Affinity Chromatography As a Method for Sample Preparation in Gas Chromatographyrmass Spectrometry

Home , Affinity chromatography

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 concentrations 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, immunoaffinity 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 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 707 85–93 wx wx wx wx wx wx wx wx wx wx wx wx wx wx wx wx wx wx MS 19,20 r NICI 14–18 r IRMS 50,51 NICI 52–57 r r C ECD 22–25 EI 33–43 ionization References r r r r NICI 69–72 NICI; FAB ESI 58–62 r r r HRMS tandem MS tandem MS 27–32 NICI 63–68 NICI r r r r r NICI 73–78 EI NICI; GC NICI 2,3 NICI 21 NICI; GC ESI r r r r r r r MS 26 MS tandem MS MS HRMSMS MS; GC 44–49 HRMS MS tandem MSMS; GC this work tandem MS MS MS MS; GC MS 79–84 MS tandem MS r r r r r r r r r r r r r r r r r r LC LC GC mass spectrometry r SPE, HPLC GC Ž. brain, cellsfood solvent extraction LC muscle GC class Matrix Sample preparation Mass spectrometry r pyrene derivatives urine PBA GC a wx 2 -nitrosoalbumin plasma Affinity chromatography GC B Boronate affinity chromatography A Protein affinity chromatography C Immunoaffinity chromatography Miscellaneous urine, blood IAC; ODS; HPLC GC Drugs urine, faeces IAC, multi-IAC GC SteroidsDNA adducts urine, bile DNA, urine IAC IAC; ODS; HPLC GC GC S 3-Nitrotyrosine-albumin() TxB and metabolites plasma urine HiTrapBlue Sepharose GC PBA; ODS; TLC GC Table 1 Affinity chromatography as a method forCompound sample preparation in gas chromatography () Amino acid derivativesBenzo plasma PBA; Matrex Gel PBA-60 GC Dihydroxy compounds() Arachidonic acid metabolites plasma; urine urine, plasma PBA; solvent extraction IAC; ODS GC GC Neutral glycolipids cells PBA; TLC FAB 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 chromatography; 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 application 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 selectively 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 phenylboronic acidŽ. PBA , immobilized on stationary support phases. The underlying mecha- nism of this technology, named boronate affinity chromatographyŽ. BAC , involves the chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable complex, from which the compounds can be recovered specifically, e.g., by alkaliwx 4 . BAC has been widely used for the selective extraction of numerous dihydroxylated low-molecular-mass and high-molecular-mass compounds, including glycated albumin and hemoglobinwx 5,6 . In recent years, affinity chromatography has been subject of reviews with reference to various aspects, including IAC and BACwx 7–13 . By now, affinity chromatography is a very useful analytical tool for the selective extraction of individual substances and numerous classes of natural and synthetic compounds for further analysis by various analytical techniques. The present paper is concerned with a special aspect of affinity chromatography, i.e., its use as an analytical procedure for sample preparation in those methods that are based on mass spectrometry, in particular on GCrMS and LCrMS. Advances in affinity chromatography and mass spectrometry have enabled the development of the most specific and sensitive analytical methods available for the most accurate quantitative determination of a variety of compounds, including drugs and their metabolites, in complex biological samples by the least labor-intensive approach. Recent methodological applications of affinity chromatography to the quantitative GCrMS of various classes of compounds are discussed in this articleŽ. Table 1 .

2. Quantitative determination by GCrMS and GCrtandem MS In general, methods involving quantitative GCrMS or GCrtandem MS, e.g., isotope dilutionrmass spectrometryŽ. IDrMS , consist of several analytical steps. These steps D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 709 include addition of the internal standard, usually a stable isotope-labelled analog of the analyte of interest, isolation of the compounds by SPE, at least one derivatization step, and GC separation followed by on-line MS analysis of the GC peaks of the analytes and the respective internal standardsŽ. Fig. 1 . When IAC is used, the SPE step may be omitted. HPLC or TLC may be required when affinity chromatography is not feasible. Quantitative determination with GCrMS instrumentsŽ e.g., with single-stage quadrupole Ž.SSQ instruments . is usually performed in the selected-ion monitoring Ž. SIM mode, because this scanning technique provides the most sensitive detection. The mass spectrometer is set to pass alternately two specific- and, as a rule, intense-ions with mass-to-charge ratios Ž.mrz corresponding to the same ion of the analyte and the internal standard, respectivelyŽ. Fig. 1 . Quantitative determination with GCrtandem MS instruments, e.g., with triple-stage quadrupoleŽ. TSQ mass spectrometers, is regularly

Fig. 1. Schematic drawing of techniques in quantitative analysis with single-stage quadrupoleŽ. SSQ and triple-stage quadrupoleŽ. TSQ instruments. Ž. A Quantitative analysis by selected-ion monitoring Ž. SIM of two ions specific for an endogenous analyte and for its stable isotope-labeled analog.Ž. B Quantitative analysis by selected-reaction monitoringŽ. SRM of two specific product ions generated by CID of the corresponding parent ions. One pair of ions is chosen for the endogenous compound, the corresponding second pair of ions is chosen for the internal standard. Q, quadrupole. 710 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 performed in the selected-reaction monitoringŽ. SRM mode. In TSQ mass spectrometers, the first quadrupole alternately selects two specific ions, the parent ions, which are subjected in the second quadrupole of the instrument to collision-induced dissociation Ž.CID or collision-activated dissociation Ž CAD . to product ions by means of a collision gas, such as argon. The third quadrupole is set to pass concomitantly two specific product ions with mrz corresponding to the same ion of the analyte and the internal standard, respectivelyŽ. Fig. 1 . Quantitative determination by GCrtandem MS is more accurate than by GCrMS due to the performance of the second mass spectrometric separation. In GCrMS, electrically uncharged gaseous molecules, coming directly from the GC column, are ionized in the ion source of the instrument, either by negative-ion chemical ionizationŽ. NICI or by electron-impact ionization Ž. EI , with NICI providing the highest sensitivity for most analytes. In LCrMS, ionization of the compounds present in HPLC effluents or other liquid phases is usually performed by electrospray ionizationŽ. ESI or atmospheric-pressure ionization Ž. API .

3. Methodological applications

3.1. Protein affinity chromatography

3.1.1. Analysis of S-nitrosoalbumin and 3-nitrotyrosine-albumin Nitric oxideŽ. NO and reactive NO derivatives, such as peroxynitrite, react with various amino acids and amino acid residues in peptides, proteins and enzymes, cysteine and tyrosine being the most reactive moieties, to form nitrosylated and nitrated derivatives. These reactions change their original physicochemical properties and biological functions. S-nitroso and S-nitro compounds are vasodilators and inhibitors of platelet aggregation, SNALB being the most abundant circulating S-nitroso compound in humanswx 94 . The circulating free amino acid 3-nitrotyrosineŽ. 3-NT and 3-nitrotyrosine residues in proteins are putative indicators of nitrosative stress in vivowx 95 . Because of this potential significance, circulating S-nitrŽ. osyl ated and tyrosine-nitrated proteins are currently subject of intense scientific research. At present, circulating S-nitrŽ. osyl ated and tyrosine-nitrated proteins are measured in toto. With very minor exceptionswx 96 , these compounds are not accessible to direct MS analysis. Their analysis by GCrMS requires their conversion to low-molecular-mass compounds. Thus, S-nitroso com- wx pounds are selectively converted by HgCl2 to nitrite 3 , and tyrosine-nitrated proteins are converted by acid-catalyzed hydrolysis or enzymic proteolysis to 3-nitrotyrosine wx97 . Both nitrite and 3-NT are readily accessible to GCrMS analysis w 3,97,98 x . Quantitative determination of S-nitroso compounds and 3-nitrotyrosine is associated with major methodological problems. Firstly, these compounds can be artifactually formed during sample treatment from ubiquitously occurring nitrite and nitratewx 98,99 . This problem can be overcome by working at neutral pH and separating tyrosine chromatographically before derivatizationwx 3,98,99 . Another major problem when measuring S-nitroso compounds and tyrosine-nitrated proteins in toto is the ignorance of the nature and concentration of the individual proteins present in the biological sample since D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 711 protein molecules regularly contain several cysteine and tyrosine residues. Thus, human albumin contains a single cysteine but 18 tyrosine molecules. While relatively pure SNALB can be readily synthesizedwx 3 , uncontrolled nitration of tyrosine residues of albumin and other proteins in vitro and in vivo leads to a mixture of not yet fully characterized 3-nitrotyrosine-albuminŽ. 3-NT-ALB and tyrosine-nitrated proteins, respectively. Thus, the number and the position of 3-nitrotyrosine in 3-NT-ALB and other proteins are still unknown. Synthesis of a pure, well-characterized 3-NT-ALB and other tyrosine-nitrated proteins has not yet been possible. These considerations have led to the opinion that quantitative analysis of specific S-nitrosylated and tyrosine-nitrated circulating proteins, such as SNALB and 3-NT-ALB, would offer a better analytical tool, yielding evident and reliably interpretable results, rather than quantitation in toto. In this area, affinity chromatography of native and modified albumin with the use of commercially available HiTrapBlue Sepharose affinity columns has enabled the highly selective and accurate quantitative determination of SNALB and 3-NT-ALB in human plasma wx2,3 . Human serum albumin, SNALB and 3-NT-ALB behave identically on HiTrapBlue Sepharose columns, while the majority of other serum and plasma proteins have no affinity to the column. Native plasmaŽ. up to 0.4-ml aliquots for 1-ml cartridges spiked with the stable isotope-labeled analogs of SNALB and 3-NT-ALB is simply diluted in 50 mM phosphate bufferŽ. pH 7 and applied to the columns preconditioned with the same buffer. From the column, albumin, SNALB and 3-NT-ALB are selectively eluted with the same buffer but of considerably higher ion strength, i.e., 1.5 M KCl, with a recovery of about 50%wx 3 . SDS-PAGE electrophoresis shows that 90% of the proteins present in the eluate is albuminŽ. Fig. 2 . By contrast, eluates from plasma extraction with common Sephadex PD-10 cartridges were found to contain up to 25% proteins distinct from albuminŽ. Fig. 2 . Five-milliliter HitrapBlue Sepharose cartridges can be used to prepare very pure15 N-labeled SNALBŽ S15 NALB. with high isotopic purity when using15 N-labeled butylnitritewx 2 . This synthetic route is superior to the use of 15 N-labeled nitrous acid because other nitrŽ. os ated albumin species are not formedwx 2 . Although HiTrapBlue Sepharose cartridges can be regenerated for reuse, they should be used once only for the measurement of endogenous SNALB and 3-NT-ALB. The GCrMS method for the quantitative determination of SNALB in human plasma is based on the conversion of the S-nitroso and S-15 N-labeled nitroso groups of SNALB 15 15 and S NALB into nitrite and N-labeled nitrite by HgCl2 , respectively, and their conversion to the pentafluorobenzylŽ. PFB derivativeswx 2,3 . Quantitation is performed by SIM of the ions with mrz of 46 for nitrite and mrz 47 for 15 N-labeled nitrite in the NICI mode. Typical GCrMS chromatograms from the quantitative analysis of SNALB in human plasma before and after addition of SNALB are shown in Fig. 3. Mean precision and accuracy of the GCrMS method within the range 0–10 mM are 95% and 99%, respectively. The limit of the quantitation of this method is 100 nM for SNALB and 0.2 nM for Sw15 Nx ALB. Since SNALB is finally detected as nitrite, the nitrite concentration in the eluate should be maintained as low as possible. Use of ammonium sulfamate to remove nitrite is one possibility to suppress interfering nitritewx 2 . At physiological concentrations, other plasma compounds such as S-nitrosoglutathione, S-nitrosocysteine, S-nitrosohemoglobin and 3-NT-ALB do not interfere with the mea- 712 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731

Fig. 2. SDS-PAGE electropherogram and gel scan chromatograms from the analysis of commercially available human serum albuminŽ. HSA; lane 1 , of freshly obtained human plasma without extraction Ž. lane 2 , and of Sw15 Nx ALB preparations after extraction by Sephadex PD-10Ž. lane 4 and HiTraBlue Sepharose cartridges Ž.lane 6 of reaction mixtures of fresh human plasma withw15 Nx nitrite in HCl acidic solutionŽ. pH 2 . Lanes 3 and 5 correspond to Sw14 Nx ALB preparations extracted by Sephadex PD-10 and HiTrapBlue Sepharose cartridges, respectively. With permission of Tsikas et al.wx 2 . D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 713

Fig. 3. Partial GCrMS chromatograms from analyses of SNALB in a 0.4-ml aliquot of a human plasma sample from a healthy volunteer, with S15 NALBŽ. 1 mM as internal standard without Ž. A and with addition of 1 mM SNALBŽ. B . Native samples were extracted with 1-ml HiTrapBlue Sepharose cartridges; eluates were r r treated with HgCl2 and subsequently with pentafluorobenzyl bromide. SIM of m z 46 for SNALB and m z 47 for15 NALB. With permission of Tsikas et al.wx 3 .

surement of SNALBwx 3 . This method has been applied to determine the SNALB levels in plasma of healthy and ill persons. In all subjects investigated, similar concentrations of SNALB were determined in plasma. In the plasma of 23 healthy humans, the mean SNALB concentration was determined as 181 nM. This level is about 40 times smaller than that previously obtained by another group by measurement of chemiluminescence wx94 . In the plasma of ill persons suffering from hepatic diseases Ž.ns40 or chronic renal failure Ž.ns6 , the mean SNALB concentration was determined as 161 and 147 nM, respectively. S-nitroso and S-nitro compounds can be distinguished by GCrMS wx100 . Thus, this method could be applied to determine simultaneously S-nitrosoalbumin and S-nitroalbumin. Tyrosine-nitrated albumin has the same affinity to HiTrapBlue Sepharose cartridges as albumin and SNALB. This offers the unique possibility to perform a single, highly selective extraction step both for SNALB and 3-NT-ALB prior to individual quantitation by GCrMS and GCrtandem MS, as described for the individual compoundswx 3,98 . w2 x Using 3-nitro- H3 tyrosine as an internal standard, which was added to the eluates of 714 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731

HiTrapBlue Sepharose affinity columns, we determined the concentration of 3-NT-ALB in the plasma of healthy people by GCrtandem MS, following proteolysis by the enzyme pronase and HPLC separation, as described previously for the free amino acid 3-nitrotyrosinewx 98 . Fig. 4 shows a characteristic chromatogram from such an analysis. Since the number of nitrated tyrosine residues in 3-NT-ALB is unknown, 3-nitrotyrosine levels are normalized to the tyrosine levels. In the plasma of six healthy persons, the mean ratio of 3-nitrotyrosine to tyrosine, both coming from albumin, was determined as approximately 1:106. Considering a dilution factor of 5 for plasma and a recovery of approximately 50% for affinity extraction and enzymic proteolysis each, the concentration of 3-NT-ALB in plasma of healthy humans is estimated to be about 20 nM. Free 3-nitrotyrosine at physiological concentrations was found not to interfere with the measurement of 3-NT-ALB. 3-Nitrotyrosine does not bind considerably to albumin nor does it interact with the HiTrapBlue Sepharose. Nitrite and nitrate also do not interfere with the measurement of 3-NT-ALB, since 3-NT-SNALB-derived 3-nitrotyrosine is completely separated from tyrosine by HPLC prior to derivatizationwx 98 .

Fig. 4. Partial chromatogram from the GCrtandem MS of 3-NT-ALB in a 0.4-ml aliquot of a human plasma w2 x sample from a healthy volunteer with 3-nitro- H3 tyrosine as an internal standard, which was added to the HiTrapBlue Sepharose column eluateŽ. 2 ml at 5 nM. SRM of mr z 379 Ž from mr z 396 . for endogenous r r w2 x 3-NT-ALBŽ. upper panel and of m z 382 Ž from m z 399 . for 3-nitro- H3 tyrosineŽ. lower panel . Endogenous 3-NT-ALB was proteolyzed to 3-nitrotyrosine by the enzyme pronase. Endogenous 3-nitrotyro- w2 xwx sine and 3-nitro- H3 tyrosine were further analyzed and derivatized as described 98 . D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 715

3.2. Boronate affinity chromatography

The principle, on which BAC is based on, is the ability of the tetrahedral anionic form of boronates to condense reversibly with 1,2- or 1,3-cis-diols to form five- or six-membered covalent complexes. When bonded-phase phenylboronic acid columns are used, compounds possessing 1,2- or 1,3-cis-dihydroxy groups, such as carbohydrates and catecholamines, capable of forming stable complexes, are selectively retained on the column from a biological matrix. Destruction of the complex, commonly performed with bases, yields the diols in the original form in a matrix poor in other compounds. As a rule, PBA columns are used only once for quantitative analyses. BAC has been widely used as a sample preparation step for numerous dihydroxy compounds, such as thromboxaneŽ. Tx B22 Ž TxB . and its major urinary metabolites, in various analytical techniques, including MSŽ. Table 1 . BAC in combination with GCrMS or GCrtandem MS has simplified, shortened, and improved the quantitative analysis of many compounds.

3.2.1. Thromboxane and metabolites Lawson et al.wx 18 have introduced BAC as a selective SPE step into the GCrMS analysis of TxB2 and its major urinary metabolites, 2,3-dinor-TxB2 Ž. Fig. 5 and 11-dehydro-TxB222 . The acyclic form of 11-dehydro-TxB and methoximated TxB but not native TxB22 and prostaglandinŽ. PG Fa , all 1,3-diols, were found to condense with the bonded-phase PBAŽ. Fig. 5wx 18 . This observation was explained as the result of the tendency of the planar phenyl groups to orient themselves so that their p orbitals align, thereby forcing the boronic acid groups too close together to admit sterically fixed cyclic

1,3-diols, such as nonmethoximated TxB22 and PGFa . The introduction of BAC and NICI permitted a more rapid, less labor-intensive and more specific analysis of

2,3-dinor-TxB22 and TxB in much smaller volumes of urineŽ. 5 vs. 300 ml than with previously used common SPE or solvent extraction and EI ionization. Nevertheless, the method by Lawson et al. still involves two TLC steps, which makes the whole method time-consuming. Later, Weber et al.wx 16 reported than one SPE step on ODS columns and a subsequent TLC step after SPE on PBA columns enable accurate quantitation of r urinary 2,3-dinor-TxB22 and TxB by simple GC MS. Omission of any chromatographic steps, with the sole exception of BAC, in the analysis of 2,3-dinor-TxB22 and TxB in smaller than 5-ml aliquots of urine was accomplished by GCrtandem MS in the NICI wx r mode 14 . A typical chromatogram from the GC tandem MS analysis of 2,3-dinor-TxB2 in a 3-ml aliquot of human urine after a single SPE step on bonded-phase PBA is shown in Fig. 6. The mean accuracy and precision of this method are 103% and 95%, respectivelywx 14 . Lorenz et al. have shown that a single SPE step on PBA columns suffices for the accurate quantification of 11-dehydro-TxB2 in human urine by GCrtandem MSwx 17 . By contrast, simple GCrMS has been shown to require a further

TLC step for sample purification in the analysis of urinary 11-dehydro-TxB2 in its wx open-ring form 15 . Methoximation of 2,3-dinor-TxB22 and TxB can be performed in urine buffered to pH 5wx 18 or pH 8.6 wx 16 , and in ODS extracts reconstituted in pyridine wx14 . The PBA column can be used either in the trigonal, neutral form wx 18 or in the wx tetrahedral anionic form 16 . Methoximated 2,3-dinor-TxB22 and TxB are eluted from 716 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 717

Fig. 6. Partial chromatograms from the analysis of 2,3-dinor-thromboxane B22Ž. 2,3-dinor-TxB in human urine by GCrtandem MS after a single boronate affinity chromatography for sample preparation. SRM of r r r r m z 240Ž. from m z 586 for endogenous 2,3-dinor-TxB2 Ž.Ž. upper panel and of m z 244 from m z 590 w2 x wx for the internal standard 2,3-dinor- H42 TxBŽ. lower panel . With permission of Tsikas et al. 14 . the PBA column by aqueous alkaline methanol. Combination of BAC with GCrMS or GCrtandem MS has found wide applicability in clinical studieswx 4 . The analytical r performance of the combination of BAC with GC tandem MS for TxB2 and its metabolites in biological fluids is comparable with that of IAC with GCrMSŽ see Section 3.3.1. .

3.2.2. Amino acid deriÕatiÕes Abnormal secretion andror metabolism of the catecholamine norepinephrineŽ. NE , an aromatic 1,2-diol, is important in the diagnosis of a number of disease states, in evaluating the etiology of neuroendocrinology disorders, as well as providing an index of overall activity of the sympathic nervous system. Monitoring plasma levels of NE allows an assessment of sympathic activity in vivo. Due to the low endogenous levels of circulating NE and the complex nature of the biological matrices, highly sensitive and selective methodologies are required for the accurate quantitative determination of NE in biological samples. Despite the use of BAC prior to GCrMS, accurate analysis of NE at

Fig. 5. Schematic drawing of the extraction procedure of methoximated 2,3-dinor-thromboxane B2 Ž 2,3-dinor- wx TxB2 . on bonded-phase phenylboronic acid. With permission of Tsikas et al. 14 . 718 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 low levels and in small plasma volumesŽ. 25–100 ml has been difficult Ž discussed in Ref.wx 19. . Combination of a semiautomated BAC with subsequent GCrtandem MS analysis in the NICI mode of the pentafluoropropionyl derivatives of NE enabled Kuhlenbeck et al.wx 19 to rapidly and accurately quantify NE in rat and dog plasma. NE was eluted from the PBA columns by using 1 M acetic acid in methanolŽ. 6:94, vrv. Dityrosine, an unusual amino acid, may be a useful marker for assessing oxidative damage to proteins. Studies involving oxidative stress may require quantitative determination of dityrosine. The utility of BAC for the isolation and analysis of dityrosine has been demonstratedwx 20 . Interaction of dityrosine with phenyl boronate has been shown to involve both specific association with the boronate moiety and hydrophobic binding to the phenyl group. This is a demonstration of an unusual interaction of a 1,4-diol with phenyl boronic acid to form seven-membered cyclic boronates. This specific interaction suggests that the phenolic groups of dityrosine, which stand in para-position, are oriented in the cis-position despite the voluminous amino acid moieties. BAC of dityrosine allows simple separation of dityrosine from tyrosine and other amino acids and should be a useful sample purification procedure in methods based on GCrMS, analogous to 3-nitrotyrosinewx 98 .

3.2.3. Miscellaneous BAC has been reported to be useful for the quantitative GCrMS analysis of carcinogenic tetrahydroxylated benzowxa pyrene derivatives w 21 x , various dihydroxy compounds such as mevalonic acidwx 22 , a cholestrol biosynthetic precursor, and aromatic 2-hydroxycarboxylic acidswx 25 , and neutral glycolipids wx 26Ž. Table 1 . In addition, PBA has been used in solution to selectively extract diols from biological samples into organic solvents as their cyclic boronates, with five- and six-membered rings. These diols include glycerol mononitrateswx 23 , which are denitrated metabolites of the drug glycerol trinitrate, and 3-chloropropanediolwx 24 .

3.3. Immunoaffinity chromatography

Bonded-phase PBA cartridges of consistent quality have been commercially available for several years. The applicability of BAC is simply limited to a minority of dihydroxy-group-possessing compounds. By contrast, immunoaffinity chromatography Ž.IAC is, in principle, applicable to every analyte for which antibodies raised against the analyteŽ. s can be prepared. This explains the wide applicability of IAC as an isolation and purification step in various analytical methods. Table 1 summarizes those publications that report the use of IAC as a sample preparation procedure in methods involving MS methodologies. Prominent classes of compounds being accessible to IAC combined with MS include arachidonic acid derivatives, steroids, DNA adducts, drugs, and toxins. Until recently, the major limitation to IAC has been the absence of commercial sources for immunoaffinity resins. In the 1990s, immunoaffinity proliferated and some firms have developed affinity resins which are now commercially available for some compounds and classes of compounds, the majority of them being drugs. However, so far, immunosorbents for only about 6% of the compounds discussed in this article are commercially available. The future viability of IAC is highly dependent on the interest D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 719 of manufacturers and on the involvement of the EC Community Bureau of Reference in preparing and supplying the necessary materialsŽ for discussion see Ref.wx 72. . In consideration of the high selectivity of IAC, commercialization of immunoaffinity resins for a larger number of analytes is still a task for industrial firms.

3.3.1. Arachidonic acid metabolites This class of physiological compounds is an example of analytes, the reliable determination of which is difficult in biological fluids and requires several purification steps and sophisticated methodologies. The most frequently used detection methods include immunoassays and GCrMSwx 4 . The major difficulty in quantifying arachidonic acid metabolites, such as prostaglandins, thromboxane, and their metabolites, is the occurrence of a high number of structurally related compounds in very low concentrations in urine and plasmawx 4 . There exist numerous commercially available immunoassays for a variety of arachidonic acid metabolitesŽ. the eicosanoids . They offer the advantage of speed and relatively low cost, but do not provide the specificity and quantitative accuracy of a well-designed assay based on GCrMS and, in particular, GCrtandem MSwx 4,31 . These considerations do not apply only to eicosanoids, but they can be in general extended to other physiological and nonphysiological compounds, such as the lysergideswx 63 . Analyte concentrations determined by immunoassays are gener- ally substantially higher than the concentrations determined by GCrMS or GCrtandem MSwx 4,63 . The specificity of immunoassays for many analytes has been improved by adding a preliminary HPLC step to the detection process. In methods based on GCrMS, HPLC andror TLC are usually required and used for sample purificationwx 4 . Alterna- tively, the specificity both of GCrMS methods and immunoassays can be improved by IAC. In GCrMS assays, use of IAC for sample purification makes superfluous other chromatographic procedures, such as TLC or HPLC, and enables reliable quantitation by simple GCrMS instead of the much more expensive GCrtandem MSwx 29,67 . Hubbard et al.wx 43 have reported for the first time the use of IAC for the quantitation r of TxB2 in human urine by GC MS. The efficiency of the combination of IAC with r GC MS for urinary TxB2 is comparable with that of the combination of BAC with GCrtandem MS, with the exception that the latter technique allows simultaneous wx quantitation of TxB22 and its major urinary metabolite, 2,3-dinor-TxB 14 . The same group has extended IAC to the quantitative determination by GCrMS of urinary and wx plasma 6-oxo-PGF1a 42 . These first promising results have prompted other eicosanoid research groups to develop IAC methods for the quantitation by GCrMS of other individual eicosanoids and groups of eicosanoids, as a rule, the primary eicosanoid together with its major urinary or circulating metabolitewx 31,32,34,36,39,40,41 . Recently, Chiabrando et al.wx 28,29 have extended IAC to the GCrMS quantitative determination of urinary isoprostanes, namely, of 8-iso-PGF2 a and its major urinary metabolites. In these methods, either the untreated biological sample or ODS extracts of the sample are applied to single-bed or mixed-bed columns, containing the immobilized antibodies against various eicosanoids. Elution is frequently performed by aqueous acetone. Immunoaffinity columns can be repeatedly used after appropriate washing. Quantitation is carried out most frequently by GCrMS after various derivatization steps, but without further chromatographic steps for sample purification. Whether quantitation 720 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 of immunoextracted eicosanoids by GCrtandem MS offers higher selectivity than GCrMS has not yet been determined. The SIM and SRM chromatograms usually contain two peaks, corresponding to the endogenous compound and its stable isotope- labeled analog, respectively, when a single-immunosorbent is used, unless the immobilized antibody shows cross-reactivity to other eicosanoidswx 29 . Immunoaffinity columns for 8-iso-PGF2 a are now commercially available. Fig. 7 shows a chromatogram from the r GC tandem MS analysis of urinary 8-iso-PGF2 a after a single direct IAC of a urine sample on a commercially available 8-iso-PGF2 a affinity column. It remains to be investigated whether combination of GCrtandem MS with a SPE step on ODS cartridges and a subsequent TLC step for the quantification of 8-iso-PGF2 a in human urinewx 101 reveals the same accuracy as a combination of GCrMS with IAC wx 29 . A special case of IAC has been presented by Hishinuma et al.wx 34 , who used a monoclonal antibody against cis-3-hexene-1-ol for the specific extraction of D17-6-oxo- r PGF1a from serum or urine and subsequent GC MS quantification. The antibody used

r Fig. 7. Partial chromatogram from the GC tandem MS analysis of urinary 8-iso-PGF2 a on a commercially w2 x available 8-iso-PGF2 a affinity column and 8-iso- H42 PGFa as internal standardŽ both from Cayman Chemical, Ann Arbor, MI, USA.Ž. . The centrifuged urine sample 5-ml aliquot , spiked with the internal standardŽ. 1 ngrml , was applied directly to the column Ž. 20 ml . Compounds were eluted with 95 vol.% ethanol, solvents were evaporated to dryness under a stream of nitrogen, and the pentafluorobenzyl ester trimethylsilyl ether derivatives were prepared. SRM of the product ions at mr z 299Ž. from mr z 569 for the endogenous compound and mr z 303Ž. from mr z 573 for the internal standard was performed as described wx101 . D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 721 recognizes the epitope characteristic for the v3-olefine structure, a partial structure of trienoic prostanoids. IAC has been used not only for GCrMS and GCrtandem MS, but also for quantitation of eicosanoids by immunoassayswx 31–33 ; in these studies, immunoassays have been validated by GCrMS.

3.3.2. Steroids Anabolic preparations, such as the androgenic 17b-19-nortestosterone, are attractive means of improving the growth rate and feed conversion of animals in livestock breeding. However, in all European Union countries, the use of growth promoters has been banned since 1988. Usually, not only urine, but also bile and muscle, is chosen as the matrix for screening for the presence of these compounds using GCrMS. For several corticosteroids, the European Union has imposed maximum residue levels in different matrices. Therefore, not only confirmation of the presence of anabolics, but also their quantitative determination in various matrices is required. In this area, IAC has been shown to be a simple, rapid and highly selective sample purification procedure prior to quantitation by HPLC and UV detection, immunoassays, and GCrMSŽ. Table 1 . The applicability and limitations of IAC in multiple-residue analysis of anabolizing and doping agents has been excellently reviewed in 1991 by van Ginkelwx 72 . At that time, van Ginkel concluded that the applicability of IAC has been limited, and he supposed that the availability of multiple-immunoaffinity chromatography materials will be the major factor determining its success or failure. In the 1990s, IAC proliferated further to steroids analysis, too. Thus, immunoaffinity materials are now available for a wider spectrum of analytes, but this spectrum is still narrow and, as a rule, immunoaffinity columns must still be laboratory-prepared for steroid analysis and also in other fields Ž.see Section 3.3.1 and below .

3.3.3. DNA adducts Interaction of alkylating agents with DNA yields a variety of reaction products. The predominant alkylation sites have been identified in the nucleophilic N-7 position of guanine and, to a lesser extent, at the O-6 and O-4 atoms of guanine and thymine, respectively. O-6-alkylguanine and O-4-alkylthymine lead to point mutation in DNA after replication. The detection of modified bases is hindered mainly by the analytical obstacle involved in measuring very low concentrations, for instance one adduct in 107 –109 parent bases. Besides immunoassays, GCrMS is frequently used to detect and measure DNA adducts in biological samples, but this technique requires extensive sample purification, analogous to that for other classes of analytes discussed in this article. In the last decade, IAC of DNA adducts has been shown to make the clean up of samples efficient and simple, and to allow highly selective and sensitive quantitation by GCrMS in the NICI mode, in analogy to other compoundsŽ. Table 1 . However, this similarity implies also analogous difficulties, especially in terms of commercial availability of prepared immunosorbents. A further difficulty is the growing number of DNA adducts, the structure of which must first be elucidated. At present, the following DNA adducts have been analyzed by a combination of IAC with GCrMS: O-6-butylguanine and other O-6-alkylguanineswx 56,57,62 , 3-methyladenine wx 59–61 and other 3-al- 722 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 kyladenineswx 58 , and ethenoadenine and ethenoguanine w 53,55 x . For a brief overview, see the article by Shuker and Bakerwx 58 .

3.3.4. Drugs Combination of IAC with MS has also been applied to the analysis of drugs. However, considering the very large number of drugs, the number of applications is rather smallŽ. Table 1 . IAC has found wide applicability for the GCrMS analysis of anabolizing and doping drugswxwx 64,65,68,69,72 and lysergide 63,70 . The use of IAC in drug analysis has been reviewedwx 63,72 . The majority of these methods do not concern therapeutically administered drugs, but the illegal use of banned drugs, such as anabolizing and doping drugs in sports and livestock production. This may explain the limited application of IAC to drug analysis. The major problems with drug residue analysis is the possibility of obtaining false-positive results. European Union guidelines are mainly concerned with the identification criteria and general analytical quality controlŽ for a review, see Ref.wx 72. . Drug identification and quantitation is mainly based on MS and immunochemical procedures. The possibility of obtaining false-positive results limits the applicability of immunoassayswx 68,72 . How- ever, advances in IAC in recent years have improved both the immunoassays and those based on MSwx 66 . In drug analysis, IAC is superior to other purification techniques, such as HPLC, with respect to analytical parameters. In most cases, a combination of IAC with GCrMS has proved to be most useful. Analogous to other analytes, e.g., arachidonic acid metabolites, the use of a highly selective method of detection, such as tandem MS, with standard SPE procedures allows as reliable a drug analysis as the combination of IAC with MSwx 63 . For some drugs, such as the lysergides, on-line immunoaffinity extraction with LCrtandem MS is feasiblewx 63 . Nevertheless, only a limited few find applications outside the laboratory which originally developed them, because the suitability of a technique is in general not solely determined by its analytical specifications but also by nonanalytical parameters, such as costs and the availability of reagents and equipment. Reagents required for IAC of drugs are in most cases commercially available. However, immunoextraction columns prepared by commercial sources are still rare. As discussed above, for only about 6% of the compounds mentioned in this article are prepared immunoextraction columns commercially available, and 40% of them are for drugs, such as the lysergides and synthetic corticosteroids. An example for the use of commercially available immunoaffinity columns for the quantitative GCrMS determination of the corticosteroids dexamethasone and betamethasone in bovine urine is illustrated in Fig. 8. Fanelli and co-workers have used an immunoaffinity gel to prepare a cartridge, which was inserted into an automatic HPLC system for on-line extraction and purificationwx 82 . By injecting urine samples directly into the system, it was possible to collect a single fraction containing the analytes of interest. Of 65 bovine urine samples, which were found to be positive with a commercial corticosteroid ELISA kit, only 21 were confirmed to be positive for dexamethasone, with concentrations ranging from 0.12 to 4.5 ngrml; the limit of quantitation of the method was 0.1 ngrml for dexamethasone for a 4-ml aliquot of urine and injection of a 8-ml aliquot into a programmable-temperature vaporizer injector. In this system, the IAC used for purification has been reported not to show any decrease in performance after D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 723

Fig. 8. Partial GCrMS chromatograms from analyses of dexamethasone and betamethasoneŽ SIM of mr z 680 and 590,wx Mq andwx M-TMSOHq , respectively. in bovine urine with flumethasone as internal standard ŽSIM of mr z 698 and 608,wx Mq andwx M-TMSOHq , respectively. after enzymatic hydrolysis with b-glucuronidaserarylsulfatase, purification with immunoaffinity extraction on commercially available dexamethasone immunoaffinity columns, and derivatization to the tetra-trimethylsilyl derivatives. Left: blank urine, spiked with 5 ngrml of dexamethasone and betamethasone and 10 ngrml of flumethasone. Right: dexamethasone-positiveŽ. 0.19 ngrml urine sample from a bovine breeding farm in Northern Italy and spiked with 5 ngrml of flumethasone. With permission of Bagnati et al.wx 82 . repeated injections of urine samples; more than 100 injections were made without decrease in the recoverieswx 82 .

3.3.5. Miscellaneous This group includes, in general, toxic andror mutagenicrcarcinogenic compounds, such as heterocyclic aromatic amines, perchloroethene, aflatoxin B1, ochratoxin A and other mycotoxins, sarin hydrolysis products, and polycyclic aromatic hydrocarbons Ž.Table 1 . Without exception, IAC applied in these publications is based on the use of laboratory-prepared immunosorbent columns, which were obtained by immobilizing, as a rule, experimentally produced antibodies raised against the respective analytes. Retained analytes were then eluted by aqueous organic solvents, such as methanol and acetone, or aqueous buffered solutions, depending on the chemical structure of the analyte. One impressive example of the utility of IAC in combination with GCrMS was presented by a Japanese groupwx 78 . On March 20, 1995, a terrorist attack with sarin, i.e., 724 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731

Fig. 9. Total ion current chromatogramŽ. upper panel and EI mass spectrum Ž. lower panel of the peak indicated by an arrow in the chromatogram in the upper panel, generated from a formalin-fixed brain tissue of 50-year-old male station employee on the Tokyo subway, who died on arrival. The EI mass spectrum was generated after immunoaffinity extraction on AChE immunoaffinity column and derivatization to the trimethysilylŽ. TMS derivative, and agrees with the EI mass spectrum of the TMS derivative of authentic methylphosphonic acid. With permission of Matsuda et al.wx 78 . D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 725 isopropylmethylphosphonofluoridate, occurred on the Tokyo subway. The sarin was mixed with organic solvents and vaporized in a closed compartment of the train. Many people inhaled the sarin gas and collapsed. Eventually, 12 people died and over 5000 were injured. One of the hydrolysis products of sarin was detected in the formalin-fixed brain tissue of four poison victims. Sarin-bound acetylcholinesteraseŽ. AChE was solubilized from the cerebellums, purified by AChE-IAC, digested with trypsin, and methylphosphonic acid was identified by GCrMSŽ. Fig. 9 . The procedure described by this groupwx 78,79 should be useful for the forensic diagnosis not only of sarin poisoning, but also of intoxication with highly toxic, protein-bound agents, and underscores the usefulness of the combination of IAC with GCrMS in forensic medicine.

4. Conclusions and prospects

Analytical methods based on MS in combination with on-line chromatographic separation, either GC, i.e., GCrMS and GCrtandem MS, or by LC, i.e., LCrMS and LCrtandem MS, have been developed to provide highly efficient and reliable analytical techniques for an almost unlimited number of compounds. Despite the excellent analytical performances of analytical approaches based on MS, sample preparation, e.g., extraction andror isolation of the analyte of interest prior to the final mass-spectrometric analysis, is still indispensable for achieving unimpeachable results. Constant advances in various fields of affinity chromatography, such as PAC, BAC, and especially IAC, over the last decade make it apparent that affinity chromatography is the best partner of MS from the analytical point of view. Affinity chromatography is still growing and becoming accessible to a constantly increasing number of analysts all over the world. However, today as in the past, there is a major limitation to affinity chromatography, in particular for nonprotein compounds, that hinders a wider and more rapid expansion of affinity chromatography in analytical chemistry, namely the limited commercial availability of prepared immunosorbents and the high primary costs. The ratio of the costs for the purchase of ODS, PBA,

HiTrapBlue Sepharose, and immunoaffinity columns for the eicosanoids 8-iso-PGF2 a and PGE2 is currently about 1:1.6:4.6:7.4. Many of these columns, in particular immunoaffinity columns, can be regenerated and reused several times without the appearance of Amemory effectsB and loss of efficiency in terms of recovery, so that the cost ratio may be shifted clearly in favor of immunoaffinity. It may, therefore, be concluded that the future viability of immunoaffinity for the analysis of low-molecular- mass analytes by MS and other technologies is highly dependent on the interest of commercial firms. Finally, it is the responsibility of the research groups and analysts to take a decision in favor of analysis reliability by incorporating in their analytical methods sample preparation procedures based on affinity chromatography.

Acknowledgements The excellent laboratory assistance of M. Bohme,¨ I. Fuchs, F. Stutzer and M.T. Suchy is gratefully acknowledged. The author thanks F.-M. Gutzki for the performance 726 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 of GCrMS and GCrtandem MS analyses. This work was supported in parts by the Deutsche ForschungsgemeinschaftŽ. Grant TS 60r2-1 .

References

wx1 Travis J, Pannell R. Selective removal of albumin from plasma by affinity chromatography. Clin Chim Acta 1973;49:49–52. wx2 Tsikas D, Sandmann J, Rossa S, Gutzki F-M, Frolich¨ JC. Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry: I. Preparation, purification, isolation, characterization and metabolism of S-w15 Nx nitrosoalbumin in human blood in vitro. J Chromatogr, B: Biomed Sci Appl 1999; 726:1–12. wx3 Tsikas D, Sandmann J, Gutzki F-M, Stichtenoth D, Frolich¨ JC. Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry: II. Quantitative determination of S-nitrosoalbumin in human plasma using S-w15 Nx nitrosoalbumin as internal standard. J Chromatogr, B: Biomed Sci Appl 1999; 726:13–24. wx4 Tsikas D. Application of gas chromatography–mass spectrometry and gas chromatography–tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans. J Chromatogr, B: Biomed Sci Appl 1998;717:201–45. wx5 Singhal RP, DeSilva SS. Boronate affinity chromatography. Adv Chromatogr 1992;31:293–335. wx6 Liu XC, Scouten WH. Boronate affinity chromatography. Methods Mol Biol 2000;147:119–28. wx7 Cutler P. Affinity chromatography. Methods Mol Biol 1996;59:157–68. wx8 West I, Goldring O. Lectin affinity chromatography. Methods Mol Biol 1996;59:177–85. wx9 Wilchek M, Chaiken I. An overview of affinity chromatography. Methods Mol Biol 2000;147:1–6. wx10 Strandh M, Andersson HS, Ohlson S. Weak affinity chromatography. Methods Mol Biol 2000;147:7–23. wx11 Chockalingam PS, Jurado LA, Robinson FD, Jarrett HW. DNA affinity chromatography. Methods Mol Biol 2000;147:141–53. wx12 Minobe S, Shibatani T, Tosa T. Affinity chromatography of pyrogens. Methods Mol Biol 2000;147: 155–62. wx13 Gordon NF, Whitney DH, Londo TR, Nadler TK. Affinity perfusion chromatography. Methods Mol Biol 2000;147:175–93. wx14 Tsikas D, Gutzki F-M, Bohme¨¨ M, Fuchs I, Frolich JC. Solid- and liquid-phase extraction for the gas chromatographic–tandem mass spectrometric quantification of 2,3-dinor-thromboxane B2 and 2,3-dinor- 6-oxo-prostaglandin F1alpha in human urine. J Chromatogr, A 2000;885:351–9. wx15 Weber C, Beetens J, De Clerck F, Tegtmeier F. Gas chromatographic–mass spectrometric determination of 11-dehydrothromboxane B2 in human urine. J Chromatogr 1992;577:1–7. wx16 Weber C, Holler M, Beetens J, De Clerck F, Tegtmeier F. Determination of 6-keto-PGF1alpha, 2,3-dinor-6-keto-PGF1alpha, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2alpha in human urine by gas chromatography–negative ion chemical ionization mass spectrometry. J Chro- matogr 1991;562:599–611. wx17 Lorenz R, Helmer P, Uedelhoven W, Zimmer B, Weber PC. A new method using simple solid phase extraction for the rapid gas chromatographic mass-spectrometric determination of 11-dehydro-thromboxane B2 in urine. Prostaglandins 1989;38:157–70. wx18 Lawson JA, Brash AR, Doran J, FitzGerald GA. Measurement of urinary 2,3-dinor-thromboxane B2 and thromboxane B2 using bonded-phase phenylboronic acid columns and capillary gas chromatography– negative-ion chemical ionization mass spectrometry. Anal Biochem 1985;150:463–70. wx19 Kuhlenbeck DL, O’Neill TP, Mack CE, Hoke SH, Wehmeyer KR. Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization. J Chromatogr, B: Biomed Sci Appl 2000;738:319–30. wx20 Malencik DA, Sprouse JF, Swanson CA, Anderson SR. Dityrosine: preparation, isolation, and analysis. Anal Biochem 1996;242:202–13. D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 727 wx21 Simpson CD, Wu MT, Christiani DC, Santella RM, Carmella SG, Hecht SS. Determination of r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzowxa pyrene in human urine by gas chromatographyr negative ion chemical ionizationrmass spectrometry. Chem Res Toxicol 2000;13:271–80. wx22 Saisho Y, Kuroda T, Umeda T. A sensitive and selective method for the determination of mevalonic acid in dog plasma by gas chromatographyrnegative ion chemical ionization–mass spectrometry. J Pharm Biomed Anal 1997;9–10:1223–30. wx23 Scharpf F, Yeates RA, Laufen H, Eibel G. Gas chromatographic assay of glycerol mononitrates in biological samples. J Chromatogr 1987;413:91–9. wx24 Rodmann LE, Ross RD. Gas–liquid chromatography of 3-chloropropanediol. J Chromatogr 1986; 369:97–103. wx25 Higa S, Kishimoto S. Isolation of 2-hydroxycarboxylic acids with a boronate affinity gel. Anal Biochem 1986;154:71–4. wx26 Uda I, Sugai A, Kon K, Ando S, Itoh YH, Itoh T. Isolation and characterization of novel neutral glycolipids from Thermoplasma acidophilum. Biochim Biophys Acta 1999;1439:363–70. wx27 Mizugaki M. Establishment of microanalysis of prostaglandin metabolites by GCrMS and its clinical application. Yakugaku Zasshi 1999;119:61–80. wx28 Chiabrando C, Valagussa A, Rivalta C, Durand T, Guy A, Zuccato E, et al. Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-prostaglandin F2alpha. J Biol Chem 1999; 274:1313–9. wx29 Bachi A, Zuccato E, Baraldi M, Fanelli R, Chiabrando C. Measurement of urinary 8-epi-prostaglandin F2alpha, a novel index of lipid peroxidation in vivo, by immunoaffinity extractionrgas chromatography–mass spectrometry: basal levels in smokers and nonsmokers. Free Radical Biol Med 1996;20:619– 24. wx30 Adatia I, Barrow SE, Stratton PD, Ritter JM, Haworth SG. Effect of intracardiac repair on biosynthesis of thromboxane A2 and prostacyclin in children with a left to right shunt. Br Heart J 1994;72:452–6. wx31 Tagari P, Callaghan DH, Black C, Yerge JA. Measurement of canine urinary thromboxanes by GC–MS and HPLC–RIA. Prostaglandins 1994;47:293–306. wx32 Hiramatsu M, Hayashi Y, Yamamoto S, Hayashi A, Sawada M, Hamanaka N, et al. Application of an alpha-sidechain length-specific monoclonal antibody to immunoaffinity purification and enzyme immunoassay of 2,3-dinor-6-keto-prostaglandin F1alpha from human urine. Prostaglandins, Leukotrienes Essent Fatty Acids 1994;50:69–79. wx33 Djurup R, Chiabrabdo C, Jorres A, Fanelli R, Foegh M, Soerensen HU, et al. Rapid, direct enzyme immunoassay of 11-keto-thromboxane B2 in urine, validated by immuonoaffinityrgas chromatography–mass spectrometry. Clin Chem 1993;39:2470–7. wx34 Hishinuma T, Shimomura K, Nishikawa M, Ohyama Y, Ishibashi M, Mizugaki M. Separation and concentration of delta17-6-keto-PGF1alpha using monoclonal antibody to omega 3-olefin structure of trienoic prostanoids. Prostaglandins 1992;44:329–38. wx35 Chiabrando C, Rivoltella L, Martelli L, Valzacchi S, Fanelli R. Urinary excretion of thromboxane and prostacyclin metabolites during chronic low-dose aspirin: evidence for an extrarenal origin of urinary thromboxane B2 and 6-keto-prostaglandin F1 alpha in healthy subjects. Biochim Biophys Acta 1992;1133:247–54. wx36 Ishibashi M, Watanabe K, Ohyama Y, Mizugaki M, Hayashi Y, Takasaki W. Novel derivatization and immunoextraction to improve microanalysis of 11-dehydrothromboxane B2 in human urine. J Chro- matogr 1991;562:613–24. wx37 Minuz P, Barrow SE, Cockcroft JR, Ritter JM. Effects of non-steroidal anti-inflammatory drugs on prostacyclin and thromboxane biosynthesis in patients with mild essential hypertension. Br J Clin Pharmacol 1990;30:519–26. wx38 Minuz P, Barrow SE, Cockcroft JR, Ritter JM. Prostacyclin and thromboxane biosynthesis in mild essential hypertension. Hypertension 1990;15:469–74. wx39 Chiabrando C, Pinciroli V, Campoleoni A, Benigni A, Piccinelli A, Fanelli R. Quantitative profiling of 6-ketoprostaglandin F1alpha, 2,3-dinor-6-ketoprostaglandin F1alpha, thromboxane B2 and 2,3-dinor- thromboxane B2 in human and rat urine by immunoaffinity extraction with gas chromatography–mass spectrometry. J Chromatogr 1989;495:1–11. 728 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731

wx40 Barrow SE, Ward PS, Sleighthol MA, Ritter JM, Dollery CT. Cigarette smoking: profiles of thromboxane- and prostacyclin-derived products in human urine. Biochim Biophys Acta 1989;993:121–7. wx41 Mackert G, Reinke M, Schweer H, Seyberth HW. Simultaneous determination of the primary prostanoids prostaglandin E2, prostaglandin F2alpha and 6-oxo-prostaglandin F1alpha by immunoaffinity chromatography in combination with negative ion chemical ionization gas chromatography–tandem mass spectrometry. J Chromatogr 1989;494:13–22. wx42 Vrbanac JJ, Eller TD, Knapp DR. Quantitative analysis of 6-keto-prostaglandin F1alpha using immunoaffinity purification and gas chromatography–mass spectrometry. J Chromatogr 1988;425:1–9. wx43 Hubbard HL, Eller TD, Mais DE, Halushka PV, Baker RH, Blair IA, et al. Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography– mass spectrometry. Prostaglandins 1987;33:149–60. wx44 McEvoy JD, McCaughey WJ, Cooper J, Kennedy DG, McCartan BM. Nortestosterone is not a naturally occurring compound in male cattle. Vet Q 1999;21:8–15. wx45 Delahaut P, Jacquemin P, Colemonts Y, Dubois M, De Graeve J, Deluyker H. Quantitative determination of several synthetic corticosteroids by gas chromatography–mass spectrometry after purification by immunoaffinity chromatography. J Chromatogr, B: Biomed Sci Appl 1997;696:203–15. wx46 Stanley SM, Smith L, Rodgers JP. Biotransformation of 17-aklylsteroids in the equine: gas chromatographic–mass spectral identification of ten intermediate metabolites of methyltestosterone. J Chro- matogr, B: Biomed Sci Appl 1997;690:55–64. wx47 Aguilera R, Becchi M, Grenot C, Casabianca H, Hatton CK. Detection of testosterone misuse: comparison of two chromatographic sample preparation methods for gas chromatographic-combustionsr isotope ratio mass spectrometric analysis. J Chromatogr, B: Biomed Sci Appl 1996;687:43–53. wx48 Bagnati R, Fanelli R. Determination of 19-nortestosterone, testosterone and trenbolone by gas chromatography–negative-ion mass spectrometry after formation of the pentafluorobenzylcarboxy- methoxime-trimethylsilyl derivatives. J Chromatogr 1991;547:325–34. wx49 van Ginkel LA, Stephany RW, van Rossum HJ, van Blitterswijk H, Zoontjes PW, Hooijschuur RC, et al. Effective monitoring of residues of nortestosterone and its major metabolite in bovine urine and bile. J Chromatogr 1989;489:95–104. wx50 Haasnoot W, Schilt R, Hamers AR, Huf FA, Farjam A, Frei RW, et al. Determination of beta-19- nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreat- ment. J Chromatogr 1989;489:157–71. wx51 van Ginkel LA, Stephany RW, van Rossum HJ, Steinbuch HM, Zomer G, Van der Heeft E, et al. Multi-immunoaffinity chromatography: a simple and highly selective clean-up method for multi-anabolic residue analysis of meat. J Chromatogr 1989;489:111–20. wx52 Hams AJ, Ranasingle A, Morinello EJ, Nakamura J, Upton PB, Johnson F, et al. Immunoaffinityrgas chromatographyrhigh-resolution mass spectrometry method for the detection of NŽ.2 ,3-ethenoguanine. Chem Res Toxicol 1999;12:1240–6. wx53 Chen HJ, Chaing LC, Tseng MC, Zhang LL, Ni J, Chung FL. Detection and quantification of 1, NŽ.6 -ethenodiamine in human placental DNA by mass spectrometry. Chem Res Toxicol 1999;12:1119– 26. wx54 Rouzer CA, Chaudhary AK, Nokubo M, Ferguson DM, Reddy GR, Blair IA, et al. Analysis of the X malondialdehyde-2 -deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatographyrelectron capturernegative chemical ionization mass spectrometry. Chem Res Toxicol 1997;10:181–8. wx55 Ravanat JL, Turesky RJ, Gremaud E, Trudel LJ, Stadler RH. Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography–mass spectrometry assay. Chem Res Toxicol 1995;8:1039–45. wx56 Airoldi L, Magagnotti C, Chiappetta L, Bonfanti M, Pastorelli R, Fanelli R. Simultaneous immunoaffinity purification of O6-methyl-, O6-ethyl-, O6-propyl- and O6-butylguanine and their analysis by gas chromatographyrmass spectrometry. Carcinogenesis 1995;16:2247–50. wx57 Airoldi L, Magagnotti C, Bonfanti M, Chappetta L, Lolli M, Medana C, et al. Detection of O6-butyl- D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 729

and O6Ž. 4-hydroxybutyl guanine in urothelial and hepatic DNA of rats given the bladder carcinogen N-nitrosobutylŽ. 4-hydroxybutyl amine. Carcinogenesis 1994;15:2297–301. wx58 Shuker DE, Bartsch H. Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography-mass spectrometry and other methods of quantitation. Mutat Res 1994;313:263–8. wx59 Prevost V, Shuker DE, Friesen MD, Eberle G, Rajewsky MF, Bartsch H. Immunoaffinity purification and gas chromatography–mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man. Carcinogenesis 1993;14:199–204. wx60 Friesen MD, Garren L, Prevost V, Shuker DE. Isolation of urinary 3-methyladenine using immunoaffinity columns prior to determination by low-resolution gas chromatography-mass spectrometry. Chem Res Toxicol 1991;4:102–6. wx61 Shuker D-E, Friesen MD, Garren L, Prevost V. A rapid gas chromatography–mass spectrometry method for the determination of urinary 3-methyladenine: application in human subjects. IARC Sci Publ 1991;105:102–6. wx62 Bonfanti M, Magagnotti C, Galli A, Bagnati R, Moret M, Gariboldi P, et al. Determination of O6-butylguanine in DNA by immunoaffinity extractionrgas chromatography-mass spectrometry. Can- cer Res 1990;50:6870–6. wx63 Reuschel SA, Eades D, Foltz RL. Recent advances in chromatographic and mass spectrometric methods for determination of LSD and its metabolites in physiological specimens. J Chromatogr, B: Biomed Sci Appl 1999;733:145–59. wx64 Dubois M, Taillieu X, Colemonts Y, Lansival B, De Graeve J, Delahaut P. GC–MS determination of anabolic steroids after multi-immunoaffinity purification. Analyst 1998;123:2511–616. wx65 Schanzer W, Delahaut P, Geaer H, Machnik M, Hornik S. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography–high-resolution mass spectrometry. J Chromatogr, B: Biomed Sci Appl 1996;687:93–108. wx66 Webb KS, Baker PB, Cassells NP, Francis JM, Johnston DE, Lancaster SL, et al. The analysis of lysergideŽ. LSD : the development of novel immunoassay and immunoaffinity extraction procedures together with an HPLC–MS confirmation procedure. J Forensic Sci 1996;41:938–46. wx67 Hooijerink H, Schilt R, van Bennekom EO, Huf FA. Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography–mass-selective detection. J Chromatogr, B: Biomed Sci Appl 1994;660:303–13. wx68 Stanley SM, Wilhelmi BS, Rodgers JP. Comparison of immunoaffinity chromatography combined with gas chromatography–negative ion chemical ionisation mass spectrometry and radioimmunoassay for screening dexamethasone in equine urine. J Chromatogr 1993;620:250–3. wx69 Stanley SM, Wilhelmi BS, Rodgers JP, Bertschinger H. Immunoaffinity chromatography combined with gas chromatography–negative ion chemical ionisation mass spectrometry for the confirmation of flumethasone abuse in the equine. J Chromatogr 1993;614:77–86. wx70 Rule GS, Henion JD. Determination of drugs from urine by on-line immunoaffinity chromatography– high-performance liquid chromatography–mass spectrometry. J Chromatogr 1992;582:103–12. wx71 Bagnati R, Paleologo Orundi M, Russo V, Danese M, Berti F, Fanelli R. Determination of zeranol and beta-zearalanol in calf urine by immunoaffinity extraction and gas chromatography–mass spectrometry after repeated administration of zeranol. J Chromatogr 1991;564:493–502. wx72 van Ginkel LA. Immunoaffinity chromatography, its applicability and limitations in multi-residue analysis of anabolizing and doping agents. J Chromatogr 1991;564:363–84. wx73 Stillwell WG, Turesky RJ, Sinha R, Tannenbaum SR. N-oxidative metabolism of 2-amino-3,8-dimethyl- imidazowx 4,5-f quinoxalineŽ. MeIQx in humans: excretion of the N2–glucoronide conjugate of 2 hydroxyamino-MeIQx in urine. Cancer Res 1999;59:5154–9. wx74 Stillwell WG, Turesky RJ, Sinha R, Skipper PL, Tannenbaum SR. Biomonitoring of heterocyclic aromatic amine metabolites in human urine. Cancer Lett 1999;143:145–8. wx75 Pahler A, Volkel W, Dekant W. Quantitation of N epsilon-Ž. dichloroacetyl -L-lysine in proteins after perchloroethene exposure by gas chromatography-mass spectrometry using chemical ionization and negative ion detection following immunoaffinity chromatography. J Chromatogr, A 1999;847:25–34. wx76 Pahler A, Parker J, Dekannnt W. Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation. Toxicol Sci 1999;48:5–13. 730 D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731

wx77 Wang JS, Shen X, He X, Zhu YR, Zhang BC, Wang JB, et al. Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qudong, People’s Republic of China. J Natl Cancer Inst 1999;91:347–54. wx78 Matsuda Y, Nagao M, Takatori T, Niijima H, Nakajima M, Iwase H, et al. Detection of the sarin hydrolysis product in formalin-fixed brain tissues of victims of the Tokyo subway terrorist attack. Toxicol Appl Pharmacol 1998;150:310–20. wx79 Nagao M, Takatori T, Matsuda Y, Nakajima M, Niijima H, Iwase H, et al. Detection of sarin hydrolysis products from sarin-like organophosphorus agent-exposed human erythrocytes. J Chromatogr, B: Biomed Sci Appl 1997;701:9–17. wx80 Pastorelli R, Restano J, Guanci M, Maramonte M, Magagnotti C, Allevi R, et al. Hemoglobin adducts of benzowxa pyrene diolepoxide in newspaper vendors: association with traffic exhaust. Carcinogenesis 1996;17:2389–94. wx81 Redig P, Shaul O, Inze D, Van Montagu M, Van Onckelen H. Levels of endogenous cytokins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells. FEBS Lett 1996;391:175–80. wx82 Bagnati R, Ramazza V, Zucchi M, Simonella A, Leone F, Bellini A, et al. Analysis of dexamethasone and betamethasone in bovine urine by purification with an Aon-lineB immunoaffinity chromatography– high-performance liquid chromatography system and determination by gas chromatography–mass spectrometry. Anal Biochem 1996;235:119–26. wx83 Awata N, Toba F, Ando M, Shimada H, Miyairi S, Kato T, et al. Immunoaffinity extraction of 4-hydroxy-2-Ž. 4-methylphenyl benzothiazole and its metabolites for determination by gas chromatography–mass spectrometry. Biol Pharm Bull 1994;17:843–5. wx84 Trickland PT, Kang D, Bowman ED, Fitzwilliam A, Downing TE, Rothman N, et al. Identification of 1-hydroxypyrene glucuronide as a major pyrene metabolite in human urine by synchronous fluorescence spectroscopy and gas chromatography–mass spectrometry. Carcinogenesis 1994;15:483–7. wx85 Friesen MD, Garren L, Bereziat JC, Kadlubar F, Lin D. Gas chromatography–mass spectrometry analysis of 2-amino-1-methyl-6-phenylimidazowx 4,5-b pyridine in urine and feces. Environ Health Per- spect 1993;99:179–81. wx86 Day BW, Naylor S, Gan LS, Sahali Y, Nguyen TT, Skipper PL, et al. Molecular dosimetry of polycyclic aromatic hydrocarbon epoxides and diol epoxides via hemoglobin adducts. Cancer Res 1990;50:4611–8. wx87 Bagnati R, Castelli MG, Airoldi L, Paleolog Oriundi M, Ubaldi A, Fanelli R. Analysis of diethylstilbe- strol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography–negative-ion chemical ionization mass spectrometry. J Chromatogr 1990;527:267–78. wx88 Manchester DK, Weston A, Choi JS, Trivers GE, Fennessey PV, Quintana E, et al. Detection of benzowxa pyrene diol epoxide-DNA adducts in human placenta. Proc Natl Acad Sci U S A 1988; 85:9243–7. wx89 Wilkes JG, Sutherland JB. Sample preparation and high-resolution separation of mycotoxins possessing carboxylic groups. J Chromatogr, B: Biomed Sci Appl 1998;717:135–56. wx90 Scott PM, Kanshere SR, Lau BP, Lewis DA, Hayward S, Ryan JJ, et al. Survey of Canadian human blood plasma for ochratoxin A. Food Addit Contam 1998;15:555–62. wx91 Visconti A, Pascale M, Centonze G. Determination of ochratoxin A in wine by means of immunoaffinity column and high-performance liquid chromatography. J Chromatogr, A 1999;864:89–101. wx92 MacDonald S, Wilson P, Barnes K, Damant A, Massey R, Mortby E, et al. Ochratoxin A in dried vine fruit: method and survey. Food Addit Contam 1999;16:253–60. wx93 Dragacci S, Grosso F, Bire R, Fremy JM, Coulon S. A French monitoring programme for determining ochratoxin A occurrence in pig kidneys. Nat Toxins 1999;74:167–73. wx94 Stamler JS, Jaraki O, Osborne J, Simon D, Keany J, Vita J, et al. Nitric oxide circulates in mammalian plasma primary as a S-nitroso adduct of serum albumin. Proc Natl Acad Sci U S A 1992;89:7674–7. wx95 Halliwell B. What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS Lett 1997;411:157–60. wx96 Ferranti P, Malorni A, Mamone G, Sannolo N, Marino G. Characterisation of S-nitrosohaemoglobin by mass spectrometry. FEBS Lett 1997;400:19–24. wx97 Frost MT, Halliwell B, Moore KP. Analysis of free and protein-bound nitrotyrosine in human plasma by D. TsikasrJ. Biochem. Biophys. Methods 49() 2001 705–731 731

a gas chromatographyrmass spectrometry method that avoids nitration artifacts. Biochem J 2000; 345:453–8. wx98 Schwedhelm E, Tsikas D, Gutzki F-M, Frolich¨ JC. Gas chromatographic–tandem mass spectrometric quantification of free 3-nitrotyrosine in human plasma at the basal state. Anal Biochem 1999;276:195– 203. wx99 Tsikas D, Raida M, Sandmann J, Rossa S, Forssmann W-G, Frolich¨ JC. Electrospray ionization mass spectrometry of low-molecular-mass S-nitroso compounds and their thiols. J Chromatogr, B: Biomed Sci Appl 2000;742:99–108. wx100 Tsikas D. Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatographyrmass spectrometry. Anal Chem 2000;72:4064–72. wx101 Tsikas D, Schwedhelm E, Fauler J, Gutzki F-M, Mayatepek E, Frolich¨ JC. Specific and rapid quantification of 8-iso-prostaglandin F2alpha in urine of healthy humans and patients with Zellweger syndrome by gas chromatography–tandem mass spectrometry. J Chromatogr, B: Biomed Sci Appl 1998;716:7–17.