INTRODUCTION:
Lamotrigine is (6-(2,3-Dichlorophenyl)-1,2,4-triazine-3,5-diamine).(1)
It blocks voltage-gated sodium channels in presynaptic
neurons and inhibits the release of glutamate. It displays neuroprotective
effects in various models and is a clinically useful anti-epileptic agent.(2)
Various analytical techniques have been employed for the determination of lamotrigine in pure and dosage forms including HPLC,(3-7)
gas chromatography,(8,9) thin-layer chromatography,(10)
capillary electrophoresis,(11,12) spectrophotometry,(13-15)
adsorptive stripping voltammetry,(16) selective
membrane electrode,(17) Immunofluorimetric
assay(18) and radioimmunoassay.(19)
Minoxidil is
(6-(piperidin-1-yl)pyrimidine-2,4-diamine 3-oxide).(1) It is a
peripheral vasodilator and it is used in scalp preparations for hair loss.(2)
Several methods were reported for the determination of minoxidil
in pure and dosage forms including HPLC,(20-22) Gas-chromatography,(23) capillary isotachophoresis,(24)
derivative spectrophotometry,(25) Integrated flow
injection-solid phase spectrophotometry,(26)
spectrophotometry(27-31) and differential
pulse polarography.(32)
Cefixime is [(6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid trihydrate].(1) It is generally
classified as third generation cephalosporin antibiotic and is given by mouth
in the treatement of susceptible infections including
gonorrhoea, otitis media, pharyngitis, lower respiratory tract infections such as
bronchitis and urinary tract infections.(2) It is reported to be
determined through many techniques including HPLC,(33-37) HPTLC(38), capillary zone
electrophoresis(39,40), voltammetry(41,42),
selective membrane electrode(43), Spectrophotometry(33,44-47)
and spectrofluorimetry( 47,48).
Minoxidil
Lamotrigine
Cefixime
2. EXPERIMENTAL:
2.1.
Apparatus
All of the spectrophotometric measurements were carried out using a
Shimadzu UV-1800 with matched 1 cm quartz cells. A Lutron
digital pH-meter was used for pH adjustment.
2.2.
Materials and reagents
All solvents and
reagents were of analytical grade and double distilled water was used throughout
the work. Lamotrigine (Delta Pharm,
10th of Ramadan city, Egypt) working solution 5 µg.ml-1 was
prepared by dissolving in least amount of DMF then completing to volume with
distilled water.
Minoxidil ((Delta Pharm, 10th
of Ramadan city, Egypt) working solution 5 µg.ml-1 was prepared by
dissolving in least amount of methanol then completing to volume with distilled
water.
Cefixime (Sigma Pharmaceuticals, Kwesna,
Egypt) working solution 5 µg.ml-1 was prepared by dissolving in
distilled water. 5 M HCl (El-Nasr Chemicals, Egypt)
was prepared by diluting 225 ml of concentrated HCl
(36%) to 500 ml.
Methyl Orange (Fluka, Switzerland) 20 mg/100 ml dissolved in and completed
to 100 ml mark with distilled water and filtered (stable for 2 weeks at least).
Methylene blue (Fluka,
Switzerland) 20 mg/100 ml dissolved in 20 ml methanol then completed to 100 ml
with distilled water (stable for 2 weeks at least).
Bromide/bromate stock solution were prepared by dissolving 100 mg
of potassium bromate (Winlab,
England) and 1.0 gm of potassium bromide (Winlab,
England) in 100 ml distilled water (stable for 10 days at least). Working
solution was freshly prepared daily by diluting 2.5 ml of stock solution to 100
ml with distilled water.
2.3.
Pharmaceutical preparations
The following
available pharmaceutical preparations were analyzed
Lamotrine® tablets labeled to contain 25 mg per
tablet. Batch No. MT1960807 (Multi Apex
Pharm. Company, Badr City).
Ximacef® Capsules labeled to contain 400 mg cefixime per capsule. Batch No.90640 (Sigma
Pharmaceuticals, Kwesna, Egypt).
Performa® lotion
5% Batch # 80163 (Delta Pharm Company, 10th of
Ramadan, Egypt).
2.3.
Procedure
2.3.1.
General spectrophotometric procedure for
determination of Lamotrigine, Minoxidil and Cefixime.
2.3.1.1. Lamotrigine
To 0.5 ml bromate working solution add 0.1-2 ml (0.5 to 20 µg/ml)
drug then acidify using 1 ml 5 M HCl and stand for 5
minutes, add 1.0 ml dye working solution then stand for 5 minutes and complete
to 10 ml mark with distilled water then measure absorbance against reagent
blank similarly prepared at 667 nm or 508 nm for methylene
blue or methyl orange, respectively.
2.3.1.2. Minoxidil
Mix 1.0 ml of bromate working solution with 0.1- 2 ml (0.5 to 20 µg/ml)
drug in 10 ml measuring flask and add 1 ml 5 M HCl
stand for 5 minutes, add 1 ml dye working solution and stand for 5 minutes and
complete to 10 ml mark with distilled water then measure absorbance against
blank similarly prepared at 667nm or 508 nm for methylene
blue or methyl orange, respectively.
2.3.1.3. Cefixime
In 10 ml
measuring flask place 1 ml bromate working solution,
0.1- 2 ml (0.5 to 20 µg/ml) drug, acidify with 4 ml 5 M HCl,
stand for 15 minutes then add 0.5 ml dye and wait for 5 minutes before diluting
to 10 ml with distilled water and measure absorbance at 747 nm or 508 nm for methylene blue or methyl orange, respectively against
reagent blank similarly prepared.
In the stated
methods calibration graph was prepared by plotting absorbance versus
concentration of drug and the concentration of the unknown was computed from
the regression equation derived from the Beer’s law data.
2.3.2.
Procedure for pharmaceutical preparations
For Lamotrine® tablets: 10
tablets were crushed and a weight equivalent to two tablets was taken,
extracted three successive times with 20 ml DMF, filtered into 100 ml measuring
flask and then completed to volume with distilled water. Then follow the same
procedures described for determination as in authentic sample.
For
Performa® lotion: Transfer 1.0 ml of lotion to 50 ml measuring
flask then complete to mark with distilled water. Then follow the same
procedures described for determination as in authentic sample.
For Ximacef® capsules contents of 10 capsules were obtained and
weight equivalent to 500 µg was taken, extracted three successive times with
methanol, filtered into 100 ml measuring flask and then completed to volume
with distilled water. Then follow the same procedures described for
determination as in authentic sample.
2.3.3 Reference
method procedure for Lamotrigine determination
(49) (according to Delta Pharm.) Amount equivalent to 10 mg was extracted with methanol
three times, filtered, washed into 100 ml measuring flask and completed to
volume with dist. Water. Known volumes were taken into 10 ml measuring flask
and completed to volume with 0.1 M HCl then
absorbance was measured at 267 nm against blank.
3. RESULTS AND DISCUSSION:
The proposed spectrophotometric methods are indirect and are based on
the determination of the residual bromine (insitu
generated) after allowing the reaction between each drug and a measured amount
of bromine to be complete. The surplus bromine was determined by reacting it
with a fixed amount of either methylene blue or
methyl orange dye. The methods rely on the bleaching action of bromine on the
dyes, the discoloration being caused by the oxidative destruction of the dyes. Lamotrigine, minoxidil or cefixime when added in increasing amounts to a fixed amount
of insitu generated bromine, consumes the latter
proportionately and there occurs a concomitant fall in the concentaration
of bromine. When a fixed amount of dye is added to the decreasing amounts of
bromine, a concomitant increase in the concentration of dye results.
Consequently, a proportional increase in the absorbance at the respective λmax is observed with increasing
concentration of each drug.
The insitu generation of bromine is carried out using a mixture of potassium bromide and
potassium bromate in presence of 5 M HCl according to the
following equation:
5Br-
+ BrO3- + 6H+ 3Br2
+ 3H2O
3.1.
Absorption spectra
The resulting
absorption spectra are due to the red colour of
residual unoxidized methyl orange at 508 nm (fig 1)
or blue colour of residual unoxidized
methylene blue at 667 nm for lamotrigine
and minoxidil and at 747 for cefixime
as both minoxidil and lamotrigine
are analyzed in presence of 1.5 ml 5N HCL while cefixime
is analyzed in presence of 4 ml 5N HCl (Fig 3).
Fig 1: Absorption spectra of methyl orange in
presence of 5µg/ml Lamotrigine(A), 5µg/ml Minoxidil
(B) and 5µg/ml Cefixime (C).
Methylene blue
exhibited two types of absorption spectra according to different volumes
of hydrochloric acid. In low HCl concentration (1 ml 5N HCl
and complete to 10 ml) it exhibits peak maximum at 666 nm and in high HCl concentration (4 ml 5N HCl
and complete to 10 ml) it exhibits peak maximum at 745 nm. A detailed
investigation regarding different forms of methylene
blue was carried out(52) proving the reasons for variation in
absorption maximum at different acidities. Fig 2
Fig 2: Absorption spectra of methylene blue against blank in weak acidic medium (A) and in strong acidic medium (B)
Fig 3: Absorption spectra of methylene blue in presence of 20µg/ml Lamotrigine(A), 10µg/ml
Minoxidil(B) and 10µg/ml Cefixime
(C)
3.2. Effect
of dye concentration
Experiments were
performed to found appropriate dye concentrations by stabilizing other
experimental conditions and using different volumes of dyes and results
illustrated that increasing dye volume above 1ml in case of lamotrigine
and minoxidil does not result in change of absorption
while only 0.6 ml were sufficient in case of Cefixime.(Fig
4 and 5)
Fig.(4) Effect of methyl
orange concentration on absorption
intensity.
Fig.(5) Effect of methylene blue
concentration on absorption intensity.
3.3. Effect
of Acidity
Different acids
were tested as a medium for bromine generation including sulphuric
acid, hydrochloric acid, nitric acid and phosphoric acid. Hydrochloric acid
produced the most precise and accurate results. Therefore, 5 M HCl was used throughout experiments and it was found that
0.5-2 ml 5 M HCl is the appropriate acid volume for
determination of Lamotrigine and increasing HCl volume above 2 ml result in a rapid decrease in
absorption. While, 1.0-4.0 ml 5 M HCl is suitable for
Minoxidil analysis. And 2.0-5.0 ml 5 M HCl is the appropriate volume in case of Cefixime. And this indicates that the specified acid
concentration for bromination reaction was not
critical.
3.3. Effect
of time
Time required to
brominates and oxidize the drug before addition of dye and time required to
irreversibly oxidize dye after its addition was studied throughout different
experiments using both of dyes. It was found that the bromination
reaction was found to be complete in 5 minutes for lamotrigine
and minoxidil and in 15 minutes in case of cefixime and after oxidation time, contact times up to 60
min had been examined and no further bromination was
detected. A contact time of 5 minutes was necessary for the bleaching of the
dye colour by the residual bromine and the colour of both dye solutions remain stable for at least two
hours after mixing with the reaction mixture.
Method
validation
The developed
methods were validated according to international conference of harmonization
guidelines. (51) The
linearity range of absorbance as a function of drug concentration (Table 1) provide
an accurate measure of sensitivity of reagents used. Calibration curves have
correlation coefficients (r) higher than 0.999 indicating good linearity. The
accuracy of the methods were determined by investigating the recovery of drugs
at concentration levels covering the specified range (three replicates of each
concentration). The results showed excellent recoveries (table 2).Intraday
precision was evaluated by calculating standard deviation (SD) of five
replicate determinations using the same solution containing pure drug at three
different levels. The SD values revealed the high precision of the methods
(values vary from 0.89 to 1.12). For inter-day reproducibility on a day-to-day
basis, a series was run, in which the standard drug solution at three levels
was analyzed each for five days. The day-to-day SD values were in the range of
1.09-1.84. The limit of detection (LD) and limit of quantitation
(LQ) were calculated.