Ionization, sample, tandem mass spectrometry

Recently, Blount et al. (2000) summarized a methodology to detect di- -butyl phthalate metabolites in urine. In humans or animals, di- -butyl phthalate is metabolized to mono- -butyl phthalate and oxidative products, which are excreted through the urine and feces. Human urine samples are processed by P-glucuronidase hydrolysis (to release the mono phthalate ester) followed by solid-phase extraction. The eluate is concentrated mono- -butyl phthalate is chromatographically resolved by reverse-phase HPLC, detected by negative ion atmospheric pressure chemical ionization (APCI) tandem mass spectrometry, and quantified by isotope dilution. 

Hsieh et al. (2006a) demonstrated the use of packed-column supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization (APCI)—tandem mass spectrometry (MS/MS) for the analysis of metabolic stability samples. For the SFC step, the mobile phase is liquid carbon dioxide with some organic modifiers added to adjust the retention time of the analytes. In this report, the primary organic modifier was methanol, and the authors described how the percentage of methanol in the mobile phase varied the retention of the analytes and affected the relative response of the test compounds in the APCI source. The authors also demonstrated that the results obtained by the SFC—APCI—MS/MS assay were equivalent to those obtained by a conventional HPLC—APCI—MS/MS assay, as shown in Figure 12.3. 

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

Specifically for triazines in water, multi-residue methods incorporating SPE and LC/MS/MS will soon be available that are capable of measuring numerous parent compounds and all their relevant degradates (including the hydroxytriazines) in one analysis. Continued increases in liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) sensitivity will lead to methods requiring no aqueous sample preparation at all, and portions of water samples will be injected directly into the LC column. The use of SPE and GC or LC coupled with MS and MS/MS systems will also be applied routinely to the analysis of more complex sample matrices such as soil and crop and animal tissues. However, the analyte(s) must first be removed from the sample matrix, and additional research is needed to develop more efficient extraction procedures. Increased selectivity during extraction also simplifies the sample purification requirements prior to injection. Certainly, miniaturization of all aspects of the analysis (sample extraction, purification, and instrumentation) will continue, and some of this may involve SEE, subcritical and microwave extraction, sonication, others or even combinations of these techniques for the initial isolation of the analyte(s) from the bulk of the sample matrix. 

More recently, liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) have been evaluated as possible alternative methods for carfentrazone-ethyl compounds in crop matrices. The LC/MS methods allow the chemical derivatization step for the acid metabolites to be avoided, reducing the analysis time. These new methods provide excellent sensitivity and method recovery for carfentrazone-ethyl. However, the final sample extracts, after being cleaned up extensively using three SPE cartridges, still exhibited ionization suppression due to the matrix background for the acid metabolites. Acceptable method recoveries (70-120%) of carfentrazone-ethyl metabolites have not yet been obtained. 

The above-mentioned method is effective in identifying the molecules of detected ions. However, because PVDF film is not permeable to light, it is difficult to observe tissue sections. To resolve this problem, we developed a method to fix tissue sections on transparent film, and then performed MS on those sections.6 We used a conductive film because we expected the ionization efficiency would increase when the electric charge accumulation on the sample was reduced. The film used for this purpose was a polyethylene terephthalate (PET) film with a thickness of 75-125 pm, having a 5 15-nm-thick layer of evaporated oxidation indium tin (ITO) upon it (ITO film). This film is used in touch-panel displays because of its high transparency and superior conductivity. We used it to perform MS/MS for tissue sections and succeeded in identifying multiple proteins from mass spectra.6 Therefore, the further development of this method will enable the application of the mass-microscopic method to observe tissue by optical microscope and to perform tandem mass spectrometry (MSn) at the observation part, simultaneously, enabling the identification of molecules included the part. 

To record a mass spectrum it is necessary to introduce a sample into the ion source of a mass spectrometer, to ionize sample molecules (to obtain positive or negative ions), to separate these ions according to their mass-to-charge ratio (m/z) and to record the quantity of ions of each m/z. A computer controls all the operations and helps to process the data. It makes it possible to get any format of a spectrum, to achieve subtraction or averaging of spectra, and to carry out a library search using spectral libraries. A principal scheme of a mass spectrometer is represented in Fig. 5.2. To resolve more complex tasks (e.g., direct analysis of a mixture) tandem mass spectrometry (see below and Chapter 3) may be applied. 

McClean S, O Kane EJ, Smyth WR 2000. Electrospray ionization-mass spectrometric characterization of selected antipsychotic drugs and their detection and determination in human hair samples by liquid chromatography-tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 740 141. 

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. 

Miao X. and C.D. Metcalfe (2003). Determination of pharmaceuticals in aqueous samples using positive and negative voltage switching microhore liquid chromatography/electrospray ionization tandem mass spectrometry. Journal of Mass Spectrometry 38 27-34. 

Volmer D.A., B. Mansoori, and S.J. Locke (1997). Study of 4-quinolone antibiotics in biological samples by short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Analytical Chemistry 69 4143 155. 

A newer approach for lipid analysis is electrospray ionization tandem mass spectrometry (ESI-MS/MS) (Welti et al., 2002). This method requires limited sample preparation and sample size to identify and quantify lipids. Fauconnier et al. (2003) used ESI-MS/MS to analyze phospholipid and galactolipid levels during aging of potato tubers. 

In the absence of tandem mass spectrometry equipment, almost equally reliable estimations of the PA concentrations can be made using gas chromatography-mass spectrometry (GC-MS). A standard quadrupole instrument, such as the one used for organic acid analysis, will be sufficient. Depending on the derivative, a choice between positive and negative ionization will have to be made. In general, a more extensive prepurification of the biological samples, will have to be realized. 

Gu, Z.-M., Zhou, D., Lewis, N.J., Wu, J., Johnson, H. A., McLaughlin, J.L., and Gordon, J. Quantitative evaluation of Annonaceous acetogenins in monthly samples of paw paw (Asimina triloba) twigs by liquid chromatography/electrospray ionization/tandem mass spectrometry. Phytochem. Anal., 10, 32, 1999. 

FAB and LSIMS are matrix-mediated desorption techniques that use energetic particle bombardment to simultaneously ionize samples like carotenoids and transfer them to the gas phase for mass spectrometric analysis. Molecular ions and/or protonated molecules are usually abundant and fragmentation is minimal. Tandem mass spectrometry with collision-induced dissociation (CID) may be used to produce abundant structurally significant fragment ions from molecular ion precursors (formed using FAB or any suitable ionization technique) for additional characterization and identification of chlorophylls and their derivatives. Continuous-flow FAB/LSIMS may be interfaced to an HPLC system for high-throughput flow-injection analysis or on-line LC/MS. 

A selective, sensitive, and rapid hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of donepezil in human plasma [32], Donepezil was twice extracted from human plasma using methyl-ferf-butyl ether at basic pH. The analytes were separated on an Atlantis HILIC Silica column with the mobile phase of acetonitrile ammonium formate (50 mM, pH 4.0) (85 15, v/v) and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r = 0.9994) over the concentration range of 0.10-50.0 ng/ ml and the lower limit of quantification was 0.1 ng/ml using 200 /d plasma sample. The CV and relative error for intra- and inter-assay at four quality control levels were 2.7% to 10.5% and —10.0% to 0.0%, respectively. There was no matrix effect for donepezil and cisapride. The present method was successfully applied to the pharmacokinetic study of donepezil after oral dose of donepezil hydrochloride (10 mg tablet) to male healthy volunteers. 

Chan, J.H., Timperman, A.T., Qin, D., Aebersold, R., Microfabricated polymer devices for automated sample delivery of peptides for analysis by electrospray ionization tandem mass spectrometry. Anal. Chem. 1999, 71, 4437 1444. 

S.-A.A. Fredriksson, L.-G. Hammarstrom, L. Henriksson and H.-A.A. Lakso, Trace determination of alkyl methylphosphonic acids in environmental and biological samples using gas chromatography/negative-ion chemical ionization mass spectrometry and tandem mass spectrometry, J. Mass Spectrom., 30, 1133-1143 (1995). 

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