Antibiotics, Vol. 11, Pages 1735: Quantifying Antibiotic Distribution in Solid and Liquid Fractions of Manure Using a Two-Step, Multi-Residue Antibiotic Extraction

C.P.: Conceptualization, Validation, Investigation, Writing—Original Draft; A.Y.: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing—Original draft preparation, Visualization; C.R.: Conceptualization, Resources, Writing—Review and Editing, Supervision; S.L.: Conceptualization, Writing—Review and Editing, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Figure 1. Recoveries from extraction of spiked manure with either EDTA McIlvaine buffer (solid bar) or 50:50 MeOH:ACN (the dotted bar). Recoveries are treatment averages (n = 3), with error bars based on ± standard error. The antibiotics examined include Ampicillin (AMP), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

Figure 1. Recoveries from extraction of spiked manure with either EDTA McIlvaine buffer (solid bar) or 50:50 MeOH:ACN (the dotted bar). Recoveries are treatment averages (n = 3), with error bars based on ± standard error. The antibiotics examined include Ampicillin (AMP), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

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Figure 2. Recoveries from the initial two-step extraction experiment with EDTA-McIlvaine (solid bar) as the first extractant followed by MeOH (dotted bar) as the second extractant. Resulting recoveries were averages (n = 3), with error bars based on ± standard error. The antibiotics examined included Ampicillin (AMP), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

Figure 2. Recoveries from the initial two-step extraction experiment with EDTA-McIlvaine (solid bar) as the first extractant followed by MeOH (dotted bar) as the second extractant. Resulting recoveries were averages (n = 3), with error bars based on ± standard error. The antibiotics examined included Ampicillin (AMP), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

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Figure 3. Extraction recoveries of antibiotics from four types of manure processed through a manure treatment system (graphed from left to right within each antibiotic): unprocessed manure (UPM), liquid separated manure (SL), solid separated manure (SS), and manure treated using a bedding recover unit (BRU). The extraction was a two-step process with an EDTA-McIlvaine buffer as the first extractant (EDTA, top half of the bar) and MeOH as the second (bottom half of the bar). Values from each extraction fraction are averages (n = 3) with ± standard error bars. The antibiotics examined include Ampicillin (AMP, not recovered), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

Figure 3. Extraction recoveries of antibiotics from four types of manure processed through a manure treatment system (graphed from left to right within each antibiotic): unprocessed manure (UPM), liquid separated manure (SL), solid separated manure (SS), and manure treated using a bedding recover unit (BRU). The extraction was a two-step process with an EDTA-McIlvaine buffer as the first extractant (EDTA, top half of the bar) and MeOH as the second (bottom half of the bar). Values from each extraction fraction are averages (n = 3) with ± standard error bars. The antibiotics examined include Ampicillin (AMP, not recovered), Ceftiofur (CEF), Penicillin-G (PEN), Benzylpenicilloic Acid (BEN), Sulfadimethoxine (SUL), Oxytetracycline (OXY), Tetracycline (TET), Chlortetracycline (CHL), Tulathromycin-A (TUL), and Tylosin Tartrate (TYL).

Antibiotics 11 01735 g003 Figure 4. EDTA-McIlvaine and MeOH extracts based on methods in Section 3.6.3 were combined using two methods: (A) the two extract fractions were mixed, then diluted to 500 mL with DI water prior to cleanup using solid phase extraction (SPE), and (B) the EDTA-McIlvaine fraction was cleaned up using SPE and then eluted into the MeOH fraction. Circled numbers refer to sequence of steps in each method. Figure 4. EDTA-McIlvaine and MeOH extracts based on methods in Section 3.6.3 were combined using two methods: (A) the two extract fractions were mixed, then diluted to 500 mL with DI water prior to cleanup using solid phase extraction (SPE), and (B) the EDTA-McIlvaine fraction was cleaned up using SPE and then eluted into the MeOH fraction. Circled numbers refer to sequence of steps in each method. Antibiotics 11 01735 g004

Table 1. Select physiochemical properties and analytical method parameters of antibiotics inves-tigated in this study, including their chromatographic retention times (RT).

Table 1. Select physiochemical properties and analytical method parameters of antibiotics inves-tigated in this study, including their chromatographic retention times (RT).

AntibioticsAcronymInternal StandardMolecular Weight
(g/mol)Precursor Ion
(m/z)Product Ion
(m/z)RT
(min)Log Kow bpKaβ-LactamsAmpicillinAMPNA349.41350.1106.1 a, 114.01.881.353.07, 7.12 cCeftiofurCEFNA523.56524.0241.0 a, 125.22.031.222.64, 3.44, 10.7 dPenicillin-GPENNA334.39335.1217.1 a, 160.02.131.832.97, 4.75 cBenzylpenicilloic AcidBENNA352.4353.1160.0 a, 128.02.12NDNDSulfonamidesSulfadimethoxineSULSUL-d6310.33311.1156.1 a, 92.12.111.631.62, 6.13 eTetracyclinesOxytetracyclineOXYDEM460.44461.2201.1 a, 98.11.89−0.93.71, 8.08, 10.15 bTetracyclineTETDEM444.3445.0410.2 a, 154.11.89−1.373.56, 7.09, 9.28 cChlorotetracyclineCHLDEM478.88479.1154.1 a, 981.94−0.623.49, 7.14, 9.28 cMacrolidesTulathromycin-ATULNAL806.1403.972.1 a, 116.11.863.698.6–9.6 eTylosin TartrateTYLROX916.112916.5174.1 a, 101.01.971.957.71cInternal StandardsSulfadimethoxine-d6SUL-d6NA316.37317.95108.0 a2.11 DemeclocyclineDEMNA464.86465.1154.1 a1.92 Nalidixic acid-d5NALNA237.27238.24104.2 a2.23 RoxithromycinROXNA837.06837.54158.1 a2.07

Table 2. Total solids (TS) of the manure used in this study, and the wet mass used in the extraction experiments based on a TS of 0.25 g/g manure for extraction. Solids are reported as average values (n = 3) with ± standard error.

Table 2. Total solids (TS) of the manure used in this study, and the wet mass used in the extraction experiments based on a TS of 0.25 g/g manure for extraction. Solids are reported as average values (n = 3) with ± standard error.

ManureTotal Solids (gsolids/gwet manure)Mass Extracted (gwet)Blank Dairy Manure (BDM)0.134 ± 0.0052Unprocessed Pit Manure (UPM)0.074 ± 0.0043.61Separated Liquid (SL)0.064 ± 0.00024.20Separated Solids (SS)0.347 ± 0.0100.770Bedding Recovery Unit (BRU)0.370 ± 0.1200.722 Table 3. Results for optimization experiment that combined extracts for one injection per sample. Method A was compared to Method B as diagrammed in Figure 4. Table 3. Results for optimization experiment that combined extracts for one injection per sample. Method A was compared to Method B as diagrammed in Figure 4. AntibioticMethod AMethod Bp-ValueRecoveries (%) ± SDOxytetracycline131 ± 1345 ± 40.0004Tetracycline114 ± 768 ± 20.0004Chlorotetracycline67 ± 354 ± 10.0021Penicillin-G66 ± 158 ± 10.0006Sulfadimethoxine56 ± 331 ± 20.0003Tylosin53 ± 247 ± 60.18Tulathromycin-A49 ± 943 ± 20.32Ceftiofur11 ± 0.36.4 ± 0.50.0002Ampicillin2.3 ± 0.17.3 ± 0.40.00003Benzylpenicilloic Acid1.3 ± 0.045.6 ± 0.40.0001

Table 4. Method performance parameters for the two-step extraction of antibiotics from manure using Method A.

Table 4. Method performance parameters for the two-step extraction of antibiotics from manure using Method A.

AntibioticsRecovery (%RSD),
n = 3Matrix EffectLinearity Fit (R2)LOD a (µg/kg)LOQ b (µg/kg)AMP2% (4%)88%0.9953.5810.8CEF11% (21%)88%0.9960.8932.71PEN57% (22%)79%0.9862.537.68BEN0.5% (31%)85%0.9884.8314.6SUL56% (8%)68%0.9990.6061.84OXY131% (17%)88%0.9998.0524.4TET114% (10%)88%0.9992.026.11CHL66.2% (6%)89%0.999721.2TUL47% (25%)57%0.9893.189.64TYL55% (3%)86%0.9960.2290.694

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