Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt

Corresponding Author: M. M. Tolba, Department of analytical chem¬istry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. Fax: ++20502247496; E-mail: manar2kareem@yahoo.com.

	ABSTRACT
	INTRODUCTION
	EXPERIMENTAL
	GENERAL PROCEDURES
	RESULTS AND DISCUSSION
	CONCLUSION
	REFERENCES

	ABSTRACT

  A simple and sensitive kinetic spectrophotometric method was established for the determination of acar¬bose and miglitol in bulk and in their pharmaceutical preparations using alkaline potassium permanganate as an oxidizing agent. the method involves determination of acarbose and miglitol by kinetic studies of their oxidation at room temperature for a fixed time of 15 minutes for acarbose and 25 minutes for miglitol. The absorbance of the colored manganate ion was measured at 610 nm. Alternatively, the kinetic decrease in the absorbance of permanganate upon addition of the studied drugs at 525 nm was also used. The absorbance¬concentration plot was rectilinear over the concentration range of 4 - 20 and 1-10 µg/ ml for acarbose and miglitol, respectively. The detection limits were 0.189 and 0.089 µg/ ml at 610 nm and 0.081 and 0.179 µg/ ml at 525 nm for acarbose and miglitol respectively. The method was successfully applied for the determination of these drugs in their dosage forms. the results obtained were in good agreement with those obtained with the reference methods.

KEY WORDS:    kinetic determination; spectophotometry; acarbose; miglitol; potassium permanganate; pharmaceuti¬cal analysis

	INTRODUCTION

  Acarbose(O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy- 3-(hydroxymethyl)-2-cyclohexen-1-yl] amino]-α -D-glucopyranosyl-(1->4)-O-α- D-glucopyranosyl-(1->4)- D-glucose) (1) is an oral alpha-glucosidase inhibitor, especially sucrase. It is given by mouth in the treatment of type 2 diabetes mellitus. It has also been studied for the treatment of reactive hypoglycemia, the dumping syndrome and certain types of hyperlipoproteinaemia (2). Miglitol (2R, 3R, 4R, -5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl)- 3,4,5-piperidine-triol) is a desoxynojirimycin derivative, also competitively inhibit glucoamylase and sucrase but has weak effects on pancreatic α-amylase (3).
  Some analytical methods have been reported for the determination of the studied drugs. The reported meth¬ods for acarbose include Gas Chromatography - Mass spectrophotometry(GC-MS) (4), High Performance Liquid Chromatography(HPLC) (5, 6) and Capillary Electrophoresis(C.E.) (6, 7). As for miglitol, the reported methods were HPLC-MS (8) and C.E. (9).
  To the best of our knowledge, no spectrophotometric methods have been reported for the analysis of acar¬bose and miglitol up till now. The results obtained were promising.
  Several analytical techniques such as, spectrophotom¬etry, chromatography, capillary electrophoresis, kinetic fluorimetry, flow injection analysis and chemilumines¬cence were utilized for selective oxidation and determina¬tion of many pharmaceutical compounds in formulations using potassium permanganate as reagent viz, benzene¬diols and 1,2,4 benzenetriol (10), perchloroethylene (11), arsenic (III) (12), methotrexate (13), propranolol (14), tet¬racyclines residues (15), psilocin and psilocybin (16), cap¬topril (17), nickel ions (18), cefprozil (19), tramadol hydro¬chloride (20), ramipril (21), isoxsuprine (22), triprolidine (23), norfloxacin (24), fungicidal ethylenebisdithiocarba¬mate (25).
  The aim of the present work was to study the reaction between the studied drugs with potassium permanganate in alkaline medium kinetically in an attempt to evaluate them in their dosage forms. The proposed method was simple and did not need sophisticated instruments or spe¬cial skill, sensitive, rapid and readily adaptable to both the bulk drug and dosage forms.

	EXPERIMENTAL

Reagents

  All chemicals used were of analytical reagent grade and the solvents were of spectroscopic grade.Potassium permanganate (Merck, Germany), 1 × 10-2 M and 7.6 × 10-3 M aqueous solutions.Sodium hydroxide (BDH, UK), 0.5 M aqueous solution.

Materials

  The different pharmaceutical preparations were pur¬chased from the commercial source in the local market. They include: Acarbose was kindly offered from Alkan Pharma S.A.E., Egypt under licence of Bayer-Leverkusen, Germany.Miglitol was kindly offered from Sigma Pharmaceutical Industries, Egypt.
  Glucobay 50 tablets, labeled to contain 50 mg acarbose/ tablet, Batch # 080. The product of Alkan Pharma S.A.E., Egypt under licence of Bayer- Leverkusen, Germany.
  Glyset tablets, labeled to contain 50mg miglitol/ tablet, Batch # 6191668. The product of Bayer Company, USA.

Stock solutions

  Stock solutions of acarbose and miglitol were pre¬pared by dissolving 100.0 mg of the studied drugs in 100 ml distilled water. Other concentrations were prepared by further dilution with distilled water. These solutions alsowere found stable for at least three days without alteration when kept in the refrigerator.

Apparatus

  UV-1601, Shimadzu recording spectrophotometer (P/ N 206-67001) equipped with kinetic accessory provided with temperature controlled cell (TCC-240A) thermo¬electric temperature. Recording range, 0-1; wavelength, 610 and 525 nm; factor 1; number of cell, 1; reaction time (min.) 15, 25 min.; cycle time, 0.1 min.

	GENERAL PROCEDURES

Construction of the kinetic calibration graphs

  Aliquot solutions containing 40-200 µg of standard acarbose and 10-100 µg of miglitol solutions were trans¬ferred into a series of 10-ml volumetric flasks. 1 ml of 0.5 M NaOH, followed by 1 or 2.5 ml of 1 × 10-2 M potassium permanaganate for acarbose and miglitol, respectively at 610 nm or 0.5 ml of 7.6 × 10-3 M for both drugs at 525 nm were added. The mixture was shaken well and completed to volume with distilled water. The increase at 610 nm or the decrease at 525 nm in the absorbance was scanned during 15 and 25 min. for acarbose and miglitol, respec¬tively at ambient temperature (25°C) against an appropri¬ate blank, prepared simultaneously. The reaction order was obtained by plotting log reaction rate ('A/'t) over the specified time period versus log concentration of the drug. The calibration graphs and the regression equations were obtained by plotting the absorbance (A) or the difference in absorbance ('A) at the specified time versus concentra¬tion of the drug in µg/ml.

Procedure for the Determination of the Studied Compounds in Dosage Forms

  An accurately weighed quantity of the mixed contents of 10 powdered tablets equivalent to 100.0 mg of the drug was transferred into a 100 ml volumetric flask. The con¬tent of the flask was completed to 100 ml with distilled wa¬ter then sonicated for 15 minutes and filtered. An aliquot of the cited solutions was taken and the above procedure was applied. The nominal content was calculated either from a previously plotted calibration graph or using the regression equation.

RESULTS AND DISCUSSION

  Oxidation of the studied drugs (Fig. 1) with KMnO4 was carried out in presence of NaOH. Trials were made to determine the drug through oxidation with KMnO4 in neutral and acidic media, but no apparent reaction prod¬ucts were observed. Potassium permanganate in alkaline medium oxidized the studied drugs and yielded the green color of manganate radical, which absorbs maximally at 610 nm (Fig. 2). The intensity of the color was increased with time, and so, a kinetic method was developed for the determination of these drugs at 610 nm. An alternative ki¬netic method for the determination of both drugs based upon measuring the decrease in the absorbance of KMnO4 at 525 nm was also developed.

 

View larger version : [in a new window] Figure 1. The structure formulae for the studied drugs

 

View larger version : [in a new window] Figure 2. Absorption spectra of the studied drugs after reaction with KMnO4 /NaOH system. (a, b) The produced manganate ions after the reaction of KMnO4 with acarbose (20µg/ml) or miglitol(10µg/ml); (c, d) Oxidation products of acarbose and miglitol, respectively; (e) KMnO4 (1 × 10-3 M).

  Other oxidants were tested to determine the studied drugs, such as 10% H2O2, potassium persulphate in alka¬line medium and potassium periodate in strong acid me¬dium but all failed to give satisfactory results In case of H2O2 and persulphate, complete decomposition of the drug was observed, as revealed by the absence of any absorb¬ing species. In case of periodate, oxidation of the drug re¬sulted in hypsochromic shift and hypochromic effect, with maximum absorbance at 236 nm. and this was in agree¬ment with the reported results of oxidation of amino-alco¬hol compounds (26).

Study of Experimental Parameters

  The different experimental parameters affecting the formation of the oxidation product were studied. Variables were optimized by changing each in turn, while, keeping all others constant.
  Effect of Kmno4 concentration. The influence of KMnO4 concentration on the absorbance of the reaction product was studied using different volumes (0.2-3.5 ml) of 1 × 10-2 M KMnO4. The reaction rate and hence maximum absorbance increased with increasing KMnO4 concentra¬tion at 610 nm. It was found that at least 1 or 2.5 ml of 1 × 10-2 M KMnO4 was adequate for the maximum absor¬bance of acarbose and miglitol, respectively as shown in Fig. 3, 0.5 ml of 7.6 × 10-3 M was sufficient for measuring the decrease in the absorbance at 525 nm. for both drugs.

 

View larger version : [in a new window] Figure 3. Effect of KMnO4 on the absorbance intensity at 610 nm

  Effect of sodium hydroxide concentration. The in¬fluence of the concentration of NaOH on the absorbance of the reaction product was studied using different volumes (0.2-1.4 ml) of 0.5 M NaOH. It was found that increasing the volume of 0.5 M NaOH would increase A or 'A of the reaction up to 1 ml for both drugs at 610 nm. or 525 nm. after that NaOH has no effect on the absorbance as shown in Figs 4 & 5.

 

View larger version : [in a new window] Figure 4. Effect of NaOH on the absorbance intensity at 610 nm.

 

View larger version : [in a new window] Figure 5. Effect of NaOH on the absorbance intensity at 525 nm.

  Effect of temperature. The effect of temperature on the reaction rate was studied, it was found that, permanganate was reduced to manganate radical at room tem¬perature (25°C) while at higher temperatures, manganese dioxide was produced. Therefore, room temperature was selected as the optimum temperature.
  Effect of time. The effect of time on the reaction between KMnO4 and the studied drugs was studied. The absorbance of the reaction mixture was increased with time and never reach maximum in a reasonable time. Quantification was therefore made at fixed times of 15 min. foracarbose and 25 min. for miglitol (Figs. 6-9).

 

View larger version : [in a new window] Figure 6. Absorption versus time graphs for the reaction between acarbose and KMnO4 at 610 nm. a) 4 µg/ml b) 5 µg/ml c) 10 µg/ml d) 15 µg/ml e) 20 µg/ml.

 

View larger version : [in a new window] Figure 7. Absorption versus time graphs for the reaction between miglitol and KMnO4 at 610 nm. a) 1 µg/ml b) 2 µg/ml c) 4 µg/ml d) 6 µg/ml e) 8 µg/ml f) 10 µg/ml.

 

View larger version : [in a new window] Figure 8. Absorption versus time graphs for the reaction between acarbose and KMnO4 at 525 nm. a) Blank b) 4 µg/ml c) 5 µg/ml d) 10 µg/ml e) 15 µg/ml f) 20 µg/ml.

 

View larger version : [in a new window] Figure 9. Absorption versus time graphs for the reaction between miglitol and KMnO4 at 525 nm. a) Blank b) 1 μg/ml c) 2 μg/ml d) 4 μg/ml e) 6 μg/ml f) 8 μg/ml g) 10 μg/ml.

Evaluation of the Kinetic Parameters
  As mentioned above, the reaction between KMnO4 and the studied drugs never reach completion and a decision was made to apply a kinetic method for their determina¬tion. Consequently, the order of the reaction and reaction rate constants were determined at 610 and 525 nm.
  The rate of the reaction was found to be dependent on acarbose and miglitol concentrations. The rates were fol¬lowed at room temperature with various concentrations in the range of 4-20 μg/ml for acarbose and 1-10 μg/ml for miglitol keeping KMnO4 and NaOH concentrations constant at the recommended levels mentioned above. The reaction rate obeys the following equation:Rate of the reaction = t A = K`[drug]n (1) where K` is the pseudo-order rate constant and n is the order of the reaction.
  The rate of the reaction may be estimated by the variable time method measurement (27), where A is the absorbance and t is the time in seconds. Taking logarithms of rates and drug concentrations (Table 1), the previous equation is transformed into: log(rate) = log = + (2)
  Plot of log reaction rate versus log drug concentration at 610 nm and 525 nm gave the regression equation, correlation coefficient, pseudo-order rate constant and order of the reaction which are indicated in Table 1. These results indicate that the reaction is pseudo first order reaction in the drug concentration.

Selection of the best kinetic method

  Several kinetic techniques were adopted for the selection of the best method. Rate constant, fixed absorbance and fixed time methods (28. 29) were tried and the most suitable analytical method was selected taking into ac¬count the applicability, the sensitivity, i.e. the slope of the calibration graph and the correlation coefficient (r).
  Rate constant method. Graphs of log absorbance versus time for acarbose and miglitol concentration in the range of 6.20 × 10-6 - 3.10 × 10-5 M and 4.83 × 10-6-4.83 × 10-5 M, respectively were plotted and all appeared to be rectilinear. Pseudo-first order rate constants (K`) corresponding to different drug concentrations (C) were calculated from the slopes multiplied by –2.303 and are presented in Table 2.

  Regression of (C) versus K` gave equations: At 610 nm K` = -1.56 × 10-3 + 11.76 C (r=0.9900) for acarbose K` = -5.10 × 10-4 + 1.78 C (r=0.9850) for miglitol At 525 nm K`= -1.01 × 10-3 + 8.17C (r=0.9590) for acarbose K`= -1.03 × 10-3 + 3.78 C (r=0.9570) for miglitol where C is the molar concentration of the drugs.
  Fixed absorbance method. Reaction times required to reach specific absorbance of redox reaction for differ¬ent concentrations of acarbose and miglitol 1.55 × 10-5 – 3.10 × 10-5 M and 1.93 × 10-5 – 4.83 × 10-5 M, respective¬ly were recorded. A preselected value of the absorbance (0.2) for acarbose and miglitol was fixed and the time was measured in seconds. The reciprocal of time (1/t) versus the initial concentration of drug was plotted. Table 3 and the following equations of the calibration graphs were obtained: At 610 nm 1/t = -2.92 × 10-2 + 1847.57 C (r=0.9650) for acarbose 1/t = -5.89 × 10-3 + 402.04 C (r=0.9960) for miglitol At 525 nm 1/t = -1.38 × 10-2 + 1010.16 C (r=0.9750) for acarbose 1/t = -7.92 × 10-4 + 87.92 C (r=0.9960) for miglitol where C is the molar concentration of the drugs.

  Fixed time method. At a preselected fixed time, which was accurately determined, the absorbance was measured. Calibration graphs of the absorbance versus initial con¬centrations of acarbose and miglitol at fixed times of 15 and 25 min., respectively were established with the regres¬sion equations and correlation coefficients assembled in Table 4.

  It is clear that the slope increases with time and the most acceptable values of the correlation coefficient (r) was chosen as the most suitable time interval for the measurement.
  As a conclusion, the fixed time method was chosen for quantification because it gave the best correlation coeffi¬cient in a reasonable time.

Quantification

  After optimizing the reaction conditions, the fixed time method was applied to the kinetic determination of 4-20 µg/ml of acarbose and 1-10 µg/ml of miglitol in raw materials.
  Analysis of the data gave the following regression equations: At 610 nm A= -7.332 × 10-3 + 2.243 × 10-2 C (r = 0.9999) for acarbose A= -3.782 × 10-3 + 7.797 × 10-2 C (r = 0.9999) for miglitol At 525 nm A= -4.250 × 10-3 + 2.799 × 10-2 C (r = 0.9999) for acarbose A= 9.787 × 10-3 + 5.939 × 10-2 C (r = 0.9998) for miglitol where A is the absorbance and C is the concentration in μg/ml.
  IStatistical evaluation of the regression line gave the values of Sy/x, Sa, Sb which are indicated with the detection limits, quantification limits, % RSD and % Er. in Table 5. These small values point out to the high precision of the proposed methods.

  Statistical analysis of the results obtained by both the proposed and reference methods (30, 31) revealed no sig¬nificant difference regarding the accuracy and precision as indicated by the Student t-test and F test (32), as shown in Table 6. The reference method (30) for acarbose in¬volved an optical rotation method given by Alkan Pharma S.A.E., Egypt under licence of Bayer- Leverkusen, Ger¬many. While, the reference method (31) for miglitol was an HPLC method using acetonitrile and 25 mM sodium dibasic phosphate as mobile phase and detection at 205 nm. The data was provided by Sigma Pharmaceutical In¬dustries, Egypt.

  The proposed methods were successfully applied for the determination of the studied drugs in their different dosage forms. The results obtained were in a good agree¬ment with the reference methods (30, 31), as shown in Table 7.

Mechanism of the reaction
  The data used in the optimization of KMnO4 concen¬tration and the data of the calibration graphs were used to calculate the stoichiometry of the reaction adopting the limiting logarithmic method (33).
  The ratio of the reaction between acarbose or miglitol and KMnO4 in alkaline medium was calculated by divid¬ing the slope of KMnO4 curve over the slope of the drug curve (Figs. 10 & 11). It was found that, the ratios were 1.012: 0.951 for acarbose and 0.943: 0.990 for miglitol pointing out to a ratio of 1:1 (KMnO4 to drug).

 

View larger version : [in a new window] Figure 10. Stoichiometry of the reaction between acarbose and KMnO4 adopting limiting logarithmic method (33). A) Variable concentrations of acarbose at constant KMnO4 concentration; b) Variable concentrations of KMnO4 at constant acarbose con¬centration.

 

View larger version : [in a new window] Figure 11. Stoichiometry of the reaction between miglitol and KMnO4 adopting limiting logarithmic method (33). A) Variable concentrations of miglitol at constant KMnO4 concentration; b) Variable concentrations of KMnO4 at constant miglitol concen¬tration.

 

View larger version : [in a new window] Figure 12. The proposed pathway for the reaction between the studied drugs and potassium permanganate in alkaline medium.

	CONCLUSION

  The proposed methods are simple, accurate, precise, sensitive, rapid, low cost and selective compared to the reference methods (30, 31).
  Furthermore, the proposed methods don’t require elaboration of procedures, which are usually associated with chromatographic methods. The proposed methods were applied successfully for determination of the stud¬ied compounds in raw material as well as in different dos¬age forms. The only limitation for this method, if used in other pharmaceutical preparations containing antioxidant which will cause interference and this can be solved by using suitable solvent extraction.
  From the above study some specific advantages in the application of kinetic methods can be expected (35): ? Selectivity due to the measurement of the evolution of the absorbance with the time of reaction instead of the measure of a concerete absorbance value. ? Possibility of no interference of other absorbent active compounds present in the commercial product, if they are exhibiting stability in the chemical reaction condi¬tions established for the proposed kinetic method. ? Possibility of no interference of the colored and /or tur¬bidity back ground of the sample.

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