A spectrophotometric method for quantitative determination of ...

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Received: 13 April 2004; Accepted: 12 November 2004. DOI : 10.1134/S1061934806010084. Cite this article as: Azam Khan, M., Iqbal, Z., Rasul Jan, M. et al.

ISSN 1061-9348, Journal of Analytical Chemistry, 2006, Vol. 61, No. 1, pp. 32–36. © Pleiades Publishing, Inc., 2006.

ARTICLES

A Spectrophotometric Method for Quantitative Determination of Lactulose in Pharmaceutical Preparations Mir Azam Khan*, Zafar Iqbal**, Mohammad Rasul Jan*, Jasmin Shah*, Waqar Ahmad***, Zia U1 Haq****, and Obaidullah***** *Department of Chemistry, University of Peshawar, Peshawar-25120, Pakistan **Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan ***Department of Pharmacy, Malakand University, Peshawar-25120, Pakistan ****Libra Pvt Ltd. Industrial Estate, Peshawar *****Drug Control Administration, Ministry of Health, Peshawar Received April 13, 2004; in final form, November 12, 2004

Abstract—A simple spectrophotometric assay for the quantification of lactulose in pharmaceutical preparations was developed. The method is based on hydrolysis of lactulose under acidic conditions. The hydrolyzed product reacts with resorcinol, giving absorption peaks at 398 and 480 nm. Both absorption wavelengths can be used for the determination of lactulose. The limit of detection of lactulose at 398 nm and 480 nm was 0.075 µg mL–1 and 0.65 µg mL–1, respectively. The calibration was linear in the range of 5–25 µg mL–1. Analytical conditions were optimized, and the method was validated for analysis of pharmaceutical preparations. The determined amount of lactulose was found to be in good agreement with labeled claims in commercial products. The proposed method is economical, convenient, and suitable for the quantification of lactulose in pharmaceutical preparations. DOI: 10.1134/S1061934806010084 1

Lactulose is a diasaccharide consisting of one molecule of galactose and one molecule of fructose. Lactulose is soluble in water and very slightly soluble in alcohol [1]. Lactulose concentrate (D-Fructose, 4-O-B-Dgloacto furonosyl-lactulose) is a solution of sugars that consists principally of lactulose with minor quantities of lactose, galactose, and traces of other related sugars and water [2]. Lactulose has applications in medicine and food technology. It is used for the prevention and treatment of chronic constipation, portal systemic encephalopathy, and other intestinal and hepatical disorders [3]. Clinically, the ratio of lactulose–mannitol excretion in urine after administration of these nonmetabolized sugars has been used to evaluate the extent of malabsorption and intestinal permeability disruption in several infectious and nutritional diseases [4]. Several methods for the detrmination of lactulose have been reported, mainly based on enzymatic hydrolysis followed by spectrophotometric determination [5], automated enzymatic reactors with flow analysis [3], enzymatic fabricated electrodes [6], and enzymatic electrochemical biosensors [7]. Other reported methods are based on chromatographic methods such as high performance anion-exchange chromatography [4], HPLC with an electrochemical detector [8], and gas liquid chromatography [9]. Most of these methods are 1 The

reported for the analysis of lactulose in dairy products only. The development of the spectrophotometric method was based on the fact that glucose, galactose, and other related sugars present in lactulose solution are aldohexoses, while fructose, the hydrolyzed product of lactulose, is ketohexose, and this difference in functional groups was exploited for the determination of lactulose in pharmaceutical preparations. The investigated method was based on the fact that hydrochloric acid hydrolyzes lactulose into fructose and glucose followed by dehydration and that subsequent reaction of the resulting product with resorcinol gives colored condensation product [10]. The aim of the present work is to develop an easy and economical spectrophotometric method for detrmination of lactulose in pharmaceutical preparations. EXPERIMENTAL Materials. All chemicals were of analytical grade and obtained as follows: resorcinol from BDH (England), hydrochloric acid from Merck (Germany), and lactulose from sigma. Instruments. Analysis was carried out using double beam UV–visual spectrophotometers (a UV-1601 Shimadzu Japan and a Hitachi U2000 spectrophotometer) and an HPLC (Perkin Elmer equipped with an RI detector).

text was submitted by the authors in English.

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A SPECTROPHOTOMETRIC METHOD FOR QUANTITATIVE DETERMINATION

Preparation of solutions. Resorcinol solution (0.05%): resorcinol (50 mg) was dissolved in concentrated hydrochloric acid and the volume was adjusted to 100 mL with concentrated hydrochloric acid. Standard lactulose solution. Standard lactulose stock solution was prepared by dissolving the lactulose in the distilled water, and then concentration was adjusted to 5000 µg/mL with the same solvent. Various dilutions were then prepared with the same solvent. Sample preparation. Sample solutions of lactulose 250 µg/mL were prepared by simple dilution using distilled water from various brands of lactulose commercial preparations—DISEC syrup (Libra Pvt. Ltd.), Floralac syrup (Platinum Pharmaceutical Pvt. Ltd.), and Lilac syrup (GETZ Pharma Pvt. Ltd.). Method of analysis. The lactulose solution (250 µg/mL, 5 mL) was mixed with the resorcinol solution (0.05%; 4 mL for studies at 480 nm and 8 mL for 398 nm) and heated in a boiling water bath for 20 min. These conditions were chosen after the optimization described below. A similar procedure was applied to the sample solution, and six replicates were done for each sample. The quantity of lactulose in each sample was calculated through application of the calibration curve. Optimization procedure. The acidic resorcinol solution was added to the standard lactulose solution and then heated in a boiling water bath for 15 min. The resulting orange–red-colored solution was scanned on a double beam spectrophotometer to obtain the absorption spectrum of the solution. The spectrum is shown in Fig. 1. Solutions with various concentrations of resorcinol (0.01–0.2%) were prepared in concentrated HCl. Then, resorcinol solution (4 mL) was mixed with lactulose solution (5 mL) and heated in a boiling water bath for 15 min. The absorbance of the resultant orange-colored solution was measured both at 398 and 480 nm. The appropriate amount of resorcinol (0.05%) was evaluated by mixing a varying volume of the resorcinol solution (1–8 mL) with lactulose solution (5 mL) and then heated and processed with a procedure similar to that described above. The lactulose sample solutions were prepared by mixing (5 mL, 250 µg/mL), with the resorcinol solution (0.05%; 8 mL for 398 nm and 4 mL for 480 nm) and heated in a boiling water bath for 5–30 min. Then the absorption of the resultant solutions was measured both at 398 and 480 nm. ESTIMATING THE ANALYTICAL CHARACTERISTICS OF THE METHOD Calibration curve. The instrumental response for lactulose was checked by measuring the absorbance of various standard solutions of lactulose. It was found to be linear in the range of 5–25 µg/mL (r = 0.999 and 0.998) at 398 and 480 nm, respectively. JOURNAL OF ANALYTICAL CHEMISTRY

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Absorbance 0.5 0.4 0.3 0.2 0.1 0

250

300

350

400

450 500 550 Wavelength, nm

Fig. 1. UV–visual absorption spectrum of the lactulose resorcinal complex.

Limit of detection. The limit of detection for lactulose was determined by preparing two dilution samples, 5 and 15 µg/mL. Each sample was analyzed ten times; then, the following equation was used to calculate the detection limits: limit of detection = standard concentration × 2(SD)/mean. The limits of detection for lactulose at 480 nm and 398 nm method were found to be 0.075 and 0.65 µg/mL, respectively. Recovery. A known quantity of lactulose was added to preanalyzed syrup samples, then mixed well by shaking, and again analyzed. The experiments were performed in triplicate, and the mean percent recovery was calculated. Precision. The precision of the method was determined by quantifying the lactulose in five replicates of the same sample of various commercial preparations. Variations between the results were determined by δ/M × 100, where δ is the standard deviation (±SD) and M is the mean concentration of lactulose in commercial preparation. The variation between the results for lactulose was ±0.18% (M 14.97 µg/mL; SD ±0.028). The low variations between the results showed that the method is appropriate for analysis of lactulose in pharmaceutical preparations. RESULTS AND DISCUSSION The presented method is based on the fact that hydrochloric acid hydrolyzes lactulose into fructose and glucose followed by dehydration and subsequent reaction of the resulting product with resorcinol. This leads to the formation of an orange–red-colored compound [10]. Resorcinol was used to quantify fructose formed from hydrolysis of lactulose; for convenience, it was dissolved in hydrochloric acid to carry out both steps, hydrolysis and reaction of resorcinol with fruc-

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MIR AZAM KHAN et al. Step-I CH2OH

CH2OH O OH

HOH2C OH

O

O

Acid hydrolysis

OH O

CH2OH HO O + OH

OH

HO CH2OH

OH

OH

OH

CH2OH Lactulose

Fructose

Galactose

Step-II HOH2C

O

OH

O

HO CH2OH

HCl –3H2O

OH Fructose

O

H

HO

Hydroxymethylfurfural

Step-III OH

O O HO

H

Red condensation product

+ OH

Hydroxymethylfurfural

Resorcinol

Fig. 2. Proposed chemical reaction for formation of colored condensation product from lactulose.

tose simultaneously. The proposed chemical reaction has three steps, as is shown in Fig. 2. In the first step, hydrochloric acid hydrolyzes lactulose into fructose and galactose. In the second step, lactulose degrades to Absorbance 0.6 0.5

480 nm 398 nm

hydroxymethylfurfural because, during dehydration of hexose sugars with concentrated acid, ketoses (fructose) are transformed into hydroxymethyl furfural much quicker than aldoses (glucose, mannose, or galacAbsorbance 0.6 0.5 0.4

0.4

480 nm 398 nm

0.3

0.3

0.2 0.2 0.1 0.1 0 0

0

1

2

3

4

5

0.01 0.02 0.03 0.04 0.05 0.10 0.20 0.25 0.30 0.40 Concentration, %

Fig. 3. Optimization of resorcinol concentration for determination of lactulose.

6 7 8 Volume, mL

Fig. 4. Optimization of resorcinol (0.05%) volume for determination of lactulose.

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A SPECTROPHOTOMETRIC METHOD FOR QUANTITATIVE DETERMINATION Absorbance 0.6

35

Absorbance 0.6

0.5 0.4

398 nm 0.4

0.3 0.2

480 nm 398 nm

0.1 0

480 nm

0.2 8 10 12 14 16 18 20 22 24 26 28 30 min

Fig. 5. Optimization of heating time for determination of lactulose.

10

0

tose). In the third step, the resorcinol reagent forms a red condensation product much more rapidly if the sugar is a ketose (fructose) as opposed to when it is an aldose (glucose, mannose, or galactose).

20 30 Concentration, µg/mL

Fig. 6: Range of linearity for determination of lactulose.

The UV–visible spectrum of the resulting colored solution showed two absorption peaks—one at 398 nm and the other at 480 nm. The spectrum is shown in Fig. 3. Both of these wavelengths are used in this study for the spectrophotometric determination of lactulose in pharmaceutical preparations.

398 nm, respectively; the results are shown in Fig. 4. Heating was chosen for uniform heat in titration flasks. The optimum heating time after mixing of the resorcinol (0.5%) with lactulose solution was 18 min for 480 nm and 20 min for 398 nm (Fig. 5).

Various parameters were optimized for the determination of lactulose. The studies showed that a resorcinol solution at 0.05% was the optimum concentration for the formation of colored complex; the results are shown in Fig. 3. It was also found that 4 and 8 mL of the resorcinol solution (0.05%) the optimum are the volumes required for color development for 480 and

The mean percent recovery of the lactulose for the present method at 398 nm was between 99.7 to 100.33%, and at 480 nm it was between 98.9 and 100.07%. The results are shown in Table 1. The instrumental response for lactulose was found to be linear in the concentration range of 5–25 µg/mL both at 398 (r = 0.999) and 480 nm (r = 0.998), as shown in Fig. 6. The

Table 1. Percent recovery of the fortified standard lactulose from commercial syrup preparations Std added (µg mL–1)

Wavelength 398 nm 398 nm 480 nm 480 nm

Std recovered (µg mL–1)

Percent recovery

7.6 9.97 7.505 9.89

100.33 99.7 98.9 100.066

7.5 10 7.5 10

Table 2. Comparison of the present method with the HPLC method of analysis for lactulose in various commercial syrup preparations Analytical Results Sample Disec syrup Floralac syrup Lilac syrup

UV–visible method

HPLC method [11]

398 nm

%

480 nm

%

Qty

%

3.27 ± 0.034 3.46 ± 0.04 3.44 ± 0.01

97.64 103.4 102.79

3.26 ± 0.033 3.48 ± 0.039 3.44 ± 0.056

97.64 104.00 103.4

3.32 ± 0.005 3.43 ± 0.030 3.39 ± 0.026

99.1 102.38 101.19

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limit of detection and limit of quantification were calculated for both selected wavelengths. The limit of quantification was 2.18 µg mL–1, and the limit of detection was 0.654 µg mL–1 for 480 nm. Similarly, the limit of quantification was 0.25 µg mL–1 and the limit of detection was 0.075 µg mL–1 for 398 nm. The presented method of analysis under the optimum conditions was applied for the determination of lactulose in local commercially available pharmaceutical preparations, and the results were compared with those obtained by existing methods (Table 2). REFERENCES 1. Tolman, K.G., in Remington, The Science and Practice of Pharmacy, Gennaro, A.R., Ed., 20th ed., Easton, PA: Mack, 2000. 2. United State Pharmacopoeia, Rockville, MD: U. S. Pharmacopeial Convention, 2000.

3. Mayer, M., Genrich, M., Kunnecke, W., and Bilitewski, U., Anal. Chem. Acta, 1996, vol. 324, p. 37. 4. Bao, Y., Silva, T.M., Guerrant, R.L., Lima, A.M., and Fox, J.W., J. Chromat. Biomed. Appl., 1996, vol. 685, p. 105. 5. Amine, A., Moscone, D., Bernardo, R.A., Marconi, E., and Pallesch, G., Anal. Chem. Acta, 2000, vol. 406, p. 217. 6. Sekine, Y. and Hall, E.A., Biosens. Bioelectron., 1998, vol. 13, p. 995. 7. Moscone, D., Bernardo, R.A., Marconi, E., Amine, A., and Palleschi, G., Analyst, 1999, vol. 124, p. 325. 8. Wring, S.A., Terry, A., Causon, R., and Jenner, W.N., J. Pharm. Biomed. Anal., 1998, vol. 16, no. 7, p. 1213. 9. Martinez-Augustin, O., Boza, J.J., Romera, J.M., and Gil, A., Clin. Biochem., 1995, vol. 28, p. 401. 10. Baum, S.J., Bowen, W.R., and Poulter, S.R., in Laboratory Exercises in Organic and Biological Chemistry, 2nd ed., New York: Macmillan, 1981, p. 110. 11. British Pharmacopoeia, London: The Stationary Office, 2002, vol. 1, part 2, p. 1015.

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