O-(pentafluorobenzyloxycarbonyl)benzoyl chloride: A novel electrophoric derivatisation reagent for amino compounds designed for negative ion chemical ionisation mass spectrometry

Article abstract of DOI:10.1002/rcm.4775

The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)benzoyl chloride, is described. The reagent was tested against selected primary and secondary amino compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation (NICI), i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting mass NICI mass spectra were expectedly dominated by dissociative resonance electron capture typically observed with these compounds. The reagent is suitable for detecting volatile primary and secondary amines with high sensitivity. Background is reduced by a shift in detected m/z and retention time, as demonstrated for the analysis of the drug methylphenidate from human plasma. Copyright

Full text of DOI:10.1002/rcm.4775

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 3320–3324
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/rcm.4775
o-(Pentafluorobenzyloxycarbonyl)benzoyl chloride: a
novel electrophoric derivatisation reagent for amino
compounds designed for negative ion chemical
ionisation mass spectrometry
*
Hans J. Leis and Werner Windischhofer
University Children’s Hospital, Division of Analytical Biochemistry and Mass Spectrometry, Auenbruggerplatz 34/2, A-8036 Graz, Austria
Received 30 July 2010; Revised 7 September 2010; Accepted 7 September 2010
The synthesis of a novel electrophoric derivatisation reagent, o-(pentafluorobenzyloxycarbonyl)ben-
zoyl chloride, is described. The reagent was tested against selected primary and secondary amino
compounds as analytical targets. The derivatives exhibit excellent mass spectral properties under
negative ion chemical ionisation (NICI), i.e. reduced fragmentation and thus high ion current for the
targeted m/z during analysis. Since the reagent bears a pentafluorobenzyl ester group, resulting mass
NICI mass spectra were expectedly dominated by dissociative resonance electron capture typically
observed with these compounds. The reagent is suitable for detecting volatile primary and secondary
amines with high sensitivity. Background is reduced by a shift in detected m/z and retention time, as
demonstrated for the analysis of the drug methylphenidate from human plasma. Copyright # 2010
John Wiley & Sons, Ltd.
In quantitative analysis of trace amounts there is a never-
ending search for ultimate sensitivity and selectivity.
Analytical assays based on mass spectrometry have emerged
as reference methods due to the well-known inherited
characteristics, allowing the use of stable isotope labeled
internal standards and the choice of various ionisation and
detection modes. In particular, negative ion chemical
ionisation (NICI) provides an extremely sensitive tool in
gas chromatography/mass spectrometry (GC/MS). Basi-
cally, using this detection mode, there are two ways to
produce negative ions from an uncharged molecule: by
resonance electron capture (REC) and dissociative resonance
electron capture.1 REC produces a molecular radical anion in
its excited state that must be stabilised to prevent electron
autodetachment, whereas dissociative REC results in non-
radical anions with a distribution of internal energies,
depending largely on the leaving group and its ability to
absorb excess internal energy, which increases with its size.2–
but, interestingly, the molecular radical anion is seen in
relative low abundance in many cases. Instead of this,
sequential loss of hydrogen fluoride (HF) from the molecular
anion is observed, sometimes displaying a complex frag-
mentation pattern in the NICI mass spectra and hence
reducing the ion current of the ‘diagnostic’ fragment chosen
for quantification.12,13 Thus, derivatives yielding low frag-
mentation under NICI with ions in the higher mass range are
ideally suited for enhancement of sensitivity. This is met by
PFB derivatives of carboxylic acids and phenols, where both,
the carboxylate or phenolate anion and PFB radical formed
by dissociative REC, are stabilised by resonance and hence
produce virtually only these ions without any further
fragmentation in many cases.1,14 Up to now, these extremely
sensitive derivatives are only available for carboxylates and
phenolates, using the PFB bromide as a reagent. It was thus
the aim of this study to develop a new reagent that produces
derivatives of amino compounds with a NICI response
similar or identical to the PFB derivatives of carboxylic acids
or phenols.
4
Besides mass-selective detection, the REC process adds
another dimension of selectivity to the analytical process.
REC is strongly dependent on the electron affinity of the
target molecule, and thus on its electronegativity, rendering
it as electrophoric. As many analytical targets possess only
low electron affinity, chemical derivatisation is usually
employed to enhance NICI response. There are many
reagents available, the most widely used compounds are
perfluoroacyl5–8 and pentafluorobenzyl9–11 (PFB) deriva-
tives. For perfluoroacylates, REC response is usually high,
EXPERIMENTAL
Chemicals and reagents
Pentafluorobenzyl alcohol was purchased from ABCR
(Kahrlsruhe, Germany). Phthalic anhydride, heptafluoro-
butyric anhydride, memantine.HCl, amantadine.HCl, meta-
mphetamine.HCl and amphetamine sulfate were supplied
by Sigma-Aldrich (Vienna, Austria). [18O2H3]-methylpheni-
date was self-synthesised as previously desribed.12 Silylation
grade pyridine was obtained through Pierce (Rockford, IL,
*Correspondence to: H. J. Leis, University Children’s Hospital,
Research Unit of Osteology and Analytical Mass Spectrometry,
Auenbruggerplatz 34/2, A-8036 Graz, Austria.
E-mail: hans.leis@medunigraz.at
Copyright # 2010 John Wiley & Sons, Ltd.

Electrophoric derivatisation reagent for NICI-MS 3321
USA). Racemic (ꢀ)-threo-methylphenidate hydrochloride
was from Medice (Iserlohn, Germany). All other substances,
solvents and reagents of analytical grade were from Merck
(Darmstadt, Germany).
i.d., SGE). The injector was operated in the splitless mode at
2808C. Helium was used as a carrier gas at a constant flow
rate of 2.0 mL/min. The initial column temperature was
1608C for 1 min, followed by an increase of 408C/min to
3158C and an isothermal hold of 10 min. For analysis of
heptafluorobutyrates, the initial column temperature was set
to 808C. The mass spectrometer transfer line was kept at
3208C. NICI was performed with methane as a moderating
gas at an electron energy of 70 eV and an emission current of
0.250 A.
Synthesis of
o-(pentafluorobenzyloxycarbonyl)benzoyl
chloride (PBBCl)
In a screw-capped reaction vial phthalic anhydride (148 mg,
1 mmol) and pentafluorobenzyl alcohol (PFB-OH, 198 mg,
1 mmol) were dissolved in benzene (300 mL) and pyridine
(100 mL) was added. The mixture was kept at 1008C for 2 h.
After acidification with concentrated HCl the intermediate
product was extracted with chloroform. The organic phase
was dried over anhydrous sodium sulfate and concentrated
under nitrogen to yield an oily residue that solidified on
standing in over 90% yield. The crude compound was
recrystallised from benzene or chloroform to yield the pure
mono-PFB ester of phthalic acid as a white solid, as judged
by straight-phase HPLC on silica (20% ether in n-hexane as
a mobile phase) and GC/MS after conversion into the
Extraction and derivatisation of
methylphenidate
Methylphenidate was extracted from spiked plasma as
previously described.12 Briefly, 1 mL of plasma was spiked
with 0.5 ng of [18O2H3]-methylphenidate internal standard
and different concentrations of methylphenidate (4.5-9-18-
36-72 pg). Plasma was made alkaline by addition of
carbonate buffer (pH 9.0) and extracted with 2.5 mL of n-
hexane for 15 min. After centrifugation, the organic layer was
brought to dryness under nitrogen and derivatised as
described above. The residue was reconstituted in 60 mL
ethyl acetate and 2 mL were subjected to GC/NICI-MS
analysis.
.
trimethylsilyl (TMS) ester (MS (EI): m/z 418 (M þ), m/z 403
. .
.
.
þ
(M þ– CH3), m/z 329 (M –TMSO ), m/z 181 (C7H2F:5þ)
Each of the above intermediate products (36.4 mg) were
treated with thionyl chloride (0.5 mL) at room temperature
for 1 h. Excess reagent was removed under nitrogen and the
oily residue dissolved in dichloromethane (10 mL), yielding a
10 mM solution of the PBBCl reagent.
RESULTS AND DISCUSSION
PBBCl reagent preparation and derivatisation
The use of a bifunctional carboxylic acid as starting
compound for the synthesis of an electrophoric derivatisa-
tion reagent provided the functionalities necessary for the
preparation of a typical acylation agent suitable for amino
compounds, as shown in Fig. 1. Alcoholysis of phthalic
anhydride with PFB-OH proceeded smoothly to yield the
mono-PFB ester of phthalic acid. Subsequent formation of the
acyl chloride occurred quantitatively to provide the reactive
group for acylation reactions. As the intermediate acid
product is solid it can be conveniently purified by crystal-
lisation. The acyl chloride reagent can be prepared freshly on
demand, although we have used reagent solutions over
several weeks without any noticeable deterioration when
stored in a freezer.
Derivatisation with PBBCl
Aqueous solutions of amphetamine sulfate, metamphetami-
ne.HCl methylphenidate.HCl, memantine.HCl and amanta-
dine.HCl) were made alkaline with carbonate buffer (pH 9.0)
and extracted with n-hexane. The solvent was removed
under nitrogen and the residue was treated with 100 mL of
reagent solution (PBBCl, 1 mM in dichloromethane) for
30 min at room temperature. Solvent was removed under
nitrogen and the residue reconstituted in 100 mL ethyl acetate
and 1 mL was subjected to analysis by GC/NICI-MS. Alter-
natively, the following procedure can be used: The dry
residue after hexane extraction was treated with 200 mL of
carbonate buffer and 200 mL of reagent solution. The mixture
was shaken for 30 min at room temperature. Thereafter, 1 mL
of n-hexane was added, the mixture vortexed and the upper
hexane phase collected. Concentration and reconstitution
was carried out as described above.
We have used the general procedure described herein to
prepare the PBB derivatives of various primary and
secondary amines without optimising reaction conditions
for each substance. The derivative formed smoothly with the
compounds tested by simply adding reagent solution at
room temperature. Some primary amines could be deriva-
tised by simply adding reagent solution to the alkaline
hexane extract. This is of particular advantage for volatile
amines that would suffer from considerable loss during
solvent evaporation. We have performed derivatisation
reactions with and without diisopropylethylamine as an
acid scavenger and found no enhancement of derivative
yield. With methylphenidate as an example we have
established reaction yields under these conditions. Thus,
reaction has already progressed to 78% after 2 min, gradually
increasing to >98% after 30 min. The PPB derivatives showed
good stability. Derivatised samples were stored under
For assessment of reaction yields, 20 ng of methylpheni-
date was treated with PBBCl as described above. After
certain time points (2-10-15-20-30 min) 50 mL of heptafluor-
obutyric anhydride (containing 2% heptafluorobutyric acid)
were added and the mixture left for an additional 30 min. A
50:50 response was estimated by derivatising equal amounts
separately (as HFB and PBB derivatives) and analysing the
combined solutions.
Gas chromatography/mass spectrometry
A Finnigan TRACE 8000 gas chromatograph coupled to a
Finnigan TRACE quadrupole mass spectrometer (Thermo-
Quest, Vienna) was used. The gas chromatograph was fitted
with a BPX5 fused-silica capillary column (30 m ꢁ 0.25 mm
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3320–3324
DOI: 10.1002/rcm

3322 H. J. Leis and W. Windischhofer
F
F
F
F
O
O
O
F
F
F
F
F
O
HO
2h, 100°C
O
F
+
OH
O
F
F
F
F
F
F
O
O
O
O
F
F
O
SOCl2
r.t., 1h
F
F
Cl
OH
O
Figure 1. Reaction scheme for the preparation of the PBBCl reagent.
various conditions and re-analysed. There was no decrease
in signal intensity when samples were analysed after storage
for 2 weeks at room temperature and 4 weeks at –208C. As the
introduced functional group is large, volatility of the PBB
derivatives is reduced. The reagent is thus well suited for the
analysis of amino compounds of low to moderate volatility.
Under the conditions employed no reaction was observed
with alcoholic and phenolic hydroxyls. Using harder
conditions (high temperatures, base catalyst), there may be
some reaction with hydroxyl groups, but we did not see any
reaction under the mild conditions described here for phenol,
2,3-dichlorophenol, pentachlorophenol, stearyl alcohol, and
butanol. Thus the range of possible interference during
analysis is reduced to amino compounds.
311
100
75
50
25
0
A
369
CH3
330
178
O
CF2 CF3
CF2
O
O
N
291
290
349
389
167
M●─
221
192
271
159
429
59
50
100 150 200 250 300 350 400 450 500 550 600
m/z
NICI-MS of PBB derivatives
NICI as a soft ionisation technique can be expected to show
reduced fragmentation and more high-mass ions as com-
pared to EI. This is, however, only true to a certain extent
when for example perfluoroacyl derivatives are measured.
These derivatives produce molecular anions by REC, and
besides delivering fragment ions in the higher m/z range up
to the molecular ion, fragmentation is quite intense which
results predominantly from the sequential loss of HF from
the molecular anion. We have observed this fragmentation
pattern frequently after derivatisation with heptafluorobu-
tyric anhydride,12,13 thus suggesting the fluoroacyl part to
mainly direct this pathway. In Fig. 2 the NICI mass spectra of
methylphenidate heptafluorobutyrate and amphetamine
pentafluorobenzoylate are shown. Fragmentation is intense
in both cases, the molecular radical anion is not (amphet-
amine) or only weakly (methylphenidate) present. The
proportion of the total ion current is very low, no matter
which fragment ion is chosen for quantification.
274
100
75
50
25
0
B
212
HN
O
F
F
F
F
289
F
309
198
M●─
213
50
100 150 200 250 300 350 400 450 500 550 600
m/z
On the other hand, fragmentation under NICI conditions
can be drastically reduced to one or two fragment ions with
Figure 2. NICI mass spectra of methylphenidate heptafluo-
robutyrate (A) and amphetamine pentafluorobenzoate (B).
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3320–3324
DOI: 10.1002/rcm

Electrophoric derivatisation reagent for NICI-MS 3323
282
100
90
80
70
60
50
40
30
20
10
0
striking intensity in certain cases: PFB derivatives of
carboxylic acids undergo dissociative REC when subjected
to NICI that leaves the carboxylate anion with high stability
and high abundance.1,14 We have observed this unique
feature also with pentafluorocarbamates and pentafluoro-
carbonates.15,16 It could thus be expected that PBB deriva-
tives would also show this fragmentation mechanism and
yield similar results. The NICI mass spectra of the tested
target compounds after PBB derivative formation are given
in Fig. 3. In any case, the carboxylate anion is present with
striking abundance. Fragmentation is extremely reduced or
absent. For the PBB derivatives of memantine, amantidine
A
HN
O
O
O
F
F
F
m/z 282
F
F
284
265
197
200
100
300
296
400
500
600
m/z
100
90
80
70
60
50
40
30
20
10
0
B
.
and amphetamine there is a fragment ion at [M ꢂ–17] with
N
low relative abundance, which is not observed in the NICI
mass spectra of the secondary amines, methamphetamine
and methylphenidate. It is likely that rearrangement under
hydrogen transfer leads to the final neutral loss of ammonia
from the primary amine derivative.
O
O
O
F
F
m/z 296
F
F
F
298
181
200
Gas chromatography of PBB derivatives
100
300
400
500
600
Besides their unique fragmentation behaviour PBB deriva-
tives of amino compounds also show certain benefits in GC
analysis. Due to the bulky PBB group retention times are
shifted dramatically compared to heptafluorobutyrates. As
the latter produce highly volatile derivatives, the retention
time of certain analytes may be right within a region of high
matrix interference when complex matrices such as blood
plasma are encountered. In Fig. 4 analysis of a plasma extract
of the drug methylphenidate is shown after derivatisation
with HFBA and PBBCl. A volume of 1 mL of human plasma
was spiked with 2 ng methylphenidate and 4.7 ng of the
internal standard, [2H318O]-methylphenidate. HFB deriva-
tives elute at a retention time of 5.80 min, whereas PBB
derivatives are seen at 10.95 min. It is evident from this
comparison that matrix interference is by far more prominent
with the heptafluorobutyrates. This may additionally be due
to the fact that HFBA also reacts with hydroxyl groups and
thus extends the range of possible interfering compounds. As
a result of reduced fragmentation, the peak area measured
for the HFB derivative in a separately derivatised 1:1 mixture
of HFB and PBB derivatives was only 14.7% of the PBB
compound. Thus, for methylphenidate, a sensitivity gain of 7
is achieved for the GC/MS detection. The shift in retention
time is even 2 min larger, as indicated in Fig. 4, since for the
heptafluorobutyrates the initial GC temperature was low-
ered to 808C. Our previously described method for the
determination of methylphenidate using HFB derivatives
had a detection limit of 72 pg/mL plasma.12 Thus, we have
established a calibration curve for this compound in
human plasma after PBB derivatisation at concentrations
below that limit. For the concentration range investigated
(4.5–72 pg/mL plasma) we have found a linear response
(r2 ¼ 0.9996) with a signal-to-noise ratio of 96 at 4.5 pg/mL.
In general, sensitivity is enhanced by the PBB reagent
described here in comparison to common fluoroacylates.
Additionally, the shift in retention time provides a region of
lower interference from the matrix. Thirdly, the selectivity of
the reagent for amino compounds also reduces interference
from biological matrices.
m/z
298
100
F
F
C
90
80
70
60
50
40
30
20
10
0
F
F
m/z 298
F
O
O
O
HN
300
281
300
100
200
400
500
600
m/z
326
100
90
80
70
60
50
40
30
20
10
0
F
F
F
D
F
F
m/z 326
O
O
O
HN
H3C
CH3
328
309
300
100
200
400
380
500
600
m/z
100
90
80
70
60
50
40
30
20
10
0
E
F
F
F
F
m/z 380
O
F
O
O
N
COOCH3
381
400
H
100
200
300
500
600
m/z
Figure 3. NICI mass spectra of PBB derivatives of amphet-
amine (A), metamphetamine (B), amantadine (C), memantine
(D), and methylphenidate (E).
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3320–3324
DOI: 10.1002/rcm

3324 H. J. Leis and W. Windischhofer
5.20
100
80
60
40
methylphenidate-HFB
m/z 369
m/z 374
RT: 5.80
MA: 1387471
20
5.65
100
80
[2H318O]-methylphenidate-HFB
60
RT: 5.80
MA: 2064397
4.92
40
20
RT: 10.98
MA: 2179016
100
80
4.58
60
m/z 380
methylphenidate-PBB
40
5.07
9.06
10.70
20
9.06
100
80
[2H318O]-methylphenidate-PBB
60
m/z 385
RT: 10.95
MA: 3577173
40
20
5
6
7
8
9
10
11
12
13
14
Time (min)
Figure 4. Chromatograms obtained after analysis of human plasma spiked with 2.0 ng/mL of methylphenidate and
4.7 ng/mL of the stable isotope labeled internal standard. Identical samples were converted into the PBB and HFB
derivatives, respectively, and analysed by GC/NICI-MS.
CONCLUSIONS
2. Williamson DH, Knighton WB, Grimsrud EP. Int. J. Mass
Spectrom. 2000; 196: 481.
In this study we report the synthesis of a new derivatisation
reagent, PBBCl. The compound is reactive towards
primary and secondary amines and displays ideal charac-
teristics for analysis by GC/NICI-MS. NICI response is
better than with the established heptafluorobutyryl deriva-
tives. The unique NICI mass spectra obtained allow
specific detection. The reagent can be prepared freshly on
demand by a simple procedure. The PBB derivatives are
sufficiently stable, as repeated injections of one prepared
sample on several consecutive days did not show any loss
of signal intensity. The reagent described herein allows
the beneficial PFB derivatisation hitherto restricted to
phenolates and carboxylic acids also to be extended to
amino compounds, making them amenable to highly
sensitive assays.
3. Chen ECM, Wenthworth WE, Desai E, Batten CF.
J. Chromatogr. 1987; 399: 121.
4. Berkout VD, Mazurkiewicz PH, Deinzer ML. Rapid Commun.
Mass Spectrom. 1999; 13: 1850.
5. Kostiainen R. Anal. Chim. Acta 1988; 204: 233.
6. Gaskell SJ, Brooks PW. Int. J. Mass Spectrom. Ion Phys. 1983;
48: 241.
7. Macfarlane RG, Midgley JM, Watson DG, Evans PD.
J. Chromatogr. 1990; 532: 13.
8. Hubbard WC, Hundley TR, Oriente A, MacGlashan DW.
Anal. Biochem. 1996; 236: 309.
9. Hubbard WC, Alley MC, McLemore TL, Boyd MR. Cancer
Res. 1988; 48: 4770.
10. Strife RJ, Murphy RC. J. Chromatogr. B 1984; 305: 3.
11. Hachey DL, Patterson BW, Reeds PJ, Elsas LJ. Anal. Chem.
1991; 63: 919.
12. Leis HJ, Fauler G, Raspotnig G, Windischhofer W. J. Mass
Spectrom. 2000; 35: 1100.
13. Leis HJ, Fauler G, Raspotnig G, Windischhofer W.
J. Chromatogr. B 2000; 744: 113.
14. Leis HJ, Leis M, Windischhofer W. J. Mass Spectrom. 1996; 31:
486.
15. Leis HJ, Windischhofer W, Fauler G. Rapid Commun. Mass
Spectrom. 2002; 16: 646.
REFERENCES
1. Leis HJ, Fauler G, Rechberger GN, Windischhofer W. Curr.
Med. Chem. 2004; 11: 1585.
16. Leis HJ, Windischhofer W, Raspotnig G, Fauler G. J. Mass
Spectrom. 2001; 36: 923.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 3320–3324
DOI: 10.1002/rcm