Research Article: Automatic Sampling and Analysis of Organics and Biomolecules by Capillary Action-Supported Contactless Atmospheric Pressure Ionization Mass Spectrometry

Date Published: June 6, 2013

Publisher: Public Library of Science

Author(s): Cheng-Huan Hsieh, Anil Kumar Meher, Yu-Chie Chen, Toby James. Athersuch.


Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the capillary inlet. The sample droplet on the well can be readily infused into the C-API capillary through capillary action. When the sample solution reaches the capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.

Partial Text

The field of atmospheric pressure ionization (API) mass spectrometry has grown quickly since the inspired work of desorption electrospray ionization (DESI) was reported by Cooks and co-workers in 2004 [1]. Furthermore, because of the improved performance of mass spectrometers, the recent development of atmospheric pressure ion sources is striking. In the past few years, many new ion sources[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14],[15], [16], [17], [18] such as one-step[1], [6], [7], [8], [9], [10], [11], [12], [13], [14],[15] and two-step [16], [17], [18] ionization methods have been reported. The main advantages of these ionization methods over conventional API techniques are their simplicity and convenience through the elimination and reduction of sample preparation steps. Combined with suitable mass analyzers, qualitative information regarding molecular weights and analyte structures can be obtained. Automatic sampling API mass spectrometry provides the possibility for conducting quantitative analysis. Previously, automatic scanning probe electrospray ion source for imaging mass spectrometry has been proposed for the analysis of small molecules [15]. Automatic sampling atmospheric pressure chemical ionization (APCI) was recently proposed for quantitative analysis, but its setup requires several pumps and valves [19].

All of the amino acids, caffeine, and creatinine were purchased from Sigma (St. Louis, MO, USA). Acetonitrile was purchased from Merck (Darmstadt, Germany). Fused silica capillary tubes [150 µm outer diameter (o.d.) and 10 µm inner diameter (i.d.)] were obtained from GL Science (Japan). Aluminium sheets (3 cm×3 cm×2 mm) and stainless steel tubes (0.8 mm o.d. and 0.5 mm i.d.) were obtained from local companies.

This study focused on the development of automatic sampling MS analysis using the C-API setup. Faster sampling speeds are helpful for high-throughput analysis. In principle, a thinner capillary tube would provide a faster sampling speed. Thus, a capillary (150 µm o.d., 10 µm i.d., 1 cm in length) was tapered and used as the sampling tube and the C-API spray emitter. Bradykinin (10 µM) in acetonitrile/deionized water (1∶1, v/v) was initially used as the model sample. The tapered capillary filled with acetonitrile/deionized water (1∶1, v/v) was placed in front of a mass spectrometer (Fig. 1). After a 4 µL sample droplet was deposited on the aluminium plate on a XY stage, the plate was moved up to immerse the capillary inlet into the small droplet (Fig. 1). Simultaneously, the mass spectrometer acquired the MS signal. The sampling speed was estimated based on the time of doubly charged bradykinin ion signal observed in the extracted ion chromatogram (EIC) at m/z 531. Figure 2A shows the EIC at m/z 531, whereas Figure 2B shows the mass spectrum obtained at the 0.2 min time point when the peak at m/z 531 just appeared. The flow rate was estimated to be ∼4 nL min−1, whereas the linear velocity was estimated to be ∼5 cm min−1. The results indicate that the thin capillary tube provided a fast sampling speed. However, capillary tips that were thinner than used herein were too fragile to be held with tweezers in the current design. Thus, the smallest capillary tube with an original i.d. of 10 µm was used as the C-API sampling tube and spray emitter in this study.

A simple automatic sampling C-API MS design has been demonstrated. Considering no external force is required in the sampling process, the setup is quite straightforward. The automatic sampling system was based on placing an aluminium sample holder on an XY moveable stage controlled by a computer. Multiple sample analyses and sampling capillary flushing can be readily conducted automatically after every run. Subnanoliter samples were sufficient for the analysis. The lowest concentration that the current approach could detect was at the attomole level. Faster sampling speeds can further benefit the real-time monitoring of chemical and biochemical reactions by reducing the delay. Sensitivity and sampling speed may be potentially improved using thinner and shorter capillaries as the C-API sampling tube and spray emitter, but the clotting problem arising in thinner capillaries because of the presence of undesirable species such as salts in the sample solution should be considered. We have demonstrated the possibility of using the current approach in quantitative analysis. However, it should be noticed that properly controlling the experimental condition such as humidity can reduce the evaporation of sample solutions during analysis. Spiking an internal standard to samples can compensate the evaporation problems. Still, the current setup may be further modified by placing the C-API tip in a chamber with humidity control to improve its analytical performance.