Simultaneous determination of ten macrolides drugs in feeds by high performance liquid chromatography with evaporation light scattering detection

Abstract

A sensitive and reproducible method based on high performance liquid chromatography with evaporation light scattering detection (ELSD) was developed for the simultaneous determination of 10 macrolides drugs such as azithromycin, tulathromycin, spiramycin, tilmicosin, tylosin, erythromycin, clarithromycin, roxithromycin, midecamycin and josamycin in feeds. Feed samples were extracted with a sodium borate buffer solution (pH 10.0) − ethyl acetate. The dry extracts were dissolved in a phosphate buffer solution (pH 8.0), and then applied to an Oasis HLB solid-phase extraction cartridge for cleanup. The residues were reconstituted in 0.5 mL of the mobile phase. By optimizing the main operational parameters of ELSD and chromatographic conditions, all target compounds were well separated on an Ecosil C₈-SH column (250 mm × 4.6 mm, 5 μm) using a gradient elution program. The calibration curves showed good linearity (r > 0.9985) in the range of 1–200 μg mL⁻¹ for ten analytes. The average recoveries of all analytes from five kinds of feeds spiked at three levels were between 60.2% and 112%, with intra-day and inter-day relative standard deviations below 11% and 15%, respectively. The limits of detection ranged from 0.4 to 0.8 mg kg⁻¹ for ten macrolides.

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1. Introduction

For many years antibiotics have been widely used in food-producing animals for treating infectious diseases or growth promotion.¹ Nevertheless, the improper or excessive usage of antibiotics as feed additives greatly contributes to the selection and transmission of antibiotic-resistant bacteria. These bacteria spread into the human body through the food chain, and their presence ultimately has consequences for human health.² Thus, the European Union decided to ban antibiotics as feed additives from January 1, 2006 onwards.³ But some antibiotics such as macrolides and tetracyclines are licensed for usage as feed additives in China, the United States and some other countries.

Macrolides are a group of weakly basic antibiotics produced by Streptomycetes and show great inhibitory activity to Gram-positive bacteria and mycoplasma. Some of them have been frequently used in feeds to gain weight and improve feed conversion ratio. Nowadays the amount of macrolides used in global feed additives is second only to tetracyclines, hence a reliable and practical analytical method is necessary for the monitoring of macrolides drugs in various feeds for animal-derived food safety.

The determination of macrolides drugs is traditionally performed by microbiological or immunological assays.^4,5 However, they are often time consuming and lack the specificity required for analytical purposes. Chromatographic techniques, which allow simultaneous analysis, are main alternatives. Numerous methods have been developed for the determination of macrolides drugs in animal tissues, urine and feeds by liquid chromatography (LC) with ultraviolet (UV), electrochemical or fluorimetric detection,^6–10 and by liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).^11–14 However, most of macrolides drugs have weak UV absorbance in the low wavelength range as it lacks a suitable chromophore.¹⁵ Therefore, a sensitive and selective UV detection of this group is difficult. Furthermore, macrolides compounds possess high oxidation potential at glassy carbon electrode, so electrochemical detection is susceptible to be interfered by reducing substances in the mobile phase and sample matrix.⁹ The fluorimetric detection needs derivatization, which is troublesome as well as easily causes decomposition of some drugs such as erythromycin.¹⁰ It seems to be an appropriate means using LC-MS or LC-MS/MS with high sensitivity and selectivity, whereas they are too expensive to be used widely in China. Along with the development of analytical instruments, the sensitivity of universal detectors is increased greatly. The evaporation light scattering detection (ELSD) response does not depend on the optical characteristics of target analytes, which eliminates the common problems associated with UV and fluorimetric detections. So it is increasingly being used to analyze the compounds that there are no UV absorptivity and fluorescence in molecules, especially nonvolatile compounds.¹⁶ Currently, there is still no report for simultaneous determination of major macrolides drugs using HPLC-ELSD.

This study focused on the development of a sensitive, reliable and practical method capable of simultaneous extracting, cleaning up and detecting ten macrolides drugs including azithromycin (AZI), tulathromycin A (TUL), spiramycin (SPM), tilmicosin (TIL), tylosin (TYL), erythromycin A (ERY), clarithromycin (CLA), roxithromycin (ROX), midecamycin (MDM), and josamycin (JOS) in feeds by high performance liquid chromatography with evaporation light scattering detection. The chemical structures of the studied drugs are shown in Fig. 1.

Fig. 1 Chemical structures of ten macrolides drugs.

2. Experimental

2.1. Materials and reagents

AZI, TUL, TIL, TYL, ERY were purchased from Sigma Chemicals Co. (St. Louis, MO, USA), SPM, CLA, ROX, JOS were granted from European Pharmacopoeia (EDQM, Strasbourg, France), and MDM was obtained from China Institute of Veterinary Drug Control (Beijing, China). All these chemicals were analytical grade ≥95% purity.

Acetonitrile, formic acid from Fisher Scientific (Fair Lawn, NJ, USA) and ammonium acetate from TEDIA (Fairfield, OH, USA) were HPLC grade. Methanol, ethyl acetate, disodium tetraborate decahydrate, disodium hydrogen phosphate dodecahydrate, sodium hydroxide, hydrochloric acid, and ammonia from Guangzhou Chemical Reagent Factory (Guangzhou, Guangdong, China) were analytical reagent grade. High purity water was obtained by a MilliQ system from Millipore (Molsheim, France). Oasis HLB solid-phase extraction (SPE) cartridge (60 mg, 3 mL) and Oasis MCX SPE cartridge (60 mg, 3 mL) were purchased from Waters Co. (Milford, MA, USA). Bond Elut-C18 SPE cartridge (200 mg, 3 mL) was purchased from Agilent Technologies Co. (Santa Clara, CA, USA).

2.2. Standard solutions

Individual stock standard solutions (1 mg mL⁻¹) were prepared by weighing approximately 10 mg of each standard into 10 mL volumetric flask and dissolving with acetonitrile, respectively. These solutions were stored at −20 °C and stable for at least 6 months.

Working standard mixture solutions of ten macrolides were prepared by mixing individual stock solutions and serially diluting to different levels with acetonitrile. These solutions were stored at 4 °C and stable for at least 1 month.

2.3. Instrumentation and chromatographic conditions

Analysis was performed on a Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA, USA), consisting of a quaternary pump solvent management system, an autosampler, and an online degasser, coupled with a Waters 2424 ELSD connected to a GA-10B Air Generator (Zhongxinghuili Technology Development Co. Ltd., Beijing, China). Data acquisition and processing, as well as instrumental control were performed by Empower Software. An Ecosil C₈-SH column (250 mm × 4.6 mm, 5 μm, Lubex, Germany) was applied for LC separation. The mobile phase was composed of acetonitrile (A) and acetate buffer solution containing 2 mM ammonium acetate and 0.1% formic acid (B) and the gradient elution was programmed as Table 1. The flow rate was set at 1.0 mL min⁻¹ and the column temperature was maintained at room temperature. The injection volume was 40 μL.

Table 1 The HPLC gradient elution program

Time (min)	A (acetonitrile, %)	Curve^a
a Gradient curve is a typical application in Waters 2695 Alliance HPLC system. Curve 6 represents a linear change, and curve 7 represents a slight concave change.
0–1	8	6
1–15	8–45	7
15–20	45	6
20–22	45–8	6
22–30	8	6

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All the analytes were monitored with ELSD. The nebulizer gas (air) pressure was set at 20 psi and the drift-tube temperature was maintained at 60 °C, meanwhile, the nebulizer temperature was 36 °C. Gain was set at 300. The time constant was set as Slow Speed.

2.4. Extraction

Accurately weigh 1 g ground and sieved feed sample into 15 mL polypropylene centrifuge tube, 5 mL of 25 mM sodium borate buffer solution (pH 10.0) was added into the tube. After vortexing for 1 min, 5 mL of ethyl acetate was added, and then the tube was ultrasonicated for 15 min and centrifuged at 8500×g for 5 min at 4 °C. The supernatant was decanted into a glass tube. The extraction was repeated with 5 mL of ethyl acetate. All the supernatants were combined and blown until near dryness at 45 °C under a gentle stream of nitrogen. The dry extracts were reconstituted in phosphate buffer solution (pH 8.0) for cleanup.

2.5. Cleanup

An Oasis HLB SPE cartridge was employed for sample cleanup. The cartridge was sequentially preconditioned with 3 mL of methanol, 3 mL of water, and 3 mL of 0.1 M phosphate buffer solution (pH 8.0). The reconstituted solution was passed through the cartridge at a flow rate of about 1 mL min⁻¹. The cartridge was successively washed with 3 mL of water and 3 mL of 40% methanol in water, and then was dried by using a low positive pressure followed by elution with 5 mL of methanol. The eluates were evaporated to dryness under a gentle stream of nitrogen at 45 °C, and the residues were re-dissolved in 0.5 mL of mobile phase. Finally, the extracts were vortexed for 30 s and filtered through a 0.22 μm syringe filter prior to HPLC-ELSD analysis.

3. Results and discussion

3.1. Optimization of separation conditions

It was observed the tailing of the chromatographic peaks of macrolides compounds in general silica-based LC stationary phases as their tertiary amines in molecules. In contrast, the performance of the endcapped C₈ and C₁₈ columns prepared from high purity silica gel is satisfactory. As a result, the Ecosil C₈-SH column was used for separation of compounds in subsequent studies.

As for mobile phase, different kinds of organic phases such as methanol and acetonitrile were tested. Chromatographic peaks of some analytes were broadened and partially overlapped when methanol was as organic phase. Conversely, good peak shapes of analytes were achieved when acetonitrile as organic phase. In order to increase the buffering capacity of the aqueous phase and easily be evaporated, different concentrations of ammonium acetate buffer (2, 5 and 20 mM) were evaluated as aqueous phase. The experimental results showed that JOS was eluted in ca. 24 min, and it was strange that JOS disappeared when the ammonium acetate solution was up to 5 or 20 mM (Fig. 2). It was suggested that the higher concentration ammonium acetate buffer affect the formation of JOS sol (droplets) when it was eluted (ca. 24 min) in high percentage of water in mobile phase. Additionally, the mobile phase containing 0.1% formic acid could effectively suppress peak tailing. So that good chromatographic resolutions and symmetrical peak shapes of ten macrolides compounds were achieved when acetonitrile was as organic phase (A) and 2 mM ammonium acetate buffer solution containing 0.1% formic acid as aqueous phase (B).

Fig. 2 HPLC-ELSD chromatograms of different concentrations of ammonium acetate buffer solution as aqueous phase (1) AZI (azithromycin); (2) TUL (tulathromycin A); (3) SPM (spiramycin); (4) TIL (tilmicosin); (5) TYL (tylosin); (6) ERY (erythromycin A); (7) CLA (clarithromycin); (8) ROX (roxithromycin); (9) MDM (midecamycin); (10) JOS (josamycin).

On the basis of the selected chromatographic column and mobile phases, the chromatographic separation was further investigated under different gradient elution conditions. The initial and final eluent compositions were optimized in order to obtain the best resolution of target analytes. The results showed that it was difficult to achieve good separation for AZI and TUL if the initial composition of acetonitrile was below 5% (Fig. 3a), and however, the response of AZI was significantly decreased if the initial composition of acetonitrile was above 10% (Fig. 3b). Therefore, the initial composition of acetonitrile was set at 8%. It was also shown that if the final composition of acetonitrile was lower than 40%, ERY and CLA could not be separated well and their responses were weak (Fig. 3c). Besides, ERY, CLA, ROX and MDM were not separated completely when the final composition of acetonitrile was higher than 50% (Fig. 3d). Thus, the final composition of acetonitrile was set at 45%.

Fig. 3 Influence of different gradient elution conditions on chromatographic separation of ten macrolides compounds (each at 20 μg mL⁻¹) (a) the initial composition of acetonitrile set at 5%; (b) the initial composition of acetonitrile set at 10%; (c) the final composition of acetonitrile set at 40%; (d) the final composition of acetonitrile set at 50%; (e) the gradient durations from the initial composition to the final composition last 13 min; (f) the initial composition of acetonitrile linearly increased to the final composition in 15 min; (g) solvent blank; (h) the optimal separation conditions. Peak identification is the same as in Fig. 2.

The gradient durations and curve were also further tested. When the gradient durations were 13 min, the peaks of AZI and TUL could not be completely separated (Fig. 3e). However, when the gradient durations were 15 min, all analytes were completely separated. In addition, if the composition of mobile phase was linearly increased (curve 6 in Table 1) from 8% to 45% in 15 min, the responses of SPM, TIL and TYL were reduced significantly (Fig. 3f). Finally, the curve 7 (see Fig. S1†), which represents a slight concave change, was selected as the gradient curve, the separation and responses of target analytes were v1ery well. The typical chromatograms of solvent blank and ten macrolides compounds are shown in Fig. 3g and h, respectively.

3.2. Optimization of ELSD parameters

The main operational parameters of the ELSD including the drift-tube temperature and nebulizer gas pressure significantly affect the signal response of analyte.¹⁷ Under the optimized gradient elution program and flow rate, the ELSD parameters were optimized by the injection of 20 μg mL⁻¹ three typical compounds including TUL, TIL and ROX at different drift-tube temperature from 40 °C to 100 °C and nebulizer gas pressure between 20 and 60 psi. As shown in Fig. S2A,† an increase in the ELSD response is observed when the drift-tube temperature rose from 50 °C to 60 °C. Then the signals gradually drop and level off. It is noticeable that baseline noise significantly increases at the temperature below 50 °C, and the compounds could not be detected while the temperature was lower than 45 °C. That is because the mobile phase could not be evaporated completely at low drift-tube temperature, leading to great baseline noise. Conversely, higher temperature might cause excessive volatilization and decomposition of compounds, and resulted in reduction of sensitivity. Accordingly, the drift-tube temperature was set at 60 °C for higher sensitivity.

Setting the drift-tube temperatures at 60 °C, the nebulizer gas pressure was optimized. The experimental results show there is a significant decrease of the ELSD response with the rise of gas pressure, due to the formation of smaller droplets that scatters less light (Fig. S2B†).¹⁸ Furthermore, the minimum pressure that the ELSD allowed to set is 20 psi. Therefore, 20 psi was selected as the optimal gas pressure, which provided the highest response for all analytes.

The parameter of the Gain controls the detector's signal amplification to ensure the detection of small peaks, but higher Gain value leads to the rise of baseline noise. The optimal Gain value was 300 in this study. Moreover, the time constant was set as Slow Speed in order to reduce baseline noise and enhance sensitivity.

3.3. Extraction

Various solvents such as methanol,^19,20 acetonitrile,^21,22 sodium borate buffer solution (pH 10.0) – ethyl acetate⁶ have been used to extract macrolides. In this study, the extraction efficiencies of the three solvents were compared by analyzing compound feed samples spiked with ten macrolides at 10 mg kg⁻¹. The results of preliminary experiments showed that three solvents had similar extraction efficiency and the extract recoveries of ten macrolides were more than 85%. But the most serious matrix interference was produced when methanol was used as the extraction solvent, and acetonitrile took second place. For verifying whether the pH 10.0 of sodium borate buffer was the optimal condition, the pH value of sodium borate buffer solution was further investigated. Because most of macrolides are considered to be unstable under acidic condition, and the compounds contain nitrogen that is neutral under alkaline pH, sodium borate buffer solution was adjusted to different pH values including 8.0, 9.5, and 10.0 in the alkaline range with 2 M sodium hydroxide. As shown in Fig. S3,† at 8.0 of pH value, the recoveries of all target analytes except MDM and JOS (above 70%) were very poor. While recoveries more than 85% were obtained for all analytes at pH 10.0. Comparison with pH 10.0, although most analytes obtained high recoveries, the recoveries of both of AZI and TUL were obviously decreased at pH 9.5.

3.4. Cleanup

The Oasis HLB cartridge,²³ Bond Elut-C₁₈ cartridge,^14,24,25 and Oasis MCX cartridge,^26,27 have been reported to cleanup macrolides drugs in extracts of feeds. Thus, three types of cartridges were applied for purifying the extracts of three solvents above, respectively. Each type of cartridge was used in its corresponding most suitable conditions. In brief, as for the C₁₈ cartridge, 5 mL of 20% acetonitrile in water was used to dissolve the extracts, 3 mL of water and 3 mL of 20% methanol in water as washing solvents and 5 mL of 5% ammonia in methanol as elution solvent; as for the MCX cartridge, 5 mL of 0.1 M hydrochloric acid was used to dissolve the extracts, 3 mL of water and 3 mL of methanol as washing solvents, and 5 mL of 5% ammonia in methanol as elution solvent. The results are given in Table S1.† Except AZI, TUL and TIL, other analytes could not be retained well on the MCX cartridge. But TIL was seriously interfered from the matrix extracts of methanol. High recoveries for TIL were obtained from acetonitrile and sodium borate buffer solution (pH 10.0) – ethyl acetate. Recovery for TYL was more than 70% only from sodium borate buffer solution (pH 10.0) – ethyl acetate.

Most analytes were retained well on the C₁₈ cartridge, but both of AZI and TUL were almost not retained. Conversely, on the HLB cartridge high recoveries were obtained for all the compounds except for TUL. Additionally, 5 mL of 5% ammonia in methanol had to be applied for eluting TUL on the HLB cartridge. Therefore, the system of sodium borate buffer solution (pH 10.0) – ethyl acetate was selected as the extraction solvent for the following experiments. Typical chromatograms of the spiked and blank compound feeds with different extraction solvents are shown in Fig. 4. Extracting with methanol showed the most matrix interference that especially affected the peaks of TIL and TYL (Fig. 4A). Obviously, extracting with acetonitrile and ethyl acetate had less matrix interference. 5 mL of 5% ammonia in methanol could elute TUL on the HLB cartridge. But the matrix interference seriously affected the peak of TYL in Fig. 4B.

Fig. 4 Chromatograms of the extracts with (A) methanol, (B) acetonitrile, (C) sodium borate buffer solution (pH 10.0) – ethyl acetate under the Oasis HLB cartridges purification (a) blank feed spiked at 10 mg kg⁻¹; (b) blank feed; (c) 5% ammonia in methanol elution. Peak identification is the same as in Fig. 2.

3.5. Performance of the method

3.5.1 Linearity. To verify linearity of the developed method, each calibration curve was established in triplicates with six concentrations. According to the published report,²⁸ the logarithm of the injected concentration is linearly correlated to the logarithm of the peak area from ELSD within a certain range of concentrations. As shown in Table 2, all the calibration curves show good linearity in the range of 1–100 μg mL⁻¹ for AZI and ERY, 1–200 μg mL⁻¹ for TIL and CLA, and 2–200 μg mL⁻¹ for TUL, SPM, TYL, ROX, MDM, and JOS. The curves are given by equations with the correlation coefficients (r) greater than 0.99 for ten macrolides except for AZI and ERY.

Table 2 Linearity, limit of detection and limit of quantification

Compound	Calibration curve^a	Correlation coefficient, r	Linear range, μg mL^−1	LOD mg kg^−1	LOQ mg kg^−1
a x is the logarithmic value for peak area in relation to the ELSD chromatogram and y is the logarithmic value for concentration (μg mL^−1) injected.
Azithromycin	y = 0.5524x − 1.7958	0.9991	1–100	0.4	1.0
Tulathromycin	y = 0.5386x − 1.5641	0.9995	2–200	0.8	1.5
Spiramycin	y = 0.6015x − 1.8841	0.9995	2–200	0.6	1.5
Tilmicosin	y = 0.5892x − 1.9912	0.9998	1–200	0.4	1.0
Tylosin	y = 0.5918x − 1.7831	0.9998	2–200	0.6	1.5
Erythromycin	y = 0.6255x − 2.1139	0.9985	1–100	0.4	1.0
Clarithromycin	y = 0.6040x − 2.0037	0.9999	1–200	0.4	1.0
Roxithromycin	y = 0.5588x − 1.7075	0.9999	2–200	0.6	1.5
Midecamycin	y = 0.5671x − 1.5782	0.9996	2–200	0.8	1.5
Josamycin	y = 0.6080x − 1.7950	0.9996	2–200	0.8	1.5

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3.5.2 Recovery. The method was firstly established in accordance with a typical complete feed sample consisting of corn gluten, soybean meal, wheat bran, bone meal, amino acids, and vitamin. Subsequently, the method developed was applied to other four kinds of feeds, including one pig and one poultry compound formula feeds, one pig premix, and one feed additive. Recovery studies spiked at three levels for ten analytes were performed to validate the method proposed. Each spiking level was repeated three times for each feed. The samples were prepared and analyzed on three consecutive days. Recoveries were calculated according to the calibration curve described in Table 2. The data are shown in Table 3. The Experiment with spiked samples showed that at the spiking level of 1 mg kg⁻¹, the average recoveries of AZI, TIL, ERY and CLA ranged from 70.4% to 112% with relative standard deviations (RSDs) less than 11%; the average recoveries of TUL, SPM, TYL, ROX MDM, and JOS ranged from 64.2% to 97.2% with RSDs of below 13% and at 1.5 mg kg⁻¹. The average recoveries of all target compounds ranged from 63.2% to 104% with RSDs below 13% at 5 mg kg⁻¹, and from 60.2% to 101% with RSDs below 15% at 20 mg kg⁻¹. In summary, the overall recoveries were between 60.2% and 112% for all ten macrolides drugs, and both of the intra-day and inter-day RSD were below 15%.

Table 3 Average recoveries and precision of ten macrolides from the spiked feeds (n = 3)^a

Compound	Spiking level, mg kg^−1	Average recovery (RSD), %
		A		B		C		D		E
		Intra-day	Inter-day	Intra-day	Inter-day	Intra-day	Inter-day	Intra-day	Inter-day	Intra-day	Inter-day
a A and B represent two kinds of pig compound feeds; C, D and E represents poultry compound feed, pig premix and feed additive, respectively.
Azithromycin	1	82.3(3.5)	81.2(5.1)	75.9(4.5)	78.4(5.3)	70.4(2.1)	72.4(5.7)	93.9(2.8)	88.9(4.6)	85.8(4.1)	83.2(4.7)
	5	78.9(4.3)	79.1(5.7)	88.7(3.3)	88.9(3.0)	73.3(2.7)	73.5(3.3)	60.9(3.8)	68.7(9.3)	71.4(4.7)	77.1(10.9)
	20	75.4(2.1)	73.9(3.0)	89.9(2.7)	91.4(2.2)	78.3(4.1)	80.1(8.5)	74.2(0.1)	76.0(3.2)	89.5(7.7)	92.0(3.8)
Tulathromycin	1.5	65.5(6.8)	64.2(3.4)	68.1(2.6)	71.0(6.9)	65.4(3.4)	64.6(2.2)	78.3(6.7)	77.2(4.5)	68.0(3.4)	70.9(3.7)
	5	70.3(5.2)	68.5(7.5)	75.2(5.9)	78.4(5.4)	61.7(6.3)	63.5(6.6)	62.7(2.2)	69.2(5.9)	67.3(6.7)	70.3(7.2)
	20	70.8(0.8)	69.0(6.7)	65.6(6.3)	69.3(8.3)	76.5(7.0)	73.7(7.8)	60.2(4.0)	63.9(4.7)	72.2(5.8)	73.5(4.2)
Spiramycin	1.5	90.2(2.2)	89.9(2.6)	73.4(1.1)	77.6(5.8)	89.7(7.8)	95.2(11.5)	80.5(3.5)	81.3(0.6)	79.2(4.5)	80.2(4.3)
	5	70.5(4.6)	74.2(7.1)	75.0(3.9)	76.4(3.8)	103.8(3.3)	98.9(7.6)	65.8(4.8)	73.8(6.9)	86.0(5.3)	85.9(1.8)
	20	98.6(7.4)	95.6(7.0)	80.1(2.5)	76.5(9.5)	91.9(6.1)	92.7(6.4)	72.2(2.7)	80.0(13.9)	74.8(3.4)	74.8(0.6)
Tilmicosin	1	88.0(4.0)	89.2(3.5)	90.4(3.2)	84.7(10.7)	76.5(3.7)	78.0(5.3)	99.6(0.3)	95.6(5.9)	95.1(1.5)	88.3(10.9)
	5	89.7(9.3)	89.1(2.2)	89(0.4)	87.9(3.2)	76.2(4.0)	77.6(3.6)	96.2(3.9)	96.7(3.1)	94.7(2.2)	90.7(3.2)
	20	95.9(1.6)	96.2(4.7)	92.3(0.4)	83.3(14.5)	83.9(5.3)	82.7(6.8)	81.1(0.7)	87.7(10.7)	90.8(4.4)	86.7(6.7)
Tylosin	1.5	94.4(3.1)	88.5(12.7)	93.5(0.8)	93.1(3.1)	85.4(5.6)	84.3(4.7)	92.5(2.1)	92.1(3.3)	89.7(4.9)	90.6(2.0)
	5	90.1(5.2)	89.6(6.8)	95.7(3.7)	96.1(4.5)	91.1(2.0)	90.5(0.9)	91.6(4.3)	93.2(4.9)	90.5(6.4)	91.5(5.3)
	20	96.2(1.9)	95.5(2.6)	101.2(5.6)	97.5(9.3)	93.4(9.8)	95.6(4.6)	98.4(0.2)	95.2(4.8)	97.3(9.7)	95.9(5.8)
Erythromycin	1	90.5(3.2)	92.1(5.0)	89.3(4.3)	90.2(3.0)	89.0(6.2)	87.3(5.3)	111.2(3.2)	105.3(8.8)	79.3(2.5)	80.1(3.3)
	5	94.3(6.3)	95.7(2.1)	98.1(3.0)	94.7(7.7)	95.3(9.3)	87.5(10.4)	96.9(3.7)	93.2(7.6)	75.4(4.4)	78.5(8.1)
	20	99.4(0.3)	96.5(6.5)	93.5(10.0)	92.2(2.1)	80.7(1.6)	82.1(3.2)	99.3(0.9)	98.0(2.0)	76.7(5.6)	77.6(5.2)
Clarithromycin	1	94.1(2.9)	93.6(3.7)	83.8(3.8)	84.0(4.5)	76.9(3.5)	77.1(4.2)	94.8(1.0)	95.7(2.3)	78.8(3.2)	77.2(4.7)
	5	78.9(0.7)	80.5(1.5)	90.3(0.9)	92.0(2.0)	77.0(4.2)	75.6(4.7)	86.6(6.0)	90.7(7.8)	80.1(0.9)	78.2(1.7)
	20	95.1(5.5)	92.2(8.2)	82.8(8.3)	90.3(11.7)	75.8(1.7)	75.9(2.0)	91(4.5)	93.8(4.3)	72.7(9.0)	73.8(8.6)
Roxithromycin	1.5	83.1(6.7)	88.7(7.9)	85.7(4.8)	87.2(5.7)	77.3(3.7)	80.3(7.5)	82.6(5.6)	85.5(6.4)	86.2(3.8)	86.1(1.3)
	5	74.5(7.9)	76.0(4.2)	89.8(0.5)	93.8(6.2)	96.5(4.8)	94.5(5.4)	90.4(4.4)	89.1(3.5)	97.8(2.6)	90.4(5.5)
	20	86.0(2.7)	80.3(10.0)	96.9(5.4)	96.3(0.9)	70.2(8.6)	78.2(10.6)	79.2(6.7)	83.7(7.7)	71.5(1.2)	72.6(2.0)
Midecamycin	1.5	90.6(3.3)	89.3(1.3)	91.3(5.5)	90.4(3.7)	93.4(3.4)	92.2(3.7)	97.2(3.3)	92.3(5.2)	71.6(1.0)	75.9(9.2)
	5	86.3(3.8)	92.1(7.7)	89.1(7.1)	88.3(8.9)	99.5(5.9)	93.4(12.3)	79(3.9)	85.2(8.2)	74.3(8.4)	78.2(10.1)
	20	92.9(6.8)	93.3(0.6)	90.4(10.5)	87.8(4.3)	89.5(4.4)	90.2(5.1)	79.4(7.7)	82.7(5.6)	77.7(2.3)	70.2(6.5)
Josamycin	1.5	71.1(0.4)	70.4(2.7)	78.2(2.9)	75.6(6.8)	76.7(2.1)	80.7(9.6)	94.2(0.8)	90.7(2.1)	82.5(3.1)	79.9(5.6)
	5	62.5(5.6)	63.2(3.2)	80.0(1.5)	74.7(11.3)	84.3(4.9)	82.5(3.8)	70.7(3.0)	78.3(11.2)	77.1(0.9)	79.6(2.9)
	20	69.1(4.3)	67.5(5.6)	75.8(5.7)	72.1(7.3)	80.5(1.0)	78.7(3.2)	83(9.2)	87.0(6.4)	78.6(5.0)	76.8(4.9)

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3.5.3 Limit of detection (LOD) and limit of quantification (LOQ). As the majority of materials in various feeds are included in the complete feed sample used to establish this method, the LOD and LOQ of the method were determined on the basis of the typical complete feed matrix. The LOD, defined as the concentration of macrolides giving a signal to noise ratio (S/N) of 3, was 0.4 mg kg⁻¹ for AZI, TIL, ERY, and CLA, 0.6 mg kg⁻¹ for SPM, TYL, and ROX, 0.8 mg kg⁻¹ for TUL, MDM, and JOS, respectively. The LOQ, based on S/N of 10, were 1 mg kg⁻¹ for AZI, TIL, ERY, and CLA, 1.5 mg kg⁻¹ for TUL, SPM, TYL, ROX, MDM, and JOS (shown in Table 2). According to the Bulletin 168 of the Ministry of Agriculture in China, the dosage range of TYL using in the pig feeds is 10–100 mg kg⁻¹ and using in the poultry feeds is 4–50 mg kg⁻¹. The dosage of TIL using as feed additive is 2 g kg⁻¹. Thus, the LOD and LOQ demonstrate that the HPLC-ELSD method is sensitive enough for the determination of macrolides in real feed samples.

3.6 Applications to real samples

100 samples including piglet, swine, poultry and fish feeds were collected from local feed markets. In the real samples, tilmicosin and tylosin were detected in two pig feeds with the concentration of 385 mg kg⁻¹ and 3.6 mg kg⁻¹. As a result, the HPLC-ELSD method developed is practical for the monitoring of macrolides drugs in feeds.

4. Conclusions

A sensitive, reliable and practical HPLC-ELSD method was developed for simultaneous determination of ten macrolides (AZI, TUL, SPM, TIL, TYL, ERY, CLA, ROX, MDM, and JOS) in feeds. The optimization of the ELSD parameters and the selection of the mobile phase are very important for multi-analyte assay. All recoveries and precisions were satisfied with the requirements for veterinary drugs analysis in feeds. The limits of detection for all compounds ranged from 0.4 to 0.8 mg kg⁻¹. Therefore, the proposed method could be suitable for the routine analysis and monitoring of macrolides drugs in pig and poultry feeds.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 31372476), the Program for Changjiang Scholars and Innovative Research Team in University (no. IRT13063) and Science and Technology Program Project of Guangzhou (2014J4100190).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12623h

‡ These authors contributed equally to this work.

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