Extt-active Spectrophotometric Determination of Nortriptyline Hydrochloride Clsing Sudan 11 , IV and Black B

A simple spectrophotometric methods has been developed for the determination of nortriptyline h!~drochloride in pure and in pharmaceutical for-mulations based on the formation of ion-pair complexes I\ it11 sudun 11 (SI1); sudan (IV) (SI\;) and sudan black B (S13~). Tlie selectivity of the method was impr-o\.ed through extraction with chloroform. The optimum conditions for complete extracted colour development were assessed. The absorbance measurements were made at 534. 596 and 649 nm for Sit , SI\. and SBB complexes, respecti\;e!!.. The calibration graph was linear in the ranges 0.5280. 0.537.5 and 0.5 3 1.0 L I ~ ml'l of the drug usiny the same reagents, respectively. The precision of the procedure was checked by calculating the relative standard deiiation of ten replicate determinations on 15 pg ml-' of nortriptyline HCI and was found to be 1.7, 1.3 and 1 5 5 % using SII, SI1, and Ssu complexes, respectively. The niolar absorptivity and Sandell sensitivity for each ion-pair were calculated. The proposed methods were successfully applied to the deterniination of pure nortriptyline HCI and in pharmaceutical formulations, and the results demonstrated that the method is equally accurate, precise and reproducible as the official method.

~~liarmacopoeia"'.The bulk drug is estimated by non-aqueous titration with perchloric acid usins I -naphtholbenzein solution as indicator, whereas the ultra violet method was used ti,r its formulations.Nortriptyline hydrochloride is commonly determifled by hi yli pel-li,~-~nance liquid chromatography (2).Visible spectrophototnetric method have also been developed for the determination of this drug using light green and orange 11"'.
The other methods include capillary electrochromatography'l' elect]-ospray ionization mass spectrometry"', colorimetry'2." and electrometry '7-14' However, these methods are subjected to interferences from common additives and excipients The purpose of this work was to develop a simple spectrophotometric 11iethod for the determination of nortriptyline in pure and in pharmaceut~cal formulntions using sudan 11, IV and black B as the coloured reagents Solvent extraction was used to improve the selectivity of the method The drug contents in pharmaceutical forn~ulations were successfully determined by the proposed methods using the standard addition method.

Appcr~zlt~ I S
The spectra were recorded with a Perkin Elmer Lambada 3B recording double beam spectrophotometer, attached with 10.0 mm quartz cells was used for the absorbance measurements during the application of the proposed method under the optimum conditions.The pH measurements were made with an Orion research model Aliquots of the drug solutions representing 5.00-375 pg of nortriptyline HCI were tl-ansferred into a series of separating funnels, 5.0 ml of pH 2.34, 3.76 and 3.08 buffer solution for ion-pairs of SII, SI\. and Sos, respectively and 3 0 ml of 10" M reagent solution were added to each separating funnel.The total volume of the aqueous phase was made upto 10 ml with water.10 ml of chloroform were added and the contents were shaken for 3 0 win atid allowed to stand for clear separation of the two layers.The chloroformic layer was passed through anhydrous sodium sulphate and the absorbance was measured at 534, 596 and 649 nm for Srl, SI\. and SBB, respectively, against a blank sample that had been treated equally.

A z~a/)~sis qj'phnr-~nnce ~riica( fonn ulations
Bulked samples were prepared by weighing 20 tablets and finding the average weight of one tablet.An amount of the bulked sample equivalent to 25 mg of the active constituents was weighed accurately and transferred to a 100 ml calibrated flask, shaken with 50 ml of water.Thenfiltered if necessary and diluted to volume with water.An aliquot of this solution containing 100 pg of the analyte was transferred to a separating hnnel and analysed as shown in the General Procedure applying the standard addition technique.

Results and Discussion
O/~/in~i.s~tr/iorrc?f'concl'iiion.s.for.ah,sorhmlce mens~r~.c.n~en~.s The proposed methods are based on the formation of ionpair complexes as a result of the reaction between nortriptyline HCI with SII, SI\. or S H ~ reagents, which give red, pink or deep red coloured products, respectively.A solvent is needed to extract the ion-pair formed to increase the selectivity of the determination.
Preliminary experiments with a number of organic solvents commonly used (1,2- dichloroethane, benzene, hexane, diethyl ether, chloroform, carbon tetrachloride) for solvent extraction showed that 1,2-dichloroethane and chloroform are useful solvents for all ion-pail-complexes.The latter was selected because of its slightly higher sensitivity and considerably lower extraction ability for the reagents.Experiments were performed in which the volume of chloroSor~n was kept constant at 10 ml, while that of the aqueous phase was varied between 2.5-25 n~l.
keeping the reagent content constant.The absorbance decreases on increasing the aqueous to chloroform phasevolume ratio.However, a ratio of 1 : 1 was chosen so as to cornprolnise between maximum absorbance and a colourless blank; thus a linear relationship between absorbance and concentration could be achieved.The molar ratio and Jab's continuous variation methods established the stoichiometry of the ion-pairs.The results obtained with these methods showed that the composition. of the ion-pair complexes were (1 : 1) (drug .reagent).The shape of the curves indicated that the complexes were labile.Hence a large excess of reagents must ;:.iways be used to enhance the formation of the co~nplex.The loparithn~ic stability constants of the ion-pair were calculated by applying the Halve!! and Manning equation to the data obtained from the molar-ratio and Jab's continuous variation methods.The results of pK values \\ere 7.05, 9 38 and 8.06 sing SII, Sl\ and S13r3, respectively.

Ah.so~p/iorr . ~I C > C I I I C I
The absorption spectra of the reaction products were recorded in the range of 400 -750 nm.Characteristic bands in the visible spectrum at A,,,,;,, 534, 596 and 649 nm for SI1.SI\. and SRHnortriptyline complexes were observed, whereas the absorbance of the drug or all reagent have a negligible absorbance at or near the A,,,,;,, regions.This absorption band was ascribed to the ion-pair complex formation.

( ' L I / I / ~ ;.! l / i o ~ I , ~I -o ~/ I . s
Under-the experimental conditions described above, linear calibration graphs were obtained over the range 0.5-28.0.0.5-37.5 and 0.5 -3 1 0 pg /ml of nortriptyl~ne HCl using SII, SI\.and SBB, respectively.For more accurate results, hnybom optirnum concentration ranges were calculated and recorded in Table ( 1 ) .The molar absorptivity and Sandell sensitivity were also recorded in Table (1).Ten replicate determinations on a standard solution that contained 15 pg 1111-I of nortriptyline HCI showed relative standard deviation of 1.7, 1.3 and 1.55 O / O for SII, SIV and S e ~.
respectively.The standard deviation of the absorbance measurements was 0.0026, 0.003 1 and 0.0029 obtained from a series of 13 blank solution.The limits of detection (K=3 ' and of determination (K= fO) of the method were established according to the TUPAC definitions (C1= KSJs where C 1 is the limits of detection, So is the standard error of blank determination, s is the slope ofthe standard curve and K is the constant related to the confidence interval)"" and the values were recorded in Table ( I ) The correlation coefficient and the factors a and b of the regression line equation: A = a + bC, for the reagents examined are summarized in Table (1).In order .todetermine the accuracy and precision of the recommended procedures, solutions containing five different concentrations of nortriplyline HCI were prepared and five absorbance measurements were made on each reaction product obtained according to the proposed method using S I ~, Sl\-and SHH.The results are given in Table 2.The over-all relative standard derivation of these 75 determinations was 1.42 % and the mean standard analytical error (SD/ n ) was 0 293 'I'irblc ( 2 ) .Acc~11.acy and I'recision of ?he proposed procedul-e ----. .

SII SIV Su15
--  As can be seen from the results presented, most of the excipients, additi\.esand degradation products normally present in pharmaceutical formulations do not interfere with the determination o f nortriptyline.The proposed method was applied to the determination of this drug in its dosage forms applying the standard addirion technique.The results are shown in Table 3 together with those obtained by the official method"' (depending on non-aqueous titration with 0.1 M perchloric acid using 1 -naptholbenzein solution as indicator).Moreover, in order to detect any losses of nortriptyline, the standard additions method was also used.showing the absence of a systematic error in the method, as the corresponding tabulated t-and Fvalues for five degrees of freedom at 95% confidence le\.elI;', Therefore, it can be concluded that there is no significant difference between the proposed method and the official procedures.Table 3 gives the results of rhese applications

60 1 A
/ digital ionalyzer pH-meter./?etrgen/.sLII ILJ M~~tericrl.~De-ionized, freshly double distilled water \vas used throughout.All reagents were of analytical grade.Stock solutions of ~X I O -' M of sudan I \ , IV and black U (Aldricll p ~d u c t ) wc1.e prepared by dissolving the appropriate weight of the pure a~~alytical reagent grade in least amount of acetone in a 100 ml calibrated flask and co~nplete to the mark with the same solvent Nortriptyline HCI stock standard solution, 250 big nil-', freshly prepared by dissolving 25 nig of pure drug (ICN Chemicals) in water in a 100-ml calibrated flask and diluted to the mark with water.Working solutions were freshly prepared by appropriate dilution with water.Acetate buffer solutions of pH values 1.63-5.66were prepared by mixing appropriate volume of 1.0 M hydrochloric or acetic acids with 1.0 M sodium acetate as reco~nmended"~).(;el ~L~I.CI/ ~I -~L * C ~/ I T C ' S

Fig (I ) :
Fig (I ): Effect of pH on the absorbance of 20 pg ml" nortriptylin complexed with 3 x 10% slI' S I , and SR13

Fig. ( 2 )
Fig.(2) shows the effect of reagent concentration i~sed.At levels of about 3.0 ml of lo-' M , the highest constant absorbance was obtained for the extracted ion-pair complexes.So this concentration was used for all further studies.hak kin^ i ~n e s ranging from 0.5 to 5.0 rnin did not produce any change in the colour intensity and so 3.0 min shaking time was selected.Reproducible absorbance readings were obtained after a single extraction with 10 ml of chlorofor~n.Repeate:d extraction did not show an increase in the recovery percent results.

**
Mean RSD = 1.42% *** Mean standard analytical error = 0.293 The interference 15 kinds of drugs and excipients on the determination of nortriptyline were examined.The drugs tested were added individually to a solution containing 15 pg ml-' of notriptyline HCI.A maximuni error of + 3.0 % in the absorbance reading was considered tolerable.The tolerance limits of galactose.glucose, lactose, sucrose, saccharin, citric acid, magnesiuni stearate, gum acacia, starch, sodium phosphate, Vc, V..\, VI312,Vrl and fluphenazinc whicb usually present in ~iortriptyline formulations do not interfere up to 250 fold molar excess The interference of the degradate products results from thermal and hydrolatic treatment with strong acid or base do not occurs up to 100 fold excess.So this drug can be determined in pure and in pharmaceutical formulations in routine analysis without interference from excipients, additives and degradation products.AI.ILI/~;.SI.Y ( ?~' ~. ' / ~~I I " M I ~C L ' I I J / C .~~/ fol"~?111/CI/lo~7,~ The et'fect of pH was examined by varying the pH values from 1.63 to 5.66 acetate buffer solutions and the results are shown in Fig (1).Maximum and constant absorbance values were obtained in the pH range 2.11-2.73,3.51-4.04and 2.78-3.38 for SII, Slv and Sno complexes, respectively.Outside these pH rar'ges, the absorbance decl-eased rapidly.Experimentally, it was found that more stable, more reproducible and faster development of the colour was achieved at pH 2.34, 3.76 and 3 .O8 for SI1, S 1" and SBB complexes, respectively.

Table ( 1
): Spectral, sensitivity and calibl-ation graph cliaracteristics of the analyte solutions.All the values are means of six determinations.

Table ( 3
). Determination of nortriptyline HCl in different pharmaceutical formulations using SII, SIv and SBB reagents.Myers Squibb Egypt Contain 10 mg per tablet The performance of the recommended method was assessed by calculation of t-and F-values.A mean values o f Student's t-and Fvalues were obtained (Table3),