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Ion-association method for the colorimetric determination of neomycin sulphate in pure and dosage forms
Spectrochimica Acta Part A 59 (2003) 663 /670 www.elsevier.com/locate/saa
Ion-association method for the colorimetric determination of neomycin sulphate in pure and dosage forms A.S. Amin a,*, Y.M. Issa b a
Chemistry Department, Faculty of Science, Benha University, Benha, Egypt Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt
b
Received 28 January 2002; accepted 20 April 2002
Abstract A simple, fairly rapid, sensitive and accurate method is described for the colorimetric determination of neomycin sulphate (NMS), based on the measurement of the absorbance of the extracted organic soluble ion-association complex formed between neomycin dictation and a bulky counter anion. Different chromotropic acid azo dyes were examined as counter ions. The effect of pH, the counter ion concentration, sequence of addition and solvents for extraction were also illustrated. The most suitable system is based on reagent VIII (pH 7.5) with chloroform as the extraction solvent. The use of other counter ions, in conjunction with their respective solvents, was found to be less sensitive. The neomycinreagent VIII system exhibits negligible or no interference when used for the determination of up to 58 mg ml 1 of NMS in the presence of several drug excipiences. The method has been used for the determination of up to 58 mg ml 1 with a good recovery (99.89/1.5%), and the precision is supported by the low relative standard deviation 0/1.35%. The sensitivity is discussed and the results are compared with the official method. The proposed method was applied successfully to the determination of NMS in pure and dosage forms, with a good precision and accuracy compared to the official one. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neomycin sulphate; Chromotropic acid azo dyes; Colorimetric determination; Pharmaceutical analysis
1. Introduction Neomycin sulphate (NMS) [1405-10-3] is a broad-spectrum antibiotic used in gastrointestinal infections, mainly in child diarrhea caused by suspensions and eye drops mixed with pharmaceutical agents such as kaolin, pectin and alumi-
* Corresponding author E-mail address: [email protected] (A.S. Amin).
nium hydroxide. These substances are utilized as antiflatulent, antiacid and antidiarrheal [1,2]. They absorb bacteria and toxins responsible for the onset of diarrhea, vomiting, nausea and cramps in various intestinal infections and coating the inflamed mucous membrane of the intestinal tract [3]. However, these excipients are capable of absorbing various antibiotics such as neomycin [3,4]. Various procedures [5,6] were suggested to the use of buffers to increase the liberated of adsorbed neomycin. On the other hand Harris [6]
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demonstrated that the interaction between neomycin and pectin may be inhibited by the addition of electrolytes. Therefore, an attempt was made to establish the optimum conditions for the release of the adsorbed neomycin and for its quantification by spectrophotometric [7 /9], flourometric [10], liquid chromatographic [11 /13] and high performance liquid chromatographic [14 /18] methods. In the present work, the use of bulky chromotropic acid azo dyes as anionic counter ions for the formation of neomycin ion-association complexes whose absorbance can be monitored upto extraction was investigated. The system was further optimized with respect to pH, counter ion concentration, sequence of addition, choice of solvent and shaking time. Key analytical characteristics of the complexes were compared. Determination of NMS in the presence of common drug, excipients was also performed.
2. Experimental 2.1. Apparatus All absorbance measurements were made on a Perkin/Elmer Lambda 3B UV /Visible spectrophotometer with matched quartz cuvettes of 10 mm path length. pH measurements of the aqueous phase were performed using an Orion research model 601 A/digital ionalyzer pH-meter. 2.2. Reagents and solutions All chemicals were of analytical-reagent grade and all solutions were freshly prepared in bidistilled water. The chromotropic acid azo dyes used were either monoazo (I /V) or bisazo (VI/VIII) compounds:
where the substituents X are as follows: p -SO2NH2
(I), o-Cl (II), p -Cl (III), p -Br(IV), p -I (V), bis p-Cl (VI), bis p -Br (VII) and bis p-I (VIII). These azo dyes were prepared with a previously recommended method [19]. 2 /10 3 M of the reagent stock solutions were prepared by dissolving the required amount of the reagent in water. Phosphate buffer solutions of different pH value 4.0 /9.0 were prepared according to the previously recommended method [20]. NMS was obtained from the Egyptian International Pharmaceutical Industries Company (EIPICO). An accurately weighed 100 mg of NMS was dissolved in water and made up to 100 ml with water in a calibrated flask to give a stock solution of 1.0 mg ml 1. The stock solution was further diluted stepwise with water to achieve the working standard solutions. 2.3. General procedure Volumes of 2.0 ml of each counter ion (chromotropic acid azo dyes) and 5.0 ml of optimum buffer solution (Table 1) were transferred into a series of 50 ml separating funnels then, aliquots of NMS containing up to 580 mg were added. The total volume of the aqueous phase was adjusted to 10 ml by the addition of water. Finally, 10 ml of extraction solvent were added to each funnel and the contents were shaken vigorously for 5.0 min and then allowed to stand for a few minutes until the two phases had completely separated. The absorbance of the separated organic layer was measured at the respective lmax (Table 1) against a reagent blank prepared similarly. At least duplicate measurements were made in all cases. 2.4. Procedure for tablets The contents of twenty tablets of NMS were crushed, powdered, weighed out and the average weight of one tablet was determined. An accurate weight equivalent to 100 mg NMS was dissolved in 20 ml distilled water then filtered. The filtrate was diluted to 100 ml with distilled water in a 100 ml calibrated flask to give 1.0 mg ml 1 stock solution. This solution was further diluted stepwise to the request concentration with water and then analysed as described under the general procedure.
Ionassociation
I-NMS II-NMS III-NMS IV-NMS V-NMS VI-NMS VII-NMS VIII-NMS a b c d e f
pH
6.5 8.0 7.5 6.0 7.5 7.0 7.5 7.5
lmax
561 546 670 608 610 658 666 607
Beer’s mg ml 1
0.4 /51 0.4 /46 0.4 /43 0.4 /41 0.4 /45 0.4 /52 0.4 /50 0.4 /58
Ringbom mg ml 1
1.6 /48 2.0 /43 2.0 /40 1.6 /38 2.0 /42 1.6 /50 2.0 /47 1.6 /55
D.L.a mg ml 1
0.08 0.10 0.08 0.07 0.10 0.08 0.06 0.04
o b /103
8.25 6.67 6.15 5.53 7.02 9.31 7.82 10.36
S.S.c mg cm 2
0.074 0.092 0.100 0.111 0.088 0.066 0.079 0.059
Quantitative parameterd a
b
r
0.008 /0.011 /0.009 0.013 /0.007 0.010 0.009 /0.012
0.013 0.011 0.010 0.009 0.012 0.015 0.013 0.017
0.9996 0.9992 0.9994 0.9990 0.9995 0.9998 0.9993 0.9998
The detection limit. Molar absorptivity, l mol 1 cm 1. Sandell sensitivity. Regression equation A /a/bC , where C is the concentration in mg ml 1. Theoretical values for t - and F -tests at five degrees of freedom and 95% confidence limits are 2.57 and 5.05, respectively. Average of six determinations.
t evalue
F e-test
Error %
RSDf %
1.28 1.05 1.46 1.18 1.34 1.51 1.20 1.09
2.41 2.13 2.71 2.27 2.59 2.82 2.44 2.20
9/1.2 9/0.9 9/0.8 9/1.4 9/1.0 9/0.7 9/1.3 9/1.1
0.93 1.16 0.84 1.19 1.07 0.78 0.95 1.02
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Table 1 Spectral characteristics, accuracy and precision of the ion-association formed with counter ions (I /VIII)
665
666
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2.5. Procedure for eye drops The contents of 5.0 bottles of eye drops were mixed and the average volume of one bottle was determined. An accurate volume equivalent to 5.0 mg of NMS was diluted with water in a 50-ml calibrated flask and completed to the mark with water. This solution was further diluted stepwise to the requisite concentration with distilled water and analysed as described under the general procedure described above. 2.6. Microbiological method The microbiological assay by agar diffusion was carried out according to the USP XXIV [21]. The extraction liquid was 0.1 M phosphate buffer solution of pH 8.0.
3. Results and discussion A preliminary investigation of the background absorption of 1.0 ml of 2 /10 3 M counter ion solution (without NMS) was conducted. The characteristics of the extract reveals that these reagents have high background absorption especially when chloroform, dichloromethane or methyl isobutyl ketone was used as the extracting solvent. For systems that are highly colored, no further investigations were conducted. The optimum conditions for the development of colorimetric procedures were established by varying the parameters one at a time and observing the effect produced on the absorbance of the colored product [22]. 3.1. Choice of organic solvent Toluene, methylene chloride, methyl isobutyl ketone, chloroform, benzene, dichloromethane, dichloro ethane, and carbontetrachloride were used as solvents in the extraction. The absorbances of 35 mg ml 1 NMS, corrected for the corresponding blanks, for counter ions (I /VIII) systems are shown in Fig. 1. It is evident that the most suitable solvent for counter ions I/V is chloroform, whereas for reagents VI /VIII systems dichloro
Fig. 1. Absorption spectra of 35 mg ml 1 of NMS ionassociation using counter ion (I /VIII).
methane and chloroform seem to be promising solvents. The chosen of chloroform for all further studies was due to the more stability of extracted colored product (1.5 h) compared with dichloro methane (30 min). 3.2. Effect of pH The pH of the aqueous phase is an extremely important factor for ion-associations [23 /25]. Here the influence of pH on the extraction was evaluated by measuring the absorbance of the ionassociate when 35 mg ml 1 of NMS was used, and that of the corresponding blanks over the pH range 2.0 /12.0, by the addition of the appropriate buffers (acetate, phosphate, universal and thiel buffer). In general, phosphate buffer solution is the best one of them and the optimum pH value for each system was optimized and recorded in Table 1. Moreover, the optimum volume of buffer solution was found to be 5.0 ml in 10 ml aqueous phase. 3.3. Counter-ion concentration The influence of the respective counter-ion concentrations on the absorbance of the extracted ion-associate was studied by fixing the NMS concentration at 35 mg ml 1 and varying the
A.S. Amin, Y.M. Issa / Spectrochimica Acta Part A 59 (2003) 663 /670
Fig. 2. Effect of counter ion of I /VIII on 35 mg ml 1 of NMS at the optimum pH, solvent, shaking time, absorbance measured at the respective lmax.
concentration of the reagent from 0.4 /6.0 mM. The results are plotted in Fig. 2, which shows that the signal remains constant at counter-ion concentration E/3.8 mM for all systems. A fixed concentration of 4.0 mM counter-ion was subsequently used for all systems in order to minimize the blank absorbance; also, no further improvement in precision was observed for concentrations of counter ion higher than 4.0 mM. 3.4. Effect of shaking time The extraction was studied by shaking different samples on a shaker and varying the shaking time from 2.0 to 10 mins for the complexes based on 35 mg ml 1 of NMS. It was found that the absorbance remained constant over this time period for all systems. A shaking time of 5.0 min was adopted for all extractions. It was further observed that the violet extracts remained stable for at least 1.5 h. 3.5. Chemistry of the colored products The formation of the extracted violet colored ion-associate complexes is based on the basic nature of the drug, which react under specified experimental conditions form ion-associate com-
667
Fig. 3. Continuous variation plots for NMS ion-associated with counter ion (I /VIII).
plexes with chromotropic acid azo dyes. The formed complexes are extractable into chloroform to obtain a soluble ion-association. The molar ratio of NMS to counter-ion was determined with the molar ratio and continuous variation methods and found to be 1:1 using counter ions I, II, III, IV, and V, whereas 2:1 ion-associate was formed for VI,VII, and VIII. The shapes of the resulting curves indicated that the ion-associates are labile. Consequently, a large excess of reagent must be always used to enhance the formation of the complex (Fig. 3). 3.6. Quantification Using 9/12 points, each point being repeated at least twice, generated the calibration graphs. The day-to-day variation in the calibration graph was found to be within acceptable limits, as well as the repetitive determination of standard solutions carried out over a period of several days, where a relative standard deviation (RSD) of not more than 1.35% was obtained. The linearity of the calibration graphs was studied under the above conditions for the respective counter ions. The analytical characteristics of the extraction are summarized in Table 1. The detection limit was
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Table 2 Effect of foreign compounds on the determination of 35 mg ml 1 NMS Foreign compounds
Kaolin, pectin Aluminium hydroxide Aluminium chlorohydroxide Sodium acetate Propylene glycol Poly vinyl alcohol Polymyxin B sulphate Hydrocortizone acetate Polysorbate Methylprednisolone acetate Bacitracin zinc Dexamethazone phosphate Benzalkonium chloride Starch Mixtureb,c a b c
Conc. mg ml 1
700 1000 900 600 500 1350 1500 800 1100 500 350 700 500 1500
NMS recovered (%)a I
II
III
IV
V
VI
VII
VIII
97.2 102.8 103.5 103.0 96.5 102.7 96.5 97.0 103.5 104.0 96.5 103.5 97.5 103.0 103.0
98.0 98.5 103.0 97.0 96.5 103.0 97.0 104.0 96.5 98.0 97.0 96.5 103.0 103.5 97.0
96.8 103.5 96.5 96.8 103.0 98.1 103.5 96.5 103.0 97.0 104.0 96.5 101.5 96.5 98.0
103.5 102.4 96.5 97.4 102.7 102.6 96.5 102.5 104.0 96.5 103.0 97.5 102.5 97.0 103.5
102.7 96.5 98.1 103.5 103.0 103.5 104.0 96.5 96.8 103.5 97.5 103.5 98.0 103.0 96.5
97.5 103.5 97.0 103.0 98.5 97.0 102.5 98.0 103.0 102.5 97.0 104.0 96.5 103.5 96.8
103.5 96.5 96.5 97.0 103.0 103.5 96.5 103.5 97.0 96.5 103.0 102.5 104.0 96.5 103.5
103.0 97.0 96.5 103.5 103.5 104.0 96.5 103.0 97.0 98.0 102.0 96.5 103.0 102.0 97.5
Mean n /3. 175 mg ml 1 each of the compounds in the first column. n /5.
calculated as described earlier [26] using the equation 3s/S where s is the standard deviation of the blank and S is the corresponding slope of the calibration graph. The upper and lower limits of the dynamic linear range of concentration were estimated from the calibration graph, noting when the points started to deviate from the straight line. For more accurate analysis, Ringbom optimum concentration ranges were determined by plotting the transmittance percentage vs the logarithmic values of NMS concentration in mg ml 1 The molar absorptivities of the ion-association systems obtained are comparable to each other pointing out that the most sensitive counter ion is reagent VIII. The reagent VIII based ion-association is not only the most sensitive but also exhibits the lowest detection limit and the widest dynamic linear concentration range (Table 1). In order to determine the accuracy and precision of the proposed procedures, solutions containing five different concentrations of NMS were prepared and analysed in quintuplicate. The relative errors and RSDs in Table 1 can be considered satisfactory, at least for the concentration levels
examined. The performance of the proposed procedures was assessed by comparison with the official method [21]. Mean values obtained in Student’s t- and F -test [27] showed the absence of any systematic error in the method (Table 1). 3.7. Interferences Interferences from some common drug excipients were examined and the results are given in Table 2. It was found that these compounds exhibit minimum interference, even when present at 50 times the level of NMS. Determination of 35 mg ml 1 NMS in a mixture containing 350 mg ml 1 each of kaolin, pectin, aluminium hydroxide, polyvinyl alcohol, sodium acetate, propylene glycol, benzalkonium chloride, polymyxin B sulphate, polysorbate, dexamethazone phosphate, hydrocortizone acetate, methylprednisolone acetate, aluminium chlorohydroxide complex, bacitracin-zinc and starch are satisfactory, with an RSD of 1.4% and a recovery of 96.5%. The RSD of six determination of 35 mg ml 1 NMS standard solution was 1.2%.
Table 3 Determination of NMS in different pharmaceutical formulations using counter ion (I /VIII) Lable claim
c
Neomycin tablets
500 mg
FML-Neod
5 mg ml 1
Predmycin-Pd
5 mg ml 1
Conjunctind
5 mg ml 1
Decadron Neomycind
3.5 mg ml 1
Neo-Cortefd
5 mg ml 1
Neo-Medrold
2.5 mg ml 1
a b c d
Founda (9/SD) I
II
III
IV
V
VI
VII
VIII
Off.
4969/0.72 t b /1.21 F b /1.78 4.979/0.56 t b /1.58 F b /2.70 5.059/0.62 t b /1.66 F b /3.10 5.079/0.50 t b /1.46 F b /2.60 3.529/0.74 t b /1.09 F b /1.65 4.939/0.48 t b /1.75 F b /2.99 2.519/0.67 t b /1.47 F b /2.18
4959/0.63 t b /1.42 F b /2.32 5.059/0.71 t b /1.17 F b /1.68 5.049/0.56 t b /1.03 F b /1.58 4.939/0.70 t b /1.89 F b /3.19 3.489/0.47 t b /1.82 F b /2.68 4.969/0.58 t b /1.35 F b /2.05 2.489/0.61 t b /1.71 F b /2.63
5039/0.52 t b /1.98 F b /3.41 5.039/0.52 t b /2.02 F b /3.13 4.949/0.76 t b /1.37 F b /2.01 4.979/0.62 t b /1.04 F b /1.73 3.479/0.45 t b /2.29 F b /4.46 5.069/0.70 t b /1.00 F b /1.41 2.479/0.63 t b /1.38 F b /2.47
4949/0.66 t b /1.33 F b /2.12 4.949/0.48 t b /2.10 F b /3.67 5.039/0.60 t b /1.49 F b /2.78 5.059/0.54 t b /1.74 F b /2.66 3.539/0.72 t b /1.10 F b /1.74 4.949/0.58 t b /1.28 F b /2.02 2.539/0.46 t b /2.32 F b /4.63
5059/0.58 t b /1.56 F b /2.73 5.0246 t b /2.22 F b /4.00 4.969/0.68 t b /1.41 F b /2.16 4.949/0.71 t b /1.11 F b /1.54 3.479/0.60 t b /1.31 F b /2.51 5.059/0.56 t b /1.34 F b /2.20 2.519/0.45 t b /2.18 F b /4.84
5049/0.76 t b /1.07 F b /1.60 4.9164 t b /1.30 F b /2.07 5.079/0.58 t b /1.72 F b /2.97 4.959/0.46 t b /2.05 F b /3.66 3.459/0.71 t b /1.13 F b /1.79 5.089/0.62 t b /1.12 F b /1.79 2.529/0.55 t b /2.02 F b /3.24
4939/0.82 t b /1.18 F b /1.37 4.9475 t b /1.07 F b /1.50 4.9466 t b /1.38 F b /2.30 5.049/0.58 t b /1.71 F b /2.32 3.499/0.45 t b /2.31 F b /4.43 4.939/0.69 t b /1.06 F b /1.45 2.479/0.60 t b /1.79 F b /2.72
5029/0.67 t b /1.26 F b /3.05 5.0649 t b /1.20 F b /1.49 5.0878 t b /1.17 F b /1.64 4.949/0.53 t b /1.64 F b /2.76 3.539/0.72 t b /1.15 F b /1.74 5.079/0.82 t b /0.98 F b /1.67 2.539/0.61 t b /1.39 F b /2.63
5119/0.97
5.1292
4.901.00
4.879/0.88
3.609/0.95
4.849/0.83
2.449/0.99
A.S. Amin, Y.M. Issa / Spectrochimica Acta Part A 59 (2003) 663 /670
Formulation
Average of six determinations. Theoretical values for t -and F -values for five degrees of freedom and 95% confidence limits are 2.57 and 5.05, respectively. Memphis Company for Pharmaceutical Industries, Egypt. Egyptian International Pharmaceutical Industries Company, Egypt.
669
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3.8. Analytical applications To establish the validity of the proposed procedures, pharmaceutical formulations were analysed using chromotropic azo dyes counter ions (I / VIII). The same batches were analysed by the USP [21] method and the results obtained were compared statistically utilizing Student’s t - and F tests. The present methods showed almost the same degree of accuracy and precise as the USP method. The proposed method have the advantage of being virtually free from interferences as well as having moderately low detection limits, and it may therefore be suitable for the determination of this drug in biological fluids using standard addition method (Table 3).
4. Conclusion Extractive colorimetric procedure for the determination of NMS was developed based on the formation of extractive ion-associate complexes with chromotropic acid azo dyes as counter ions. The proposed procedure is fairly rapid, simple, sensitive, precise, accurate and promises good prospects for the determination of NMS in pharmaceutical formulations. Additionally, the method uses cheaper procedures than the microbiological method. The chromotropic acid azo dyes are commercially available by many chemical companies.
References [1] L.S. Goodman, A. Gilman, The Pharmacological Basis of Therapeutics, Macmillan, New York, 1985, pp. 1150 / 1166.
[2] A. Wade, Martindale’s Extra Pharmacopeia, 27th ed., Pharmaceutical Press, London, 1978, p. 1135, 1155 /1160. [3] H.H. Zedan, M. Salama, W.M. Abdel-Radman, Egypt J. Pharm. Sci. 23 (1982) 19. [4] K. Florey, Analytical Profiles of Drug Substances, vol. 8, Academic Press, New York, 1979, pp. 339 /488. [5] F.S. Ghazy, A.A. Kassem, S.H. Shalaby, Pharmazie 39 (1984) 821. [6] W.A. Harris, Australas J. Pharm. 52 (1971) 569. [7] S.M. Abu-El-Wafa, M.A. El-Ries, F.M. Abou-Attai, R.M. Issa, Anal. Lett. 22 (1989) 13. [8] A.S. Amin, Spectrosc. Lett. 29 (1996) 1003. [9] M.R.C. Marques, E.R.M. Hackmann, T. Saito, Anal. Lett. 22 (1989) 621. [10] B. Gala, A. Gomez Hens, D. Perez bendito, Anal. Chim. Acta 347 (1995) 310. [11] E. Adams, R. Schepers, E. Roets, J. Hoogmartens, J. Chromatogr. 741 (1996) 233. [12] E. Adams, R. Schepers, L.W. Gathu, R. Kibaya, E. Roets, J. Hoogmartens, J. Pharm. Biomed. Anal. 15 (1997) 505. [13] M.H. Vega, G.M. Garica, E.R. Gesche, R.F. Saelzer, J. Planar Chomatogr. Mod. TLC 1 (1992) 62. [14] E. Roets, E. Adams, I.G. Muriithi, J. Hoogmartens, J. Chromatogr. 1 (1995) 131. [15] W. Funk, T. Kuepper, A. Wirtz, S. Netz, J. Planar Chromatogr. Mod. TLC 3 (1994) 10. [16] J.A. Reid, J.D. Macneil, J. AOAC. Int. 82 (1999) 61. [17] V.K. Agrawal, J. Liq. Chromatogr. 13 (1990) 2475. [18] B. Shaikh, J. Jachson, G. Guyer, W.R. Ravis, J. Chromatogr. Biomed. Appl. 109 (1991) 571. [19] A.S. Amin, Y.M. Issa, Anal. Lett. 30 (1997) 69. [20] H.T.S. Britton, Hydroden Ions, fourth ed., Chapman and Hall, London, 1952. [21] ‘United State Pharmacopoeia’, 24th ed., United States pharmacopoeial convention, Rockville, MD 2000. [22] D.L. Massart, B.G.M. Vandeginste, S.N. Deming, Y. Michotte, L. Kaufman, Chemometrics, A Text Book, Elsevier, Amsterdam, 1988. [23] S.L. Bhongade, A.V. Kasture, Talanta 40 (1993) 1525. [24] M. Oue, K. Kimura, T. Shono, Analyst 113 (1988) 551. [25] B. Saad, S.M. Sultan, F.E.O. Suliman, Talanta 44 (1997) 53. [26] C.M. Legua, P.C. Falco, A.S. Cabeza, Anal. Chem. Acta 283 (1993) 635. [27] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, third ed., Ellis Horwood, Chichester, 1993.
Ion-association method for the colorimetric determination of neomycin sulphate in pure and dosage forms A.S. Amin a,*, Y.M. Issa b a
Chemistry Department, Faculty of Science, Benha University, Benha, Egypt Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt
b
Received 28 January 2002; accepted 20 April 2002
Abstract A simple, fairly rapid, sensitive and accurate method is described for the colorimetric determination of neomycin sulphate (NMS), based on the measurement of the absorbance of the extracted organic soluble ion-association complex formed between neomycin dictation and a bulky counter anion. Different chromotropic acid azo dyes were examined as counter ions. The effect of pH, the counter ion concentration, sequence of addition and solvents for extraction were also illustrated. The most suitable system is based on reagent VIII (pH 7.5) with chloroform as the extraction solvent. The use of other counter ions, in conjunction with their respective solvents, was found to be less sensitive. The neomycinreagent VIII system exhibits negligible or no interference when used for the determination of up to 58 mg ml 1 of NMS in the presence of several drug excipiences. The method has been used for the determination of up to 58 mg ml 1 with a good recovery (99.89/1.5%), and the precision is supported by the low relative standard deviation 0/1.35%. The sensitivity is discussed and the results are compared with the official method. The proposed method was applied successfully to the determination of NMS in pure and dosage forms, with a good precision and accuracy compared to the official one. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Neomycin sulphate; Chromotropic acid azo dyes; Colorimetric determination; Pharmaceutical analysis
1. Introduction Neomycin sulphate (NMS) [1405-10-3] is a broad-spectrum antibiotic used in gastrointestinal infections, mainly in child diarrhea caused by suspensions and eye drops mixed with pharmaceutical agents such as kaolin, pectin and alumi-
* Corresponding author E-mail address: [email protected] (A.S. Amin).
nium hydroxide. These substances are utilized as antiflatulent, antiacid and antidiarrheal [1,2]. They absorb bacteria and toxins responsible for the onset of diarrhea, vomiting, nausea and cramps in various intestinal infections and coating the inflamed mucous membrane of the intestinal tract [3]. However, these excipients are capable of absorbing various antibiotics such as neomycin [3,4]. Various procedures [5,6] were suggested to the use of buffers to increase the liberated of adsorbed neomycin. On the other hand Harris [6]
1386-1425/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 0 2 ) 0 0 2 1 6 - 0
664
A.S. Amin, Y.M. Issa / Spectrochimica Acta Part A 59 (2003) 663 /670
demonstrated that the interaction between neomycin and pectin may be inhibited by the addition of electrolytes. Therefore, an attempt was made to establish the optimum conditions for the release of the adsorbed neomycin and for its quantification by spectrophotometric [7 /9], flourometric [10], liquid chromatographic [11 /13] and high performance liquid chromatographic [14 /18] methods. In the present work, the use of bulky chromotropic acid azo dyes as anionic counter ions for the formation of neomycin ion-association complexes whose absorbance can be monitored upto extraction was investigated. The system was further optimized with respect to pH, counter ion concentration, sequence of addition, choice of solvent and shaking time. Key analytical characteristics of the complexes were compared. Determination of NMS in the presence of common drug, excipients was also performed.
2. Experimental 2.1. Apparatus All absorbance measurements were made on a Perkin/Elmer Lambda 3B UV /Visible spectrophotometer with matched quartz cuvettes of 10 mm path length. pH measurements of the aqueous phase were performed using an Orion research model 601 A/digital ionalyzer pH-meter. 2.2. Reagents and solutions All chemicals were of analytical-reagent grade and all solutions were freshly prepared in bidistilled water. The chromotropic acid azo dyes used were either monoazo (I /V) or bisazo (VI/VIII) compounds:
where the substituents X are as follows: p -SO2NH2
(I), o-Cl (II), p -Cl (III), p -Br(IV), p -I (V), bis p-Cl (VI), bis p -Br (VII) and bis p-I (VIII). These azo dyes were prepared with a previously recommended method [19]. 2 /10 3 M of the reagent stock solutions were prepared by dissolving the required amount of the reagent in water. Phosphate buffer solutions of different pH value 4.0 /9.0 were prepared according to the previously recommended method [20]. NMS was obtained from the Egyptian International Pharmaceutical Industries Company (EIPICO). An accurately weighed 100 mg of NMS was dissolved in water and made up to 100 ml with water in a calibrated flask to give a stock solution of 1.0 mg ml 1. The stock solution was further diluted stepwise with water to achieve the working standard solutions. 2.3. General procedure Volumes of 2.0 ml of each counter ion (chromotropic acid azo dyes) and 5.0 ml of optimum buffer solution (Table 1) were transferred into a series of 50 ml separating funnels then, aliquots of NMS containing up to 580 mg were added. The total volume of the aqueous phase was adjusted to 10 ml by the addition of water. Finally, 10 ml of extraction solvent were added to each funnel and the contents were shaken vigorously for 5.0 min and then allowed to stand for a few minutes until the two phases had completely separated. The absorbance of the separated organic layer was measured at the respective lmax (Table 1) against a reagent blank prepared similarly. At least duplicate measurements were made in all cases. 2.4. Procedure for tablets The contents of twenty tablets of NMS were crushed, powdered, weighed out and the average weight of one tablet was determined. An accurate weight equivalent to 100 mg NMS was dissolved in 20 ml distilled water then filtered. The filtrate was diluted to 100 ml with distilled water in a 100 ml calibrated flask to give 1.0 mg ml 1 stock solution. This solution was further diluted stepwise to the request concentration with water and then analysed as described under the general procedure.
Ionassociation
I-NMS II-NMS III-NMS IV-NMS V-NMS VI-NMS VII-NMS VIII-NMS a b c d e f
pH
6.5 8.0 7.5 6.0 7.5 7.0 7.5 7.5
lmax
561 546 670 608 610 658 666 607
Beer’s mg ml 1
0.4 /51 0.4 /46 0.4 /43 0.4 /41 0.4 /45 0.4 /52 0.4 /50 0.4 /58
Ringbom mg ml 1
1.6 /48 2.0 /43 2.0 /40 1.6 /38 2.0 /42 1.6 /50 2.0 /47 1.6 /55
D.L.a mg ml 1
0.08 0.10 0.08 0.07 0.10 0.08 0.06 0.04
o b /103
8.25 6.67 6.15 5.53 7.02 9.31 7.82 10.36
S.S.c mg cm 2
0.074 0.092 0.100 0.111 0.088 0.066 0.079 0.059
Quantitative parameterd a
b
r
0.008 /0.011 /0.009 0.013 /0.007 0.010 0.009 /0.012
0.013 0.011 0.010 0.009 0.012 0.015 0.013 0.017
0.9996 0.9992 0.9994 0.9990 0.9995 0.9998 0.9993 0.9998
The detection limit. Molar absorptivity, l mol 1 cm 1. Sandell sensitivity. Regression equation A /a/bC , where C is the concentration in mg ml 1. Theoretical values for t - and F -tests at five degrees of freedom and 95% confidence limits are 2.57 and 5.05, respectively. Average of six determinations.
t evalue
F e-test
Error %
RSDf %
1.28 1.05 1.46 1.18 1.34 1.51 1.20 1.09
2.41 2.13 2.71 2.27 2.59 2.82 2.44 2.20
9/1.2 9/0.9 9/0.8 9/1.4 9/1.0 9/0.7 9/1.3 9/1.1
0.93 1.16 0.84 1.19 1.07 0.78 0.95 1.02
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Table 1 Spectral characteristics, accuracy and precision of the ion-association formed with counter ions (I /VIII)
665
666
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2.5. Procedure for eye drops The contents of 5.0 bottles of eye drops were mixed and the average volume of one bottle was determined. An accurate volume equivalent to 5.0 mg of NMS was diluted with water in a 50-ml calibrated flask and completed to the mark with water. This solution was further diluted stepwise to the requisite concentration with distilled water and analysed as described under the general procedure described above. 2.6. Microbiological method The microbiological assay by agar diffusion was carried out according to the USP XXIV [21]. The extraction liquid was 0.1 M phosphate buffer solution of pH 8.0.
3. Results and discussion A preliminary investigation of the background absorption of 1.0 ml of 2 /10 3 M counter ion solution (without NMS) was conducted. The characteristics of the extract reveals that these reagents have high background absorption especially when chloroform, dichloromethane or methyl isobutyl ketone was used as the extracting solvent. For systems that are highly colored, no further investigations were conducted. The optimum conditions for the development of colorimetric procedures were established by varying the parameters one at a time and observing the effect produced on the absorbance of the colored product [22]. 3.1. Choice of organic solvent Toluene, methylene chloride, methyl isobutyl ketone, chloroform, benzene, dichloromethane, dichloro ethane, and carbontetrachloride were used as solvents in the extraction. The absorbances of 35 mg ml 1 NMS, corrected for the corresponding blanks, for counter ions (I /VIII) systems are shown in Fig. 1. It is evident that the most suitable solvent for counter ions I/V is chloroform, whereas for reagents VI /VIII systems dichloro
Fig. 1. Absorption spectra of 35 mg ml 1 of NMS ionassociation using counter ion (I /VIII).
methane and chloroform seem to be promising solvents. The chosen of chloroform for all further studies was due to the more stability of extracted colored product (1.5 h) compared with dichloro methane (30 min). 3.2. Effect of pH The pH of the aqueous phase is an extremely important factor for ion-associations [23 /25]. Here the influence of pH on the extraction was evaluated by measuring the absorbance of the ionassociate when 35 mg ml 1 of NMS was used, and that of the corresponding blanks over the pH range 2.0 /12.0, by the addition of the appropriate buffers (acetate, phosphate, universal and thiel buffer). In general, phosphate buffer solution is the best one of them and the optimum pH value for each system was optimized and recorded in Table 1. Moreover, the optimum volume of buffer solution was found to be 5.0 ml in 10 ml aqueous phase. 3.3. Counter-ion concentration The influence of the respective counter-ion concentrations on the absorbance of the extracted ion-associate was studied by fixing the NMS concentration at 35 mg ml 1 and varying the
A.S. Amin, Y.M. Issa / Spectrochimica Acta Part A 59 (2003) 663 /670
Fig. 2. Effect of counter ion of I /VIII on 35 mg ml 1 of NMS at the optimum pH, solvent, shaking time, absorbance measured at the respective lmax.
concentration of the reagent from 0.4 /6.0 mM. The results are plotted in Fig. 2, which shows that the signal remains constant at counter-ion concentration E/3.8 mM for all systems. A fixed concentration of 4.0 mM counter-ion was subsequently used for all systems in order to minimize the blank absorbance; also, no further improvement in precision was observed for concentrations of counter ion higher than 4.0 mM. 3.4. Effect of shaking time The extraction was studied by shaking different samples on a shaker and varying the shaking time from 2.0 to 10 mins for the complexes based on 35 mg ml 1 of NMS. It was found that the absorbance remained constant over this time period for all systems. A shaking time of 5.0 min was adopted for all extractions. It was further observed that the violet extracts remained stable for at least 1.5 h. 3.5. Chemistry of the colored products The formation of the extracted violet colored ion-associate complexes is based on the basic nature of the drug, which react under specified experimental conditions form ion-associate com-
667
Fig. 3. Continuous variation plots for NMS ion-associated with counter ion (I /VIII).
plexes with chromotropic acid azo dyes. The formed complexes are extractable into chloroform to obtain a soluble ion-association. The molar ratio of NMS to counter-ion was determined with the molar ratio and continuous variation methods and found to be 1:1 using counter ions I, II, III, IV, and V, whereas 2:1 ion-associate was formed for VI,VII, and VIII. The shapes of the resulting curves indicated that the ion-associates are labile. Consequently, a large excess of reagent must be always used to enhance the formation of the complex (Fig. 3). 3.6. Quantification Using 9/12 points, each point being repeated at least twice, generated the calibration graphs. The day-to-day variation in the calibration graph was found to be within acceptable limits, as well as the repetitive determination of standard solutions carried out over a period of several days, where a relative standard deviation (RSD) of not more than 1.35% was obtained. The linearity of the calibration graphs was studied under the above conditions for the respective counter ions. The analytical characteristics of the extraction are summarized in Table 1. The detection limit was
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668
Table 2 Effect of foreign compounds on the determination of 35 mg ml 1 NMS Foreign compounds
Kaolin, pectin Aluminium hydroxide Aluminium chlorohydroxide Sodium acetate Propylene glycol Poly vinyl alcohol Polymyxin B sulphate Hydrocortizone acetate Polysorbate Methylprednisolone acetate Bacitracin zinc Dexamethazone phosphate Benzalkonium chloride Starch Mixtureb,c a b c
Conc. mg ml 1
700 1000 900 600 500 1350 1500 800 1100 500 350 700 500 1500
NMS recovered (%)a I
II
III
IV
V
VI
VII
VIII
97.2 102.8 103.5 103.0 96.5 102.7 96.5 97.0 103.5 104.0 96.5 103.5 97.5 103.0 103.0
98.0 98.5 103.0 97.0 96.5 103.0 97.0 104.0 96.5 98.0 97.0 96.5 103.0 103.5 97.0
96.8 103.5 96.5 96.8 103.0 98.1 103.5 96.5 103.0 97.0 104.0 96.5 101.5 96.5 98.0
103.5 102.4 96.5 97.4 102.7 102.6 96.5 102.5 104.0 96.5 103.0 97.5 102.5 97.0 103.5
102.7 96.5 98.1 103.5 103.0 103.5 104.0 96.5 96.8 103.5 97.5 103.5 98.0 103.0 96.5
97.5 103.5 97.0 103.0 98.5 97.0 102.5 98.0 103.0 102.5 97.0 104.0 96.5 103.5 96.8
103.5 96.5 96.5 97.0 103.0 103.5 96.5 103.5 97.0 96.5 103.0 102.5 104.0 96.5 103.5
103.0 97.0 96.5 103.5 103.5 104.0 96.5 103.0 97.0 98.0 102.0 96.5 103.0 102.0 97.5
Mean n /3. 175 mg ml 1 each of the compounds in the first column. n /5.
calculated as described earlier [26] using the equation 3s/S where s is the standard deviation of the blank and S is the corresponding slope of the calibration graph. The upper and lower limits of the dynamic linear range of concentration were estimated from the calibration graph, noting when the points started to deviate from the straight line. For more accurate analysis, Ringbom optimum concentration ranges were determined by plotting the transmittance percentage vs the logarithmic values of NMS concentration in mg ml 1 The molar absorptivities of the ion-association systems obtained are comparable to each other pointing out that the most sensitive counter ion is reagent VIII. The reagent VIII based ion-association is not only the most sensitive but also exhibits the lowest detection limit and the widest dynamic linear concentration range (Table 1). In order to determine the accuracy and precision of the proposed procedures, solutions containing five different concentrations of NMS were prepared and analysed in quintuplicate. The relative errors and RSDs in Table 1 can be considered satisfactory, at least for the concentration levels
examined. The performance of the proposed procedures was assessed by comparison with the official method [21]. Mean values obtained in Student’s t- and F -test [27] showed the absence of any systematic error in the method (Table 1). 3.7. Interferences Interferences from some common drug excipients were examined and the results are given in Table 2. It was found that these compounds exhibit minimum interference, even when present at 50 times the level of NMS. Determination of 35 mg ml 1 NMS in a mixture containing 350 mg ml 1 each of kaolin, pectin, aluminium hydroxide, polyvinyl alcohol, sodium acetate, propylene glycol, benzalkonium chloride, polymyxin B sulphate, polysorbate, dexamethazone phosphate, hydrocortizone acetate, methylprednisolone acetate, aluminium chlorohydroxide complex, bacitracin-zinc and starch are satisfactory, with an RSD of 1.4% and a recovery of 96.5%. The RSD of six determination of 35 mg ml 1 NMS standard solution was 1.2%.
Table 3 Determination of NMS in different pharmaceutical formulations using counter ion (I /VIII) Lable claim
c
Neomycin tablets
500 mg
FML-Neod
5 mg ml 1
Predmycin-Pd
5 mg ml 1
Conjunctind
5 mg ml 1
Decadron Neomycind
3.5 mg ml 1
Neo-Cortefd
5 mg ml 1
Neo-Medrold
2.5 mg ml 1
a b c d
Founda (9/SD) I
II
III
IV
V
VI
VII
VIII
Off.
4969/0.72 t b /1.21 F b /1.78 4.979/0.56 t b /1.58 F b /2.70 5.059/0.62 t b /1.66 F b /3.10 5.079/0.50 t b /1.46 F b /2.60 3.529/0.74 t b /1.09 F b /1.65 4.939/0.48 t b /1.75 F b /2.99 2.519/0.67 t b /1.47 F b /2.18
4959/0.63 t b /1.42 F b /2.32 5.059/0.71 t b /1.17 F b /1.68 5.049/0.56 t b /1.03 F b /1.58 4.939/0.70 t b /1.89 F b /3.19 3.489/0.47 t b /1.82 F b /2.68 4.969/0.58 t b /1.35 F b /2.05 2.489/0.61 t b /1.71 F b /2.63
5039/0.52 t b /1.98 F b /3.41 5.039/0.52 t b /2.02 F b /3.13 4.949/0.76 t b /1.37 F b /2.01 4.979/0.62 t b /1.04 F b /1.73 3.479/0.45 t b /2.29 F b /4.46 5.069/0.70 t b /1.00 F b /1.41 2.479/0.63 t b /1.38 F b /2.47
4949/0.66 t b /1.33 F b /2.12 4.949/0.48 t b /2.10 F b /3.67 5.039/0.60 t b /1.49 F b /2.78 5.059/0.54 t b /1.74 F b /2.66 3.539/0.72 t b /1.10 F b /1.74 4.949/0.58 t b /1.28 F b /2.02 2.539/0.46 t b /2.32 F b /4.63
5059/0.58 t b /1.56 F b /2.73 5.0246 t b /2.22 F b /4.00 4.969/0.68 t b /1.41 F b /2.16 4.949/0.71 t b /1.11 F b /1.54 3.479/0.60 t b /1.31 F b /2.51 5.059/0.56 t b /1.34 F b /2.20 2.519/0.45 t b /2.18 F b /4.84
5049/0.76 t b /1.07 F b /1.60 4.9164 t b /1.30 F b /2.07 5.079/0.58 t b /1.72 F b /2.97 4.959/0.46 t b /2.05 F b /3.66 3.459/0.71 t b /1.13 F b /1.79 5.089/0.62 t b /1.12 F b /1.79 2.529/0.55 t b /2.02 F b /3.24
4939/0.82 t b /1.18 F b /1.37 4.9475 t b /1.07 F b /1.50 4.9466 t b /1.38 F b /2.30 5.049/0.58 t b /1.71 F b /2.32 3.499/0.45 t b /2.31 F b /4.43 4.939/0.69 t b /1.06 F b /1.45 2.479/0.60 t b /1.79 F b /2.72
5029/0.67 t b /1.26 F b /3.05 5.0649 t b /1.20 F b /1.49 5.0878 t b /1.17 F b /1.64 4.949/0.53 t b /1.64 F b /2.76 3.539/0.72 t b /1.15 F b /1.74 5.079/0.82 t b /0.98 F b /1.67 2.539/0.61 t b /1.39 F b /2.63
5119/0.97
5.1292
4.901.00
4.879/0.88
3.609/0.95
4.849/0.83
2.449/0.99
A.S. Amin, Y.M. Issa / Spectrochimica Acta Part A 59 (2003) 663 /670
Formulation
Average of six determinations. Theoretical values for t -and F -values for five degrees of freedom and 95% confidence limits are 2.57 and 5.05, respectively. Memphis Company for Pharmaceutical Industries, Egypt. Egyptian International Pharmaceutical Industries Company, Egypt.
669
670
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3.8. Analytical applications To establish the validity of the proposed procedures, pharmaceutical formulations were analysed using chromotropic azo dyes counter ions (I / VIII). The same batches were analysed by the USP [21] method and the results obtained were compared statistically utilizing Student’s t - and F tests. The present methods showed almost the same degree of accuracy and precise as the USP method. The proposed method have the advantage of being virtually free from interferences as well as having moderately low detection limits, and it may therefore be suitable for the determination of this drug in biological fluids using standard addition method (Table 3).
4. Conclusion Extractive colorimetric procedure for the determination of NMS was developed based on the formation of extractive ion-associate complexes with chromotropic acid azo dyes as counter ions. The proposed procedure is fairly rapid, simple, sensitive, precise, accurate and promises good prospects for the determination of NMS in pharmaceutical formulations. Additionally, the method uses cheaper procedures than the microbiological method. The chromotropic acid azo dyes are commercially available by many chemical companies.
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[2] A. Wade, Martindale’s Extra Pharmacopeia, 27th ed., Pharmaceutical Press, London, 1978, p. 1135, 1155 /1160. [3] H.H. Zedan, M. Salama, W.M. Abdel-Radman, Egypt J. Pharm. Sci. 23 (1982) 19. [4] K. Florey, Analytical Profiles of Drug Substances, vol. 8, Academic Press, New York, 1979, pp. 339 /488. [5] F.S. Ghazy, A.A. Kassem, S.H. Shalaby, Pharmazie 39 (1984) 821. [6] W.A. Harris, Australas J. Pharm. 52 (1971) 569. [7] S.M. Abu-El-Wafa, M.A. El-Ries, F.M. Abou-Attai, R.M. Issa, Anal. Lett. 22 (1989) 13. [8] A.S. Amin, Spectrosc. Lett. 29 (1996) 1003. [9] M.R.C. Marques, E.R.M. Hackmann, T. Saito, Anal. Lett. 22 (1989) 621. [10] B. Gala, A. Gomez Hens, D. Perez bendito, Anal. Chim. Acta 347 (1995) 310. [11] E. Adams, R. Schepers, E. Roets, J. Hoogmartens, J. Chromatogr. 741 (1996) 233. [12] E. Adams, R. Schepers, L.W. Gathu, R. Kibaya, E. Roets, J. Hoogmartens, J. Pharm. Biomed. Anal. 15 (1997) 505. [13] M.H. Vega, G.M. Garica, E.R. Gesche, R.F. Saelzer, J. Planar Chomatogr. Mod. TLC 1 (1992) 62. [14] E. Roets, E. Adams, I.G. Muriithi, J. Hoogmartens, J. Chromatogr. 1 (1995) 131. [15] W. Funk, T. Kuepper, A. Wirtz, S. Netz, J. Planar Chromatogr. Mod. TLC 3 (1994) 10. [16] J.A. Reid, J.D. Macneil, J. AOAC. Int. 82 (1999) 61. [17] V.K. Agrawal, J. Liq. Chromatogr. 13 (1990) 2475. [18] B. Shaikh, J. Jachson, G. Guyer, W.R. Ravis, J. Chromatogr. Biomed. Appl. 109 (1991) 571. [19] A.S. Amin, Y.M. Issa, Anal. Lett. 30 (1997) 69. [20] H.T.S. Britton, Hydroden Ions, fourth ed., Chapman and Hall, London, 1952. [21] ‘United State Pharmacopoeia’, 24th ed., United States pharmacopoeial convention, Rockville, MD 2000. [22] D.L. Massart, B.G.M. Vandeginste, S.N. Deming, Y. Michotte, L. Kaufman, Chemometrics, A Text Book, Elsevier, Amsterdam, 1988. [23] S.L. Bhongade, A.V. Kasture, Talanta 40 (1993) 1525. [24] M. Oue, K. Kimura, T. Shono, Analyst 113 (1988) 551. [25] B. Saad, S.M. Sultan, F.E.O. Suliman, Talanta 44 (1997) 53. [26] C.M. Legua, P.C. Falco, A.S. Cabeza, Anal. Chem. Acta 283 (1993) 635. [27] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, third ed., Ellis Horwood, Chichester, 1993.