Multiclass Comparative Analysis of Veterinary Drugs, Mycotoxins, and Pesticides in Bovine Milk by Ultrahigh-Performance Liquid Chromatography-Hybrid Quadrupole-Linear Ion Trap Mass Spectrometry.

A multiclass and multiresidue method for simultaneously screening and confirming veterinary drugs, mycotoxins, and pesticides in bovine milk was developed and validated with ultrahigh-performance liquid chromatography-hybrid quadrupole-linear ion trap mass spectrometry (UHPLC-Qtrap-MS). A total of 209 targeted contaminants were effectively extracted using an optimized QuEChERS method. Quantitative and qualitative confirmation were achieved simultaneously by multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scan mode. The validation results exhibited a good sensitivity with the LOQs of 0.05-5 µg/kg, which was satisfactory for their MRLs in China or EU. The recoveries of in-house spiked samples were in the range of 51.20-129.76% with relative standard deviations (RSD) between replicates (n = 3) 0.82% and 19.76%. The test results of 140 milk samples from supermarkets and dairy farms in China showed that cloxacillin, aflatoxin M1, acetamiprid, and fipronil sulfone were found with lower concentrations. Combined with the residue results from the literature, penicillin G and cloxacillin (beta-lactams), enrofloxacin and ciprofloxacin (fluoroquinolones), and sulfamerazine (sulfonamides) were more frequently detected in different countries and need to receive more attention regarding their monitoring and control.

Is implantation of autologous chondrocytes superior to microfracture for articular-cartilage defects of the knee? A systematic review of 5-year follow-up data.

BACKGROUND: Autologous chondrocyte implantation (ACI) and microfracture are two of the main surgical treatment options for articular cartilage lesions of the knee. Consensus regarding the best clinical options to repair knee cartilage lesions is lacking. We undertook a systematic review to clarify the clinical efficacy of ACI and microfracture at minimum mean 5-year follow-up. METHODS: A literature search was conducted using the MEDLINE, Embase and Cochrane Library databases up to August 2018. Only comparative clinical studies of ACI and microfracture for the treatment of articular cartilage lesions of the knee with level I/Ⅱ evidence were included. Clinical outcomes and the prevalence of treatment failure from each study were extracted and compared. The methodological quality of the included studies was analyzed by means of the PEDro scale. RESULTS: Five comparative studies (three randomized controlled trials and two prospective cohort studies) met our eligibility criteria. ACI and microfracture elicited significant improvement in clinical outcomes after 5 years. However, better clinical results with significant differences were found with modified versions of ACI (ACI with a modified collagen membrane [ACI-C] or matrix-applied chondrocyte implantation [MACI]) than with microfracture as determined by the Knee Injury and Osteoarthritis Outcome Score, activities of daily living assessment, Tegner Activity Scale score, and the International Knee Documentation Committee objective and subjective scores. No significant difference was observed in the treatment failure rate between these two methods within a particular study. CONCLUSIONS: Currently, the best-available evidence suggests that some clinical outcomes of articular cartilage lesions of the knee treated with modified versions of ACI (ACI-C or MACI) can significantly improve patient outcomes at the mid-term follow-up of 5 years compared with those obtained using microfracture.

Simultaneous determination of 58 pesticides and relevant metabolites in eggs with a multi-functional filter by ultra-high performance liquid chromatography-tandem mass spectrometry.

A sensitive method for the simultaneous determination of 58 pesticides and relevant metabolites in eggs was developed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) after clean-up with a multi-functional filter (MFF) based on quick, easy, cheap, effective, rugged, and safe method (QuEChERS). The egg sample was extracted with 5 ml water and 10 ml 1% acetic acid in acetonitrile and then salt out with sodium chloride. The extracted solution was filtered directly through an MFF containing 50 mg PSA, 50 mg C18, and 150 mg magnesium sulphate before UHPLC-MS/MS analysis. The clean-up and filter procedures were integrated using the MFF to substantially improve the work efficiency. Good linearity was shown for each analyte, and all the correlation coefficients exceeded 0.99. The recoveries in the eggs at the five spiked levels were 74.4%-115.2%, and the relative standard deviations (RSDs) were less than 15.3%. The limit of detection (LOD) and the limit of quantitation (LOQ) of 58 pesticides and 8 metabolites in eggs were 0.1-1.0 µg/kg and 0.2-5.0 µg/kg, respectively. The decision limit (CCα) and detection capacity (CCß) were 3.4-111.1 µg/kg and 6.8-122.1 µg/kg, respectively. This method has also been successfully applied in the determination of actual eggs samples. This developed method is more effective and faster in the monitoring of pesticide residue in eggs compared to the traditional analytical method.

Inhibitory effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced femoral head necrosis.

Apoptosis and DNA oxidative damage serve significant roles in the pathogenesis of steroid­induced femoral head necrosis. Vitamin E demonstrates anti­apoptotic and anti­oxidant properties. Therefore, the present study investigated the effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at an early stage of steroid­induced femoral head osteonecrosis. Japanese white rabbits were randomly divided into three groups (steroid, vitamin E­treated, and control groups), each comprising 12 rabbits. Those in the steroid group (group S) were initially injected twice with an intravenous dose of 100 µg/kg Escherichia coli endotoxin, with a 24 h interval between the two injections, and then with an intramuscular dose of 20 mg/kg methylprednisolone, three times at intervals of 24 h in order to establish a rabbit model of osteonecrosis. The vitamin E treated group (group E) received the same treatment as group S, and were administered 0.6 g/kg/d vitamin E daily from the beginning of modeling. The control group (group C) was injected with normal saline at the equivalent dosage and times as the aforementioned two groups. Two time points, weeks 4 and 6 following the completion of modeling, were selected. Osteonecrosis was verified by histopathology with hematoxylin-eosin staining. The apoptosis rate of osteonecrosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The apoptosis expression levels of caspase­3 and B­cell lymphoma 2 (Bcl­2), and DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry. At weeks 4 and 6 following the completion of modeling, the vacant bone lacunae rates of group E were 15.87±1.97 and 25.09±2.67%, respectively, lower than the results of 20.02±2.21 and 27.79±1.39% for group S; and the osteocyte apoptosis indexes of group E were 20.99±2.95 and 33.93±1.62%, respectively, lower than the results of 26.46±3.37 and 39.90±3.74% from group S. In addition, the Bcl-2 expression at week 4 in the femoral head tissues of group E was higher compared with group S; and the proportion of Bcl­2­positive cells of group E was 9.81±1.01%, higher compared with group S at 8.26±1.13%. The caspase­3 staining data at week 4 in femoral head tissues demonstrated that in the 12 femoral heads of group S, four were negative (32%) and eight were positive (68%); in group E, five were negative (45%) and seven were positive (55%); and in group C, 11 were negative (95%) and one was positive (5%). In addition, the DNA oxidative damage rate at week 4 in the bone marrow hemopoietic cells of group E was (7.24±1.44%), lower compared with group S (11.80±1.26%), and higher compared with group C (5.75±1.47%). Vitamin E is effective in intervening in apoptosis through decreasing caspase­3 expression and upregulating Bcl­2 expression, and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid­induced femoral head necrosis in rabbit models.

[Rapid screening and confirmation of 205 pesticide residues in rice by QuEChERS and liquid chromatography-mass spectrometry].

A method for rapid screening and confirmation of 205 pesticide residues in rice was developed by combining QuEChERS and high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (LC-Q-TRAP/MS). The rice samples were extracted with acetonitrile, and then cleaned up with primary secondary amine (PSA), anhydrous magnesium sulfate (MgSO4) and C18 adsorbent. Finally, the samples were detected by LC-Q-TRAP/MS in multiple reaction monitoring with information-dependent acquisition of enhanced product ion (MRM-IDA-EPI) mode followed with database searching. A total of 205 pesticide residues were confirmed by retention times, ion pairs and the database searching using EPI library, and quantified by external standard method. All the pesticides showed good linearities with linear correlation coefficients all above 0.995. The limits of quantification (LOQs) for the 205 pesticides were 0.5-10.0 µg/kg. The average recoveries of the 205 pesticides ranged from 62.4% to 127.1% with the relative standard deviations (RSDs) of 1.0% - 20.0% at spiked levels of 10 µg/kg and 50 µg/kg, and only 20 min were needed for the analysis of an actual rice sample. In brief, the method is fast, accurate and highly sensitive, and is suitable for the screening and confirmation of pesticide residues in rice.

[Expression of human bone morphogenetic protein 7 gene in adipose-derived stem cells and its effects on osteogenic phenotype].

OBJECTIVE: To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phenotype. METHODS: Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, and CD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Lipofectamine 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkaline phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. RESULTS: ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P < 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P < 0.05). CONCLUSION: The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.

An improved on-line solid phase extraction coupled HPLC-MS/MS system for quantification of sifuvirtide in human plasma.

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1-6250 ng/mL, and the correlation coefficients (r(2)) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from -7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC-MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).