Technological and scientific progress has led to the development of numerous
synthetic drugs. It is therefore imperative to dispose of analytical methods
to determine these drugs both in the quality control manufacturing phase of
the pharmaceutical formulations, pure form and their determination in the human
body. The statins (or HMG-CoA reductase inhibitors) formed a class of hypolipidemic
drugs used to lower cholesterol levels in people with or at risk of cardiovascular
disease. They lower cholesterol by inhibiting the enzyme HMG-CoA reductase,
which is the rate-limiting enzyme of the mevalonate pathway of chloestrol synethesis.
Simvastatin (SVT) [1S [1α, 3α.7β, 8β (2S*, 4S*), 8αβ]
1, 2, 3, 7, 8, 8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran2yl)
ethyl]-1-naphthalenyl-2,2-dimethylbutanoate, atorvastatin (AVT) [R (R*,R*)]
(fluorophenyl) β, δ-dihydroxy-5-(1-methyl)-3-phenyl-4 [(phenylamino)carbonyl]1H-pyrrole-1-heptanoic
acid, calcium salt (2:1) trihydrate and pravastatin (PST)1-naphthalene-heptanoicacid,
1, 2, 6, 7, 8, 8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy
[1S [1α (βS*, δSβ (R*), 8aα] monosodium salt, are selective
and competitive inhibitors of HMG-CoA reductase. Literature survey revealed
that HPLC methods (Kim et al., 2004; Malenovic
et al., 2004; Jemal et al., 2000;
Zhang et al., 2004; Srinivasu
et al., 2002; Cermola et al., 2006;
Altuntas et al., 2004; Miao
and Metcalfe, 2003; Van-Pelt et al., 2001;
Dohalsky et al., 2006; Ma
et al., 2007; Seshachalam and Kothapally, 2008;
Shah et al., 2007; Erturk
et al., 2003; Jamshidi and Nateghi, 2007;
Zhu and Neirinck, 2003; Mulvana
et al., 2000; Deng et al., 2008;
Chaudhari et al., 2007), electrophoresis (Kocijan
et al., 2005), UV spectroscopic (Erk, 2002,
2004, 2003; Siavash
et al., 2007; Nagaraj and Rajshree, 2007)
and electrochemical methods (Lovric and Nigovic, 2006;
Ozkan et al., 2003; Nigovic,
2006) have been reported for the analysis of these drugs and their metabolites
in biological fluids. So far no visible spectrophotometric method was reported
for the quantitative determination of these drugs in pharmaceutical dosage forms.
The present study describes development of simple, accurate, precise and reproducible
method for the determination of statin drugs in tablet dosage forms. Extractive
spectrophotometric procedures are popular for their sensitivity in the assay
of drugs and therefore, ion-pair extractive spectrophotometry has received considerable
attention or the quantitative determination of many pharmaceutical compounds.
| Fig. 1: The structures of statin drugs under investigation
Quantitative analysis of the drugs applying extractive spectrophotometric methods
is mainly founded on such factors as type of solvent extraction, nature of coordination
agents, acidity of the solution, concentration of reagents, temperature, time
of reaction and extraction. The structures of statin drugs under investigation
are given in Fig. 1.
MATERIALS AND METHODS
Reagents: All chemicals and reagents used throughout this study were
of analytical grade. Reagents used included ammonium molybdate (Mallinchrodt
Chemical works, New York), ammonium-thiocyanate (Winlap,U.K.) and ascorbic acid
(EL-Nasr.Co., Egypt). Solvents were always HPLC or spectroscopic grade. Doubly
distilled water was used to prepare all solutions. Freshly prepared solutions
were always employed.
Materials: Pharmaceutical grade atorvastatin calcium (AVT), simvastatin (SVT) and pravastatin sodium (PST) were used without further purification and were supplied by Egyptian Co. for chemical and pharmaceuticals, S.A.E, Egypt.
Apparatus: All the spectral analyses were made using Perkin-Elmer 601 spectrophotometer and quarts cell of 1 cm optical length was used. Automatic pipettes (Socorex Swis 200 μL and 200-1000 μL) were used.
Preparation of standard solutions: Stock solutions of AVT, SVT and PST containing 1 mg mL-1, were prepared by dissolving 100 mg of drugs in 100 mL methanol in a calibrated measuring flask.
Stock solutions of pharmaceutical preparations as a pure base form of drugs: An accurately weighed amount, equivalent to 100 mg of each drug from composite of 20 powdered tablets, was transferred into a 100 mL calibrated flask and diluted to the mark with the appropriate solvent, sonicated for 20 min and filtered off through a Whidtman No.1 filter paper to obtain solutions of 1000 μg mL-1. Further dilutions were made to obtain sample solution. All measurements were made at room temperature (25±1°C).
Batch measurements: The ion-pair distribution ratio was determined
at room temperature by shaking equal volumes (5 mL) of the organic and aqueous
phases of a given composition in a 50 mL separating funnel for 15 min. After
the phases were separated by gravity, an aliquot of the organic phase was used.
A calibration curve for spectrophotometric determination of the drugs was prepared
by taking known amounts of standard solution of the drugs forming the complex
and measuring the absorbance of the organic phase vs. reference solution at
470 nm for all the drugs under investigation.
RESULTS AND DISCUSSION
Anionic thiocyanate complexes of Mo (V) formed ion associates with the positively
charged drugs, the ion association complex, with two oppositely charged ions,
behaved as a single unit held together by an electrostatic force of attraction.
Spectral characteristics: In an attempt to increase the sensitivity
of the assay, the absorption spectra of the extracted Mo (V)-thiocyanateBSVT,
Mo (V)-thiocyanate BPST and Mo (V)-thiocyanateBAVT ion-pairs in 1, 2-dichloroethane
are scanned against blank reagent at different wavelengths ranged from 400 to
550 nm as shown in Fig. 2. Maximum absorbance with highest
sensitivity is accomplished at 470 nm for all the drugs under study.
| Fig. 2: Absorption spectra Mo (V)-statins ion-pairs
|Fig. 3: Effect of sulphuric acid concentration on the spectra
of the ion-pairs of simvastatin, atorvastatin and pravastatin drugs at λmax
= 470 nm
Optimization of variables: Optimum conditions necessary for rapid and quantitative formation of coloured ion-pair complexes with maximum stability and sensitivity were established by a number of preliminary experiments. Extraction of Mo (V)-thiocyanate complex with drugs from sulphuric, hydrochloric and nitric acid solutions in 1, 2-dichloroethane was investigated It is found that, maximum absorbance and molar absorpitivity (ε) of the 1, 2- dichloroethane extract are obtained by using sulphuric acid. from Fig. 3. It is obvious that, 1.6-2 mL of 4M H2SO4 is suitable for the ionBpairs formations. The effect of ascorbic acid concentration on the formation and extraction of the ion-pairs is examined by varying the ascorbic acid concentration in the aqueous phase. The absorbance of the extracted ion-pairs is increased by increasing the concentration of ascorbic acid first and then decreased later which means that 1x104 and 3x104 μg mL-1 of ascorbic acid for SVT, PST and AVT drugs, respectively, are sufficient for complete conversion of Mo (IV) to Mo (V) (Fig. 4a).
Optimum conditions are fixed by varying one parameter at a time while keeping other parameters constant and observing the effect on the absorbance at 470 nm. The effect of ammonium molybdate concentration is studied by extracting the coloured ion-pair species at different concentrations of ammonium molybdate varied from 5 to 160 μg mL-1. The absorbance of the extracted ionBpairs is increased by increasing the molybdate concentration up to 50 μg mL-1 for AVT, SVT or PST drugs, respectively (Fig. 4b). The effect of adding different concentrations of ammonium thiocyanate ranged from (5x103 to 7x104 μg mL-1) on the formation of ionBpairs is performed. The results obtained showed that, 25x103 to 40x103 μg mL-1 of ammonium thiocyanate are sufficient to give sensitive and reproducible results for microdetermination of SVT, PST and AVT drugs. In the presence of excess thiocyanate concentration over Mo (V), it is possible to obtain ion-pair species having various numbers of coordinate thiocyanate anions, so the colour of the aqueous solutions of Mo (V) thiocyanate is unstable which makes the quantitative measurements somewhat difficult (Fig. 4c).
The effect of time on the formation of the ionBpairs is studied carefully and
illustrated in Fig. 5 which shows the increase of absorbance
with time up to 15, 15 and 10 min for SVT, PST and AVT drugs, respectively.
Then the extract gradually lost its orange red colour, so all measurements are
made within a few minutes after phase separation because the variation of the
absorbance is not significant after the first 20 min.
|Fig. 4: The effect of variable concentrations of different
reactants on the ion-pairs (a) effectof ascorbic acid concentration (b)
effect of Mo (VI) concentration and (c) effect of SCN concentration
|Fig. 5: Effect of time on the spectra of the ion-pairs at
λmax = 470 nm
|Fig. 6: Effect of temperature on the spectra of the ion-pairs
at λmax = 470 nm
Absorbance-temperature curve represents the reaction of AVT, SVT or PST drugs
with Mo (V)-thiocyanate at λmax = 470 nm, at the temperature
range from 0 to 50°C (Fig. 6).
Figure 6 shows that the absorbance is generally increased by temperature increase and reached a maximum value at 27°C (room temperature) for SVT, PST and AVT drugs, respectively and slightly decreased above this temperature due to dissociation of the formed ion-pair complexes. Therefore, the temperature chosen is 25±2°C as the best temperature range for microdetermination of the drugs under study in pure and in pharmaceutical forms which confirmed the fact that ion-pairs are more stable at room temperature.
The effect of organic extracting solvents is investigated. A number of organic solvents such as 1, 2-dichloroethane, chloroform, petroleum ether, dichloromethane and ethyl acetate are studied for extraction of the complexes in order to provide an applicable extraction procedure. 1, 2-dichloroethane is preferred as it gives the highest absorbance of coloured extracts and have the highest molar absorptivity values.
Stoichiometry of the formed ion-pairs: The composition of ion-pairs
is determined by the contineous variation and the molar ratio methods using
equimolar solution to check the ratio between Mo (V) and SVT, PST and AVT drugs
to select the optimum conditions for their microdetermination. The results indicate
that a 1:1 Mo (V): AVT, Mo (V): SVT or Mo (V): PST ion-pairs are formed through
the electrostatic attraction between positive protonated drugs, AVT+,
SVT+ or PST+ and thiocyanate negative complex [Mo (SCN)6]¯
as shown by the proposed strucures given in Fig. 7.
| Fig. 7: The proposed structures of (a) SVT (b) PST and (c)
AVT-Mo (V)-thiocyanate ion-pairs
|Table 1: Analytical characteristics, precision and accuracy
of the proposed method
|Ya = ax+b, where x is the concentration in μg
Linearity and range: Beers law range, molar absorptivity, regression
equation and correlation coefficient determined for each drug are given in Table
1. A linear relationship is found between the absorbance at λmax
and the concentration of the drug in the range of 10-280, 10-150 and 10-180
μg mL-1 for SVT, PST and AVT, respectively, Regression analysis
of Beers plots at λmax reveals a good correlation. The
graphs show negligible intercepts and are described by the regression equation
obtained by the Least-squares method. The correlation coefficients are found
between 0.9983-0.9987 indicating good linearity. The high molar absorptivity
values of 32.27x103, 99.02x103 and 53.81x103
L mol-1 cm-1 of the ion-pairs of SVT, AVT and PST, respectively,
indicate the high sensitivity of the method.
Validation of the methods: Examined samples were prepared and tested
at four levels of drug using the proposed procedures. The complete set of validation
assays is performed for the drug as determined by the proposed method. The results
obtained for the pure drug are given in Table 2. The precision
and accuracy of the methods were tested by analyzing five replicates of the
drug. The standard deviation, relative standard deviation, recovery and 95%
confidence limits of different amounts tested were determined from the calibration
curve, as recorded in Table 2.
|Table 2: The Inter- and Intra-day precision and accuracy data
for simvastatin, atorvastatin and pravastatin determination obtained by
the proposed method, n = 5
|Table 3: Spectrophotometric microdetermination of simvastatin,
atorvastatin and pravastatin drugs in their pharmaceutical preparations
using the Mo(V)-thiocyanate method
|No. of replicates (n ) =5, #Standared F-values
at 95 % confidence level = 6.39, #Standared t-values at 95% confidence
level = 2.77, *standard deviation values using proposed method, ** Standard
deviation values using official method
The accuracy of the method is indicated by the excellent recovery (98.50-100.2),
(98.80-102.0) and (98.50-100.1) for SVT, AVT and PST, respectively and the precision
is supported by the low standard deviation, (SD = 0.013 to 0.057, 0.019 to 0.064
and 0.011 to 0.073 for SVT, PST and AVT drugs, respectively) and relative standard
deviation (RSD% = 0.24 to 0.40, 0.22 to 0.32 and 0.20 to 0.45 % for SVT, PST
and AVT drugs, respectively).
Precision, accuracy and specificity: Day-to-day precision and accuracy were evaluated by analyzing five samples of three different concentrations, which are prepared and analyzed on the same day (Table 2). Sample-to-sample variability is assessed using five samples o three different concentrations analyzed on four different days over a period of a week. These results show the accuracy and reproducibility of the assay. Thus, it was concluded that there were no significant intra-day or inter-day differences.
Interferences in pharmaceutical analysis: It is important to test the selectivity towards the excipients and fillers added to the pharmaceutical formulations. The concentration of the drugs in the dosage forms is calculated from the appropriate calibration graphs. There is no shift in the absorption maximum due to the presence of other constituents of the dosage forms. No interferance from common excipents, which indicates the selectivity of the proposed method.
Application: The validity of the proposed method is tested by the determination of SVT, AVT and PST drugs in dosage forms manufactured in the local companies. Table 3 shows the results obtained during the determination of SVT, AVT and PST drugs in the dosage forms. The results are compared with those obtained applying the official method (reported in the European pharmacopoeia) for SVT and PST drugs and official method for AVT drug. The results obtained are compared statistically by t-test and F-test with those obtained by official method on the sample of the same batch. It is obvious from this table that the percentage recoveries, SD and RSD values obtained applying the proposed method are very close to those obtained by the official method. The t-test and F-test values obtained at the 95% confidence level and degree of freedom (n = 4) did not exceed the theoretical tabulated value indicating that there is no significant difference between accuracy and precision of the proposed and the official methods.
A rapid, simple, sensitive and accurate extractive spectrophotometric method
has been developed which can be used for determination of SVT, AVT and PST drugs
in pharmaceutical formulations. There are some methods available for this determination,
which use UV-spectrophotometry; however, these have several disadvantages: the
cost of equipment, manner of performance, time required, difficulty of reaction
conditions and analytical procedures. We described the extractive spectrophotometric
method for determination of SVT, AVT and PST drugs based on the formation of
ion pair with thiocyanate of Mo (V). The method makes use of simple reagents,
which an ordinary analytical laboratory can afford and validation showed them
to be suitable for routine determination of SVT, AVT and PST drugs in its formulations.
The commonly used additives such as starch, silicon dioxide, magnesium stearate,
glucose, glycerin, talc, sodium lauryl sulphate and sodium saccharin do not
interfere with the assay procedures. The main The main advantages of these procedures
are low cost of reagents, apparatus used and short time of analysis .the method
are Characterized by good precision, reproducibility of determination and high
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