Food Analytical Methods

, Volume 9, Issue 8, pp 2210–2222 | Cite as

Quantification of Lactulose and Epilactose in the Presence of Lactose in Milk using a dual HPLC analysis

  • Eva Rentschler
  • Beatrice Kuschel
  • Manuel Krewinkel
  • Wolfgang Claaßen
  • Claudia Glück
  • Bo Jiang
  • Wanmeng Mu
  • Timo StresslerEmail author
  • Lutz Fischer


The valuable lactose derivatives lactulose and epilactose can be derived from lactose either by the Lobry de Bruyn-Alberda van Ekenstein transformation during heat treatments or by enzymatic conversion using cellobiose 2-epimerases (EC The chromatographic determination of lactose, lactulose, and epilactose in milk is challenging, due to the variable ratio of the three saccharides and their similar retention properties. In this work, a dual high-performance liquid chromatography (HPLC) analysis for the quantification of lactose, lactulose, and epilactose in milk samples was developed and validated. The samples originated from an enzymatic lactose conversion using the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Application of this enzyme led to the formation of high lactulose concentrations (28.0 g/L) in milk. The dual HPLC analysis utilized a combination of two chromatographic separation techniques, configured in two parallel systems. After precolumn derivatization, the samples were analyzed as follows: Method 1 determined the concentration of lactose and epilactose using a C18 column with an ion-pair reagent as eluent, coupled with a UV detector. Method 2 determined the concentration of lactulose using a trimodal stationary phase (hydrophilic interaction, anion- and cation-exchange properties) with acetonitrile/ammonium formiate buffer as eluent, coupled with an evaporative light scattering detector. Both methods were validated in terms of linearity, precision and recovery. The revealing detection limits in the milk samples were 3.32 mg/L for lactose, 4.73 mg/L for epilactose and 139 mg/L for lactulose. The dual HPLC analysis presented allows accurate lactose, lactulose, and epilactose separation in complex food matrices such as milk.


Precolumn derivatization Ion-pair HPLC HILIC/IEX HPLC Lactulose Epilactose Milk 



Many thanks go to Michael Merz and Prof. Jörg Hinrichs (Institute of Food Science and Biotechnology) for helpful discussions.

Compliance with Ethical Standards

Conflict of interest

Eva Rentschler declares that she has no conflict of interest. Beatrice Kuschel declares that she has no conflict of interest. Manuel Krewinkel declares that he has no conflict of interest. Wolfgang Claaßen declares that he has no conflict of interest. Claudia Glück declares that she has no conflict of interest. Bo Jiang declares that he has no conflict of interest. Wanmeng Mu declares that he has no conflict of interest. Timo Stressler declares that he has no conflict of interest. Lutz Fischer declares that he has no conflict of interest. This article does not contain any studies with human or animal subjects.

Supplementary material

12161_2016_405_MOESM1_ESM.pdf (265 kb)
ESM 1 (PDF 264 kb)


  1. Abbott SR (1980) Practical aspects of normal-phase chromatography. J Chromatogr Sci 18:540–550CrossRefGoogle Scholar
  2. Aït-Aissa A, Aïder M (2014) Lactulose: production and use in functional food, medical and pharmaceutical applications. Practical and critical review. Int J Food Sci Technol 49:1245–1253CrossRefGoogle Scholar
  3. Alpert AJ (1990) Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J Chromatogr A 499:177–196CrossRefGoogle Scholar
  4. Alpert AJ, Shukla M, Shukla AK, Zieske LR, Yuen SW, Ferguson MA, Mehlert A, Pauly M, Orlando R (1994) Hydrophilic-interaction chromatography of complex carbohydrates. J Chromatogr A 676:191–22CrossRefGoogle Scholar
  5. Anumula KR (1994) Quantitiative determination of monosaccharides in glycoproteins by HPLC with highly sensitive fluorescence detection. Anal Biochem 220:275–283CrossRefGoogle Scholar
  6. Borch RF, Bernstein MD, Durst HD (1971) Cyanohydridoborate anion as a selective reducing agent. J Am Chem Soc 93:2897–2904CrossRefGoogle Scholar
  7. Brons C, Olieman C (1983) Study of the high-performance liquid chromatographic separation of reducing sugars, applied to the determination of lactose in milk. J Chromatogr A 259:79–86CrossRefGoogle Scholar
  8. Cataldi TRI, Angelotti M, Bufo SA (1999a) Method development for the quantitative determination of lactulose in heat-treated milks by HPAEC with pulsed amperometric detection. Anal Chem 71:4919–4925CrossRefGoogle Scholar
  9. Cataldi TRI, Campa C, Angelotti M, Bufo SA (1999b) Isocratic separations of closely-related mono-and disaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection using dilute alkaline spiked with barium acetate. J Chromatogr A 855:539–550CrossRefGoogle Scholar
  10. Charlesworth JM (1978) Evaporative analyzer as a mass detector for liquid chromatography. Anal Chem 50:1414–1420CrossRefGoogle Scholar
  11. Chávez-Servín JL, Castellote AI, López-Sabater MC (2004) Analysis of mono- and disaccharides in milk-based formulae by high-performance liquid chromatography with refractive index detection. J Chromatogr A 1043:211–215CrossRefGoogle Scholar
  12. Cortacero-Ramírez S, Segura-Carretero A, Cruces-Blanco C, Hernáinz-Bermúdez de Castro M, Fernández-Gutiérrez A (2004) Analysis of carbohydrates in beverages by capillary electrophoresis with precolumn derivatization and UV detection. Food Chem 87:471–476CrossRefGoogle Scholar
  13. De Block J, Merchiers M, Van Renterghem R, Moermans R (1996) Evaluation of two methods for the determination of lactulose in milk. Int Dairy J 6:217–222CrossRefGoogle Scholar
  14. Erich S, Anzmann T, Fischer L (2012) Quantification of lactose using ion-pair RP-HPLC during enzymatic lactose hydrolysis of skim milk. Food Chem 135:2393–2396CrossRefGoogle Scholar
  15. Fischer K, Wacht M, Meyer A (2003) Simultaneous and sensitive HPLC determination of mono- and disaccharides, uronic acids, and amino sugars after derivatization by reductive amination. Acta Hydrochim Hydrobiol 31:134–144CrossRefGoogle Scholar
  16. Förster-Fromme K, Schuster-Wolff-Bühring R, Hartwig A, Holder A, Schwiertz A, Bischoff SC, Hinrichs J (2011) A new enzymatically produced 1-lactulose: a pilot study to test the bifidogenic effects. Int Dairy J 21:940–948CrossRefGoogle Scholar
  17. Gama MR, da Costa Silva RG, Collins CH, Bottoli CBG (2012) Hydrophilic interaction chromatography. TrAC Trends Anal Chem 37:48–60CrossRefGoogle Scholar
  18. GDCh (2005) Positionspapier der lebensmittelchemischen Gesellschaft zu den Angaben “laktosefrei” und “laktosearm”. Lebensmittelchemie 59:45–46Google Scholar
  19. Grill E, Huber C, Oefner P, Vorndran A, Bonn G (1993) Capillary zone electrophoresis ofp-aminobenzoic acid derivatives of aldoses, ketoses and uronic acids. Electrophoresis 14:1004–1010CrossRefGoogle Scholar
  20. Hase S (1996) Precolumn derivatization for chromatographic and electrophoretic analyses of carbohydrates. J Chromatogr A 720:173–182CrossRefGoogle Scholar
  21. Hase S, Hara S, Matsushima Y (1979) Tagging of sugars with a fluorescent compound, 2-aminopyridine. J Biochem 85:217–220Google Scholar
  22. Hernández-Hernández O, Montañés F, Clemente A, Moreno FJ, Sanz ML (2011) Characterization of galactooligosaccharides derived from lactulose. J Chromatogr A 1218:7691–6CrossRefGoogle Scholar
  23. ICH (1996) Validation of analytical procedures: text and methodology Q2 (R1).Google Scholar
  24. Jandera P (2011) Stationary and mobile phases in hydrophilic interaction chromatography: a review. Anal Chim Acta 692:1–25CrossRefGoogle Scholar
  25. Karlsson G, Winge S, Sandberg H (2005) Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection. J Chromatogr A 1092:246–249CrossRefGoogle Scholar
  26. Kazarian AA, Taylor MR, Haddad PR, Nesterenko PN, Paull B (2013) Ion-exchange and hydrophobic interactions affecting selectivity for neutral and charged solutes on three structurally similar agglomerated ion-exchange and mixed-mode stationary phases. Anal Chim Acta 803:143–153CrossRefGoogle Scholar
  27. Kazarian AA, Nesterenko PN, Soisungnoen P, Burakham R, Srijaranai S, Paull B (2014) Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography. J Sep Sci 37:2138–2144CrossRefGoogle Scholar
  28. Kim Y-S, Oh D-K (2012) Lactulose production from lactose as a single substrate by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Bioresour Technol 104:668–672CrossRefGoogle Scholar
  29. Ko J-H, Huang H, Kang G-W, Cheong W-J (2005) Simultaneous quantitative determination of monosaccharides including fructose in hydrolysates of yogurt and orange juice products by derivatization of monosaccharides with p-aminobenzoic acid ethyl ester followed by HPLC. Bull Korean Chem Soc 26:1533–1538CrossRefGoogle Scholar
  30. Kohler M, Haerdi W, Christen P, Veuthey J-L (1997) The evaporative light scattering detector: some applications in pharmaceutical analysis. TrAC Trends Anal Chem 16:475–484CrossRefGoogle Scholar
  31. Krewinkel M, Kaiser J, Merz M, Rentschler E, Kuschel B, Hinrichs J, Fischer L (2015) Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose. J Dairy Sci 98:3665–3678CrossRefGoogle Scholar
  32. Kubica P, Kot-Wasik A, Wasik A, Namieśnik J, Landowski P (2012) Modern approach for determination of lactulose, mannitol and sucrose in human urine using HPLC–MS/MS for the studies of intestinal and upper digestive tract permeability. J Chromatogr B 907:34–40CrossRefGoogle Scholar
  33. Lamari FN, Kuhn R, Karamanos NK (2003) Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis. J Chromatogr B Anal Technol Biomed Life Sci 793:15–36CrossRefGoogle Scholar
  34. Levene H (1960) Robust tests for equality of variances. In: Olkin I (ed) Contributions to Probability and Statistics: Essays in Honor of Harold Hotelling. Stanford Univ. Press, Palo Alto, CA, pp 278–292Google Scholar
  35. Liu X, Pohl CA (2010) HILIC behavior of a reversed-phase/cation-exchange/anion-exchange trimode column. J Sep Sci 33:779–786CrossRefGoogle Scholar
  36. Liu X, Pohl CA (2012) Comparison of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases and their use in active pharmaceutical ingredient and counterion determinations. J Chromatogr A 1232:190–195CrossRefGoogle Scholar
  37. Liu X, Pohl C, Woodruff A, Chen J (2011) Chromatographic evaluation of reversed-phase/anion-exchange/cation-exchange trimodal stationary phases prepared by electrostatically driven self-assembly process. J Chromatogr A 1218:3407–3412CrossRefGoogle Scholar
  38. Liu X, Pohl C, Chen J (2014) HILIC/anion-exchange/cation-exchange multimodal media. Pat. Appl. Publ. 13/918,481:Google Scholar
  39. Manzi P, Pizzoferrato L (2013) HPLC determination of lactulose in heat treated milk. Food Bioprocess Technol 6:851–857CrossRefGoogle Scholar
  40. Márquez-Sillero I, Cárdenas S, Valcárcel M (2013) Comparison of two evaporative universal detectors for the determination of sugars in food samples by liquid chromatography. Microchem J 110:629–635CrossRefGoogle Scholar
  41. Martinez-Castro I, Olano A (1980) Influence of thermal processing on carbohydrate composition of milk. Formation of epilactose. Milchwissenschaft 35:5–8Google Scholar
  42. Martinez-Castro I, Calvo MM, Olano A (1987) Chromatographic determination of lactulose. Chromatographia 23:132–136CrossRefGoogle Scholar
  43. Mathews BT, Higginson PD, Lyons R, Mitchell JC, Sach NW, Snowden MJ, Taylor MR, Wright AG (2004) Improving quantitative measurements for the evaporative light scattering detector. Chromatographia 60:625–633CrossRefGoogle Scholar
  44. Meyer A, Raba C, Fischer K (2001) Ion-pair RP-HPLC determination of sugars, amino sugars, and uronic acids after derivatization with p-aminobenzoic acid. Anal Chem 73:2377–2382CrossRefGoogle Scholar
  45. Montilla A, Moreno FJ, Olano A (2005) A reliable gas capillary chromatographic determination of lactulose in dairy samples. Chromatographia 62:311–314CrossRefGoogle Scholar
  46. Moreno FJ, Montilla A, Villamiel M, Corzo N, Olano A (2014) Analysis, structural characterization, and bioactivity of oligosaccharides derived from lactose. Electrophoresis 35:1519–34CrossRefGoogle Scholar
  47. Mourey TH, Oppenheimer LE (1984) Principles of operation of an evaporative light-scattering detector for liquid chromatography. Anal Chem 56:2427–2434CrossRefGoogle Scholar
  48. Mu W, Li Q, Fan C, Zhou C, Jiang B (2013) Recent advances on physiological functions and biotechnological production of epilactose. Appl Microbiol Biotechnol 97:1821–1827CrossRefGoogle Scholar
  49. Nelofar A, Laghari AH, Yasmin A (2010) Validated HPLC-RI method for the determination of lactulose and its process related impurities in syrup. Indian J Pharm Sci 72:255–258CrossRefGoogle Scholar
  50. Nishimukai M, Watanabe J, Taguchi H, Senoura T, Hamada S, Matsui H, Yamamoto T, Wasaki J, Hara H, Ito S (2008) Effects of epilactose on calcium absorption and serum lipid metabolism in rats. J Agric Food Chem 56:10340–10345CrossRefGoogle Scholar
  51. Oefner PJ, Chiesa C (1994) Capillary electrophoresis of carbohydrates. Glycobiology 4:397–412CrossRefGoogle Scholar
  52. Olano A, Calvo MM (1989) Kinetics of lactulose, galactose and epilactose formation during heat-treatment of milk. Food Chem 34:239–248CrossRefGoogle Scholar
  53. Olano A, Corzo N (2009) Lactulose as a food ingredient. J Sci Food Agric 89:1987–1990CrossRefGoogle Scholar
  54. Panesar PS, Kumari S (2011) Lactulose: production, purification and potential applications. Biotechnol Adv 29:940–948CrossRefGoogle Scholar
  55. Pereira da Costa M, Conte-Junior CA (2015) Chromatographic methods for the determination of carbohydrates and organic acids in foods of animal origin. Compr Rev Food Sci Food Saf 14:586–600CrossRefGoogle Scholar
  56. Petuely F (1957) Bifidusflora bei Flaschenkindern durch bifidogene Substanzen (Bifidusfaktor). Dtsch Med Wochenschr 79:174–179Google Scholar
  57. Prodolliet J, Hischenhuber C (1998) Food authentication by carbohydrate chromatography. Zeitschrift für Leb und -forsch A 207:1–12CrossRefGoogle Scholar
  58. Reimerdes EH, Rothkitt KD (1985) Ionenchromatographische Bestimmung von Lactulose und Epilactose neben Lactose und anderen Kohlenhydraten in Milch und Milchprodukten. Z Lebensm Unters Forsch 181:408–411CrossRefGoogle Scholar
  59. Rentschler E, Schuh K, Krewinkel M, Baur C, Claaßen W, Meyer S, Kuschel B, Stressler T, Fischer L (2015) Enzymatic production of lactulose and epilactose in milk. J Dairy Sci 98:6767–75CrossRefGoogle Scholar
  60. Schuster-Wolff-Bühring R, Fischer L, Hinrichs J (2010) Production and physiological action of the disaccharide lactulose. Int Dairy J 20:731–741CrossRefGoogle Scholar
  61. Schuster-Wolff-Bühring R, Michel R, Hinrichs J (2011) A new liquid chromatography method for the simultaneous and sensitive quantification of lactose and lactulose in milk. Dairy Sci Technol 91:27–37Google Scholar
  62. Seki N, Saito H (2012) Lactose as a source for lactulose and other functional lactose derivatives. Int Dairy J 22:110–115CrossRefGoogle Scholar
  63. Seki N, Hamano H, Iiyama Y, Asano Y, Kokubo S, Yamauchi K, Tamura Y, Uenishi K, Kudou H (2007) Effect of lactulose on calcium and magnesium absorption: a study using stable isotopes in adult men. J Nutr Sci Vitaminol (Tokyo) 53:5–12CrossRefGoogle Scholar
  64. Sharma P, Sharma BC, Puri V, Sarin SK (2008) An open-label randomized controlled trial of lactulose and probiotics in the treatment of minimal hepatic encephalopathy. Eur J Gastroenterol Hepatol 20:506–511CrossRefGoogle Scholar
  65. Shen X, Perreault H (1998) Characterization of carbohydrates using a combination of derivatization, high-performance liquid chromatography and mass spectrometry. J Chromatogr A 811:47–59CrossRefGoogle Scholar
  66. Silva AS, Valente IM, Nunes C, Coimbra MA, Guido LF (2013) Determination of aldoses, deoxy-aldoses and uronic acids content in a pectin-rich extract by RP-HPLC-FLD after p-AMBA derivatization. Chromatographia 76:1117–1124CrossRefGoogle Scholar
  67. Sjöberg J, Adorjan I, Rosenau T, Kosma P (2004) An optimized CZE method for analysis of mono- and oligomeric aldose mixtures. Carbohydr Res 339:2037–43CrossRefGoogle Scholar
  68. Suzuki T, Nishimukai M, Shinoki A, Taguchi H, Fukiya S, Yokota A, Saburi W, Yamamoto T, Hara H, Matsui H (2010a) Ingestion of epilactose, a non-digestible disaccharide, improves postgastrectomy osteopenia and anemia in rats through the promotion of intestinal calcium and iron absorption. J Agric Food Chem 58:10787–10792CrossRefGoogle Scholar
  69. Suzuki T, Nishimukai M, Takechi M, Taguchi H, Hamada S, Yokota A, Ito S, Hara H, Matsui H (2010b) The nondigestible disaccharide epilactose increases paracellular Ca absorption via Rho-associated kinase-and myosin light chain kinase-dependent mechanisms in rat small intestines. J Agric Food Chem 58:1927–1932CrossRefGoogle Scholar
  70. Troyano E, Olano A, Fernandez-Diaz M, Sanz J, Martínez-Castro I (1991) Gas chromatographic analysis of free monosaccharides in milk. Chromatographia 32:379–382CrossRefGoogle Scholar
  71. Tseng H-M, Gattolin S, Pritchard J, Newbury HJ, Barrett DA (2009) Analysis of mono-, di- and oligosaccharides by CE using a two-stage derivatization method and LIF detection. Electrophoresis 30:1399–405CrossRefGoogle Scholar
  72. Verhaar LAT, Van der Aalst MJM, Beenackers JAWM, Kuster BFM (1979) Ion-exchange chromatography of lactose-lactulose isomerization mixtures using a boric acid-borate eluent. J Chromatogr A 170:363–370CrossRefGoogle Scholar
  73. Vorndran AE, Grill E, Huber C, Oefner PJ, Bonn GK (1992) Capillary zone electrophoresis of aldoses, ketoses and uronic acids derivatized with ethylp-aminobenzoate. Chromatographia 34:109–114CrossRefGoogle Scholar
  74. Wang J, Hu X, Tu Y, Ni K (2006) Determination of spectinomycin hydrochloride and its related substances by HPLC-ELSD and HPLC-MSn. J Chromatogr B Analyt Technol Biomed Life Sci 834:178–82CrossRefGoogle Scholar
  75. Watanabe J, Nishimukai M, Taguchi H, Senoura T, Hamada S, Matsui H, Yamamoto T, Wasaki J, Hara H, Ito S (2008) Prebiotic properties of epilactose. J Dairy Sci 91:4518–4526CrossRefGoogle Scholar
  76. Zhang K, Dai L, Chetwyn NP (2010a) Simultaneous determination of positive and negative pharmaceutical counterions using mixed-mode chromatography coupled with charged aerosol detector. J Chromatogr A 1217:5776–84CrossRefGoogle Scholar
  77. Zhang Z, Yang R, Wang H, Ye F, Zhang S, Hua X (2010b) Determination of lactulose in foods: a review of recent research. Int J Food Sci Technol 45:1081–1087CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Eva Rentschler
    • 1
  • Beatrice Kuschel
    • 1
  • Manuel Krewinkel
    • 1
  • Wolfgang Claaßen
    • 1
  • Claudia Glück
    • 1
  • Bo Jiang
    • 2
  • Wanmeng Mu
    • 2
  • Timo Stressler
    • 1
    Email author
  • Lutz Fischer
    • 1
  1. 1.Department of Biotechnology and Enzyme Science, Institute of Food Science and BiotechnologyUniversity of HohenheimStuttgartGermany
  2. 2.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina

Personalised recommendations