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Name of the ingredient
2'-Fucosyllactose (AAN# 119914)
Definition of the ingredient
2'-Fucosyllactose is a purified, white to off-white amorphous powder produced through a microbial fermentation process that involves the use of a genetically modified strain of Escherichia coli K-12. After purification, 2'-Fucosyllactose is finally obtained by crystallisation and/or by spray drying.
Chemical name: O-6-Deoxy-alpha-L-galactopyranosyl-(1-2)-beta-D-galactopyranosyl-(1-4)-D-glucose
Molecular formula: C18H32O15
CAS Number: 41263-94-9
| Test | Method reference | Acceptance criteria |
|---|---|---|
| Description | ||
| Appearance, visual | ISO 6658 | Powder, agglomerates, or powder with agglomerates |
| Colour, visual | ISO 6658 | white to off-white |
| Characteristics | ||
| pH (20°C, 5% solution) | Ph. Eur. 2.2.3 | 3.2 – 7.5 |
| Water | Karl-Fischer (Ph. Eur. 2.5.12) | ≤ 7.0 % (w/w) |
| Identification | ||
| HPLC | HPLC[1], [2], [3] | Retention time of main peak correspond to the retention time of the 2'-FL standard ±3%. |
| Specific optical rotation | Method as stated in the Ph. Eur. monograph Lactose monohydrate | -55° to -62° |
| NMR | Ishizuka et al., 1999[4] van Leeuwen et al., 2014[5] | Characteristic signals of 2'-FL should be identified in the 1H NMR spectrum |
| MS | MS[6] | Base molecular ion peak match molecular weights of 2'-FL |
| HPAEC | HPAEC[7] | Retention time of the main peak correspond to the retention time of the 2’-FL standards ±3% |
| HPAEC-PAD-MS | HPAEC-PAD-MS[8] | Retention time of the main peak corresponds to retention time of 2'-FL standard ±3% and base molecular ion peak matches its molecular weight. |
| Assay | ||
| 2'-fucosyllactose (2’-FL) (water free) | HPLC[1,2,3] or HPAEC[7] | 75.0 - 102.0% (w/w) |
| Test | Method reference | Acceptance criteria |
|---|---|---|
| Residual solvents | ||
| Acetic acid (as free acid and/or sodium acetate) | Boehringer Mannheim/R-Biopharm Test Kit, Cat. No. 10 148 261 035 (UV method) | ≤0.5 % |
| Incidental metals and non-metals | ||
| Lead | ICP-MS[9] | |
| Cadmium | ICP-MS[9] | ≤0.01 mg/kg |
| Mercury | ICP-MS[9] | ≤0.02 mg/kg |
| Arsenic | ICP-MS[9] | ≤0.1mg/kg |
| Molybdenum | ICP-MS[9] | ≤300 mg/kg |
| Selenium | ICP-MS[9] | ≤15 mg/kg |
| Other organic or inorganic impurities or toxins | ||
| Residual Endotoxin | Ph Eur 2.6.14 | ≤10 EU/mg |
| Residual Protein | Bradford Assay | ≤0.01 % |
| Sulfated ash | Ph. Eur. 2.4.14 | ≤1.5% (w/w) |
| Difucosyllactose (DFL) | HPLC[2,3] or HPAEC[7] | ≤20.0% (w/w) |
| Sum of 2'-Fucosyllactose and Difucosyllactose (water free) | HPLC[2,3] or HPAEC[7] | ≥85.0% (w/w) |
| D-lactose | HPLC[2,3] or HPAEC[7] | ≤10.0% (w/w) |
| L-fucose | HPLC[2,3][10] | ≤2.0% (w/w) |
| 2'-Fucosyl-D-lactulose | HPLC[1,2,10] | ≤2.0% (w/w) |
| Sum of HiMS (2'-FL + DFL + D- Lactose + L-Fucose + 3-Fucosyllactose) (water free) | HPLC[1,2,10] or HPAEC[7] | ≥92.0% (w/w) |
| Other carbohydrates (3'-fucosyllactose, fucosylgalactose and others) |
HPLC[2,3] or HPAEC[7] | ≤6.0% (w/w) |
| Notes | ||
|
*Only if acetic acid is used in manufacture. †Only if Na2MoO4 is used in manufacture. ‡Only if Na2SeO3 is used in manufacture. §2-Fucosylgalactose, 3-Fucosyllactose, Glucose, Galactose, Mannitol, Sorbitol, Galactitol, Trihexose, Allo-lactose, and other structurally related carbohydrates. |
||
Footnotes
| [1] | HPLC: Column: TSKgel Amide-80 (4.6 x 150 mm); Mobile phase: Water/Acetonitrile (Isocratic); Flow rate: 1.1 mL/min; Column temperature: 25oC; Detector: RID. |
|---|---|
| [2] | HPLC: Precolumn: XBridge BEH Amide (5 x 3.9 mm, 3.5 μm,); Separating column: 3 x XBridge Amide (3.5 μm, 250 x 4.6 mm); Mobile phase: Water/Acetonitrile/Triethylamine 69:31:0.1 (V/V/V); Flow rate: 1.2 mL/min; Column temperature: 60°C; Detector: RID. |
| [3] | HPLC: Column: Waters Spherisorb NH2 (3 µm, 250 mm x 4.6 mm), Mobile phase: ACN/Water (4:1); Flow rate: 1.8 mL/min; Column temperature: 35oC; Detector: RID. |
| [4] | Ishizuka, Y., Nemoto, T., Fujiwara, M., Fujita, K., Nakanishi, H. (l999) Three-Dimensional Structure of Fucosyllactoses in an Aqueous Solution, Journal of Carbohydrate Chemistry 18(5), 523-533. |
| [5] | van Leeuwen, S. S., Schoemaker, R. J. W., Gerwig, G. J., van Leusen-van Kan, E. J. M., Dijkhuizen, L., Kamerling, J. P. (2014) Rapid milk group classification by 1H NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples. Glycobiology 24(8), 728-739. |
| [6] | MS: Sample preparation: Sample was dissolved in Water/Acetonitirile 1:1 (V/V); Dry temperature: 280C; End plate offset: 500 V, 90 nA; Capillary: 3000 V, 160 nA; Nebuliser pressure: 4 bar; Dry gas: 8.0 L/min; Ionisation: ESI negative; Mode: Full scan MS; Calibration: with Na-formate cluster solution. |
| [7] | HPAEC: Column: Carbopac PA-210 (4 x 150 mm with 4 x 30 mm guard column); Mobile phase: 500mM NaOH/Water/100mM NaOH (Gradient); Flow rate: 0.8 mL/min; Column temperature: 31oC; Detector: PAD. |
| [8] | HPAEC-PAD-MS: Column: Dionex CarboPac PA-100 (250 mm x 2 mm) with CarboPac PA-100 guard column (50 mm x 2 mm); Mobile phase: Water/0.1M NaOH/0.1M NaOH + 0.3M NaOAc/0.1M NaOH + 1M NaOAc (Gradient); Flow rate: 0.2 mL/min; Column temperature: 35oC; MS mode: ESI (+ve) |
| [9] | ICP-MS: EN 13805:2002 or EPA-6020A:2007 or EN 15763:2009. |
| [10] | HPLC: Column: Asahipak NH2P-50 4E (4.6 x 250 mm); Mobile phase: Water/Acetonitrile (Isocratic); Flow rate: 1.1 mL/min; Column temperature: 30oC; Detector: cCAD. |
Key to abbreviations
EU = Endotoxin units
EN = European norms
EPA = United States Environmental Protection Agency
HPLC = High-pressure liquid chromatography
HPAEC = High performance anion exchange chromatography
ICP = Inductively coupled plasma
ISO = International organisation for standardisation
MS = Mass spectrometry
NMR = Nuclear magnetic resonance
PAD = Pulsed amperometric detection
Ph Eur = European pharmacopoeia