1. INTRODUCTION:
Metoprolol, which named (RS)-1-(Isopropylamino)-3-[4-(2-methoxyethyl)phenoxy] propan-2-ol is a β1-adrenoreceptor antagonist (β1-blocker),is widely used in the treatment of hypertension, angina pectoris and cardiac dysrhythmias (Figure1) [1,2], it could be as tartrate or succinate salt.
Metoprolol tartrate possess one asymmetric carbon, giving a pair of enantiomers, (𝑆)- and (𝑅), the drug is marketed as racemic mixture. but, it has been demonstrated that the (S)-enantiomer has greater affinity (50–500 folds) for binding to the β-adrenergic receptors than R enantiomer [3,4].
Figure 1. Structure of Metoprolol enantiomers
[1]
The survey of literature revealed HPLC methods for determination of metoprolol enantiomers using chiral stationary phases[5,6],electrophoresis [7,8].
Cyclodextrins are chiral cyclic oligosaccharides obtained from starch through enzymatic degradation. Their structures are composed of α-(l,4)-linked d-glucose units arranged toroidally, the three main types of cyclodextrins are α-,β-,and γ-cyclodextrin, which contain six, seven, and eight glucose units, respectively. β-cyclodextrine and its derivatives are the most widly used in chiral recognition techinques because their cavity can fit into a large number of molecules [9].
Cyclodextrins have many advantages: available relatively at low cost, have good solubility in aqueous buffers, UV transparency, and good stability, for these unique characterities they can be used as chiral selectors added to running buffer in electrophoresis technique, with very little succesful tries to add cyclodextrins to mobile phase as chiral selectros in HPLC [10].
This paper describes the development of an inexpensive, simple and accurate RP-HPLC method to determine metoprolol tartrate enantiomers by adding chiral selector(methyl beta cyclodextrine M-β-CD) to the mobile phase using classical C18 column which is available in most laboratories and relatively inexpensive
The developed method was also used to determine the enantiomers of the drug in commercially available tablets of metoprolol tartrate.
2. MATERIALS AND METHODS:
2.1. Chemicals and reagents:
Standard of racemic metoprolol tartrate, sc-205751, standard of (S)-metoprolol sc-212886 (Santa cruz Biotechnology), (M-β-CD) Methyl beta cyclodextrine, 98.7)%Degree of substitution: 3.0-9.0( (Zibo Qianhui Biological Technology CO, LTD). Glacial acetic acid and HPLC methanol (Panreac, EU.Quimica.SA). Triethylamine( TEA) (Scharlau Chemie S.A.,Barcelona-Spain). Water for HPLC (Chem-lab NV,belgium).
2.2. Instruments:
HPLC system (Jasco, Japan) consisted of PU-980 intelligent pump, Jasco UV-970 intelligent detector,manual injector, column C18 (250X4.6, 5µm solutionicle size) Nucleodur Macherey Nagel ,Germany. Spectrophotometer Hitachi U-1800, Quartz cuvettes, Sartorius sensitive analytical balance Ag,d=0.1 mg, Germany. Ultrasonic bath (Transsonic 310, ,εlma®) Germany. pH meter Orion model 410 A. 0.45 µ cellulose nitrate filters (Sartorius stedim) ,Germany.
2.3. Chromatographic conditions:
Chromatographic separation was conducted in the reversed phase operating mode, with a mobile phase consists of aqueous solution contains chiral selector M-β-CD (1.5 g/l)/methanol with the volumetric ratio of 86:14 respectively , (aqueous solution :add to one liter of HPLC water 3.3 ml TEA the adjust the pH on 3.1 using glacial acetic acid), The final pH value of mobile phase was adjusted by using glacial acetic acid. The mobile phase was filtered using 0.45 µm disposable filter, and degassed by ultrasonic vibration prior to use. The measurements were carried out in an isocratic elution mode at ambient temperature with a flow rate of 0.5 mL/min, an injection volume of 20 𝜇L and UV detection at 274 nm. Each experiment was run in triplicate and each solution was prepared freshly.The column should be conditioned with mobile phase for two hours prior to injection.
2.4. Samples preparation:
Mobile phase without chiral selector should be prepared for the dilution and dissolving the samples.
Racemic metoprolol tartrate stock solution (1000 mg/l) was prepared by dissolving 50 mg of drug to 50 ml mobile phase with the aid of ultrasonication .mobile phase is the solvent for all dilution procedures to prepare a standard series of concentrations (182.5-1000 mg/l).
For commercial tablets , Twenty tablets of labeled claim 50 mg of racemic metoprolol tartrate were weighed accurately. An average weight of each tablet was determined. An accurately weighed quantity of powder equivalent to 100 mg of metoprolol tartrate was transferred into 100ml volumetric flask and sonicated for 10 min with 50 ml of mobile phase then dilut to the volume with the same solvent. The contents were mixed well then filtered rejecting the first portion of filtrate, The prepared solution concentration is 1000 mg/l, Aliquots of the tablets solution were prepared by dilution the last solution. Then the concentration of the each enantiomer was calculated using calibration curve.
3. RESULTS:
3.1.Method development:
3.1.1.Absorption spectra:
Metoprolol tartrate shows maximum absorption at 274 nm using mobile phase wich contains M-β-CD as a solvent Figure 2.
Figure 2.. Metoprolol tartrate(70mg/l) spectra using mobile phase as a solvent
3.1.2. Enantioseparation of metoprolol tartrate:
chromatographic conditiones were applied using mobile phase without any chiral selector (figure 3) showed one peak at retention time tR of 18.44 min, then the same conditions were applied using mobile phase contained M-β-CD (1.5 g/l) as a chiral selector showed two peaks at retention time of tR1=22.8min, tR2=31.1min ,resolution Rs=4.407 (figure 4)
Figure 3. Chromatogam of metoprolol tartrate (600mg/l), Mobile phase: aqueous solution/methanol of 86:14 (v/v/v), 0 g/l M -𝛽-CD, pH of 3.1 , flow rate of 0.5 mL/min, detection wavelength of 274nm,and injection volume of 20 𝜇L
Figure 4. Chromatogram of metoprolol tartrate enantiomers, Mobile phase: aqueous solution /methanol of 86:14 (v/v),1.5 g/l M -𝛽-CD, pH of 3.1 , flow rate of 0.5 mL/min, detection wavelength of 274nm,and injection volume of 20 𝜇L,racemic metoprolol tartrate (182.5, 500,800,1000 mg/l)
3.1.2.1. Determination of the Elution Order of (R)-metoprolol and (S)-metoprolol:
An aliquot of racemic metoprolol tartrate and (S)-metoprolol were dissolved in the mobile phase, and the mixture was submitted to chromatographic analysis under the above mentioned conditions. The two elution peak areas were compared, the later peak had a larger area, corresponding to (S)-metoprolol, as shown in figure 5.
Figure 5. Determination of elution order of metoprolol tartrate enantiomers
3.1.2.2. Effect of mobile phase composition on resolution:
The most suitable mobile phase composition for enantiomeric resolution of metoprolol tartrate is (aqueous solution :add to one liter of HPLC water 3.3 ml TEA the adjust the pH on 3.1 using glacial acetic acid) 86%: methanol 16%) and the other conditions are (M-𝛽-CD 1.5 g/l ,pH of 3.1, flow rate 0.5 ml/min, detection wavelength at 274nm, at room temperature) as shown in table 1.
Table 1: Effect of mobile phase composition on resolution
|
Rs (resolution) |
Composition of mobile phase (Aqueous solution : methanol) |
|
1.5 |
80:20 |
|
4.407 |
86:16 |
|
4.019 |
90:10 |
3.1.2.3. Effect of chiral selector concentration on resolution:
Table2 shows the effect of M -𝛽-CD concentration on resolution and the other conditions are (pH of 3.1, flow rate 0.5 ml/min, detection wavelength at 274nm, room temperature).
Table 2: Effect of M -𝛽-CD concentration on resolution
|
Concentration of M -𝛽-CD (g/l) |
Rs (resolution) |
|
1 |
No separation |
|
1.5 |
4.407 |
|
2 |
3.64 |
3.1.2.4. Effect of mobile phase pH on resolution:
Tables 3 shows the effect on the USP resolution(Rs) of variation in final pH value of mobile phase and the other conditions are (M -𝛽-CD 1.5 g/l, flow rate 0.5 ml/min, detection wavelength at 274nm, at room temperature).
Table 3: Effect of pH on resolution
|
Rs (resolution) |
pH of mobile phase |
|
|
2.7 |
|
4.407 |
3.1 |
|
3.98 |
3.5 |
3.1.3. Method validation [11,12]:
The proposed method was validated through the examination of System Suitability, linearity ,accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ).
3.1.3.1. System Suitability:
The system suitability was determined by calculating theoretical plates (N), retention factor, separation factor, resolution factor and tailing factor of enantiomers peaks resulted for the HPLC method analysis of racemic metoprolol tartrate. N was calculated as N=16(Rt/W)2, the retention factor (k) was calculated as (k=t R−t M/t M ), where, tR refer to the retention time of the analyte and tM refer to the elution time of the non-retained components. The selectivity factor (α) was calculated as the ratio of retention factors, α=k2/k1. The resolution factor (Rs) was calculated as Rs= tR2-tR1/0,5(w1+w2), where, tR1, tR2 refer to the retention time of the first and second enantiomers; w1 and w2 are the peak widths for the first and second eluting enantiomers, respectively. Tailing factor (T) was calculated as T=W0.05/2f where, W0.05 is the width of peak at 5% height from base line and f is the distance from peak maximum to the leading edge of the peak ,the previous parameters are shown in table 4.
Table 4 :System suitability:
|
|
N |
T |
tR |
K |
α |
Rs |
|
R enantiomer |
2386 |
1.7 |
22.8 min |
3.75 |
1.458 |
4.407 |
|
S enantiomer |
4392 |
0.9 |
31.1 min |
5.47 |
3.1.3.2. Linearity:
Linearity was examined by conducing triplicate injections at six concentration levels of racemic metoprolol tartrate (ranging from 182.5 to 1000 mg/L). Calibration curves of each enantiomer were linear over the tested concentration range. Regression equation of R enantiomer is y=10004-519663 with regression coefficients (R2) of 0.999, and y=12649x-716087 with R2of 0.9969 for (𝑆)-enantiomer as shown in figures 6,7.
Figur 6. Calibration curve of R enantiomer of metoprolol tartrate
Figure 7. Calibration curve of S enantiomer of metoprolol tartrate
3.1.3.3.Accuracy and precesion:
Three concentrations of racemic metoprolol tartrate were taken(182.5, 500,800)
Precision for both intra-and inter-day and accuracy (as recovery%) for the method were shown in Table 5, all of the %RSDs for intra- and inter-day assays were less than 2%, indicating that the chromatographic conditions and the method were valid and reproducible.