[Determination of paraquat and diquat residues in urine samples based on solid-phase extraction and ultra performance liquid chromatography-high resolution mass spectrometry].

Determining the presence of paraquat (PQ) and diquat (DQ) in urine samples through physical and chemical testing is challenging. As PQ and DQ have characteristics such as high molecular polarity and good water solubility, they are difficult to be retained by conventional reversed-phase columns. Most of the methods in the literature use hydrophilic interaction chromatography (HILIC) for the retention of PQ and DQ, but they often require high concentrations of buffer salts as the mobile phase, which increase the contamination of the mass spectrometer. In view of the above problems, a rapid and accurate analysis method was developed for the determination of PQ and DQ residuals in urine samples based on weak cation exchange (WCX) solid-phase extraction (SPE) and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) in this study. Urine samples were first diluted with phosphate buffer (pH=6.86) and pretreated using the WCX SPE method. Chromatographic separation was performed on a Syncronis HILIC column (100 mm×2.1 mm, 1.7 µm). An electrospray ion source in the positive (ESI+) mode and full mass-data dependent MS2 (full mass-ddMS2) mode was used for quantification by matrix-matched external standard method. In this study, the concentration of ammonium formate in the mobile phase in the HILIC mode was effectively reduced to 10 mmol/L by the continuous optimization of the chromatographic conditions. MS optimization results indicated that the molecular ion (M+·) of PQ and DQ had the strongest response. In addition, sample pretreatment conditions were also optimized. The obtained results indicated that the hydrophobic polytetrafluoroethylene (PTFE) filter membrane, acetonitrile-water (1∶1, v/v) as a fixing solution, and polypropylene vials were suitable for PQ and DQ analysis. Under the optimal conditions, the linearity of PQ and DQ was good with correlation coefficients (r2) greater than 0.998. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.2 µg/L and 0.6 µg/L, respectively. Mean spiked recoveries of PQ and DQ at the four spiked levels (1.0, 20.0, 100.0, and 200.0 µg/L) were in the range of 85.8%-101% and 80.3%-86.9%, with the RSDs of 0.8%-5.1% and 0.9%-4.2%. The established method was employed for the analysis and confirmation of PQ and DQ for clinical poisoning cases. In one case, a 23-year-old male who had taken approximately 20 mL of pesticide orally was confirmed as DQ poisoning by the developed method. DQ concentration monitoring of the urine samples was conducted for this case during the clinical treatment process. The patient was successfully discharged from the hospital after five times of blood perfusion and other treatments until the DQ concentration was low in the urine samples. In conclusion, the method developed in this study based on WCX SPE-UPLC-HRMS can be used for the confirmation of poisoning cases and concentration monitoring during clinical treatment, providing strong technical support for clinical precision treatment. The method is rapid, simple, sensitive, and accurate, and it is suitable for the detection of PQ and DQ in urine samples.

[Simultaneous determination of nine estrogens in bullfrogs using filtered solid phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry].

Due to the harmful effects of estrogens and their prevalence in animal foods, accurate analysis of estrogen levels in animal foods is imperative in order to effectively assess food safety risks and ensure consumer safety. Therefore, a rapid and accurate method based on PRiME HLB solid phase extraction (SPE) cartridge purification and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine nine estrogen residues in bullfrogs. The nine estrogens included estriol (E3), 17ß-estradiol (ß-E), 17α-estradiol (α-E), 17α-ethinylestradiol (EE2), estrone (EI), diethylstilbestrol (DES), dienestrol (DE), hexestrol (HEX), and dienestrol diacetate (DD). This study optimized the mobile phase system, extraction solvent, and SPE cartridges. Because estrogens present weak alkalinity, adding a small amount of alkaline substance to the mobile phase benefits estrogen ionization into the ionic state, eliminates the peak trailing phenomenon, and enhances the signal response of estrogens to improve sensitivity. Estrogens have one or more hydroxyl groups in their chemical structures. According to the principle of similar solubility, polar solvents are chosen as extraction solvents. Based on the complex matrix composition of meat samples, SPE is required for purification to reduce matrix effects. The liquid chromatographic conditions were optimized, and the 0.5 mmol/L ammonium fluoride aqueous solution-acetonitrile system as mobile phases showed better sensitivity than the ammonium acetate aqueous solution-acetonitrile system and the ammonia-acetonitrile system for the nine estrogens. When acetonitrile was used as the extraction solvent, the extraction rates of all nine estrogens exceeded those of methanol and ethyl acetate and increased by 15%-40%. By focusing on the matrix purification effect of four different SPE cartridges, the results showed that the matrix purification ability of the PRiME HLB cartridge outperformed that of the HLB, C18, and Silica SPE cartridges. After purification by the PRiME HLB cartridge, the recoveries of all compounds were in the range of 70%-125%, and the DD recovery was increased from 47% to 74%, whereas the HEX recovery was reduced from 180% to 123%. Therefore, the PRiME HLB SPE cartridge was selected as the cleanup material for this experiment. Finally, the sample was extracted using acetonitrile, purified by PRiME HLB SPE cartridge, and separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm) with a mobile phase of 0.5 mmol/L ammonium fluoride aqueous acetonitrile solution at a flow rate of 0.3 mL/min. The detection was conducted in positive and negative ion switching mode (ESI+/ESI-) and multiple reaction monitoring (MRM) scanning, and it was quantified using a matrix-matched external standard method. Under the optimal experimental conditions, the linear ranges were 0.5-100.0 µg/L for E3, ß-E, α-E, EI, DE, HEX, and DD, and 1.0-100.0 µg/L for EE2 and DES. The nine estrogens showed good linearity in all linear ranges, with correlation coefficients of 0.9953-0.9994. The limits of detection were 0.17-0.33 µg/kg, and the limits of quantification were 0.5-1.0 µg/kg. The recoveries of the nine estrogens spiked at the three spiked levels of low (2.0 µg/kg), medium (10.0 µg/kg), and high (80.0 µg/kg) were 107.4%-125.3%, 67.0%-123.3%, and 65.1%-128.2%, respectively. The relative standard deviations were 1.9%-17.6%. The method established in this study was applied to detect nine estrogen residues in 50 commercially available bullfrog samples, and the results showed that HEX, EI, and DES were detected in few samples. The method is simple, rapid, sensitive, and reproducible, and can be used for the simultaneous, rapid and accurate determination of large quantities of samples.

[Rapid determination of four phenolic environmental estrogen residues in cooking oil by ultra-performance liquid chromatography-high resolution mass spectrometry coupled with solid-phase extraction].

A fast, sensitive and accurate method for the determination of trace bisphenol S (BPS), bisphenol F (BPF), bisphenol A (BPA) and 4-nonylphenol (4-NP) in cooking oil samples was developed by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) coupled with solid-phase extraction (SPE). Cooking oil samples were extracted by acetonitrile, then the supernatant was purified by SLC SPE cartridges. The chromatographic separation was carried out on a Waters ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 µm) with a linear gradient elution procedure using 0.05% (v/v) triethanolamine aqueous solution and methanol as mobile phases. The quantification analysis was operated in a negative electrospray ion (ESI-) source mode under the selected ion monitoring (SIM) mode with internal standard method. The four target analytes showed good linearity with correlation coefficients (r) greater than 0.999. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) were in the ranges of 0.03-0.11 µg/kg and 0.10-0.36 µg/kg, respectively. The recoveries of the four target analytes spiked in oil samples were in the range of 86.3%-96.1% at spiked levels of 1.0, 10.0 and 80.0 µg/kg, respectively, while the relative standard deviations (RSDs) were in range of 2.2%-8.8% (n=6). No significant matrix interference was found in this method. The proposed method is simple and fast. It can be applied for the rapid determination of trace BPS, BPF, BPA, and 4-NP in cooking oil samples.

Low local blood perfusion, high white blood cell and high platelet count are associated with primary tumor growth and lung metastasis in a 4T1 mouse breast cancer metastasis model.

It was originally thought that no single routine blood test result would be able to indicate whether or not a patient had cancer; however, several novel studies have indicated that the median survival and prognosis of cancer patients were markedly associated with the systemic circulation features of cancer patients. In addition, certain parameters, such as white blood cell (WBC) count, were largely altered in malignant tumors. In the present study, routine blood tests were performed in order to observe the change of blood cells in tumor-bearing mice following the implantation of 4T1 breast cancer cells into the mammary fat pad; in addition, blood flow in breast tumor sites was measured indirectly using laser Doppler perfusion imaging (LDPI), in an attempt to explain the relevance between the blood circulation features and the growth or metastasis of breast cancer in mice model. The LDPI and blood test results indicated that the implantation of 4T1 breast cancer cells into BALB/c mice led to thrombosis as well as high WBC count, high platelet count, high plateletcrit and low blood perfusion. Following implantation of the 4T1 cells for four weeks, the lung metastatic number was determined and the Pearson correlation coefficient revealed that the number of visceral lung metastatic sites had a marked negative association with the ratio of basophils (BASO%; r=-0.512; P<0.01) and the mean corpuscular hemoglobin was significantly correlated with primary tumor weight (r=0.425; P<0.05). In conclusion, the results of the present study demonstrated that tumor growth led to thrombosis and acute anemia in mice; in addition, when blood BASO% was low, an increased number of lung metastases were observed in tumor-bearing mice.

Cytotoxicity enhancement in MDA-MB-231 cells by the combination treatment of tetrahydropalmatine and berberine derived from Corydalis yanhusuo W. T. Wang.

AIM: Our previous works have demonstrated that Chinese herb medicine yanhusuo (Corydalis yanhusuo W. T. Wang) has strong anti-cancer proliferation effect in MDA-MB-231 cells. The goal of this study was to find out the synergic cytotoxicity effect of three natural compounds, tetrahydropalmatine (THP), berberine (Ber), and dehydrocorydaline (DHC), isolated from C. yanhusuo W. T. Wang. MATERIALS AND METHODS: The IC50 of THP Ber and DHC in single use, as well as in combination use at fixed ratios and doses was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Isobologram, combination index and modified coefficient of drug interaction (CDI) methods were used for evaluation the combination effects of THF! Ber, and DHC in different ratio and concentration. RESULTS: The results indicated that the combination of THP and Ber shown the strongest anti-cancer cell proliferation effect at the ratio of 2:3 (Ber: THF the average CDI value was 0.5795). DHC and THP have additive cytotoxicity in MDA-MB-231 cells. However, there wasn't any synergistic effect between Ber and DHC, and it even exhibited antagonistic effect when the percentage of DHC was >50%. CONCLUSION: Our findings suggested that the combination of THP and Ber might be beneficial for anti-proliferation of MDA-MB-231 breast cancer cells through a significant synergy effect.