Spectrophotometric Determination of Lactulose in Pharmaceutical Preparations Using Acid Hydrolysis

by Mir Azam Khan, Mohammad Rasul Jan, Jasmin Shah, Zafar Iqbal, and Hamayun Khan
Lactulose concentrate U.S.P. (4-O-B-D-Galactopyranosyl D-fructose) is a sugar solution consisting principally of lactulose and minor quantities of lactose, galactose, and traces of other related sugars and water.1 Lactulose is a disaccharide that contains one molecule of galactose and one molecule of fructose. It may be prepared by epimerization of lactose in limewater medium. Lactose is a disaccharide containing one molecule of galactose and one molecule of glucose. The commercially available lactulose syrup is a light yellow to dark yellow, viscous, sweet liquid. Lactulose is soluble in water and slightly soluble in alcohol.2 Lactulose nds application in medicine and food technology. It is utilized in infant formula and in the prevention and treatment of chronic constipation, portal systemic encephalopathy, and other intestinal and hepatic disorders. 3 Lactulose is formed during milk heat treatment and has been proposed by the International Dairy Federation and European Commission as an analytical index to distinguish ultrahigh-temperature (UHT) milk from in-container sterilized milk.4 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.5 All of the important applications of lactulose in various fields demonstrate the need for simpler, reliable methods for quantitative determination in biological uids, pharmaceutical preparations, and dairy products. Several reported methods have been based mainly on enzymatic hydrolysis followed by spectrophotometric determination,4 an automated enzymatic reactor with ow analysis,3 an enzymatic fabricated electrode,6 and an enzymatic electrochemical biosensor.7 Other techniques center on chromatography, such as high-performance anion exchange chromatography,5 HPLC with electrochemical detection,8 and gasliquid chromatography. 9,10 All of the above-mentioned methods are time consuming, difcult to use, and expensive. Moreover, most of the reported analytical methods for lactulose apply to dairy products only. This paper describes an easy spectrophotometric method for the determination of lactulose in pharmaceutical preparations. The method is based on acid hydrolysis of lactulose without the use of enzymes. The technique is precise and accurate, and is suitable for pharmaceutical preparations without interfering with other excipients.

Experimental
Instrumentation
Absorbance was measured using a UV-1601 UV-VIS spectrophotometer and analytical balance model LIBROR EB420H, (Shimadzu, Tokyo, Japan). The effect of temperature was determined with a thermostatic water bath (Thermostat, China National Machinery and Equipment, Beijing, China).

Chemicals and reagents

All chemicals used were of analytical reagent-grade purity: 1. Sulfuric acid (6N). Sulfuric acid 6N (Merck KGaA, Darmstadt, Germany) was prepared by diluting 40.8 mL concentrated sulfuric acid to 250 mL distilled water. 2. Standard lactulose solution. Standard lactulose stock solution (5000 g/mL) was prepared in distilled water. Through dilution with distilled water, 1000 g/mL and nally 500 g/mL working standard lactulose solutions were prepared. 3. Sample lactulose preparation. Sample lactulose soluAMERICAN LABORATORY JULY 2004 41

ACID HYDROLYSIS continued

tion (500 g/mL) was prepared in distilled water from Disec syrup (Libra private Ltd., N.W.F.P, Pakistan), Floralac syrup (Platinum Pharmaceutical private Ltd., Karachi, Pakistan), and Lilac syrup (GETZ Pharma private Ltd., Karachi, Pakistan).

Procedure
A calibration graph was constructed by transforming standard lactulose solution (525 g/mL lactulose) into glass-stoppered flasks to which 10 mL of 6N sulfuric acid was added. The flasks were heated for 70 min in a boiling water bath. The solutions were cooled and diluted to 100 mL with distilled water. The absorbance was measured at 284 nm using 1-cm quartz cells. The same procedure was applied for blank and lactulose in the pharmaceutical preparations. To check the reproducibility of the method, the percent recovery test was applied for the determination of lactulose. A preanalyzed sample of lactulose was taken and a known standard was added. The drug samples were fortified at levels ranging from 7.5 to 12.5 g/mL of standard drug.

Figure 1

Acid hydrolysis of lactulose.

Results and discussion

The determination of lactulose is based on the assumption that this compound is the only source of fructose in pharmaceutical preparations, and fructose is produced only by the hydrolysis of lactulose2 (Figure 1). The disaccharides may be hydrolyzed to monosaccharides through acid hydrolysis. In the case of lactose, the resulting monosaccharides are primarily glucose and galactose. If lactulose is used in the initial feedstock, then fructose and galactose will be the resulting monosaccharides. 11 Lactulose, on reacting with sulfuric acid through heating, will hydrolyze into fructose and galactose followed by rearrangement in the molecule to produce a compound with absorb42 JULY 2004 AMERICAN LABORATORY

Figure 2

Absorption spectra of the reaction product after acid hydrolysis of lactulose.

Figure 3

Effect of acid normality on hydrolysis of lactulose.

ance in the ultraviolet region. The mixture provides maximum absorbance at 284 nm (Figure 2), which is useful for the determination of lactulose spectrophotometrically. Various parameters were optimized for the determination of lactulose.

Effect of acidity on the hydrolysis of lactulose

An increase in absorbance was determined in the range of 16N sulfuric acid. Above 6N, constant absorbance was obtained, as shown in Figure 3. Other mineral acids were tested and found to be unsatisfactory. The volume of 6N sulfuric acid was studied in the range of 520 mL. Constant absorbance was obtained in the range of 1020 mL. Hence, 10 mL of 6N sulfuric acid was used for the acid hydrolysis of lactulose (Figure 4).

Figure 4

Effect of volume of 6N sulfuric acid on the acid hydrolysis of lactulose.

Effect of heating time on the acid hydrolysis of lactulose

The effect of time on the acid hydrolysis of lactulose at a temperature of 100 C was studied from 30 to 90 min; the results are shown in Figure 5. The maximum concentration of hydrolyzed product formed at a heating time of 70 min.

Figure 5

Effect of heating time on the acid hydrolysis of lactulose.

Analytical data
Beers law was observed in the range of 0.525 g/mL of lactulose following acid hydrolysis (Figure 6). The molar absorptivity for the hydrolyzed product was found to be 5.3 104 L/mol/1cm. The standard deviation and coefficient of variation of the absorbance were found to be 0.05 and 5.37% for 0.5 g/mL lactulose. The limit of detection and limit of quantification were 0.156 and 0.5 g/mL, respectively.

Effect of interfering species

Figure 6 Range of linearity for the determination of lactulose.

The effect of lactose on the deterAMERICAN LABORATORY JULY 2004 43

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Table 1

Recovery efciencies obtained in the fortied drug samples (n = 3)

Standard added (g/mL) 12.5 10 7.5 Total found (g/mL) 23.22 20.45 18.06 Recovered (g/mL) 12.95 10.18 7.79 Percent recovery 103.6% 2.2% 101.8 3.1% 103% 5.9%

Known sample (g/mL) 10.27 10.27 10.27

Table 2

Application of investigated spectrophotometric method for the determination of lactulose in pharmaceutical preparations
Label claim/5 mL 3.35 g of lactulose 3.35 g of lactulose 3.35 g of lactulose Found/5 mL 3.27 0.0815 g 3.325 0.034 g 3.33 0.076 g
2. 3. 4. 5. 6. 7. 8.

Sample name Disec syrup Lilac syrup Floralac syrup

mination of lactulose using the proposed method was studied by adding a known amount of lactose to a solution containing 10 g/mL lactulose. No significant interference was observed.

Percent recovery
The recoveries of standard drug from the fortied drug samples varied between 101.8 5.9% and 103.6 2.2% (Table 1).

Application to pharmaceutical preparations

The investigated method was applied to the determination of lactulose in local, commercially available pharmaceutical preparations. The results are shown in Table 2. The quantity of lactulose using the method provides reliable results following the acid hydrolysis of lactulose.

9. 10. 11.

1. USP-24 NF-19. United States Pharmacopeial Convention Inc., Rockville, MD, 2000. The Science and Practice of Pharmacy, 20th ed. Gennaro AR, ed. Easton, PA: Mack Publishing Co., 2000:1271. Mayer M, Genrich M, Kunnecke W, Bilitewski U. Anal Chim Acta 1996; 324:3745. Damascene AA, Bernardo RA, Marconi E, Palleschi G. Anal Chim Acta 2000; 406:21724. Bao Y, Silva TMJ, Guerrant RL, Lima AAM, Fox JW. J Chromatogr B 1996; 685:10512. Sekine Y, Hall EAH. Biosens Bioelectron 1998; 13:9951005. Moscone D, Bernardo RA, Amine E, Palleshi G. Analyst 1999; 124:3259. Wring SA, Terry A, Causon R, Jenner WN. J Pharmaceut Biomed Anal 1998; 16:121324. Martinez O, Julio A, Boza J, Romera JM, Gil A. Clin Biotech 1995; 28:4015. De Block J, Merchiers M, Rentergham RV, Moermants R. Int Dairy J 1996; 6:21722. U.S. patent appl 20020169344. Kind Code: Nov 14, 2002.

Percentage 98.0 2.40% 99.25 1.01% 99.4 2.26%

and reproducible. It has demonstrated potential for the detection of lactulose in bulk and pharmaceutical preparations. The method can find wide application in the pharmaceutical industry, and can easily be modified for the analysis of lactulose in other fields, such as the dairy industry.

References

Conclusion
The method described here is very simple, precise,

Mr. Azam Khan, Dr. Rasul Jan, and Dr. Shah are with the Department of Chemistry, University of Peshawar, N.W.F.P., Pakistan; tel./fax: +92 91 9216652; e-mail:jasminshah2001@yahoo.com. Dr. Iqbal is with the Department of Pharmacy, University of Peshawar, N.W.F.P., Pakistan. Mr. Khan is with the Department of Chemical Engineering, Kyunpook, National University, Taegue, South Korea.

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