Increasing ions: solvent-assisted inlet ionisation boosts sensitivity

Inlet ionisation

The growth of atmospheric pressure ionisation techniques in mass spectrometry is continuing at a rapid pace, with two particular groups dominating the scene. The first comprises the swathe of ambient ionisation processes that have been devised since desorption electrospray ionisation (DESI) and direct analysis in real time (DART) were announced in 2004.

During these processes, the sample is subjected to ionisation in the open air by a variety of mechanisms and the ions are transported to the mass spectrometer vacuum for analysis. Typically, only a small proportion of ions survive the journey through the air to the inlet.

The second atmospheric pressure ionisation mode was first reported in 2010. Initial examples involved laser irradiation of sample-matrix mixtures, in a similar way to matrix-assisted laser desorption/ionisation (MALDI). However, there was a key difference in that the laser did not induce ionisation.

Instead, the laser produced droplets of the matrix/analyte sample which were drawn into a heated transfer tube that connected atmospheric pressure to the vacuum inlet of the mass spectrometer. It was in the pressure drop region in this tube that ionisation occurred, hence the name inlet ionisation. There was a strong dependence of ion yield on the temperature of the inlet capillary.

Sample transfer was also accomplished by sonification, laser-induced acoustic desorption, air-gun pellet-induced shock waves, and even using a spatula and, in each case, multiply charged ions of peptides and proteins were produced and detected.

In inlet ionisation, a far greater proportion of ions reach the mass spectrometer because the perilous transfer of ions from atmospheric pressure to the vacuum is avoided. So, detection limits down to amol levels for peptides and low fmol levels for proteins have been achieved routinely.

Now, inlet ionisation has been extended to liquid samples in a technique called solvent-assisted inlet ionisation (SAII), reported by Charles McEwen, Vincent Pagnotti and Nicholas Chubatyi of the University of the Sciences, Philadelphia.

Solvent-assisted inlet ionisation in practice

The sample solutions were held in a polypropylene vial into which was placed a fused silica transfer capillary. The other end was positioned within the heated ion transfer tube that linked atmospheric pressure to the first vacuum stage of the mass spectrometer.

The solution flow rate through the capillary was governed by its length, internal diameter, temperature and the distance it protruded into the heated capillary. Typical flow rates of 10-48 µL/min were employed during system evaluation but low flows down to 2 µL/min were also successful. These flow rates were higher than those employed in nano-electrospray ionisation and led to a more stable, rugged procedure.

The inlet system was interfaced with a high-resolution Fourier transform mass spectrometer operating at a mass resolution of 100,000 and produced remarkable sensitivities for a range of analytes from small drug molecules to proteins.

For instance, for a solution of insulin in aqueous acetonitrile, multiply charged ions were clearly observed by SAII with a detection limit in the low amol region compared with 3.44 fmol/µL for electrospray ionisation under the same solution concentration, flow rate and instrument tune conditions.

Solutions of the drug ciprofloxacin hydrochloride in water produced a strong protonated molecule in SAII from 14 fmol of sample and a linear calibration curve was obtained from 830 fM to 830 nM (830 zmol/µL to 830 fmol/µL). Arginine had a detection limit of a few ppt in aqueous solutions.

These low sensitivities are expected to decrease further when selected reaction monitoring is employed.

SAII operates without applied voltages, laser irradiation or an ion source enclosure and has the advantage of easier sample handling than electrospray ionisation. However, it is not all plain sailing.

Peptides and proteins produce a high number of sodium ion adducts. In some cases, the number of adducts is far greater then the number of charges on the analytes, suggesting that sodium ions replace some acidic protons. This effect was removed for a few scans by washing the inlet tube but a longer lasting solution to the problem is clearly required.

In addition, salts, strong acids and strong bases cause ionisation suppression and low concentrations of analyte induce similar signal suppression to that found in electrospray ionisation. The researchers declared that they are taking steps to overcome these problems.

The technique holds great promise. McEwen concluded that "at this early stage of development, the simplicity and sensitivity of this method without use of ion funnels or special optics suggests that inlet ionisation methods will be an important addition to future mass spectrometry development."

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