Download Review: Derivatization in mass spectrometry—6. Formation of mixed

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. Mass Spectrom. 9, 421
(2003).
J.M. Halket and V.G. Zaikin, “Derivatization in mass
spectrometry—3. Alkylation(arylation)”, Eur. J. Mass
Spectrom. 10, 1 (2004).
V.G. Zaikin and J.M. Halket, “Derivatization in mass
spectrometry—4. Formation of cyclic derivatives”, Eur. J.
Mass Spectrom. 10, 555 (2004).
J.M. Halket and V.G. Zaikin, “Derivatization in mass
spectrometry—5. Specific derivatization of monofunctional
compounds”, Eur. J. Mass Spectrom. 11, 127 (2005).
B. Rozynov, “Characterization of N-formyl amino acids,
amino acid methyl esters and N-formyl amino acid methyl
esters by conventional electron impact mass spectrometry”,
Poster presented on the 52th ASMS Conference on Mass
Spectrometry and Allied Topics, Nashvill, USA (2004).
D.E. Matthews, E. Ben-Galim and D.M. Bier, “Determination
of stable isotopic enrichment in individual plasma amino acids
by chemical ionization mass spectrometry”, Anal. Chem. 51,
80 (1979).
C. Deng, C. Shang, Y. Hu and X. Zhang, “Rapid diagnosis
of phenylketonuria and other aminoacidemias by quantitative
analysis of amino acids in neonatal blood spots by gas
chromatography-mass spectrometry”, J. Chromatogr. B 775,
115 (2002).
A. Pons, C. Richet, C. Robbe, A. Herrmann, P. Timmerman,
G. Huet, Y. Leroy, I. Carlstedt, C. Capon and J.-P. Zanetta,
“Sequential GC/MS analysis of sialic acids, monosaccharides,
and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives”, Biochemistry 42, 8342 (2003).
P. Dallakian and H. Budzikiewicz, “Gas chromatographychemical ionization mass spectrometry in amino acid analysis
of pyoverdins”, J. Chromatogr. A 787, 195 (1997).
K.R. Leimer, R.H. Rice and C.W. Gehrke, “Complete mass
spectra of N-trifluoroacetyl-n-butyl esters of amino acids“, J.
Chromatogr. 141, 121 (1977).
J.M. Halket, K. Blau and S. Down (Eds), GC/MS Companion
Handbook of Amino Acid Derivatives, Part II – Trifluoroacetyl,
n-Butyl Ester Derivatives. HD Sciences, Nottingham, UK
(1990).
NIST/EPA/NIH Mass Spectral Library, v. 2.0 and 2.0d.
National Institute of Standards and Technology, Gaithersburg,
USA (2002 and 2005).
631
14. M.F. Schulman and F.P. Abramson, “Plasma amino acid
analysis by isotope ratio gas chromatography mass spectrometry computer techniques”, Biomed. Mass Spectrom.
2, 9 (1975).
15. V.G. Zaikin and V.V. Luzhnov, “Use of cycloalkylcarbonyl
derivatives for the determination of amino acid methyl esters
by gas chromatography-mass spectrometry”, J. Anal. Chem.
(Russian) 57, 605 (2002).
16. A. Brückner and A. Schieber, “Determination of free Damino acids in mammalian by chiral gas chromatographymass spectrometry”, J. High Res. Chromatogr. 23, 576 (2000).
17. P. Dallakian, J. Voss and H. Budzikiewicz, “Assignment of
the absolute configuration of the amino acids of pyoverdins by
GC/MS”, Chirality 11, 381 (1999).
18. B. Glaser and W. Amelung, “Determination of 13C natural
abundance of amino acid enantiomers in soil: methodological
considerations and first results”, Rapid Commun. Mass
Spectrom. 16, 891 (2002).
19. A. Royer, S. Beguin, J.C. Tabet, S. Hulot, M.A. Reding and
P.Y. Communal, “Determination of glyphosate and aminomethylphosphonic acid residues in water by gas chromatography with tandem mass spectrometry after exchange ion
resin purification and derivatization. Application on vegetable
matrixes”, Anal. Chem. 72, 3826 (2000).
20. Z.H. Kudzin, D.K. Gralak, J. Drabowicz and J. Łuczak, “Novel
approach for the simultaneous analysis of glyphosate and its
metabolites”, J. Chromatogr. A 947, 129 (2002).
21. M.W. Dunkan and A. Poljak, “Amino acid analysis of peptides
and proteins on the femtomole scale by gas chromatography/
mass spectrometry”, Anal. Chem. 70, 890 (1998).
22. K. Blau and J.M. Halket (Eds). Handbook of Derivatives
for Chromatography. John Wiley & Sons, Chichester, UK
(1993).
23. O. Vandenabeele-Trambouze, C. Rodier, M. Dobrijevic,
D. Despois, R. Sternberg, C. Vidal-Madjar, M.F. GrenierLoustalot and F. Raulin, “Identification of amino acids
by capillary gas chromatography. Application to Martian
samples”, Chromatographia 53, S332 (2001).
24. P. Husek, “Chloroformates in gas chromatography as general
purpose derivatizing agents”, J. Chromatogr. B 717, 57
(1998).
25. J. Wang, Z.-H. Huang, D.A. Gage and J.Th. Watson, “Analysis
of amino acids by gas chromatography—flame ionization
detection and gas chromatography—mass spectrometry:
Simultaneous derivatization of functional groups by an
aqueous-phase
chloroformate-mediated
reaction”,
J.
Chromatogr. 663, 71 (1994).
26. A. Namera, M. Yashiki, M. Nishida and T. Kojima, “Direct
extract derivatization for determination of amino acids in
human urine by gas chromatography and mass spectrometry”,
J. Chromatogr. B 776, 49 (2002).
27. M. Pan, T.J. Mabry, P. Cao and M. Moini, “Identification of
nonprotein amino acids from cycad seeds as N-ethoxycarbonyl
ethyl ester derivatives by positive chemical-ionization gas
chromatography-mass spectrometry”, J. Chromatogr. A 787,
288 (1997).
632
28. A. Derieux, S. Rochut, M.-C. Papillon and C. Pepe,
“Identification des colles protéiques présentes dans les œuvres
d’art par couplage GC/SM à trappe d’ions”, C.R. Acad. Sci.
Paris, Chimie/Chemistry 4, 295 (2001).
29. R. Mateo-Castro, J.V. Gimeno-Adelantado, F. Bosh-Reig, A.
Domenech-Carbo, M.J. Casas-Catalan, L. Osete-Cortina, J. De
la Cruz-Canizares and M.T. Domenech-Carbo, “Identification
by GC-FID and GC-MS of amino acids, fatty acids and bile
acids in binding media used in works of art”, Fresenius J.
Anal. Chem. 369, 642 (2001).
30. J. Pietzsch, U. Julius and M. Hanefeld, “Stable isotope ratio
analysis of amino acids: the use of N(O,S)-ethoxycarbonyl
trifluoroethyl ester derivatives and gas chromatography/mass
spectrometry”, Rapid Commun. Mass Spectrom. 11, 1835
(1997).
31. J. Pietzsch, S. Kopprasch and R. Bergmann, “Analysis of 3chlorotyrosine as a specific marker of protein oxidation: the
use of N(O,S)-ethoxycarbonyltrifluoroethyl ester derivatives
and gas chromatography/mass spectrometry”, Rapid Commun.
Mass Spectrom. 17, 767 (2003).
32. P. Cao and M. Moini, “Quantitative analysis of fluorinated
ethylchloroformate derivatives of protein amino acids and
hydrolysis products of small peptides using chemical ionization
gas chromatography-mass spectrometry”, J. Chromatogr. A
759, 111 (1997).
33. P. Cao and M. Moini, “Quantitative analysis of fluorinated
ethylchloroformate derivatives of non-protein amino
acids using positive and negative chemical ionization gas
chromatography-mass spectrometry”, J. Chromatogr. A 710,
303 (1995).
34. T.G. Sobolevsky, A.I. Revelsky, I.A. Revelsky, B. Miller and
V. Oriendo, “Electron ionization mass spectra of N(O,S)isobutoxycarbonyl isobutyl esters of amino acids”, Eur. J.
Mass Spectrom. 8, 447 (2002).
35. T.G. Sobolevsky, A.I. Revelsky, I.A. Revelsky, B. Miller and V.
Oriendo, “Simultaneous determination of fatty, dicarboxylic
and amino acids based on derivatization with isobutyl
chloroformate followed by gas chromatography-positive ion
chemical ionization mass spectrometry”, J. Chromatogr. B
800, 101 (2004).
36. T.G. Sobolevsky, A.I. Revelsky, B. Miller, V. Oriendo,
E.S.Chernetsova and I.A. Revelsky, “Comparison of silylation
and esterification/acylation procedures in GC-MS analysis of
amino acids”, J. Sep. Sci. 26, 1474 (2003).
37. R.S. Borisov, A.A. Richkov, B.V. Vas’kovsky and V.G. Zaikin,
“Differentiation of leucine and isoleucine in the form of
derivatives by gas chromatography/electron ionization-mass
spectrometry”, Mass Spectrometry (Russian) 1, 199 (2004).
38. J.T. Simpson, D.S. Torok and S.P. Markey, “Pentafluorobenzyl
chloroformate derivatization for enhancement of detection
of amino acids or alcohols by electron capture negative ion
chemical ionization mass spectrometry”, J. Am. Soc. Mass
Spectrom. 6, 525 (1995).
39. J.T. Simpson, D.S. Torok, J.E. Girard and S.P. Markey, “Analysis
of amino acids in biological fluids by pentafluorobenzyl
chloroformate derivatization and detection by electron capture
Formation of Mixed Derivatives of Polyfunctional Compounds
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
negative ionization mass spectrometry”, Anal. Biochem. 233,
58 (1996).
C. Rodier, R. Sternberg, F. Raulin and C. Vidal-Madjar,
“Chemical derivatization of amino acids for in situ analysis of
Martian samples by gas chromatography”, J. Chromatogr. A
915, 199 (2001).
C. Haberhauer-Troyer, G. Alvarez-Llamas, E. Zitting, P.
Rodriguez-Gonzalez, E. Rosenberg and A. Sanz-Medel,
“Comparison of different chloroformates for the derivatization of seleno amino acids for gas chromatographic analysis”,
J. Chromatogr. A 1015, 1 (2003).
A.P. Vonderheide, M. Montes-Bayon and J.A. Caruso, “Solidphase microextraction as a sample preparation strategy for
the analysis of seleno amino acids by gas chromatographyinductively coupled plasma mass spectrometry”, Analyst 127,
49 (2002).
F. Montigon, J.J. Boza and L.B. Fay, “Determination of 13Cand 15N-enrichment of glutamine by gas chromatography/
mass spectrometry and gas chromatography/combustion/
isotope ratio mass spectrometry after N(O,S)-ethoxycarbonyl
ethyl ester derivatization”, Rapid Commun. Mass Spectrom.
15, 116 (2001).
S. Casal, M.B. Oliveira and M.A. Ferreira, “Gas
chromatographic quantification of amino acid enantiomers
in food matrices by their N(O,S)-ethoxycarbonyl
heptafluorobutyl ester derivatives”, J. Chromatogr. A 866,
221 (2000).
K.R. Kim, J.H. Kim, E.-J. Cheong and C.-M. Jeong, “Gas
chromatographic amino acid profiling of wine samples for
pattern recognition”, J. Chromatogr. A 722, 303 (1996).
K.R. Kim, J.-H. Kim, C.-H. Oh and T.J. Mabry, “Capillary
gas chromatography of protein amino acids as N(O,S)-isobutoxycarbonyl tert-butyldimethylsilyl derivatives in aqueous
samples”, J. Chromatogr. 605, 241 (1992).
H. Kataoka, S. Matsumura, H. Koizumi and M. Makita,
“Rapid and simultaneous analysis of protein and non-protein
amino acids as N(O,S)-isobutoxycarbonyl methyl ester derivatives by capillary gas chromatography”, J. Chromatogr. A
758, 167 (1997).
V. Maurino, C. Minero, E. Pellizzetti, S. Angelino and M.
Vincenti, “Ultratrace determination of highly hydrophilic
compounds by 2,2,3,3,4,4,5,5-octafluoropentyl chloroformatemediated derivatization directly in water”, J. Am. Soc. Mass
Spectrom. 10, 1328 (1999).
P. Campins-Falco, R. Herraez-Hernandez, A. SevillanoCabeza and I. Trumpler, “Derivatization of amines in
solid-phase extraction supports with 9-fluorenylmethyl
chloroformate for liquid chromatography”, Anal. Chim. Acta
344, 125 (1997).
C. Minero, M. Vincenti and E. Pelizzetti, “Determination of
ethylene glycol in aqueous matrix by direct derivatization
with hexyl chloroformate”, Ann. Chim. 83, 511 (1993).
C. Minero, M. Vincenti, S. Lago and E. Pelizzetti, “Determination
of trace amounts of highly hydrophilic compounds in water by
direct derivatization and gas chromatography-mass spectrometry”, Fresenius J. Anal. Chem. 350, 403 (1994).
V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005)
52. K. Biemann and S.A. Martin, “Mass spectrometric determination of the amino acid sequence of peptides and proteins“,
Mass Spectrom. Rev. 6, 1 (1987).
53. D.R. Knapp, Handbook of analytical derivatization reactions.
John Wiley & Sons, New York, USA (1979).
54. R.A. Day, H. Falter and J. Lehman, “N-Terminal groups in
mass spectrometry of peptides: A study including some new
and useful derivatives”, J. Org. Chem. 38, 782 (1973).
55. Ju.A. Ovchinnikov, A.A. Kirjushkin, V.A. Gorlenko and
B.V. Rozinov, “Mass spectrometric determination of amino
acid sequence in peptides. Desulphurization and complete Nmethylation of sulphur-containing peptides”, J. Gen. Chem.
(Russian) 41, 660 (1971).
56. P. Cao and M. Moini, “Rapid derivatization and gas
chromatography/mass spectrometry analysis of dipeptides in
aqueous solution”, Rapid Commun. Mass Spectrom. 11, 349
(1997).
57. P. Capitan, T. Malmezat, D. Breuille and C. Obled, “Gas
chromatographic-mass spectrometric analysis of stable
isotopes of cysteine and glutathione in biological samples”, J.
Chromatogr. B 732, 127 (1999).
58. V.G. Zaikin, R.S. Borisov, B.V. Vas’kovsky and L.Yu. Sklyarov,
“Derivatization of dipeptides by alkyl chloroformate/alkanol
for GC/EI-MS analysis: differentiation of isomers”, Poster
presented on the 52th ASMS Conference on Mass spectrometry and Allied Topics, Nashville, USA (2004).
59. K. Biemann, F. Gapp and J. Seibl, “Application of mass spectrometry to structure problems. I. Amino acid sequence in
peptides“, J. Am. Chem. Soc. 81, 2274 (1959).
60. H. Nau, H.-J. Forster and J.A. Kelley, “Polypeptide sequencing
by a gas chromatograph-mass spectrometer computer system.
II. Characterization of complex mixtures of oligopeptides
as trimethylsilylated polyamino alcohols”, Biomed. Mass
Spectrom. 2, 326 (1975).
61. H.J. Nau, “Gas chromatography-mass spectrometry of
permethylated peptides and their reduced and trimethylsilylated
derivatives”, J. Chromatogr. 121, 376 (1976).
62. R.A. Clare, D.S. Davies and T.A. Baillie, “The analysis of
terbutaline in biological fluids by gas chromatography electron
impact mass spectrometry”, Biomed. Mass Spectrom. 6, 31
(1979).
63. K. Jacob, W. Vogt, M.J. Knedel and G. Schwertfeger,
“Quantitation of adrenaline and noradrenaline from human
plasma by combined gas chromatography-high-resolution
mass fragmentography”, J. Chromatogr. 146, 221 (1978).
64. W.B. Martin, J.A. Hurlbut, J.M. Storey, K.C. Faul and T.L.
Thomas, “A simple two-stage derivatization method for GC/
MS analysis of ephedra-alkaloids”, Proceedings of ASMS
Conference on Mass Spectrometry and Allied Topics 1467
(1998).
65. L. Damasceno, R. Ventura, J. Ortuno and J. Segura,
“Derivatization procedures for the detection of β2-agonists by
gas chromatographic/mass spectrometric analysis”, J. Mass
Spectrom. 35, 1285 (2000).
66. A.S. Christophersen, E. Hovland and K.E. Rasmussen, “Glass
capillary column gas chromatography of phenolalkylamines
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
633
after flash-heater derivatization using a double injection technique“, J. Chromatogr. 234, 107 (1982).
D.K. Crockett, E.L. Frank and W.L. Roberts, “Rapid analysis
of metanephrine and normethanephrine in urine by gas
chromatography-mass spectrometry”, Clin. Chem. 48, 332
(2002).
M. Preu, D. Guyot and M. Petz, “Development of a gas
chromatography-mass spectrometry method for the analysis of
aminoglycoside antibiotics using experimental design for the
optimization of the derivatization reactions”, J. Chromatogr.
A 818, 95 (1998).
J. Vink, H.J.M. van Hal and C.J. Timmer, “Determination of
nanongram amounts of the antiarrhythmic drug Org 6001 in
biological fluids and tissues using selected ion monitoring”,
Biomed. Mass Spectrom. 7, 592(1980).
N.U. Olsson, P. Kaufmann and S. Dzeletović, “Preparation
and gas chromatographic-mass spectrometric analysis of Nacetyl-O-trimethylsilyl derivatives of long-chain base residues
of natural sphingomyelin”, J. Chromatogr. B 698, 1 (1997).
C.H. Hocart, J. Wang and D.S. Letham, “Derivatives of
cytokinins for negative ion mass spectrometry”, J. Chromatogr.
A 811, 246 (1998).
F. Saltron, Y. Berthoz, R. Rues, N. Auguin and L. Belhade,
“Structural elucidation of clenbuterol- and mabuterol-like
compounds in complex matrices using silylated and nbutylboronate derivatives by gas chromatography/electron
impact and chemicalk ionization mass spectrometry”, J. Mass
Spectrom. 31, 810 (1996).
J. Lee, K. Kim and D.-S. Lho, “Analysis of cyclic boronated and
trimethylsilylated phenolalkylamines by gas chromatography
and electron impact mass spectrometry”, Rapid Commun.
Mass Spectrom. 13, 1491 (1999).
C.J.W. Brooks, C.G. Edmonds and S.J. Gaskell, “Derivatives
suitable for GC-MS”, Chem. Phys. Lipids 21, 403 (1978).
J.M. Halket, P.J. Watkins, A. Przyborowska, B.L. Goodwin,
A. Clow, V. Glover and M. Sandler, “Isatin (indole-2,3-dione)
in urine and tissues: detection and determination by gas chromatography-mass spectrometry”, J. Chromatogr. Biomed.
Appl. 562, 279 (1991).
A. Pons, J. Popa, J. Portoukalian, J. Bodennec, D. Ardail, O.
Kol, M.J. Martin-Martin, P. Hueso, P. Timmerman, Y. Leroy
and J.P. Zanetta, “Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion”,
Anal. Biochem. 284, 201 (2000).
Z. Mielniczuk, S. Alugupalli, E. Mielniczuk and L. Larsson,
“Gas chromatography-mass spectrometry of lipopolysaccharide 3-hydroxy fatty acids: comparison of pentafluorobenzoyl
and trimethylsilyl methyl ester derivative”, J. Chromatogr.
623, 115 (1992).
S. Perwaiz, D. Forrest, D. Mignault, B. Tuchweber, M.J.
Phillip, R. Wang, V. Ling and I.M. Yousef, “Appearance of
atypical 3α,6β,7β,12β-tetrahydroxy-5β-cholan-24-oic acid in
spgp knockout mice”, J. Lipid Res. 44, 494 (2003).
M. Zamaitis, S. Poloyac and R. Frye, “Identification of
omega hydroxyl fatty acids in biological samples as their
634
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
Formation of Mixed Derivatives of Polyfunctional Compounds
pentafluoropropyl derivatives by gas chromatography/mass
spectrometry with positive and negative ion detection”, Rapid
Commun. Mass Spectrom. 16, 1411 (2002).
D.J. Harvey, J.M. Tiffany, J.M. Duerden, K.S. Pandher and L.S.
Mengher, “Identification by combined gas chromatographymass spectrometry of constituent long-chain fatty acids
and alcohols from the meibomian glands of the rat and a
comparison with human meibomian lipids”, J. Chromatogr.
414, 253 (1987).
G. Gutnikov, “Fatty acid profiles of lipid samples”, J.
Chromatogr. B 671, 71 (1995).
D.L. Chance, K.O. Gerhardt and T.P. Mawhinney, “Gasliquid chromatography-mass spectrometry of hydroxyl fatty
acids as their methyl esters tert-butyldimethylsilyl ethers”, J.
Chromatogr. A 793, 91 (1998).
C.T. Hou, H.W. Gardner and W. Brown, “12,13,16-Trihydroxy9(Z)-octadecenoic acid, a possible intermediate in the
bioconversion of linoleic acid to tetrahydrofuranyl acids
by Clavibacter sp. ALA2”, J. Am. Oil Chem. Soc. 78, 1167
(2001).
S. Steffenrud, P. Borgeat, H. Salari, M.J. Evans and M.J.
Bertrand, “Gas chromatography-mass spectrometry of
monohydroxyeicosatetraenoic acids as their methyl esters
trimethylsilyl, allyldimethylsilyl, and tert-butyldimethylsilyl
ethers”, J. Chromatogr. 416, 219 (1987).
P. Wheelan, J.A. Zirrolli and R.C. Murphy, “Analysis of
hydroxyl fatty acids as pentafluorobenzyl ester, trimethylsilyl
ether derivatives by electron ionization gas chromatography/
mass spectrometry”, J. Am. Soc. Mass Spectrom. 6, 40 (1995).
H.J. Leis, G. Fauler, G. Raspotnig and W. Windischhofer,
“Quantitative gas chromatographic/mass spectrometric
analysis of morphine glucuronides in human plasma by
negative ion chemical ionization mass spectrometry”, J. Mass
Spectrom. 37, 395 (2002).
M.R. Anari, R.W. Burton, S. Gopaul and F.S. Abbott,
“Metabolic profiling of valproic acid by cDNA-expressed
human cytochrome P450 enzymes using negative-ion
chemical ionization gas chromatography-mass spectrometry”,
J. Chromatogr. B 742, 217 (2000).
C. Tsaconas, P. Padieu, G. Maume, M. Chessebeuf, N. Hussein
and N. Pitoizet, “Gas chromatography-mass spectrometry of
isobutyl ester trimethylsilyl ether derivatives of bile acids and
application to the study of bile sterol and bile acid biosynthesis in rat liver epithelial cell lines“, Anal. Biochem. 157,
300 (1986).
M.J. Casas-Catalan, M.T. Doménech-Carbo, R. Mateo-Castro,
J.V. Gimeno-Adelantado and F. Bosch-Reig, “Characterization
of bile acids and fatty acids from ox bile in oil paintings by gas
chromatography-mass spectrometry”, J. Chromatogr. A 1025,
269 (2004).
R. Edenharder and J. Slemz, “Gas chromatographic and
mass spectrometric analysis of bile acids as trifluoroacetylhexafluoroisopropyl and heptafluorobutyryl derivatives”, J.
Chromatogr. 222, 1 (1981).
J.D.J. van den Berg, K.J. van den Berg and J.J. Boon,
“Determination of the degree of hydrolysis of oil paint samples
using a two-step derivatization method and on-column GC/
MS”, Progr. Org. Coating 41, 143 (2001).
92. H. Miyazaki, M. Ishibashi and K. Yamashita, “Use of
new silylating agents for separation and identification of
prostaglandins by gas chromatography-mass spectrometry”,
J. Chromatogr. 153, 83 (1978).
93. T. Obata, Y. Sakurai, Y. Kase, Y. Tanifuji and T. Horiguchi,
“Simultaneous
determination
of
endocannabinoids
(arachidonylethanolamide and 2-arachidonylglycerol) and
isoprostane (18-epiprostaglandin F2α) by gas chromatographymass spectrometry-selected ion monitoring for medical
samples”, J. Chromatogr. B 792, 131 (2003).
94. B. Sjoquist, E. Oliw and I. Lunden, “Mass fragmentographic
determination of prostaglandin F2α in human and rabbit urine”,
J. Chromatogr. 163, 1 (1980).
95. C. Fischer, “A PGE2-derivative for quantitative gas
chromatographic-mass spectrometric measurement in the
selected ion monitoring mode”, Biomed. Mass Spectrom. 11,
114 (1984).
96. A.G. Smith and C.J.W. Brooks, “Gas chromatography chemical
ionization mass spectrometry of prostaglandin F two alpha
cyclic boronate derivative”, Biomed. Mass Spectrom. 4, 258
(1977).
97. C. Thevenon, M. Guichardant and M. Lagarde, “Gas
chromatographic-mass spectrometric measurement of 15-deoxyΔ12,14-prostaglandin J2, the peroxisome proliferators-activated
receptor γ ligand, in urine”, Clin. Chem. 47, 768 (2001).
98. C. Signorini, M. Comporti and G. Giorgi, “Ion trap tandem
mass spectrometric determination of F2-isoprostanes”, J.
Mass Spectrom. 38, 1067 (2003).
99. M. Scirmai, O. Beck, N. Stephansson and M.M. Halldin,
“A GC-MS study of three major acidic metabolites of Δ1tetrahydrocannabinol“, J. Anal. Toxicol. 20, 573 (1996).
100. D. Rosenthal, T.M. Harvey, D.R. Brine and M.E. Wall,
“Comparison of gas chromatography mass spectrometry
methods for the determination of Δ9-tetrahydrocannabinol in
plasma“, Biomed. Mass Spectrom. 5, 312 (1978).
101. L. Karlsson, J. Jonsson, K. Abert and C. Roos, “Determination
of Δ9-tetrahydrocannabinol-11-oic acid in urine as its pentafluoropropyl-, pentafluoropropionyl derivative by GC/MS
utilizing negative ion chemical ionization“, J. Anal. Toxicol.
7, 198 (1983).
102. R.A. Chalmers, J.M. Halket and G.A. Mills (Eds),
GC/MS Companion, Organic Acid Derivatives. HD Science,
Nottingham, UK (1993).
103. S.H. Lee, S.O. Kim and B.C. Chung, “Gas chromatographicmass spectrometric determination of urinary oxoacids using O(2,3,4,5,6-pentafluorobenzyl)oxime-trimethylsilyl ester derivatization and cation-exchange chromatography”, J. Chromatogr.
B 719, 1 (1998).
104. K.W. Waddell. I.A. Blair and J. Wellby, “Combined capillary
column gas chromatography negative ion chemical ionization
mass spectrometry of prostanoids”, Biomed. Mass Spectrom.
10, 8 (1983).
105. R. Navarro-Gonzalez, A. Negron-Mendoza and G.
Albarran, “Analysis of keto acids as their methyl esters
V.G. Zaikin and J.M. Halket, Eur. J. Mass Spectrom. 11, 611–636 (2005)
of
2,4-dinitrophenylhydrazone
derivatives
by
gas
chromatography and gas chromatography-mass spectrometry”,
J. Chromatogr. 587, 247 (1991).
106. F.R. Seymour, E.C.M. Chen and J.E. Stouffer, “Identification
of ketoses by using their peracetylated oxime derivatives: a
G.L.C.-M.S. approach”, Carbohydr. Res. 83, 201 (1980).
107. R.M. Thompson and D.A. Cory, “Mass spectrometry of
some ultraviolet absorbing derivatives of sugars and related
alditols: identification in biologic fluids after separation by
high performance liquid chromatography”, Biomed. Mass
Spectrom. 6, 117 (1979).
108. N. Hirota, T. Furuta and Y. Kasuya, “Determination of cortisol
in human plasma by capillary gas chromatography-mass
spectrometry using [2H5] cortisol as an internal standard”, J.
Chromatogr. 425, 237 (1988).
109. L.A. Shakerdi, J.M.C. Connell, R. Frazer and A.M. Wallace,
“Analysis of biological samples by gas chromatography-mass
spectrometry without a reference stanbdard: measurement
of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone
excretion rate in human subjects”, J. Chromatogr. B 784, 367
(2003).
110. B.K. Yap, G.A.R. Johnston and R. Kazlauskas, “Routine
screening and quantitation of urinary corticosteroids using
bench-top gas chromatpgraphy-mass spectrometry detection”,
J. Chromatogr. 573, 183 (1992).
111. C.J.W. Brooks, “Some aspects of mass spectrometry in
research on steroids”, Philos. Trans. Roy. Soc. London A 293,
53 (1979).
112. D. Kury and U. Keller, “Trimethylsilyl-O-methyloxime
derivatives for the measurement of [6,6-2H2]-D -glucose
enriched plasma samples by gas chromatography-mass
spectrometry”, J. Chromatogr. 572, 302 (1991).
113. O. Pelletier and S. Cadieux, “Quantitative determination
of glucose in serum by isotope-dilution mass spectrometry following gas-liquid chromatography with fused silica
column“, Biomed. Mass Spectrom. 10, 130 (1983).
114. M.C. Dumasia, “In vivo biotransformation of 17αmethyltestosterone in the horse revisited: identification
of 17-hydroxymethyl metabolites in equine urine by gas
chromatography/mass spectrometry”, Rapid Commun. Mass
Spectrom. 17, 320 (2003).
115. R. Meatherall, “GC-MS confirmation of codeine, morphine,
6-acetylmorphine, hydrocodone, hydromorphone, oxycodone,
and oxymorphone in urine”, J. Anal. Toxicol. 23, 177 (1999).
116. D. de Boer, S.N. Bensink, A.R. Borggreve, R.D. van Ooijen
and R.A.A. Maes, “Profiling 19-norsteroids. I. Tandem mass
spectrometric characterization of the heptafluorobutyryl
ester and pentafluorobenzyloxime heptafluorobutyryl ester
derivatives of 19-nortestosterone using collisionally activated
dissociation”, J. Mass Spectrom. 30, 497 (1995).
117. D. de Boer, S.N. Bensink, A.R. Borggreve, R.D. van Ooijen
and R.A.A. Maes, “Profiling 19-norsteroids. II. Tandem mass
spectrometric characterization of the heptafluorobutyryl
ester and pentafluorobenzyloxime heptafluorobutyryl ester
derivatives of 19-norandrosterone using collisionally activated
dissociation”, J. Mass Spectrom. 30, 505 (1995).
635
118. P.-Y. Shu, S.-H. Chou and C.-H. Lin, “Determination of
corticosterone in rat and mouse plasma by gas chromatographyselected ion monitoring mass spectrometry”, J. Chromatogr.
B 783, 93 (2003).
119. W.C. Hubbard, C. Bickel and R.P. Schleimer, “Simultaneous
quantitation of endogenous levels of cortisone and cortisol in
human nasal and bronchoalveolar lavage fluids and plasma via
gas chromatography-negative ion chemical ionization mass
spectrometry”, Anal. Biochem. 221, 109 (1994).
120. Y.-S. Kim, H. Zhang and H.-Y. Kim, “Profiling neuresteroids
in cerebrospinal fluids and plasma by gas chromatography/
electron capture negative chemical ionization mass
spectrometry”, Anal. Biochem. 277, 187 (2000).
121. A. Loidl-Stahlhofen, W. Kern and G. Spiteller, “Gas
chromatographic-electron impact mass spectrometric
screening procedure for unknown hydroxyaldehydic
lipid peroxidation products after pentafluorobenzyloxime
detivatization”, J. Chromatogr. A 673, 1 (1995).
122. V.N. Reinhold, F. Wirtz-Peitz and K. Biemann, “Synthesis,
gas-liquid chromatography, and mass spectrometry of per-Otrimethylsilyl carbohydrate boronates“, Carbohydr. Res. 37,
203 (1974).
123. J.M. Halket, I. Ganschow and B.P. Lisboa, “Gas
chromatographic-mass spectrometric properties of boronate
esters of 24R,25-dihydroxycholecalciferol”, J. Chromatogr.
192, 434 (1980).
124. R.D. Coldwell, D.J.H. Trafford, M.J. Varley, H.L.J. Makin
and D.N. Kirk, “The measurement of vitamins D2 and D3 and
seven major metabolites in a single sample of human plasma
using gas chromatography/mass spectrometry”, Biomed.
Environ. Mass Spectrom. 16, 81 (1988).
125. T. Doco, M.A. O’Neill and P. Pellerin, “Determination of
neutral and acidic glycosyl-residue compositions of plant
polysaccharides by GC-EI-MS analysis of the trimethylsilyl
methyl glycoside derivatives”, Carbohydrate Polym. 46, 249
(2001).
126. M. Ishibashi, M. Itoh, K. Yamashita, H. Miyazaki and
H. Nakata, “Diethylhydrogensilyl cyclic diethylsilylene
derivatives in gas chromatography-mass spectrometry of
hydroxylated steroids. II. Pregnanes with a hydroxylated 17βside-chain”, Chem. Pharm. Bull. 34, 3298 (1986).
127. M. Ishibashi, T. Irie and H. Miyazaki, “Diethylhydrogensilylcyclic diethylsilylene derivatives in the gas chromatographymass spectrometry of hydroxylated steroids. IV.
Hydrocortisone”, J. Chromatogr. 399, 197 (1987).
128. H. Nakata, M. Ishibashi, M. Itoh and H. Miyazaki,
“Diethylhydrogensilyl cyclic diethylsilylene derivatives in gas
chromatography/mass spectrometry of hydroxylated steroids.
III. Fragmentation of 5β-pregnane-17,20,21-triol derivatives”,
Org. Mass Spectrom. 22, 23 (1987).
129. G. Biancotto, R. Angeletti, P. Traldi, M. Silvestri, M. Saccon
and F. Guidugli, “Determination of 17β-estradiol in bovine
plasma: development of a highly sensitive technique by ion
trap gas chromatography-tandem mass spectrometry using
negative chemical ionization”, J. Mass Spectrom. 37, 1266
(2002).
636
130. D.D. Fine, G.P. Breidenbach, T.L. Price and S.R. Hutchins,
“Quantitation of estrogens in ground water and swine lagoon
samples using solid-phase extraction, pentafluorobenzyl/
trimethylsilyl derivatization and gas chromatographynegative ion chemical ionization tandem nass spectrometry”,
J. Chromatogr. A 1017, 167 (2003).
131. M. Claeys, C. Van Hove, A. Duchateau and A.G. Herman,
“Quantitative determination of 6-oxo-PGF1α in biological
fluids by gas chromatography mass spectrometry”, Biomed.
Mass Spectrom. 7, 544 (1980).
132. B. Mayer, R. Moser, H.-J. Leis and H. Gleispach, “Rapid
separation of arachidonic acid metabolites by silicic acid
chromatography for subsequent quantitative analysis by gas
chromatography-mass spectrometry”, J. Chromatogr. 378,
430 (1986).
133. R.J. Strife and J.R. Simms, “Mass spectrometry/mass
spectrometry of prostaglandins: Daughter ion spectra of
derivatized and isotope-labeled E and D prostanoids”, Anal.
Chem. 60, 1800 (1988).
134. I. Morita, M. Kawamura and S.-L. Murita, “Quantitative
determination of 6-ketoprostaglandin F1α in biological fluids
by capillary gas chromatography-chemical ionization mass
spectrometry”, J. Chromatogr. 221, 361 (1980).
135. T. Obata, “Strategy and principles of the simultaneous analysis
of prostanoids by gas chromatography/mass spectrometry/
selected-ion monitoring”, Anal. Chim. Acta 465, 379 (2002).
136. J.J. Vrbanac, T.D. Eller and D.R. Knapp, “Quantitative
analysis of 6-keto-prostaglandin F1α using immunoaffinity
purification and gas chromatography-mass spectrometry“, J.
Chromatogr. 425, 1(1988).
137. J.M. Rosenfeld, Y. Moharir and R. Hill, “Direct solid-phase
isolation and oximation of prostaglandin E2 from plasma and
quantitation by gas chromatography with mass spectrometric
detection in the negative-ion chemical ionization mode”, Anal.
Chem. 63, 1536 (1991).
138. H. Schweer, C.O. Meese, B. Watzer and H.W. Seyberth,
“Determination of prostaglandin E1 and its main plasma
metabolites 15-keto-prostaglandin Eo and prostaglandin Eo by
gas chromatography/negative ion chemical ionization triplestage quadrupole mass spectrometry”, Biol. Mass Spectrom.
23, 165 (1994).
Formation of Mixed Derivatives of Polyfunctional Compounds
139. W. Hammes, U. Büchsler, P. Kinder and H. Bökens,
“Simultaneous determination of prostaglandin E1,
prostaglandin E0 and 15-keto-prostaglandin E0 in human
plasma by gas chromatography/negative ion chemicalionization tandem mass spectrometry”, J. Chromatogr. A 847,
187 (1999).
140. B. Watzer, H.W. Seyberth and H. Schweer, “Determination
of misoprostol free acid in human breast milk and serum by
gas chromatography/negative ion chemical ionization tandem
mass spectrometry”, J. Mass Spectrom. 37, 927 (2002).
141. M. Ishibashi, K. Watanabe, Y. Ohyama, M.M. Mizugaki and
N. Harima, “Dimethylisopropylsilyl ether derivative in gas
chromatography/mass spectrometry of 2,3-dinor-6-ketoprostaglandin F1a”, Chem. Pharm. Bull. 37, 539 (1989).
142. D.A. Herold, J. Savory, M. Kinter, R. Ross and M.R. Wills,
“Determination of urinary prostanoids by capillary gas
chromatography/high-resolution mass spectrometry”, Anal.
Chim. Acta 197, 149 (1987).
143. A.-N. Söderling, H. Ryberg, A. Gabrielsson, M. Lärstad, K.
Torén, S. Niari and K. Caidahl, “A derivatization assay using
gas chromatography/negative chemical ionization tandem
mass spectrometry to quantify 3-nitrotyrosine in human
plasma”, J. Mass Spectrom. 38, 1187 (2003).
144. V. Pitthard and P. Finch, “GC-MS analysis of monosaccharide
mixtures as their diethyldithioacetal derivatives: application to
plant gums used in art works”, Chromatographia (Supplement)
53, S-317 (2001).
145. M. Preu and M. Petz, “Development and optimization of a
new derivatization procedure for gas chromatographic-mass
spectrometric analysis of dihydrostreptomycin. Comparison
of multivariate and step-by-step optimization procedures”, J.
Chromatogr. A 840, 81 (1999).
146. T.A. Sasaki, J.M. Wilkins, J.B. Forehand and S.C. Moldoveanu,
“Analysis of heterocyclic amines in mainstream cigarette
smoke using a new NCI GC-MS technique”, Anal. Letters 34,
1749 (2001).
Received: 27 May 2005
Accepted: 28 July 2005
Web Publication: 2 December 2005