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How to Increase LC-MS Sample Throughput with Multiplexing HPLC Systems

Multiplexing technology enables parallel HPLC systems to synchronize to a single mass spectrometer.

by Thermo Fisher Scientific
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Problem: Many LC-MS-based laboratories must analyze an increasing number of samples, which requires extensive sample purification, high throughput, and increased labor.  

Solution: Multiplexing technology enables parallel HPLC systems to synchronize to a single mass spectrometer. With multiplexed Thermo Scientific Aria™ systems, each HPLC operates independently, permitting multiple HPLC methods to run simultaneously. This enhanced analytical feature reduces costly downtime associated with traditional single channel LC systems whose detectors are idle more than 75 percent of the time. Multiplexed HPLC systems ensure the maximum utilization of the mass spectrometer, increasing productivity without compromising data quality or sensitivity.  

In drug discovery, drug development, and clinical research applications, it is common to carry out repeated direct injection of complex samples, such as biological fluids. TurboFlow™ technology allows direct injection of these complex samples into mass spectrometers, simplifying sample preparation. Fast, efficient separations of complex sample matrices and compounds of interest are achieved because the system retains small molecules and filters out proteins and larger materials by diffusion, size exclusion, and column chemistry.  

To demonstrate the advantages of TurboFlow and multiplexing technologies, a liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method with on-line sample extraction has been developed for the determination of Angiotensin I. (Singh, et. al., 2007 AACC Annual Meeting, San Diego, CA)  

Plasma Renin Activity (PRA) is used to screen for high blood pressure of renal origin. The kidney protease renin produces the peptide Angiotensin I (Ang I) from a plasma protein. Ang I is a decapeptide with the following sequence: Asp-Arg-Val- Tyr-Ile-His-Pro-Phe-His-Leu. The molecular weight of Angiotensin I is 1296.5 Da. Today, many laboratories determine PRA in patient samples by measuring the concentration of Ang I using a radioimmunoassay (RIA) method. Historically, this analysis involved significant sample purification prior to RIA or HPLC qualification of Ang I in the buffered plasma samples.  

Chromatographic Conditions 

• System: Thermo Scientific Aria TLX-4 HTLC System 

• TurboFlow Column: Cyclone, 50 x 1.0 mm 

• Analytical Column: C18, 33 x 4.6 mm, 5 microns 

• Cycle Time (injection to injection): 1.5 minutes 

• Sample: Plasma in pH 6 maleate buffer with enzyme inhibitors  

PRA can be determined by measuring the concentration of Angiotensin I using a LC-MS/MS TurboFlow method. The new assay was evaluated by analyzing 59 patient samples. The new method correlates well (R = 0.95) to the existing RIA method and eliminates the need for sample purification, providing an overall throughput of 40 samples per hour.  

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