Supplementary content
Analysis of antibiotic residues
Plant material, soil and liquid manure were analysed by means of HPLC-MS/MS.
I) Plant material
Preparation of stock and standard solutions
AMO, AMA, Diketo, ENR, CIP, MON, DC, epi-DC, DMC, epi-DMC, OTC, epi-OTC, TC, epi-TC, CTC,
epi-CTC and iso-CTC were purchased as commercially available products. Usually, stock solutions
(1.0 g/L) were prepared in methanol. Stock solutions of epi-iso-CTC (approx. 200 mg/L) were prepared
by partial epimerisation of dissolved iso-CTC. For this purpose 2 mL of iso-CTC solution (1.0 g/L) were
added to 3 mL of methanol and 5 mL of mobile phase A (double distilled water /acetonitrile/ formic
acid: 1800/200/2 (v/v/v), see below.) This mixture was heated at 60° C (thermostat block, 60 min).
The exact concentrations of both epimers were determined by the difference based on the initial
concentration of the iso-CTC standard. The concentrations of the keto-enol tautomers of epi-CTC
were analogously calculated. To obtain the appropriate standard solutions for external calibration in
the range of 10 – 2000 µg/L, aliquots of the defrosted stock solutions were diluted with the mobile
phase A. Standard solutions were freshly prepared on a daily basis.
Stock solution of CIP was prepared in acidic acetonitrile solution (formic acid 2% v/v). Stored at -30°C,
all stock solutions were stable for at least six months.
Extraction and clean up
Roots and green parts of red cabbage (stems, old and young leaves), stored at -80°C, were
homogenised in a Mortar Grinder (Grindomix GM200, Retsch). Solid-liquid extractions were performed
with 5 g for old leaves, young leaves and stems and with 3 g for roots. For each tissue type, duplicate
extractions were carried out and the extraction procedure was followed in parallel for duplicates. The
water contents of roots ranged from 46-56 % and of green parts from 53-63 %.
Solid-Liquid Extraction
The solid-liquid extraction consists of two extraction stages applying the following buffer:
KH2PO4 buffer (pH 2.0)
The buffer was prepared at 0.01 mol/L KH2PO4 using double distilled water. The pH was adjusted to
pH 2.0 with trichloroacetic acid (20% w/w).
Extraction stage 1
Weighed amounts of homogenate were placed in 50 mL centrifuge glasses, to which 20 ml KH2PO4
1
buffer were added. Further, they were centrifuged for 10 min (4000 rotations per minute (rpm), 2325 g)
and subsequently sonicated (5 min). The supernatants were transferred to fresh centrifuge glasses.
Extraction stage 2
10 ml of buffer solutions were again added to the remaining residues, suspended for 5 min using ultrasonification, centrifuged for 10 min and the resulting supernatants were combined with the supernatant
from the first extraction stage. The combined extracts were further treated by the solid phase
extraction (SPE).
Solid Phase Extraction (SPE)
Bond Elut C 18 cartridges (3 cc, 200 mg) were conditioned with 3 mL methanol, followed by an equal
volume of water and dilute trichloroacetic acid (2 % w/w). The whole extract (30 mL) was passed
slowly through the column (~35 – 40 drops per minute). The effluent was discarded. After washing
with 2 mL of trichloroacetic acid (2 % w/w), 2 mL of water and again 2 mL of trichloroacetic acid (2 %
w/w), elution was performed with 3 mL of acetonitrile. The eluates were evaporated to dryness at 30
°C under a stream of nitrogen. The remaining residues were dissolved in 1 mL of mobile phase A (see
below) and the resulting concentrates were analysed by HPLC-MS/MS.
HPLC-MS/MS-Equipment
Degasser: SCM 1000 Vakuum Membrane Degasser, HPLC-gradient pump SpectraSYSTEM P 4000,
injection system: AS 3000, autosampler (10 ºC), column oven (heated to 30 °C; photodiode arraydetector: UV 6000 LP (190 – 800 nm) linked to the ESI ion trap mass spectrometer LCQ Advantage
(ThermoFisher Scientific).
Injection volume: 20-40 μL; flow: 0.4 mL/min; guard column: YMC-Pack ODS-AM 10 x 3.0 mm i.d., S-5
μm; analytical column: YMC-Pack ODS-AM 150 x 3.0 mm i.d., S-5 μm.
Xcalibur software (version 2.0) was used to perform the analysis.
For the chromatographic separation of the analytes extracted from plant material, the following
gradient program with the mobile phases A, B and C (composition see below) was composed (Table
S1):
mobile phase A: water / acetonitrile/ formic acid: 1800 / 200 /2 (v/v/v)
mobile phase B: water / acetonitrile/ formic acid: 800 / 1200 /2 (v/v/v)
mobile phase C: acetonitrile
2
Table S1: HPLC-gradient program for plant material
Time [min]
Mobile phase A [%]
Mobile phase B [%]
Mobile phase C [%]
0.00
90
10
0
1.50
80
20
0
10.00
60
40
0
12.00
0
100
0
12.50
0
0
100
25.00
0
0
100
26.00
90
10
0
35.00
90
10
0
Mass spectrometry
Mass spectrometric analysis was carried out in the ESI positive ion mode by multiple reaction
monitoring of molecular ion adducts [M+H]+ and confirming product ions.
The adjusted instrumental parameters were as follows:
Tune-Page-Parameters:
Mass Range: 80 – 2000 [m/z]; Sheath Gas Flow Rate: 47 [arb]; Auxiliary Gas Flow Rate: 0 [arb]; Ion
Spray Voltage: 5 kV; Capillary Temperature: 250 ºC; Capillary Voltage: 9 V; Tube Lens Offset: - 5.0 V;
Multipole
1
Offset:
-
1.5
V;
Lens
Voltage:
-
32
V;
Multipole
2
Offset:
-5.5
3
Table S2: Chromatographic and mass spectrometric parameters (plant material)
Collision
energy
[%]
Product ions
[m/z]
Abbreviation
Precursorion
[m/z]
Enrofloxacin
ENR
360.2
42
Ciprofloxacin
CIP
332.1
44
Monensin
MON
693.4
45
479.2 (100 %)
22.80
Amoxicillin
AMO
365.8
45
207.9 (100 %)
348.7 (100 %)
3.09
Amoxicilloic acid
AMA
384.0
44
366.9 (100 %)
340.0 (10-20 %)
323.0 (50-70 %)
2.70
Amoxicillin-diketopiperazine
Diketo
365.8
30
159.9 (100 %)
207.0 (10 %)
6.74
Tetracycline
TC
445.0
34
epi-Tetracycline
e-TC
445.0
34
Doxycycline*
DC
445.0
34
428.0 (100 %)
14.43
epi-Doxycycline*
e-DC
445.0
34
428.0 (100 %)
13.71
Oxytetracyclin
OTC
461.1
44
epi-Oxytetracyclin
e-OTC
461.1
44
Demeclocyclin
DMC
465.0
40
epi-Demeclocyclin
e-DMC
465.0
40
Chlortetracycline
CTC
479.1
40
epi-ketoChlortetracycline
e-keto-CTC
479.1
40
epi-enolChlortetracycline
e-enol-CTC
479.1
40
iso-Chlortetracycline*
iso-CTC
479.1
40
462.0 (100 %)
10.33
epi-isoChlortetracycline*
e-iso-CTC
479.1
40
462.0 (100 %)
462.8 (< 5 %)
8.52
Analyte
(relative peak
intensity)
245.1 (< 5 %)
316.1 (100 %)
317.0 (10-20 %)
288.1 (100 %)
314.1 (< 5 %)
409.9 (30%)
426.9 (100%)
427.9 (10 %)
409.9 (40-50 %)
426.9 (100 %)
427.9 (30 %)
425.8 (100 %)
442.9 (60-80 %)
443.9 (15-20 %)
426.0 (100 %)
444.0 (95 %)
442.9 (40-60 %)
448.0 (100 %)
430.0 (10-20 %)
448.0 (100 %)
430.0 (10-20 %)
461.8 (100 %)
443.8 (55-100 %)
461.9 (100 %)
262.7 (< 2 %)
444.0 (< 2 %)
461.9 (100 %)
443.9 (50-80 %)
461.2 (10 %)
Retention**
time
[min]
9.60
8.30
8.74
7.29
7.50
7.14
10.59
9.14
13.00
9.00
11.45
* for screening (Commission decision 93/256/EEC)
**The retention times are given for the chromatogram of a standard solution (5 mg/L of each
compound).
4
Validation
The confirmation strategy relies on the use of two selected reaction monitoring signals (SRM) (Table
S2). Positive findings were confirmed via the relative intensity ratios of the two mass transitions, in
accordance to requirements described in the Draft SANCO/1085/2000 and Commission Decision
2002/657/EC in conjunction with Council Directive 96/23/EC. (See also:
Commission Decision
93/257/EEC and 93/256/EEC, Kaufmann et al. 2015).
The confirmation of drug residues (ENR and tetracyclines) in vegetables and wheat by high resolution
mass spectrometry was demonstrated in an earlier study (Grote et al. 2009, Meric 2010).
Quantification is based on the summed signal intensities of corresponding transitions (TIC). The ratio
of analyte peak area versus analyte concentration was utilised to construct the calibration lines. The
evaluation of the LC/MS-data was supported by applying the analytic software “Valoo” ( “analytic
software”, Leer, Germany).
By matrix-calibration of analytes over a range of 10 – 2000 µg/kg (fresh weight, fw) assay linearity was
determined. For all analytes (except AMO and AMO metabolites, see comment in Table S3 and S4)
the linear calibration functions ranged from LOQ (Table S4) to 2000 µg/kg fw. Coefficients of
determination were throughout higher than 0.98.
As a criteria for the measuring precision of the analytical procedure, the relative standard deviations
(srel) for the determination of antibiotics in spiked red cabbage leaves (150 µg/kg fw, six replicates,
matrix-calibration) were determined. The obtained srel values between 4.9 and 10.7% comply with the
Commission Decision 2002/657/EC (Table S3).
The parameter selectivity was determined by comparison of retention times and product-ion intensities
of the analytes in external standard solution and in extracts of spiked red cabbage leaves. The
retention times differ from each other not more than 3.8 %. Based on the relative intensity of the actual
base peak, the maximum tolerance with regard to product ion intensities was not exceeded. Thus, the
requirements of the Commission Decision 2002/657/EC are fulfilled.
Limits of detection (LOD) were estimated according to the signal-to-noise ratios (S/N = 3:1) and limits
of quantification (LOQ) according to S/N = 1:10. The calculation takes into account the standard
deviation of six measurements of plant matrix blanks (extracts of control plants) and the slopes of the
regression lines (matrix –calibrations).
LOD and LOQ values calculated for the analytes in red cabbage matrix are summarised in Tab. S4.
5
Table S3: Recovery ± standard deviation (SD) of analytes from spiked red cabbage leaves
(except AMO, AMA and Diketo*)
(Sample weight: 3 g (fresh weight, fw), spiking 150 µg/kg fw, matrix calibration method)
Spiked analytes
AMO*
AMA*
Diketo*
CTC/ e-keto-/e-enol-CTC
iso-CTC/ e-iso-CTC
TC/ e-TC
DMC /e-DMC
DC/ e-DC
ENR
CIP
MON
Recovery ± SD [%]
(Epimers and tautomers included)
77.0 ± 6.3
55.8 ± 7.2
91.3 ± 4.2
93.2 ± 7.3
84.8 ± 7.6
100.0 ± 5.4
100.0 ± 9.8
97.3 ± 9.9
98.6 ± 10.7
89.6 ± 8.0
45.1 ± 4.9
*Due to the rapid conversion of these compounds in contact with plant materials, the recovery-values were
calculated by analysing spiked extraction solutions (hydrogenphosphate buffer pH 2.0)
6
Table S4: Limits of detection (LOD) and limits of quantification (LOQ) of spiked analytes
in red cabbage leaves (except AMO, AMA and Diketo*)
Analytes
LOD [µg/kg fw]
LOQ [µg/kg fw ]
AMO*
6.7
14.3
AMA*
5.9
10.4
Diketo*
6.2
12.4
CTC/e-keto-/e-enol-CTC
3.1
10.5
iso-CTC/ e-iso
3.7
12.1
TC/e-TC
4.7
9.2
DMC/e-DMC
5.3
15.2
DC/e-DC
7.9
20.4
ENR
3.2
11.0
CIP
3.5
11.9
MON
7.2
24.8
*Due to the rapid conversion of these compounds in contact with plant materials, the LOD and LOQ-values
were calculated by analysing spiked extraction solutions (hydrogenphosphate buffer pH 2.0)
Further details of the development of the analytical methods are described elsewhere (Chowdhury
2012).
II) Soil samples
Preparation of stock and standard solutions
Stock solutions of ENR, CIP, CTC, e-CTC and iso-CTC (with the exception of e-iso-CTC) were
prepared as individual solutions. For this purpose 10.0 mg of the solid (powder) was weighed into a 10
mL volumetric flask. In the case of tetracyclines it was filled up with methanol. Acetonitrile was used as
solvent for enrofloxacin. Double distilled water, acidified by addition of 20 µL of hydrochloric acid
(25% w/w) was used for ciprofloxacin.
The preparation of e-iso-CTC was carried out analogously to the above-mentioned method for plant
materials. The calibration solutions (10-1500 µg/L) were prepared daily by diluting the stock solutions
with mobile phase A.
7
Extraction and clean up
Dry soil samples were stored at ambient temperature. Duplicate extractions using 5 g of soil material
were performed in parallel. Two different buffer solutions were used for the extraction stages:
Mg(NO3)2 /NH3 buffer (pH 8.5) (for ENR and CIP-determination)
The final solution contained 1.6 mol/L Mg(NO3)2 and 0.3 mol/L NH3. In detail, 410 g of Mg(NO3)2 *
6.H2O were weighed into a 1000 mL volumetric flask and dissolved in 700 mL of water. Then 20 mL of
ammonia solution (25% w/w) were added. The pH was adjusted to 8.5 with hydrochloric acid (25 %
w/w). Water was used to make up the final volume of 1000 mL.
NH3/NH4Cl/EDTA buffer (pH 10.0) (for ∑CTC / iso-CTC-determination)
The final solution contained 4.7 mol/L NH3, 1.0 mol/L NH4Cl and 0.1 mol/L EDTA-Na2 dihydrate. In
detail, 54.0 g of ammonium chloride were weighed into a 1000 mL-volumetric flask and dissolved in
300 mL of water. Then 37.224 g EDTA-Na2 dihydrate and 350 mL of ammonia solution (25% w/w)
were added and filled up to 1000 mL with water.
Extraction stage 1
Of each soil sample 5 g were suspended in 10 mL of Mg(NO3)2/NH3 buffer (pH 8.5) for the
determination of ENR and CIP, or NH3/NH4Cl/EDTA (pH 10) for the determination of the sum of CTC
(∑CTC)*. Afterwards the suspension was equilibrated for 30 min on a horizontal shaker (275 rpm) and
centrifuged for 10 min at 4000 rpm (2325 g). The supernatant was transferred to another 50 ml
centrifuge glass.
*NB: Under the ammoniacal extraction conditions applied, CTC converts completely to epi-iso-CTC
and iso-CTC. Therefore, CTC is quantified as the sum of epi-iso-CTC and iso-CTC, denoted as ∑CTC
(Vockel 2005).
Extraction stage 2
The solid residue was again treated with 10 mL of extractant, slurried with a glass rod and then
shaken for 30 min. The suspension was centrifuged for 10 min at 4000 rpm (2325 g) and the
supernatant was added to the first supernatant. Hydrochloric acid (25% w/w) was added carefully to
the combined extraction solutions, to adjust to ~pH 3 (for ENR and CIP) or to ~ 4 (for ∑CTC / iso-CTC)
and again centrifuged for 10 minutes at 4000 rpm. These extracts were subsequently treated by solid
phase extraction.
8
Solid Phase Extraction (SPE)
Oasis HLB Bond-cartridges (6 cc, 200 mg) were conditioned with 5 mL methanol and 5 mL water. The
whole soil extract (~30 mL) was passed slowly through the column. The effluent was discarded. After
washing twice with 3 mL of methanol/water: 5/95 (v/v), elution was performed with 6 mL methanol. The
eluate was evaporated to dryness at 30 °C under a stream of nitrogen. The remaining residues were
dissolved in 500 µL of mobile phase A (water/acetonitrile/formic acid: 1800/200/2 v/v/v). The resulting
concentrates were analysed by HPLC-MS/MS.
HPLC-MS/MS -analysis
Table S5: HPLC-gradient program for soil samples
The mobile phase A, B and C used for the gradient system were composed as follows:
A: water / acetonitrile/ formic acid 1800 /200 /2 (v/v/v)
B: water/ acetonitrile/ formic acid
800 /1200 /2 (v/v/v)
C: methanol
Time [min]
Mobile phase A [%]
Mobile phase B [%]
Mobile phase C [%]
0.00
90
10
0
1.00
80
20
0
10.00
60
40
0
12.30
50
50
0
17.00
0
100
0
21.00
0
100
0
22.30
50
50
0
24.00
90
10
0
35.00
90
10
0
Mass spectrometry
Mass spectrometric analysis was carried out in the ESI positive ion mode by multiple reaction
monitoring (MRM) of molecular ion adducts [M+H]+ and confirming product ions.
Tune-Page-Parameters:
Mass Range: 80 – 2000 [m/z]; Sheath Gas Flow Rate: 45 [arb]; Auxiliary Gas Flow Rate: 0 [arb]; Ion
Spray Voltage: 5 kV; Capillary Temperature: 250 ºC; Capillary Voltage: 9 V; Tube Lens Offset: - 5.0 V;
Multipole 1 Offset: - 2.5 V; Lens Voltage: - 16 V; Multipole 2 Offset: - 5.5 V.
For identification of the analytes see Table S6.
9
Table S6: Chromatographic and mass spectrometric parameters (soil samples)
PrecursorAnalyte
Abbre-
ion
Collision
energy
viation
[m/z]
(%)
Product-ions
Retention time
(m/z) (relative
[min]
peak intensity)
245.2 (< 5 %)
Enrofloxacin
ENR
360.2
40
316.2 (100 %)
8.7
317.0 (10-20 %)
Ciprofloxacin
CIP
332.1
40
288.1 (100 %)
7.5
314.1 (25 %)
409.9 (30 %)
Tetracycline
TC
1445.0
35
426.9 (100 %)
8.3
427.9 (10 %)
409.9 (40-50 %)
epi-Tetracycline
e- TC
445.0
35
426.9 (100 %)
7.0
427.9 (30 %)
444.0 (55-100 %)
Chlortetracycline
CTC
479.1
40
461.8 (100 %)
12.3
444.0 (50-80 %)
epi-Chlortetracycline
e-CTC
479.1
40
461.8 (100 %)
10.5
Chlortetracycline*
e-iso-CTC
479.1
40
462.0 (100 %)
8.0
iso-Chlortetracycline*
iso-CTC
479.1
40
462.0 (100 %)
9.7
epi-iso-
* for screening (Commission decision 93/256/EC)
10
Validation
For analytical background information see the Validation chapter in “Analyses of plant materials”
LOD were estimated according to the signal-to-noise ratios (S/N = 3:1). For ENR and CIP the LOD
values are at 1 µg/kg and for ∑CTC at 3 µg/kg soil (air dried).
LOQ (S/N = 10:1) values determined were: for ENR and CIP ~3 µg/kg and for CTC ~5 µg/kg soil.
Recovery data of ∑CTC and fluoroquinolones (ENR, CIP) from spiked soil are presented in Table 4 of
the publication.
III) Liquid manure
Extraction and HPLC-MS/MS analysis of antibiotics in liquid manure and validation of the procedure
was done previously in our laboratory as described by Vockel 2005, Grote et al. (2006) and Grote et
al. (2007a). Briefly, pig slurry, originally antibiotic free, was sampled before spiking with CTC and ENR.
Before application, the spiked manure was also sampled. The samples (1 or 5 g) were homogenised,
treated with 30 mL of aqueous McIllvain-buffer pH 4.1 (0.1 mol EDTA dissolved in 620 mL of 0.1 mol/L
citric acid and 380 mL of 0.2 mol/L sodium dihydrogenphosphate) and analysed by HPLC-MS/MS.
References
Chowdhury F (2012) Untersuchungen zur Aufnahme von Veterinärantibiotika in Rotkohl und Möhren Verbreitungspfade und Verbraucherrisiken. Dissertation, Universität Paderborn
Commission Decision 93/256/EEC of 14 April 1993 laying down the methods to be used for detecting
residues of substances having a hormonal or a thyrostatic action
Commission Decision 93/257/EEC of 15 April 1993 laying down the reference methods and the list of
national reference laboratories for detecting residues
Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC
concerning the performance of analytical methods and the interpretation of results (Text with EEA
relevance)
Council Directive 96/23/EC of 29 April 1996 measures to monitor certain substances and residues
thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and
Decisions 89/187/EEC and 91/664/EEC
Draft SANCO/1085/2000 Comission decision of 4 Decembre 2000 laying down performance criteria
for the analytical methods to be used for certain substances and residues thereof in live animals and
animal products according to Council Directive 96/23/EC
Grote M, Vockel A, Schwarze A, Mehlich A, Freitag M (2006) Antibiotika-Aufnahme von Nutzpflanzen
aus Gülle-gedüngten Böden – Ergebnisse eines Modellversuchs. J Verbr Lebensm 1:38–50
Grote M, Schwake-Anduschus C, Michel R, Stevens H, Heyser W, Langenkämper G, Betsche T,
Freitag M (2007a) Incorporation of veterinary antibiotics into crops from manured soil;
Landbauforschung Völkenrode 57:25-32
Grote M, Meric DH, Langenkämper G, Hayen H, Betsche T, Freitag M (2009) Untersuchungen zum
Transfer pharmakologisch wirksamer Substanzen aus der Nutztierhaltung in Porree und Weißkohl. J
Verbr Lebensm 4:287-304
11
Kaufmann A, Butcher P, Maden K, Walker S, Widmer M (2015) Reliability of veterinary drug residue
confirmation: high resolution mass spectrometry versus tandem mass spectrometry. Anal Chim Acta
856:54-67
Meric D H (2010) Untersuchungen zur Aufnahme und zum Transport antibiotisch wirksamer Stoffe in
Getreide- und Gemüsepflanzen, Dissertation, Universität Paderborn
Vockel A (2005) Bestimmung von Chlortetracyclinrückständen in biologischen Proben aus der
landwirtschaftlichen Tierhaltung mit HPLC-UV-MS/MS- Methodenentwicklung und Anwendung in
Medikationsstudien. Dissertation, Universität Paderborn
12