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Improved sensitivity for characterization of sulfonamides and trimethoprim in honey using QuEChERS extracts with LC-MS/MS. Hernando Escobar1; Jeffrey H.Dahl1; Eddie Medina1; Christopher T. Gilles1. 1Shimadzu Scientific Instruments, Columbia, MD.USA
Stock standard solutions of each compound were
prepared by dissolving weighed amounts in
methanol alkaline 5mM with NH4OH then diluting
to 100 µg/mL with mobile phase A:B 50:50 and
a final dilution to 1000 ng/mL using matrix extracts
from honey without SAs and TMP spiked . Table 1
shows the concentrations at each 17 levels used
to build calibration curves for external calibration
method. For recovery each level was prepared
using matrix matched extracts from honey without
SAs and TMP spiked for each QuEChERS SPE
cleanup sorbent. (Figure 2).
Mass Spectrometry:
ESI was used in positive mode, spray voltage was 4.5 kV, desolvation line
temperature was 250ºC, nebulization gas was 3.0 L/min, heating gas flow
10L/min, interface temperature was 300ºC, heater block was 400 ºC, and drying
gas 10 L/min.
To implement sulfonamide quantitation, MRM transitions were optimized using a
0.5 µg mixture of SAs, 1µL injections at 400uL/min. Three transitions from parent
ions and fragments were selected using the optimization tool software.
3. Results
A natural food as honey is subject of diverse restrictions for contaminants presence as the case of antibiotics including sulfonamides. It is a worldwide practice by
Beekeepers to use antibiotics as therapeutic agents to treat bee bacterial brood diseases, however the use of sulfonamides (SA) and trimethoprim (TMP) in
beekeeping practice is prohibited in EU. There are no a worldwide MRL’s for sulfonamides in honey in the EU, but a minimum required performance level (MRPL)
was set for analytical methods at a level of 10 µg/kg of sulfonamides, encouraging the development of reliable laboratory methods for detection in diverse foods
included honey. SAs and TMP posing threats to public health causing allergic reactions and increasing microorganism’s drug resistance. Considering MRLs and
MRPL of SAs and TMP in food products from animals tend to be continually reduced to preserve human health safety, HPLC-MS/MS is an effective strategy to
characterize and accurately measure those antibiotics. The present method is selective, fast, and very sensitive for 20 sulfonamides and trimethoprim.
1. Introduction
For Research Use Only. Not for use in diagnostic procedures.
2. Materials and MethodSample preparation
5 grams of honey were acidified with 5mL 2.0M HCl vortexed 1min and
sonicated 15 minutes before to spike with standard mixture of 21 sulfonamides
and trimethoprim at 10 ppb and 50 ppb. Extraction were performed using
QuEChERS method with extraction salts and diverse clean-up sorbents
following manufacturer’s procedure (Supelco Supel™ QuE Citrate,
PSA/C18, Zsep/C18, Zsep and Zsep+) with a final 1:5 extract dilution
using methanol to inject in the LCMS system. A multiple reaction monitoring
MRM method was optimized for quantitation for each sulfonamide compound
using a Shimadzu Nexera UHPLC with an LCMS-8060 fast-scanning triple
quadrupole mass spectrometer model equipped with software Labsolution
LCMS version 5.65 and electrospray ionization ESI.
Table 1. Conc. levels
to define cal. curves.
Authentic SAs standards were fully characterized by HPLC and MS/MS
with an MRM optimized assay. Figure 2 shows better performance for
QuEChERS extractions with SPE sorbet PSA/C18 reporting limit of
quantification LOQ as low as of 0.015 ppb (15ppt) for some SAs and
trimethoprim using calibration curves diluted with HPLC buffers and as
low as 0.03 ppb (30ppt) when calibrators were dissolved in matrix
extracted from commercial organic honey free of drugs diluted 1:5 in
MeOH. The recovery of SA compounds and trimethoprim (Figure 2)
spiked on that honey reported a range of 85 to 122% for 10µg/kg of
spiked compounds and a range of 99 to 122% when spiked with
50µg/Kg. Only sulfanilamide presented a poor recovery, reporting 10%
when 10µg/kg was spiked but an acceptable recovery of 58% when 50
µg/Kg was spiked, suggesting a matrix effect for lower level should be
considered and also an easy loss by the sulfanilamide double amino
group strong interaction with the primary secondary amine PSA sorbent.
LOQs are described in Figure 1. Calibration curves of standards in
matrix extracted from honey were linear with r2 > 0.990 in the tested
range as shown in Figure 3.
LC-MS/MS with appropriate selection of QuEChERS SPE sorbent as
extraction method provides a fast, simple, accurate and high sensitive
method for analysis of sulfonamide drugs and trimethoprim in honey
with also excellent recovery range improved from previous studies with
matrix matched calibration.
4.ConclusionsLC conditions
A Kinetex 2.6µ PFP 100 Å column (100 × 2.1 mm) was used
at 30ºC, flow rate of 0.5 mL/min, and 5 μL injection volume. A
binary gradient of 10% methanol, 0.3% formic acid (mobile
phase A) and methanol, 0.3% formic acid (mobile phase B)
was used with the gradient program described in Table 1
Table 2. HPLC gradient used.
1 1000 10 2.0
2 500 11 1.0
3 250 12 0.5
4 125 13 0.24
5 62.5 14 0.12
6 31.3 15 0.06
7 15.6 16 0.03
8 7.8 17 0.015
9 3.9
Concentration levels, ppb
Time (min) %B
0.0 2
0.5 8
5.0 30
5.01 80
5.2 5
5.5 80
6.0 80
6.01 2
8.0 2
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Figure 3: Representative calibration curves using standard drug
mixture with matrix matched added. High degree of linearity was
observed over the concentration range 1000-0.03 ppb with values of
r2 ≥ 0.990 for all analytes. Chemical structures are represented for
each sulfonamide compound and trimethoprim.
Figure 1: Criteria for LOQ selection was S/N >10 ; area RSD% < 5 (n=3) ;
reference ions 30% allowance and area calculated accuracy >90%.
Figure 2. Average (n=2) recovery of honey spiked with 10µg/Kg
(Figure 2A) and 50µg/Kg (Figure 2B) using diverse SPE sorbents.
A
B