Determination of Two Major Sialic Acids in Dairy Products by Electrophoretic Stacking Technology with Contactless Conductivity Detection

Food Analytical Methods

Yu Wang • Jiaxing Kong • Zheyan Chen • Dan Luo • Jiannong Ye • Qingcui Chu

Department of Chemistry, East China Normal University, Shanghai 200241, China


Table S1

Molecular structures and dissociation constants of NGNA, NANA and seven carbohydrates in water at 25 ℃.

Compound / Molecular structure / pKa / Ref.
NGNA / / 2.75 / [36]
NANA / / 2.60 / [36]
Gluconic acid / / 3.86 / [37]
Glucose / / 12.28 / [38]
Fructose / / 12.03 / [38]
Galactose / / 12.39 / [38]
Lactulose / / / / /
Lactose / / 11.98 / [38]
Sucrose / / 12.51 / [38]

Fig. S1. The concentration effects of (A) Na2HPO4 and (B) CTAB on the migration times of NGNA, NANA and seven carbohydrates: (A) a. 20 mM Na2HPO4 – 30 mM NaOH – 0.8 mM CTAB, b. 15 mM Na2HPO4 – 30 mM NaOH – 0.8 mM CTAB, c. 10 mM Na2HPO4 – 30 mM NaOH – 0.8 mM CTAB, d. 5 mM Na2HPO4 – 30 mM NaOH –0.8 mM CTAB, e. 1 mM Na2HPO4 – 30 mM NaOH –0.8 mM CTAB; (B) a. 0.6 mM CTAB – 30 mM NaOH – 10 mM Na2HPO4, b. 0.8 mM CTAB – 30 mM NaOH – 10 mM Na2HPO4, c. 1.0 mM CTAB – 30 mM NaOH – 10 mM Na2HPO4.

Other experimental conditions: capillary tube, 15.0 μm id × 360 μm od, effective length of 86.5 cm; excitation frequency for C4D, 900 kHz; peak-to-peak voltage for C4D, 40 Vpp; separation voltage, -18 kV; injection time, 8 s (at -18 kV); peak identifications: 1. gluconic acid, 2. NGNA, 3. NANA, 4. fructose, 5. glucose, 6. lactulose, 7. galactose, 8. lactose and 9. sucrose; and concentration of analytes: 20 µg mL-1 each.

Fig. S2. Effects of different dilution ratios for sample hydrolysate on the peak area of NANA (as the molecular model).

Optimum hydrolysis conditions: hydrolysis solvent, 30 mM H2SO4; temperature, 85 ℃; stirring rate, 800 rpm; and hydrolysis time, 60 min. Electrophoretic conditions: capillary tube, 15.0 μm id × 360 μm od, effective length of 86.5 cm; excitation frequency for C4D, 900 kHz; peak-to-peak voltage for C4D, 40 Vpp; running buffer, 20 mM NaOH - 10 mM Na2PO4 / 0.8 mM CTAB, separation voltage, -18 kV; injection time, 8 s (at -18 kV); and the concentration of standard NANA was 0.10 μg mL-1.

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Table S2

Comparison of this method and the reported methods for determination of SAs in biological samples and dairy products.

Methods / Samples / Injection volume / Analytes / LODs
(ng mL-1) / Recoveries
(%) / Refs.
HPLC-UV/MS / breast milk and milk-based products / 5 μL / NANA / 124 / 90-97 / Wu et al. 2016
HPLC-UV / milks and milk-based products / 10 μL / NGNA
NANA / 0.54
1.56 / 90.2-108
87.0-102 / Veronique et al. 2010
HPLC-UV / milk / 10 μL / NANA / 250 / 89.0-98.8 / Feng et al. 2008
SPE/μ-LC-LIF / human biofluids / 1 μL / NGNA
NANA / 0.3-0.7
0.1-0.8 / 93.00-99.10
92.00-98.90 / Orozco-Solano et al. 2013
HPLC-FD / saliva / 20 μL / NANA / 2.67 / / / Tatsuhiro et al. 2013
HPLC-FD / infant formulas / / / NGNA
NANA / 3
30 / 109
95 / Salcedo et al. 2011
SPE/LC-MS / serum and urine / 100 μL / NGNA
NANA / 0.04-0.05
0.03-0.05 / 96.1-99.6
95.2-99.1 / Priego-Capote et al. 2014
HPLC-MS / fetuin / 20 μL / NGNA
NANA / /
/ / 95.3-102.3
93.7-103.6 / Allevi et al. 2008
HPLC-MS/MS / tumor and lymph node / 20 μL / NGNA
NANA / <3.3
<3.1 / 93.6-102.4 / Wang et al. 2015
HPLC-MS/MS / infant formula / 5 μL / NANA / 100 / 91.8-112.4 / Li et al. 2015
HPLC-MS/MS / human cerebrospinal fluid / 35 μL / NANA / 139 / 91-105 / van der Ham et al. 2010
Table S2 (Continued)
Methods / Samples / Injection
volume / Analytes / LODs
(ng mL-1) / Recoveries
(%) / Refs.
GC-MS / / / / / NANA / / / / / Wachsmuth et al. 2011
GC-MS / / / / / NANA / / / / / Sassaki et al. 2008
CZE-UV / glycoproteins and blood sera / / / NANA / 619 / 96 / Strousopoulou et al. 2002
CE-UV / glycoproteins / / / NANA / 309 / / / Che et al. 1999
CE-AD / saliva / / / NANA / 500 / 91.8-109 / Zheng et al. 2016
CE-LIF / bovine and human plasma / / / NGNA
NANA / /
/ / /
/ / Szabo et al. 2012
CE-LIF / glycoprotein / 25 nL / NANA / 0.0028 / / / Ijiri et al. 2010
HPAEC-PAD / breast milk, milk-based products / 25 μL / NGNA
NANA / 1.8
3.0 / 88-101
108-115 / Tang et al. 2008
HPAEC-PAD / glycoproteins / / / NGNA
NANA / /
/ / /
/ / Rohrer et al. 2013
HPAEC-PAD / glycosaminoglycans, alginate, fucoidan, and glycans / 25 μL / NGNA
NANA / 49
46 / /
/ / Zhagn et al. 2012
CE-direct UV / serum / / / NANA / 2969 / 95.6±8.4 / Dong et al. 2001
Dong et al. 2000
Wang et al. 2009
CZE-indirect UV / sea urchin, sake / 6 nL / NGNA
NANA / /
/ / /
/ / Soga et al. 2001
Table S2 (Continued)
Methods / Samples / Injection volume / Analytes / LODs
(ng mL-1) / Recoveries
(%) / Refs.
CZE-indirect UV / Α1-acid glycoprotein / 5 nL / NANA / 7700 / / / Goto et al. 2015
CE- indirect UV / food samples / 6 nL / NGNA
NANA / /
/ / /
/ / Soga et al. 2000
CE- indirect UV / glycoproteins / 6 nL / NGNA
NANA / /
/ / /
/ / Soga et al. 1998
CZE-indirect UV / orange juice / / / NANA / / / / / Vorndran et al. 1992
CE-MS / glycoproteins and plasma / / / NANA, NGNA / 2000 / / / Ortner et al. 2008
CE-C4D / Milk, milk powder / 0.75 nLa / NGNA
NANA / 2.0
1.7-2.2 / 94-107
93-104 / In this work
a The injection volume (Vi) for the electrokinetic injection mode in CE was calculated by the equation of . In this equation, d is the inside diameter of the capillary; is the speed of electroomsmotic flow, calculated by the equation of , therein leff is the effective length of the capillary, is the migration time of the neutral species; is the injection time.

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Table S3

Precisions of two SAs in different sample matrices a.

Matrix / Analytes / Added amount (μg mL-1) / Intraday precision
(RSD%, n=7) / Interday precision
(RSD%, n=5)
Peak area / Migration time / Peak area / Migration time
Milk / NGNA / 0.050 / 2.1 / 1.5 / 5.0 / 3.6
0.20 / 2.5 / 1.0 / 4.5 / 3.7
1.0 / 1.8 / 1.1 / 2.7 / 2.7
NANA / 0.050
0.20
1.0 / 2.5
2.1
1.9 / 1.3
1.1
0.9 / 5.5
4.9
2.8 / 3.8
3.0
2.6
Milk powder / NGNA / 0.050 / 2.6 / 1.4 / 4.2 / 3.9
0.20 / 3.3 / 0.7 / 4.4 / 2.1
1.0 / 3.5 / 1.0 / 3.7 / 2.0
NANA / 0.050
0.20
1.0 / 3.6
3.2
3.1 / 1.1
0.9
0.8 / 5.0
4.0
4.4 / 2.4
1.8
2.0

a The experimental conditions were the same as those in Fig. S2.

Table S4

Recovery data from different sample matrices (n=3)a.

Matrix / Added amount
(μg mL-1) / Recovery (%, RSD%)
NGNA / NANA
Milk No.1 / 0.050 / 94 (2.9) / 98 (1.4)
0.20 / 98 (2.2) / 97 (2.3)
1.0 / 102 (2.2) / 93 (2.0)
Milk powder No.1 / 0.050 / 99 (3.0) / 95 (1.1)
0.20 / 106 (2.2) / 103 (1.4)
1.0 / 107 (1.8) / 104 (3.3)

a The experimental conditions were the same as those in Fig. S2.

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