LC/MS techniques

LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

Each screening center has medicinal and synthetic chemistry expertise in order to optimize hits identified from HTS campaigns and develop them into chemical probes. Specific capabilities vary, however typical strategies employed include parallel synthesis, computational and informatics analysis, and analytical capabilities such as LC/MS techniques. The structures of novel compounds that are prepared, their synthetic protocols, analytical data and biological data are all available, and samples of final probes developed are deposited into the MLSMR. A Working Group comprised of chemists from each center meets regularly to share information, best practices, and insure optimal use of resources. 

LC-MS at present is only complementary to GC-MS. The LC-MS technique is applied for confirmations of positive results obtained by different immunochemical methods and for confirmation of the intake of drag(s). Nevertheless, the number of studies in which procedures for screening a single group of drugs, for example, benzodiazepines, amphetamines, fentanyl analogs (Fig. 16.1), and pesticides, or a subject... 

Depending on the toxicological or criminalistic problems to be solved and/or the availability of the sample, different matrices must be analyzed by using GC-MS or LC-MS techniques. 

Applications of GC-MS or LC-MS techniques to forensic toxicology considerably reduced the volume of specimens necessary for analytical procedure. Recently, the volumes range from 0.2 to 1.0 mL. 

J.D. Henion, A comparison of direct liquid introduction LC/MS techniques employing microbore and conventional packed columns, J. Chromatogr. Sci., 18 (1980) 101-115. 

The present chapter discusses the results of a series of three small-scale interlaboratory exercises performed within the framework of the European Union-funded PRISTINE project (ENV4-CT97-0494), employing HPLC-fluorescence (FL) and LC-MS techniques for the determination of ionic and non-ionic surfactants. 

Hunt et al.150 proposed the incorporation of LC-MS techniques to eliminate other identity assays required in the QC lab. A LCQ ion trap mass spectrometer was added downstream of the UV spectrometer. In addition to a UV trace, a total ion current chromatogram was generated. Comparison to a reference chromatogram was performed to define identity without extensive interpretation of the mass spectral data. This LC-MS method eliminated the need for other identity assays such as N-terminal sequencing and was validated as a release assay for three proteins.150... 

As synthetic steps, the Michael additions of nitrogen nucleophiles were followed by nucleophilic substitutions of the chlorine atom with a primary amine and, finally, alkylations of the then secondary amino group with various alkyl bromides were performed just as previously developed for the chloro ester 1-Me in solution (see, e.g. Schemes 25,27,36 etc.). With differently substituted pyra-zoles as Michael addends, different primary amines and alkyl bromides, combinatorial libraries consisting of 8, 24 and 84 compounds were thus successfully prepared in ca. 60% yield and proved by the LC-MS technique to contain all the individual compounds in about equal amounts (Scheme 80) [127]. 

J. Yinon, L.D. Betowski, R.D. Voyksner, LC-MS techniques for the analysis of dyes. In Application ofLC-MS in Environmental Chemistry, D. Barcelo (Ed.), Elsevier, Amsterdam, the Netherlands, 1996, pp. 187-218. 

In atmospheric pressure chemical ionization (APCI) a similar interface to that used for ESI is used. A corona discharge is used to ionize the analyte in the atmospheric pressure region. The gas-phase ionization in APCI is more effective than ESI for analyzing less polar species. Both ESI and APCI are complementary methods that are well-suited for LC/MS techniques. 

The power of GC-MSand LC-MS techniques lies in their potential ability to take a mixture of many different compounds and provide structural information on each component. There is some overlap in the types of compounds that may be analysed using these two techniques, but, in the main, the role of both techniques may be considered to be complementary. In which circumstances then do we use GC-MS and in which do we use LC-MS As a general rule, if the analyte is non-polar and therefore volatile, we would probably use GC-MS. On the other hand, if the analyte is polar and/or non-volatile, the most appropriate technique will probably be LC-MS. 

FAB ionization has been used in combination with LC/MS in a technique called continuous-flow FAB LC/MS (Schmitz et al., 1992 van Breemen et al., 1993). Although any standard HPLC solvent can be used, including methyl-ferf-butyl ether and methanol, the mobile phase should not contain nonvolatile additives such as phosphate or Tris buffers. Volatile buffers such as ammonium acetate are compatible at low concentrations (i.e., <10 mM). Continuous-flow FAB has also been used in combination with MS/MS (van Breemen et al., 1993). The main limitationsof continuous-flow FAB compared to other LC/MS techniques for carotenoids, such as ESI and APCI, are the low flow rates and the high maintenance requirements. During use, the 3-nitrobenzyl alcohol matrix polymerizes on the continuous-flow probe tip causing loss of sample signal. As a result, the continuous-flow probe must be removed and cleaned approximately every 3 hr. 

M Hadj-Mahammed, BY Meklati, Qualitative determination of polymethoxylated flavones in Valencia orange peel oil and juice by LC-UV/Vis and LC-MS techniques. Lebensm Wiss Technol 20 111-114, 1987. 

Thulin, C. D., and Walsh, K. A. (1995). Identification of the amino terminus of human filaggrin using differential LC/MS techniques Implications for profilaggrin processing. Biochemistry 34, 8687-8692. 

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