LC-MS mobile phases

Tables 3 and 4 show the gas-phase acid-base scale in both positive and negative ion modes for LC/MS mobile-phase solvents [13,37]. Particularly for APCI, the mobile phase components should be considered gas-phase reagents to promote either protonation or deprotonation of the analyte ion. In this manner, appropriately volatile mobile-phase components can be tailored to the assay.

The ubiquitous nature of the sodium cation often leads to Na cationiza-tion and the presence of [M + Na]+ in the mass spectrum in addition to, or in place of, [M + H]" ". Efforts to either remove or add Na+ from the sample result in changes in the relative abundances of the [M + H]+ and [M+ Na]" ". Addition will often cause replacement of other labile protons with additional sodium atoms. Addition of lithium or potassium can replace the Na+, with the appropriate mass changes, to conhrm the identity of the Na" " adducts. Ammonium cationization will often occur from LC-MS mobile phases containing an ammonium buffer salt. Acetonitrile solvent adducts will also often form. 

Mobile-phase additives are known to play an important role in the efficient separation of chromatographic peaks in LC-MS. Mobile-phase additives used for LC-MS studies are most often volatile molecules that can be easily evaporated in the LC-MS interface to prevent ionization suppression and/or detection contamination. The most commonly used mobile-phase additives in LC-MS analysis are the small organic acids, acetic and formic acids, together with their ammonium salts. Trifluoroacetic acid (TFA) is also widely used in LC-MS analysis despite reports of ionization suppression in the ESI mode. Some studies suggest that the drawback of TFA use can be overcome for some neutral analytes by using minute concentrations of the acid in the mobile phase. A number of studies presented here used TFA as the mobile-phase additive, and in all those instances, ESI was the ionization interface. However, formic acid was the additive most commonly used, followed closely by TFA and then by the ammonium salts of both formic and acetic acid. The type of glucuronide conjugate (O vs. N) did not appear to influence the type of mobile-phase additive used. 

The key for LC-MS mobile phases is simplicity. Additives other than formic acid should not be used unless absolutely necessary. For pantothenic acid analysis, the use of 0.1% LC-MS grade formic acid in the mobile phase is advised (mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile) (Chen, et al. 2007 and Chen et al. 2009). 

Fig. 3.58. LC-MS-MS analysis of an effluent sample taken through an analytical method involving SPE. LC conditions mobile phase acetonitrile-0.01 g/100 ml ammonium acetate in water (90 10, v/v) flow rate 0.30 ml/min sample volume 20 pi. The left-hand traces show the MS-MS ion chromatograms, while the corresponding right-hand spectra show the presence of the daughter ion indicative of each dye. RT = retention time. Reprinted with permission from W. F. Smyth et al. [128].

The first bioanalytical application of LC-GC was presented by Grob et al. (119). These authors proposed this coupled system for the determination of diethylstilbe-strol in urine as a replacement for GC-MS. After hydrolysis, clean-up by solid-phase extraction and derivatization by pentafluorobenzyl bromide, the extract was separated with normal-phase LC by using cyclohexane/1 % tetrahydrofuran (THE) at a flow-rate of 260 p.l/min as the mobile phase. The result of LC-UV analysis of a urine sample and GC with electron-capture detection (ECD) of the LC fraction are shown in Ligures 11.8(a) and (b), respectively. The practical detection limits varied between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of... 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

One of the functions of an LC-MS interface is to remove the mobile phase and this results in buffer molecules being deposited in the interface and/or the source of the mass spectrometer with a consequent reduction in detector performance. Methods involving the use of volatile buffers, such as ammonium acetate, are therefore preferred. 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

Another ion profile often encountered from background ions is one in which the intensity increases or falls at a regular rate throughout the analysis. This often occurs in LC-MS during gradient elution when the ion is associated with only one component of the mobile phase. 

When optimum experimental conditions have been obtained, all of the mobile phase is removed before the analyte(s) are introduced into the mass spectrometer for ionization. As a consequence, with certain limitations, it is possible to choose the ionization method to be used to provide the analytical information required. This is in contrast to the other LC-MS interfaces which are confined to particular forms of ionization because of the way in which they work. The moving belt can therefore provide both electron and chemical ionization spectra, yielding both structural and molecular weight information. 

Figure 4.9 Schematics of electrospray LC-MS interfaces with (a) a heated capillary and (b) a heated block to allow high mobile-phase flow rates. From applications literature published by (a) Thermofinnigan, Kernel Hempstead, UK, and (b) Micromass UK Ltd, Manchester, UK, and reproduced with permission.

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 this study, the effect of mobile-phase flow rate, or more accurately, the rate of flow of liquid into the LC-MS interface, was not considered but as has been pointed out earlier in Sections 4.7 and 4.8, this is of great importance. In particular, it determines whether electrospray ionization functions as a concentration-or mass-flow-sensitive detector and may have a significant effect on the overall sensitivity obtained. Both of these are of great importance when considering the development of a quantitative analytical method. 

Table 5.16 LC-MS-MS signal responses" obtained from wheat forage matrix samples using various mobile-phase additives (injection volumes of 50 p,l). From Choi, B. K., Hercules, D. M. and Gusev, A. I., LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices , Fresenius J. Anal. Chem., 369, 370-377, Table 2, 2001. Springer-Verlag GmbH Co. KG. Reproduced with permission...

The testing of impnrities in active pharmacentical ingredients has become an important initiative on the part of both federal and private organizations. Franolic and coworkers [113] describe the utilization of PLC (stationary phase — silica gel and mobile phase — dichloromethane-acetonitrile-acetone (4 1 1, v/v)) for the isolation and characterization of impurities in hydrochlorothiazide (diuretic drug). This drug is utilized individually or in combination with other dmgs for the treatment of hypertension. The unknown impurity band was scraped off the plate and extracted in acetonitrile. The solution was filtered and used for LC/MS and NMR analysis. The proposed procedure enabled the identification of a new, previonsly nnknown impurity. It was characterized as a 2 1 hydrochlorothiazide-formaldehyde adduct of the parent drug substance. 

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