Improved sensitivity for characterization of sulfonamides and trimethoprim in honey using QuEChERS extracts with LC-MS/MS. Hernando Escobar 1 ; Jeffrey H.Dahl 1 ; Eddie Medina 1 ; Christopher T. Gilles 1 . 1 Shimadzu 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 Method Sample 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.Conclusions LC 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 pixabay.com 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 r 2 ≥ 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