Suspect Screening and Semi-Quantification of Macrolide Antibiotics in Municipal Wastewater by High-Performance Liquid Chromatography

An LCMS-8040 HPLC-MS/MS system (Shimadzu, Kyoto, Japan) consisting of an LC-30 “Nexera” liquid chromatograph and triple quadrupole mass analyzer was used in liquid chromatography–tandem mass spectrometry analyses. The HPLC system consisted of a DGU-A5 vacuum degasser, two LC-30AD chromatographic pumps, an LC-30AC autosampler and a CTO-30A column thermostat. The system control and data analysis were performed using LabSolutions 5.56 software (Shimadzu, Kyoto, Japan).

Chromatographic separation was achieved in a gradient elution mode on a Nucleodur PolarTec column (Macherey-Nagel, Duren, Germany), 150 × 2.0 mm, particle size 1.8 μm, with a reverse-phase sorbent with embedded amide groups providing additional selectivity for polar compounds. The sample injection volume was 2 μL, mobile phase flow rate 0.3 mL min⁻¹ and column thermostat temperature 40 °C. The mobile phase consisted of solvent A (deionized water with 0.1% formic acid and 5 mM ammonium formate) and solvent B (acetonitrile with 0.1% formic acid). The gradient elution was programmed as follows: 0–2 min—10% B, 2–10 min—linear ramp to 50% B, 10–15 min—50% B.

Mass spectrometry detection was carried out in MRM and PrecIS modes using electrospray ionization in a positive ion mode (ESI+). Ion source parameters: spraying, drying and curtain gas (nitrogen) flow rates—2 and 7.5 L min⁻¹, respectively; desolvation line and heat block temperature—300 °C and 400 °C, respectively; ESI capillary voltage—4.5 kV. A collision-induced dissociation (CID) with argon as a collision gas was used for obtaining tandem mass spectra. The conditions for mass spectrometric detection of macrolides in MRM mode were optimized in an automatic regime by the flow injection of analyte standard solutions (Table 1).

Compound	Molecular Weight, Da	Precursor Ion, m/z	Product Ion, m/z	Q1 Bias, V	Collision Energy, eV	Q2 Bias, V
Azithromycin	748.5	749.5	158	−33.9	41.3	−27.4
			83 *	−37.1	53.0	−33.9
Spiramycin	842.5	843.5	174	−14.5	34.5	−14.5
			142 *	−14.5	38.5	−17.8
Erythromycin	733.5	734.5	158	−33.9	32.7	−27.4
			83 *	−33.9	49.8	−33.9
Midecamycin	813.5	814.5	109	−21.0	45.2	−43.5
			174 *	−21.0	33.9	−17.8
Clarithromycin	747.5	748.5	158	−33.9	32.0	−14.5
			83 *	−33.9	50.2	−33.9
Josamycin	827.5	828.5	109	−21.0	43.6	−17.8
			174 *	−21.0	35.9	−17.8

Liquid chromatography–high-resolution mass spectrometry (HPLC-HRMS) analyses were carried out using an HPLC-HRMS system consisting of the same HPLC system as described in Section 2.3 and an Orbitrap ID-X high-resolution “tribrid” mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an OptaMax NG ion source with ESI probe. Chromatographic separation was carried out within the same parameters as described in Section 2.3. In mass spectrometry detection, the following parameters were applied: spraying, drying and curtain gas flow rates—50, 10 and 3 arb. units, respectively; transfer line temperature—325 °C; ESI capillary voltage—4.5 kV. The spectra were scanned in an m/z range of 200–1200 using an orbital ion trap mass analyzer with resolving power set to 120,000 (M/ΔM, at m/z 200). To maintain the highest accuracy in determining m/z (<3 ppm), internal EASY-IC mass scale calibration was used. Xcalibur software (Thermo Scientific, Waltham, MA, USA) was used to control the instrument and acquire HRMS chromatograms.

In the study of the analytes’ mass spectrometry fragmentation and in attributing the peaks in the obtained tandem mass spectra (Section 3.1), the flow injection of analyte solutions was used with the further determination of the elemental compositions of the product ions based on their accurate masses. A higher energy collisional dissociation (HCD) similar to CID in triple quadrupole instruments was used. The fragmentation pathways were proposed using ACD Fragmenter software (ACD/Labs, Toronto, ON, Canada).

The quantification of the target analytes in both MRM and PrecIS detection modes was carried out by an external standard method with calibration curves constructed using the calibration solutions of the six macrolides (Section 2.1). The same approach was applied to the semi-quantitative assessment of the detected macrolides or their metabolites by HPLC-MS/MS with PrecIS detection using the calibration curves obtained for the standards structurally close to the corresponding analytes. Limits of detection (LOD) and quantification (LOQ) were determined using the signal-to-noise ratio criteria of 3 and 10, respectively, and then refined in the analysis of the solution with analyte concentration close to LOQ. The full method validation, involving an estimation of matrix effects, intra-day and inter-day accuracy and precision, cannot be considered applicable in the case of the screening studies in the absence of the corresponding analytical standards.