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V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 611 Review Derivatization in mass spectrometry—6. Formation of mixed derivatives of polyfunctional compounds Vladimir G. Zaikin Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninsky prospect 29, 119991 Moscow, Russia. E-mail: [email protected] John M. Halket Drug Control Centre, King’s College London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, UK. E-mail: [email protected] The review describes chemical transformations of multi-functional compounds (amino acids and peptides, amino alcohols, amino thiols, hydroxy acids, oxo acids, oxo alcohols, compounds containing simultaneously three or more different groups etc.) by using step-wise or one-step modification or protection of functional groups. Some chemical aspects of mixed derivatization performed for improving the physical–chemical properties and mass spectral characteristics are discussed. Application of mixed derivatization to qualitative and quantitative analysis of various multifunctional compounds mainly in biological fluids and other matrices by gas chromatography/mass spectrometry in electron ionization, chemical ionization, negative-ion chemical ionization and selected ion monitoring modes is considered. Keywords: amino acids, amino alcohols, amino ketones, amino thiols, carbohydrates, chemical ionization, electron ionization, gas chromatography/mass spectrometry, hydroxy acids, hydroxyl-oxo-carboxyl-containing compounds, mixed derivatives, oxo acids, oxo alcohols, peptides, polyols, prostaglandins, selected ion monitoring, steroids Introduction Various methodological approaches can be applied to the chemical transformation of compounds containing multiple functional groups. One of them involves the protection or modification of one type of functional group by using the reactions described in the foregoing reviews,1–5 the remaining groups being unaffected. A further approach is based on the preparation of cyclic derivatives involving at least two (sometimes not identical) functional groups. 4 However, protection or modification of all polar groups, with the aid of appropriate common derivatization methods, is the most common approach. In this case, successive employment of suitable reactions, which are selective to particular groups, DOI: 10.1255/ejms.773 gives rise to mixed derivatives. The application of some of these approaches to particular types of multi-functional compounds will be considered in the review. Mixed derivatives of amino acids and oligopeptides Amino acids Unprotected amino acids having zwitterionic character possess low volatility and, on heating up to 250°C, decompose thermally and are converted into diketopiperazines. Although individual amino acids can sometimes be analyzed with the aid of a direct probe inlet, they must be converted into volatile derivatives for investigation by gas chromatography(GC) and ISSN 1356-1049 © IM Publications 2005 612 Formation of Mixed Derivatives of Polyfunctional Compounds gas chromatography/masss pectrometry (GC/MS). Silylation enables protection of both amino and carboxyl groups and could be considered as the most convenient method of derivatization of amino acids.1 However, the method has a disadvantage, because there are two hydrogen atoms in the amino group to be displaced by silyl groups and thus silylation can lead to a mixture of silyl derivatives which complicates the investigation of amino acid mixtures by GC/MS. In the case of diamino acids, the situation is even more complicated. For example, silylation of lysine can be accompanied by the introduction of from three to five silyl groups. However, due to steric hindrance, only one bulky tert-butyldimethylsilyl group can be introduced to each nitrogen atom. The polarity of amino acids may be decreased by protection of either the carboxyl group or the amino group. In this case, the carboxyl is usually transformed into an ester group whereas the amino group is converted into an N-acyl group. Best results can be obtained, however, when both functional groups are blocked. Alkyl esters of N(O,S)-acylamino acids Alkylation of the carboxyl group and acylation of the amino (and additional OH and SH) groups was practically the first, it is still the most common derivatization procedure for the analysis of amino acids by GC/MS. The following sequence of reactions is most commonly employed, particularly where R″=CF3: of amino acids by GC/chemical ionization (CI)-MS in selected ion monitoring (SIM) mode.7 The [M + H]+ ion base peaks were chosen for the analysis performed at the nanomole level. One of the recent applications of such derivatives is quantitative analysis of amino acids for rapid diagnosis of phenylketonuria and other amino acidaemias with the use of GC/electron ionization (EI)-MS.8 N-Heptafluorobutyryl isoamyl esters appeared to be efficient derivatives for the determination of glycoprotein amino acids in mixtures with sialic acid and monosaccharides by GC/EI-MS.9 Amino acid analysis of pyoverdins (siderophores produced by the fluorescent group of the bacterial genus Pseudomonas) by GC/ CI-MS was accomplished after preliminary isopropylation and N(O,S)- trifluoroacylation or pentafluoropropionylation. As a result, a number of proteinic, non-proteinic and artifact amino acids were identified.10 It should be noted that trace levels of amino acids can be determined in the form of n-butyl or menthyl esters of N-benzoyl-, N-pentafluorobenzoyl-, Ntrifluoroacetyl derivatives. The complete EI mass spectra for a large number of trifluoroacetylated amino acid n-butyl esters have been reported;11,12 some of them can also be found in the NIST/EPA/ NIH Mass Spectral Library.13 Major fragmentation pathways of the derivatives are shown in the following scheme: O CF3 C N + CH R'' R' CF3+ O -RCH=C=O O R O R O R"COCl R'OH/HCl H2N OH H2N OR' R R"CONH CF3 O N CH CH C O + HC C O C4H9 R R'' R' R OR' CF3 C -O-C4H9 + OH O N CH CH C R'' R' The preparation of such derivatives is usually accomplished step-wise. In the first stage, esterification of the carboxyl group is carried out with alcohol in the presence of acids.3 The reaction usually proceeds on heating of the amino acid and corresponding alcohol (most frequently CH3OH, C2H5OH, n-C4H9OH etc.) with 3N HCl at 150°C for 10– 15 min. In the next stage, acylation of the amino group is carried out with the aid of acyl chloride or anhydride at room temperature.2 Cases are known where these reactions proceed simultaneously in the presence of a mixture of alkylating and acylating reagents and pyridine. It should be noted that N-formyl amino acid methyl esters can also be used for the identification of amino acids. In addition, such derivatives are interesting in that N-formyl amino acids can be included in the structures of some peptides and proteins. Some features of the electron ionisation (EI) mass spectra of N-formyl methyl ester derivatives have been reported.6 For derivatization of amino acids, esterification with n-BuOH, n-PrOH, iso-PrOH or iso-BuOH and acylation with CF3COCl, C2F5COCl or C3F7COCl (or the respective anhydrides) are most frequently used. For example, N-acetyl npropyl ester derivatives were employed for the determination C - C4H7 O OH CF3 C R +. O C O C4H9 O N CH CH C C4H9 O CF3 C N + CH CH R'' R' R'' R' R R -C4H8 + R CF3 C + R' + O O N CH CH C R'' R' OH R N-Trifluoroacetyl amino acid n-butyl esters possess good gas chromatographic properties and may be used for the quantitative determination of amino acids in hydrolyzates of proteins and peptides by GC/MS in SIM mode at picomole levels.14 The base ions [M – COOC4H9]+ were recommended for detection of glycine, alanine, valine, leucine/isoleucine, proline, aspartic acid and histidine. Further characteristic ions were used for determination of other amino acids. N-Pentafluoropropionyl- and N-heptafluorobutyryl amino acid alkyl esters reveal similar EI-induced fragmentation pathways. For illustration, Figure 1 presents the EI mass spectra of such derivatives of methionine. Our investigations 15 demonstrated that Ncycloalkylcarbonyl (cycloalkyl = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl) derivatives of amino acid methyl esters were also helpful derivatives for the analysis of amino acids by GC/MS. The main decomposition pathways of these V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 613 Figure 1. EI mass spectra of (a) N-pentafluoropropionyl methionine butyl ester, (b) N-heptafluorobutyryl-methionine butyl ester (V.G. Zaikin, private collection). derivatives under EI resemble those observed for N-alkanoyl derivatives (Figure 2). Successive N(O,S)-acylation and esterification have been applied to the quantitative determination of D-amino acids in body fluids (urine, blood plasma, blood serum, milk) of mammals (hamster, horse, bovine, sheep, pig, dog).16 For the analysis, amino acids isolated from these samples were esterified with isopropanol (heating 1 h at 100°C in the presence of HCl) and then acylated with (C2F5CO)2O or (CF3CO)2O (20 min. at 100°C). Derivatized enantiomers were separated on a Chirasil–L–Val capillary column and detected by MS in SIM mode. The same approach was used for the combined determination of D- and L-amino acids that are frequently present in bacterial peptides. In this case, esterification was accomplished with MeOH, EtOH, PrOH, i-PrOH, BuOH or hexafluoroisopropanol in the presence of CH3COCl, whereas for acylation (CF3CO)2O was used.17 A further application of the same derivatization is in the stable carbon isotope analysis of amino acid enantiomers in soil samples with the aid of gas chromatography-combustion/ isotope ratio mass spectrometry. In this case, the conversion of the enantiomers to N(O,S)-pentafluoropropionyl isopropyl 614 Formation of Mixed Derivatives of Polyfunctional Compounds Figure 2. EI mass spectra of (a) N-cyclopropylcarbonyl-methionine methyl ester, (b) N-cyclobutylcarbonyl-methionine methyl ester, (c) N-cyclopentylcarbonyl-methionine ester and (d) N-cyclohexylcarbonyl-methioninge methyl ester (V.G. Zaikin, private collection). V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) esters was suggested.18 However, it was found that isotope discrimination during derivatization and combustion could alter the true 13C value of the underivatized amino acids by up to ten delta units. Derivatization via esterification and N-acylation has been used for the quantitative determination of glyphosate, the active ingredient of some commercial herbicides and its principal metabolite, aminomethylphosphonic acid, by GC/MS-MS.19 One-step derivatization by a mixture of 2,2,3,3,4,4,4-heptafluoro-1-butanol and (CF3CO)2O (1 h, 92– 97°C) gave simultaneous N-acylation and esterification of the phosphonic residue and carboxyl group (if present). EI mass spectra of the derivatives reveal abundant ions originating from cleavages as shown for aminomethylphosphonic acid derivatives: 612 O CF3 O CF3CF2CF2CH2COCO-CH2-N-CH2-P +H OCH2CF2CF2CF3 OCH2CF2CF2CF3 460 584 For quantification of both compounds, however, the analytical conditions for MS/MS detection were optimized and sums of intensities of the most representative ions were chosen: m/z 440, 321, 261 for glyphosate and 283, 223 and 181 for the metabolite. In another case,20 simultaneous determination of glyphosate and its metabolites was achieved after their preliminary derivatization by means of CF3COOH–(CF3CO)2O (10 min at 30–40°C) followed by reaction with CH3C(OCH3)3 (1,5 h at 100°C): 615 groups.22 The reaction proceeds quantitatively on heating of amino acid with n-C4H9OH/3N HCl for 15 min at 150°C and then with BSTFA and CH3CN for 90 min at 150°C. Such an approach has found an interesting application for the separation and detection of enantiomeric amino acids. Ntert-Butyldimethylsilyl derivatives of pentafluoropropionylisopropyl esters of amino acids were separated on chiral columns coated with octakis(2,6-di-O-pentyl-3-O-butyryl)τ-cyclodextrin and N-propanoyl-L-valine-tert-butylamidelinked polydimethylsiloxane and the enantiomeric derivatives detected by GC/MS.23 Alkyl esters of N(O,S)-alkoxycarbonyl-amino acids A one-step method for protection of all existing functional groups in amino acids using a mixture of alkylchloroformate (ROCOCl) and alcohol has been suggested.24 This derivatization has been of considerable interest, as it can be performed directly in aqueous media and the resulting derivatives readily extracted by organic solvents. For example, in the case of ethylchloroformate and ethanol, one can obtain the N(O,S)-ethoxycarbonyl amino acid ethyl ester. The amino acid (or mixture of amino acids) was added to a mixture of H2O, C2H5OH and pyridine, then ethylchloroformate was added and the reaction mixture was vortexed for 5 to 10 s. By changing the nature of the alcohol and chloroformate, one can prepare different esters of N(O. S)-mono- or di(alkoxycarbonyl)amino acids.25 In the absence of other functional groups in the amino acid, the reaction looks as follows: R O O + H2N OH R'O-C + Cl R O HN OR" O R"-OH R'O H2N-CHR-PO(OH)2 CH3C(OCH3)3 CF3COOH/(CF3CO)2O CF3CO-NH-CHR-PO(OH)2 CF3CO-NH-CHR-PO(OCH3)2 Quantification of the analyte was made by GC/MS and GC/CI-MS An ultra-sensitive method for the determination of amino acids in hydrolyzates of peptides and proteins at the femtomole level involved acylation with heptafluorobutyric anhydride (in CH3CN; 15 min at 50°C) followed by esterification with PFBz bromide in acetone (15 min at 50°C). Free OH or SH groups in amino acids were also protected by silylation with BSTFA/TMCS. For quantitative analysis, isotope dilution GC/negative ion chemical ionization (NICI)-MS was used.21 Alkyl esters of N(O,S)-silylamino acids The derivatization of amino acids may also be accomplished by successive esterification of the carboxyl group and silylation of amino and other free OH or SH groups. The resulting products contain O-alkyl and N-silyl This one-step method was used for the direct extraction– derivatization and quantitative determination of amino acids in human urine by GC/MS.26 The derivatization procedure involved the addition of alkylchloroformate and a mixture of alcohol–pyridine (4 : 1) and chloroform to the urine sample and gently shaking the mixture for 1 min. The organic layer was taken for analysis by GC/MS. The proposed method, however, did not succeed in the derivatization of threonine, serine, asparagine, glutamine and arginine. Four different alkylchloroformates (alkyl = Me, Et, Pr or Bu) and four alcohols (methanol, ethanol, n-propanol and n-butanol) were examined for derivatization in order to achieve a good gas chromatographic separation. Both alkyl groups appeared to influence the retention time, especially in the case of aspartic and glutamic acids. When methanol was used for protection of the carboxyl group, the derivatives of these acids were not overlapped with any other derivatives. The use of propanol, however, resulted in overlapping of both derivatized acids with the corresponding derivatives of phenylalanine and cysteine. 616 Twelve non-protein amino acids (in addition to the main protein acids) were identified as N-ethoxycarbonyl ethyl ester derivatives in a number of species of cycad seeds by GC/CI-MS in positive mode. A novel non-protein amino acid named cycasindene was also discovered.27 A further application of this approach is the investigation of organic binders used by artists. The method allows a fast and effective identification of amino acids in very small amounts of protein by ion trap GC/MS after hydrolysis of proteinic binders (such as casein, albumin, gelatine, etc.) and treatment of the resulting amino acids with ethyl chloroformate/ethanol.28 In another case,29 the same procedure was employed to determine amino acids, fatty acids and bile acids simultaneously in the binding media in works of art. Derivatization with a mixture of ethyl chloroformate and 2,2,2-trifluoroethanol was used for analysis of protein amino acids by GC/EI-MS.30 The same approach and standard GC/MS conditions were applied for the rapid and sensitive determination of 3-chlorotyrosine (in the form of N(O,S)ethoxycarbonyl-trifluoroethyl ester), a highly specific marker of myeloperoxidase-catalyzed protein oxidation.31 Quantitative analysis of 16 protein amino acids by GC/CIMS was performed using the same derivatization (in the presence of pyridine and chloroform as the solvent).32 It was found that derivatization efficiencies ranged from 90 to 99% and extraction efficiencies using chloroform were close to 100%. Quantification of all amino acids (except arginine) was made by the [M + H]+ ion monitoring. The linear dependence of the ion intensity on concentration was in the range from zero to three orders of magnitude. Twenty one non-protein amino acids were also derivatized by ethyl chloroformate and 2,2,2-trifluoroethanol in the presence of pyridine and detected by GC/CI-MS.33 In the positive CI mode (reagent gas, methane), the mass spectra of the derivatives showed characteristic [M – 19]+, [M + 1]+, [M + 29]+ and [M + 41]+ peaks whereas [M – 1]– and [M + 35]– peaks were observed in the NICI mass spectra. For quantification, positive CI mode was used, the detection limits being mostly in the femtomole range. Selected EI mass spectral data for N(O,S)isobutoxycarbonyl isobutyl esters of 17 protein amino acids has been presented.34 It was shown that the [M – C4H9OCO]+ ions were prominent in the majority of the spectra. The presented data can assist in choosing appropriate ions for quantitative determination of particular amino acids by GC/ MS in SIM mode. For simultaneous determination of 13 amino acids (histidine and arginine were not derivatized) and 13 mono- and 6 dicarboxylic acids by GC/MS, derivatization with isobutyl chloroformate/isobutanol was suggested.35 It was shown that in the positive ion CI (isobutane) mass spectra of the compounds, the [M + H]+ ion peaks usually dominate. Detection limits by total ion current (TIC) and SIM were estimated. The same derivatization procedure was applied to analysis of a lyophilized microbial culture of Escherichia coli.36 It should be noted that isobutoxycarbonyl isobutyl Formation of Mixed Derivatives of Polyfunctional Compounds esters and n-butoxycarbonyl n-butyl esters of amino acids reveal similar fragmentation patterns under EI [compare Figure 3(a) and (b)]. However, as expected, the former derivatives are more mobile under GC conditions and, hence, are more frequently used in analytical practice. Various alkyl chloroformates and alcohols have been tested to find the derivatives enabling isomeric leucine and isoleucine to be distinguished.37 Only methoxycarbonyl derivatives revealed some quantitative differences in their EI mass spectra (Figure 4). For all such derivatives of leucine having any ester alkyl groups (from methyl to octadecyl), the intensity of the peak at m/z 102 was always greater than that of the m/z 115 peak, whereas the opposite picture was observed for isoleucine derivatives. Note that the derivatives of these isomers can also be differentiated by GC retention times. Quantitative analysis of amino acids in biological fluids (plasma, whole blood) at the femtomole level can be achieved by using simultaneous derivatization with pentafluorobenzyl chloroformate and ethanol followed by GC/NICI-MS.38,39 NICI mass spectra are dominated by the [M – CH2C6F5]– ions that can be used for quantitation of amino acids. I t s h o u l d b e n o t e d t h a t d e r iva t i z a t i o n w i t h alkylchloroformate/alkanol was considered to be the most promising for the in situ analysis of amino acids in Martian samples. 40 It may also be efficient for qualitative and quantitative determination of seleno amino acids (such as selenomethionine, selenoethionine and selenocystine) in plants and animals by GC, GC/MS and even GC/inductively coupled plasma (ICP) analysis.41,42 One other application of the same derivatization approach is to measure 13C and 15N enrichments of glutamine in plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry. It was found that N(O,S)-ethoxycarbonyl ethyl ester derivatives were very stable at 20°C, even after five days storage and allowed reproducible and accurate stable isotope enrichment determination.43 Similar to N(O,S)-acyl amino acid alkyl esters, N(O,S)alkoxycarbonyl alkyl ester derivatives can be used for separation and determination of enantiomeric D- and Lamino acids on chiral GC stationary phases. Various combinations of chloroformates and alcohols were tested to get the best resolution and to decrease the analysis time: ethyl chloroformate and ethanol, heptafluorobutanol or trichloroethanol, isobutyl chloroformate and isobutanol or heptafluorobutanol.44 It should be noted that amino acid profiling in wine samples by GC/MS was efficiently performed after treatment of aqueous samples with isobutyl chloroformate followed by solid-phase extraction and derivatization with N-methylN-tert-butyldimethylsilyl trifluoroacetamide. The former reaction permitted the protection of amino and hydroxyl and thiol groups (if present) whereas silylation was used to protect carboxyl groups. The derivatives possessed good gas chromatographic properties and 17 amino acids were identified in wine samples.45 The EI mass spectra of V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 617 Figure 3. EI mass spectra of (a) N-isobutoxycarbonyl-methionine isobutyl ester, (b) N-butoxycarbonyl-methionine butyl ester (V.G. Zaikin, private collection). N(O,S)-isobutoxycarbonyl amino acid tert-butyldimethylsilyl esters have been described.46 In conclusion, it should be noted that N-alkoxycarbonyl alkyl ester derivatives of amino acids can also be prepared in two stages. For example, amino acids were treated with isobutyl chloroformate and the resulting N(O,S)-isobutoxycarbonyl derivatives were methylated with diazomethane.47 Note also, that for derivatization 2,2,3,3,4,4,5,5-octafluoropentyl chloroformate,48 9-fluorenylmethyl chloroformate (especially for LC/MS analysis49 and hexyl chloroformate50,51 have been recommended. Oligopeptides Much work has been done during the last 10–15 years on structure elucidation of peptides and even native proteins with the use of new mass spectrometric techniques based on matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI) and Fourier transform (FT) MS that eliminated the limitation on molecular weight and volatility of analyzed compounds. In principle, the latter methods do not require additional derivatization before analysis to measure the spectra of native peptides or proteins and are considered to be the best choice in 618 Formation of Mixed Derivatives of Polyfunctional Compounds Figure 4. EI mass spectra of (a) N-methoxycarbonyl-leucine pentyl ester, (b) N-methoxycarbonyl-isoleucine pentyl ester (V.G. Zaikin, private collection). such investigations. However, specific derivatization of the carboxyl or (primarily) amino terminus allows a great amount of structural information to be extracted from such mass spectra (see our forthcoming Review 8). Despite the great success of these new methods in the various investigations of proteins and peptides (amino acid sequence, conformations, intermolecular interactions etc.), the application of traditional mass spectrometric methods to the determination of simple peptides (for example, in dipeptidase hydrolyzates and biological fluids), peptidomimetics, etc. is being explored. In this section we have decided to remind the reader of some derivatization approaches that were used in the investigation of small peptides by conventional EI and CI mass spectrometry. The presence of highly polar groups (commonly, terminal NH2 and COOH as well as amide groups) in unprotected peptides promotes the formation of intermolecular hydrogen bonds and, hence, their low volatility and thermal stability. In addition, peptides possess zwitterionic characteristics. All this dictated the necessity of their preliminary chemical modification before mass spectrometric analysis (direct insertion probe MS, GC/MS). The earlier derivatization approaches involved V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) acylation of the amino terminus (as well as additional OH, SH and NH2 groups present in some amino acid residues) and esterification of end carboxyl groups.52 The EI mass spectra of resulting derivatives contain some information regarding the amino acid sequence in peptides. In mass spectrometric practice, protection of carboxyl groups by esterification with methanol or ethanol and of amino groups by conversion in Nformyl, -acetyl, -trifluoroacetyl, -heptafluorobutyryl, -benzoyl, -caproyl, -stearyl, -benzenesulfonyl,-dansyl etc. was the most popular. Derivatization usually started from esterification in the presence of HCl and finished by acylation with acyl chlorides, acid anhydrides or N-acylsuccinimides. The opposite order of derivatization can be also used. To protect amino groups in alkyl esters of peptides, the formation of Schiff’s bases is also used. In such reactions, aldehydes and ketones are involved as carbonyl compounds. Among such known derivatives are benzylidene-, 4dimethylamino-, 4-methoxy-, 4-nitro-, 4-cyanobenzylidene-, pyridylmethylidene-, 4-dimethylamino- and 2-hydroxy-1naphthylidene, cinnamylidene etc. derivatives: a H2NCHRCONHCHR1CONH...CHRnCOOCH3 + R CORb a R n NCHRCONHCHR1CONH...CHR COOCH3 b R The main features of the EI mass spectra of the latter derivatives are very abundant M+• ions that are 10 to 100 times as high as the same ions in the spectra of N-acyl methyl esters. Although N-acyl derivatives of alkyl esters are rather volatile, they are rarely used for the investigation of peptides containing more than eight amino acid units. To increase volatility in this case, permethylation of amide N-atoms in peptides was suggested. This reaction is usually carried out by the treatment with CH3I and Ag2O or NaH and CH3SOCH2–:53 CH3J/CH I/ 3SOCH2 n CH3CONHCHRCONHCHR1CONH...CHR COOCH3 n CH3CONCHRCONCHR1 CON...CHR COOCH3 CH3 CH3 CH3 This approach is applicable to peptides containing ornithine, tryptophan and/or tyrosine units but can be accompanied by the formation of ammonium and sulffonium salts in the case of peptides containing side amino and sulfide groups of the corresponding amino acids. It should be noted that, when using direct probe inlet, peptides containing from 10 to 12 amino acid residues may be investigated in the form of N-acyl methyl ester derivatives whereas peptides comprised of 15 units may be analyzed in the form of permethyl N-acyl methyl ester derivatives. However, using GC/MS, only di- and tripeptides can usually be investigated as N-acyl methyl esters. To determine the amino acid sequence in the original peptides by EI mass spectrometry, the ions formed due to 619 the following cleavages from N-acyl methyl ester derivatives are suitable: R-CO-NH-CH-CO-NH-CH-CO-NH-CH-CO-...-NH-CH-CO-OCH3 R1 R2 R3 4 R However, their relative intensities depend on the nature of both the N-acyl group and the amino acid residue that can provide additional fragmentation complicating the mass spectra. For the investigation of peptides, N-dansyl alkyl ester derivatives are suitable.54 Their EI mass spectra reveal intense M+• peaks and peaks of the ions characterizing the amino acid sequence and resulting from the cleavages shown for N-acyl methyl ester derivatives. Such derivatives may also be used for LC/MS analysis. Some amino acids provide extremely low peptide volatility, even after protection of the amino and carboxyl groups. For example, very low thermal stability may be due to the presence of arginine. To solve the problem, the guanidine side chain of arginine can be converted to an ornithine residue by reaction with aqueous hydrazine: NH-CH-CO (CH2)3 NH-C-NH2 N2H4 NH-CH-CO (CH2)3 NH2 NH arginine residue ornithine residue The resulting ornithine-containing peptides can be Nacylated and esterified to form derivatives whose EI mass spectra reveal characteristic fragmentation patterns allowing determination of the amino acid sequence. Very complicated EI mass spectra do not permit the sequence determination of N-acyl methyl ester derivatives of peptides containing cystine, cysteine and methionine. At the same time, desulfurization of the derivatives on Raney Ni in DMFA (20°C, two days) gives rise to the conversion of cystine(cysteine) and methionine residues to alanine and α-aminobutyric acid units, respectively. Such an approach increased the volatility and thermal stability of the resulting N-acyl ester derivatives whose EI mass spectra permitted the reliable determination of the amino acid sequence.55 Similar to amino acids, peptides can also be derivatized by Husek’s method with a mixture of alkylchloroformate and alkanol in aqueous media. Untill now however, this approach has only been applied to dipeptides and simple tripeptides. For example, treatment of a dipeptide mixture with ethylchloroformate/trifluoroethanol/pyridine in water solution gave rise to N-ethoxycarbonyl trifluoroethyl ester derivatives that were sufficiently separated by GC.56 Only positive ion CI was used in the work for detection and no information about the sequence of the dipeptides could 620 Formation of Mixed Derivatives of Polyfunctional Compounds be deduced from the spectra that revealed ions [M + H]+, [M + C2H5]+ and [M + C3H5]+ as the most intense peaks. Derivatization with ethylchloroformate/methanol was successfully applied to the tripeptide glutathione ( L-γglutamyl-L-cysteinylglycine).57 The EI mass spectrum of the resulting N,S-ethoxycarbonyl methyl ester derivative showed characteristic peaks enabling the amino acid sequence to be established. The potential of Husek’s reagents for derivatization of dipeptides and the determination of amino acid sequence in dipeptides in mixtures by GC/MS was recently elucidated in more detail.58 The main impact was made on mass spectrometric differentiation of isomeric dipepides. The effect of the alkyl group in both reagents on the GC and mass spectral properties was also investigated. Isomeric pairs of dipeptides (Leu–Ile/Ile–Leu, Ala–Ile/Ile–Ala. Gly–Pro/Pro– Gly, Ile–Gly/Gly–Ile, Leu–Gly/Gly–Leu, Leu–Ala/Ala–Leu, Gly–Hyp/Hyp–Gly) as well as single dipeptides (Gly–Gly, Gly–Phe, Ala–Pro, Gly–Met, Ala–Val, Gly–Leu) were (a) investigated. For symmetrical derivatization, ROCOCl and ROH (R=Me, Et, Pr, Bu in both cases) were chosen. As for amino acids, derivatization of dipeptides with alkyl chloroformate and alcohol in aqueous media gave rise to conversion of terminal amino and carboxylic groups into N-alkoxycarbonyl and alkyl ester groups, respectively. Additional hydroxyl groups (for example, in hydroxyproline) were transformed into anhydride groups. The EI mass spectra of the derivatives showed no, or only negligible, M+• peaks. The main EI-induced fragmentation of the derivatives is due to “amine”type cleavages (a1) or scission of the peptide bond (b1). It allows the unambiguous distinction of isomeric dipeptides with different sequences [compare Figures 5(a) and (d)]. O R' O R N N O O a1 (d) EI O R'' b1 EI M+ǜ (m/z 302) M+ǜ [MH]+ (b) [MH]+ (e) CI (c) R (f) CI CI-CID CI-CID [MH]+ [MH]+ Figure 5. Mass spectra of N-ethoxycarbonyl-L-isoleucyl-L-alanine ethyl ester and N-ethoxycarbonyl-L-alanyl-L-isoleucine ethyl ester measured under (a) and (d) EI, (b) and (e) CI, (c) and (f) CI-CID conditions, respectively.58 V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 621 about 15 amino acids can be investigated with this inlet system. Only penta- or hexapeptides can be analyzed by GC/ MS after conversion in amino alcohols. It should be noted, however, that such an approach is not suitable for peptides containing arginine, glutamine and asparagine residues. Mixed derivatives of amino alcohols and amino ketones Scheme 1. CI mass spectra of the derivatives revealed mainly the [M + H]+ ion peaks enabling the molecular mass to be established [Figures 5(b) and (e)]. MS/MS spectra recorded for the [M + H]+ ions generated under CI conditions showed simultaneously the precursor ion peaks as well as peaks characteristic of the EI mass spectra [Figure 5(c) and (f)]. Hence, these CID spectra are more advantageous for structure elucidation. There were only negligible differences in the EI mass spectra of dipeptide derivatives containing isomeric leucine and isoleucine residues. For example, pronounced low molecular weight ion peaks at m/z 69 are only observed in the spectra of isoleucine-containing derivatives. A rather efficient method for the chemical transformation of peptides was introduced by Biemann who recognized that the reduction of carbonyls in amide groups and the terminal ester group in peptides, pre-derivatized by esterification and N-acetylation (trifluorocetylation), by LiAlH4 gave rise to polyaminoalcohols that exhibit interpretable EI mass spectra.59 The volatility and gas chromatographic properties of the latter compounds in Scheme 1 can be improved by silylation of the terminal hydroxy group. The EI mass spectra of the resulting derivatives are exclusively informative. They reveal the most intense peaks due to β-cleavages allowing the amino acid sequence to be determined for the original peptide: R1 Z1 R2 Z2 R3 A2 A3 OH Me3SiO H N N BSTFA HO SiMe3 (C3F7CO)2NMe Me3SiO Synephrine Me3SiO COC3F7 N Me3SiO Z3 CH3 CH3-CH2-NH-CH-CH2-NH-CH-CH2-NH-CH-CH2-O-Si A1 To derivatize amino alcohols, amino ketones and amino thiols, one of the common derivatization methods, described in reviews,1–4 can be chosen and applied to one or both types of functional group. On the basis of the same common reactions, however, mixed derivatives can be prepared. Taking into account the low hydrolytic stability of N-silyl derivatives, it is possible to develop the method of selective introduction of silyl group to alcoholic or phenolic hydroxyl and acyl residue to amino group. This is achieved by the successive action of a silylating agent (for example, MSTFA, BSTFA or TMSIM) and acylating reagent (for example, trifluoroacetic anhydride) to replace the N-TMS group by a fluorinated acyl group.62 In a typical case, the phenolic amine is dissolved in acetonitrile and trimethylsilyl-imidazole is added. After stirring for 3 h at 60°C, the mixture is cooled and trifluoroacetic anhydride is added (60°C, 3 h). A similar two-step derivatization was used, for example, for the detection and determination of catecholamines, 63 ephedra-alkaloids (ephedrine, norephedrine, pseudoephedrine and other analogs) 64 or other β2-agonists65 by GC/MS: CH3 CH3 [M-CH3] + This approach may be used with success for the analysis of peptide mixtures, formed from acid or enzyme hydrolysis of proteins, by GC/MS.60 It should be noted that the method of reduction of N-acylated esters can be applied to the products of N-methylation of peptides. Further silylation of the products gives rise to highly volatile derivatives.61 By using a direct insertion probe, peptides containing 10–12 amino acid units can be analyzed in the form of amino alcohols. When using permethylated amino alcohols, peptides containing The EI mass spectra of such mixed derivatives show sufficient differences in fragmentation pattern due to ease of β-cleavage near the aliphatic trimethylsilyloxy group that permits definitive identification of individual ephedraalkaloids. The EI mass spectrum of the N-trifluoroacetylO,O,O-tris(trimethylsilyl) derivative of terbutaline is given in Figure 6 as an example. Note that such derivatization can be accomplished on-line in capillary GC/MS. For example, analysis of phenolalkylamines was performed using Otrimethylsilylation in the injection port of a gas chromatograph by co-injection with MSTFA followed by on-column acetylation with N-methylbis(trifluoroacetamide).66 622 Formation of Mixed Derivatives of Polyfunctional Compounds Figure 6. EI mass spectrum of N-trifluoroacetyl-O,O,O-tris(trimethylsilyl)-terbutaline (reproduced with permission from NIST/EPA/ NIH Library). The same protecting groups were introduced simultaneously in the quantitative analysis of metanephrine and normetanephrine in urine by isotope dilution GC/MS. Their conjugates were acid hydrolyzed and the resulting unconjugated drugs stirred with a mixture of MSTFA and N-methyl-bis-heptafluorobutyramide at room temperature.67 Two-step derivatization with TMSIM and heptafluorobutyrylimidazole also appeared to be an efficient approach for the analysis of some aminoglycoside antibiotics (kanamycin and gentamicin) by GC/MS.68 For the quantitative determination of 3α-amino-5αandrostan-2β-ol-17-one (antiarrhythmic drug Org 6001) in human plasma by isotope dilution GC/MS (SIM mode), simultaneous N-formylation-O-silylation was employed.69 Reaction was accomplished by heating with tert-butyldimeth ylchlorosilane in dimethylformamide at 120°C for 1.5 h. For SIM measurement, the prominent [M – 57]+ ion was selected: and silylation of the hydroxyl. Such an approach was used, for example, for the determination of cytokinins.71 The EI mass spectra of the N-pentafluorobenzyl-tertbutyldimethylsilyl derivatives reveal very abundant [M – 57]+ ions and some additional ions that can be used for structure elucidation. O O HO t-BuMe2SiCl/HCONMe2 Si O H2N NH O H Step-wise N-acylation with acetic anhydride and silylation with TMSI was employed for the detection, structure determination and profiling of long-chain amino alcohols in egg yolk, bovine milk and bovine brain sphingomyelin by GC/MS. Acetylation is most likely to have protected the OH groups as well and further action of TMSIM resulted in the displacement of O-acetyl groups by O-TMS.70 Another possible way to derivatize amino alcohols involves alkylation of the amino group with C6F5CH2Br A corresponding sequence of reactions may be used to protect hydroxyl with silyl groups and to convert the amino group into a Schiff’s base. For example, catecholamines and C6F5CHO were heated in CH3CN for 1 h at 60°C and the product was treated with BSA to convert all OH groups to O-TMS derivatives.22 The convenient two-step derivatization by formation of cyclic boronates and a phenolic silyl ether was used for structure determination and characterization of phenolalkylamines belonging to the β2-agonist series by GC/MS. 72,73 In the first stage, reaction with substituted phenyl-, 4-fluoro(chloro or bromo)phenylboronic acid was accomplished by stirring at 20°C. Further silylation can be performed on-line by co-injection of the boronate derivative and MSTFA into the GC/MS system: V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) EI mass spectra of the derivatives revealed intense (sometimes, base) M+• peaks as well as rather pronounced peaks of the following ions: [M – H] + , [M – CH 3 ] + , [M – RBO] +• , [M – RBO – NR 1 ] + , [Me 3 SiOC 6 H 4 CH 2 ] + , [M – Me3SiO]+, [M – Me3SiOC6H4]+. Formation of cyclic methyl(phenyl)boronates, at the expense of two OH groups, followed by acetylation of the amino group is a possible derivatization approach for the determination of sphingosines by GC/MS. These derivatives show rather intense M+• peaks in their EI mass spectra. At the same time, spectra of N-acetyl-O-trimethylsilyl derivatives prepared by successive silylation and acetylation reveal only negligible M+• peaks and very pronounced fragment ion peaks allowing the structure to be determined:74 NHCOCH3 R-CH-CH-CH2 O NHCOCH3 O R-CH-CH-CH2OSiMe3 B CH3 Me3SiO Mixed derivatization of amino ketones, such as biogenic amines, can involve oximation of the carbonyl group followed by silylation of all free OH groups. This procedure was used for quantification of isatin (2,3-isoindoledione), a monoamine oxidase inhibitor, by GC/MS:75 N O Si N O Si Mixed derivatives of hydroxy acids In addition to silylation or formation of cyclic derivatives, providing the protection of both types functional groups,1,4 compounds containing alcoholic hydroxyl and carboxyl groups can be converted to mixed derivatives. The latter may be prepared, for example, by esterification of the carboxyl group (in the simplest case, by methylation with CH2N2 or CH3OH in the presence of HCl, BF3 or other acids) followed 623 by acylation of the hydroxyl group by an acyl chloride or carboxylic acid anhydride. Hydroxy fatty acids, for instance, are easily identified by GC/MS if they are converted into heptafluorobutyryloxymethyl esters.76 The EI mass spectra of such derivatives are rather informative revealing peaks at m/z 69 [CF3]+, 119 [C 2 F 5 ] + and 169 [C 3 F 7 ] + originating from the heptafluorobutyryl moiety. The location of the original OH groups is determined by using a specific fragmentation regulated by the following rule. If the chain length is n and the OH group is located at position p, then there is a prevalent fragmentation between p-1 and p-2 that produces intense fragments at 14(n – p + 1). For quantitation of 3-hydroxy fatty acids as chemical markers for the determination of lipopolysaccharides, they were converted to O-pentafluorobenzoyl-methyl esters. Detection of the derivatives was made with the use of GC/NICI-MS.77 Successive methylation (CH2N2) and acetylation (Ac2O) followed by GC/MS analysis was used for identification and quantitation of atypical 3α,6β,7β,12β-tetrahydroxy5β-cholan-24-oic acid as well as other mono-, di-, tri- and tetrahydroxy bile acids in bile, serum and urine. 78 For quantification of the former acid, the ions at m/z 444 and 384 in its EI mass spectrum were selected. Ions at m/z 253 and 368, 255 and 370, 257 and 372, 217 and 374 were used for quantitative detection of cholic, chenodeoxycholic (and deoxycholic), lithocholic and 5β-cholanic acids, respectively. The same sequence of reactions can be used for the derivatization of phenolic acids. Sometimes, however, simultaneous acylation and esterification is employed. Phenolic acid is treated with a mixture of an alcohol and (C2F5CO)2O for 15–40 min at 60–75°C. It should be noted, however, that in the course of treatment with diazomethane, both carboxyl and phenolic OH groups can be methylated. The same situation is observed if methylation is carried out by means of tetramethylammonium hydroxide. Not only phenolic acids can undergo simultaneous esterification and acylation. Such a one-step procedure was used, for example, for the derivatization of 20hydroxyeicosatetraenic and 12-hydroxylauric acids with (C2F5CO)2O and C2F5CH2OH. Reaction was accomplished by heating a mixture of substrate and both of these reagents at 60°C for 45 min in a capped tube. 79 The procedure was used to demonstrate that both acids are formed from arachidonic and lauric acids, respectively, in kidney microsomal incubations. It is worth mentioning that subterminal fatty acids containing secondary alcoholic groups do not derivatize under the same conditions. For identification of these, fluorinated derivatives of omega hydroxyl fatty acids with positive and negative ion EI mass spectra are suitable (to register the latter, an ion trap was used with helium gas to thermolize the electrons). Positive ion mass spectra of the derivatives reveal a number of peaks characteristic of their particular structures: 624 Formation of Mixed Derivatives of Polyfunctional Compounds Figure 7. EI mass spectrum of 12-dimethylsilyloxy-octadecanoic acid methyl ester (reproduced with permission from NIST/EPA/NIH Library). F 379 365 351 F O F F O F F F O 365 O F F F 205 379 312 O 261 O F F O F F F O 261 345 F F F F F 205 The other common mixed derivatization of hydroxy acids involves esterification of the carboxyl group and silylation of the hydroxyl.77,80,81 EI mass spectra of such derivatives reveal rather negligible M+• peaks but have intense fragment ion peaks enabling the position of the OH group to be determined (Figure 7): +. CH3(CH2)nCH(CH2) COOCH3 m OSiMe3 . M+ + CH3(CH2) CH=OSiMe3 n + Me3SiO=CH(CH2) COOCH3 m It should be noted that such derivatizations can be used for the analysis of unsaturated fatty acids by GC/MS. In this case, acids are first converted into alkyl esters, then hydroxylated to form a vicinal diol grouping at the former double bond and, finally, the OH groups formed are silylated.5 Rather stable derivatives amenable to GC/MS can be prepared from hydroxy fatty acids by successive methylation with CH2N2 or CH3OH/acid and silylation with MTBSTFA and tert-butyldimethylsilylimidazole.82 EI mass spectra of the derivatives reveal prominent [M – 57]+ ion peaks and fragment ions indicating the location of secondary hydroxyl groups along the aliphatic chain from the ω-2 carbon to carbon number 5 in the original acids. Some additional information regarding chain length and the degree of unsaturation can also be deduced from such spectra. Valuable structural information was also extracted from the EI mass spectra of derivatives prepared by methylation followed by pertrimethylsilylation of polyhydroxy-unsaturated fatty acids.83 Monohydroxyeicosatetraenoic acids were successfully analyzed qualitatively and quantitatively by GC/MS in the form of their methyl ester trimethylsilyl, allyldimethylsilyl and tert-butyldimethylsilyl ethers.84 Successive esterification with C6F5CH2Br (20 min at 20°C in the presence of diisopropylethylamine) and silylation with BSTFA was used for structure elucidation of saturated and unsaturated hydroxy acids by GC/MS.85 These derivatives are interesting in that they can be investigated by both EI and NICI. The latter facilitates determination of the molecular weight whereas EI mass spectra provide a considerable amount of structural information because the main fragmentation is directed by the TMSO group. The most abundant fragment ions are observed when the site of unsaturation is two carbon atoms removed from the TMSO group: 181 F F F O O Me3Si F F O 461 Morphine glucuronides whose molecules include a carboxyl group as well as carbohydrate and phenolic OH groups were quantified in human plasma by GC/CI-MS (isotope dilution, monitoring of [M + H]+ ion) V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) after preliminary esterification with C6F5CH2Br followed by silylation with BSTFA.86 The second stage of derivatization can involve reaction with MTBSTFA, as was made, for example, for the profiling of valproic acid metabolites by GC/NICI-MS.87 In the bile acid series, conversion of the carboxyl group into an ester group (for example, carbomethoxy or isobutoxycarbonyl groups) may be followed by silylation of the alcoholic OH groups:88 COOH COOCH3 1. CH2N2 2. BSTFA HO H OH Me3SiO H OSiMe3 Such a procedure has been used, for example, for the characterization of ox bile, traditionally used in painting and consisting of bile acids (cholic, deoxycholic, lithocholic, chenodeoxycholic), fatty acids, hydroxyl fatty acids and cholesterol by GC/MS.89 It is interesting that ethyl chloroformate was used for the esterification; further silylation was accomplished with TMSIM. In further work,90 bile acids were analyzed by GC/EIMS after conversion of the carboxyl group into methyl or hexafluoroisopropyl esters followed by transformation of the OH groups into trimethylsilyloxy or trifluoroacetoxy groups. The EI mass spectra of the derivatives did not show any molecular ion peaks, the highest m/z corresponding to [M – CF3COOH]+•. The same principle was used as the basis for an elegant method for the determination of the degree of hydrolysis of unsaturated triacylglycerols and their oxidation products present in linseed oil-based paints. 91 The procedure included transethylation of glycerides by EtONa leading to a simultaneous hydrolysis of the glycerol–acyl bond and formation of ethyl esters. Free OH groups in the latter were further silylated with BSTFA and the resulting derivatives analyzed by GC/MS. The described methods for preparation of mixed derivatives, namely, the conversion of carboxyl groups into ester groups and acylation or silylation of alcoholic OH groups, is widely used for improving of physical–chemical properties of prostaglandins. In addition, EI mass spectra of derivatives are rather characteristic and useful for structure elucidation:92 Me3SiO COOCH3 Me3SiO Me3SiO m/z 513 The same sequence of reactions was adopted for simultaneous determination of endocannabinoids and 625 isoprostane in blood by GC/MS in SIM-mode. 93 For quantitation of prostanoids bearing only hydroxyl and carboxyl (for example, prostaglandins F2α and E2) in the form of similar derivatives, GC/EI-MS can be used. The [M – Me3SiOH]+• ions and tetradeuterio analogs as internal standards were involved in the measurement.94,95 Quantitative analysis of some similar prostaglandins can be accomplished by GC/EI-MS in a SIM mode by applying other fragment ions. For example, ions [M – C5H11 – Me3SiOH]+ (loss of pentyl radical as shown in the above structure) appeared to be more suitable for simultaneous determination of prostaglandins F2α, E1α, E2α and 19-hydroxy-F2α and - E2α in the semen of fertile men. Because prostaglandins can contain a few hydroxyl groups in addition to the carboxyl group, more complicated derivatization methods can be applied. For example, the analysis of prostaglandin F2α can be achieved with the aid of the following derivative, prepared as a result of three successive reactions:96 HO COOH HO 1. CH2N2 2. n-C4H9B(OH)2 3. BSTFA OH O COOCH3 C4H9-B O OSiMe3 As for the above mentioned compounds, the presence of the 15-trimethylsilyoxy group stimulates the EIinduced elimination of the C5H11 radical and the resulting characteristic ions can be used for quantitative analysis by GC/MS. Prostaglandins containing only carboxyl and hydroxyl groups have been determined in urine by GC/NICI-MS after esterification with pentafluorobenzyl bromide and further silylation with BSTFA.97 On the basis of the same procedure, a highly sensitive and selective method for the quantitative determination of 8-epi-15(R and S)-PGF2α in biological samples by GC/NICI-MS was described.98 Prostaglandins F1, F2 and F3 can be distinguished as similar derivatives by GC/CI-MS. Note that a comparative study of some of the abovementioned derivatization methods was performed for the analysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine.99 Esterification of the carboxyl group with CH2N2 followed by silylation with BSTFA or acylation with trifluoroacetic anhydride of phenolic and other hydroxyl groups as well as esterification with 2,2,2-trifluoroethanol and acylation with pentafluoropropionic anhydride appeared to be the most suitable approaches especially for quantitative determination of the drug metabolite by GC/MS in the NICI mode.100,101 626 Formation of Mixed Derivatives of Polyfunctional Compounds Mixed derivatives of oxo acids Even though the carbonyl group in oxo acids is less polar than carboxyl or hydroxyl groups, protection of both functional groups before the analysis by GC/MS is desirable. In the course of development of derivatization methods for the oxo acid series, problems may arise due to formation of side products. For example, if the convenient CH2N2 method of protecting the polar carboxyl group in α-keto acids is employed in the first derivatization step, not only esterification but addition of diazomethane to the oxo group may also take place: O O CH2N2 R-C-COOH O R-C-COOCH3 + R COOCH3 This is the reason why primary protection of the carbonyl group followed by modification of carboxyl group seems to be a more preferable procedure. The carbonyl group can be converted to an oxime with hydroxylamine. The second stage usually involves silylation providing protection for the OH groups of both carboxyl and oxime groups (if present):102 O 1. H2NOH.HCl 2. BSTFA R-C-COOH NOSiMe3 R-C-COOSiMe3 The first stage in this sequence can involve alkyl ethers of hydroxylamine, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine being one of the popular reagents. This reaction, followed by esterification of the free carboxyl group with MSTFA-TMCS was used, for example, for the GC/ MS determination of urinary oxoacids.103 Oximation was accomplished by adding the substituted hydroxylamine in a weakly acidic media (pH 2–3) at room temperature for 2 h. The EI mass spectra of such mixed derivatives showed M+• peaks, as well as peaks for the ions [M – CH3]+, m/z 181 [C6F5CH2]+, m/z 73, m/z 75 and m/z 255 [Me3Si=O+C6F5]. F F R CH2ONH2.HCl F R-C-(CH2) -COOH + n O F (CH2) -COOH n F N F CH2- F F F F R (CH2) -COOSIMe3 n F MSTFA/TMCS N A further method for step-wise derivatization of oxo acids is the formation of methyl esters of 2,4dinitrophenylhydrazone derivatives. 105 The oxoacid was allowed to interact with 2,4-dinitrophenylhydrazine in 15% aqueous HClO4 (30 min at 20°C). After evaporation, the residue was dissolved in 1.6 M HCl in CH3OH and heated at 70°C for 1 h. Mixed derivatives of oxo alcohols Many steroids and prostanoids, isolated from biological samples or prepared synthetically, contain hydroxyl as well as carbonyl groups. This is the reason that many derivatization methods in the oxo alcohol series have been developed for both. In many cases the physical–chemical properties of such polar compounds can be improved by one of the methods used to block the OH groups. Protection of both functional groups, however, is more desirable. One of the ways to derivatize oxo alcohols is similar to that employed for keto acids and involves conversion of the carbonyl group into an oxime group followed by silylation of all the OH groups. The formation of oximes results in a greater thermal stability than the underivatized steroids and prevents the formation of enol-ethers during the subsequent silylation procedure. When hydroxylamine is used for oxime formation, such silylation leads to the protection of all OH groups, including the hydroxyl in the oxime group.53 In the case of aldoses and ketoses, derivatization with hydroxylamine followed by peracetylation of all OH groups appeared to be an efficient methodology. The identification of the derivatives was achieved using both EI and CI (reagent gas, ammonia) GC/MS. 106 For the mass spectrometric determination of mono- and disaccharides, the use of perbenzoyl O-benzyloxime derivatives was suggested.107 When methoxylamine was used for oxime formation, silylation was employed only for the protection of native OH groups. Such methodology was used, for example, for the measurement of urinary 18-hydroxytetrahydro11-dehydrocorticosterone (main urinary metabolite of 18-hydroxycorticosterone) excretion rate in human by GC/EI-MS. 108,109 It should be noted that tertiary OH groups are silylated with difficulty.110 The EI mass spectra of methoxime-TMS-ethers of steroids usually reveal [M – OCH 3] + ions as well as the ions resulting from elimination of (CH3)3SiOH at different fragmentation stages (Figure 8). Some characteristic fragments resulting from cleavage of ring D are also observed:111 F CH2F CH2R F C=NOCH3 R1 R2 F In the case of prostanoid oxo acids, oximation with methoxyamine followed by esterification with PFB bromide was recommended to achieve very sensitive detection by GC/MS.104 CH2R -e Me3SiO + C=NHOCH3 C-R1 CHR2 V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 627 Figure 8. EI mass spectrum of 3,20-bis(O-methyloxime) of 17,21-bis(trimethylsilyloxy)-pregn-4-ene-3,11,20-trione (reproduced with permission from NIST/EPA/NIH Library). This two-step derivatization procedure was used for the measurement of D -glucose in plasma samples by GC/EI-MS112 and for the quantitative determination some monosacharides by stable-isotope-dilution GC/MS.113 In the case of corticosteroids, oximation proceeds quantitatively by treatment with CH3ONH2.HCl in pyridine at 60–80°C for 30 min. Excess of pyridine was evaporated and the residue was treated with MSTFA at 70–80°C for 15 min to 2 h:110,114 O O OH OH CH3ON 1) CH3ONH2.HCl/C5H5N 2) MSTFA-TMSI O OSiMe3 OSiMe3 hydroxylamine for 14 h at 60°C. The resulting product was heated with N-heptafluorobutyryl-imidazole for 30 min. at 60°C: F Me OH H F Me OH ONH2.HCl F H F F F F O N ON 19-Nortestosterone F F F F F F F F F F O F F Me O N F F F F F H O F F ON O cortisone CH3ON It should be noted that such oximes were eluted as syn– anti isomer pairs. Their EI mass spectra revealed the major [M – CH3O]+ ions that were used for the determination of corticosteroids in human urine by GC/MS in the ng mL–1 concentration range. The formation of methoxime may be followed by the acylation of native OH groups.115 An oxoalcohol was treated with a solution of CH 3ONH 2.HCl in pyridine at 22°C for 15 min. Then the mixture was treated with propionic anhydride at 56°C for 15 min. For mass spectrometric characterization and profiling of 19-norsteroids by electron-capture NICI/SIM, their step-wise conversion to pentafluorobenzyloxime-heptafluorobutyryl derivatives was used.116,117 The first stage was accomplished by heating a steroid oxo alcohol with O-pentafluorobenzyl F F F As previously discussed,1 some silylating agents can promote enolization of carbonyl groups and, hence, both the original and enolic hydroxyls in oxo alcohols can be protected by silyl groups. If there is no problem with the analysis of such silyl ethers of enols, which frequently arise in the form of a mixture of isomers and are not always formed quantitatively, oxo alcohols may be derivatized only by silylation.53 However, preliminary protection of a keto group by oximation removes the problem and further silylation results in the formation of the derivatives suitable for GC/MS analysis. Such an approach (oximation with pentafluorobenzylhydroxylamine.HCl followed by silylation with TMSIM) was used, for example, for the quantitative determination of corticosterone in rat and mouse plasma by GC/MS (SIM):118 628 Formation of Mixed Derivatives of Polyfunctional Compounds C6F5 N (CH3)3Si C6F5 O O Si(CH3)3 N Di(pentafluobenzyloxime)-di(trimethylsilyl) derivative of corticosterone The same procedure was also employed in the simultaneous determination of cortisone and cortisol in human nasal and bronchoalveolar lavage fluids and in plasma by GC/NICI-MS.119 Of course, oximation of the keto group may be followed by tert-butyldimethylsilylation of hydroxyls. Abundant ions [M – 57]+ in EI mass spectra of such derivatives can be used for sensitive determination of various oxo alcohols. A further derivatization procedure was used for profiling allopregnanolone and related neurosteroids in cerebrospinal fluids and plasma by GC/NICI-MS. 120 The following sequence of reactions was employed: oximation of carbonyl groups with carboxymethoxylamine.HCl (60°C for 45 min), esterification of the carboxyl group in the carboxymethoxime moiety with pentafluorobenzyl bromide and silylation of free OH groups with BSTFA. The intense [M – 181]– ions were selected for detection and quantitative determination of the above-mentioned steroids. Pentafluorobenzyloximation followed by trimethylsilylation was used for the analysis of unsaturated hydroxy aldehydes in aldehydic lipid peroxidation products.121 In this case, however, GC/EI-MS was used and structures of the products were established on the basis of their EI-induced fragmentation patterns. In specific cases, the derivatization is accomplished in a few stages. For example, steroids of the 17,21-dihydroxy-20oxo series, containing additional OH groups in other rings, are firstly transformed into cyclic boronates, then the 20-oxo group is converted to an oxime and, finally, free OH groups are silylated:53 O OH OH 1.n-C4H9B(OH)2 2.H2NOCH3 3.BSTFA CH3ON O O B-C4H9 HO Me3SiO the expense of the former followed by silylation or acylation of the latter groups. In the case of steroids containing a vicinal diol group, selective formation of a cyclic methylboronate ether is possible (exposure to CH3B(OH)2 at 60°C for 30 min). Further reaction with BSTFA gives rise to protection of the remaining OH groups.22 The same methodology can be used for the determination of carbohydrates and sugars.122 nButylboronate derivatives can significantly improve the mass spectral characteristics of 24,25-dihydroxy vitamin D when followed by silylation.123 Such mixed derivatives have been employed in routine GC/MS assays for 24,25- and 25,26dihydroxy vitamin D in human plasma.124 Monosaccharide molecules having hydroxyl groups of a different nature can be mixed derivatized in a specific way. Reaction with CH3OH/CH3COCl gives rise to the corresponding methyl glycosides. Further, their treatment with silylating agent results in protection of the remaining hydroxyl groups. The derivatives thus prepared are suitable for determining various saccharides (including acidic) by GC/EIMS.125 A further approach to the mixed derivatization of polyols, for example, in the steroid and prostaglandin series is reaction with N,O-bis(diethylhydrogensilyl)trifluoroacetamide in the presence of BSTFA.126,127 In this case, closely disposed hydroxyls form cyclic adducts whereas silyl ethers are formed for remote OH groups. Some EI mass spectral features of such derivatives in a steroid series have been elucidated with the aid of labeling experiments, metastable ion analysis and accurate mass measurements:128 O Si O H Si O Being rather acidic, phenolic hydroxyls can be protected with the same groups that are used for acids. Conversion of other alcoholic hydroxyls can be achieved with the aid of any reagent employed for alcohols. For example, 17β-estradiol was derivatized in two stages: reaction with C6F5CH2Br in AcCN (45 min, 60°C) gives rise to etherification of the phenolic 3-OH group whereas further treatment with BSTFA/TMCS permitted the protection of the 17-OH group. Quantitative analysis of this compound (limit of quantification 20 pg mL–1) in bovine plasma was accomplished with GC/NICI-MS/MS:129 OH Mixed derivatives of polyols and compounds containing both phenolic and alcoholic hydroxyls 1) C6F5CH2Br 2) BSTFA/TMCS F HO O 17 -Estradiol In addition to the one-step methods used for the protection of hydroxyl groups,1,2 polyols can also be mixed derivatized. If a polyol contains a closely disposed diol grouping and remote OH groups, it can be converted to a cyclic boronate ether at OSiMe3 F F F F Essentially the same derivatization (except that silylation was performed with TMSIM) and mass spectrometric V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) 629 Figure 9. EI mass spectrum of 6-ketoprostaglandin F1α methyloxime methyl ester tris(trimethylsilyl) derivative (reproduced with permission from NIST/EPA/NIH Library) methodology were used for quantitation of 17β-estradiol, estrone, 17α-ethynylestradiol and 16α-hydroxy-17β-etradiol (estriol) in ground water and swine lagoon samples.130 Mixed derivatives of compounds containing simultaneously hydroxy, oxo and carboxyl groups. Hydroxyl, oxo and carboxyl groups are frequently present at the same time in prostanoid molecules. Usually, a sequence of three reactions is used for derivatization of prostaglandins A and E. For example, the mass spectrometric (GC/MS, SIM mode) quantitative determination of 6-oxo-PGF1α as well as arachidonic acid metabolites was accomplished after successive methoximation, methylation and silylation (the EI mass spectrum is shown in Figure 9):131–133 N O COOH HO OH HO OH N COOCH3 OH CH2N2/ether OH OMe HO OH COOH CH3ONH2/pyridine OH N OMe BSTFA/pyridine Me3SiO Me3SiO OMe COOCH3 OSiMe3 Attempts to derivatize the prostanoid following the conventional sequence by carrying out the methyl ester formation first, resulted in poor yield of the derivative. Quantitation was performed with the hexadeuterated analog as internal standard and GC/MS in SIM mode, monitoring the [M – Me 3 SiOH – CH 3 O] + fragment ion. The same derivative and internal standard were used for quantitative determination of 6-ketoprostaglandin F1α in biological fluids by GC/CI-MS (gas reagent NH3) in SIM mode. The base peak of the [M + H – Me3SiOH]+ ion was chosen for measurement at the nanogram level.134 A similar derivatization approach, namely, methoximation of the carbonyl group with CH3ONH2, methylation of carboxyl group with CH2N2 and silylation of free hydroxyl groups with dimethylisopropylsilyl-imidazole has been recommended for the simultaneous analysis of prostanoids and thromboxane in clinical specimens (plasma, urine, cerebrospinal fluid and culture medium by GC/MS (SIM-mode).135 Deuterated analogs were used as internal standards. In another case (prostaglandin E 1 , 6-keto-PGF 1α ), methoximation of the carbonyl group (at 20°C for 16 h in the presence of pyridine) was followed by esterification with pentafluorobenzyl bromide (at 40°C for 15 min in the presence of diisopropylethylamine) and silylation (BSTFA). The NICI mass spectrum of the derivative showed an abundant ion [M – CH2C6F5]– that was used for quantification allowing a picogram detection limit to be achieved.136–138 Note that the presence of the TMSO group has little influence on fragmentation in the NICI mode. A different sequence of reactions, namely esterification with C6F5CH2Br in the presence of diisopropylethylamine, methoximation and silylation with BSTFA, was used for the simultaneous quantitative determination of prostaglandins E 1 and E 0 and 15-keto-prostaglandin E 0 in human plasma. 139 The NICI mass spectra of the derivatives showed a very abundant [M – CH2C6F5]– ion, the collisionally-induced dissociation of which gave rise to characteristic ions [M – CH2C6F5 – 2(CH3)3SiOH]– (for PGE1) and [M – CH2C6F5 – (CH3)3SiOH]– (for PGE0 and 15keto-PGE0) that were used for quantification. The use of pentafluorobenzylhydroxylamine for oximation has, of course, also been suggested. Such a reaction was 630 Formation of Mixed Derivatives of Polyfunctional Compounds included in a three-stage derivatization for the determination of misoprostol acid and 15-methyl-PGE2 in serum and breast milk samples.140 The procedure included successive esterification with C6F5CH2Br (in the presence of diisopropylethylamine), oximation with C6F5CH2ONH2.HCl and silylation (mixture of hexamethyldisilazane and TMCS). For quantitative determination, GC/NICI-MS/MS and the product ions [M – 2(CH3)3SiOH – C6F5CH2OH]– (for misoprostol acid) and [M – 2(CH3)3SiOH – C6F5CH2OH – CO2]– (for 15-methylPGE2) were used. Determination of 2,3-dinor-6-keto-prostaglandin F1α was accomplished after step-wise conversion of the compound into the n-propylamide (reaction with n-propylamine), Omethyloxime (reaction with CH 3ONH 2.HCl) and trisdimethylisopropylsilyl ethers (reaction with dimethylisopropylsilylimidazole).141 The EI mass spectrum of the derivatives revealed the M+• peak of low intensity, the base peak [M – 43]+ and many fragment ion peaks characteristic of the structure. Various kinds of oximation and esterification can be followed by silylation of free OH groups with a tertbutyldimethylsilyl-containing agent. The EI mass spectra of the resulting derivatives show abundant [M – 57] + peaks permitting the highly selective and sensitive SIMmicroanalysis of prostaglandins in biological tissues and fluids to be made.84,142 Miscellaneous mixed derivatization Some other uncommon mixed derivatization procedures can be employed for the analysis of multi-functional compounds. For example, aromatic amino acids can be successively diazotated to convert the amino group into an OH group and then esterified (methylated). Employed for anthranilic acid, this sequence of reactions gives rise to the methyl ester of salicylic acid that may be further silylated.53 Multi-step derivatization was suggested for the quantitative determination of 3-nitrotyrosine in human plasma by GC/NICI-MS in SIM mode.143 The sequence of reactions included reduction of 3-nitrotyrosine by dithionite to 3aminotyrosine, acylation with heptafluorobutyric anhydride, partial hydrolysis with HCl and, finally, methylation with trimethylsilyldiazomethane: NH2 NH2 OH O NHCOC3F7 OH O reduction OCOC3F7 A rather specific mixed derivatization was suggested for the determination of aldoses and uronic acids in polysaccharide hydrolysates from plant gums used in art works by GC/MS.144 The first stage was based on the finding that the reaction of aldoses and uronic acids with C2H5SH and CF3COOH proceeded through intermediate formation of acyclic aldehyde forms that gave rise to diethyldithioacetals in high yield. Hydroxyl groups were further protected by trimethylsilylation or acetylation (Ac2O). The paper includes retention indices and EI mass spectra of monosaccharide standard derivatives: H EtSH/H+ HO OH OH 3-nitrotyrosine OH NHCOC3F7 O NHCOC3F7 OCOC3F7 O OSiMe3 O O A two-step derivatization was tested and optimized for the sensitive determination of dihydrostreptomycin (a semisynthetic amino-glycoside antibiotic which is commonly used in meat production) by GC/EI-MS.145 The procedure involved the simultaneous cyclization of guanidine groups with hexafluoroacetone and trimethylsilylation of OH groups (by TMSIM). The EI mass spectrum of the resulting derivative revealed abundant fragment ions characterizing the derivatized streptidine as well as the derivatized dihydrostreptose and Nmethylglucosamine that could be used in SIM measurements: CF3 CF3 NH H2N NH HO NH OH N H OH OHH2N HO SiMe3 NH OSiMe3 O O O TMSI, CF3COCH2COCF3 SiMe3 CF3 N H O O Me3SiO HO NH HO CF3 CH3 H3C OSiMe3 O O Me3SiO OH O O NH H3C O SiMe3 CH3 SiMe3 A further example of mixed derivatization was found in the analysis of heterocyclic amines in mainstream cigarette smoke by GC/NICI-MS.146 Amino groups in heterocyclic compounds such as 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-1-methyl-6-phenylimidazo[4,5-d]pyridine were heptafluorobutylated and methylated in two steps. Using this derivatization procedure, the detection level of the compounds mentioned was as low as 0.5 ng/cigarette: (F7C3CO)2O N CH3 N NH2 N N N O NHCOC3F7 OMe H N N O C3F7 OMe Me3SiCHN2 NHCOC3F7 OH OSiMe3 Me3SiO O OH NHCOC3F7 OH partial hydrolysis HMDS O HFBA NH2 SEt EtS OH OH OH CH3 NO2 SEt EtS COOH O OH CH3 (CH3)2N-CH(OCH3)2 N N CH3 O N C3F7 V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005) We believe that many other mixed derivatization procedures have been and will be developed for the analysis of particular multi-functional compounds by GC/MS and other mass spectrometric techniques. Mixed derivatization for “soft” ionization mass spectrometry will be discussed in our forthcoming review. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. J.M. Halket and V.G. Zaikin, “Derivatization in mass spectrometry—1. Silylation”, Eur. J. Mass Spectrom. 9, 1 (2003). V.G. Zaikin and J.M. Halket, “Derivatization in mass spectrometry—2. Acylation”, Eur. J. 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