Pressure buffer ionization

In order to combine reversed-phase LC with atmospheric pressure chemical ionization (APCI)-MS (125), a commercially available heated nebulizer interface that can handle pure aqueous eluents at flow rates up to 2 ml/min in addition to nonvolatile buffers has been used (126). The heated nebulizer inlet probe consists... 

Liquid chromatography creates a huge volume of gas when solvent vaporizes at the interface between the column and the mass spectrometer.22 Most of this gas must be removed prior to ion separation. Nonvolatile mobile-phase additives (such as phosphate buffer), which are commonly used in chromatography, need to be avoided when using mass spectrometry. Pneumatically assisted electrospray and atmospheric pressure chemical ionization are dominant methods for introducing eluate from liquid chromatography into a mass spectrometer. 

An atmospheric pressure chemical ionization (APCI-LC-MS) (Sciex API 150 EX) method was developed for the determination of zaleplon and zolpidem in the whole blood. After single-step LLE, the hypnotics were separated by gradient-elution with an ammonium formate buffer/acetonitrile eluent on an Inertsil ODS-3 column. Methaqualone was used as IS. The recovery was higher than 70% for both hypnotics and the IS. The method was successfully applied to forensic cases [11]. 

The most recent products of this evolution are electrospray ionization (ESI)18 and atmospheric pressure chemical ionization (APCI).19 These techniques enable the routine on-line analysis of a wide variety of compounds using a variety of separation conditions (i.e., solvents, additives or buffers, flow rates, etc.). 

Ephedrine and pseudoephedrine have been measured in guinea pig plasma using HPLC with fluorescence detection, following precolumn derivatization with 5-dimethylamino-napthalene-1-sulfonyl chloride in acetonitrile. The mobile phase was 0.6% phosphate buffer (pH 6.5)-methanol (3 8 v/v). Jacob et al. developed an LC-atmospheric pressure chemical ionization MS-MS method for the quantitaion of various alkaloids found in ephedra-containing dietary supplements and also in plasma and urine from subjects using these supplements. Using this method, the concentrations of ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine and caffeine were determined in low nanogram quantities in plasma and urine. The analytical cycle time for this method was 12 min. 

The development of new ionization strategies usually opens the way to new fields of application. In particular, soft ionization techniques such as atmospheric pressure chenaical ionization (APCI) are interesting analytical tools for inorganic/organic MS. The mechanisms for the generation of reagent ions and the ionization pathways are similar for the most common APCI sources. First, the buffer gas is ionized by a beam of electrons accelerated in a high electric field. Then, a series of reactions. 

Under special conditions sulfur cations with up to 56 atoms have been observed [209]. Evaporation of liquid sulfur and cooling the vapor in an atmosphere of a cold buffer gas (He) at low pressures followed by adiabatic expansion into the vacuum of a mass spectrometer and El ionization produced mass spectra of clusters of sulfur molecules with m/e ratios up to ca. 1800. The intensity pattern shows that the species (Ss)h are most abundant n = 1-7) followed by (Sy)(S8)n-i clusters and (S6)(Ss)h-i clusters. The latter have the same mass as (Sy)2(S8) -2 clusters see Fig. 34. Thus, the composition of the clusters reflects the composition of hquid sulfur near the melting point which contains Sg, Sy and Se molecules as the majority species [34, 210]. 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

In high-pressure biochemistry the buffer system should be chosen with care. As should be clear from Section IV,A, ionizations are followed by negative volume changes and therefore increase with pressure. The variation of pH with pressure depends on the volume of ionization AVZ, which can differ as much as —30 cm3 mol-1 from one buffer to another. 

If it is necessary to use one specific buffer, it is of course easy to calculate the pH at a given pressure when AVZ is known, and from a calibration curve of reaction rate versus pH correct for the rate change due to the pH perturbation. It seems comforting that there is a buffer such as Tris which keeps a pressure-independent pH. But one must be aware that pOH is not equally independent. Water also ionizes at high pressure, governed by... 

When using PFT with a neutral selector, it is quite difficult to avoid any entrance of the chiral selector into the ionization source, particularly at a high pH, where EOF is important. The use of BGE at low pH and/or coated capillary to minimize EOF is therefore mandatory. However, the coaxial sheath gas, which generally assists the ionization process, leads to an aspirating phenomenon of the chiral selector in the MS direction. Javerfalk et al. were the first to apply PFT with a neutral methyl-/i-CD for the separation of racemic bupivacaine and ropivacaine with a polyacrylamide-coated capillary and an acidic pH buffer (pH 3). Cherkaoui et al. employed another neutral CD (HP-/1-CD) with a PVA-coated capillary for the analysis of amphetamines and their derivatives. To prevent a detrimental aspiration effect, analyses were carried out without nebulization pressure. Numerous other studies presented excellent results such as the enantioselective separation of adrenoreceptor antagonist drugs using tandem mass spectrometry (MS/MS) the separation of clenbuterol enantiomers after solid-phase extraction (SPE) of plasma samples or the use of CD dual system for the simultaneous chiral determination of amphetamine, methamphetamine, dimethamphetamine, and p-hydroxymethamphetamine in urine. 

Nilsson, S. L., Andersson, G., Sjoeberg, P. J. R., Bylund, D., Petersson, P., Joernten-Karlsson, M., and Markides, K. E. (2003). Phosphate buffers in capillary electrophoresis/mass spectrometry using atmospheric pressure photoionization and electrospray ionization. Rapid Commun. Mass Spectrom. 17, 2267-2272. 

---