Improvement in the sensitivity of an LC/MS/MS method can generally be achieved by reducing the analytical scale of the analysis. However, reducing the analytical scale significantly to nano-flow reduces the throughput, stability, and robustness as a trade-off for greater sensitivity. Nexera Mikros, a Shimadzu microflow LC/MS system, offers a balanced solution to increased sensitivity with less compromise to throughput and robustness, using flow rates from 1 to 500 μL/min and column I.D. from 0.1 to 1 mm. It achieves stable and sensitive quantitative analysis through robust performance of microflow solvent delivery, optimized ion source design, the unique UF-Link column connection, and system variety that supports a wide range of applications.
Sensitivity remains a challenge for many vitamin D metabolites, such as 1,25(OH)2 di-hydroxyvitamin D and 24,25(OH)2 di-hydroxyvitamin D, because they lack easily ionizable functional groups and are in low-abundance. As shown below, microflow LC/MS/MS analysis of standard solutions of the aforementioned vitamin D analytes resulted in a three- to five-fold improvement in the signal-to-noise ratio when compared to semi-microflow analysis.
The increased sensitivity achievable by microflow LC/MS/MS, compared to semi-microflow, resulted in a five-fold increase in the signal-to-noise ratio for Verapamil and a four-fold increase in the signal-to-noise ratio of Nor-Verapamil.
nSMOL (nano-surface and molecular orientation limited proteolysis) is a proprietary, ground-breaking process that enables selective proteolysis of the Fab region of antibodies using trypsin-immobilized nanoparticles. Fab-derived peptides are quantified via MRM measurement on a high-sensitivity mass spectrometer. The Nexera Mikros system achieved signal intensities 12 times higher than with conventional flow rates, with a corresponding lower limit of detection of 0.025 μg/mL and good linearity. The Nexera Mikros system is ideal for low-level quantitation of peptides by LCMS.
The following is an example of antibody drug analysis in plasma. The plasma spiked with trastuzumab was pretreated using nSMOL Antibody BA Kit.
In the analysis of signature peptides deriving from trastuzumab, Nexera Mikros provides a dynamic range of 0.00763 – 62.5 μg/mL, good linearity with R2 > 0.99, and excellent accuracy of 101.0% (average).
The Nexera Mikros system delivers the improvements you expect from a low-flow system with the ruggedness of HPLC. A plasma blank was protein precipitated, diluted (3x), and injected 1,500 times consecutively. During the consecutive injections, QC sample (40 ppt of nortriptyline) was injected once every 50 injections of plasma blank. Peak areas and retention times of QC sample are plotted below. With 4.38% RSD area repeatability and 0.25% RSD retention time stability, extremely stable operation was demonstrated, without loss of peak shape common in high-throughput analyses of complex matrices.
Shimadzu’s Micro-ESI LCMS source has been designed for optimal sensitivity and ease of use. An X–Y adjustable stage and viewing camera allow for easy ESI spray needle adjustment. The column oven is mounted on the LCMS to minimize the volume between the exit of the column and the ESI needle.
The Micro-ESI probe also features an ESI spray angle optimized for microflow rates, and is available for triple quadrupole mass detection mass spectrometryand quadrupole time-of-flight mass spectrometry, respectively.
Mounted to the Micro-ESI source is the CTO-Mikros column oven. Stable column temperature, essential to retention time reproducibility, is ensured and columns up to 150 mm in length can be accommodated. To preserve the optimal source adjustment, the CTO-Mikros features an innovative snap-in column locking system, the UF-Link.
When using microfow rates, even the smallest of dead volumes can cause significant diffusion and lead to loss of peak shape and sensitivity. Shimadzu’s UF-Link allows any user to correctly install a microflow column without disrupting the ESI spray needle position. A sure-sealing adapter allows the column to lock into the oven and make a zero-dead volume connection with the ESI needle. Any column up to 150 mm in length with standard fittings can be accommodated.
