الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
 |  | from | 200 |  |  |
| | | from | 200 | | |
المستخلص
The study described three analytical techniques for the analysis of three therapeutical ly important and widely used antiviral drugs. These drugs were acyclovir (ACV), amantadine hydrochloridc (AMD), and ribavirin (RBV). The drawbacks encountered in the analytical methods reported in literatures of the analysis of these drugs were behind the interest in selection of these drugs to be the targets of the present study. The methods developed in the present study include spectrophotometric, spectrofluorimetric, and thin-layer chromatographic methods.
I. SPECTROPHOTOMETRIC METHODS
1.1. General oxidation-based methods
Oxidation-based spectrophotometric methods were developed and validated for determination of the investigated drugs via oxidation With different inorganic oxidants: cerium ammonium sulphate, potassium permanganate, ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate. The oxidation reactions were performed in perchloric acid medium for cerium ammonium sulphate and in sulphuric acid medium for the other reagents. In case of cerium ammonium sulpnate and potassium permanganate, the concentration of the drug in its samples was determined by measuring the decrease in the absorption intensity of the coloured reagents at 315 and 525 nm for cerium ammonium sulphate and potassium permanganate, respectively. In case of other reagents, the concentration was determined by measuring the intensity of the developed coloured reaction products at Xmax: 675, 780, 595, 595, 475, and 475 nm for reactions with ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate, respectively. Different variables affecting the reaction conditions were carefully studied and optimized for each particular reagent with the
investigated drugs. Under the optimum conditions, linear relationships with good correlation coefficients (0.9984-0.9998) were found between the readings and the concentrations of the drug in the range of 3.0-1700 U-g/ml. The molar absorptivities were correlated with the oxidation potential of the oxidants used. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 2%. The proposed methods were successfully applied to the analysis of the investigated drugs in pure and dosage forms with good accuracy and precisions; the recovery percentages were 97.5-101,5 ± 0.40-1.23%. The results obtained by the proposed spectrophotometric methods were comparable with those obtained by the official method stated in the United States Pharmacopoeia.
1.2. Methods for amantadine HCI analyses
For AMD, two additional methods were also developed and validated. The first method was based on the charge-transfer complexation reaction between the amantadine base as an electron donor and iodine as a c-acceptor. The second method was based on the reaction of N-alkylvinylamine formed from the interaction of the free amino group in amantadine. molecule and acetaldehyde with chloranil to give coloured vinylamino-substituted benzoquinone (enamine) derivative. The coloured reaction products of these reactions were measured at 290 or 365 nm for the charge-transfer method, and at 325 or 685 nm for enamine formation-based method. The variables affecting the reactions were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9993-0.9998) were found between the readings and the corresponding concentration of the drug in the ranges of 2-40p,g/mt. The limits of detection ranged from 0.16-0.75 jag/ml. The precision of the methods was satisfactory; the values of relative standard deviations did not exceed 2%. The proposed methods were successfully applied to the analysis of AMD in its capsules with good accuracy and precisions; the label claim percentages ranged from 99.8 -100.5± 0.45-1.22%. The results
(rophotometric methods were comparable with those obtained by the official \
■pectrophotometric methods were comparable with those obtained by the official
I method.
j II.SPECTROFLUORIMETRIC METHOD
■In this part, a simple and sensitive fluorimetric method for determination of ACV,
I AMD, and RBV has been developed. The method was based on the oxidation of
these drugs by cerium(IV) in presence of perchloric acid and subsequent
I monitoring the fluorescence of the produced cerium(III) at ^excitation 255 and
Emission 35 5 mil. Different variables affecting the reaction conditions such as the
concentrations of cerium(iV), type and concentration of acid, reaction time,
temperature, and the diluting solvents were carefully studied and optimized.
Under the optimum conditions, linear relationships with good correlation
coefficients (0,9978 - 0.9996) were found between the relative fluorescence
intensity and the concentrations of the investigated drugs in the range of 50 -
1400 ng/ml. The assay limits of detection and quantitation were 20 - 49, and 62 -
160 ng/ml, respectively. The precision of the method was satisfactory; the values
of relative standard deviations did not exceed 1.58%. No interference could be
observed from the excipients commonly present in dosage forms. The proposed
method was successfully applied to the analysis of the investigated drugs in pure,
pharmaceutical dosage forms, plasma and urine samples spiked with AMD or
RBV, with good accuracy and precisions. The recovery percentages ranged from
99.2 - 101.2 ± 0.48 - 1.30%. The results obtained by the proposed fluorimetric
method were comparable with those obtained by the official United States
Pharmacopoeia.
III. THIN-LAYER CHROMATOGRAPHIC METHOD
A validated stability-indicating thin-layer chromatographic (TLC) method of the analysis of AMD and RBV in bulk and capsule forms has been developed. The degradation products for RBV can be selectively and accurately estimated in both raw material and capsules onto one precoated silica gel TLC plate 60 F 254. The developing systems used were n-hexane:methanol:diethyl aminc (80:40:5 v/v/v), and chloroform:methanol:glacial acetic acid (60:15:15 v/v/v) for AMD and RBV respectively. The separated bands were detected as brown to brownish-red spots after spraying with modified Dragendorffe s solution for AMD and as bluish-green spots after spraying with anisaldehyde for RBV. The optical densities of the separated spots were found to be linear in proportion to the drug concentration. The stability of two drugs was studied under accelerated conditions in order to provide a rapid indication of difference that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of light, acid-base hydrolysis, boiling, and oxidation. The stress testing revealed that AMD was stable under all the tested conditions; however RBV was vulnerable by these conditions and gave four degradation products. These methods show enough selectivity, sensitivity, accuracy, precision, and robustness to satisfy International Conference of Harmonization/Federal Drug Administration regulatory requirement (ICH). The developed methods could also be used for purity testing of two drugs in raw materials and capsules content uniformity testing and stability testing of capsules. Linear relationships with good correlation coefficients (0.9994, 0.9980 for AMD and RBV respectively) were found between the readings and the concentrations of the drugs in the range of 5-50 and 5-40 (ig/spot of AMD and RBV respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 4.73%. The proposed methods were successfully applied to the analysis of the investigated drugs in pure and dosage forms with good accuracy and precisions; the recovery percentages were 98.99 ± 0.98 and 98.75 ± 1.5% of
He proposed TLC methods were comparable with those obtained by the official United States Pharmacopoeia. As well, the proposed TLC methods could be applied to the analysis of both AMD and RBV in spiked plasma and urine with adequate selectivity and minor pretreatment (protein precipitation) for the sample.
In conclusion, all the developed methods could be successfully applied to the analysis of the investigated drugs in pure and pharmaceutical dosage forms. The iluorimetric method, because of its high sensitivity, could be applied to the analysis of plasma or urine samples spiked with AMD or RBV, after proper extraction step by methylene chloride. The method was not applicable to the determination of ACV because of its high water-solubility, and the failure to be extracted from its aqueous samples with water-immiscible organic solvent. The TLC-densitometric method offered high selectivity to be applied as stability-indicating (rather than the other methods), whereas it was able to separate the major drug from the possible degradation products. It also, had the adequate selectivity for application to the analysis of AMD and RBV spiked in biological fluids (plasma and urine), however, the sensitivity was not adequate for the real samples, whereas the normal concentrations of both drugs are usually below the limits of detection of the proposed method.
A summary for the methods applied in the analyses of the cited antiviral drugs were presented in table 41.
The thesis includes 41 tables and 49 figures, summary for the contents in Arabic and in English, in addition to a list of 186 references used through out the thesis.
We 41. Summary for the methods used in the analyses of the investigated antiviral drugs.
Method Acyclovir Amantadinc HC1 Ribavirin
1 Spectrophotometry
la-Ce(NH4)2(SO4)3 Range a e*103 4-12 14.03 3-12 16.79 4-12 14.89
Ib-KMnO4 Range
£ x\{) 5-40 4.97 5-40 3.44 5-60
3.25
|c-(NH4)2MoO4 Range
e*103 15-90 1.58 5-35
4.82
|d-NH4VO3 Range
e xlO3 100-800
0.23 200-1300
0.14 100-700
0.32
e- CrO, Range 100-1300
0.15 100-800
0.17 100-1400 0.13
f-K:Cr20, Range 100-1700 0.12 100-1100 0.13 100-1300 0.13
Range e*103 ’ - 100-500 0.39
h- KICX, Range e*103 . . 100-550 0.31
i- Charge-transfer complex
Range 2-16
e*103 8.2
j- Enamine formation
Range 2-10
e*103 - 11.8
II. Spectrofluorimetry
Range 0.50-1.20 0.50-1.20 0.50-1.40
III. Thin-layer chromatography
a- Modified Dragendorff s
Range 5-50
b- Anisaldehyde
Range 5-40
:onccntration ug/ml.