Simple extraction method requiring no cleanup procedure for the detection of minocycline residues in porcine muscle and milk using triple quadrupole liquid chromatography-tandem mass spectrometry

Abstract

A versatile analytical method was developed for simple detection of minocycline residues in porcine muscle and milk using liquid chromatography-triple quadrupole tandem mass spectrometry (LC/MS/MS) with electrospray ionization. Samples were extracted with a mixture of acetonitrile and ethyl acetate (2:1, v/v), and then defatted using n-hexane. No cleanup procedure was deemed necessary. Minocycline was separated on a reversed-phase analytical column using a combination of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) as the mobile phase. Matrix-matched calibration showed good linearity over a concentration range of 10–60 μg/kg with a determination coefficient (R ²) of 0.9727. Fortified porcine and milk samples having concentrations equivalent to and double the limit of quantification (LOQ = 10 ng/g), respectively, yielded recovery ranges between 83.02 and 8.03 % and relative standard deviations <18 %. Samples collected from a large market located in Seoul, Korea, tested negative for minocycline residue. These results show that a combination of acetonitrile and ethyl acetate can effectively extract minocycline from porcine muscle and milk without solid-phase extraction, a step usually required for cleanup before analysis. The developed method is simple, sensitive, and can be extrapolated to other food animal products that are rich in protein and fats.

Introduction

Antimicrobial agents are used therapeutically against bacterial infections; however, they may also be used inappropriately as both human (e.g., in response to demands from patients, rather than medical necessity) and veterinary medicines (e.g., as growth promoter for livestock) (Khachatourians 1998). Tetracyclines (TCs) are broad-spectrum antibiotics active against Gram-positive and -negative bacteria, Rickettsia, Chlamydia, Mycoplasma, Plasmodium, Spirochetes, and Mycobacteria (Chopra and Roberts 2001). They are a bacteriostatic agent, exerting their mechanism of action by binding to the bacterial 30S ribosomal subunit, thereby inhibiting protein synthesis (Nagarakanti and Bishburg 2015). Tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), doxycycline (DC), and minocycline (MNC) are five members of the tetracycline (TC) group that are commonly used in animals that are meant for human consumption, not only for the prevention and treatment of certain diseases but also to fraudulently promote growth (Samanidou et al. 2005). Minocycline (7-dimethylamino-6-dimethyl-6-deoxytetracycline, Fig. 1) is a second-generation semi-synthetic TC analogue. In humans, minocycline has non-antimicrobial properties that are independent of its antimicrobial activity (Nagarakanti and Bishburg 2015), such as anti-inflammatory (Giuliani et al. 2005), anti-apoptotic, and immunomodulatory activities (Popovic et al. 2002), as well as neuroprotective effects (Song et al. 2004). The extensive use of veterinary antibiotics may result in the accumulation of residues in various tissues and biological fluids, which in turn may pose hazardous effects and cause allergic reactions in humans. Moreover, extended exposure to residual levels of antibiotics is a major contributor to the emergence of antibiotic-resistant genes (Chopra and Roberts 2001; Michalova et al. 2004). Antibiotic-resistant genes may be passed between animal, soil, and human bacteria by horizontal gene transfer (Kobashi et al. 2007; Forsberg et al. 2012); the increasing contamination of soil with antibiotic-resistant genes may, therefore, contribute to the worldwide problem of antibiotic resistance (Khachatourians 1998; Kyselková et al. 2015). Additionally, antibiotic residues in milk and meat may result in unsellable products (Nisha 2012) because of spoilage of dairy products and shrinkage and toughening of cooked meat (Akinwumi et al. 2012). To preserve consumers’ health, maximum residue limits (MRLs) have been established in the European Union (EU) for veterinary drugs in foods of animal origin (Council Regulation (EEC) No 2377/90). Nevertheless, the corresponding MRL for minocycline in food animal products has not been established by the EU (COMMISSION REGULATION (EU) No 37/2010), US Food and Drug Administration (2014), Japan (The Japan Food Chemical Research Foundation 2015), Codex (2012), or the Korea Ministry of Food and Drug Safety (MFDS 2015).

Fig. 1

Chemical structure of minocycline

To ensure the safety of the food supply, efficient methods are required for monitoring residual levels of veterinary drugs in animal products. Porcine muscle and milk contain considerable amounts of fats, which create a lipophilic environment. Such conditions can interfere with the extraction of compounds for testing, and may result in poor recovery. Because of the complexity of animal food products, and the low limit of quantification (≤10 ppb) prescribed by the MFDS for drugs with no MRL, samples must be extracted from fatty materials prior to analysis to ensure sensitive, selective, and specific detection of the target compounds. Residual amounts of minocycline and other TCs have been determined in bovine muscle using a high-performance liquid chromatographic method with diode array detection (Samanidou et al. 2005), and in raw milk using a luminescence-based microbial method (Kurittu et al. 2000a, b). An equivalent study has not been carried out for porcine muscle, while tandem mass spectrometry has not been applied for testing raw milk. The present study aims to develop an analytical method for accurate quantification of minocycline residue in porcine muscle and milk using LC/MS/MS.

Materials and methods

Chemicals and reagents

Minocycline hydrochloride (CAS Number: 13614-98-7), HPLC-grade formic acid (purity: 98 %), and oxalic acid (purity: 99 %) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Organic solvents (analytical grade), including acetonitrile (ACN, purity: 100 %) and methanol (purity: 99.9), were obtained from J. T. Baker Chemicals (Phillipsburg, NJ, USA). Ethyl acetate (purity: 99.9) was supplied by Tedia (Fairfield, Ohio, USA). EDTA (ethylenediaminetetraacetic acid) was obtained from Katayama Chemical Industries Co., LTD. (Osaka, Japan). Membrane and syringe filters were purchased from Merck Millipore (Tullagreen, Carrigtwohill, Co. Cork, Ireland). Ultra-high purity water was produced by a Milli-Q (Millipore, Bedford, MA, USA) water purification system and used for preparation of the mobile phase.

Porcine muscles and milk samples were obtained from a local supermarket in Seoul, Republic of Korea.

Standard preparation

A standard stock solution of minocycline (1000 μg/mL) was prepared by weighing 10 mg (AG 285, METTLER TOLEDO, Seoul, Republic of Korea) into a 15 mL conical tube (Falcon, Corning Science Mexico S. A. de C.V., Tamaulipas, Mexico) followed by dissolution (Witeg^® Labmax-HF^® dispenser, Germany) with 10 mL methanol. Intermediate and working standard solutions were prepared by further dilution with methanol. Because the compound is light-sensitive (Dybvig and Cassell 1987), all standard solutions were stored at −20 °C in the dark.

Sample preparation and extraction procedure

The Ministry of Food and Drug Safety Guideline for determination of hazardous materials in agricultural and marine products (2014) was followed with some minor modifications in extraction and defatting solvents. Chopped porcine muscle (5 g) and homogenized milk (5 mL) samples were mixed with the standard solutions (0.5 mL) in a conical tube, to which 0.2 g EDTA and 0.2 g oxalic acid were added and vortex-mixed (BenchMixer™ Multi-Tube Vortexer, Benchmark Scientific, NJ, USA) for 10 min. After adding acetonitrile: ethyl acetate (2:1, v/v) (20 mL) to the mixture, it was again vortex-mixed for 15 min. Subsequently, the mixture was centrifuged at 2600 g (Union 32 R Plus, Hanil Science Industrial Co., Ltd., Incheon, Republic of Korea) for 15 min at 4 °C, and the supernatant was transferred to a 50 mL conical tube containing 20 mL n-hexane. The mixture was then vortex-mixed for 5 min and centrifuged at 2600 g for 15 min at 4 °C. The lower layer was transferred to a 15 mL conical tube and the solvents were evaporated under nitrogen gas at 40 °C (TurboVap^®RV, Caliper Life Sciences, Hopkinton, USA). Finally, the concentrated residue (approximately 0.5 mL) was reconstituted with 1 mL of 0.1 % formic acid in acetonitrile and filtered through a 0.45 μm syringe filter (MILLEX^®-LCR, Merck Millipore Corporation, Merck KGaA, Darmstadt, Germany) before analysis.

LC/MS/MS conditions and analysis

Analysis was performed using an Agilent series 1100 HPLC instrument (Agilent Technologies, CA, USA), equipped with a G1311A quart pump, a G1313A autosampler, a G1322A degasser, a G1316A column oven, and an API 3200™ mass spectrometer (Applied Biosystems, NY, USA). Triple quadrupole tandem mass spectrometric (MS/MS) analysis was carried out using an electrospray ion source in positive (ESI+) and negative (ESI−) modes. Data acquisition was performed in multiple reaction monitoring (MRM) mode, and the ABI software (version 1.4.2) was used for data management and control. The ion spray voltage and capillary temperature were set at 5.5 kV and 350 °C, respectively. Nitrogen was used as the collision gas and a pressure of 50 psi was used for ion source gas 1 (GS1) and ion source gas 2 (GS2). Optimization of precursor ions, product ions, declustering potential, and collision energy was carried out via direct injection of the drug standard solution (0.1 µg/mL) into the mass spectrometry instrument. First, the voltage of the [M + H]⁺ fragment of the voltage was optimized to the precursor ions, and the high-intensity transition was used for quantification; the other low-intensity transition was used for confirmation. The optimization parameters are shown in Table 1. Chromatographic separation was performed using a Waters XBridge™ C18 reversed-phase analytical column (2.1 × 100 mm; 3.5 μm particle size; Waters, Milford, CT, USA). The binary mobile phase system consisted of (A) 0.1 % formic acid in water and (B) 0.1 % formic acid in acetonitrile (1: 1, v/v). The sample (10 μL) was injected at a flow rate of 0.3 mL/min.

Table 1 The MRM parameters of LC/MS/MS analysis for minocycline

Results and discussion

Sample preparation

Although homogenized samples have been essential for the preparation of solid matrices (Cho et al. 2011), they can be replaced with small chopped samples, as reported in our previous studies (Park et al. 2015; Zhang et al. 2016). The effective isolation of TC residues from biological matrices is difficult because of their tendency to bind with sample proteins and chelate with metal ions. To overcome these problems and free the compound from the tissues, EDTA and oxalic acid were used in a preparatory step, prior to sample extraction (Gajda et al. 2013). Since sample preparation requires an extraction step with a suitable solvent system, we first used acetonitrile (the most commonly employed solvent) (Cho et al. 2011) alone and in combination with 0.1 % formic acid (Park et al. 2015). However, both solvent systems failed to extract minocycline with satisfactory and consistent recoveries (44–56 %). It has been reported that ethyl acetate can be used as an extraction solvent for the determination of TC residues in animal products (Haagsma and Mengelers 1989; Cooper et al. 1998). We found that a combination of acetonitrile and ethyl acetate in 2:1 (v/v) ratio for extraction/deproteinization, followed by liquid–liquid purification using n-hexane, gave good recovery, and this combination is used herein. Notably, n-hexane was used for defatting and no further cleanup procedure was implemented. Generally, cleanup procedures (solid-phase extraction, SPE) are required after sample preparation for extracting TCs from animal tissues (Nikolaidou et al. 2008; Gajda et al. 2013; Tölgyesi et al. 2014). Although cleanup can remove interfering co-extracted compounds, which increases the lifetime of the chromatographic column and improves detection, it is unnecessary if the extraction procedure effectively removes such compounds. This, in turn, will simplify sample preparation, save time, and be more cost-effective than existing processes (Park et al. 2015). To improve the transparency of the mixture, centrifugation was applied to precipitate and remove proteins and to accumulate fat droplets. Evaporation of the sample extract to complete dryness was avoided, as this could result in considerable loss of TCs (Moats and Harik-Khan 1996).

LC/MS/MS optimization conditions

Both positive and negative ESI modes were tested to find the mode with the highest intensity response. The negative mode resulted in lower intensity than the positive one; hence, the positive ESI mode was used as the MS/MS condition in this work.

The composition of mobile phases has been shown to influence the performance of the ionization process in LC/MS/MS methodology (Park et al. 2015). Herein, three buffers were tested to select the best conditions for separating minocycline on an Xbridge column. The mobile phases included (i) 0.1 % formic acid in water/0.1 % formic acid in methanol (1: 1, v/v), (ii) 0.1 % formic acid in water/0.1 % formic acid in acetonitrile (1: 1, v/v), and (iii) 0.1 % formic acid + 10 mM ammonium formate in water/0.1 % formic acid + 10 mM ammonium formate in methanol (1: 1, v/v). We found that 0.1 % formic acid in water/0.1 % formic acid in acetonitrile gave the highest intensity, whereas 0.1 % formic acid in water/0.1 % formic acid in methanol gave the lowest intensity. As a result, the 0.1 % formic acid in water/0.1 % formic acid in acetonitrile buffer solution was used as the mobile phase.

Specificity and multiple reaction monitoring (MRM) chromatograms

The specificity for minocycline detection was determined by analyzing blank samples of porcine muscle and milk (in triplicates) to evaluate possible endogenous interference. We found no interference peaks near the retention times of the target analyte. A representative MRM chromatogram of the analyte is shown in Fig. 2.

Fig. 2

MRM chromatograms of minocycline in blank, spiked, and market samples

Matrix effects and linearity

Matrices rich in protein and fat can affect analyte detection in ESI–MS analysis, resulting in signal enhancement or suppression. Matrix effects can be variable, undeterminable, and are often related to the concentration levels of the co-extracted materials. To assess this effect, the peak area responses of the analyte obtained from solvent calibration were compared with the obtained matrix-matched standard at the same concentration according to the following equation:

$${\text{Matrix effect }}\left( \% \right) \, = \frac{{{\text{peak area of standard in matrix}} - {\text{peak area of standard in solvent}}}}{\text{peak area of standard in solvent}} \times 100$$

The matrix effect is very mild and has little or no effect on minocycline quantification in porcine muscle and milk. For accuracy, a matrix-matched calibration was used for quantification throughout the study.

According to the MFDS guidelines, standard calibration should be performed at six concentration levels equivalent to 1, 2, 3, 4, 5, and 6 times the LOQ (n = 4), which are 10, 20, 30, 40, 50, and 60 μg/kg, respectively. Linearity was evaluated using least-squares linear regression and a calibration curve was prepared by plotting the peak area as a function of drug concentration. The linearity was satisfactory, with a determination coefficient (R ²) of 0.9727 (Table 2).

Table 2 Performance parameters for LC/MS/MS evaluation of minocycline in porcine muscle and milk

Limits of detection (LOD) and quantification (LOQ)

The LOD and LOQ were estimated to be 3 and 10 times the signal-to-noise ratio, respectively. As shown in Table 2, the LOD and LOQ values are 3 and 10 μg/kg, respectively. These limits are considerably low and demonstrate that the method is sensitive enough to detect the levels of drugs below the MRLs. However, the MRL for minocycline has not yet been established by any regulatory agency.

Recovery

The accuracy of the developed method was determined based on the extraction recovery of the analyte. Extraction efficiency was determined by analyzing sets of control samples added before and after extraction with the standard solution. The samples were spiked at 1 × and 2 × the LOQ. For the porcine muscle and milk samples, the mean recoveries for intra-day samples and accuracies (presented as RSDs) were 90.53–98.03 % and 1.50–17.86, respectively. The inter-day recoveries and accuracies (as RSDs) were 83.02–94.74 % and 6.51–15.32, respectively. The obtained recoveries were satisfactory based on the accepted criteria set by the Codex Alimentarius Commission (1993) (spiking concentration: >1 to ≤10 μg/kg; recovery range: 60–120 %). The RSDs were in compliance with the 2002/657/EC decision (2002), and thus the proposed method has acceptable accuracy and reproducibility.

Method application

To examine the applicability of the developed method, porcine muscle and milk samples were purchased from a local supermarket in Seoul, Korea. The market samples were extracted and analyzed according to the above methods. Residual levels of minocycline were not detected in any of the tested samples (Fig. 2).

In conclusion, the method reported herein enables the determination of residual minocycline levels within satisfactory performance parameters. Addition of EDTA and oxalic acid during sample preparation can improve the recovery of minocycline in protein-rich samples. Additionally, a mixture of acetonitrile and ethyl acetate (2:1, v/v) can effectively extract the compound from animal-based food products. The simplicity of this method is demonstrated by the absence of a cleanup procedure. None of the tested market samples contained detectable amounts of minocycline. This method can likely be extended for use with other animal products such as chicken, beef, eggs, and fish.