Application Note: 376

Mechanism of [M+H]+ Formation in Atmospheric Pressure Photoionization Mass Spectrometry

Amin Kamel and Kevin Colizza, Department of Exploratory Medicinal Sciences, Pzer PGRD, Groton, CT; Patrick Jeanville, Thermo Electron Corporation, Miami, FL

Overview
Key Words TSQ Quantum Ultra AM APPI Impurity identication To investigate an impurity of acetonitrile (propionitrile) as a possible proton donor in the absence of dopant by assuming photo-initiated isomerization of priopionitrile.

Introduction
Atmospheric Pressure Photoionization (APPI) is a novel ionization technique recently introduced by Robb et al,[1] Syage and his co-workers.[2] Two approaches of APPI have emerged: direct APPI and dopant-assisted APPI. The mechanisms of ion formation in the positive, as well as negative ion mode have been reported for both.[3-6] The initial reaction in APPI is formation of a radical cation of the dopant, which may ionize the analyte through a charge exchange reaction if the ionization energy (IE) of the radical cation of the dopant is higher than that of the neutral analyte. Alternatively, the dopant radical cation can ionize solvent molecules by proton transfer and the protonated solvent molecules can then protonate the analyte. In this study, an impurity in crude acetonitrile, propionitrile, was identied with high accuracy mass measurement and its possible involvement in the mechanism of [M+H]+ formation was investigated assuming photoinitiated isomerization of propionitrile.

Experimental Conditions
Chemicals and Reagents
Formic acid and ammonium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile was acquired from Burdick and Jackson (Muskegon, MI), and HPLC grade water was purchased from J.T. Baker (Phillipsburgh, NJ). Althiazide and Bendroumethazide were provided by Pzer Global Research and Development, Groton, CT. Propionitrile-d5 was purchased from C/D/N Isotopes (Pointe-Claire, Quebec, Canada). All chemicals were used as received. Sample preparation: Stock solutions of althiazide and bendroumethazide were in HPLC grade acetonitrile. The stock solutions were then diluted with acetonitrile to give working standard solutions at a concentration of 1.0 ng/L. No further sample preparation was required.

Sample analysis: Flow-injection analyses were performed on a Surveyor HPLC System (San Jose, CA). The isocratic method employed two distinct solvent compositions: 1). 50:50 (HPLC grade H2O) and (HPLC grade acetonitrile), and 2). 50:50 (0.1% formic acid) and (HPLC grade acetonitrile) at a ow rate of 200 L/min. Working standards (althiazide and bendroumethazide) were prepared at a concentration of 1.0 ng/mL in acetonitrile for use in this study. 5.0 L manual injections were made onto the system. The entire LC efuent from the sample injections (3 replicates) was directed to the Ion Max source equipped with an APPI interface on a TSQ Quantum Ultra AM. For dopant-assisted APPI, dopant solutions (Anisole and Acetone) were infused via syringe pump as a sheath liquid at a ow rate of 20 L/min. MS Conditions: The TSQ Quantum Ultra AM was calibrated in normal and high resolution modes with a solution of 1,3,6-Polytyrosine. Accurate mass calibration of the instrument was performed with a 50 pmol/L solution of ammoniated polyethylene gycols (PEGs). Accurate mass measurements were made in the Q3 SIM mode, employing internal calibrants. Instrument conditions employed were as follows: Ionization mode and source: Positive APPI Krypton Lamp Energy: 10 and 10.6 eV photons Vaporizer temperature: 475C Sheath gas: 47 Auxiliary gas: 25 Ion Transfer Tube Temperature: 70C Ion Transfer Tube offset: 35 V Tube Lens offset: 77V Collision energy: 22eV Scan Width: 0.2 Da Scan Rate: 200 ms Q3 Resolution (SIM Mode): 0.1 Da FWHM Accurate Mass Mode: Internal Data processing: Average (5) Micro Scans: 2

With acetone as a dopant, protonated acetone and a high abundance of protonated analytes were observed in Vaporizing ca. 2 10 M of althiazide and bendroumetthe APPI mass spectra; their corresponding radical cation hazide under APPI in ACN (no dopant) produced their species were absent and/or not detected (Figure 3). corresponding protonated species, Figure 1. These test However, when anisole was used as a dopant both procompounds showed no response in the positive ion mode tonated analytes and protonated anisole and their ESI mass spectra. corresponding radical cation species were observed in In addition to protonated acetonitrile, its dimers, the APPI mass spectra (Figure 4). and acetonitrile/water clusters, we identied protonated Most organic molecules have ionization energies (IE) [8] with high propionitrile, an impurity in crude acetonitrile, in the range of 7-10 eV[1]. The Kr lamp employed under accuracy mass measurement and a corresponding mass these experimental conditions produces ca. 10.4 eV error of <5 mmu (Figure 2). Propionitrile dimer and prophotons. Therefore, when considering the high IE of ACN pionitrile/water clusters were also observed (Figure 1). and propionitrile as compared to that of Kr lamp (IE of ACN and [Propionitrile+H]+ propionitrile are 12.2 eV [7] and + C3H5N [ACN+H] 11.8 eV,[9] respectively), one pos[Propionitrile+H2O+H]+ 42.1 56.1 10 sible mechanism for the formation of protonated althiazide and ben8 [ACN+H2O+H]+ droumethazide could be attrib[2ACN+H]++ 6 [2Propionitrile+H]+ uted to the reaction between their 4 111.0 83.1 M+. ions; produced by direct 60.1 2 81.16 95.12 97.10 photon ionization and neutral 54.12 43.1 84.12 0 molecules as follows: 3 4 5 6 7 8 9 10 11 12 M + hv M+. [Bendroflumethiazide+H]+ M+. + S [M+H]+ + [S-H].

Results and Discussion

-6

Relative Abundance

100 Relative Abundance

Cl

422.0

80
S

H N N H S O

O S NH2 O O

H2N O F F

O S

O S

O NH N H

[Althiazide+H]+ 383.9 40
60 20 0 380 385 390 395
385.96

Althiazide

Bendroflumethiazide

423.00 424.00 404 428 436

400

405

410

415

420

425

430

435

440

Figure 1: APPI mass spectra of althiazide and bendroumethazide in ACN (no dopant)

Accurate mass measurements utilizing the elemental composition calculator produced an elemental formula of C3H6N, with a corresponding mass error of 4.854 mmu Note: The theoretical accurate mass of propionitrile (protonated) is 6.0495 m/z

[ACN+H2O+H]+
100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

60.0444

[ACN+H]+
42.0338

Relative Abundance

Other possible mechanisms, as reported by Marotta and Tradldi[7] include proton transfer in the absence of dopant by assuming photo-initiated isomerization of acetonitrile. Similarly, the role of propionitrile in the production of [M+H]+ was investigated in the absence of dopant using CD3CD2CN. The formation of [M+D]+ species was observed suggesting a possible role of propionitrile in the protonation process. From the ndings of Marotta and Tradldi,[7] direct photoionization of propionitrile and/or water and subsequent formation of their corresponding protonated species can also be excluded due to their high IEs (11.8 and 12.6 eV, respectively). Furthermore, the production of protonated propionitrile under direct photoionization could

[Propionitrile+H]+ C 3H 6N
56.0543
42.0 42.1 56.0 56.1 60.0 60.1

m/z

m/z

m/z

Figure 2: Accurate mass determination of propionitrile

Page 2 of 6

100 Relative Abundance 80 60 40 20

59.14

[Acetone+H]+

60.14

0 30 40 50 60 70 80 90 100
+

110

120

100 Relative Abundance 80 60 40 20 0 380 385 390 395 400 405 410

[Bendroflumethiazide+H] 422.0

[Althiazide+H]+ 383.96
385.94 387.96 423.02 426.96

415

420

425

430

435

440

Figure 3: APPI mass spectra of althiazide and bendroumethazide in ACN: H2O (acetone dopant)

100 Relative Abundance 80 60 40 20 0 30 40 50 60 70 80 90 100

[Anisole]+. 108.8 [Anisole+H]+

be attributed to photo-initiated isomerization of propionitrile. Nguyen and co-workers have studied the isomerization of propionitrile and the most stable species have been identied using ab initio molecular orbital calculations.[9] Figure 5 shows possible isomeric structures (neutral and radical cation) of propionitrile and their corresponding relative energies based on ab initio molecular orbital calculations at the QCISD(T)/UMP2 level with the 6-31G(d,p) are summarized in Table 1. The radical cations a+., b+. and e+. have similar low energies and are the most stable forms among the possible isomers of propionitrile (Table 1). The ionization energies (IE) of propionitrile and its most stable isomers are summarized in Table 2. The low IE of isomers a+., b+. and e+ make them susceptible to
Structure radical cation E (UQCISD(T)/6-31G(d,p) kJ.mol-1

106.12 105.08

109.08
115.08 118.08

110
+

120

[Althiazide]+.
100 Relative Abundance 80 60 40 20 0 380 385

[Bendroflumethiazide+H] 421.98 [Bendroflumethiazide]+.

421.00
387.96

[Althiazide+H]+

383.98 383.07 385.92

390 395 400 405 410 m/z 415

423.02 424.96

420

425

430

435

440

Figure 4: APPI mass spectra of althiazide and bendroumethazide in ACN:H2O (anisole dopant)

a+. b+. c+. d+. e+. f+. g+. h+. i+. j+. k+. l+. m+. n+. o+.

0 -1 27 43 6 81 50 228 247 147 96 447 99 134 131

Table 1: Calculated relative energy (E, kJ.mol-1) of possible [C3H5N]+. isomers with respect to the energy of structures a+, b+, and e+ representing the lowest determined values (Nguyen et al, J. Phys. Chem. A 1999, 103, 938-948)

Page 3 of 6

H H C H
H

hv

H C H

H H

H H C H C N C H

hv

a+
H H

Structure
H C C N C

IE (eV)

eH C N C C H H

H H C H N C C H

hv

11.8

H H C H C H
H H H C C H N C H hv e-

H H eH hv C H
H H H C C H N C H

C N C H

N C H

c+
H

H H C H C N C H

7.1

+
H C H N

a
H H C C H

H H H C C H
H eH C C H H N C H hv

H eC N H H H hv C C H C N H

8.0

e+
H H

b
H C C H C N H

H H C C H H N C H

f+

NA (expected to be <10 eV based on its calculated E, see Table 1)

H eH C C H H
H C C H H hv N C e-

H H C C hv H H C N H

g+

Table 2: Ionization energy (IE, eV) of propionitrile and its most stable isomers (Nguyen et al, J. Phys. Chem. A 1999, 103, 938-948)

H H

H H

H C C H H N C

h+

H H

H C C H H H C C H C N H
H C C H N H hv ehv

H C eN hv eH hv H H

H C C H H H C C C N H
H C H C C H N H

C N

i+ j+

H H

H C H

k+

H C H H C

H eN C H hv H H

H C C

H N C H

l+
Figure 5: Possible isomeric structures (neutral and radical cation) of propionitrile and their corresponding relative energies based on ab initio molecular orbital calculations at the DE QCISD(T)/UMP2 level with the 6-31G(d,p) are summarized in Table 1. The radical cations a+., b+. and e+. have similar low energies and are the most stable forms among the possible isomers of propionitrile (Table 1). The ionization energies (IE) of propionitrile and its most stable isomers are summarized in Table 2.

H C H C H
H H C N

H H eC H C N H
H H C N H C H C H

C N H
H C H C H hv

m+

hv

H
e-

n+

H C H C H

H e N C H hv
-

H C H C H

H N C H

o+

Page 4 of 6

photoionization by the Kr radiation. Once ionized, these radical cation species can react with a neutral molecule of propionitrile to form the corresponding protonated species as proposed in Scheme 1.

H H H H C C N C H

H
C

N C C
H

H
H

H
H

C C
H H

N C

C
H

a+

[1,3]-hydrogen shift

H C H C N H

H H C H

H H H H C N C C H

H C C

H N C
H H C H N C C H H H

C C H H

N C

[1,3]-hydrogen shift

H H H C H N H C C H

H H H C C H C N H

H H

H C C H H N
C

H H H C C H
H

H C N H

C C
H

N C H

e+

[1,3]-hydrogen shift H H H

C C H C N

H H

Scheme 1: Proposed mechanisms for the formation of protonated propionitrile at m/z 56 in the APPI of propionitrile

Page 5 of 6

Conclusions
An impurity in crude acetonitrile, propionitrile was identied with high accuracy mass measurement on the TSQ Quantum Ultra AM with a corresponding mass error of <5 mmu. Theoretical data from the literature supported the assumption that one possible mechanism, among others, for the formation of [M+H]+ could be attributed to photoinitiated isomerization of propionitrile and/or other impurities. The most stable isomers of propionitrile (based on their calculated IE and DE) were assumed to undergo proton transfer. In addition to the immense data reported in the literature regarding APPI, these data and further studies may help understand different mechanisms involved in the formation of [M+H]+ in the APPI process.
References
Damon B. Robb, Thomas R. Covey, and Andries P. Bruins, Anal. Chem. 2000, 72, 3653-3659 2 Syage DB, Evans MD. Spectroscopy 2001, 16:15 3 Kauppila et al, Anal. Chem. 2002, 74, 5470-5479 4 Benter et al, Rapid commun. Mass Spectrom. 2005; 19: 326-336 5 Syage DB, J. Am. Soc. Mass Spectrom 2004, 15, 1521-1533 6 Bruins et al, Rapid Commun. Mass Spectrom. 2004; 18: 808-815 7 Ester Marotta and Pietro Traldi, Rapid Commun. Mass Spectrom. 2003; 17: 2846-2848 8 Kenny, G and Miles, H., UK patent application 1992. 9 Nguyen et al, J. Phys. Chem. A 1999, 103, 938-948.
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