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Utility of 2,3-dichloro-5,6-dicyano-p-benzoquinone in assay of codeine, emetine and pilocarpine
Talanta, Vol. 32, No. 10, pp. 1002-1004, 1985 Printed in Great Britain. All rights reserved
0039-9140/x5 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
UTILITY OF 2,3-DICHLORO-5,6-DICYANO-pBENZOQUINONE IN ASSAY OF CODEINE, EMETINE AND PILOCARPINE MOHAMED E. ABDEL-HAMID, MOHAMED S. MAHROUS Faculty (Received
of Pharmacy, 11 January
and
University
MOHAMED ABDEL-SALAM, MAGDI
M. ABDEL-KHALEK
of Alexandria,
Alexandria,
1985. Revised 24 Aprrl 1985. Accepted
Egypt
15 May
1985)
Summary-A simple and sensitive spectrophotometric method for the assay of codeine, emetine and pilocarpine IS described, based on the interaction of these drugs (as n-electron donors) with 2,3-dichloro-5,6-dicyano-p-benzoquinone (as n-acceptor) to give a highly coloured radical anion which exhibits maximum absorption at 460 nm. Formation of the radical anion has been established by electron spin resonance measurements. Beer’s law is obeyed for the alkaloids investigated. The assay results are
in accord with pharmacopoeia1 assay results. The procedure is sufficiently sensitive to permit unit dose assay of the individual alkaloids in pharmaceutical formulations. Codeine phosphate (cough-sedative), emetine hydrochloride (anti-amoebic) and pilocarpine nitrate (antimydriatic) are widely used in pharmaceutical practice. The BP compendium’ describes an acid-base titration for the analysis of codeine phosphate tablets and emetine hydrochloride injections, and the USP* reports a calorimetric method for the assay of pilocarpine eye drops. Several methods have been described for the assay of these alkaloids in the and and include colorimetric3-9 formulations, chromatographic’G’2 methods. Substituted quinones such as 2,5-dichloro-p benzoquinone,13 7,7,8,8-tetracyanoquinodimethane’4,’5 and p-chloranil’6 have been used as n-acceptors with various donors to form charge-transfer complexes Application of 2,3-dichloro-5,6and radicals. dicyano-p-benzoquinone (DDQ) for the detection and determination of some medicinal drugs containing the imidazoline ring has recently been described.” The present work describes the utility of DDQ reagent for the spectrophotometric determination of codeine, emetine and pilocarpine in dosage forms. In addition, the nature of the orange-red coloured chromogen was established by electron spin resonance spectroscopy. EXPERIMENTAL Instruments A Perkin-Elmer Model 550 S spectrophotometer with matched l-cm quartz cells and a Varian Model E 12 electron spin resonance spectrometer were used. Reagents Pharmaceutical grade codeine phosphate, pilocarpine nitrate (Merck) and emetine hydrochloride (B.D.H.) were used as working standards. DDQ solution, 0.2% was freshly prepared in methanol. All reagents used were of analytical grade. Standard solutions
An accurately weighed amount of the drug salt (codeine phosphate.
emetine
hydrochloride
and pilocarpine
nitrate)
equivalent to 0.1 g of the base was dissolved m about 20 ml of water. The solution was quantitatively transferred to a separatory funnel, made alkaline with ammonia solution and shaken with five 20-ml portions of chloroform. The extracts were pooled in a lOO-ml standard flask and diluted to volume with chloroform, to provide a standard I-mg/ml solution of the base. Determination
of codeine
For calibration serial volumes of standard base solution in the range 0.24.9 ml were transferred into a series of lo-ml standard flasks. The solvent was removed by immersing the flasks in a water-bath at 70”. The residue was dissolved in 5 ml of methanol and 1 ml of DDQ solution was added. The volume was made up to 10 ml with methanol and the absorbance at 460 nm was measured against a reagent blank similarly prepared. For determination of codeine phosphate in tablets, 10 tablets were powdered and thoroughly mixed. An accurately weighed quantity equivalent to -0.13 g of codeine phosphate (0.1 g of codeine base) was transferred into a separatory funnel containing 20 ml of water. The base was extracted as described above and the extracts were transferred into a lOO-ml standard flask and diluted to volume with chloroform. Exactly 0.6 ml of the diluted solution was then treated by the calibration procedure. Derermination
of emeiine
Ten ml of standard emetine base solution were transferred into a 50-ml standard flask and diluted to volume with chloroform. For calibration serial volumes of 0.3-l. 1 ml of this solution were treated as described above for codeine. For the determination of emetine in injections, 5 injections were mixed and an accurately measured volume -0.115 g of emetine hydrochloride (0.1 g of containing emetine base) was transferred into a separatory funnel containing 20 ml of water. The base was extracted and diluted as above, and 1 ml of this solution was analysed by the procedure for calibration. Determination
of pilocarpine
The cahbration procedure was the same as that for codeine. For the assay of pilocarpme in eye drops, the free base from an accurately measured volume containing _ 0.13 g of pilocarpine nitrate (0.1 g of pilocarpine base) was extracted and diluted as described for codeine phosphate tablets, and
1002
SHORT COMMUNICATIONS
0.6 ml of the resulting solution was assayed by the cali-
1003
08
bration procedure. Electron spin resonance The DDQ radical anion was prepared by mixing 10e4M solution of the drug base (codeine, emetine, pilocarpine) in methanol with 10e4M DDQ solution in the same solvent. The electron spin resonance spectra were run with the solution protected by a nitrogen atmosphere.
07
06 RESULTS AND DISCUSSION In spite of their structural differences, codeine, emetine and pilocarpine react with DDQ in methanol to give an intensely orange-red product which exhibits absorption maxima at 460, 520 and 560 nm (Fig. 1). The absorption bands are similar to those of
05
the DDQ radical anion obtained by reduction with 04 iodide.14 Further support of this assignment was t provided by the detection of the DDQ radical by 6 0 electron spin resonance measurements. The ESR 6 spectrum for the DDQ radical anion is shown in Fig. ;: 03 2. The spectrum displays 5 bands with relative in- a tensities 1: 2: 3 : 2: 1. The observed lines are due to the hypefine interactions caused by two equivalent 02 nitrogen nuclei. Analysis of the spectrum gave a value of 0.55 f 0.01 gauss for the coupling constant (ah). The g-factor value, as calculated from the spectrum, was 2.0050. The band-width (AH = 0.39 0 1 gauss) was greater than that for the TCNQ radical anion.‘* This is because the coupling in TCNQ occurs between the free electron and the hydrogen and nitrogen nuclei, whereas in DDQ it takes place I L I between the free electron and the nitrogen and 400 500 600 chlorine nuclei. The chlorine-interaction accounts for Wavelength the relatively large band width for the DDQ radical Fig. 1. Absorption spectra of (----) codein+DDQ reaction anion. product, measured against reagent blank, (-) DDQ On the basis of the ESR investigations, it is as- radical anion obtained by iodide reduction method, sumed that a charge-transfer complex is formed by measured against methanol. Codeine concentration = 9 mg/lOO ml; DDQ concentration = 0.2 mg/ml. the interaction of the alkaloids as n-donors and DDQ as n-acceptor. In polar solvents (e.g., methanol, a+A -----+[D...A] -D+.+A-. acetonitrile), complete electron transfer from the charge-transfer complex radical ions donor- to the acceptor moiety takes place (see below) with the formation of the DDQ radical anion as The spectral properties of the coloured radical and predominent chromogen. also the influence of various factors on the colour
Fig. 2. ESR spectrum of DDQ radical anion in methanol.
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COMMUNlCATlONS
Table 1. Assay results (6 replicates) for dosage forms of codeine phosphate, emetine hydrochloride and pilocarpine nitrate
Preparation
Nominal concentration*, mg/lOO ml
Found,? % Proposed method
Official methods’,*
r
Codeine phosphate@ tabletsi
7.68
98.6 k 1.1
98.0 f 1.2
0.95
Emetine hydrochloride@ injections #
2.30
99.0 & 0.6
98.6 * 0.7
1.09
Pilocarpine@ eye drops1
7.81
96.1 k 0.8
96.6 f 0.8
0.98
*The stated amounts of codeine phosphate, emetine hydrochloride and piloearpine nitrate are equivalent to 6, 2, and 6 mg of the corresponding base Per 100 ml, respectively. tMean and standard deviation. §For p = 0.05 the critical f-value is 2.23. tLabelled to contain 30 ma of codeine nhosnhate ner tablet (Adco Co., Eavnt). $ Labelled to contain 30 rng of emetine hydrochloride per injection (Misr co.; Egypt). YLabelled to contain 3% of pilocarpine nitrate (Alexandria Co., Egypt). development, were studied to determine the optimal conditions for the assay. Maximum absorption was obtained when 1 ml of 0.2% DDQ solution was used in the total volume of 10 ml. Non-polar solvents (benzene, carbon tetrachloride, chloroform) were found to be unsuitable, whereas polar solvents (methanol, acetonitrile) were considered as ideal solvents as high yields of the DDQ radical were obtained in these media. The reaction was found to be fast, the colour attaining maximum intensity in 5 min and then remaining stable for at least 30 min. Under the conditions described, the calibration graphs for measurement at 460 nm are linear over the concentration ranges (for the solution measured) 2-9 mg/lOO ml (codeine), 0.62.2 mg/lOO ml (emetine) and 2-8 mg/lOO ml (pilocarpine). The regression equations derived for our calibration systems by the least-squares method” were A = -0.01
f 0.08C
A =0.00+0.31C
(codeine)
official methods are equally accurate. The proposed method is simpler, faster and more sensitive than the official procedures. These advantages favour its application in the analysis and quality-control of pharmaceutical formulations containing these alkaloids. However, other drugs with basic centres are expected to give similar reactions with DDQ, so the method is limited to the assay of single-drug formulations. Acknowledgement-The authors wish to thank the Institute of Chemistry, University of Tiibingen, West Germany, for
measuring trhe ESR spectra; they are also grateful to Prof. K. A. Kovar, Institute of Pharmaceutical Sciences, University of Tiibingen for his valuable suggestions and discussion. REFERENCES pp. 605, 570. HM Stationary Office, London, 1980. 2. U.S. PharmacoDeia, 20th Revision, p. 628. Mack Publishing Company, Easton, Pa. 1980: 3. A. 0. Gettler and I. Sunshine. Anal. Chem.. 1951. 23. 1. British Pharmacopoeia,
779.
(emetine) 5.
A = -0.01
+O.llC
(pilocarpine)
with regression coefficients of 0.9991, 0.9979 and 0.9970, respectively; C is the concentration of the drug in mg/lOO ml. The slopes of the calibration curves reflect the degree of formation of the DDQ radical anion. The much higher slope for emetine is probably due to the presence of two basic centres in its molecule. The molar absorptivities for codeine, emetine and pilocarpine were 2.5 x lo’, 1.54 x lo4 and 2.2 x lo3 1 .mole-’ .cm-‘, respectively. The relative standard deviations (7 replicates) for the determination of the alkaloids investigated were all 0.2%. The method was applied to determination of codeine phosphate in tablets, emetine hydrochloride in injections and pilocarpine nitrate in eye drops, the official methods being used for comparative assays. The results are presented in Table 1. The performance of the method was assessed by a t-test. At the 95% confidence level. the calculated t-value did not exceed the tabulated value, indicating that the proposed and
and M. Abdel-Hady, Pharmazie, 1975, 30, 60. A. M. Taha, A. K. S. Ahmed, C. S. Gomaa and H. El-Fattatry, J. Pharm. Sci., 1974, 63, 1853. C. Gomaa and A. Taha, ibid., 1975, 64, 1398. A. Taha and C. S. Gomaa, ibid., 1976, 65, 986. M. E. Abdel-Hady, M. A. Abdel-Salam, N. A. AbdelSalam and Y. A. Mohamed, Planra Med., 1978,34,430. P. S. Agarwal and M. A. Elsayed, Analyst, 1981, 106, 1157. W. F. Bayne, L. C. Chu and F. T. Tao, J. Pharm. Sci., 1976, 65, 1724. R. W. Frei, W. Santi and M. Thomas, J. Chromatog., 1976, 116, 365. B. Aaroe. J. Remme and B. Salvesen, Meddr. Horsk Farm. Selsk, 1975, 31, 274; Anal. Abstr., 1976, 30, 6E4. H. Abdine, M. A. Elsayed, I. Chaaban and M. E. Abdel-Hamid, Analyst, 1978, 103, 1227. A. Taha and G. Riicker, Arch. Pharm., 1977,310,485. K.-A. Kovar, W. Mayer and H. Auterhoff, ibid., 198 1, 314, 447. S. Belal, M. A. Elsayed, M. E. Abdel-Hamid and H. Abdine, J. Pharm. Sci., 1981, 70, 127. K.-A. Kovar and M. Abdel-Hamid. Arch. Pharm.. 1984, 317, 246. M. E. Abdel-Hamrd, Disserlafion, Tiibingen, 1983. M. R. Spiegel, Theory and Problems of Probability and Statistics, pp. 215, 259. McGraw-Hill, New York, 1975.
4. Y. A. Mohamed
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
0039-9140/x5 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd
UTILITY OF 2,3-DICHLORO-5,6-DICYANO-pBENZOQUINONE IN ASSAY OF CODEINE, EMETINE AND PILOCARPINE MOHAMED E. ABDEL-HAMID, MOHAMED S. MAHROUS Faculty (Received
of Pharmacy, 11 January
and
University
MOHAMED ABDEL-SALAM, MAGDI
M. ABDEL-KHALEK
of Alexandria,
Alexandria,
1985. Revised 24 Aprrl 1985. Accepted
Egypt
15 May
1985)
Summary-A simple and sensitive spectrophotometric method for the assay of codeine, emetine and pilocarpine IS described, based on the interaction of these drugs (as n-electron donors) with 2,3-dichloro-5,6-dicyano-p-benzoquinone (as n-acceptor) to give a highly coloured radical anion which exhibits maximum absorption at 460 nm. Formation of the radical anion has been established by electron spin resonance measurements. Beer’s law is obeyed for the alkaloids investigated. The assay results are
in accord with pharmacopoeia1 assay results. The procedure is sufficiently sensitive to permit unit dose assay of the individual alkaloids in pharmaceutical formulations. Codeine phosphate (cough-sedative), emetine hydrochloride (anti-amoebic) and pilocarpine nitrate (antimydriatic) are widely used in pharmaceutical practice. The BP compendium’ describes an acid-base titration for the analysis of codeine phosphate tablets and emetine hydrochloride injections, and the USP* reports a calorimetric method for the assay of pilocarpine eye drops. Several methods have been described for the assay of these alkaloids in the and and include colorimetric3-9 formulations, chromatographic’G’2 methods. Substituted quinones such as 2,5-dichloro-p benzoquinone,13 7,7,8,8-tetracyanoquinodimethane’4,’5 and p-chloranil’6 have been used as n-acceptors with various donors to form charge-transfer complexes Application of 2,3-dichloro-5,6and radicals. dicyano-p-benzoquinone (DDQ) for the detection and determination of some medicinal drugs containing the imidazoline ring has recently been described.” The present work describes the utility of DDQ reagent for the spectrophotometric determination of codeine, emetine and pilocarpine in dosage forms. In addition, the nature of the orange-red coloured chromogen was established by electron spin resonance spectroscopy. EXPERIMENTAL Instruments A Perkin-Elmer Model 550 S spectrophotometer with matched l-cm quartz cells and a Varian Model E 12 electron spin resonance spectrometer were used. Reagents Pharmaceutical grade codeine phosphate, pilocarpine nitrate (Merck) and emetine hydrochloride (B.D.H.) were used as working standards. DDQ solution, 0.2% was freshly prepared in methanol. All reagents used were of analytical grade. Standard solutions
An accurately weighed amount of the drug salt (codeine phosphate.
emetine
hydrochloride
and pilocarpine
nitrate)
equivalent to 0.1 g of the base was dissolved m about 20 ml of water. The solution was quantitatively transferred to a separatory funnel, made alkaline with ammonia solution and shaken with five 20-ml portions of chloroform. The extracts were pooled in a lOO-ml standard flask and diluted to volume with chloroform, to provide a standard I-mg/ml solution of the base. Determination
of codeine
For calibration serial volumes of standard base solution in the range 0.24.9 ml were transferred into a series of lo-ml standard flasks. The solvent was removed by immersing the flasks in a water-bath at 70”. The residue was dissolved in 5 ml of methanol and 1 ml of DDQ solution was added. The volume was made up to 10 ml with methanol and the absorbance at 460 nm was measured against a reagent blank similarly prepared. For determination of codeine phosphate in tablets, 10 tablets were powdered and thoroughly mixed. An accurately weighed quantity equivalent to -0.13 g of codeine phosphate (0.1 g of codeine base) was transferred into a separatory funnel containing 20 ml of water. The base was extracted as described above and the extracts were transferred into a lOO-ml standard flask and diluted to volume with chloroform. Exactly 0.6 ml of the diluted solution was then treated by the calibration procedure. Derermination
of emeiine
Ten ml of standard emetine base solution were transferred into a 50-ml standard flask and diluted to volume with chloroform. For calibration serial volumes of 0.3-l. 1 ml of this solution were treated as described above for codeine. For the determination of emetine in injections, 5 injections were mixed and an accurately measured volume -0.115 g of emetine hydrochloride (0.1 g of containing emetine base) was transferred into a separatory funnel containing 20 ml of water. The base was extracted and diluted as above, and 1 ml of this solution was analysed by the procedure for calibration. Determination
of pilocarpine
The cahbration procedure was the same as that for codeine. For the assay of pilocarpme in eye drops, the free base from an accurately measured volume containing _ 0.13 g of pilocarpine nitrate (0.1 g of pilocarpine base) was extracted and diluted as described for codeine phosphate tablets, and
1002
SHORT COMMUNICATIONS
0.6 ml of the resulting solution was assayed by the cali-
1003
08
bration procedure. Electron spin resonance The DDQ radical anion was prepared by mixing 10e4M solution of the drug base (codeine, emetine, pilocarpine) in methanol with 10e4M DDQ solution in the same solvent. The electron spin resonance spectra were run with the solution protected by a nitrogen atmosphere.
07
06 RESULTS AND DISCUSSION In spite of their structural differences, codeine, emetine and pilocarpine react with DDQ in methanol to give an intensely orange-red product which exhibits absorption maxima at 460, 520 and 560 nm (Fig. 1). The absorption bands are similar to those of
05
the DDQ radical anion obtained by reduction with 04 iodide.14 Further support of this assignment was t provided by the detection of the DDQ radical by 6 0 electron spin resonance measurements. The ESR 6 spectrum for the DDQ radical anion is shown in Fig. ;: 03 2. The spectrum displays 5 bands with relative in- a tensities 1: 2: 3 : 2: 1. The observed lines are due to the hypefine interactions caused by two equivalent 02 nitrogen nuclei. Analysis of the spectrum gave a value of 0.55 f 0.01 gauss for the coupling constant (ah). The g-factor value, as calculated from the spectrum, was 2.0050. The band-width (AH = 0.39 0 1 gauss) was greater than that for the TCNQ radical anion.‘* This is because the coupling in TCNQ occurs between the free electron and the hydrogen and nitrogen nuclei, whereas in DDQ it takes place I L I between the free electron and the nitrogen and 400 500 600 chlorine nuclei. The chlorine-interaction accounts for Wavelength the relatively large band width for the DDQ radical Fig. 1. Absorption spectra of (----) codein+DDQ reaction anion. product, measured against reagent blank, (-) DDQ On the basis of the ESR investigations, it is as- radical anion obtained by iodide reduction method, sumed that a charge-transfer complex is formed by measured against methanol. Codeine concentration = 9 mg/lOO ml; DDQ concentration = 0.2 mg/ml. the interaction of the alkaloids as n-donors and DDQ as n-acceptor. In polar solvents (e.g., methanol, a+A -----+[D...A] -D+.+A-. acetonitrile), complete electron transfer from the charge-transfer complex radical ions donor- to the acceptor moiety takes place (see below) with the formation of the DDQ radical anion as The spectral properties of the coloured radical and predominent chromogen. also the influence of various factors on the colour
Fig. 2. ESR spectrum of DDQ radical anion in methanol.
SHORT
COMMUNlCATlONS
Table 1. Assay results (6 replicates) for dosage forms of codeine phosphate, emetine hydrochloride and pilocarpine nitrate
Preparation
Nominal concentration*, mg/lOO ml
Found,? % Proposed method
Official methods’,*
r
Codeine phosphate@ tabletsi
7.68
98.6 k 1.1
98.0 f 1.2
0.95
Emetine hydrochloride@ injections #
2.30
99.0 & 0.6
98.6 * 0.7
1.09
Pilocarpine@ eye drops1
7.81
96.1 k 0.8
96.6 f 0.8
0.98
*The stated amounts of codeine phosphate, emetine hydrochloride and piloearpine nitrate are equivalent to 6, 2, and 6 mg of the corresponding base Per 100 ml, respectively. tMean and standard deviation. §For p = 0.05 the critical f-value is 2.23. tLabelled to contain 30 ma of codeine nhosnhate ner tablet (Adco Co., Eavnt). $ Labelled to contain 30 rng of emetine hydrochloride per injection (Misr co.; Egypt). YLabelled to contain 3% of pilocarpine nitrate (Alexandria Co., Egypt). development, were studied to determine the optimal conditions for the assay. Maximum absorption was obtained when 1 ml of 0.2% DDQ solution was used in the total volume of 10 ml. Non-polar solvents (benzene, carbon tetrachloride, chloroform) were found to be unsuitable, whereas polar solvents (methanol, acetonitrile) were considered as ideal solvents as high yields of the DDQ radical were obtained in these media. The reaction was found to be fast, the colour attaining maximum intensity in 5 min and then remaining stable for at least 30 min. Under the conditions described, the calibration graphs for measurement at 460 nm are linear over the concentration ranges (for the solution measured) 2-9 mg/lOO ml (codeine), 0.62.2 mg/lOO ml (emetine) and 2-8 mg/lOO ml (pilocarpine). The regression equations derived for our calibration systems by the least-squares method” were A = -0.01
f 0.08C
A =0.00+0.31C
(codeine)
official methods are equally accurate. The proposed method is simpler, faster and more sensitive than the official procedures. These advantages favour its application in the analysis and quality-control of pharmaceutical formulations containing these alkaloids. However, other drugs with basic centres are expected to give similar reactions with DDQ, so the method is limited to the assay of single-drug formulations. Acknowledgement-The authors wish to thank the Institute of Chemistry, University of Tiibingen, West Germany, for
measuring trhe ESR spectra; they are also grateful to Prof. K. A. Kovar, Institute of Pharmaceutical Sciences, University of Tiibingen for his valuable suggestions and discussion. REFERENCES pp. 605, 570. HM Stationary Office, London, 1980. 2. U.S. PharmacoDeia, 20th Revision, p. 628. Mack Publishing Company, Easton, Pa. 1980: 3. A. 0. Gettler and I. Sunshine. Anal. Chem.. 1951. 23. 1. British Pharmacopoeia,
779.
(emetine) 5.
A = -0.01
+O.llC
(pilocarpine)
with regression coefficients of 0.9991, 0.9979 and 0.9970, respectively; C is the concentration of the drug in mg/lOO ml. The slopes of the calibration curves reflect the degree of formation of the DDQ radical anion. The much higher slope for emetine is probably due to the presence of two basic centres in its molecule. The molar absorptivities for codeine, emetine and pilocarpine were 2.5 x lo’, 1.54 x lo4 and 2.2 x lo3 1 .mole-’ .cm-‘, respectively. The relative standard deviations (7 replicates) for the determination of the alkaloids investigated were all 0.2%. The method was applied to determination of codeine phosphate in tablets, emetine hydrochloride in injections and pilocarpine nitrate in eye drops, the official methods being used for comparative assays. The results are presented in Table 1. The performance of the method was assessed by a t-test. At the 95% confidence level. the calculated t-value did not exceed the tabulated value, indicating that the proposed and
and M. Abdel-Hady, Pharmazie, 1975, 30, 60. A. M. Taha, A. K. S. Ahmed, C. S. Gomaa and H. El-Fattatry, J. Pharm. Sci., 1974, 63, 1853. C. Gomaa and A. Taha, ibid., 1975, 64, 1398. A. Taha and C. S. Gomaa, ibid., 1976, 65, 986. M. E. Abdel-Hady, M. A. Abdel-Salam, N. A. AbdelSalam and Y. A. Mohamed, Planra Med., 1978,34,430. P. S. Agarwal and M. A. Elsayed, Analyst, 1981, 106, 1157. W. F. Bayne, L. C. Chu and F. T. Tao, J. Pharm. Sci., 1976, 65, 1724. R. W. Frei, W. Santi and M. Thomas, J. Chromatog., 1976, 116, 365. B. Aaroe. J. Remme and B. Salvesen, Meddr. Horsk Farm. Selsk, 1975, 31, 274; Anal. Abstr., 1976, 30, 6E4. H. Abdine, M. A. Elsayed, I. Chaaban and M. E. Abdel-Hamid, Analyst, 1978, 103, 1227. A. Taha and G. Riicker, Arch. Pharm., 1977,310,485. K.-A. Kovar, W. Mayer and H. Auterhoff, ibid., 198 1, 314, 447. S. Belal, M. A. Elsayed, M. E. Abdel-Hamid and H. Abdine, J. Pharm. Sci., 1981, 70, 127. K.-A. Kovar and M. Abdel-Hamid. Arch. Pharm.. 1984, 317, 246. M. E. Abdel-Hamrd, Disserlafion, Tiibingen, 1983. M. R. Spiegel, Theory and Problems of Probability and Statistics, pp. 215, 259. McGraw-Hill, New York, 1975.
4. Y. A. Mohamed
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.