Seminar2019 Paper 6 in Cell Solid Liquid Extraction.Pdf

Journal of Chromatography A, 1488 (2017) 10–16 Contents lists available at ScienceDirect Journal of Chromatography A jo urnal homepage: www.elsevier.com/locate/chroma Development and application of an in-cell cleanup pressurized liquid extraction with ultra-high-performance liquid chromatography-tandem mass spectrometry to detect prohibited antiviral agents sensitively in livestock and poultry feces a b b c b a Huizhen Wu , Jianmei Wang , Hua Yang , Guoqin Li , Yinhuan Zeng , Wei Xia , a,∗ b,∗ Zuguang Li , Mingrong Qian a College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, 310014, China b Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, MOA Key Laboratory for Pesticide Residue Detection, Hangzhou, 310021, China c Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China a r a t b i c l e i n f o s t r a c t Article history: An in-cell cleanup pressurized liquid extraction was developed to analyze prohibited antiviral agents in Received 12 September 2016 livestock and poultry feces. Extraction and cleanup were integrated into one step. The extraction was per- Received in revised form ◦ formed using methanol-acetonitrile (1:1, v/v) with 0.5% glacial acetic acid at 90 C, and 0.75 g of PSA was 24 December 2016 used as the adsorbent during the extraction procedure. Under optimal conditions, the average recoveries Accepted 26 January 2017 −1 for 11 antiviral drugs were 71.5–112.5% at three spiked levels (20, 40, and 100 ␮g kg ). The detection Available online 27 January 2017 limits and detection quantitations of the analysis method for the eleven antiviral drugs were 0.6–1.4 and −1 1.4–4.7 ␮g kg , respectively. Finally, the method was applied to analyze amantadine, oseltamivir and Keywords: its metabolites oseltamivir acid in duck feces based on an experiment of an oral dose of two antiviral Antiviral agents drugs in duck. The amantadine, oseltamivir and oseltamivir acid can be detected in feces within approx- Pressurized liquid extraction Livestock and poultry feces imately four weeks after amantadine and oseltamivir were orally administered. The results indicate that the residue analysis in feces is a noninvasive method to monitor inhibited antiviral agents efficiently in livestock and poultry breeding. © 2017 Published by Elsevier B.V. 1. Introduction object to monitor the antiviral-drug usage during the process of livestock and poultry breeding. Therefore, a sensitive method to Antiviral agents such as amantadine, rimantadine, moroxydine, analyze the antiviral agents in livestock and poultry feces must be oseltamivir and acyclovir have been banned in poultry farming established. in China to prevent interference with the preventive effect of Several analyte methods have been reported for the determi- vaccinum. Nevertheless, these antiviral agents remain illegally nation of the residues of these compounds in solid samples, most occasionally used for the pretreatment of avian influenza in poul- of which focus on animal-derived food [1,3,9–13]. Solid-phase try farming in China [1,2]. Low levels of amantadine have been extraction [1,3,11,13] and QuEChERS method [9,10] have been detected in poultry muscle and liver [1,3,4]. Some measures should reported for sample preparation. The pressurized liquid extraction be proposed to monitor these residues in the livestock and poul- (PLE) method is a simple, rapid and promising sample preparation try cultivation process to guarantee the safety and quality of animal method and has been widely applied for multi-residue determi- foods. It is reported that after the administration of antiviral agents, nation in complex matrices including animal and environment some of them are excreted in non-metabolized form [5] or as active samples. Hussen et al. [14] have packed adsorbents in the extrac- metabolites in feces [6–8]. Thus, animal feces can be selected as an tion cells to combine the extraction and purification into one step for the analysis of organochlorine pesticides in soil. This method has been efficiently applied to determine multi-pesticides [14–19], persistent and emerging organic pollutants [17,18,20–28]. Never- ∗ Corresponding authors. theless, only a few studies focused on the possibility of extending E-mail addresses: [email protected] (Z. Li), [email protected] (M. Qian). http://dx.doi.org/10.1016/j.chroma.2017.01.070 0021-9673/© 2017 Published by Elsevier B.V. H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16 11 this approach to residue analysis in feces [29]. As far as we know, To separate the antiviral agents, gradient elution was performed this method has not been applied for antiviral residues detec- on a BEH-HILIC HPLC column (2.1 × 100 mm, 1.7 ␮m, Waters) at ◦ −1 tion in agricultural foods or environment samples. This work is to 30 C. The mobile phases were acetonitrile (A) and 5 mmol L develop a simple and sensitive method to analyze antiviral agents ammonium acetate water with 0.1% formic acid (B). The starting in livestock and poultry feces using ultra-high-performance liq- mobile-phase composition was 5% B. After an isocratic step at 5% uid chromatography-tandem mass spectrometry (UPLC–MS/MS) to B for 2 min, B was linearly increased to 45% in 7 min, decreased trace the misuse. Then, the regulatory authorities can take appro- to 5% in 3 min, and held constant for 3 min; the total cycle time −1 priate actions to prevent the abuse of antiviral drugs. was 13 min. The mobile-phase flow rate was 0.2 mL min , and the injection volume was 2 ␮L. 2. Materials and methods For the mass spectrometer, the positive electrospray ioniza- tion mode and multiple-reaction monitoring (MRM) mode were 2.1. Reagents and materials used. The optimal conditions for the sample analysis were as fol- lows: ion-spray voltage: 5000 V; curtain gas: 40 psi (nitrogen); ion ◦ HPLC gradient grade acetonitrile and methanol were purchased source temperature: 500 C; ion source gas 1 and ion source gas 2: from Merck (Germany)and HPLC grade glacial acetic acid was pur- 50 psi. The MRM transition parameters for the quantification are chased from Scharlau (Spain). The sorbents were purchased from presented in Table 1. Agela Technologies (China): C18 (40 ␮m particle size), primary sec- ondary amine (PSA) (40–60 ␮m particle size), silica gel (70–90 ␮m 2.4. Quantification and method validation particle size) and Florisil (60–100 mesh). Diatomaceous earth with 80–100 mesh was purchased from Thermo Scientific (USA). Neutral Concentrations of the amantadine, rimantadine, memantine, alumina was from Aladdin Industrial (China). Purified water with somantadine, acyclovir, imiquimod and ganciclovir in the sam- resistivity higher than 18.2 M cm was obtained by a Milli-Q water ples were performed using internal standard method, which were purification system (Millipore, USA). added to the sample post extraction. The calibration curves (three The standards of amantadine and oseltamivir acid were replicates) were using the levels 0.5, 1.0, 2.0, 5.0, 10, 20, 50 −1 ␮ purchased from Anpel Laboratory Technologies (Shanghai) Inc and 100 g L with a constant internal standard concentration −1 ␮ (China). Rimantadine, memantine, oseltamivir phosphate, acy- of 20 g L . For oseltamivir, oseltamivir acid, moroxydine and clovir, imiquimod, ganciclovir, arbidol and the internal standards, arbidol, the concentrations were determined using the matrix amantadine-d15, rimantadine-d4, memantine-d6 and acyclovir- match calibration method. The linear portion of the standard curve d4 were purchased from Toronto Research Chemicals (Canada). was used to quantify antiviral agents in the extracts; if the concen- Moroxydine was purchased from Sigma (USA). Somantadine was trations were above the linear portion, a higher internal standard purchased from J & K Scientific (USA). was added to the samples, and the extracts were subsequently The stock solutions were prepared as individual diluted and analyzed. −1 100–200 ␮g mL solutions in acetonitrile or acetonitrile aqueous All generated data from the analysis were subject to strict qual- ◦ solution. They were stored in 4 C without light and found to ity assurance and quality control (QA/QC) procedures. With each be stable for at least two month. Work standard solutions were batch of six samples to be analyzed, a solvent blank, a procedure freshly prepared by diluting the stock solutions with acetonitrile. blank and a spiked sample were sequentially run to ensure accurate Corrections were made for salt (HCl or H3PO4 salts) and purity. and precise measurement. A blank injection was performed after 3 sample injections to ensure that there was no carry-over. A check 2.2. In-cell cleanup pressurized liquid extraction procedure standard injection was performed after 20 injections to confirm the instrument performance, and the computed concentration must be ± The antiviral agents from livestock and poultry feces were within 20% of the expected value. extracted by PLE using an ASE 350 system (Thermo Scientific, USA). The limit of detection (LOD) and limit of quantitation (LOQ) for The sample purification was simultaneously performed with the the method were estimated from the data of the recovery test with extraction (in-cell cleanup) by placing the sorbent inside the cell the lowest spiked concentration where an S/N could be 3 and 10, (PSA). At the bottom of the 34-mL stainless steel vessel was a cellu- respectively. lose filter; from down to up, there were 2 g of diatomaceous earth, The recovery studies were carried out in blank samples spiked 0.75 g of PSA, 0.5 g of freeze-dried sample and 1 g of diatomaceous with a suitable volume of working standard solution. Then they earth. Subsequently, the cells were tightly closed and inserted into were allowed to stand at room temperature for 1 h and main- ◦ the cell tray for the extraction and cleanup.

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