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Sample Prep Guidelines and Protocols

Many issues with mass spectrometry analyses can be traced to inadequate or improper sample prep. Users should consider a variety of factors including sample concentration, purity, solvent solubility, and presence of incompatible components or additives. All samples must be homogenously dissolved. Stop if you notice the presence of precipitates, aggregates or cloudiness.

Samples should be at a reasonable concentration that is within the instrument's analytical range and suitable for detection without overloading the system. Overloaded samples can precipitate or clog the flow path, contaminate the inlet source, saturate the detector and/or accumulate on the internal components that can foul detector components leading to a dramatic loss in performance and requiring extensive maintenance.
Sample concentrations in the micromolar (µM) to nanomolar (nM) range are suitable for most applications. Concentrations in the milimolar (mM) range are typcially too concentrated for high-end analytical instruments. Follow the basic rule: less is more.
For LC-MS applications:
  • Range: 1 nM to 10 µM.  Optimal: 10 µM (Do not exceed 50 µM without prior consultation with staff).
For GC-MS applications:
  • Range: 10 nM to 100 µM. Optimal: 50 µM  (Do not exceed 500 µM without prior consultation with staff). 
Solvents and solubility
Most organic analytes are soluble at low concentrations (10 µM or below) in common LC-MS solvent systems (water, methanol, acetontirile). Our default sample buffer is 90% dH2O:10% acetonitrile. Check to ensure your analyte is soluble in this buffer or another similar buffer and compatible with the solvents being used. If necessary prepare stock concentrations (100X) in a high % organic solvent, then dilute into sample buffer. If unsure, ask.

Do not use solvents immiscible with water (hexane, dichloromethane, choroform, ethylacetate etc.) on an LC-MS system. This can cause a rapid rise in back pressure and damage columns.

For GC-MS applications, the preferred sample solvent is dichloromethane (DCM). Alternative solvents inlcude hexane or ethylacetate. Do not use water, methanol or acetonitrile. If you compounds are readily soluble in these, you are likely on the wrong system.
High concentrations of non-volatile salts are incompatible with electrospray ionization ion sources. Common non-volatile salts encountered include sodium chloride, sodium phosphate buffers (e.g. PBS) or salts present in organic matrices (serum or tissue extracts are >110 mM NaCl). As droplets evaporate, these salts will accumulate on the inlet cone and possibly further into the instruments ion path. They may also cause ion suppression of analytes leading to an inconsistent or low sensiitvity signal.
Total non-volatile salt concentrations should be less than 10 mM. Simple techniques to meet this threshold are either dilution of the sample with water (10 to 50-fold) or where possible a liquid-liquid swing extraction into a non-polar solvent followed by evaporation and resuspension into an appropriate sample buffer.
Volatile salts or buffer additives may be used. These include:
  • Formic acid
  • Acetic acid
  • Ammonium buffers (NH4.formate, NH4.acetate or ammounium bicarbonate).