Ion-pair Spectrophotometric Determination of Dibucaine

Ragi M. Shubietah*", Ali Z.Abu 2uhrib and Bashar A. JShalidb a: Faculty of Pharmacy, b: Department of Postgraduate Studies; An-Najah National University, P.O.Box 7, Nablus, Palestine, via Israel Abstract: A simple and sensitive spectrophotometric determination of dibucain has been established. It is based on the formation of colored ion-pair complexes between Dibucaine and each of Orange 11, Orange G, Bromothymol blue and Bromocresol green. The extract with each of the previous dyes in chlorofonn exhibited a specific wavelength of maximum absorbance, and all these wavelengths lied in the range 41 6 to 500 nm. The linear range extended from 1.5 to 60 ppm. The optimum conditions were selected after studying many variables such as pH, shaking time, temperature and dye concentrations. The method was also selective for the analyte and the drug excipients did not interfere.

This drug is about 15 times more potent than procaine and 5 times more potent than cocaine in producing local anesthesia, but it is also considerably more poisonous (4).It is used for temporary relief of painful sunburn, minor burns, scrapes, scratches, nonpoisonous insect bites, external hemorrhoidal pain, and as an injectable preparation for spinal anestl~esia'~~~'.Dibucaine is readily absorbed through the mucous membranes and should not be used around the eyes or mouth.David et a1 reported a new method for the determination of dibucaine in biological fluids using gas cliromatography/mass spectrometer.The detection limit was in the range of 1-80 ng/mL of serum.Igrashi, et nl  Moha~lled el a1 (I0' reported a spectrophotornetric method for the determination of Dibucaine and eight related drugs.Beer's law was obeyed for 5-30 pg/mL Dibucaine and the detection limit was in the r a g e 1-3.7 pg Iml.The aim of our work here is to develop a new spectrophotomctric method for the determination of Di bucaine in pl~arn~aceutical formulat ions.It was bnscd on the for~natioll of ion-pairs between Dibucaine and the positively charged dyes (e.g.BTB, BCG, OrgG and Org 11); the produced colored ion-pair can be extracted to an organic phase and determined spectrophotometrically.The sensitivity and selectivity of the suggested method was compared with other previously reported methods.

Experimental
Absorbance measurements were carried out using a UV-2 UNICAM UV-VIS spectrophotometer with I-cm quartz cells.The pH measurements were carried out using a (HANNA, model HI 8424) p1-I-meter.The temperature was controlled using Techne, circulator v-85 A .Dibucaine, Procaine, Prilocaine, Prilocainamide, Orange 11, and Orange G were obtained from SIGMA company.Bromothymol blue (BTB) and bromocresol green (BCG) were obtained from BDH.All materials were used without further purification.

Diblrraine stock solutio17 (1.0~1 o-~M..
A 1 .0 ~1 0 " ~ standard aqueous solution of Dibucaine was freshly prepared by dissolving an appropriate amount of pure drug (exactly 0.0379 g Dibucaine) in I00 ml of doubly distilled water in 100-mI volumetric flask.All solutions were prepared weekly and stored in the refrigerator.
Orange 1 1 and oi.ange G sohrtions ( I .Oh 1 o-'M) Dye solutions were prepared by dissolving exactly 0.03503g and 0.04524g of orange I1 and orange G, respectively in a 100-ml doubly distilled water.The stock solutions were freshly prepared every two weeks.
A 1 .0 ~1 0 " ~ of each BTB and BCG solutions were prepared by dissolving exactly 0.0624 g and 0.0698 g of BTB and BCG, respectively in 2 ml of 0.1 M sodium hydroxide, followed by the addition of 20 ml ethanol (96%) then the volume was completed to 100 ml using doubly distilled water.

Recommended procedure
A measured amount of dye

Effect of pH and solvents
The effect of pH of the aqueous phase on the extraction efficiency of Dibucaine ion-pairs were studied in the pH range 1.7-1 0.0 for solutions prepared as described in the general procedure.The results obtained indicated that the absorbance of the organic phase for all the systems was maximum within the pH range 1.7 -4.0.
A further increase in the pH affected a remarkable decrease in the absorbance.
Dibucaine ion-pair complexes with OrgII, OrgG, BTB and BCG showed absorption maxima at 486, 500,418 and 41 8 nm, respectively.In the present work, pH 1.75 was selected as optimum for further work when using OrgII and OrgG, and pH 3.00 when using BTB and BCG.
In the proposed methods, chloroform was found to be the

Beer's Imu n~zd sensitivity
The calibration graphs were plotted under the optimum conditions recornmended in the general procedure.The graphs obey Beer's law (Linear relationship between the absorbance and the Dibucaine concentration) over the concentration range 0.8 -15 ppm, 3.7-Glppm, 1.5-17ppn1 and 1.5 -1 8 ppm for Orange 11, Orange G, BTB, and BCG systems, respectively.Fig 1 shows this data for orange I1 dye complex with Dibucaine.The Molar absorptivities (E) were calculated from the linear part of the curve and found to be 2 .0 ~ 10' L mol-' cm-I, 5 7 x 10' L mol-' cm", 1 6 x 10' L mol-' cm-I, and 2 . 2 ~ 1 O'L mol-' cm" for Orange 11, Orange G. BTB, and BCG systems, respectively.The relative standard deviations (RSD) were calculated for each of the above ion-pair complexes, and found to be 0.71% and 0.65% for Orange I1 and Orange G, respectively, and 0.55 % for BTB and BCG These values indicated the ~o o d reproducibility of the proposed methods The con~position of the ion-pair con~plexes between Dibucaine and the investigated dyes were determined using the molar ratio method(").In this method, a series of solutions containing different concentrations of the dye were prepared.The concentration of Dibucaine and all other conditions were kept constant.The absorbance of tlle fornled cornplexes were measured at A, , , for each cornplex and plotted versus the mole ratio dye1Dibucaine.Figure 2 indicates that the ion-pair con~plexes have a 1: 1 stoichiometric ratio.

Table (2)
Analytical characteristics for the ion-pair formation methods

Parameter
Org I1 method Org G method BTB method BCG method Amount of buffer (ml)

Conlpnrisoit o f the proposed nretllod with otlrer pirblislted rnetllods
Very few spectropl~otometric and chromatographic methods and only a single electrochemical method have been reported for the determination of Dibucaine in pharmaceutical formulations.It was found that the suggested method is highly sensitive and competes well with other methods.Table 3 shows comparison between the proposed method and other reported methods.

Determination of Dibucaine in Sheriproct Ointment
An oi~lt~nent called Sheriproct, manufactured in Madrid-Spain, was obtained and analyzed as mentioned in the recommended procedure.OrgII dye was used at pH 1.75.
After extraction with 25 ml of chloroform, a solution of this ointment containing 30 p g Dibucainelml.The absorbance was then measured at 486 nm versus a similar preparation without ointment (blank).From the calibration graph, it was observed that the assay has a minimum of 93% and a maximum of 96% of the labeled amount.The RSD for six different n~easurements at the same level was about 1.5%.

Coilclrrsiorr
The use of the proposed method for quantitative determination of Dibucaine allows a selective and quantitative analysis.The results also showed that there is no significant interference from many of the excipients and similar drugs from the same family.The method is applicable to the analysis of trace amounts of Dibucaine in pharmaceutical formulations with a satisfactory reproducibility.Figure 2

( 7 '
reported a method for determination of Dibucaine and its ~netabolites in human urine by high performance liquid chromatography with fluorescence detector.Other worker~'~'re~orted an HPLC method also for the drug determination in serum.A single method has been reported in the literature for the electrochemical determination of Dibucaine by its reduction on a mercury electrode(1 o -~ to 1 o -~M ) 191.
( 4 .0 ~ I O -~M in the case of orange 11, BTB and BCG, and 8 .0 ~ IO"M in the case of orange G) was transferred into a 100-ml separato~y funnel, then 5ml of BR-buffer solution of the desired pH were added.An accurate amount of solution containing Dibucaine in the range 1.5-60 pg/nll was added and followed by 20 in1 of organic solvent.The mixture was shaken vigorously for 30 -60 s and allowed separating into two phases.The organic phase was collected in a 25-1111 volumetric flask and the volunle was completed to the mark with the organic solvent.Blank solution was prepared at the same time under the same conditions.The absorbance of the formed co~nplex was measured at 486, 500, 4 18 and 4 16 nm wavelengtl~s for orange 11, orange G, BTB and BCG, respectively.The amount of Di bucainc was detern~i~~ed from the already constructed calibration curves.Ion-pair Spectrophotometric Determination of Dibucaine most suitable for extraction of Dibucaine-OrgII and Dibucaine-OrgG ion-pairs, while dichloromethane was found to be the most suitable for extraction of Dibucaine-BTB and Dibucaine-BCG ion-pairs.A single extraction for 60 s with 30 ml organic solvent was found to have excellent extraction efficiency.BR buffer gave maximum absorbance and was chosen as optimum.The stability of the ion-pair complexes in the organic layer was determined by measuring the absorbance ovel a period of time.The colour of the Dibucaine-dye ion-pair developed instantly and the intensity remained almost constant for at least 120 hours.

R. M. S h u b i e t a h et alFigure 1
Figure1

Table (
Effect of type of organic solvent on the percent of extraction of Dibucaine in presence of OrgII, OrgG, BTB, and BCG dyes at the optimum conditions.