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Affinity Chromatography As a Method for Sample Preparation in Gas Chromatographyrmass Spectrometry J. Biochem. Biophys. Methods 49Ž. 2001 705–731 www.elsevier.comrlocaterjbbm Review Affinity chromatography as a method for sample preparation in gas chromatographyrmass spectrometry Dimitrios Tsikas) Institute of Clinical Pharmacology, HannoÕer Medical School, Carl-Neuberg-Strasse-1 30625 HannoÕer, Germany Abstract Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometryŽ. MS technology, e.g., GCrMS and LCrMS and their variants, GCrtandem MS and LCrtandem MS, are best suited both for qualitative and quantitative analyses. GCrMS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GCrMS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentra- tions in complex matrices require further chromatographic techniques, such as high-performance liquid chromatographyŽ. HPLC and thin-layer chromatography Ž. TLC for sample purification. In AbbreÕiations: API, atmospheric pressure ionization; BAC, boronate affinity chromatography; CAD, collision-activated dissociation; CID, collision-induced dissociation; CrIRMS, combustionrisotope ratio mass spectrometry; EI, electron impact; ESI, electrospray ionization; FABrMS, fast atom bombardmentrmass spectrometry; GC, gas chromatography; GCrECD, gas chromatographyrelectron capture detection; GCrMS, gas chromatographyrmass spectrometry; GCrtandem MS, gas chromatographyrtandem mass spectrometry; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; IAC, im- munoaffinity chromatography; IDrMS, isotope dilutionrmass spectrometry; LC, liquid chromatography; LCrMS, liquid chromatographyrmass spectrometry; LCrtandem MS, liquid chromatographyrtandem mass spectrometry; MS, mass spectrometry; mr z, mass-to-charge ratio; NE, norepinephrine; NICI, negative-ion chemical ionization; NO, nitric oxide; 3-NT, 3-nitrotyrosine; 3-NT-ALB, 3-nitrotyrosine-albumin; ODS, octadecylsilica; PAC, protein affinity chromatography; PBA, phenylboronic acid; PFB, pentafluorobenzyl; PG, prostaglandin; SIM, selected-ion monitoring; SNALB, S-nitrosoalbumin; SPE, solid-phase extraction; SRM, selected-reaction monitoring; SSQ, singe-stage quadrupole; TLC, thin-layer chromatography; TSQ, triple-stage quadrupole; Tx, thromboxane. ) Tel.: q49-511-532-3959; fax: q49-511-532-2750. E-mail address: [email protected]Ž. D. Tsikas . 0165-022Xr01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved. PII: S0165-022XŽ. 01 00230-5 706 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 recent years, affinity chromatographyŽ. e.g., boronate and immunoaffinity chromatography has been developed to a superior technique for sample preparation of numerous classes of compounds in GCrMS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GCrMS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Gas chromatographyrmass spectrometry; Affinity chromatography; Phenyl boronic acid; Arachi- donic acid derivates; Nitrosylated and nitrated proteins; Drugs 1. Introduction Initially, mass spectrometryŽ. MS and gas chromatography Ž. GC and, later, liquid chromatographyŽ. LC have developed independently. In the last two decades, the desire to combine high resolution, provided by high-performance liquid chromatography Ž.HPLC and, in particular, by capillary GC, with the exquisite accuracy and sensitivity provided by MS has led to the development of the most efficient analytical technologies presently available, i.e., LCrMS and GCrMS and variations of them, e.g., LCrtandem MS and GCrtandem MS. LCrMS is largely used to analyze polar, thermally labile and high-molecular-mass compounds, such as peptides and proteins. On the other hand, GCrMS is preferably used to analyze low-molecular-mass compounds. As a rule, these compounds are in the majority polar, and their analysis by GCrMS requires chemical conversion, i.e., derivatization of the compounds into nonpolar, volatile and thermally stable derivatives amenable to GC analysis. For LCrMS, and in particular for GCrMS, isolation of an analyte of interest from its matrix is an absolute requirement. This procedure aims at isolating the compound from the matrix, e.g., by extraction, for the purpose of injection andror derivatization, and at removing other compounds which may interfere with the subsequent analysis. Therefore, the efficiencyŽ. e.g., selectivity of the extraction step, which is usually the first step in all analytical methods, may decisively determine the quality of the total analysis. For instance, simple solvent extraction or nonspecific solid-phase extractionŽ. SPE of an analyte on straight or reversed-phase materials, such as octadecylsilicaŽ. ODS , entails further time-consuming chromatographic steps, such as HPLC andror thin-layer chro- matographyŽ. TLC , andror more sophisticated and expensive techniques, such as tandem MS. For the last two decades, intensive research in the area of SPE has led to the development of novel SPE materials, suitable for selective extraction of analytes from various matrices. One possibility of gaining selectivity is to increase the affinity of a certain compound or a class of compounds for a moiety appropriately immobilized on a support. This has led to the development of affinity chromatography. In classical affinity chromatography, the molecule to be isolated is specifically and reversibly adsorbed on a complementary binding substanceŽ. ligand , covalently attached to an insoluble support, from which the substance of interest can be recovered specifically. Today, affinity chromatography provides a unique and powerful role in separation technology as the only technique which enables purification of an analyte on the basis of biological Table 1 Affinity chromatography as a method for sample preparation in gas chromatographyrmass spectrometry Compoundrclass Matrix Sample preparation Mass spectrometryrionization References ()A Protein affinity chromatography D. Tsikas S-nitrosoalbumin plasma Affinity chromatography GCrMSrNICIwx 2,3 3-Nitrotyrosine-albumin plasmaŽ. HiTrapBlue Sepharose GCrtandem MSwx this work r ()B Boronate affinity chromatography J. Biochem. Biophys. Methods 49 2001 705–731 r r r wx TxB2 and metabolites urine PBA; ODS; TLC GC MS; GC tandem MS NICI 14–18 Amino acid derivatives plasma PBA; Matrex Gel PBA-60 GCrtandem MSrNICI; FABrMSwx 19,20 Benzowxa pyrene derivatives urine PBA GCrMSrNICIwx 21 Dihydroxy compounds plasma; urine PBA; solvent extraction GCrMSrNICI; GCrECDwx 22–25 Neutral glycolipids cells PBA; TLC FABrMSwx 26 ()C Immunoaffinity chromatography Arachidonic acid metabolites urine, plasma IAC; ODS GCrMS; GCrtandem MSwx 27–32 GCrHRMSrNICIrEIwx 33–43 Steroids urine, bile IAC; ODS; HPLC GCrHRMSwx 44–49 muscle GCrMSrNICI; GCrCrIRMSwx 50,51 () DNA adducts DNA, urine IAC GCrMS; GCrHRMSrNICIwx 52–57 LCrtandem MSrESIwx 58–62 Drugs urine, faeces IAC, multi-IAC GCrMSrEIrNICIwx 63–68 SPE, HPLC GCrtandem MSrNICIwx 69–72 LCrMSrESI Miscellaneous urine, blood IAC; ODS; HPLC GCrMSrNICIwx 73–78 brain, cells solvent extraction LCrMSwx 79–84 food wx85–93 707 708 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 function or individual chemical structure. High selectivity and high capacity make this technique ideally suited for the isolation of a specific compound from complex biological matrices. Purification of enzymes is the oldest and best-known application of affinity chro- matography; however, protein affinity chromatographyŽ. PAC is not the subject of the present article. Based on the fact that serum or plasma albumin has the unique ability to bind dyes very tightly, Sepharose-dye conjugates have been developed for the selective adsorption of albumin in plasmawx 1 . Since other serum or plasma proteins do not significantly bind to Sepharose-dye conjugates, affinity chromatography of albumin has been initially used to remove albumin selectively from plasma, i.e., to obtain starting material for the isolation of other minor plasma proteinswx 1 . The recognition that cysteine-34-nitrŽ. osyl ated albumin, i.e., S-nitrosoalbumin Ž SNALB . and S-nitroalbumin, and tyrosine-nitrated albumin, i.e., 3-nitrotyrosine-albuminŽ. 3-NT-ALB , and other proteins endogenously occur in the human organism has extended the field of applica- tion of affinity chromatography to these modified albumin moleculeswx 2,3 . Another affinity-chromatographic technology is based on the use of chemically immobilized antibodies against the compounds to be extracted from a biological fluid. For instance, this technique, named immunoaffinity chromatographyŽ. IAC , has been used to selec- tively extract various arachidonic acid derivates from biological fluids prior to GCrMS analysiswx 4 . A third kind of affinity chromatography is based on the use of phenyl- boronic acidŽ. PBA , immobilized on stationary support phases. The underlying mecha- nism
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