Analytica Chimica Acta 378 (1999) 299311

Determination of metformin in pharmaceutical preparations using

potentiometry, spectrouorimetry and UVvisible spectrophotometry
Saad S.M. Hassana,*, Wagiha H. Mahmouda, Mohamed A.F. Elmosallamyb,
Abdel Hammeed M. Othmana
a

Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt

Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt

Received 24 February 1998; received in revised form 25 June 1998; accepted 8 July 1998

Abstract
New, simple and convenient potentiometric, spectrouorimetric and spectrophotometric methods are described for the
determination of metformin in pharmaceutical preparations. The potentiometric technique is based on preparation of PVC
membrane sensors incorporating metforminreineckate, and metformintungstosilicate ion-pairs as electroactive species with
dioctylphthalate and o-nitrophenyloctylether as plasticizers, respectively. A membrane consisting of carboxylated PVC
plasticized with dibutylsebacate is also prepared and tested. These sensors give rapid Nernstian response for 101105 M
metformin at pH range 511. The metformintungstosilicate based sensor is used in a ow-through sandwich cell for ow
injection potentiometric determination of metformin. Graphite sensors coated or doped with metformintungstosilicate-PVC,
Cu-diethyldithiocarbamate and Ni-diethyldithiocarbamate are also prepared and used for monitoring the titration of the drug
with tetraphenyl borate (TPB), Cu2 and Ni2 ions, respectively. The spectrouorimetric method depends on the reaction of
metformin with chrysenequinone in alkaline medium to give a Schiff's base, which upon hydrolysis gives the free base. The
latter in the presence of 1-naphthol gives a uorescent product with excitation and emission maxima at 450 and 520 nm,
respectively. The uorescenceconcentration relationship is linear over the range 20200 mg ml1 metformin. The proposed
spectrophotometric technique involves reaction of metformin with Cu2 in basic medium to form a Cumetformin complex.
The complex is dissolved in cyclohexylamine and its maximum absorption at 540 nm is measured. Beer's law is obeyed over
the range 0.52 mg ml1 metformin. Various cations, some nitrogenous compounds or drug excipients cause no interferences.
Results obtained by these techniques are comparable and compare favorably with data obtained using the British
Pharmacopoeia method. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Metformin; Potentiometry; Spectrouorimetry; Spectrophotometry; PVC membranes; Pharmaceutical analysis; Flow injection
analysis; Graphite sensors

1. Introduction

*Corresponding author. Fax: +202-4831836; e-mail:

iupac15@asunet.shams.eun.eg

Metformin (1,1-dimethylbiguanide) (I) is an oral

hypoglycaemic agent, clinically used in the treatment
of diabetes patients [1,2].

0003-2670/99/$ see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S0003-2670(98)00500-5

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S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

Metformin has been determined by titration in nonaqueous medium with perchloric acid using different
indicators [3]. Potentiometric titration of the drug with
perchloric acid in glacial acetic acid and mercury(II)
acetate has been developed [1]. Conductometric titration of metformin with copper sulfate has been
described [4]. Poly(vinyl chloride) (PVC) matrix
membrane sensors based on the use of ion association
complexes of metformin with tetraphenylborate [5],
tungstophosphate [6] and molybdophosphate [7] have
been used for direct potentiometric determination of
metformin in pharmaceutical preparations.
Spectrophotometric determination of metformin is
based on reaction with NaOCl followed by NaOH and
ZnSO4 to give a yellow color [8]. Spectrophotometric
determination of metformin in biological uids has
been also suggested by reactions of metformin with
biacetyl and 1-naphthol in 50% alkaline ethandiol [9],
and bromothymol blue in phosphate buffer [10].
Nuclear magnetic resonance (n.m.r.) spectrometry
[11], gas chromatography [12,13], and liquid chromatography [1417] have also been described for determination of metformin in pharmaceutical preparations
and biological uids.
In the present work, novel metformin sensors are
described based on the use of the ion association
complexes of metformin with reineckate and tungstosilicate as ion exchangers in a plasticized PVC matrix.
Carboxylated PVC as a native ionic polymer without
drug ion-pairs is also prepared and tested. These
sensors display stable, fast and linear response over
a wide range of concentrations and pH values, and are
used in a ow-through cell for continuous measurements. Graphite rods doped or coated with copperdiethyldithiocarbamate (Cu-DDC), nickel-diethyldithiocarbamate (Ni-DDC) and metformintungstosilicate-PVC are used for monitoring the potentiometric
titration of metformin in pharmaceutical preparations
with Cu2, Ni2 or tetraphenylborate. A spectrouorimetric method based on the reaction of metformin
with chrysenequinone and 1-naphthol in alkaline med-

ium to form a uorescent product is used for the

determination of metformin in pharmaceutical formulations. Metformin is also determined spectrophotometrically by reaction with copper sulfate in
cyclohexylamine. The results obtained by the developed techniques are in good agreement and compared
favorably with the standard non-aqueous titration
method of the British Pharmacopoeia.
2. Experimental
2.1. Apparatus
All potentiometric measurements were carried out
at 2518C with an Orion Model 901 microprocessor
Ionalyzer using metformintungstosilicate, reineckate
and PVC-carboxylated membrane sensors, in conjunction with an Orion double junction Ag/AgCl reference
electrode (Model 90-02) containing 10% (w/v)
sodium nitrate solution in the outer compartment. A
Ross glass electrode (Orion 81-02) was used for all pH
measurements. A laboratory-made ow-through sandwich cell [18,19] equipped with metformintungstosilicate-PVC membrane was prepared as previously
described and incorporated in a single ow injection
(FI) system, with an Omnit injection valve and a
peristaltic pump. The potentiometric output was measured with an Orion microprocessor Ionalyzer pHmillivoltmeter and recorded with a strip chart recorder
(Linear 1200). A Perkin-Elmer Lambda 15 UV/VIS
spectrophotometer and a Perkin-Elmer LS50 uorescence spectrometer were used.
2.2. Reagents and materials
All chemicals were of analytical reagent grade
unless otherwise stated, and doubly distilled deionized
water was used throughout. Poly(vinyl chloride) powder, PVC-COOH powder, dioctylphthalate (DOP), 2nitrophenylphenylether (NPPE), tetrahydrofuran
(THF), ammonium reineckate, tungstosilicic acid,
copper(II) sulfate pentahydrate, nickel(II) sulfate
hexahydrate and sodium diethyldithiocarbamate trihydrate (Na DDC) were obtained from Sigma
(St. Louis, MO). Pure metformin (purity not less
than 99.5%) was provided by El-Nasr Pharm. Cyclohexylamine, n-butylamine, diethylamine, ethylene

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

diamine, nitrobenzene, carbon tetrachloride, chrysenequinone, 1-naphthol, hydroquinone, potassium

hydroxide, dioxan and ethanol were obtained from
Fluka AG (Buchs, Switzerland) and Merck AG
(Darmstadt, Germany). Dosage forms containing metformin were obtained from local drug stores.
2.3. Metformin ion-pairs
Metformintungstosilicate, and reineckate ionpairs were prepared by mixing 15 ml of 1102 M
aqueous metformin solution with 50 ml of 1102 M
tungstosilicic acid and ammonium reineckate, respectively. The mixture was shaken well, the precipitates
were ltered off through a G4 sintered glass crucible,
washed with deionized doubly distilled water, dried at
about 1008C for 30 min and ground to a ne powder.
2.4. Metformin poly(vinyl chloride) sensors
A 10 mg portion of metformin ion-pair was mixed
in a glass Petri dish (5 cm diameter) with 0.19 g of
PVC powder and 0.35 g of 2- nitrophenylphenylether.
With the carboxylated-PVC membrane, 0.2 g of the
polymer was mixed with 0.35 g of dibutylsebacate.
The cocktail was dissolved in 5 ml of tetrahydrofuran,
the Petri dish was covered with lter paper, and left
overnight to allow slow evaporation of the solvent at
room temperature. A master PVC membrane
(0.1 mm thick) was obtained, sectioned with a cork
borer (10 mm diameter) and glued to polyethylene
tubing as previously described [20,21]. The electrode
was lled with a mixture of an equal volume of
aqueous 1102 M metformin hydrochloride and
sodium chloride solutions. An Ag/AgCl internal reference wire electrode (ca. 1.0 mm diameter) was used.
The sensor was conditioned by soaking in 1102 M
metformin for 2 h and stored in the same solution
when not in use.
2.5. Cu-DDC, Ni-DDC and metformin
tungstosilicate-PVC graphite sensors
A rod of spectrographic graphite (0.6 mm in diameter and 16.0 mm long) was forced inside a polyethylene tube. The end of the graphite rod was washed
with acetone, dried in air for 23 h, dipped into a
beaker containing 10 ml of 0.1 M copper sulfate or

301

0.1 M nickel sulfate for about 3 h and then dipped in

another beaker containing 0.1 M sodium diethyldithiocarbamate for about 3 h. The rod was washed
with puried water several times, and allowed to dry.
The graphite metformintungstosilicate PVC was prepared by mixing the ion-pair with PVC powder and 2nitrophenylphenylether in 5 ml of THF; the graphite
electrode was coated with a thin lm of the cocktail
and then left to dry.
2.6. Sensor calibrations
The sensors were calibrated by transferring 10 ml
aliquots of 0.11011106 M aqueous solutions of
metformin hydrochloride to 50 ml beakers followed
by immersing the metformin-PVC membrane sensor
in conjunction with a double junction Ag/AgCl reference electrode in the solution. The potential readings
were recorded after stabilization to 0.2 mV and the
e.m.f. was plotted as a function of the logarithm of the
metformin concentration. The calibration graphs were
used for subsequent determination of unknown metformin concentrations. The potentiometric selectivity
pot:
were determined by the separate
coefcients KMF;B
solution technique using 1102 M metformin and
interferents [21].
2.7. Flow injection measurements
A laboratory-made ow-through sandwich potentiometric cell, equipped with a metformintungstosilicate ion-pair PVC membrane, was fabricated and
used in a single stream FI system. The metformin
detector was prepared and conditioned as described
previously [18,19]. The cell was assembled, connected
to the FI system and placed in conjunction with a
single-junction Ag/AgCl electrode in a Petri dish lled
with the carrier electrolyte solution. A carrier solution
consisting of 5103 M sodium acetate was propelled
through the cell by a peristaltic pump at 3.3 ml min1.
Successive 20 ml aliquots of 101105 M standard
metformin hydrochloride solutions were injected into
the owing stream using an Omnit injection valve.
PTFE tubing (0.8 mm i.d.) was used; the tubing distance between the injection valve and the detector was
about 10 cm. Both the metformin sensor and reference
electrode were connected to a pH-millivoltmeter,
which was attached to a strip chart recorder to record

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S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

the FI signals. The waste from the Petri dish was

continuously removed by the peristaltic pump. At least
three signals for each sample were recorded and their
average potential changes were measured. Flow injection determination of metformin dosage forms was
similarly carried out using the same carrier solution.
The obtained signals were compared with a calibration
plot for 101105 M pure metformin solutions under
the same conditions.
2.8. Potentiometric measurements

25 ml volumetric asks, followed by 2.5 ml of 0.2 M

copper(II) sulfate and 12.5 ml of cyclohexylamine.
The solution was completed to the mark with puried
water. The mixture was shaken for 15 min, ltered off
on a G4 sintered glass crucible and the absorbance of
the colored reaction mixtures was measured at
5452 nm in a 1.0 cm quartz cuvette against a reagent
blank.
2.11. Determination of metformin in pharmaceutical
preparations

Aliquots (28 ml) of metformin solution containing

(33120 mg ml1) were transferred to 50 ml beakers.
The metformin-TS-PVC, Cu-DDC or Ni-DDC graphite sensor in conjunction with an Orion double
junction Ag/AgCl reference electrode was immersed
in the solution and titrations were carried out with
standard 1102 M tungstosilicic acid, NaTPB or
0.1 M CuSO4 or NiSO4 using the above mentioned
sensors, respectively. The e.m.f. was recorded after
potential stabilization to 0.5 mV. The change in the
potential reading was measured and used for calculation of metformin content in pharmaceutical preparations. The titration curves were plotted and the
equivalence points were determined.

Ten tablets of the drug were weighed and pulverized. A portion equivalent to one tablet (500850 mg)
was accurately transferred to a 50 ml beaker, dissolved
in the least amount of puried water and ltered to
remove insoluble components. The ltrate was transferred to a 500 ml volumetric ask, completed to the
mark with water and the content determined by the
proposed potentiometric, spectrouorimetric or spectrophotometric methods.

2.9. Spectrofluorimetric measurements

Plasticized PVC membrane sensors incorporating

metforminreineckate (MFRE), and metformin
tungstosilicate (MFTSA) ion-pairs were prepared
with suitable plasticizers and electrochemically
evaluated under static and hydrodynamic modes of
operation according to IUPAC recommendations
[22].

Aliquots (0.21.2 ml) of 1102 M metformin

solution covering the range 0.2200 mg ml1 were
transferred into 10 ml volumetric asks, followed
by 5 ml of chrysenequinone solution (0.04%, w/v in
dioxan-water, 1:4 by vol.), 2.0 ml of 1-naphthol solution (2%, w/v in ethanol), 0.5 ml of hydroquinone
solution (0.01%, w/v in ethanol) and 1.0 ml of 3 M
potassium hydroxide. The solutions were completed
to the mark with ethanol, shaken well and left to stand
at room temperature for 15 min. The uorescence of
the resulting solutions was measured at their maximum emission at 520 nm using their maximum excitation wavelength of 450 nm. A calibration graph was
constructed.
2.10. Spectrophotometric measurements
Aliquots (0.28.0 ml) of metformin hydrochloride
solutions containing (0.416 mg) were pipetted into

3. Results and discussion

3.1. Potentiometric measurements

3.1.1. Performance characteristics of the sensors

The membranes were prepared using casting solutions of composition 2:70:28 wt% of metformin ionpair, 2-nitrophenylphenylether and PVC, respectively.
The carboxylated-PVC sensor was prepared using a
casting solution of the composition 70:30 wt% of
dibutylsebacate and carboxylated-PVC powder,
respectively. Table 1 summarizes the response characteristics of three different sensor assemblies for each
system under static conditions. It was found (Fig. 1)
that sensors based on MFRE and MFTSA ion-pairs
are almost identical in terms of calibration slope and
detection limit. The two sensors exhibited Nernstian

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

303

Table 1
Performance characteristics of metformin-PVC membrane sensors
Parameter

MFRE

MFTSA

Carboxylated PVC

Slope (mV/decade)
Intercept (mV)
Correlation coefficient (r) (n6)
Lower limit of linear range (M)
Lower limit of detection (M)
Response time for 1102 M (s)
Working range of pH

59.90.5
325.20.2
0.996
9.5105
5.2105
10
511

60.00.3
125.30.4
0.997
5.1105
5.5106
30
511

55.00.6
320.10.2
0.996
8.5105
6.9106
20
511

response over the range 5.5105101 M metformin

with cationic slopes of 59.90.5, and 60.00.3 mV/
decade and lower limits of detection of approximately
5.2105, and 5.5106 M, respectively.
The response times of the sensors were tested for
1102 and 1103 M metformin; the sequence of
measurements was from low to high concentrations.
The time required for the sensors to reach values
within 0.5 mV of the nal equilibrium potential

was measured. The response times of MFRE and

MFTSA sensors to 1102 M metformin were 10
and 30 s, respectively. Both sensors have a potential
stability and uctuation of the calibration slope not
exceeding 0.6 mV/decade over a period of four weeks.
After that, the calibration slope and the linear range of
response gradually decreased probably due to leaching
of the ion-pair from the membrane. The response time
was also increased to 1.52 min.

Fig. 1. Potentiometric response of: (A) metformintungstosilicate (MFTSA); (B) carboxylated-PVC; and (C) metforminreineckate (MF
RE) polymeric membrane sensors.

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S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

In contrast, the metformin membrane sensor based

on carboxylated-PVC exhibited a near-Nernstian
slope of 55.00.6 mV/decade over the range
8.5105101 M metformin. The lower limit of
detection was 6.9106 M metformin, and the
dynamic response time for 1102 M was 20 s. Carboxylated-PVC membranes offered an attractive solution of the leaching problems commonly experienced
with polymeric membrane sensors containing electroactive drug/ion-pairs. Self-regeneration of this membrane upon soaking in the test solution resulted in an
increase of the life span, selectivity, stability and
durability of the sensor.
3.1.2. Effect of pH
The response of metforminreineckate, and tungstosilicate-based sensors was practically unaffected by
pH change over the range 511. At pH values lower
than 4, the potential readings sharply increased for
sensors based on metforminreineckate and tungstosilicate ion-pairs, due to the interference by H ions.
At pH values >pH 10 a slight decrease of the potentials
of tungstosilicate and reineckate-based sensors was
noticed. In contrast, the potential response of the
carboxylated-PVC based sensor was stable over the
pH range 511. At pH<4, a slight decrease of the
sensor potential was obtained, probably due to a
change in the membrane phase. At pH>10, there
was a slight increase in the potential probably due
to interference by Na. The pHmV plots revealed
that the three developed sensors have the same pH (5
11) working range.
3.1.3. Effect of temperature
The response characteristics of the metformin sensor based on a carboxylated-PVC membrane plasticized with DBS were examined at temperatures
ranging 25608C using 11021105 M metformin test solutions. The isothermal coefcient of the
sensor (dE0/dG) was determined. Calibration plots at
different temperatures were made and the intercept
value (E0) of the plots at zero metformin concentration
was plotted vs. (T-25). The intercept of the resulting
straight line, which represents the isothermal coefcient according to the relation: E0T25(dE0/dG) (T25), was 1.2 mV. This fairly low isothermal coefcient
value of the sensor within the experimental temperature range revealed a reasonable thermodynamic ionic

exchange process at the membrane/drug solution

interface and good mechanical and thermal stability
up to 558C.
3.1.4. Effect of foreign ions
pot:
Potentiometric selectivities KMF;B
of reineckate,
tungstosilicate and carboxylated-PVC metformin
membrane-based sensors were determined using the
separate solutions method [21], with 1102 M aqueous solutions of metformin and the foreign compounds at pH ca. 5. The results obtained (Table 2)
show reasonable selectivity toward metformin in the
presence of many inorganic cations, amines, amides
and amino acids. Pharmaceutical excipients and diluents commonly used in drug formulations (e.g. glucose, lactose, maltose, mannitol, starch, talc powder
and magnesium stearate) at concentrations as high as a
10100 fold molar excess over metformin did not
interfere. The metformintungstosilicate membrane
sensor was, in general, more selective than the other
two sensors, but the carboxylated-PVC-based membrane sensor showed better selectivity in the presence
of some amino compounds such as urea, aniline and 2diethylaminoethanol.
3.1.5. Flow injection analysis (FIA)
Potentiometric measurements of metformin under
hydrodynamic operation were examined using a owTable 2
pot:
Potentiometric selectivity coefficients KMF;B
of metformin PVC
membrane sensors
Interferent (B)

Na
NH
4
Ca2
2
Ba
Co2
Cu2
Urea
Aniline
Guanidine
Glutamic acid
n-Butylamine
t-Butylamine
Dicyandiamide
2-Diethylaminoethanol

pot:
KMF;B

MFRE

MFTSA

Carboxylated
PVC

6.2103
7.1103
9.2103
5.3103
4.2103
2.3102
4.1103
3.5102
5.2102
9.2103
8.3102
7.5102
8.1103
6.2102

4.9103
4.8103
8.2103
1.1103
6.5103
1.3102
3.2103
2.7102
3.2102
1.6103
5.9102
9.0102
1.8102
8.2102

4.2103
4.3103
7.5103
5.2103
2.6102
3.0102
1.2103
2.6102
4.1102
2.6103
4.1101
5.9102
3.1102
7.2102

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

through detector incorporating a MFTSA-PVC

matrix membrane. Different concentrations of metformin were injected in a carrier stream consisting of
5103 M sodium acetate at 3.3 ml min1 and the
peak heights were measured and compared with the
standard. Reproducibility was evaluated from
repeated injection of 20 ml of 1104 M solution of
metformin. The results showed a wide range of hydrodynamic measurements (1.6 mg ml11.6 mg ml1)
without any signicant baseline drift or change in
the response time. The relative standard deviation
of the potential was 0.5%. The calibration slope
was 50 mV/decade and the limit of detection was
20 mg ml1 metformin.
3.1.6. Potentiometric titrations
Metformin (MF) reacts with some anions such as
tetraphenylborate (TPB) and some cations such as
Cu2 and Ni2 to form white, red and orange precipitates, due to the formation of [MFH TPB],
[(MF)2Cu]and [(MF)2Ni] complexes, respectively.

305

Graphite rods coated with a plasticized PVC

membrane incorporating metformintungstosilicate
(MFTSA), or saturated with copper-diethyldithiocarbamate (Cu-DDC) and nickel-diethyldithiocarbamate (Ni-DDC) were prepared and used as sensors to follow the titrations of metformin with TPB,
Cu2 and Ni2 ions, respectively. The potential
breaks (Figs. 24) ranged from 80 to 140 mV for
1:1 (metformin:TPB), and 2:1 (metformin:Cu2 or
Ni2) reactions.
3.1.7. Potentiometric determination of metformin in
dosage forms
The results obtained for determining metformin in
solution (50350 mg ml1) by direct potentiometry
using MFTSA, MFRE and PVC-COOH membrane
sensors show average recoveries of 99.4%, 98.9% and
98.5% with mean standard deviations of 0.2%, 0.5%
and 0.6%, respectively, (Table 3). Potentiometric
titrations of 315 mg of metformin using graphite
based Cu-DDC, Ni-DDC and MFTSA PVC mem-

Fig. 2. Typical potentiometric titration curves of: (A) 4.0; (B) 6.0; and (C) 8.0 ml of 0.1 M metformin with standard solution of 1102 M
NaTPB using an MFTSA PVC graphite sensor.

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S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

Fig. 3. Typical potentiometric titration curves of: (A) 2.0; (B) 4.0; (C) 6.0; and (D) 8.0 ml of 0.1 M metformin with standard 0.1 M CuSO4
using a Cu-DDC graphite sensor.

brane sensors were carried out with CuSO4, NiSO4

and NaTPB solutions as titrants, respectively. The
average recoveries were 98.3%, 98.2% and 97.9%
with mean standard deviations of 0.3%, 0.6% and
0.7%, respectively (Table 3). Flow injection analysis
for metformin in pharmaceutical preparations using
the MFTSA detector gave results with an average
recovery of 99.8% and a mean standard deviation of
0.3%. No interferences occurred from excipients and
diluents used in pharmaceutical preparations (e.g.
glucose, maltose, lactose, starch, acacia, Tween-80,
magnesium stearate and carboxy methylcellulose).

3.2. Spectrofluorimetric measurements

3.2.1. Nature and conditions of the reaction
It has been reported that 9,10-phenanthraquinone
reacts with biguanide compounds in an alkaline medium to form colored complexes [23]. In this study, the
reaction of metformin with chrysenequinone was
examined in an attempt to obtain a uorophore with
high sensitivity. Due to the rigidity of the chrysenequinone reagent, it is possible to assume that the
reaction of metformin with chrysenequinone in alkaline medium proceeds to give the corresponding

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

307

with time probably due to oxidation. Addition of

0.01% hydroquinone stabilizes the uorophore intensity for at least 3 h at room temperature.
3.2.2. Spectrofluorimetric determination of
metformin in dosage forms
The uorescenceconcentration relationship is linear over the range 20200 mg ml1 metformin. The
precision was studied with a series of ten determination of 50 mg ml1 metformin. The average recovery
and standard deviation were 99.3% and 0.3%,
respectively (Table 3). Excipients and diluents used
in pharmaceutical preparations (e.g. acacia, starch,
talc, Tween-20, sucrose, lactose and magnesium stearate) did not interfere. Amounts present at concentrations as high as a 100-fold wt. excess over metformin
did not affect the intensity, stability and rate of reaction.
3.3. Spectrophotometric measurements

Fig. 4. Typical potentiometric titration curves of: (A) 2.0; (B) 4.0;
(C) 6.0; and (D) 8.0 ml of 0.1 M metformin with standard 0.1 M
NiSO4 using an Ni-DDC graphite sensor.

Schiff's base (A) which upon partial hydrolysis in the

medium yields the free base (B), and with further
reaction with 1-naphthol develops a strong uorescent
reaction product (C). The proposed chrysenequinone
reaction scheme shown in Fig. 5 is in a good agreement with that suggested by other workers for the
colorimetric reaction of 9,10-phenanthraquinone with
biguanide drugs [24].
Experimental parameters affecting the uorescence
intensity of the reaction product including the concentration of chrysenequinone (0.020.08%, w/v), 1naphthol (18%, w/v) and KOH (14 M) were examined. Maximum uorescence intensity was obtained at
520 nm, by using an excitation wavelength of 430 nm
and 0.04% (w/v) chrysenequinone, 2% 1-naphthol and
3 M KOH (Fig. 6). Under these working conditions,
however, the uorophore gradually loses its intensity

3.3.1. Nature and conditions of the reaction

Reaction of Cu(II) with biguanides is known to give
the complexes [Cu(MF2Big)2]X2 and [Cu(MF2
Big)X2], where XCl, Br, OH, NO
3 , NCS ,

NCO or N
3 [25]. In the present work, metformin
was allowed to react with Cu2 in a strongly alkaline
medium to form a water-insoluble pink complex. The
complex is precipitated along with copper hydroxide.
Trials were made for quantication of metformin by
extraction of its copper complex with some waterimmiscible organic solvents such as benzene, nitrobenzene, carbon tetrachloride, methyl isobutylketone,
butanone, isobutyl alcohol, cyclohexane, and chloroform. None of these solvents was suitable for quantitative extraction of the Cumetformin complex.
Attempts were next made to use water-miscible
organic solvents to effect dissolution of coppermetformin complex. It was found that some aliphatic
amines such as diethylamine, n-butylamine and cyclohexylamine could dissolve the Cumetformin complex completely to give a violet colored solution.
Cyclohexylamine gave the maximum absorbance
compared to the other amines. The basicity of cyclohexylamine is sufcient for complex formation without a need for addition of alkali. Aqueous ammonia
was not suitable to dissolve the complex because it

Five measurements.

Glucophage, 500 mg/tablet

(Cid., Egypt)
Glucophage retard, 850 mg/tablet
(Cid., Egypt)
Metformin, 500 mg/tablet
(EL-Nasr Pharm. Chem., Egypt)

Drug

98.90.4
98.70.5
98.90.3

99.40.2

98.80.6

99.10.4

98.60.5

98.50.7

98.50.6

98.90.3

98.70.2

98.30.3

98.10.7

98.40.6

98.20.6

Ni-DDC

Cu-DDC

MFTSA

PVC-COOH

Graphite sensors

PVC-membrane sensors
MFRE

Potentiometric titration

Direct potentiometry

Mean metformin recovery ( standard deviation) (%)a

99.10.2

98.10.6

97.90.7

MFTSA

99.70.5

99.30.2

99.80.3

FIA

99.10.6

98.80.2

99.30.3

98.60.3

98.80.4

98.50.6

98.40.5

98.50.7

96.90.8

SpectroSpectroNon-aqueous
fluorimetry photometry titrimetry (BP)

Table 3
Determination of metformin in some pharmaceutical preparations using manual and flow injection potentiometry, spectrofluorimetry, spectrophotometry and non-aqueous titrimetry

308
S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

309

Fig. 5. Reaction scheme of metformin with chrysenequinone in the presence of 1-naphthol in an alkaline medium.

partially extracted Cu(II) from the metformincopper

complex.
The absorption spectrum of the coppermetformin
complex solution in cyclohexylamine shows maximum absorbance at 5452 nm, whereas the copper(II)
blank solution in cyclohexylamine displays maximum
absorbance at 593 nm (Fig. 7). The effect of cyclohexylamine concentration (1070%, v/v) on the maximum absorption was examined. Maximum
absorption was obtained at 40% cyclohexylamine;
the color was stable for at least 8 h.
The stoichiometry of the reaction of Cu(II)
and metformin was determined by Job's method of
continuous variations at 545 nm. The mole ratio of
Cu(II)/metformin against absorbance plot showed a
maximum absorbance at a mole fraction of 0.5, indicating the formation of 1:1 metformin:Cu(II) com-

plex. The stability constant of the complex was

spectrophotometrically determined by the method of
Harvey and Manning [26] and found to be
6.93103 M1.
3.3.2. Spectrophotometric determination of
metformin in dosage forms
Under the optimum experimental conditions, Beer's
law was obeyed for metformin over the range 0.3
2.6 mg ml1. The molar absorptivity of the Cumetformin complex (max8103 l mol1cm1) reects
the sensitivity of the method. The relative standard
deviation was found to be less than 0.6%. The proposed method was applied for the determination of
metformin in some pharmaceutical preparations. The
results obtained (Table 3) show an average recovery of
98.60.4%.

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S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

Fig. 6. Fluorescence spectra of 1.2103 M metformin: (A) excitation; and (B) emission spectra.

Fig. 7. Absorption spectra of the reaction product of 1102 M metformin with 0.2 M copper(II) sulfate and 40% (v/v) cyclohexylamine (A),
and 0.2 M copper(II) sulfate with 40% cyclohexylamine (blank) (B).

S.S.M. Hassan et al. / Analytica Chimica Acta 378 (1999) 299311

4. Conclusions
A statistical comparison of the results obtained by
the proposed methods and those obtained by the
British Pharmacopoeia method shows good correlation. The proposed potentiometric, spectrouorimetric
and spectrophotometric methods give comparable
results and did not suffer from any interferences by
many pharmaceutical additives, diluents and active
ingredients commonly used in drug formulations. The
spectrouorimetric method has the advantages of high
accuracy and direct determination of metformin in
dosage forms at room temperature without prior
separation. The potentiometric methods described in
this work offer many advantages over the other proposed spectrouorimetric and spectrophotometric
techniques in terms of low limit of detection
(8.6 mg ml1), wide range of concentration assay
(8.6 mg ml1165 mg ml1), and wide pH working
range (511). The use of ow injection potentiometry
enables a large sample throughput rate (ca. 80
samples h1) and application to small sample volumes
(ca. 20 ml). Previously reported spectrophotometric
methods involved time-consuming extraction steps
[9,27]. The n.m.r. [11] and conductometric [4] procedures were used for determining milligram quantities
of metformin. The advantages offered by the present
methods suggest their use for the routine analysis of
metformin in pharmaceutical preparations.
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