A rapid, specific and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determination of isofraxidin, rosmarinic acid and kaempferol-3-O-glucuronide in rat plasma using warfarin as an internal standard (IS). Separation was conducted on a Thermo Hypersil GOLD C column with linear gradient elution using methanol and water. Mass spectrometric detection was conducted using selected reaction monitoring (SRM) via an electrospray ionization (ESI) source. All analytes exhibited good linearity within their concentration ranges (r > 0.9990). The lower limits of quantitations of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide were 1.31, 0.67 and 0.92 ng/mL, respectively. Intra- and inter-day precisions of these investigated components exhibited an RSD within 11.7%, and the accuracy ranged from -12.5 to 15.0% at all QC levels. The developed method was successfully applied to a pharmacokinetic study of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide in rats after oral administration of Herba Sarcandrae Extract.
The close relationships
of miRNAs with human diseases highlight
the urgent needs for miRNA detection. However, the accurate detection
of a target miRNA in mixed miRNAs of high sequence homology presents
a great challenge. Herein, a novel method called target-protection
rolling circle amplification (TP-RCA) is proposed for this purpose.
The protective probe is designed so that it can form a fully complementary
duplex with the target miRNA and can also mismatch duplexes with other
nontarget miRNAs. These duplexes are treated with a single strand-specific
nuclease. Consequently, only the target miRNA in a perfect-match duplex
can resist the cleavage of nuclease, whereas the nontarget miRNAs
in mismatched duplexes will be digested completely. The protected
target miRNA can be detected using RCA reactions. MicroRNA let-7 family
members (let-7a–let-7f) and nuclease CEL I were used as proof-of-concept
models to evaluate the feasibility of the TP-RCA method under different
experimental conditions. The experimental results show that the TP-RCA
method can unambiguously detect the target let-7 species in mixtures
of let-7 family members even though they may differ by only a single
nucleotide. This TP-RCA method significantly improves the detection
specificity of miRNAs.
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