1. DEVELOPMENT OF A HILIC-MS METHOD FOR THE ANALYSIS OF GOAT COLOSTRUM OLIGOSACCHARIDES
Inmaculada Calvillo1, Ana Isabel Ruiz-Matute1*, Cipriano Carrero-Carralero1, Francisco Javier Moreno2, Alfonso Clemente3, María Luz Sanz1
Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, Madrid (Spain)
Instituto de Investigación en Ciencias de la Alimentación (CSIC-UAM, Nicolás Cabrera 9, Madrid (Spain)
Estación experimental del Zaidín (CSIC), Profesor Albareda 1, Granada (Spain) *
INTRODUCTION
Currently, there is a high interest in the study of human milk oligosaccharides considering their beneficial activities such as anti-inflammatory properties or modulation of the immune system. Goat milk contains higher amount of oligosaccharides (GMO) than cow or sheep milk and it is known to be composed by fucosylated and sialylated oligosaccharides, which makes it similar in composition to human milk [1, 2].
Due to their complexity and structural diversity, GMO characterization presents a challenging analytical task and appropriate chromatographic and spectrometric methods should be developed. Scarce studies have been conducted for the characterization of GMO structures [1,2] and even less regarding those from goat colostrum.
OBJECTIVE
Considering that concentration of these biologically active oligosaccharides is supposed to be higher in colostrum, the objective of this work was to develop a new methodology using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) which allows the separation and characterization of goat colostrum oligosaccharides.
MATHERIALS AND METHODS
GMO EXTRACTION
HILIC-MS ANALYSIS
COLUMNS
REMOVAL OF FAT AND PROTEINS2
STATIONARY PHASES:
Ethylene bridge hybrid with trifunctionally-bonded amide phase (BEH X-Bridge column) (2.1  150 mm) (Waters)
Sulfoalkylbetaine zwitterionic (ZIC®-HILIC column) (4.6  150 mm) (Sequant)
GOAT MILK COLOSTRUM
Size-exclusion chromatography (SEC)
Agilent 1200 series HPLC
ESI quadrupole HP-110 MS
BioGel P2 (Bio-Rad)
ORGANIC MODIFIERS (OM)
Acetonitrile
Methanol
CHEMICAL ADDITIVES
FLOW
REMOVAL OF MONO- AND DISACCHARIDES
0.1% Formic acid
0.1% Acetic acid
5 mM Ammonium acetate
0.1% Ammonium hydroxide
0.2 to 0.4 mL min-1
OLIGOSACCHARIDES
GRADIENTS
80:20 to 50:50 (OM:water; v/v)
85:15 to 50:50 (OM:water; v/v)
90:10 to 50:50 (OM:water; v/v)
RESULTS
ZIC®-HILIC column
BEH X-Bridge column
POSITIVE IONIZATION MODE
POSITIVE IONIZATION MODE
NEGATIVE IONIZATION MODE
min 10 20 30 40 50
5000
10000
15000
20000 4 5 6 2 1 3 7 8
AMMONIUM HYDROXIDE
AMMONIUM HYDROXIDE
AMMONIUM HYDROXIDE
min 5 10 15 20
4000
8000
12000
16000 25 30 35 4 2 3 7 1 8 6
min 5 10 15 20 25 30 35
20000
60000
100000
140000 3 6 2
SIM
SIM
SIM
FORMIC ACID
min 20 25 30 35 40 45
20000
40000
60000
80000 2 3 4 5
SIM
TABLE 1. Molecular ion adducts registered for colostrum goat milk oligosaccharides ([M+Na]+ in positive ionization mode and [M-H]- in negative ionization mode)
Peak no
CARBOHYDRATE
[M+Na]+
[M-H]- 1
α-2´-Fucosyl-lactose
511
487 N 2
α-3´- Galactosyl-lactose or β-6´-Galactosyl-lactose
527
503 3
3´-Sialyl-lactose or 6´- Sialyl-lactose
656
632 A 4
Galactotetraose
689
665 5
6´-Sialyl-lactosamine
697
673 6
Glycolyl-neuraminyl-lactosamine
713 7
Pentagalactosamine
850
826 8
N-Acetyl-glucosaminyl-hexosyl-lactose
730
706
Negative ionization mode only allowed the monitorization of acid GMO
Broad peaks with poor symmetry were observed in the HILIC profile obtained using ZIC®-HILIC column.
Acid GMO were less retained under basic conditions.
Broader peaks were obtained at a flow rate of 0.2 mL/min
The use of ammonium acetate negatively affected to the GMO ionization. Moreover, basic conditions allowed the detection of a higher number of oligosaccharides.
min 10 15 20 25 30 35 40
40000
80000
120000 1 2 3 5 6 4
ACETIC ACID
SIM
HILIC-MS analysis of colostrum goat milk allowed the determination of acid and neutral oligosaccharides, similar to those found in human milk [1].
Eight molecular ion adducts corresponding to acid (A) and neutral (N) GMO were recorded in the selected ion monitoring (SIM) mode to optimize the HILIC method (Table 1).
Acid GMO eluted before neutral GMO under all assayed conditions.
min 15 20 25
2000
4000
6000 30 35 3 5 2 1 6
SIM
AMMONIUM ACETATE
CONCLUSIONS
BEH amide column working under basic conditions and 0.4 mL/min flow rate has been found to be a useful tool for the analysis of GMO.
This methodology will be applied for further characterization of GMO from several colostra using LC-QTOF MS.
REFERENCES:
ACKNOWLEDGMENTS:
[1] Meyrand M, Dallas DC, Caillat H, Bouvier F. (2013) Small Ruminant Research 113, 411.
[2] Martinez-Ferez A, Guadix E, Guadix EM. (2006) Journal of Membrane Science 276, 23.
This work was funded by Junta de Andalucía (project POII ) and CSIC (project i-link0827).