Determination of Sulfonamides in Chicken Muscle by Pulsed Direct Current Electrospray Ionization Tandem Mass Spectrometry

A simple and rapid approach for the simultaneous detection of trace amounts of six sulfonamides in chicken muscle was developed using pulsed direct current electrospray ionization tandem mass spectrometry (pulsed-dc ESI-MS/MS). The pretreatment of chicken muscle samples consisted of two steps: acetonitrile extraction and <i>n</i>-hexane delipidation. Sulfonamides do not need to be derivatized or chromatographed prior to pulsed-dc ESI-MS/MS. The factors affecting the performance of pulsed-dc ESI-MS/MS were studied. Under optimum conditions, the quantitative performance of pulsed-dc ESI-MS/MS was validated according to European Union Decision 2002/657/EC, and the sensitivity of pulsed-dc ESI-MS/MS was 3 times higher than that of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The limits of detection obtained by pulsed-dc ESI-MS/MS were in the range of 0.07–0.11 μg/kg. The proposed method was simple, rapid, and sensitive, and was successfully used for quantitation and rapid screening of sulfonamides in real chicken muscle samples

PCR primers.

<p>PCR primers.</p

Role of differentiated CSCs (DCSCs) in myocardial functional recovery following I/R.

<p>A, Expression of Sca-1, CD29 and CD44 in DCSCs was determined by Flow cytometry assay. B, RT Real-time PCR analysis indicated transcription levels of cardiac specific transcription factors and genes in DCSCs. C, Western blot assay demonstrated increased expression of Gata4 and sarcomeric (SM) α-actin in DCSCs compared to un-induced CSCs. D, Expression of SM α-actin (green) and Gata4 (red) was observed in DCSCs by Immunofluoresence assay (Magnification 400X). Nucleus was stained with DAPI (blue). Myocardial functional recovery at end of reperfusion was represented as % of equilibration (Eq) in groups treated with vehicle, CSC and DCSC (E) or media control, CSC CM and DCSC CM (F). Mean ± SEM, n = 5–6/group, *p<0.01, **p<0.001 vs. Vehicle or Media C, #p<0.01, ##p<0.001 vs. CSC or CSC CM. CM: conditioned medium.</p

CSCs expressed much higher mRNA (A) and protein levels (B) of SDF-1 compared to production of VEGF, HGF and IGF-1.

<p>Mean ± SEM, N = 4 individual experiments, ***p<0.001 vs. SDF-1.</p

The Akt pathway in CSC-induced cardioprotection following I/R.

<p>A, Western blot assay revealed myocardial Akt activation in groups of Vehicle, CSCs, media control and CSC CM after I/R. B, The use of AMD3100 in CSC CM or SDF-1 siRNA in CSCs did not change myocardial Akt activation. Shown are representative immunoblots of p-Akt and T-Akt (one lane/group), and densitometry data of p-Akt represented as % of T-Akt. C, Inhibition of the Akt signaling by LY294002 did not affect CSC-improved post-ischemic LVDP, +dP/dt and –dP/dt. Results are mean ± SEM, n = 4-6/group, *p<0.05, **p<0.01 vs. control or LY294002 alone at the corresponding time point.</p

CSC-derived SDF-1 in the attenuation of cellular injury.

<p>A, Remaining LDH levels in cardiac tissue were determined after I/R. B, Western blot analysis indicated cleaved caspase-3 levels after I/R. Shown is representative immunoblots in each groups (one lane/group). Bar graph represents relative levels of Caspase-3 P20 (% of GAPDH). C, Myocardial hydrogen peroxide (H2O2) production was analyzed after I/R. A–C: Mean ± SEM, N = 4–5/group, *p<0.05, **p<0.01 vs. Vehicle. D, Cardiomyocyte (H9c2) viability and LDH levels in supernatant were determined after 24-hr of hypoxia in groups of vehicle and CSC CM with or without AMD3100. Mean ± SEM, N = 3, *p<0.05 vs. vehicle.</p

Determination of dominant paracrine factor(s) in CSCs.

<p>A, Cytokine antibody array was performed in conditioned medium (CM) from CSCs and DCSCs. Each number represented the fold increase of cytokine expression compared to the negative control (medium). B, CSC- and DCSC-secreted SDF-1 was determined in supernatants by ELISA. C, The SDF-1 expression in CSCs and cardiomyocytes was analyzed in cell lysates using ELISA. Mean ± SEM, N = 3–6/groups, ***p<0.001 vs. CSC. UD: Undetectable.</p

CSC-derived SDF-1 in mediating acute protection following I/R.

<p>A, Changes of LVDP and +/− dP/dt following I/R in groups of media control and CSC CM with or without AMD3100, an inhibitor of the SDF-1 receptor. B, Myocardial functional recovery at end of reperfusion was shown as % of Eq (equilibration). Mean ± SEM, n = 5–6/group, *p<0.05, **p<0.01, ***p<0.001. CM: conditioned medium.</p

Myocardial STAT3 in CSC-mediated acute cardioprotection following I/R.

<p>A, Myocardial activation of STAT3 was determined in groups of Vehicle, CSCs, media control and CSC CM after I/R by Western blot assay. B, AMD3100 or SDF-1 siRNA neutralized CSC-induced STAT3 activation compared to their counterparts. Representative immunoblots of p-STAT3 and T-STAT3 were shown (one lane/group) and bar graph represents relative levels of p-STAT3/T-STAT3 ( %) in A and B. C, Stattic abolished CSC-mediated acute protection as demonstrate by unimproved LVDP and +/− dP/dt following I/R. D. Remaining LDH in myocardial tissue after I/R. E. Cardiac caspase-3 levels were determined in hearts treated with vehicle, stattic and CSC+stattic after I/R by Western blot. Shown are representative immunoblots (2 lanes/group). Results are Mean ± SEM, N = 4–5/group, *p<0.05 vs. vehicle or media control.</p

Identification of cardiac stem cell (CSC) characteristics.

<p>A, Flow cytometry assay indicated expression of cell surface markers in CSCs. B, Expression of cell surface markers in mesenchymal stem cells (MSCs). C, RT Real-time PCR data showed mRNA levels of cardiac specific transcription factors in CSCs, MSCs and cardiomyocytes (isolated from adult mouse heart as a positive control). Mean ± SEM, n = 3 individual experiments. UD: Undetectable.</p