Swine Muscle Research Articles

Development of A Real‐Time PCR Assay for the Detection ofTrichinella Spiralisin Muscle Tissue of Swine and Derivatives

AbstractTrichinellosis is an emergent zoonosis in several regions of the world and it is considered a public health problem. Trichinellosis represent one of the most important zoonotic diseases in Argentina. The purpose of this study was to develop a real‐time PCR assay with internal control to detectTrichinella spiralisDNA in samples of swine muscles and its derivatives. PCR amplification of DNA from muscle samples was performed by real‐time PCR with an internal porcine DNA amplification control. The developed PCR assay specifically detectsT. spiralisshowing no amplification with otherTrichinellagenotypes and showed an estimated sensitivity of 0.024 larvae per gram in swine muscle samples. From the 21 samples analyzed, five samples negative by enzymatic digestion were positive by real‐time PCR, which demonstrates that this technique is capable of detectingT. spiralisin cases when enzymatic digestion cannot. In this work, a new molecular assay with internal control forT. spiralisdetection was successfully developed.Practical ApplicationsTrichinellosis is considered a public health problem in Argentina and also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts have focused on the control ofTrichinellaor the elimination ofTrichinellafrom the food chain. In our country, most human infections are caused byTrichinella spiralis, which is transmitted mainly by the ingestion of raw or undercooked infected swine meat or its derivative products. New controls in order to improve food safety for consumers should be developed. This new real‐time PCR assay with internal control shows a substantial increase in diagnostic sensitivity in comparison with muscle artificial digestion and the possibility to avoid false negative results. This specific PCR forT. spiralismay be useful for detection of infection at early stages in humans and food animals.

Determination and surveillance of hydrocortisone and progesterone in livestock products by liquid chromatography-tandem mass spectrometry

A simple analytical method for the determination of hydrocortisone and progesterone in bovine, swine, and chicken muscle and eggs was developed. Hydrocortisone and progesterone were extracted with acetonitrile and subsequently cleaned-up using an Oasis® HLB mini-cartridge. The method was validated in accordance with Japanese guidelines and exhibited trueness from 86.6% to 104.3% and precision (relative standard deviations (RSDs) of repeatability and within reproducibility were under 8.7% and 11.7%, respectively). The method was applied to 103 bovine muscle, 137 swine muscle, 69 chicken muscle and 52 egg samples that were commercially available in Tokyo, Japan. The hydrocortisone concentration was 0.9–41.2 µg kg−1 in all bovine muscle samples, with an average of 7.7 µg kg−1 and a median of 6.2 µg kg−1. The progesterone concentration in 50 samples exceeded the limit of quantification (LOQ) and reached a maximum of 95.4 µg kg−1. Hydrocortisone was also detected in all swine muscle samples at concentrations of 2.0–56.0 µg kg−1. Its average and median concentrations amounted to 13.1 and 11.3 µg kg−1, respectively. Twenty-three samples contained progesterone levels surpassing the LOQ, with a maximum concentration of 107.0 µg kg−1. No chicken muscle samples contained any of the analytes. The progesterone concentration was 15.5–200.0 µg kg−1 in all egg samples, with an average of 95.4 µg kg−1 and a median of 90.5 µg kg−1.

Time- and space-resolved spectroscopic characterization of laser-induced swine muscle tissue plasma

The spatial-temporal evolution of muscle tissue sample plasma induced by a high-power transversely excited atmospheric (TEA) CO2 pulsed laser at vacuum conditions (0.1–0.01Pa) has been investigated using high-resolution optical emission spectroscopy (OES) and imaging methods. The induced plasma shows mainly electronically excited neutral Na, K, C, Mg, H, Ca, N and O atoms, ionized C+, C2+, C3+, Mg+, Mg2+, N+, N2+, Ca+, O+ and O2+ species and molecular band systems of CN(B2Σ+–X2Σ+), C2(d3Πg–a3Πu), CH(B2Σ−–X2Π; A2Δ–X2Π), NH(A3Π–X3Σ−), OH(A2Σ+–X2 Σ+), and CaOH(B2Σ+–X2Σ+; A2Π–X2Σ+). Time-resolved two-dimensional emission spectroscopy is used to study the expanded distribution of different species ejected during ablation. Spatial and temporal variations of different atoms and ionic excited species are reported. Plasma parameters such as electron density and temperature were measured from the spatio-temporal analysis of different species. Average velocities of some plasma species were estimated.

Time and space-resolved spectroscopic and imaging study of a laser-produced swine muscle tissue plasma

SynopsisWe investigated the optical emission and imaging features of plasmas produced by a high-power transversely excited atmospheric CO2 laser pulses on a swine muscle tissue sample in different vacuum conditions. The analyzed plasma shows electronically excited neutral Na, K, C, Mg, H, Ca, N, and O atoms, ionized C+, C2+, C3+, Mg+, Mg2+, N+, N2+, Ca+, O+, and O2+ species and molecular band systems of CN, C2, CH, NH, OH and CaOH. Time-resolved 2D emission spectroscopy is employed to study the expanded distribution of several species ejected during ablation. The expansion of the plume front was analyzed using Shock wave and Drag models.

Validation of Perfusion Quantification with 3D Gradient Echo Dynamic Contrast-Enhanced Magnetic Resonance Imaging Using a Blood Pool Contrast Agent in Skeletal Swine Muscle.

The purpose of our study was to validate perfusion quantification in a low-perfused tissue by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with shared k-space sampling using a blood pool contrast agent. Perfusion measurements were performed in a total of seven female pigs. An ultrasonic Doppler probe was attached to the right femoral artery to determine total flow in the hind leg musculature. The femoral artery was catheterized for continuous local administration of adenosine to increase blood flow up to four times the baseline level. Three different stable perfusion levels were induced. The MR protocol included a 3D gradient-echo sequence with a temporal resolution of approximately 1.5 seconds. Before each dynamic sequence, static MR images were acquired with flip angles of 5°, 10°, 20°, and 30°. Both static and dynamic images were used to generate relaxation rate and baseline magnetization maps with a flip angle method. 0.1 mL/kg body weight of blood pool contrast medium was injected via a central venous catheter at a flow rate of 5 mL/s. The right hind leg was segmented in 3D into medial, cranial, lateral, and pelvic thigh muscles, lower leg, bones, skin, and fat. The arterial input function (AIF) was measured in the aorta. Perfusion of the different anatomic regions was calculated using a one- and a two-compartment model with delay- and dispersion-corrected AIFs. The F-test for model comparison was used to decide whether to use the results of the one- or two-compartment model fit. Total flow was calculated by integrating volume-weighted perfusion values over the whole measured region. The resulting values of delay, dispersion, blood volume, mean transit time, and flow were all in physiologically and physically reasonable ranges. In 107 of 160 ROIs, the blood signal was separated, using a two-compartment model, into a capillary and an arteriolar signal contribution, decided by the F-test. Overall flow in hind leg muscles, as measured by the ultrasound probe, highly correlated with total flow determined by MRI, R = 0.89 and P = 10−7. Linear regression yielded a slope of 1.2 and a y-axis intercept of 259 mL/min. The mean total volume of the investigated muscle tissue corresponds to an offset perfusion of 4.7mL/(min ⋅ 100cm3). The DCE-MRI technique presented here uses a blood pool contrast medium in combination with a two-compartment tracer kinetic model and allows absolute quantification of low-perfused non-cerebral organs such as muscles.

Multi-residue Determination of Polar Veterinary Drugs in Livestock and Fishery Products by Liquid Chromatography/Tandem Mass Spectrometry.

Residues of 37 polar veterinary drugs belonging to six families (quinolones, tetracyclines, macrolides, lincosamides, sulfonamides, and others) in livestock and fishery products were determined using a validated LC-MS/MS method. There were two key points in sample preparation. First, extraction was performed with two solutions of different polarity. Highly polar compounds were initially extracted with Na2EDTA-McIlvaine's buffer (pH 7.0). Medium polar compounds were then extracted from the same samples with acetonitrile containing 0.1% formic acid. Secondly, cleanup was performed using a single SPE polymer cartridge. The first extracted solution was applied to the cartridge. Highly polar compounds were retained on the cartridge. Then, the second extracted solution was applied to the same cartridge. Both highly and medium polar compounds were eluted from the cartridge. This method satisfied the guideline criteria for 37 out of 37 drugs in swine muscle, chicken muscle, bovine muscle, prawn, salmon trout, red sea bream, milk, and honey; 35 out of 37 in egg; and 34 out of 37 in flounder. The LOQ ranged from 0.1 to 5 μg/kg. Residues were detected in 24 out of 110 samples and analyzed using the validated method.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

Development of a Rapid Method for the Confirmatory Analysis of Flunixin Residue in Animal Tissues Using Liquid Chromatography–Tandem Mass Spectrometry

Flunixin is approved for use in veterinary medicine as a nonsteroidal anti-inflammatory agent. There is a need for the control of residues and development of methods to monitor its compliance with legislation. However, few methods have been reported for the analysis of flunixin in the animal liver, kidney, and fat. Therefore, the aim of this work was to develop a relatively rapid sample preparation and sensitive using liquid chromatography–tandem mass (LC-MS/MS) confirmatory method to detect flunixin residues in the liver, kidney, muscle, and fat of swine and chicken. After acid hydrolysis, the sample was extracted with ethyl acetate. The extract was finally evaporated to dryness and reconstituted in a water/methanol mixture, and determination was carried out by LC-MS/MS. Flunixin was detected using positive electrospray ionization in selected reaction monitoring (SRM). Estimated limit of quantification of the method was 0.5, 0.5, 0.25, and 0.05 μg/kg for the liver, kidney, muscle, and fat, respectively. The method was validated in animal tissues in terms of selectivity, linearity, trueness, precision, decision limit (CCα), and detection capability (CCβ). All the trueness values fell within a range between 73.6 and 84.8 %. Precision values for all levels of concentration tested showed excellent relative standard deviation (RSD < 15 %). The CCα and CCβ values have been established for each tissue. A relatively rapid and sensitive LC-MS/MS method for the quantitative determination of flunixin in different animal tissues was developed and validated. The method is suitable for monitoring the flunixin residues in animal tissues.

DETERMINATION OF FLORFENICOL AMINE IN SWINE MUSCLE BY HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROSCOPY

Nuflor (florfenicol) Premix for Swine is approved by the U.S. Food and Drug Administration (FDA) and the same regulatory agencies from many other countries for control of swine respiratory disease. A simple and fast LC-MS/MS method for the assay of florfenicol amine, the marker residue of florfenicol in swine muscle is described. A 24-hr strong acid hydrolysis of tissue residues converts florfenicol and its known metabolites into florfenicol amine and also fully releases florfenicol amine from florfenicol residues covalently bound to the swine muscle. The majority of fatty acids and lipids are removed via hexane extraction. Then, the pH of the extracted hydrolysate is adjusted to 11 and florfenicol amine is extracted by ethyl acetate. A portion of ethyl acetate extract is dried under nitrogen and reconstituted with 1 mL acetonitrile. Dispersive solid-phase extraction is used for cleanup in which 100 mg primary secondary amine sorbent is mixed with the extract. After the centrifugation, the supernatant is diluted 20-fold for a hydrophilic interaction LC-MS/MS analysis. No matrix effect is observed in the LC-MS/MS assay and the recovery is nearly 100%. In the comparison with the current US FDA Center for Veterinary Medicine regulatory surveillance method, the new method is simple, fast, and accurate. The validated method is used to investigate the depletion of florfenicol from the swine administrated with Nuflor (florfenicol) Premix.

Assessment of heavy metal residues in Brazilian poultry and swine tissue

The aim of this study was to investigate the presence of arsenic, lead and cadmium residues in samples of liver, kidney and muscle of poultry and swine during the years from 2002 to 2008. A total of 1978 samples were analyzed: 1031 of poultry and 947 of swine from Brazilian slaughterhouses. The samples were analyzed at the National Agricultural Laboratory using the atomic absorption spectrometry technique. In poultry, the arsenic residues were detected in 53.6% of liver samples, although no results have exceeded the MRL. In kidneys, 39.7% of the samples showed measurable residues and there was no violation of the limits. Regarding lead, there were 5 contaminated liver samples and 24 contaminated kidney samples (1.5 and 3.6% respectively). In muscle tissue there was only one sample with residues. For cadmium, 3.8% of the samples were found below MRL. Cadmium residues were found in 110 kidney samples (16.3% of tested), but only one exceeded the legislation limits. In swine, arsenic residues were detected in 15.3% of liver samples, although no results exceeded the MRL. In kidneys, 14.2% of samples showed measurable residues. However, there was no violation of limits. Regarding lead levels, 7 liver samples and 18 kidney samples were contaminated (2.6 and 2.7% respectively). Cadmium was found below MRL in 14.9% of the analyzed samples. In kidneys, 448 samples (67.8% of the total tested) had cadmium residues and 4 samples exceeded the legislation limits. During the seven years of study (2002-2008), only 5 (0.25%) out of 1978 tested samples violated the Brazilian law. However, even with low levels of violation, monitoring is essential, since the number of samples with residues was often greater than the number of violations, and in some cases, such as cadmium in the kidneys, the majority of samples contained quantifiable residues and with levels often near the limits.

Determination of 13 Quinolone Antibiotic Residues in Swine Muscle by UPLC-MS/MS

A method was developed for the simultaneous determination of 13quinolone(QNs)antibiobtics in swine muscle by ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).The analytes were extracted from the sample using Na2EDTA-Mcllvaine buffer solution and phosphate buffer with ultrasonic bath and purified by solid-phase extraction(SPE)cartridge.Sample was analyzed in electrospray positive ion and multiple reaction monitoring(MRM)mode.Satisfied recoveries(64.55%—117.62%) of all the drugs are demonstrated in 5.0,50.0and 100.0μg/kg spiked levels with the RSD for intra-day and inter-day of 13analytes less than 14.28%and 12.81%.Limit of detection(LOD)and limit of quantitation(LOQ)are 1.0μg/kg and 5.0μg/kg.The method is sim-ple,accurate,sensitive and suitable for the determination of 13quinolone(QNs)antibiobtics in swine muscle.

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry

This paper presents LC-MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0μgkg(-1). Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45°C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04μgkg(-1) to 2.11μgkg(-1) and the detection capability (CCβ) ranged from 1.46μgkg(-1) to 2.89μgkg(-1). The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.

Reasoned opinion on the modification of the existing MRLs for spinosad in small fruit and berries and several commodities of animal origin

In accordance with Article 6 of Regulation (EC) No 396/2005 the evaluating Member State (EMS) Belgium compiled an application to modify the existing MRLs for spinosad in animal origin commodities as a result of the intended use on kale, for which no amendment of the existing MRL was proposed. Belgium proposed to raise the existing MRLs in several commodities of swine and ruminant origin. In accordance with Article 6 of Regulation (EC) No 396/2005 the EMS United Kingdom received an application from the Horticultural Development Company to modify the existing MRLs for spinosad in the group of small fruit and berries. The United Kingdom proposed to raise the existing MRLs to 1 mg/kg. The EMSs drafted the evaluation reports in accordance with Article 8 of Regulation (EC) No 396/2005, which were submitted to the European Commission and forwarded to EFSA. According to EFSA the data are sufficient to derive a MRL proposal of 1.5 mg/kg for the proposed use on kale. Thus, no amendment of the existing MRL is required. A MRL of 1.5 mg/kg is proposed for the intended indoor use on the group of small fruit and berries. No modification of the MRLs recommended under the Article 12 review for the commodities of animal origin were found necessary except for swine liver (0.7 mg/kg), sheep and goat fat (3 mg/kg), sheep and goat liver (1.5 mg/kg), swine muscle (0.1 mg/kg), bovine muscle (0.3 mg/kg), sheep and goat muscle (0.2 mg/kg), poultry muscle (0.2 mg/kg). Adequate analytical enforcement methods are available to control the residues of spinosad on the products under consideration. Based on the risk assessment results, EFSA concludes that the proposed amendment of the MRLs will not result in a consumer exposure exceeding the toxicological reference value and therefore is unlikely to pose a consumer health risk.

Development of a liquid chromatography–tandem mass spectrometry with ultrasound-assisted extraction method for the simultaneous determination of sudan dyes and their metabolites in the edible tissues and eggs of food-producing animals

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of sudan I, sudan II, sudan III, sudan IV, and their metabolites such as 4-aminoazobenzene and ortho-aminoazotoluole in 12 animal derived foods (including the muscle and liver of swine, muscle, liver and skin of chicken and duck, muscle and skin of fish, as well as the eggs of hen and duck). Sample preparation procedure included ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Aluminum B cartridges. The detection and quantification of the 6 sudan dyes and their metabolites were performed by a reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS). The CCαs and the CCβs of various samples varied from 0.03μg/kg to 0.12μg/kg, 0.09μg/kg to 0.19μg/kg, respectively. The recoveries of spiked sample from 0.2μg/kg to 0.8μg/kg ranged from 61.9% to 87.4% with the relative standard deviations of less than 19.1%. Performances of the whole analytical procedure meet the criteria established by the European Commission for mass spectrometric detection.

Determination and Confirmation of Parent and Total Ractopamine in Bovine, Swine, and Turkey Tissues by Liquid Chromatography with Tandem Mass Spectrometry: Final Action 2011.23

A multilaboratory study of AOAC Official Method 2011.23 was performed to satisfy requirements for Final Action status through the AOAC expert review panel process. The study included nine collaborating laboratories from the United States, Canada, Brazil, and The Netherlands. Five incurred residue materials (bovine muscle, bovine liver, swine muscle, swine liver, and turkey muscle) were analyzed by each laboratory as blind duplicates for parent and total ractopamine content. After removal of invalid data, the parent and total ractopamine methods demonstrated acceptable reproducibility (RSDR 11.4-42.4%, HorRatR 0.34-2.01) based on AOAC criteria. The method was awarded Final Action status by the Official Methods Board on October 4, 2012.

Determination of Pentachlorophenol Residue in Meat and Fish by Gas Chromatography–Electron Capture Detection and Gas Chromatography–Mass Spectrometry with Accelerated Solvent Extraction

A novel analytical method, using gas chromatography-electron capture detection (GC-ECD) and GC-mass spectrometry detection (MS), was developed for the qualitative and quantitative measurement of pentachlorophenol in meat and fish. The analyte was extracted by methanol-2% trichloroacetic acid (3/1, v/v) with accelerated solvent extraction (ASE). The eluted fraction was evaporated and derivatized with acetic anhydride-pyridine (1/1, v/v) for GC-ECD analysis and GC-MS confirmation. The parameters for extraction pressure, temperature and cycle of ASE, cleanup, derivatization and analysis procedure were optimized. The averaged decision limits and detection capability of the method were in the ranges of 0.25-0.41 and 0.49-1.01 µg/kg in the muscle and liver of swine and bovine and in the muscle of carp and finless eel, respectively. Spiked recoveries from levels of 0.5-2.0 µg/kg were found to be more than 71.1%, with relative standard deviation less than 14.7% in GC-ECD and GC-MS. This rapid and reliable method can be used for the characterization and quantification of residues of pentachlorophenol in animal and fish tissues.

Does resveratrol improve insulin signaling in chronically ischemic myocardium?

BackgroundResveratrol is a naturally occurring polyphenol believed to be cardioprotective. We previously demonstrated that resveratrol improves insulin signaling and glucose metabolism in liver and skeletal muscle of swine with metabolic syndrome. Although resveratrol has metabolic benefits in peripheral tissues, its effect on insulin signaling in ischemic myocardium (IM) is unclear. Therefore, we developed a clinically relevant swine model of metabolic syndrome and chronic myocardial ischemia to investigate the effects of resveratrol on insulin signaling in cardiac tissue. Materials and methodsThirteen male Yorkshire swine were fed a high-cholesterol diet for 4 wk then underwent surgical placement of an ameroid constrictor to their circumflex artery to induce chronic myocardial ischemia. The high-cholesterol control group was given no drug (n = 7). The experimental group was provided the same diet and received supplemental resveratrol (100 mg/kg/d) (n = 6). Tissue was harvested 7 wk after ameroid placement for western blot and histological analyses. ResultsIn IM, there was no significant difference between the two groups in the insulin signaling markers studied. In nonischemic myocardium, there was a significant decrease in phosphorylated AMP-activated protein kinase α (P = 0.021) in the group treated with resveratrol; otherwise, there were no significant differences between the groups. Immunostaining for glucose transporter 4 and Periodic acid-Schiff staining for myocardial glycogen stores was similar between the groups. ConclusionsResveratrol has complex effects on glucose metabolism. Although prior studies demonstrated that resveratrol supplementation improves insulin sensitivity in peripheral tissues, in chronically IM, there are no significant alterations.

Purification of swine carbonic anhydrase isoenzymeIIIand measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA-III) in swine plasma and urine have not been previously determined or reported. CA-III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA-III from swine muscle tissues and determined CA-III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA-III in the tissues of female swine (age, 3months) and plasma of young swine (age, 1-5months) and adult female pigs (age, 2-3years) were determined using the ELISA system for swine CA-III. The mean (±SD) levels of CA-III in the skeletal muscles were 3.8±3.2mg/g (wet tissue), and in the plasma, 230±193ng/ml at 1month, 189±208ng/ml at 2months, 141±148ng/ml at 3months, 78±142ng/ml at 4months and 53±99ng/ml at 5months. The mean level of CA-III in the plasma samples from 2- to 3-year-old pigs was 18±60ng/ml. CA-III in the plasma samples was found to decrease from 1month until 3years of age (p<0.01). We performed far-western blotting to clarify the cause of the observed decrease in CA-III in plasma. Our results demonstrated that CA-III is bound to the transferrin and albumin. In addition, we determined that the levels of CA-III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.

Matrix effects in the determination of β‐receptor agonists in animal‐derived foodstuffs by ultra‐performance liquid chromatography tandem mass spectrometry with immunoaffinity solid‐phase extraction

Matrix effects in determination of three β-receptor agonists including salbutamol (SAL), clenbuterol, and terbutaline in animal-derived foodstuffs were studied by ultra-performance LC-MS/MS with cleanup of immunoaffinity SPE column (IAC). Some animal tissue samples including pig liver, swine muscle, and fish muscle were hydrolyzed by the mixed enzyme solution or HCl solution, and the cleanup efficiencies with SAL IAC, MCX SPE column, and C(18) -SCX tandem columns were examined and compared by using spiked experiments. The results showed that the matrix effects in the determination of SAL and terbutaline can be eliminated with SAL IAC cleanup, and the average recoveries of SAL were 77.4~81.5%, 79.0~80.3%, and 85.0~87.2% in pig liver, swine muscle, and fish muscle, respectively. The decision limit (ccα) and detection capability (ccβ) for SAL in pig liver were 0.02 and 0.05 μg/kg, respectively.

Matrix effects in the simultaneous determination of fenicol antibiotics in swine muscle and casings by ultra performance liquid chromatography-tandem mass spectrometry

Animal tissue samples were extracted with ethyl acetate or alkalized ethyl acetate, and purified with MCX or LC-18 solid-phase extraction columns, and the matrix effects (ME%) in the simultaneous determination of four fenicol antibiotics including chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) were studied by ultra performance liquid chromatography-tandem mass spectrometry. ME% for FFA in swine muscle and casings extracted by ethyl acetate with LC-18 column cleanup were 11.1% and 10.4%, and in swine muscle extracted by ethyl acetate and alkalized ethyl acetate with MCX column cleanup ME% were 8.1% and 16.1%, respectively, showing strong signal suppression; while ME% for FF in swine muscle extracted by ethyl acetate and alkalized ethyl acetate with LC-18 column cleanup were 148.5% and 146.9%, and in swine muscle extracted by ethyl acetate and alkalized ethyl acetate with MCX column cleanup were 234.3% and 150.9%, showing strong signal reinforcement. Deuterated chloramphenicol-d5 could only be used as an internal standard to eliminate matrix effects for CAP determination. It may be that the most effective method to eliminate matrix interference in the simultaneous determination of the four analytes is quantified by using matrix-matched solutions. Under the optimum conditions of sample pretreatment and quantitative analysis, the decision limits (ccα as specified in European Directive 2002/657/EC) for FFA, TAP, CAP and FF in swine muscle were determined to be 0.4, 0.05, 0.02 and 0.02 μg kg−1, and 0.5, 0.1, 0.04 and 0.03 μg kg−1 in casings; the detection capabilities in swine muscle were determined to be 0.7, 0.09, 0.05 and 0.05 μg kg−1, and 1.2, 0.19, 0.12 and 0.07 μg kg−1 in casings, respectively.