Linli Cheng

Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography-tandem mass spectrometry

A reliable and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) confirmation method has been developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in chicken muscle. Samples were extracted with basic ethyl acetate, defatted with hexane, and cleaned up on Oasis MCX cartridges. LC separation was achieved on a XTerra C(18) column with gradient elution using a mobile phase composed of acetonitrile and water at a flow rate of 0.20 mL/min. The analysis was carried out on a triple-quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via electrospray interface operated in the positive and negative ionization modes, with deuterated chloramphenicol-d5 (d(5)-CAP) as the internal standard. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for each analyte with one precursor ion and two product ions. Limits of detection (LODs) were 0.1 microg/kg for CAP, 0.2 microg/kg for FF and 1 microg/kg for TAP and FFA in chicken muscle. Linear calibration curves were obtained over concentration ranges of 0.3-20 microg/kg for CAP, 0.5-20 microg/kg for FF and 3-100 microg/kg for TAP and FFA in tissues. Mean recoveries of the 4 analytes ranged from 95.1% to 107.3%, with the corresponding intra- and inter-day variation (relative standard deviation, R.S.D.) less than 10.9% and 10.6%, respectively. The decision limit (CCalpha) and detection capability (CCbeta) of the method were also reported.

In vitro and in vivo metabolite profiling of valnemulin using ultraperformance liquid chromatography-quadrupole/time-of-flight hybrid mass spectrometry.

Valnemulin, a semisynthetic pleuromutilin derivative related to tiamulin, is broadly used to treat bacterial diseases of animals. Despite its widespread use, metabolism in animals has not yet been fully investigated. To better understand valnemulin biotransformation, in this study, metabolites of valnemulinin in in vitro and in vivo rats, chickens, swines, goats, and cows were identified and elucidated using ultraperformance liquid chromatography–quadrupole/time-of-flight hybrid mass spectrometry (UPLC-Q/TOF-MS). As a result, there were totally 7 metabolites of valnemulin identified in vitro and 75, 61, and 74 metabolites detected in in vivo rats, chickens, and swines, respectively, and the majority of metabolites were reported for the first time. The main metabolic pathways of valnemulin were found to be hydroxylation in the mutilin part (the ring system) and the side chain, oxidization on the sulfur of the side chain to form S-oxides, hydrolysis of the amido bond, and acetylization in the amido of the side chain. In addition, hydroxylation in the mutilin part was proposed to be the primary metabolic route. Furthermore, the results revealed that 2β-hydroxyvalnemulin (V1) and 8α-hydroxyvalnemulin (V2) were the major metabolites for rats and swines and S-oxides (V6) in chickens.

Development of a rapid competitive indirect ELISA procedure for the determination of deoxynivalenol in cereals

Abstract A rapid and sensitive method based on competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed and validated for the detection of DON (deoxynivalenol) in cereals with the confirmation of the reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Within this method, the IC50 (half maximal inhibitory concentration of a substance) of the DON-specific monoclonal antibody (MAb) we produced was 61.10 ng/mL. The limit of detection (LOD) value, measured by IC10, was 6.12 ng/mL, which is below the maximum residue levels (MRLs) established for DON in various cereals. In spiked samples, the recoveries ranged from 79.3% to 115.2% for maize and from 74.3% to 118.3% for wheat. This method was compared with the UPLC-MS/MS method by testing four concentrations. The correlation coefficient for the two methods was 0.9884 in a linear-regression analysis. The results illustrated the reliability of the ciELISA method for the determination of cereal samples.

Hapten-antibody recognition studies in competitive immunoassay of α-zearalanol analogs by computational chemistry and Pearson Correlation analysis.

The molecular recognition of hapten–antibody is a fundamental event in competitive immunoassay, which guarantees the sensitivity and specificity of immunoassay for the detection of haptens. The aim of this study is to investigate the correlation between binding ability of one monoclonal antibody, 1H9B4, recognizing and the molecular aspects of α‐zearalanol analogs. The mouse‐derived monoclonal antibody was produced by using α‐zearalanol conjugated to bovine serum albumin as an immunogen. The antibody recognition abilities, expressed as IC50 values, were determined by a competitive ELISA. All of the hapten molecules were optimized by Density Function Theory (DFT) at B3LYP/ 6‐31G* level and the conformation and electrostatic molecular isosurface were employed to explain the molecular recognition between α‐zearalanol analogs and antibody 1H9B4. Pearson Correlation analysis between molecular descriptors and IC50 values was qualitatively undertaken and the results showed that one molecular descriptor, surface of the hapten molecule, clearly demonstrated linear relationship with antibody recognition ability, where the relationship coefficient was 0.88 and the correlation was significant at p < 0.05 level. The study shows that computational chemistry and Pearson Correlation analysis can be used as tool to help the immunochemistries better understand the processing of antibody recognition of hapten molecules in competitive immunoassay. Copyright

A specific UPLC-ESI-MS/MS method for analysis of cyadox and its three main metabolites in fish samples

A rapid and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of cyadox (CYX) and its three major metabolites, quinoxaline-2-carboxylic acid (QCA), 1-desoxycyadox (1-DCYX) and 1,4-bisdesoxycyadox (BDCYX) in fish. Samples were extracted with acetonitrile–0.5 M hydrochloric acid (9 + 1) and cleaned up using Oasis MAX cartridges. The total time taken for separation by UPLC was less than 5 min. The four target compounds were then determined by ESI-MS/MS. At the fortified levels of 2–50 μg kg−1 in fish, recoveries of all the compounds ranged from 80.2% to 88.2%, with a relative standard deviation of 6.8 to 14.6%. Limits of detection for CYX, QCA, 1–DCYX and BDCYX were 0.52, 1.07, 0.69 and 0.38 μg kg−1, respectively.

Ultra-performance liquid chromatography-tandem mass spectrometry determination and depletion profile of flunixin residues in tissues after single oral administration in rabbits.

An ultra-performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS) method was developed for the detection of flunixin residues in rabbit tissues. The samples were extracted with acidic acetonitrile, defatted with n-hexane, and then purified by HLB solid-phase extraction cartridge. Analysis was carried out on UPLC-ESI-MS/MS working with multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the method were 0.3-0.8μgkg(-1) and limits of quantification (LOQs) were 1.0-3.0μgkg(-1) in rabbit tissues, respectively. In all fortified samples at a concentration range of 1.0-300.0μgkg(-1), mean recoveries were 61.7-115.7% with relative standard deviations (RSDs) below 16%. Residue depletion of flunixin in rabbit was conducted after oral administration at a dose of 5mgkg(-1) of body weight. The average concentrations for flunixin measured 2h post-administration in kidney and intestine were significantly higher than in liver, heart and muscle. The concentrations for flunixin in all rabbit tissues were below the LOD or not detected in all tissues after 96h administration of drug. A minimum withdrawal time of 21h was indicated for residue levels in heart, liver, kidney, intestine and muscle below the maximum residue limits (MRLs).

Analysis of mequindox and its two metabolites in swine liver by UPLC-MS/MS

A new ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of mequindox (MEQ) and its two metabolites (1-desoxymequindox and 1, 4-bisdesoxymequindox, 1-DMEQ and BDMEQ) in swine liver. This method was validated on the basis of Commission Decision 2002/657/EC. Target analytes were extracted from swine liver with acid acetonitrile, purified by an Oasis MAX cartridge, and separated by UPLC. The chromatography total time was less than 8 min with gradient elution of 0.1% formic acid in methanol. Data acquisition was carried out by electrospray ionization tandem mass spectrometry (ESI/MS/MS) operated in a multiple reaction monitoring mode. At fortified levels from 2 to 100 μg kg−1 in swine liver, recoveries of target analytes ranged from 80% to 85% with the intra-day coefficient of variation (CV) being ≤ 14.48% and inter-day CV ≤ 14.53%, respectively. The limit of detection (LOD) ranged from 0.58 to 1.02 μg kg−1 and the limit of quantification (LOQ) range was 1.93 to 3.40 μg kg−1 for each analyte. The result indicated that this method was specific, sensitive, and suitable for the quantification and conformation of MEQ and its two metabolites in swine liver.

Analysis of Quinocetone and its Four Metabolites in Swine Liver by Ultra-performance Liquid Chromatography Quadrupole Time-of-flight Mass Spectrometry

A new method using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/ Q-TOF-MS) was developed for determination of quinocetone (QCT) and its major metabolites, 1–desoxyquinocetone (1– DQCT), 4–desoxyquinocetone (4–DQCT), 1,4–bisdesoxyquinocetone (DQCT) and methyl–3–quinoxaline–2–carboxylic acid (MQCA) in swine liver. Tissue samples were extracted by a two-step method. Firstly, all analytes except MQCA were extracted with ethyl acetate. Then a hydrochloric acid solution was added in the remained sediment for hydrolysis of combined MQCA. The MQCA in the acid extract was transferred to ethyl acetate by liquid-liquid extraction (LLE) approach. All the ethyl acetate extract were combined, evaporated, resolved in a phosphate buffer, and cleaned up using a Oasis MAX cartridges. The chromatographic separation of all the analytes was achieved in less than 5 min using UPLC. The identification and confirmation by accurate mass measurement and their different fragmentation pathways were performed on ESI-Q-TOF-MS (MS/MS). The quantitation was carried out in TOF mode using narrow window extracted ion chromatogram of each compound. In the range of 1-100 µg·kg–1, the calibration curve equations of MQCA, QCT, 4-DQCT, 1-DQCT and DQCT were y=0.7878x+10.49, y=4.656x+42.244, y=5.5825x+24.919, y=8.491x+21.195 and y=10.733x+160.72, respectively. And the relative coefficients of all calibration curves were higher than 0.98. The limit of detection and the limit of quantification were 1.4–4.8 µg·kg –1 and 4.6–15.9 µg·kg–1, respectively. The established method was validated for determination of incurred swine liver samples in an actual residue study.

Development of a GC-MS/MS method for determination of organochlorine pesticide residues in wild Ligusticum chuanxiong and chestnut

A gas chromatographic-tandem mass spectrometric method (GC-MS/MS) has been developed for sensitive and reliable detection of 8 organochlorine pesticide residues in wild L. chuanxiong and chestnut. The target compounds were extracted from both samples and simultaneously rudely cleaned up by water-acetone and dichloromethane. Then, the obtained dichloromethane phase was evaporated to dry, treated with H2SO4, centrifuged, evaporated and redissolved in 0.5 mL of benzene for GC-MS/MS detection. When spiked from 5 to 500 μg/kg, the average recoveries of organochlorine pesticides in two kinds of sample ranged from 74.2 to 110.9% with the intra-day RSD of 0.1 to 10.1% and the inter-day RSD of 0.7 to 10.5%. The limit of detection of eight compounds varied from 0.13 to 1.2 μg/kg in two kinds of samples.