Supporting Information
Determination of fluoroquinolones in bovine milk samples using a MWCNTbased pipette tip solid phase extraction step prior to capillary electrophoretic
separation.
Valeria H. Springera,b*, Johan Jackséna, Patrik Eka, Adriana G. Listab, Åsa Emmera*
a
KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of
Chemistry, Analytical Chemistry, Teknikringen 36, SE-100 44 Stockholm, Sweden
b
Analytical Chemistry Section, INQUISUR (UNS-CONICET), Av. Alem 1253, B8000CPB Bahía
Blanca, Buenos Aires, Argentina
*Corresponding author. Fax: +54 291 4595160 (V.H. Springer). Fax: +46-8-108425 (Å. Emmer).
E-mail addresses: aae@kth.se (Å. Emmer), valeria.springer@uns.edu.ar (V.H. Springer)
1
Figures
Figure S1. Chemical structures of the studied fluoroquinolones.
Figure S2. SPE scheme with a photograph of the miniaturized SPE tip inserted. All
conditions for analyte extraction are mentioned in Section 3.1.4.
2
Figure S3. Electropherograms obtained when different buffer solutions were used for CE
separation of selected FQs. A) 20 mM ammonium formate containing 0.05 % (w/v) Tween
20 at pH 3.0, B) 20 mM potassium dihydrogenphosphate and 2 mM CTAB at pH 3.0, C) 20
mM ammonium dihydrogenphosphate and 2 mM CTAB at pH 3.0.
Peaks: OFL, ofloxacin; CIP, ciprofloxacin; NOR, norfloxacin
CE instrument: Hewlett Packard HP
3D
CE capillary electrophoresis system, effective
capillary length: 71 cm (Palo Alto, CA, USA).
3
Figure S4. Comparison between electropherograms of milk samples spiked with 100 µg L1 of each analyte under optimal SPE-CE-UV conditions. A) Full-cream milk, B) Infant
formula milk and C) Skimmed milk. Peaks: 1) OFL, 2) CIP, 3) NOR.
CE instrument: Beckman Coulter MDQ P/ACE (Palo Alto, CA, USA).
4
Tables
Table S1. Main composition of the analyzed commercial milk samples
Main composition
(per 100 mL)
Milk samples
Infant formula
Skimmed
Full-cream
Carbohydrates (g)
7.1
4.6
4.8
Proteins (g)
1.5
3.0
3.0
Total fat (g)
3.7
1.5
3.0
Vitamin A (µg)
85
64
60
Vitamin D (µg)
1.1
1.0
0.63
Vitamin B1(mg)
0.06
Vitamin B2 (mg)
0.14
Vitamin B3 (mg)
0.56
Vitamin B6 (mg)
0.06
Vitamin B9 (µg)
7.4
Vitamin B12 (µg)
0.29
Vitamin C (mg)
6.4
Vitamin D (µg)
1.1
Vitamin E (mg)
0.46
Vitamin K (µg)
4.5
5
Table S2. Miniaturized solid phase extraction protocol
Rinsing steps
Volume
Number of cycles
MeOH
500 μL
4
Water
500 μL
3
1000 μL
20
500 μL
2
500 μL
5
Loading of sample
Aqueous solution (pH 6)
Rinsing step
Water
Elution
2% (v/v) FA in MeOH
Table S3. Migration times and resolution between peaks under optimal conditions of SPECE-UV method
Migration time (min)*
OFL
20.710 ± 0.038
CIP
21.002 ± 0.039
NOR
21.202 ± 0.043
Resolution (Rs)*
Rs OFL/CIP
1.173 ± 0.094
Rs CIP/NOR
0.898 ± 0.055
* Standard solution of FQs (100 µg L-1 each analyte), 𝑥̅ ± SD (n=10)
Table S4. Comparison between the proposed method and different methodologies to
determine FQs in milk samples
6
Analytes
Ciprofloxacin, Chloramphenicol,
Ampicillin, Tetracycline, and
Sulfamethoxazole
Ofloxacin, Pefloxacin, Difloxacin
and Enrofloxacin
Ciprofloxacin, Enrofloxacin,
Sarafloxacin, Oxolonic acid and
Flumequine
Norfloxacin, Ciprofloxacin,
Ofloxacin and Gatifloxacin
Ciprofloxacin and Enrofloxacin
Norfloxacin, Enrofloxacin,
Danofloxacin, Ciprofloxacin,
Sulfadoxine, Sulfadimethoxine,
Sulfamerazine, Sulfamethoxazole, Sulfachlorpyridazine,
Sulfathiazole and Ceftiofur.
Ciprofloxacin, Norfloxacin and
Ofloxacin
Sample pretreatment
Analysis
LODs
Ref.
- Protein precipitation with
trichloroacetic acid solution.
- Liquid – liquid extraction
(LLE) and evaporated to
dryness.
- SPE (solid phase extraction)
step.
CE-UV
12 µg L-1
(Ciprofloxacin)
16
HPLC - ECL
6 µg L-1
(Ofloxacin)
37
LC - FD
17 µg L-1
(Ciprofloxacin)
38
- Deproteination with 10%
HClO4 solution. The protein
was removed after
centrifugation for 3 min.
- Deproteination with 5% (w/v)
metaphosphoric acid.
- On – line column clean up.
- Addition of 1 mL 50 g/L lead
acetate solution followed by
shaking and mixture with
phosphate buffer (pH 7).
Separation of supernatant by
centrifugation at 4ºC.
- SALLUE (Salting-out assisted
liquid–liquid ultrasonic
extraction) : Addition of 10 mM
Al3+ solution, acetonitrile and
ammonium sulfate before
sonication during 10 min.
- Protein precipitation with 2 M
HCl solution followed by
centrifugation.
- SPE step with HLB cartridge
- Protein precipitation with 10
% trichloroacetic acid solution
and water. Consecutive
centrifugation steps and
filtration.
- Derivatization procedure for
sulfonamide compounds
-LLE with methanol: water
(70:30) mixture containing
0.1% formic acid followed by
centrifugation
- Protein precipitation with 3 M
HCl solution followed by
centrifugation step. The pH
was adjusted to 6 and the
aqueous phase was
centrifugated.
- µ-SPE with 5 mg MWCNTs
(multiwalled carbon nanotubes)
Spectrofluorometry
9 µg L-1
(Norfloxacin)
10 µg L-1
(Ciprofloxacin)
39
12 µg L-1
( Ofloxacin)
CE-UV
30 µg L-1
(Ciprofloxacin)
19
8 µg L-1
LC-FD/DAD
( Norfloxacin
and
Ciprofloxacin)
8
11.6 µg L-1
(Ofloxacin)
CE-UV
8.1 µg L-1
(Ciprofloxacin)
This
method
7.5 µg L-1
(Norfloxacin)
7
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