LC/MS/MS methods are now widely recognized as gold standard for the accurate quantitation of small molecules in biological fluids and results more suitable and reliable compared to immunoassay based approaches. For metabolites in biological fuids, the concentration level of the target component is often low, therefore more sensitive analysis is required than semimicro-flow LC/MS analysis, which is superior in analytical throughput, stability and robustness. Generally, in LC/MS analysis, improvement of sensitivity can be expected by reducing the analytical scale which is defined by the total flow rate and column size. On the other hand, by reducing the analytical scale to nano-flow, a well skilled analysts who have special technics in nano-flow analysis is required, and throughput, stability and robustness are reduced by trade-offs. LC/MS analysis on a micro-flow scale using 0.1 to 1 mm I.D. column with 1 to several μL/min has the excellent balance of sensitivity, throughput, and robustness. Nexera Mikros, a Shimadzu microflow LC/MS system provide the stable and sensitive quantitative analysis by not only improved sensitivity through stable performance of micro-flow solvent delivery and optimized ion source design, but also utilization of the unique UF-Link column connection and system variety that support a wide range of applications.

► Improved Detection Limits with Microflow
► Incomparable Microflow Ruggedness
► Easy, Familiar Operation

Features
Incomparable Microflow Ruggedness

Realize the Benefits of Microflow While Enjoying the Ruggedness and Reliability of HPLC
The Nexera Mikros system delivers the improvements you expect from a low-flow system with the ruggedness of HPLC.

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Applications

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Microflow Liquid Chromatography Mass Spectrometry System
Incomparable Microflow Ruggedness
Realize the Benefits of Microflow While Enjoying the Ruggedness and Reliability of HPLC
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.

*Results obtained under our application conditions.

Nexera Mikros Applications

High Sensitivity Analysis for the Bioanalysis
While the results of quantification of vitamin D metabolites using LC/MS/MS are more reliable than other methods, in metabolite measurement, the vitamin D is lack of easily ionizable functional groups and pose assay sensitivity challenges mainly for quantification of low-abundance metabolites such as 1,25(OH)2　di-hydroxyvitamin D and 24,25(OH)2 di-hydroxyvitamin D. To overcome this sensitivity problem, It is effective to perform analysis with micro-flow LC/MS/MS. The comparison between micro-flow LC/MS/MS and semimicro-flow LC/MS/MS for the analysis of standard solutions of Vitamin D metabolites shown a 3 to 5 fold improvement in the ratios of signal/noise.

High Sensitivity Analysis for the Bioanalysis
While the results of quantification of vitamin D metabolites using LC/MS/MS are more reliable than other methods, in metabolite measurement, the vitamin D is lack of easily ionizable functional groups and pose assay sensitivity challenges mainly for quantification of low-abundance metabolites such as 1,25(OH)2　di-hydroxyvitamin D and 24,25(OH)2 di-hydroxyvitamin D. To overcome this sensitivity problem, It is effective to perform analysis with micro-flow LC/MS/MS. The comparison between micro-flow LC/MS/MS and semimicro-flow LC/MS/MS for the analysis of standard solutions of Vitamin D metabolites shown a 3 to 5 fold improvement in the ratios of signal/noise.

Monoclonal Antibody Bioanalysis
— Nexera™ Mikros and nSMOL™ Antibody BA Kit —
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 intra-day reproducibility of this trastuzumab-derived-peptide analysis is summarized in the table below.
These results show an excellent reproducibility, with both of accuracy and precision within 20% at LLOQ and within 15% even at other concentrations.

*1: This curve is drawn on logarithmic scale.
*2: QC sets 1 and 2 were analyzed over a 2-day period, with each set of concentrations analyzed five times.

Microflow Liquid Chromatography Mass Spectrometry System

Robust and Easy-to-Use Microflow ESI Source
Micro-ESI™ Sources
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.

CTO-Mikros™ + UF-Link™: Easy, Dependable Column Installation
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.

LC-Mikros™: Innovative Microflow Pump Design
Shimadzu LC pumps are the industry standard for long life and high performance. The LC-Mikros solvent delivery pump has completely new direct-drive engineering to deliver micro to semi-micro flow rates ranging from 1 to 500 μL/min. UHPLC system pressure tolerance of 80 MPa (11,600 psi) allows the use of every column type.
The LC-Mikros pump is designed for microflow gradient elution, even at low percentages. In the example below, excellent gradient stability is shown using mobile phase compositions ranging from 1% to 90%. The LC-Mikros delivers stable retention times essential to the chromatographer.

SIL-40C XR: Low-Volume Direct Injection Autosampler
The SIL-40C XR autosampler delivers you excellent accuracy and reproducibility as well as extremely low carry-over. Its injection volume ranges from 0.1 to 50 μL and your precious sample has never wasted due to the total injection method SIL-40C XR adapted.
It contributes to produce highly accurate results even if you had to use low injection volume (less than 1 μL). In case of micro flow rate with narrow column, optimum injection volume also gets lowered. It is also difficult to increase the injection volume, especially, if you would use a sample solvent with high elution power. In this case, sample must be diluted to weaken the sample solvents. Highly accurate micro volume injection capability of SIL-40C XR allows you to use samples with high organic solvent contents without dilution.
System volume and gradient delay are minimized with the newly developed “programmable loop cut-off mechanism,” which automatically diverts the sample loop from the flow line using a high-pressure valve as soon as the sample in needle is drained. This is extremely important in minimizing microflow analysis run times.

Columns for Microflow Analysis

Shim-pack™ MC C18
Versatility
Shim-pack MC C18 column with 1.9 μm UHPLC particle achieves excellent separation and peak shape.
The high pressure rating of 70 MPa allows these columns to be compatible with a range of mobile phase flow rates from 1 to dozens of μL per minute.
The metal coordinative adsorption is suppressed by deactivating the column parts in contact with the solution.
Both small and large molecule compounds, such as peptides, can be analyzed.
Excellent durability can be achieved even if the biological sample analysis.

Shim-pack MC PLONAS Series
Superior Performances with Superficially Porous Particles (SPP)
Shim-pack MC PLONAS 2.7 μm columns reduce the back pressure yet maintain the performance of a sub 2 μm fully porous particle column. The pressure limit is 40 MPa.
Achieve lower LODs and LOQs, better resolution, and greater reproducibility using the unique superficially porous particle found in Shim-pack MC PLONAS.
Greater dimensional offerings allows for higher efficiency with better resolving power for challenging applications.
An array of pore sizes designed to meet the needs of any application.
Increased ruggedness because of larger porosity frits resulting in less clogging compared to competitor columns.
Excellent column-to-column and lot-to-lot reproducibility.

Shim-pack Trap Columns

Trap Columns Wide Portfolio
Shim-pack MCT C18/C8
70 MPa pressure limitation and a unique modification on the column inner surface protects against absorption of the molecule.

Shim-pack MCT LC18/LC8
A small-volume cartridge column with 40 MPa pressure limit. Useful in reducing the gradient delay for a low flow rate.

Nexera Mikros: Trap and Elute Configuration
The trap and elute system can be constructed by using a dedicated piping kit (228-71751-42).

Direct Injection System
Ideal for sample volume-limited analyses. This system provides rapid and highly sensitive micro LCMS analysis without sample loss.

Trap and Elute System
Ideal for analyses with larger injection volumes or when some degree of sample cleanup is desirable.

Make-Up Flow System
The addition of a post-column make-up flow pump allows mobile phase modifiers to be added to ensure the highest ionization efficiency in the MS source.