Chromatographic and chromato-mass spectral characterization of amino acids derivatives formed via the interaction with dimethyl acetal of dimethylformamide

Article abstract of DOI:10.1134/S1070363215080204

A series of amino acids have been converted into the derivatives via interaction with dimethylformamide dimethyl acetal for gas chromatography identification. The derivatives have been for the first time characterized with mass spectra and retention indices corresponding to the standard nonpolar polydimethylsiloxane stationary phases. Basic features of mass spectroscopic fragmentation of the derivatives have been stated; the rules for interpretation of their gas chromatography retention indices have been figured out, including the additive scheme elements.

Full text of DOI:10.1134/S1070363215080204

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 8, pp. 1920–1928. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © I.G. Zenkevich, T.I. Pushkareva, 2015, published in Zhurnal Obshchei Khimii, 2015, Vol. 85, No. 8, pp. 1365–1373.
Chromatographic and Chromato–Mass Spectral
Characterization of Amino Acids Derivatives
Formed via the Interaction with Dimethyl Acetal
of Dimethylformamide
I. G. Zenkevich and T. I. Pushkareva
St. Petersburg State University, Universitetskii pr. 26, St. Petersburg, 198504 Russia
e-mail: izenkevich@mail15.com
Received March 5, 2015
Abstract—A series of amino acids have been converted into the derivatives via interaction with dimethyl-
formamide dimethyl acetal for gas chromatography identification. The derivatives have been for the first time
characterized with mass spectra and retention indices corresponding to the standard nonpolar polydimethyl-
siloxane stationary phases. Basic features of mass spectroscopic fragmentation of the derivatives have been
stated; the rules for interpretation of their gas chromatography retention indices have been figured out,
including the additive scheme elements.
Keywords: amino acid, dimethylformamide, dimethyl acetal, mass spectrum, gas chromatography retention
index, identification
DOI: 10.1134/S1070363215080204
Organic compounds containing several functional
groups with active hydrogen atoms are often insuf-
ficiently volatile and thermally stable to be directly
analyzed by means of gas chromatography. Analysis of
such compounds is possible after conversion in the
more volatile derivatives via substitution of the active
hydrogen atoms with less polar covalently bound
fragments [1–3].
Dimethylformamide dialkyl acetals act as
alkylating agents with respect to many compounds
containing active hydrogen atoms (carboxylic acids,
phenols, thiols, etc.) [7]. The corresponding reactions
have been considered to yield the alkyl ester and
dimethylformamide [11].
RCO2H + (CH3)2N–CH(OCH3)2
→ RCO2CH3 + CH3OH + (CH3)2N–CHO.
Amino acids present a typical example of com-
pounds requiring the modification for performing their
gas chromatography analysis. The direct gas chromato-
graphy analysis of free amino acids has been reported
in the literature (for example, [4]); however, such
studies seem to be faulty [5]. A few methods for amino
acids modification are known [3, 6], the preferred one
being the single-stage substitution of hydrogen atoms
of the COOH and NH2 groups under the action of
dimethylformamide dialkyl acetals (CH3)2N–CH(OR)2
(R = CH3, С2Н5, С3Н7, tert-С4Н9, etc.) [7]. Using the
simplest representative of this class, dimethyl-
formamide dimethyl acetal, is the most convenient in
view of further gas chromatography analysis. The
pioneering examples of the described approach have
been given in [8–10].
Our studies, however, revealed that dimethyl-
formamide (the retention index for standard nonpolar
polydimethylsiloxane stationary phases being of
749±16) was not detected in noticeable amount in the
starting reagent (the major component content being of
≈93 %) and in the reaction mixtures. Hence, we
suggested that the intermediate monoalkyl acetals can
be decomposed into dimethylamine and (in the case of
DMF dimethyl acetal) methyl formate.
RCO2H + (CH3)2N–CH(OCH3)2
→ RCO2CH3 + [(CH3)2N–CH(OH)OCH3]
→ (CH3)2NH + HCO2CH3.
Retention indices of the both products for the standard
nonpolar phases are low (dimethylamine 383±15, methyl
1920

CHROMATOGRAPHIC AND CHROMATO–MASS SPECTRAL CHARACTERIZATION
1921
formate 386±10); therefore, they did not interfere with
detection of the target reaction products and were not
separated from the solvent signal (CH2Cl2, 515±7).
lists the gas chromatography retention indices (RI) of
the substrates and the products, including the
experimental values RIexp determined in this work, the
reference values RIref, and the reference mass spectra.
In all the cases the substrates signals were not detected
in the reaction mixture chromatogram, thus confirming
the quantitative modification of the substrates in the
corresponding derivatives.
DMF dialkyl acetals act as synthetic equivalents of
carbonyl compounds in the reactions with primary
amines, forming the Schiff’s bases (1,1-dialkyl
formamidines or dimethylaminomethylene N-DMAM
derivatives of primary amines) [12].
It is generally believed [3] that the presence of the
p–π conjugation (–N=CH–N<) in the N-DMAM
derivatives explains the high intensity of the molecular
ion signals in the mass spectra. However, this is only
true in the cases of the presence of the only
conjugation system in the molecule (i.e., in the cases of
the aromatic amino acids derivatives Ar–N=CH–N<);
for example, Irel(М) 80% for the aniline derivative. The
presence of several alternative centers of the charge
localization in the molecular ions regularly decreases
the Irel(М) values [16] (11% for the monoethanolamine
derivative and ~0.1% for certain amino acids). The
strongest signals in the mass spectra were those at m/z
44 (NMe2) and [М – 44] or (in certain cases) at m/z =
91 (С7Н7) and m/z = 71 (Me2NCHN) (the benzylamine
derivative) and m/z 85 [М – СН2ОН] (the ethanol-
amine derivative) (Table 1).
RNH2 + (CH3)2N–CH(OCH3)2
→ RN=CH–N(CH3)2 + 2СН3ОН.
Secondary amines gave the N-formyl derivatives
under the similar conditions [10]. Such reactions are
believed to involve formation of the corresponding di-
alkyl acetals via transamination, followed by hydrolysis.
R2NH + (CH3)2N–CH(OCH3)2
→ (CH3)2NH + [R2N–CH(OCH3)2] → R2N–CHO.
Majority of amino acids contain primary amino
groups and therefore form the derivatives of their
methyl esters (N-DMAM-Me) in the reaction with
DMF dimethyl acetal [2, 7]. Such derivatives have
been used in gas chromatography analysis for about 30
years but have not been systematically characterized
with the mass spectral and chromatographic (retention
indices on the standard stationary phases) parameters
so far (in particular, the relevant data have been absent
in the latest version of NIST mass spectra database
[13]), meaning that each analysis of amino acids in the
form of the N-DMAM-Me derivatives should include
modification of the reference amino acids samples,
whereas the analytical devices are used as simple
comparators. Such state of the art does not comply
with the potential of contemporary chromato–mass
spectrometry; therefore, in this work we obtained the
reference mass spectra of the products of amino acids
interaction with DMF dimethyl acetals and determined
the gas chromatography retention indices of the
products on the standard nonpolar stationary phases.
The obtained data enable analysis of other amino acids
as well.
Gas chromatography retention indices of all the
prepared N-DMAM derivatives were determined using
the standard nonpolar polydimethylsiloxane stationary
phase BPX-1 and the more polar RTX-5 phase contain-
ing 5% of phenyl groups (the phases similar to the latter
ones are referred to as “semi-standard” [13]) (Table 1).
The average difference between the RI values of the
prepared N-DMAM derivatives determined for the two
stationary phases was of 14±4 (Table 1), typical of the
low-polar compounds. Another important gas
chromatography parameter was the average difference
between RI values of the substrate and its N-DMAM
derivative. The latter parameter reflected the changes
of the chromatography retention properties due to the
substrate modification and gave a base for the simple
additive scheme allowing for estimation of the
retention index of the reaction product from the
relevant substrate parameters [17]. The data in Table 1
show that for the scheme below the discussed value
was of 396±16.
The possibility to prepare the derivatives under the
action of DMF dimethyl acetal was verified using sets
of acids (decanoic, butanedioic, and citric ones) and
amines (aniline, benzylamine, and monoethanolamine)
as examples. Direct gas chromatography analysis was
possible in the cases of monocarboxylic acids and
primary amines, allowing for determination of the
conversion degree from the areas of peak correspond-
ing to the reaction substrates and products. Table 1
CH3
N
N
ХNH2
CH3
Х
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

1922
ZENKEVICH, PUSHKAREVA
Table 1. Mass spectra and gas chromatography retention indices of products of the interaction of selected carboxylic acids
and primary amines with dimethylformamide dimethyl acetala
Product
Substrate (RI)
RIref
[13–15]
RIexp
m/z ≥ 40 (Irel ≥ 2%)
Decanoic acid (1378±10)
Butanedioic acid (no data)
Citric acid (no data)
1313±1
1008±3
1424±2
1312±7 Not required. Retention index is sufficient for identification
1004±8 Not required. Retention index is sufficient for identification
1424±3 Not required. Retention index is sufficient for identification
Aniline (958± 13)
1366±2
1384±3
1391±10 149 (8), 148 (80) [М], 147 (49), 134 (3), 133 (31), 132 (5), 131 (4), 120
(14), 119 (2), 118 (3), 107 (5), 106 (54), 105 (7), 104 (33) [M – N(CH3)2],
92 (2), 91 (6), 79 (10), 78 (11), 76 (5), 75 (2), 74 (8), 73 (10), 65 (3), 64
(2), 63 (2), 57 (7), 52 (4), 51 (34), 50 (8), 45 (22), 44 (100) [N(CH3)2], 43
(4), 42 (25), 41 (3), 39 (4)
Benzylamine (994±10)
1396±2
1407±2
1408
163 (5), 162 (46) (М), 161 (9), 147 (9), 134 (3), 132 (2), 120 (8), 119
(2), 118 (6), 117 (3), 106 (13), 104 (2), 103 (2), 92 (6), 91 (65)
[C6H5CH2], 90 (5), 89 (7), 85 (5), 84 (4), 83 (2), 81 (2), 79 (3), 77 (4),
71 (26) [(CH3)2NCHN], 65 (17), 63 (4), 58 (4), 57 (18), 51 (5), 50 (2),
46 (6), 45 (17), 44 (100) [N(CH3)2], 43 (3), 42 (34), 41 (4), 39 (6)
2-Aminoethanol (675±23)
1052±2
No data 116 (11) (М), 98 (3), 86 (4), 85 (61) [M – CH2OH], 72 (3), 71 (3), 58
1064±2
(2), 57 (4), 46 (4), 45 (14), 44 (100) [N(CH3)2], 43 (6), 42 (34), 41 (7)
a
Retention indices determined for the standard nonpolar phase BPX-1 are given in italics; other values correspond to the RTX-5 phase
containing 5% of phenyl groups.
According to that estimation, the expected RI value
of the N-DMAM derivative of 2-phenylethylamine
(RI = 1079±11) was of 1475±19.
the primary amine rather than the starting amino acid
derivative. Indeed, the mass spectrum contained the
signals at m/z = 44, 70, and 71 corresponding to the
(CH3)2N–CH=N– fragment, but the RI value (1036±2)
was lower than that for Ser-N-DMAM-Me (1233±2);
therefore, the product could not be the threonine de-
rivative. The component structure could be elucidated
from the mass spectroscopy and chromatography data
taking advantage of the algorithm discussed in [18]. To
do so, the experimentally determined RI value should
be reduced by the increment corresponding to the
–NH2 → –N=CH–N(CH3)2 conversion, that gave the
RI value of the starting primary amine, (1036±2) –
(396±16) ≈ 640±16, the amine molecular mass being
55 Da lower than that of the N-DMAM derivative. The
mass spectrum of the unknown component contained
the strong signals at m/z 112 and (112 – 15) = 97;
however, they could not be assigned to the М+· and
[М – СН3]+ ions, since the RI values of all the amines
with М = 112 – 55 = 57 were much lower than
640±16. The only suitable solution was 1-aminopro-
panol-2 that could be formed via threonine decar-
boxylation. The refence RI value of the suggested
solution (654±13) practically coincided with the
estimation made (640±16, RTX-5); the agreement was
Table 2 gives the retention indexes and the
reference mass spectra of the products of modification
of 15 amino acids RCH(NH2)CO2H via the reaction
with DMF dimethyl acetal. The scheme of the
conversion for the substrates containing primary amino
groups is given below.
RCH(CO2H)NH2 + (CH3)2N–CH(OCH3)2
→ RCH(CO2CH3)–N=CH–N(CH3)2.
In certain cases, the reaction mixtures contained the
non-identified components or the methyl esters (for
example, in the case of tryptophane).
Since the both functional groups of amino acids
were involved in the reaction with DMF dimethyl
acetal, the target products were designated as X-N-
DMAM-Me (X being the code of the corresponding
amino acid). In the cases of glycine and histidine, the
reaction products were not detected owing to insolub-
ility of the amino acids in the reaction mixture.
The only detected product of the reaction with
threonine was assigned to the N-DMAM derivative of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

CHROMATOGRAPHIC AND CHROMATO–MASS SPECTRAL CHARACTERIZATION
1923
Table 2. Mass spectra and gas chromatography retention indices of products of the interaction of selected α-amino acids with
dimethylformamide dimethyl acetala
Amino acid (М) RI(BPX-1)/RI(RTX-5)
Reaction product: m/z ≥ 40 (Irel ≥ 2%)
Glycine (75→144)
Alanine (89→158)
–
1143±3/1176±2
Ala-N-DMAM-Me: 158 (6) [M], 100 (6), 99 (100) [M – CO2CH3], 98 (2), 83
(3), 72 (8), 71 (2), 59 (2), 58 (3), 57 (5), 56 (12), 55 (4), 54 (2), 46 (4), 45 (5),
44 (99) [NMe2], 43 (4), 42 (22)
Serine (105→174)
–/1025±4
Not identified: 119 (0.3), 101 (11), 86 (4), 84 (3), 71 (12), 70 (100), 69 (16),
59 (7), 56 (5), 55 (2), 54 (2), 51 (2), 49 (2), 45 (3), 44 (10), 43 (15), 42 (50),
41 (9), 40 (9)
–/1046±3
Not identified: 118 (3), 102 (14), 76 (3), 75 (100) [CH(OMe)2], 74 (5), 59 (2),
58 (4), 47 (20), 44 (3), 43 (6), 42 (59), 41 (2)
1233±2/1280±2
Ser-N-DMAM-Me: 173 (0.1) [M – H], 156 (18) [M – H2O], 141 (2), 125 (2),
124 (13), 98 (3), 97 (29) [M – H2O – CO2CH3], 96 (24), 95 (3), 84 (2), 81 (2),
72 (3), 71 (3), 68 (2), 57 (5), 56 (5), 55 (2), 54 (16), 53 (2), 52 (2), 45 (6), 44
(100) [NMe2], 43 (9), 42 (34), 41 (4)
Proline (115→157)
Valine (117→186)
1332±2/1389±2
1264±2/1288±2
–/1036±2
Methyl ester of N-formylproline: 157 (5) [M], 129 (8), 106 (3), 99 (7), 98
(100) [M – CO2CH3], 86 (2), 77 (5), 73 (2), 71 (6), 70 (90) [M – CO2CH3 –
CO], 69 (3), 68 (14), 55 (2), 51 (3), 50 (2), 45 (2), 44 (10), 43 (29), 42 (11), 41
(26), 39 (7), 38 (2)
Val-N-DMAM-Me: 186 (4) [М], 144 (6), 143 (77) [M – C3H7], 142 (3), 128
(5), 127 (61) [M – CO2CH3], 115 (2), 100 (2), 86 (2), 84 (4), 83 (6), 82 (2), 73
(4), 72 (6), 71 (4), 69 (2), 68 (2), 59 (4), 58 (2), 57 (13), 56 (4), 55 (8), 46
(15), 45 (6), 44 (100) [NMe2], 43 (4), 42 (21), 41 (8), 39 (3)
N-DMAM derivative of 1-amino-2-propanol: 113 (5), 112 (66), 111 (7), 98
(3), 97 (40), 96 (2), 85 (6), 84 (8), 83 (7), 82 (4), 80 (3), 71 (10), 70 (24), 69
(6), 68 (29), 67 (6), 58 (5), 57 (8), 56 (25), 55 (2), 54 (4), 46 (5), 45 (14), 44
(100) [NMe2], 43 (11), 42 (67), 41 (51), 40 (6), 39 (20), 38 (2)
Threonine
(119→188)
Leucine (131→200)
1342±2/1380±2
Leu-N-DMAM-Me: 200 (0.1) [М], 157 (7), 156 (39) [M –NMe2], 144 (9), 143
(18)
[M – C4H9], 142 (7), 141 (73) [M – CO2CH3], 125 (2), 112 (15), 100 (2), 99
(20), 98 (3), 97 (6), 96 (2), 87 (2), 86 (2), 85 (41), 84 (9), 83 (5), 82 (2), 73
(5), 72 (11), 71 (5), 70 (5), 69 (3), 59 (2), 58 (2), 57 (9), 56 (6), 55 (5), 54 (5),
53 (2), 46 (13), 45 (6), 44 (100) [NMe2], 43 (11), 42 (25), 41 (10), 39 (3)
Ile-N-DMAM-Me: 199 (0.1) [M – H], 157 (3), 156 (28) [M – NMe2], 144 (7),
143 (74) [M – C4H9], 142 (4), 141 (39) [M – CO2CH3], 115 (2), 113 (3), 112
(7), 111 (2), 100 (2), 99 (2), 97 (2), 96 (2), 85 (5), 84 (7), 83 (5), 74 (2), 73
(28), 72 (6), 71 (5), 70 (3), 69 (7), 68 (3), 59 (2), 58 (2), 57 (8), 56 (3), 55 (3),
46 (14), 45 (6), 44 (100) [NMe2], 43 (6), 42 (23), 41 (13), 39 (3)
nor-Leu-N-DMAM-Me: 200 (0.2) [M], 157 (5), 156 (34) [M – NMe2], 144
(2), 143 (14) [M – C4H9], 142 (6), 141 (73) [M – CO2CH3], 140 (3), 112 (5),
100 (2), 99 (8), 98 (3), 97 (4), 96 (2), 85 (15), 84 (5), 83 (4), 73 (7), 72 (6), 71
(7), 70 (2), 69 (4), 68 (2), 59 (2), 58 (2), 57 (7), 56 (4), 55 (4), 53 (3), 46 (10),
45 (6), 44 (100) [NMe2], 43 (11), 42 (22), 41 (9), 29 (2)
Isoleucine
(131→200)
1344±2/1380±2
1390±2/1426±2
1460±2/1516±2
Norleucine
(131→200)
Asparaginic acid
(133→216)
Asp-N-DMAM-Me2: 216 (10) [M], 185 (2), 184 (5), 158 (6), 157 (68) [M –
CO2CH3], 156 (18), 155 (5), 143 (11), 125 (4), 116 (2), 115 (27), 100 (3), 99
(37), 98 (3), 97 (9), 87 (4), 84 (2), 83 (9), 74 (4), 73 (2), 72 (4), 71 (2), 70 (2),
69 (2), 59 (6), 57 (11), 56 (6), 55 (6), 54 (16), 46 (5), 45 (7), 44 (100) [NMe2],
43 (5), 42 (25), 41 (3)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

1924
ZENKEVICH, PUSHKAREVA
Table 2. (Contd.)
Amino acid (М) RI(BPX-1)/RI(RTX-5)
Reaction product: m/z ≥ 40 (Irel ≥ 2%)
Lysine (146→270)
1961±2/1994±13
Lys-bis-N-DMAM-Me: 270 [M], 226 (3), 211 (3) [M – CO2CH3], 200 (3), 199
(8), 198 (4), 166 (5), 165 (4), 157 (6), 156 (2), 154 (2), 144 (3), 143 (6), 140
(4), 139 (19), 137 (2), 129 (2), 128 (31), 127 (17), 125 (4), 114 (4), 113 (4),
112 (18), 111 (9), 100 (3), 99 (15), 98 (4), 97 (6), 96 (3), 94 (3), 86 (13), 85
(29), 84 (11), 83 (5), 82 (3), 73 (15), 72 (10), 71 (11), 70 (100) [Me2N–CN],
69 (7), 68 (4), 67 (3), 59 (2), 58 (7), 57 (14), 56 (6), 55 (3), 54 (2), 46 (34), 45
(8), 44 (82) [NMe2], 43 (7), 42 (28), 41 (7)
Glutamic acid
(147→230)
1578±2/1637±2
1581±2/1638±2
Glu-N-DMAM-Me2: 230 (11) [M], 199 (10), 198 (4), 172 (4), 171 (48) [M –
CO2CH3], 170 (20), 169 (4), 158 (2), 157 (29), 155 (11), 143 (12), 141 (2),
139 (8), 128 (2), 112 (9), 111 (100) [M – CO2CH3 – HCO2CH3], 100 (2), 99
(4), 98 (3), 97 (10), 96 (2), 95 (2), 85 (7), 84 (7), 83 (5), 82 (2), 74 (2), 73
(16), 72 (6), 71 (8), 70 (9), 69 (2), 68 (5), 59 (6), 58 (3), 57 (18), 56 (6), 55 (4),
54 (3), 46 (21), 45 (8), 44 (88) [NMe2], 43 (7), 42 (38), 41 (12), 40 (2), 39 (3)
Methionine
(149→218)
Met-N-DMAM-Me: 218 (5) [M], 203 (3), 186 (2), 174 (2), 171 (2), 160 (2),
159 (15) [M – CO2CH3], 158 (7), 157 (81) [M – CH3SCH2], 145 (3), 144 (40),
143 (21) [M – (CH2)2SCH3], 128 (5), 115 (2), 113 (4), 112 (61), 111 (60) [M –
CO2CH3 – CH3SH], 104 (3), 100 (2), 99 (10), 98 (3), 97 (23), 87 (3), 85 (9),
84 (36), 83 (5), 75 (2), 74 (2), 73 (4), 72 (6), 71 (6), 70 (5), 69 (2), 68 (4), 63
(2), 62 (2), 61 (43) [CH3SCH2], 59 (3), 58 (4), 57 (18), 56 (5), 55 (3), 54 (4),
47 (3), 46 (9), 45 (11), 44 (100) [NMe2], 43 (4), 42 (36), 41 (11),
40 (2), 39 (3)
Histidine
–
(155→224)
Phenylalanine
(165→234)
1692±2/1732±2
Phe-N-DMAM-Me: 235 (4), 234 (27) [M], 176 (3), 175 (20) [M – CO2CH3],
162 (11), 161 (100) [M – PhCH2], 134 (8), 132 (2), 131 (3), 121 (12), 120
(15), 119 (4), 118 (3), 117 (3), 105 (3), 104 (12), 103 (11), 102 (3), 99 (29), 92
(2), 91 (36), 90 (4), 89 (4), 80 (9), 79 (2), 78 (6), 77 (12), 76 (2), 73 (3), 72
(22), 51 (5), 46 (4), 45 (6), 44 (90) [NMe2], 43 (4), 42 (27), 41 (2), 39 (2)
Arg-bis-N-DMAM-Me: 298 [M], 250 (1), 248 (1), 212 (5), 211 (2), 197 (2),
187 (2), 186 (13), 185 (4), 184 (32), 168 (2), 157 (6), 153 (2), 152 (5), 168 (2),
157 (6), 153 (2), 152 (5), 151 (4), 143 (6), 142 (7), 141 (8), 140 (4), 129 (3),
128 (28), 126 (6), 125 (28), 124 (6), 114 (3), 113 (3), 111 (5), 109 (3), 101 (3),
100 (36), 99 (46), 98 (63), 97 (17), 96 (6), 85 (22), 84 (6), 83 (11), 82 (7), 81
(2), 80 (6), 75 (2), 73 (23), 72 (41), 71 (27) [(Me2NCHN], 70 (13), 69 (3), 68
(8), 67 (2), 59 (3), 58 (6), 57 (15), 56 (8), 55 (6), 54 (4), 53 (2), 46 (21), 45
(12), 44 (100) [NMe2], 43 (22), 42 (37), 41 (9), 40 (8)
Arginine
(174→298)
1842±2/1909±2
Tyrosine
(181→250)
–/1987±4
Tyr-N-DMAM-Me: 250 [M], 191 (9) [M – CO2CH3], 144 (6), 143 (53)
[Me2NCHNCHCO2CH3], 107 (2), 96 (6), 91 (4), 84 (3), 83 (7), 79 (2), 72 (7),
71 (2), 68 (2), 58 (2), 57 (10), 56 (3), 53 (3), 46 (6), 45 (4), 44 (100) [NMe2],
43 (3), 42 (22), 41 (3), 39 (3)
1994±2/2038±5
Tyr-N-DMAM-Me2: 264 [M], 205 (5) [M – CO2CH3], 144 (8), 143 (72)
[Me2NCHNCHCO2CH3], 133 (2), 122 (2), 121 (9), 103 (11), 100 (2), 91 (2), 84
(2), 79 (2), 78 (6), 77 (4), 76 (2), 74 (2), 73 (5), 72 (5), 71 (6), 64 (2), 57 (11),
56 (2), 53 (2), 51 (2), 46 (9), 45 (7), 44 (100) [NMe2], 42 (21), 41 (3), 40 (2)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

CHROMATOGRAPHIC AND CHROMATO–MASS SPECTRAL CHARACTERIZATION
1925
Table 2. (Contd.)
Amino acid (М) RI(BPX-1)/RI(RTX-5)
Reaction product: m/z ≥ 40 (Irel ≥ 2%)
Tryptophane
(204→273)
2161±9/–
Methyl ester of tryptophane: 218 [M], 202 (2), 201 (12), 181 (2), 170 (2), 169
(2), 155 (2), 131 (13), 130 (100) [(3-indolyl)СН2], 128 (3), 115 (3), 103 (4),
102 (4), 78 (2), 77 (5), 75 (3), 44 (4), 43 (3)
2327±12/2321±15 Trp-N-DMAM-Me: 273 [M], 214 (5) [M – CO2CH3], 169 (5), 145 (2), 144
(30), 143 (100) [Me2NCHNCHCO2Me], 142 (2), 131 (3), 130 (26), 129 (2),
128 (2), 117 (2), 116 (2), 115 (8), 112 (12), 107 (5), 103 (4), 102 (2), 100 (2),
89 (2), 84 (6), 83 (4), 77 (6), 73 (2), 72 (4), 58 (2), 57 (7), 45 (3), 44 (58)
[NMe2], 43 (2), 42 (20)
–/2374±17
N-formyl-Trp-N-DMAM-Me: 301 [M], 242 (4) [M – CO2CH3], 169 (2), 144
(10), 143 (100) [Me2NCHNCHCO2Me], 142 (2), 130 (7), 115 (6), 107 (2),
103 (2), 102 (3), 83 (4), 77 (3), 72 (5), 57 (4), 46 (5), 45 (2), 44 (50) [NMe2],
42 (12)
a (“–”) Products not detected.
absolute after correcting for the RI difference for the
BPX-1 and RTX-5 phases (14±4 index units). The
signal at m/z = 112 could then be assigned to the [М –
Н2О]+ ions; such signals were found in the mass
spectra of serine methyl ester and the Ser-N-DMAM-
Me derivative (Table 2).
acids, the Irel of the signal of the derivative molecular
ion was of 0.1–0.2%. Figure 1 gives a spectrum of nor-
Leu-N-DMAM-Me as a representative example of the
latter case.
One of the limitations of mass spectrometry
analysis of amino acids and peptides is the complicated
identification of the isomers, first of all, the leucine–
isoleucine pair. Retention indexes of their N-DMAM
derivatives were almost the same (1342±2 and
1344±2), but their mass spectra were significantly
different: for leucine Irel (m/z = 143) >> Irel (m/z =
141), and for isoleucine the given relation was the
opposite. On the contrary, the isoleucine–norleucine
pair could not be distinguished using the given mass
spectral feature, but their RI values were significantly
The average RI(RTX-5) – RI(BPX-1) difference for
the N-DMAM derivatives of amino acids was of
46±14 index units (Table 2), meaning that those com-
pounds were more polar than the N-DMAM
derivatives of monofunctional primary amines. The
molecular ion peaks were reliably detected (Irel = 4–
11%) only in the mass spectra of the alanine, valine,
asparaginic acid, glutamic acid, methionine, and
phenylalanine derivatives. In the cases of other amino
Irel, %
50
75
100
125
150
175
200
m/z
Fig. 1. Mass spectrum of N-DMAM derivative of norleucine methyl ester (М = 200).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

1926
ZENKEVICH, PUSHKAREVA
Since DMF dialkyl acetals could react with phenols
and thiols as well, it was not surprising to detect two
components in the products of DMF dimethyl acetal
reaction with tyrosine, the first one corresponding to
the normal N-DMAM-Me derivative (М = 250), and
the second being formed via additional methylation of
the phenolic hydroxyl group (М = 264). Signals of the
molecular ions were absent in the both derivatives
mass spectra, but the [M – CO2CH3] signals at m/z = 191
and 205, respectively, were reliably detected (Table 2).
The combined use of mass spectra and gas
chromatography retention indexes for identification of
the N-DMAM derivatives of the amino acids opens
unique possibilities for chromato–mass spectral data
interpretation. Let us consider a hypothetical substitu-
tion of the –CH(CO2CH3)–N=CH–N(CH3)2 fragment
with a simpler moiety (methyl group) taking advantage
of the approach described in [17].
200
400
600
800
1000
RI (RCH3)
Fig. 2. Linear relation between retention indices of
N-DMAM-Me derivatives of amino acids and those of the
derivatives containing methyl group instead of the
–CH(CO2CH3)–N=CHN(CH3)2 fragment.
CO2CH3
different (typically of the isomers with different
number of branching in the butyl group).
R
R
CH
CH3
N
CHN(CH3)2
Major signals in the mass spectra of the N-DMAM-
Me derivatives of the studied amino acids were
assigned to the ions corresponding to dimethylamino
group [NMe2]+ (m/z = 44, Irel = 50–100%), elimination
of the methoxycarbonyl group [M – CO2CH3] = [M –
59], the [M – R] fragments (R being the α-substituent
of the amino acid) (m/z = 143), and, in certain cases,
[М – NMe2]+. In the presence of OH group in the R
fragment (serine and threonine), the signals of the [M –
H2O]+· ions and the corresponding secondary ions
became the characteristic ones. The arginine derivative
(judging from the RI value of 1842±1, the bis-N-
DMAM-Me derivative was formed, via the amino
group and the guanidine fragment) revealed the hardly
interpreted mass spectrum; the lysine behavior was
similar.
For convenience of the further analysis, we will
discuss the parameters of the linear regression given
below rather than the average ΔRI value for that
reaction.
RI [R–CH(CO2CH3)–N=CH–N(CH3)2]
= a RI [R–CH3] + b,
a = 0.96±0.04, b = 906±27, r = 0.992, S0 = 32, N = 10;
RI [R–CH3]
(1)
= a RI [R–CH(CO2CH3)–N=CH–N(CH3)2] + b, (2)
a = 1.03±0.04, b = –(921±67), r = 0.992, S0 = 33, N = 10.
The generalized standard deviation S0 value reflects
the accuracy of the result; it should be of ±32–33 index
units on the average. The plot of the linear regression (1)
is given in Fig. 2.
The behavior of the amino acids containing
secondary amino groups or the NH groups in aromatic
systems (proline and tryptophane) was in agreement
with the known chemical properties of DMF dimethyl
acetal [10]. Formation of the N-DMAM products was
impossible for those substrates, they were converted
into the N-formyl derivatives. For example, the only
product in the case of proline (М 115) was a compound
with M = 157 (ΔМ = 42), corresponding to methylation
of the carboxyl group (ΔМ = 14) and N-formylation
(ΔМ = 28). In the case of tryptophane, two products
were detected: the N-DMAM-Me derivative and the
secondary product of its N-formylation (Table 2).
Equations (1) and (2) formally describe the
mutually inverse relations. The first one should be
used to estimate the RI values of the N-DMAM
derivatives from the RI data for their simple analogs
RCH3. On the contrary, the second one allows
estimation of RI values of such simple analogs from
the data for the N-DMAM derivatives of amino acids.
Combined application of the equations will allow
estimation of RI and verification of the experimental
data. Let us give the examples.
Example 1. Estimation of retention indices of the
N-DMAM derivatives of cycteine HSCH2CH(NH2)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

CHROMATOGRAPHIC AND CHROMATO–MASS SPECTRAL CHARACTERIZATION
1927
CO2H. Since the reaction with compound I allowed
methylation of cycteine at the SH group, ethanethiol
(HSCH2CH3, RI = 502±8) and methyl ethyl sulfide
(CH3SCH2CH3, RI = 605±10) should be taken as the
amino acid structural analogs. Using the Eq. (1), the
following estimations could be made.
during 1–2 h. The sealing was necessary to prevent
losses of volatile DMF dimethyl acetal (bp 102–108°C
[5]). The reaction course was visually monitored by
the amino acid dissolution. The so prepared mixtures
were directly used for the gas chromatography
analysis; the chromato–mass spectrometry analysis
was performed using the mixtures diluted with
dichloromethane in the 1:500 ratio.
(0.96±0.04)(502±8) + (906±27) ≈ 1388±35
(Cys-N-DMAM-Me),
Chromatographic analysis of the reaction mixtures
was performed using a Khromatek-Kristall 5000.2
chromatograph equipped with a flame ionization
detector and a BPX-1 column (standard nonpolar poly-
dimethylsiloxane phase) with the length of 10 m and
the inner diameter of 0.53 mm (the stationary phase
film thickness was of 2.65 µm). The analysis condi-
tions were as follows: temperature program of 70 to
250°C at 5 deg/min, 5.1 mL/min of nitrogen as carrier
gas, splitting 6.0 : 1, evaporator temperature 250°C,
specimen volume 0.2–0.3 µL.
(0.96±0.04)(605±10) + (906±27) ≈ 1487±37
(Cys-N-DMAM-Me2).
The accuracy of the results was not very high, but
simplicity of the method makes it attractive for
preliminary estimation of the retention indices.
Example 2. The molecular ion signal was not
detected in the mass spectrum of the lysine derivative
with RI = 1961±2 (Table 2), and the mass spectrum
was not informative enough to confirm the derivative
structure. Let us resolve the issue using the additional
chromatography data.
Chromato–mass spectrometry analysis was
performed using a Shimadzu 2010 Plus device in the
EI ionization mode, the interface and the ion source
temperature was of 200°С. An RTX-5 MS column
with length of 30 m and inner diameter of 0.32 mm
(the stationary phase film thickness was of 0.25 µm).
The analysis conditions were as follows: temperature
program of 70 to 200°C at 5 deg/min, 1.83 mL/min of
helium as carrier gas, splitting 11.7 : 1, evaporator
temperature 200°C, specimen volume 0.2–0.3 µL.
Lysine [H2N(CH2)4CH(NH2)CO2H] interaction
with DMF dimethyl acetal could result in formation of
the bis-N-DMAM-Me derivative (CH3)2N–CH=N–
(CH2)4CH(CO2CH3)–N=CH–N(CH3)2. In order to
verify that, let us first estimate the retention index for
its structural analog taking advantage of Eq. (2) (the
errors were omitted for clarity): RI [(CH3)2N–CH=N–
(CH2)4CH3] = 1.03·1961 – 921 ≈ 1099. Then, using the
ΔRI increment for the (CH3)2N–CH=N–(CH2)4CH3 →
H2N(CH2)4CH3 conversion, the retention index for 1-
pentanamine could be estimated, RI = 1099 – 396 ≈
703. The reference RI value for 1-pentanamine was of
717±9, and the results agreement unambiguously
confirmed the derivative structure.
The linear-logarithmic retention indices of the
reaction products were determined using a solution of
a mixture of С9–С17 n-alkanes with the odd number of
the carbon atoms in n-hexane as internal reference.
ACKNOWLEDGMENTS
The presented examples confirmed the high value
of gas chromatography retention indices for interpreta-
tion of the chromato–mass spectroscopy results,
including those obtained during analysis of derivatives
of amino acids prepared via their modification with
dimethylformamide dimethyl acetal.
The experiment was carried out using the equip-
ment installed in the Resource Center of Institute of
Chemistry, St. Petersburg State University. Authors
acknowledge the assistance of the Center collaborators.
EXPERIMENTAL
REFERENCES
Dimethylformamide dimethyl acetal (Aldrich) with
the major component content of 93% was used. The
derivatives of the amino acids (pure grade, 1–3 mg)
were prepared via the interaction with 50 µL of DMF
dimethyl acetal in sealed glass ampoule with length of
40–50 mm and inner diameter of 2 mm upon heating at
80–100°C in the air thermostat of the chromatograph
1. Drozd, J., Chemical Derivatization in Gas Chromato-
graphy. J. Chromatogr. Library, Amsterdam: Elsevier,
1981, vol. 59.
2. Handbook of Derivatives for Chromatography, Blau, K.
and Halket, J.M., New York.: J. Wiley&Sons, 1993.
3. Encyclopedia of Chromatography, New York: Taylor &
Francis, 2010, vol. 1.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015

1928
ZENKEVICH, PUSHKAREVA
4. Suh, H.-J., Kim, S.-R., Lee, K.-S., Park, S., and Kang, S.C.,
J. Photochem. Photobiol. (B), 2010, vol. 99, p. 67. DOI:
10.1016/j.jphotobiol.2010.02.005.
12. Kataoka, H., J. Chromatogr. (A), 1996, vol. 733, p. 19.
13. The NIST 14 Mass Spectral Library (NIST14/2014/
EPA/NIH). Software/Data Version (NIST14); NIST
Standard Reference Database, no. 69, 2014, National
Institute of Standards and Technology, Gaithersburg,
MD 20899; http://webbook.nist.gov.
5. Zenkevich, I.G., Anal. Bioanal. Chem., 2013, vol. 405,
p. 3075. DOI: 10.1007/s00216-013-6751-2.
6. Sunozova, E.V., Trubnikov, V.I., and Sakodynskii, K.I.,
Gazovaya khromatografiya aminokislot (Gas Chromato-
graphy of Amino Acids), Mosocw: Khimiya, 1976.
14. Osek, J., Osczapowicz, J., and Drzewinski, W., J. Chro-
matogr., 1986, vol. 351, p. 177.
7. Chromatography Catalog and Handbook, Rockford:
Pierce Biotechnology Inc., 2003.
8. Thenot, J.-P., Horning, E.C., Stafford, M., and Horning, M.G.,
15. Dolecka, E., Raczynska, E.D., and Drapala, T., J. Chem.
Soc., Perkin Trans. 2, 1988, p. 257. DOI: 10/1039/
p29880000257.
16. Zenkevich I.G. and Ioffe, B.V., Interpretatsiya mass-
spektrov organicheskikh soedinenii (The Interpretation
of the Mass Spectra of Organic Compounds),
Leningrad: Khimiya, 1986.
Anal. Lett., 1972, vol. 5, no. 4, p. 217.
9. Thenot, J.-P. and Horning, E.C., Anal. Lett., 1972,
vol. 5, no. 8, p. 519.
10. Thenot, J.-P., Ruo, T.I., and Bouwsma, O.J., Anal. Lett.,
17. Zenkevich I.G., Zh. Org. Khim., 1992, vol. 29, no. 9,
1980, vol. 13, no. 9, p. 759.
p. 1827.
11. Meierhenrich, U., Thiemann, W.H.P., and Rosenbauer, H.,
J. Anal. Appl. Pyrolysis, 2001, vol. 60, p. 13. DOI:
10.1016/S0165-2370(00)00103-0.
18. Zenkevich, I.G., Chemom. Intell. Lab. Syst., 2004,
vol. 72, p. 233. DOI: 10.1016/j.chemolab.2003.12.016.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 8 2015