The analytical method presented above allows for rapid, high-throughput detection and quantitation of up to 36 TOrCs including pharmaceuticals, personal care products and steroid hormones using UHPLC-MS/MS.
Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.
This technique has a high degree of sensitivity, and the amine specific isobaric reagents of iTRAQ allow the identification and quantitation of up to eight different samples simultaneously [ 17, 19, 20].
Simulation studies in mouse-sized phantoms have shown that attenuation can degrade quantitation accuracy by up to −18%99mTcmTc or In) or −41%% (I) [ 160].
Quantitation was performed for up to 40 cycles of melting at 95°C for 15 s, annealing at 50°C for 1 min, and extension at 73°C for 15 s, the optimal conditions determined empirically.
Next, we aimed to demonstrate the quantitative precision, accuracy, and dynamic range of the 12-plex DiLeu reagents for bottom-up protein quantitation by labeling a complex mixture of S. cerevisiae lysate tryptic peptides and analyzing by high-resolution LC MS.
The detector's response was linear (R 2 > 0.999) and reliable for morphine quantitation from 0.005 up to 1 mg/mL.
Furthermore, these results were backed up by the quantitation of real-time RT-PCR results.
Sample preparation and instrumentation methods were as published, with a 2 pg/mg limit of detection and an 8 pg/mg limit of quantitation, and the method was linear up to 2000 pg/mg 17.
All assays were set up for a relative quantitation method in which mean Ct values from MMTV-Wnt1 samples were used as calibrators for data analysis for MMTV-Wnt/ILK samples.
