Confirmation of Pesticide Residues by Liquid Chromatography

Chapter 3

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 6, 2017 | http://pubs.acs.org Publication Date: February 22, 1990 | doi: 10.1021/bk-1990-0420.ch003

Confirmation of Pesticide Residues by Liquid Chromatography/Tandem Mass Spectrometry Thomas Cairns and Emil G. Siegmund Department of Health and Human Services, Food and Drug Administration, Mass Spectrometry Service Center, Los Angeles District Laboratory, 1521 West Pico Boulevard, Los Angeles, CA 90015

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The production of a protonated molecule ion, [MH] , for a pesticide under investigation is often the principle ion produced under the soft ionization conditions determined by the various LC/MS methods. While this ion is preferred for primary identification purposes, the lack of fragment ions places the burden of proof of presence on a single ion. Product ion spectra derived from protonated molecule ions can usually provide the additional information needed to satisfy the criteria for confirmation. Cases histories discussed for organophosphorus pesticides, methylureas, and carbamates indicate a strong trend towards the increased reliance of LC/MS/MS to satisfy the criteria for confirmation.

Most applications of L C / M S to pesticide analysis involves soft ionization methods because of the operational characteristics of the various interface types employed. Only two devices can offer the option of producing spectral data under EI conditions, the Moving Belt Interface (MBI) and the more recently introduced Particle Beam. However, production of a protonated molecule ion or adduct ion provides a primary identification method based on molecular weight. The presence of a single ion is not sufficient for confirmation of presence unless high resolution measurements substantiate the molecular formula at the correct retention time. Under low resolution conditions it has been experimentally accepted that a minimum of three structurally significant ions are required for proof of presence (1). Since most applications are performed on low resolution instruments, the need for additional structural evidence for confirmation is necessary. With the availability of M S / M S instruments, the degree of specificity by soft ionization methods has been extended via precursor/product ion experiments to provide the final evidence for confirmation. In some cases, reaction product ion monitoring provides a screening method for generic classes of compounds such as organophosphorus pesticides (2,3). This chapter not subject to U.S. copyright Published 1990 American Chemical Society Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

3. CAIRNS & SIEGMUND

Confirmation of Pesticide Residues

41


CASE HISTORIES Organophosphorus Pesticides (OP). Because of the thermal lability of many pesticides belonging to this general class, analysis by HPLC is the method of choice for primary screening of residues. For confirmation purposes, however, L C / M S techniques often lack sufficient structural evidence to be conclusive. In the case of dimethoate (Fig. 1) the protonated molecule ion at m/z 230 under methane CI conditions is accompanied by two major fragment ions, m/z 88 and 199. Under incurred residue conditions, however, the utility of the ion at m/z 88 for confirmation purposes is unrealistic, since the background spectrum generated by the MBI doers not allow ions below an m/z value of 100 to be used with confidence particularly at ng levels injected on column. With only two potential ions for confirmation, the burden of proof of presence becomes a critical issue. A solution to this problem can be found in the product ion spectrum generated by using the protonated molecule ion as the precursor ion (Fig. I). In particular, the product ion at m/z 125 is a clear indication of the presence of a dimethoxyphosphorothionate. The appearance of another product ion at m/z 171 is further evidence of a dithionate entity (Scheme I). The structural significance of these product ions derived from the protonated molecule ion without the interference of background ions (sample matrix plus interface effects) has permitted the confirmation of dimethoate beyond the minimum criteria. In the reverse mode, product ions can be used for structural elucidation work. In the case of etrimphos (Fig. 2), an unknown compound encountered in a pesticide residue sample, its identity could not have been inferred either by the EI or CI spectrum since both lacked fragment ions for structural detective work. However, the product ion spectrum derived from the protonated molecule yielded an indication that the compound could be a dimethoxyphosphorothionate or dithionate from the presence of the ion at m/z 125 (cf. dimethoate). The additional presence of ions at m/z 109 and 143 in conjunction with m/z 125 was strong evidence (Fig. 2) that the compound was a dimethoxyphosphorothionate. Additional experimental work on the M S / M S of a larger number of organophosphorus pesticides has revealed that generic product ions exist to characterize the phosphates, i.e. phosphorothiolates, phosphorothionates, and phosphorodithioates. Therefore, a screening method based on certain product ions such as m/z 125 to detect the presence of dimethoxyphorothionates can be formulated. This selected product ion monitoring (SPIM) approach (3) has several advantages over the conventional EI and CI spectral characterization. First, the ability to preferentially screen without extensive sample clean-up can be an advantage over the labor intensive sample work-up thereby saving considerable time and effort. Second, the interfering sample matrix background and the interface generated background have been eliminated. Third, the level of sensitivity of detection for these compounds has been improved by utilizing the generic product ion approach to target those samples requiring additional analysis. Therefore, this screening approach gives two vital criteria - the retention window containing the potential OP and its molecular weight inferred from the protonated molecule ion that yielded the product ion.

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

42

LIQUID CHROMATOGRAPHY/MASS

Methane Chemical Ionization

SPECTROMETRY

199 CH30 SCH2CONHCH N

x

3

230 DIMETHOATE


88

258 l)„l,l

Collision-activated Dissociation

l,il,.|,JJl

i

270

,,

125

47 42

,ι,,,,ΐι,,^

Precursor I

79 157

171

230

Figure 1· Mass spectral data for dimethoate: top. methane CI; bottom, product ion spectrum using the protonated molecule ion, [M + Η J , m/z 230 as the precursor ion.

Scheme 1. Product ion fragmentation pathway of the protonated molecule ion at m/z 230 for dimethoate using argon in the collision cell.



Β

3

O ^ S

Ν

143 164

»Λ CH2CH3

200

233

265

Figure 2. Mass spectral data for etrimphos: A , EI mass spectrum; B, CI mass spectrum; C , product ion spectrum using the protonated molecule ion, [M + Η) , m/z 293 as the precursor ion.

100

C H

C H 3 C K / 0 - ^ \

Ο—CH2CH3

300

"Γ"

292

300

NT 292


G


44

UQUID

CHROMATOGRAPHY/MASS

SPECTROMETRY

Neburon [l-n-butyl-3-(3,4-dich1orophenyl)-l-methylureal. The mass spectra obtained for neburon with the MBI under methane CI and ammonia CI are illustrated in Figure 3. For confirmation purposes in residue samples, ion values below m/z 100 are impractical to use because of interfering background ions. Admittedly in this case the presence of two chlorine atoms gives the added advantage of observing the appropriate chlorine ratio measurement in both the protonated molecule ion and chlorine-containing fragment ions (m/z 188 and 239 under methane CI). These spectra were obtained using a vaporizer temperature of 170° C and a source temperature of 130 C to eliminate temperature dependence phenomenon (4). Neburon was then examined under TSP conditions (Fig. 4). Normally the vaporizer temperature is set for maximum sensitivity by observing the resultant intensity of the reagent gas spectrum, i.e. [NH ] when using ammonium acetate as buffer. At high vaporizer temperatures, severe thermal decomposition was encountered as evidenced from the complete disappearance of the protonated molecule ion at m/z 275. With the lowering of the temperature to 125° C, a mass spectrum was obtained displaying only two prominent fragment ions, m/z 205 and 222. These ions were consistent with the thermal degradation of neburon to dichlorophenylisocyanate (molecular weight 187) followed by ionization of the reagent species | N H f and | N H . N H ] to give m/z 205 and 222 respectively. Therefore, the product ion spectrum derived using the protonated molecule ion, m/z 275, was used as confirmatory proof of presence. The ions used for confirmation were the [MH] + and [M + H + 2] to indicate the presence of two chlorines and the product ion spectrum of [ M H | to give m/z 205 and 88. 4

+

4

4

3

+

Carbaryl [1-naphthyl-N-methylcarbamate]. The thermal instability of carbamates has necessitated their analysis by HPLC (5). For confirmation purposes, the CI mass spectra (methane and ammonia) obtained on the MBI interface exhibit m/z 145 as the base peak corresponding to the protonated 1-naphthol moiety. Under ammonia conditions, the mass spectrum of carbaryl exhibited a protonated molecule ion at m/z 202 together with an ammonium adduct ion at m/z 219. However, these ions were of such low relative abundance as to preclude them from consideration as precursor ions for M S / M S experiments. Under the circumstances it was concluded that carbaryl did not display thermal degradation and that the ion at m/z 145 was produced by fragmentation of the protonated molecule ion. Using the protonated l-naphthol moiety, the product ion spectrum (Fig. 5) exhibited product ions to indicate a structural disassembly of the ion atom by atom. Similar experiments with the protonated molecule ion derived from injecting 1-naphthol did not reveal the same product ion spectrum. Therefore, for proof of presence the use of a fragment ion derived under methane CI rather than the protonated molecule ion was found to be satisfactory since it was demonstrated that 1-naphthol would not yield the same product ion spectrum. While G C / M S methods can detect and confirm to residue levels as low as 10 pg injected, the sensitivity levels obtained by M S / M S methods are generally one order of magnitude higher (100 pg) due to loss of ions in the collision cell.


45

Confirmation ofPesticide Residues

S

>

CAIRNS &SIEGMUND

X Relative Abundance

n

e

C

M H | 275

I

+

1 !

1

1 4

8

8

I ' ' ' ' • ι 100

\

239 li

, m/z

S

•

1

" i 200 Ammonia C I

X Relative Abundance


Metha

88

I •

1•

MH

+

11 ,.

!

1 · .

300 275

Neburon 114 100

m/z

200

300

Figure 3. Mass spectral characterization of neburon via the moving belt interface: top, under methane CI conditions; bottom, under ammonia CI conditions.

100-1

ι

Vaporizer Temperature ° C 70

H

IΓ\ i

ϊ

ï Tii

65 60 55

I T T m/z 205

4— S

77

m/z 222 32-,

MH m/z 275

2.3-,

[M + NH4[j m/z 292

+

jl

^ , ί\, ,11

/Ν,

λ

Ί

Λ-JL

411.8

TIC J

L

100

Scan Number

500

Figure 4. Multiple ion detection chromatograms for neburon via thermo spray |100ng injections into the solvent system, 50% methanol-water with 0.1M ammonium acetate] at various vaporizer settings.



LIQUID CHROMATOGRAPHY/MASS

Methane Chemical Ionization

SPECTROMETRY

145

OCONHCH3

CARBARYL

173 185

m/z

100

Collision-activated Dissociation ,00Ί

Confirmation of Pesticide Residues by Liquid Chromatography

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