Development and Validation of UHPLC-MS/MS Assay for Therapeutic Drug Monitoring of High-dose Methotrexate in Children with Acute Lymphoblastic Leukemia.

PURPOSE: Precise and timely detection of methotrexate (MTX) concentration played a key role in high-dose MTX individualization therapy in acute lymphoblastic leukemia (ALL) children to avoid serious adverse effects or nonresponse. This report described a sensibility and validation of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for therapeutic drug monitoring (TDM) of methotrexate concentration in children's plasma. METHODS: One-step protein precipitation of samples was accomplished by adding 200 µL of acetonitrile to 100 µL of plasma sample. The separation of plasma samples was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column with gradient elution using a mobile phase constituted of acetonitrile and 1% formic acid. The detection was executed by electrospray ionization (ESI) of triple quadrupole tandem mass spectrometer (TQMS) in the multiple reaction monitoring (MRM) mode with the transitions m/z 455.2 → 307.9 for methotrexate and m/z 458.2 → 311.2 for IS, separately. Linear concentration range of the calibration curve was 44-11,000 nmol/L and 44 nmol/L was the lower limit of quantification. RESULTS: The methotrexate elution time was at 1.577 min, and the overall running time was only 3.3 min. The intra- and interday precision for all the analysis results was within 11.24%, and mean recoveries rate of methotrexate exceeded 87.98%. CONCLUSION: The described and fully validated UHPLC-MS/MS method was successfully applied in clinical TDM after infusion of high-dose methotrexate 1-5 g/m2 to 41 childpatients.

River Valleys Shaped the Maternal Genetic Landscape of Han Chinese.

A general south-north genetic divergence has been observed among Han Chinese in previous studies. However, these studies, especially those on mitochondrial DNA (mtDNA), are based either on partial mtDNA sequences or on limited samples. Given that Han Chinese comprise the world's largest population and reside around the whole China, whether the north-south divergence can be observed after all regional populations are considered remains unknown. Moreover, factors involved in shaping the genetic landscape of Han Chinese need further investigation. In this study, we dissected the matrilineal landscape of Han Chinese by studying 4,004 mtDNA haplogroup-defining variants in 21,668 Han samples from virtually all provinces in China. Our results confirmed the genetic divergence between southern and northern Han populations. However, we found a significant genetic divergence among populations from the three main river systems, that is, the Yangtze, the Yellow, and the Zhujiang (Pearl) rivers, which largely attributed to the prevalent distribution of haplogroups D4, B4, and M7 in these river valleys. Further analyses based on 4,986 mitogenomes, including 218 newly generated sequences, indicated that this divergence was already established during the early Holocene and may have resulted from population expansion facilitated by ancient agricultures along these rivers. These results imply that the maternal gene pools of the contemporary Han populations have retained the genetic imprint of early Neolithic farmers from different river basins, or that river valleys represented relative migration barriers that facilitated genetic differentiation, thus highlighting the importance of the three ancient agricultures in shaping the genetic landscape of the Han Chinese.

A UHPLC-MS/MS method coupled with liquid-liquid extraction for the quantitation of phenacetin, omeprazole, metoprolol, midazolam and their metabolites in rat plasma and its application to the study of four CYP450 activities.

Drug-drug interactions (DDIs) are thought to be associated with the inhibition of cytochrome P450 activities. The cocktail method with analysis of the metabolism of two or more probe drugs is used to determine CYP450 activities. In this study, we established a UHPLC-MS/MS method for simultaneous quantitation of four CYP450 probe drugs (phenacetin, omeprazole, metoprolol and midazolam) and their metabolites (acetaminophen, 5'-hydroxy omeprazole, α-hydroxy metoprolol and 1'-hydroxy midazolam) in rat plasma. Sample preparation by plasma protein precipitation was combined with a liquid-liquid extraction method. The separation was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column with a gradient elution, using water containing 0.1% formic acid (A) and acetonitrile (B) in a run time of only 3.0 min. Detection was conducted with a 6420 series triple-quadrupole tandem mass spectrometer, using ESI in positive ion mode with multiple reaction monitoring (MRM). The calibration curves were linear over the concentration range 10-5000 ng/mL for phenacetin, omeprazole, metoprolol and midazolam, and 1-500 ng/mL for their metabolites. Intra- and inter-day precisions were within 15%, and the accuracies were in the range of 87-112%. The method was successfully applied to the pharmacokinetic study of probe drugs/metabolites and DDIs with 3-n-butylphthalide (NBP) after administration of a single oral dose of phenacetin, omeprazole, metoprolol and midazolam in rats.

[Two new flavonoid glycosides from leaves of Moringa oleifera].

Two new flavonoid glycosides, quercetin-3-O-(4-O-crotonyl)-ß-D-glucopyranoside (1) and quercetin-3-O-[6-O-(2E)-pentenoyl]-ß-D-glucopyranoside (2), along with nine known ones, isoquercetin (3), astragalin (4), quercetin-3-O-(6-O-acetyl)-ß-D-glucopyranoside (5), kaempferol-3-O-(6-O-acetyl)-ß-D-glucopyranoside (6), quercetin-3-O-(6-O-crotonyl)-ß-D-glucopyranoside (7), kaempferol-3-O-(6-O-crotonyl)-ß-D-glucopyranoside (8), vitexin (9), isovitexin (10), and isorhamnetin-3-O-ß-D-glucopyranoside (11), were isolated from the leaves of Moringa oleifera by various chromatographic technologies. Their structures were elucidated by spectroscopic methods including UV, IR, MS, and NMR. In addition, compounds 7 and 8 were isolated from this plant for the first time.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All.

An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, ß-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

As a non-covalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence-based dye for detecting proteins both in 1-D and 2-D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which similars to that of glutaraldehyde (GA)-silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

A method for sensitive staining of DNA in polyacrylamide gels using basic fuchsin.

BACKGROUND: PAGE is a widely used analytical method to resolve components of a DNA mixture based on their size. Various DNA visualization methods including fluorescence, visible dye and silver have been used for the detection of gel-separated DNA, with each having different advantages and disadvantages in terms of sensitivity, safety and simplicity. RESULTS: A fast and sensitive visible dye-based staining method for DNA in polyacrylamide gels using basic fuchsin (BF) is described. As low as 10-20 pg of DNA can be visualized within 10 min; the sensitivity is fourfold more sensitive than that of SYBR® Gold stain, the most sensitive commercial fluorescent probe, but similar to silver staining kit from GE Healthcare. In addition, the mechanism studies suggest that the interaction of BF with DNA is mainly contributed by non-intercalative binding mode. CONCLUSION: By comprehensive studies of this visible dye-based protocol, we concluded that BF stain is a fast and sensitive method currently available for detecting DNA in polyacrylamide gels.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.