Chromatography focus on


Why Ultrapure Water is Important for Nano-LC/MS Applications Maricar Tarun1 , Stéphane Mabic2 Paramus, NJ, USA , David P. Budac3 and Mark J. Hayward3, 1 Merck Millipore, Billerica, MA, USA2 Merck Millipore, Saint-Quentin-en-Yvelines, France3 Lundbeck Research,


Nano-flow LC (nano-LC) is an important tool in proteomics research and in the measurement of low level biomarkers. To avoid the common problem of emitter clogging, only the highest purity solvents and reagents should be used.


Connecting a liquid chromatography (LC) system to a mass spectrometer with an electrospray ionisation (ESI) source, LC/MS or LC/MS/MS (MS/MS for tandem mass spectrometry), offers today’s laboratories superior sensitivity and dynamic range over other detectors, and provides far more molecularly-specific information.1


However,


minute sample quantities, complex matrices, and very low concentrations (pg/mL) make analysis of endogenous biomolecules found in vivo a challenge. An excellent solution is provided by nanoflow LC (nano-LC) coupled to nano-electrospray ionisation (nanospray), which typically utilises 75 µm ID columns. Nano-LC requires minimal sample loading and results in more concentrated peaks. The very low flow rate (nL/min range) is optimal for nanospray sources. Nanospray ionisation increases ionisation efficiency, leading to better sensitivity.2-8


Nano-LC/MS emitters/sprayers have tips with inner diameters of ~1-30 µm that are prone to clogging.4,9


It is therefore important to choose not only the most robust


emitters, but also the highest purity solvents and reagents. This work uses the separation of neuropeptides by nano-LC/MS to demonstrate that using freshly produced ultrapure water as a solvent component in nano-LC/MS does not clog emitters. Neuropeptides are important biomarkers of neurological disorders that are present in very low concentrations (< 100 pg/mL) in central nervous system tissues as well as peripheral fluids (for example, cerebrospinal fluid and plasma), making their detection and quantification quite difficult. The low detection limits, low sample requirement, and increased sensitivity of nano-ESI MS make it ideal for the analysis of biological samples where sample availability is limited and analyte concentration is extremely low.


Experimental


The nano-LC/MS system consisted of a Waters nanoACQUITY UPLC system with a Quattro Premier XE MS/MS equipped with a nanospray source (Waters, Milford, MA) operated at a flow of 900 nL/min. A Waters Atlantis dC18 (75 µm x 100 mm, 3 µm) analytical column; PicoTip SilicaTip, 360 µm OD, 10 µm tip ID emitters (New Objective, Woburn, MA); and a methylated monolithic silica MonoSpray, 360 µm OD, 50 µm tip ID (GL Sciences, Torrance, CA) were used.


The mobile phase in the gradient elution was composed of (A) ultrapure water from a Milli-Q Gradient system with an A10 monitor (EMD Millipore, Billerica, MA) containing 0.2% acetic acid (BDH Aristar Ultra acetic acid from VWR, West Chester, PA) and 1% acetonitrile (B&J acetonitrile UV, Honeywell Burdick & Jackson, Morristown, NJ), and (B) 100% acetonitrile. The gradient elution is given in Table 1.


The neuropeptide biomarker standards used were angiotensin, leu-enkephalin and met-enkephalin. Different concentrations of these standards were injected to monitor changes in retention times, peak areas, calibration curves, and the physical state of the emitter. Injection volume was 1 µL.


An Olympus BH-2 microscope was used to take photomicrographs of emitters. Results and Discussion


Emitter clogging is the most common problem associated with nano-LC/MS experiments.3,4,9


. In this work, the aqueous mobile phase was made from freshly


delivered ultrapure water obtained from a water purification system (Figure 1). The system includes reverse osmosis, which removes significant amounts of ions, organics, particulates, and bacteria. This is followed by electrodeionisation (Elix module), a technology specifically dedicated to ion removal using ion exchange resins that are continuously regenerated by an electric current. High grade ion exchange resins, synthetic activated carbon, and UV photooxidation further reduce the concentration of ions and organics. This combination of technologies results in ultrapure water with a consistent quality: ion-free, and with the lowest possible levels of organics (thus avoiding accumulation in reversed-phase columns that could later elute as extraneous peaks in the chromatogram). When water is delivered using a 0.22 µm membrane final filter, this filter also retains bacteria and particulates (> 0.22 µm) that could clog emitters.


Table 1. Gradient profile for the separation of neuropeptides Time


0.00 0.50 2.50 2.60 3.00


%A 90.0 90.0 50.0 50.0 10.0


Time 3.01 3.50 3.51 5.00


%A 0.0 0.0


90.0 90.0


Figure 1. Schematic showing the technologies used to produce ultrapure water suitable for nano-LC/MS work. The 0.22 µm point-of-use final filter retains particulates that could clog emitters.


Keeping ionic contamination of solvents to a minimum is important. Ions such as Na+ and K+


Figure 2A shows the difference in the mass spectrum of a bradykinin standard dissolved in fresh ultrapure water and directly infused onto a mass spectrometer, and when it is dissolved in ultrapure water spiked with sodium ions (Figure 2B).


form adducts with peptides, complicating data analysis and reducing sensitivity.


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