Extraction of ions from solutions under atmospheric pressure as a method for mass spectrometric analysis of bioorganic compounds (2008)

The basic features of mass spectrometry, such as high sensitivity, molecular mass determination capacity, and acquisition of structural information, are not used practically to analyze non-volatile, polar, and thermally unstable compounds, in particular the majority of bioorganic substances. This is because it is impossible to evaporate these compounds without decomposition under ordinary heating. It is necessary to use non-destructive methods of evaporation and ionization of the substances in the ion source of the mass spectrometer or, alternatively, chemical protection of polar and labile groups. Existing methods of 'soft' ionization require obligatory sample preparation procedures. This fact limits the application of these methods.1 Recently, certain progress has been achieved in this field of mass spectrometry applying the generation of the ions directly from solutions under atmospheric pressure.2-4 In this paper we report a method called the extraction of dissolved ions under atmospheric pressure (EDIAP). This method allows the mass spectra of non-volatile and thermolabile bioorganic compounds to be recorded with controllable fragmentation or clustering of the quasi-molecular ion. Control of the mass spectrum allows unique information about the molecular mass of a substance, its structural features, and the nature of interaction between the ions and the neutral solvent molecules to be obtained. Moreover, the kinetics and mechanisms of ion-molecule reactions can be also investigated. These numerous tasks can be solved using only the ion source invented at the Institute of Analytical Instrumentation of USSR Academy of Sciences. The developed ion source is schematically shown in Fig. 1. The ion source has been installed in a MX 1320 double focusing mass spectrometer with accelerating potential of 2.5 kV. Commercially available pumps have been used in the differential pumping system. The pump speed was 5 L/s in the fore-vacuum chamber and 700 L/s in the high-vacuum chamber. The core elements of the ion source. 1 – A metal capillary which is used to infuse the sample into the ionization zone; 2, 3 – Orifice plates. These form the gas-dynamic stream. The metal capillary is placed coaxially to the plate orifices while the first plate is a counter electrode of the electrohydrodynamical spraying system. A differential pumping system provides the necessary pressure distribution in all system chambers. The ion-optical parameters of the intermediate chamber provide the ion beam focusing onto the orifice of the second plate. The potential applied between the orifice plates (ΔU) varies from 0 up to 900 V. 4 – Focusing lenses. 5 – Mass spectrometer inlet. 6 - An ion current monitor. Samples pass at a flow rate of 1–10−4 µL/s through a thin metal capillary (needle) at high voltage (2–4 kV). A potential is applied between the capillary and the first orifice plate. An inhomogeneous electric field transforms a flow of liquid into a flow of charged substance-containing micro-droplets. The mechanism of the extraction of ions from these micro-droplets is as follows. In the atmospheric pressure chamber and intermediate chamber of the differential pumping system the charged micro-droplets undergo repeated collisions with the gas molecules. As a result micro-droplets are supplied with a continuous non-destructive flow of energy required for evaporation of the solvent. As the surface charge of the micro-droplets increases, the Rayleigh limit is achieved, and a droplet breaks up into smaller droplets. The repetition of this process results in the formation of charged solvated molecules, so-called clusters. The breakage of the micro-droplets and the extraction of ions can also be caused by boiling up the drops under abrupt gas expansion in the differential pumping system. Moreover, this process can be promoted by the breakage of droplets at the pressure shocks in the supersonic stream, as well as by zero evaporation of the ions from the surface of a charged droplet. The number of solvent molecules in a cluster formed depends on the energy accepted by the cluster during the collisions with the gas molecules. The collision energy varies depending on the change in potential, ΔU, between the orifice plates in the intermediate chamber of the pumping system. Consequently, the cluster ions can be destroyed if increased ΔU potential is applied. This permits the recording of mass spectra constituted only of quasi-molecular ions. Further increase of the ΔU potential forms the mass spectra of the characteristic fragment ions. To demonstrate the universality of the developed method in terms of the substance type and the characteristic ion formation, a number of dissolved substances have been analyzed. Among the non-volatile and thermally unstable compounds which were analyzed are sugars, antibiotics, amino acids, peptides, nucleosides, steroids, etc. To obtain the mass spectrum, 10–100 nmol of a compound is required depending on the difference between proton (cation) affinities of a target analyte and a solvent molecule. HPLC grade methanol, water, acetonitrile, and their mixtures have been used as the solvents. The molecular mass can be easily determined via quasi-molecular ion inspection of the mass spectrum. A quasi-molecular ion arises from the interaction of a neutral molecule and a proton ([M+H]+) or a singly charged cation ([M+Kt]+). The injection of solutions into the mass spectrometer was carried out under atmospheric pressure without heating. The generally accepted method to test the ‘softness’ of ionization is to obtain the mass spectrum of sucrose. At the same time, the spectrum must contain the peak characterizing the molecular mass of sucrose. Using the EDIAP method we have obtained the mass spectrum of an aqueous methanolic solution of sucrose resulting in the formation of the quasi-molecular ion at m/z 343 corresponding to the protonated disaccharide molecule, [M+H]+. At the same time, the addition of potassium chloride leads to the formation of the [M+K]+ ion. Also it has to be noted that no fragmentation of the investigated compound occurs under these conditions. The nature of the low-mass ions in the mass spectrum is due to the formation of clusters like Sn or Kt+ · Sn, where Kt+  NH, H3O+, CH3OH, etc.; S  CH3OH, H2O, etc. As an example, Fig. 2 shows the mass spectrum of erythromycin dissolved in aqueous 95% methanol. To analyze erythromycin, the medicament of the same name was used without any purification. This fact explains the presence of the contaminant peaks at m/z 742, 756, and 772 along with the erythromycin quasi-molecular ion at m/z 734. Mass spectrum of erythromycin. Traditionally, unsubstituted amino acids are considered to be compounds which are difficult to analyze by mass spectrometry.5 As a rule, the thermal instability of amino acids such as arginine, cysteine, and creatine is a challenge during their identification. For instance, the molecular ion of arginine cannot be registered at all in both electron and chemical ionization mass spectra. Only the special technique of rapid evaporation enables the detection of the sufficiently intensive peak of the quasi-molecular ion along with fragment ions and ionized products of arginine pyrolysis. Figure 3 shows the mass spectrum of a 10−2 M aqueous solution of arginine hydrochloride. The spectrum was recorded in different modes of the invented ion source. Mass spectrum of arginine at the different potentials applied between the diaphragms in the fore-vacuum chamber. In the 'cluster ions' mode (ΔU = 0–100 V) the mass spectrum represents a distribution of the ions in the so-called 'aqueous series' of MH+(H2O)n ions, where MH+ = H3O+, NH, ArgH+. The maximum value of n is a function of ΔU. At ΔU = 30 V the arginine molecule produces short aqueous series of the cluster ions. The intensity of the cluster ions decreases as the molecular mass of the cluster increases. Moreover, no peaks corresponding to arginine fragmentation or decomposition were detected in this mode. Although sometimes the formation of clusters helps to achieve better accuracy of the molecular mass measurements, it certainly can hinder the analytical use of mass spectrometry. Using the invented ion source these cluster ions can be easily removed applying higher ΔU values. In the 'molecular ions' mode (ΔU = 100–250 V) redistribution of peak intensities in the mass spectrum takes place. Arginine appears in the form of the stable quasi-molecular ion at ΔU = 150 V (Fig. 3) and no fragmentation occurs. Using this ionization mode unique information about the molecular mass and, consequently, the molecular formula can be obtained. Individual heavy bioorganic molecules and complex mixtures of natural or synthetic origin can be analyzed. In particular, a mixture of 19 common non-modified amino acids comprised of natural proteins has been analyzed. The result is presented in Fig. 4. All amino acids produce the quasi-molecular ions making the mass spectrum of such a complex mixture easy to interpret. Mass spectrometric analysis of the mixture of amino acids in the ‘molecular ions’ mode. It has also been established that the mass spectrometric behavior of oligopeptides and their mixtures is similar to that of amino acids when the 'molecular ions' mode is used. The mass range of the analyzed peptides is limited by the mass spectrometer (m/z 1500) rather than by the ionization technique. To identify the molecular weight of 2,4-dinitrophenyl-Gly-Gly-Phe-Arg-OH, only 10−11 moles of the substance are required. This proves the method to be highly sensitive. This is confirmed by the inspection of the intensity redistribution between the m/e 116 and m/e 70 peaks against the ΔU value. Such a controllable dissociation seems to be very perspective for the amino acid sequence determination of peptides. For instance, the most abundant peaks in the mass spectrum of glycylleucine at ΔU = 350 V are m/e 189 (52%), m/e 132 (100%), and m/e 86 (63%). In fact, these peaks represent the quasi-molecular ions of the dipeptide, the protonated C-terminal amino acid, and the characteristic fragment ion of leucine [LeuH-46]+, respectively. Thus, the developed method for the generation of ions directly from the liquid phase allows non-volatile and thermally unstable substances to be analyzed by mass spectrometry without preliminary chemical protection of polar and labile groups. Information about the molecular mass and structural features of a substance can be obtained in such a manner. Moreover, the EDIAP method satisfies the requirements of the 'liquid chromatograph/mass spectrometer' interface.6 The latter enables the EDIAP method to form the basis for an instrumental platform of LC/MS/computer possessing broad structural and analytical potentialities.