct injection of the drug without
tedious and complicated procedures involving protein precipitation
method and without consuming the time during extraction procedures.

⁎
Corresponding author.
E-mail address: dr_samah157@yahoo.com (S.A.E.A. Mohamed).

https://doi.org/10.1016/j.microc.2019.04.079
Received 7 January 2019; Received in revised form 29 April 2019; Accepted 29 April 2019
Available online 01 May 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
S.A.E.A. Mohamed and F.F. Belal
2. Experimental
2.1. Apparatus
⁎ Merck Hitachi Chromatograph (L-7100) equipped with Rheodyne
injector (20 μL loop), UV detector (L-7400) and a solvent degasser
(L-7612), Darmstadt, Germany.
⁎ Consort NV P901 pH Meter.
2.2. Materials
– Methocarbamol with purity of 100.15% was provided by Sigma
Pharmaceutical Company, Cairo, Egypt.
– Paracetamol with purity of 100.12% was provided by Amoun
Pharmaceutical Company, Cairo, Egypt.
– Diclofenac sodium with purity of 100.6% was provided by EPICO,
10th of Ramadan City, Egypt.
– Ibuprofen with 99.66% purity was provided by Kahira
Pharmaceutical Company, Cairo, Egypt.
– Methorelax® tablets (325 mg paracetamol and 400 mg metho-
carbamol), methoquick® tablets (50 mg diclofenac and 500 mg
methocarbamol) and ibuflex® tablets (400 mg ibuprofen and 750 mg
methocarbamol).
– Methanol, acetonitrile, n-propanol and ethanol, all are products of
Sigma-Aldrich (Germany).Sodium dodecyl sulphate (SDS), ortho-
phosphoric acid 85% and triethylamine (Riedel-deHäen, Germany).
– Human plasma was obtained from Mansoura University Hospitals
(Mansoura, Egypt) and was kept frozen until used after gentle
thawing.
2.3. Chromatographic conditions
The chromatographic separation was performed using C8 column
Fig. 1. Micellar HPLC method for simultaneous determination of MET with PAR, DCF and IBU.
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Microchemical Journal 148 (2019) 262–269
(250 mm × 4.6 mm i.d., 5-μm particle size), Separation was performed
using isocratic mobile phase system composed of 0.12 M SDS, n-pro-
panol (15%), triethylamine (0.3%),adjusted to pH 6 by 0.02 M ortho-
phosphoric acid and pumped in a rate of 1 mL per minute. The detec-
tion wavelength was set at 220 nm.
2.4. Standard solutions preparation
400 μg/mL methanolic standard solutions of MET, PAR, DCF and
IBU were prepared separately. All solutions were stable for more than
1 week on keeping in the refrigerator.
2.5. Procedures
2.5.1. Construction of calibration graphs
Concentrations ranging from 5 to 200 μg/mL for MET, 5–80 μg/mL
for PAR and 5–100 μg/mL for both DFC and IBU were transferred into
10 mL volumetric flasks, completed to the volume with mobile phase,
mixed well and injected into the chromatograph. The calibration graphs
were constructed using peak area versus the concentration of the drug
(μg/mL). Then, the corresponding regression equations were derived.
2.5.2. Analysis of synthetic mixtures
Aliquots of MET in combination with PAR in a ratio of (1.23:1, w/
w) or with DCF in a ratio of (10:1, w/w) or with IBU in a ratio of
(1.87:1, w/w) were transferred into 10 mL flasks completed to the vo-
lume with mobile phase and injected into the chromatograph. The
percent found for each drug was calculated based on calibration graph
or the derived regression equation.
2.5.3. Analysis of combined tablets
An accurately weighed quantity of mixed contents of 10 tablets of
methorelax® or methoquick® or ibuflex® tablets equivalent to one tablet
S.A.E.A. Mohamed and F.F. Belal Microchemical Journal 148 (2019) 262–269

Table 2
Numbe r of Theoretical Plates (NTP)

3500
Analytical performance data for the determination of the studied drugs by the
(NTP) of PAR proposed method.
(NTP) of MET Parameter PAR MET DCF IBU
3000
(NTP) of DCF
(NTP) of IBU Linearity range (μg/mL) 5–80 5–200 5–100 5–100
2500 Intercept (a) 58.87 −13.84 −110.54 −16.81
Slope (b) 54.37 42.22 118.55 27.53
Correlation coefficient (r) 0.9999 0.9999 0.9999 0.9999
2000 S.D. of residuals (Sy/x) 19.24 37.08 53.38 17.28
S.D. of intercept (Sa) 13.03 20.40 33.93 10.99
S.D. of slope (Sb) 0.31 0.22 0.69 0.22
1500
S.D. 1.68 1.55 1.48 1.78
3 4 5 6 7
% RSD 1.67 1.54 1.47 1.79
pH
% Error 0.75 0.63 0.66 0.79
LOD (μg/mL) 0.79 1.59 0.94 1.31
LOQ (μg/mL) 2.39 4.83 2.86 3.99
5
(K) of MET
(K) of PAR Table 3
Retention factor (K)

4
(K) DCF Application of the proposed method for the analysis of PAR, MET, DCF and IBU
3 (K) IBU in pure form.
Drug Proposed method Comparison method
2
[14,15]
a
Conc.added (μg/ %Found
1 mL)

0 PAR 5.0 102.30 101.56

0 5 10 15 20 25 10.0 98.42 98.02
% of n-propanol 20.0 98.59 100.78
40.0 101.21
80.0 99.80
Mean ± SD 100.06 ± 1.68 100.12 ± 1.86
2.0 t 0.04 (2.44)b
(Tf) of MET F 1.22 (6.94)b
MET 5.0 101.77 101.35
(Tf) of PAR
Tailing factor (Tf)

1.8 10.0 101.80 98.65

(Tf) DCF 20.0 102.77 100.45
(Tf) IBU 40.0 99.99
1.6 100.0 98.49
200.0 100.34
Mean ± SD 100.86 ± 1.55 100.15 ± 1.38
1.4 t 0.66 (2.36)b
F 1.27 (19.29)b
DCF 5.0 102.15 102.43
1.2 10.0 101.26 98.57
0.00 0.05 0.10 0.15 0.20
20.0 101.24 100.81
SDS concentration (M) 40.0 98.28
100.0 100.21
Mean ± SD 100.63 ± 1.48 100.60 ± 1.94
Fig. 2. Study the effect of pH, % of n-propanol and SDS concentration on
t 0.02 (2.44)b
chromatographic behavior of the studied drugs. F 1.71 (6.94)b
IBU 5.0 99.36 97.96
10.0 98.71 102.00
20.0 97.47 99.01
Table 1 40.0 102.30
Optimum system suitability parameters for separation of the studied drugs. 100.0 99.75
Mean ± SD 99.52 ± 1.78 99.66 ± 2.10
Drug Number of theoretical plates Tailing factor (Tf) Resolution (RS)
t 0.10 (2.44)b
(N)
F 1.38 (6.94)b

PAR 2850 1.51 (PAR/MET) = 1.91

Figures between parentheses are the tabulated t and F values at p = 0.05 [24].
MET 2940 1.36 (MET/DCF) = 6.17
DCF 2400 1.50 (DCF/IBU) = 1.72 Each result is the average of three separate determinations.
IBU 2300 1.48

( ),
2
where: Number of theoretical plates
tR
(N) = 5.54 Wh/2
Resolution
2 tR described before under construction of calibration graphs and the
(R) = Tf = W0.05 / 2f.
W 1+ W2 nominal content of each tablet was calculated.

2.5.4. Analysis of MET in spiked human plasma

of each was transferred into a 100 mL volumetric flask, add 80 mL of One milliliter aliquots of human plasma sample were transferred
methanol, sonication for thirty minutes, completed to the volume with into 10 mL volumetric flasks, spiked with different concentrations of
methanol and filtered. Different concentrations were analyzed as MET over the concentration range of 4–7.5 μg/mL. Complete the flask

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Table 4 Table 5
Precision results for the determination PAR, MET, DCF and IBU by the proposed Assay results for the determination of different concentrations of MET in syn-
HPLC method. thetic mixture with PAR, DCF and IBU.
Drug Intra-day precision Inter-day precision Synthetic Proposed method Comparison method
mixture [14,15]
Conc. Conc. % Found Conc. Conc. % Found
added found added found Conc. taken (μg/mL) % Founda % Founda
(μg/mL) (μg/mL) (μg/mL) (μg/mL)
MET PAR MET PAR MET PAR
PAR 10.0 9.95 99.57 10.0 9.97 99.78
20.0 20.28 101.40 20.0 20.42 102.12 MET/PAR 20.0 16.25 101.91 98.00 101.35 101.56
40.0 39.84 99.62 40.0 40.62 101.56 (1.23: 1, 40.0 32.5 100.40 101.90 98.65 98.02
X ± S. D. 100.20 ± 1.04 101.15 ± 1.22 w/w) 80.0 65.0 100.61 99.65 100.45 100.78
%R.S.D. 1.04 1.21 Mean 100.97 99.85 100.15 100.12
%Error 0.60 0.70 ± S.D. 0.82 1.96 1.38 1.86
MET 20.0 20.32 101.60 20.0 20.47 102.36 tb MET 0.89 PAR 0.17
40.0 39.36 98.42 40.0 39.82 99.55 Fb 2.82 1.11
100.0 98.22 98.22 100.0 100.44 100.44 MET/DCF MET DCF MET DCF MET DCF
X ± S. D. 99.41 ± 1.90 100.78 ± 1.44 (10: 1, 50.0 5.0 98.72 101.20 101.35 102.43
%R.S.D. 1.91 1.43 w/w) 100.0 10.0 101.60 99.10 98.65 98.57
%Error 1.10 0.82 200.0 20.0 99.70 100.15 100.45 100.81
DCF 10.0 9.80 98.05 10.0 9.89 98.97 Mean 100.01 100.15 100.15 100.60
20.0 21.31 101.58 20.0 19.68 98.43 ± S.D. 1.46 1.05 1.38 1.94
40.0 39.37 98.44 40.0 40.48 101.21 tb MET 0.12 DCF 0.35
X ± S. D. 99.36 ± 1.94 99.54 ± 1.47 Fb 1.13 3.40
%R.S.D. 1.95 1.48 MET/IBU MET IBU MET IBU MET IBU
%Error 1.12 0.86 (1.87: 1, 30.0 16.0 101.34 101.36 101.35 97.96
IBU 10.0 10.17 101.75 10.0 10.09 100.96 w/w) 60.0 32.0 98.61 98.22 100.90 102.00
20.0 19.65 98.27 20.0 19.61 98.06 90.0 48.0 99.97 100.45 100.45 99.01
40.0 39.80 99.50 40.0 39.60 99.01 Mean 99.97 100.01 100.90 99.66
99.84 ± 1.77 99.34 ± 1.48 ± S.D. 1.37 1.62 0.45 2.10
X ± S. D.
tb MET 1.11 IBU 0.23
%R.S.D. 1.77 1.49
Fb 9.20 1.68
%Error 1.02 0.86

*N. B. Each result is the average of three separate determinations. N. B. Each result is the average of three separate determinations.
The tabulated values of t-test equal 2.77 and F-test equal 19 at p = 0.05 [24] in
all cases.
to the volume with mobile phase, filter by micro disk filter, mix well
and inject into the chromatograph. Construction of calibration graph
studied (Fig. 2). Two types of columns were tried C8 and C18, the last
was performed and % recoveries were calculated using the derived
one causes long retention times of the four drugs. So, C8 was used. The
regression equation.
wavelength of 220 nm was sufficient for estimation of four drugs with
suitable sensitivity. Different pH values of mobile phase were studied
3. Results and discussion
ranging from 3.5 to 6, the best one was pH 6. Different concentrations
of SDS (0.05–0.15 M) were studied. The concentration of 0.12 M was
The use of micellar mobile phase as an alternative chromatographic
chosen as the high concentration causes overlap between IBU and DCF
approach has several advantages in comparison to conventional chro-
while low concentration of SDS causes longer retention times and peak
matographic separation techniques for screening pharmaceutical ana-
broadening. The percentage of n-propanol (8–15%) in mobile phase
lysis. The main target of the present work was to develop a new HPLC
was studied, 15% give good resolution and symmetrical peaks in ade-
methodology with green and eco-friendly properties to determine MET
quate time. While, the replacements of n-propanol by other organic
in its three combined dosage forms using a single chromatographic
modifiers like n-butanol, methanol and acetonitrile was studied. In case
conditions and a single run in a short time, less than 11 min (Fig. 1). It is
of using n-butanol, an overlap between IBU and DCF peaks was ob-
clear that the difference in hydrophilicity and lipophilicity properties
served. While using methanol or acetonitrile, both IBU and DCF were
between the studied drugs made the separation difficult, as Log P(octanol/
retained for 20 min. Finally, the flow rate of 1 mL/min was the best one
water) values of the studied drugs are equal to 0.6, 0.5, 4.5 and 4.0 for
regarding the peak symmetry and suitable retention times. Fig. 2 and
MET, PAR, DCF and IBU respectively [20]. As reported before, one of
Table 1 summarized the optimum system suitability parameters for the
the advantages of the micellar mobile phase system is the applicability
proposed method.
to solve problems of separation of drugs which differ in their polarity
using low percent of organic modifier. The separation was performed
due to the existence of different types of interactions of the analytes 4. Method validation
with micelles in the mobile phase and the column surface altered by the
adsorption of surfactant molecules, e.g. electrostatic, hydrophobic and The validation criteria of the method were evaluated based on ICH
steric interactions, permitting the separation of compounds of different recommendations [23] including:
nature [21,22].
The optimization of chromatographic conditions to allow the se- 4.1. Linearity and range
paration and quantification of the four studied drugs and obtain high
values of number of theoretical plates and symmetrical peaks were The calibration curves of drug concentrations in microgram range

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Table 6
Assay results for the determination of MET in combined tablets with PAR, DCF and IBU.
Dosage form Proposed method Comparison method [14,15]

a
Conc. taken (μg/mL) % Found % Founda

MET PAR MET PAR MET PAR

Methorelax® tablets 20.0 16.25 98.12 99.10 100.11 99.79

(400 mg MET + 325 mg PAR) 40.0 32.5 101.75 101.23 99.55 99.40
(1.23: 1, w/w) 80.0 65.0 101.25 99.29 100.90 101.17
Mean 100.37 99.87 100.19 100.12
± S.D. 1.97 1.18 0.68 0.93
tb MET 0.15 PAR 0.28
Fb 8.41 1.60
Methoquick® tablets (500 mg MET+ 50 mg DCF) MET DCF MET DCF MET DCF
(10: 1, w/w) 50.0 5.0 98.81 99.55 99.83 100.50
100.0 10.0 101.18 100.45 100.22 99.69
200.0 20.0 99.60 99.84 100.50 101.62
Mean 99.86 99.95 100.18 100.60
± S.D. 1.21 0.46 0.34 0.97
tb MET 0.44 DCF 1.06
Fb 12.86 4.45
Ibuflex® tablets (750 mg MET+ 400 mg IBU) MET IBU MET IBU MET IBU
(1.87: 1, w/w) 30.0 16.0 100.50 99.52 100.01 99.98
60.0 32.0 99.50 100.49 99.55 100.50
90.0 48.0 100.16 99.84 100.90 98.48
Mean 100.05 99.95 100.15 99.65
± S.D. 0.51 0.49 0.69 1.05
tb MET 0.20 IBU 0.44
Fb 1.82 4.50

Each result is the average of three separate determinations.

The tabulated values of t-test equal 2.77 and F-test equal 19 at p = 0.05 [24] in all cases.

Table 7 4.2. LOD and LOQ

Application of the proposed HPLC method for determination of MET in spiked
human plasma: Detection and Quantitation limits were calculated statistically ac-
Parameter Conc. added Conc. found % Recovery cording to ICH guidelines, the values are summarized in Table 2.
(μg/mL) (μg/mL)

MET in spiked human plasma 4.0 3.88 97.00

4.3. Accuracy and precision
5.0 5.30 106.00
6.0 5.785 96.41
7.5 7.54 100.53 The method was found to be accurate by comparing the results with
Mean 99.98 the results obtained by the comparison HPLC methods [14,15] in Tables
S.D. 4.40
3 and 5 using F& t-tests [24]. The repeatability of the developed method
% RSD 4.40
(intraday and interday precision) was evaluated by injecting three
different concentrations of standard solution on the same day for the
intra-day study (n = 3) and the following two consecutive days for
inter-day precision (n = 9) and calculate % RSD which is acceptable to
against peak area were rectilinear over concentration ranges as cited in be less than 2% (Table 4).
Table 2. The high values of correlation coefficients (close to one) of the
four drugs point out the good linearity of the calibration graphs.

Table 8
The penalty points for determination of MET binary mixtures by the reported HPLC methods and the proposed method.
Reagents/ Penalty points
instruments
Ref [11] Ref [12] Ref [13] Ref [14] Ref [15] Ref [16] Ref [17] Ref [18] Proposed HPLC method

Hazardousness

Propanol ….. ….. ….. ….. ….. ….. ….. ….. 0

Methanol 6 6 ….. ….. 6 6 ….. 6 …..
Acetonitrile ….. ….. 8 8 8 …… 8 ……. …..
SDS ….. ….. ….. ….. ….. ….. ….. ….. 0
TEA ….. ….. ….. ….. ….. ….. ….. ….. 0
Occupational hazard ….. ….. ….. ….. ….. ….. ….. ….. …..
Waste 3 3 3 3 3 3 3 1 3
Total penalty points 91 91 89 89 83 91 89 93 97

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S.A.E.A. Mohamed and F.F. Belal
Fig. 3. Micellar HPLC determination of MET (100 μg/mL) and PAR (81.25 μg/
mL) in Methorelax® tablets.
4.4. Robustness
The method proved to be robust by checking the effect of minor
changes of pH of mobile phase ( ± 1), percent of n-propanol ( ± 1%)
and conc. of SDS ( ± 0.01 M). No significant effect was demonstrated.
4.5. Specificity
The proposed method was specific as the successful application of
the method for simultaneous determination of MET in combined three
commercial tablets without interference from common excipients,
(Table 6).
4.6. System suitability testing
The system suitability of the method was proved based on chro-
matographic parameters including; number of theoretical plates, re-
solution and tailing factor, the optimum parameters were summarized
in Table 1 and Fig. 2.
5. Pharmaceutical application
The method was used successfully for the estimation of MET si-
multaneously in its combined tablets with PAR, DCF and IBU and the
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Microchemical Journal 148 (2019) 262–269
Fig. 4. Micellar HPLC determination of MET (150 μg/mL) and DCF (15 μg/mL)
in Methoquick® tablets.
results were in good agreement with the comparison methods [14,15]
results as shown in Figs. 3–5 and Table 6.
6. Application of the method for the estimation of MET in spiked
human plasma
The main advantage of the proposed method is the simplicity of
quantitation of MET in spiked human plasma by direct injection method
without the need to long time of extraction procedures, Fig. 6 and
Table 7.
7. The assessment of the method greenness using eco-scale profile
The analytical Eco-scale is a comprehensive approach used to
evaluate the greenness of the analytical procedures. It is one of several
green metrics. It is based on calculating the penalty points and com-
paring the parameters and steps for the analytical process [25]. MET
has been determined previously by eight HPLC methods [11–18]. These
chromatographic methods were mainly based on using methanol or
acetonitrile as a main component in mobile phase composition. The
U.S. Environmental Protection Agency considered them as hazardous
solvents due to their inherent toxicity, particularly for acetonitrile, its
detoxification is carried out by chemical treatment (mainly through
combustion) produces highly toxic compounds [26].The comparison
between the analytical Eco-scale values of the reported methods and the
proposed method is cited in Table 8. The higher Eco-scale score of the
proposed method was obtained as we avoid the use of hazardous sol-
vents like acetonitrile and methanol by using lower percent and greener
solvent (n-propanol). The penalty points of the proposed method was
97, this value proves that the proposed method is an excellent green
method of analysis and more environmentally friendly.
S.A.E.A. Mohamed and F.F. Belal Microchemical Journal 148 (2019) 262–269

Fig. 5. Micellar HPLC determination of MET (90 μg/mL) and IBU (48 μg/mL) in Ibuflex® tablets.

Fig. 6. Application of micellar HPLC method for determination of MET (7.5 μg/mL) in spiked human plasma.

8. Conclusions References

The proposed method is the first micellar HPLC system for si-
multaneous estimation of MET in its combined dosage forms with PAR,
DCF and IBU. It is the first isocratic mobile phase system for quantifi-
cation of the four drugs in a single run using the same chromatographic
conditions in a short time. Moreover, the micellar system is an eco-
nomic and safe according to applying the Eco-scale profile of the ana-
lytical procedures. Finally, the simplicity and safety of the method al-
lows the determination of MET in spiked human plasma without
tedious extraction procedures.
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