teorico 14 Espectrometría de masa IV

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ESPECTROMETRIA DE 
MASA (4º)
Acoplamiento de cromatografía con 
espectrometría masa
•Cromatograma de iones totales (TIC)
•Monitoreo selectivo de iones (SIM)
•Espectro de masa 
Análisis cuantitativo con CG/MS: estándar interno
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Ion-pair reversed phase high performance liquid chromatography (IP-RP-HPLC)-
UV chromatogram (A) and liquid chromatography-mass spectrometry (LC-MS) 
total ion chromatogram (TIC) (B) of standards of GTI-2040, 3′ N-1, 3′ N-2, and 
3′N-3. UV was set at 260 nm. 
UV vs TIC
Gas chromatography/mass spectrometry analysis of urine organic acids from a 
patient with methylmalonic acidemia, showing total ion content (top) and the mass 
spectra corresponding to methylmalonic acid (bottom) 
TIC – full spectra
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Total ion chromatogram (TIC) and 
the full scan mass spectra of GTI-
2040 and major metabolites (M1-
M5) from the plasma extract of a 
patient (A) shows the TIC of GTI-
2040 and M1 to M5 metabolites; 
(B) shows the mass spectrum of 
M1, the putative 3′N-1 metabolite 
with retention time (RT) of 14.8, 
which contains an ion envelope 
including [M-6H],6- [M-5H],5- and 
[M-4H]4- ions; (C) shows the 
mass spectrum of M2, the 
putative 3′N-2 metabolite at RT 
14.2 minutes, which contains the 
most abundant ion of [M-3H]3-; and 
(D) shows the mass spectrum of 
M3, the putative 3′N-3 metabolite at 
RT 12.9 minutes containing the 
most abundant ion of [M-3H]. 
Total ion chromatogram and SIM chromatogram of the six chlorinated 
phenoxy acid herbicides. (A) Total ion chromatogram was obtained 
using DBA as ion-pairing reagent. Eluent A (water) and B (acetonitrile) 
containing 5 mmol l 1 DBA. Linear gradient from 30% B/A to 85% B/A in 
15 min, 15 min re-equilibration. (B) SIM chromatogram was obtained by 
in-tube SPME-LC/ESI-MS of river water spiked with 0.05 ng ml 1. 1, 
MCPA; 2, 2,4-D; 3, Dichlorprop; 4, 2,4,5-T; 5, 2,4-DB; 6, 2,4,5-TP.
Analyst, The
DOI: 10.1039/b100380l 
TIC - SIM
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The liquid chromatography (LC) –MS data set is visualized as a total ion chromatogram
('peptide trace'), in which peptide-ion intensity is shown as a function of peptide retention
time (part b).
To detect Ac–Lys residues in this experiment, certain distinct features of this PTM were
considered in the post-run data analysis (part c). Acetylation of a Lys residue in a peptide
leads to a mass increment of +42 Da, which can be detected by MS. A mass increment
of +42 Da is also evident for the modified Lys residue using MS/MS.
In some cases, further information is available in the mass spectra because chemically
modified amino-acid residues generate modification-specific fragment ions or they eliminate
modification-specific neutral moieties (neutral loss). In the case of Ac–Lys residues, a
diagnostic ion signal at the mass-to-charge ratio (m/z) 126.1 is generated from the modified
peptides. Therefore, extraction of the spectra that contain the m/z 126.1 ion signal from the
LC–MS data set (part b) will retrieve the subset of data that originated from Ac–Lys peptides
('Ac–Lys trace') (part c).
The MS/MS spectrum of a candidate Ac–Lys peptide at m/z 528.8 that elutes at 36 min (part
c) indeed contains the m/z 126.1 signal (part d). The ladder-like y-ion series of peptide-
fragment ions facilitated the assignment of the amino-acid sequence and the modified sites
(part d).
This MS/MS spectrum allowed the assignment of two Ac–Lys residues in the histone protein
(part e).
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Elucidación de Compuestos desconocidos presentes en agua ambiental por utilización de Espectrometría de Masa
Instrumental utilizado:
• SPE: Preconcentración para Muestras diluidas
• HPLC: Convencional
• ESI: Poca Fragmentación. [M+H]+
• QTOF: Detector híbrido con posibilidad de MS/MS
R = 5000 
Preparación de las muestras: 0,1% Acido Fórmico
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Muestra 1: Agua Superficial, El Clot
Cromatograma. Modo Positivo
Tiempo de Retención: 28,11 min.
Área: 363 u.a.
Área Mínima: 100 u.a.
Espectro de Masa del pico de TR = 28,11 min.
PARAMETRO M M+1 M+2 M+3 M+4
Masa 297,0567 298,0599 299,0540 300,0570 301,0516
Abundancia (cuentas) 1084 187 735 133 152
Abundancia Relativa (%) 100 17,3 67,8 12,3 14,0
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Desviaciones aceptables
N° Cuentas Error (%)
< 60 > 50
60 – 200 ± 20
>200 ± 10
PARAMETRO M M+1 M+2 M+3 M+4
Masa 297,0567 298,0599 299,0540 300,0570 301,0516
Abundancia (cuentas) 1084 187 735 133 152
Error Aceptado (%) --- ± 20 ± 10 ± 20 ± 20
Abundancia Relativa (%) 100 17,3 67,8 12,3 14,0
Tolerancias (%) --- 13,8-20,7 61,0-74,6 9,8-14,7 11,2-16,8
Determinadas 
empíricamente con 
patrones para el sistema 
utilizado
Búsqueda de Composiciones Elementales
(Elemental Composition, MassLynx Software)
Parámetro Valor
Error ±7 mu
C 0 – 50
H 0 – 100
O 0 – 20
F 0 – 20
P 0 – 3
Cl
Br
S
[M+H]+ m/z = 297,0567
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Abundancia Relativa M M+2
Compuesto Incógnita 100 61,0 – 74,6
Cl 100 31,98
2 Cl 100 63,93
S 100 4,44 
2 S 100 8,89
2 Cl, 2 S 100 72,82
Patrón Isotópico
Búsqueda de Composiciones Elementales
(Elemental Composition, MassLynx Software)
Parámetro Valor
Error ±7 mu
C 0 – 50
H 0 – 100
O 0 – 20
F 0 – 20
P 0 – 3
Cl 2
Br 0
S 0 – 2
[M+H]+ m/z = 297,0567
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N ° Composición [M+H]+ Error (mu) % M+1 % M+2 % M+3 % M+4
1 C5H17N6OFPCl2 0,4 8,1 64,5 5,2 10,5
2 C14 H15N2OCl2 0,6 16,6 65,4 10,7 11,2
3 C11H16N2O2FCl2 -0,6 13,7 65,2 8,9 11,0
4 C4H13N8F2Cl2 1,0 7,6 64,4 4,9 10,5
5 C12H20O2PCl2 -1,1 10,0 65,0 6,5 10,9
6 C4H16N8FSCl2 -1,3 8,4 68,7 5,7 13,3
7 C10H19F3PCl2 1,3 13,7 69,3 9,3 13,7
8 C8H16N6PCl2 1,6 11,3 64,5 7,3 10,6
9 C8H17N2O3F2Cl2 -1,7 10,0 65,0 6,5 10,9
10 C9H15N2F4Cl2 1,9 11,0 64,5 7,0 10,6
11 C7H12N8FCl2 2,1 10,9 64,5 7,0 10,6
12 C3H15N8O4Cl2 -2,6 6,7 65,0 4,3 10,9
13 C11H19N2OSCl2 -2,8 14,1 69,5 9,6 13,8
14 C6H16N4O4FCl2 3,4 8,5 65,1 5,5 11,0
15 C2H15N10OSCl2 3,9 6,2 64,7 4,0 10,7
16 C4H11N12Cl2 -4,0 9,0 64,3 5,8 10,5
... ... ... ... ... ... ...
30 C7H14N4OF3Cl2 7,0 9,5 64,6 6,1 10,6
Composiciones Elementales para [M+H]+ m/z = 297,0567
Valores Calculados de Abundancias Relativas
N° Composición Error (mu) % M+1 % M+2 % M+3 % M+4
1 C5H17N6OFPCl2 0,4 8,1 64,5 5,2 10,5
2 C14 H15N2OCl2 0,6 16,6 65,4 10,7 11,2
Muestra 0,0 13,8 – 20,7 61,0-74,6 9,8-14,7 11,2-16,8
Comparación de las Abundancias Isotópicas Relativas
Composiciones Elementales Experimentales vs. Calculadas
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Composiciones Elementales Compatibles
N° Composición Error (mu) % M+1 % M+2 % M+3 % M+4
2 C14 H15N2OCl2 0,6 16,6 65,4 10,7 11,2
19 C6H16N6FCl2 -4,6 18,0 65,5 11,6 11,2
Muestra 0,0 13,8 – 20,7 61,0-74,6 9,8-14,7 11,2-16,8
Espectro MS/MS del [M+H]+ m/z = 297,0567
Ión m/z = 255,0146
Pérdida de propeno: C3H6m = 42,0415 u (Dm = -5,5 mu)
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Ambas Composiciones Elementales pueden perder Propeno
Pérdida de Propeno
2) C14 H15N2OCl2 - C3H3 = C11H12N2OCl2
19) C6H16N6FCl2 - C3H3 = C6H13N6FCl2
Merk Index: C14H15N2OCl2 Enilconazol, [M+H]+ m/z = 297,0561
Funguicida veterinario
N
Cl
Cl
O
N
Búsqueda en Base de Datos
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Confirmación de la Estructura
• Idéntico tiempo de retención para el cromatograma 
de un patrón comercial
• Idéntico Espectro de Masa del pico cromatográfico.
• Idéntico Espectro MS/MS del [M+H]+ m/z = 297
• Perdida de propeno factible.
• Concentración estimada: 50 mg/L
N
Cl
Cl
O
N