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Fast atom bombardment mass spectrometry of β-lactam antibiotics
International Journal of Mass Spectrometry and Ion Physics, 46 (1983) 431-434 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
431
FAST ATOM BOMBARDMENT MASS SPECTROMETRY OF B-LACTAM ANTIBIOTICS
3.L.GOWER
Beecham Pharmaceuticals,
Chemotherapeutic
Research Centre, Brockham Park,
Surrey RH3 7A3, England.
ABSTRACT The advantages and disadvantages of Fast Atom Bombardment mass spectrometry (FAB-MS) are described with reference to penicillins, cephalosporins and clavA comparison of FAB spectra with Field Desorption (FD) and Desulanic acid. orption Chemical Ionization (DCI) spectra is given together with brief comments on mass measurement of ions in FAB mass spectra, paper chromatography FAB-MS and the use of metastable scanning techniques with FAB-MS.
INTRODUCTION UnLil
recently
the
mass spectrometric analysis of penicillinsand
other 8-
lactam antibiotics as their free acids or alkali metal salts depended on derivatization to form either methyl or trimethylsilyl esters. ins such
as benzyl penicillin
the derivatization
With simple penicill-
procedure proved effective
a single ester was formed that was amenable to gas chromatography. complicated
need
With more
examples such as ampicillin or amoxycillin problems with mixtures
of products and the inability to gas chromatograph the
and
these products have led to
to find alternative methods of mass spectrometric analysis.
DCI-MS (ref.1) and FAB-MS
(ref.2) have both shown useful molecular weight and
structural information on penicillins and cephalosporins. spectra showing mqlecular weight information. MS of penicillins
Recently
and cephalosporins
FD-MS also gives
Since the original work on FAB-
a more detailed study has been
carried out
and some of the results are presented here.
FAB-MS With
of
B-LACTAM ANTIBIOTICS
free
acids e.g. ampicillin, the positive ion spectra show strong proton-
ated molecular ions CM]' often with relatively intense dimeric species [2M+H]+. Fragment ions are usually intense but obviously dependent on the side-chain structure, dominating ment
The negative ion spectra are often more intense with the molecular
ion observed in both
ion region. penicillins
A particularly and
common
[M-H]-
and intense
Q
frag-
cephalosporins _involves cleavage
across both rings to give
0020-7381/83/0000-0000/$03.00
ions
1983 Elsevier Scientific Publishing Company
432
I’i
R-C-NH
The FAB mass spectra of 8-lactam antibiotics as their alkali metal salts are usually, as might be expected, more intense than the free acid spectra with cationized species [M+X]' and [ZM+Xj+dominating the positive ion spectra. The negative ion spectra show intense ions for the two ring cleavage structure
mentioned above and for the carboxylate anion [M-Xl-and dimeric ion [2M-XI-. Losses of small molecules i.e. C02, CH3COOH are also common.
Molecular weight
determination of unknown penicillins has been aided by cation exchange using excess alkali metal ions in the glycerol e.g. Li+. The positive ion and negative ion FAB mass spectra of potassium clavulanate
(I> in general show the same characteristics
as the other 8-lactam antibiotics.
The positive ion FAB mass spectrum is dominated by the cationized molecular ion [M+K]+ at m/z 276 and dimeric species [ZM+K]+ at m/z 513.
The trimeric species
[3M+K]+ at m/z 750 and tetrameric species at m/z 987 [4M+K]+ are also intense. Glycerol adduct ions e.g., [M+K+glycerbl]+ at m/z 36Band
[2M+K+glycerol]+ at
m/z 605 are present.
Several significant fragment ions are seen in the mass
range up to m/z 250.
Comparison of the spectra with the spectra of the sodium
and lithium salts and MIKES studies of the spectra of the potassium salt are currently being carried out in order to assign structures to these ions. The base peak in the negative ion spectrum is the anion [M-K]- at m/z 198 with-an intense ion for the dimeric species [2M-K]- at m/z 435. molecular ion [M-H]- is present at m/z 236.
The pseudo
A major fragment ion at m/z 136
probably represents loss of carbon dioxide and water from the anion [M-K]-.
COMPARISON OF FAB, DCI, AND FD MASS SPECTRA OF AMPICILLIN
(II)
The positive ion FD mass spectrum of ampicillin is very simple consisting of the protonated molecular ion [MH]+ at m/z 350.
At high emitter currents a
few Low intensity fragment ions were detected but were very transitory. emitter loading produced very few spectra.
Each
Both positive and negative ion FAB
433
mass spectra are relatively simple with the negative ions spectrum being dominated by the two ring cleavage ion at m/z 207 and the @i-H] -ion at m/z 348. Loss of CQOH from this pseudomolecular
ion gave the other major fragment ion.
positive ion spectrum is more complex
The
with
side-chain fragment ions at
m/z 91 [PhCH2]*, and m/z 106 [PhCHNH2]+, together with the thiazolidine cleavage ion at m/z 160 dominating the low mass region of the spectrum and the protonated molecular ion [MH]+ at m/z 350 being
the major high mass ion.
The ammonia positive ion desorption CI spectra is complex with the protonated molecule ion and the ammonia adduct ion at m/z 350 and 367 respectively being observed.
Several intense fragment ions are observed several of which must
arise from complex rearrangements as previously described, (ref.1). In contrast, the methane negative ion desorption
CI spectrum is relatively
simple
with
molecular anion [Ml- at m/z 349 and several other minor fragment ions. base
a
The
peak in the spectra is the thiazolidine anion at m/z 159. The information provided by the three ionization methods is complementary
with FDMS being of most use with complex mixtures and DC1 in those problems where the
fragmentation
information is of value in the structural determination
of unknowns. The FAB mass spectra are the most useful with molecular weight and useful fragmentation information.
The fact that the ionization method produces long
lived species enabling mass measurement and metastable analysis to be carried out is of great importance. Work
on a more detailed comparison between the FD,
FAB,
DC1
and laser des-
orption mass spectra of penicillins will be published elsewhere.
ACCURATE MASS MEASUREMENT AND METASTABLE ANALYSIS Using peak matching on a VG-ZAB mass spectrometer the protonated and cationized molecular ions of a series polymer
of penicillins
have
been measured.
ions and ions from standard penicillins were used
Glycerol
as reference ions and
results with accuracies of 10 p.p.m. were routinely obtained. There were regions in the mass range where no suitable reference ion could be found using standard penicillins but a set of quaternary ammonium salts has been
434
built up to cover these gaps, The ed
stable
on FAB mass
phalosporins,
ion currents resulting from FAB enable MIKES spectra.
MIKES
scans
on
the
CMHl+
and
scans
[MH]-
to
be
obtain-
ions from two ce-
cephaloglycine and cephalexin have been reported previously
(ref
3).
Metastable
scanning methods are of particular importance In deciding the or-
igin of fragment ions especially when
there is the possibility that an ion may
have come from a protanated molecule on [M+H]+,
a cationized ion e.g. [M+K]+
or a glycerol attachment ion e.g. [M+K+glycerol]+.
PAPER
CHROMATOGRAPHY/FAB-MS
Recent
work has concentrated on paper
ed by FAB-MS.
chromatography
of penicillins follow-
Using Whatman No 1 paper and a butanol:ethanol:water
solvent
system a simple mixture of ampicillin and amoxycillin was chromatographed. spots were detected and each was cut from turn on the FAB probe. matrix
A
mixture of
the
methanol
Two
paper chromatogram and placed in and
glycerol was used as the
and both positive and negative FAB spectra were obtained on both spots
confirming the presence of ampicillin and amoxycillin.
The spectra were comp-
licated by what appears to be methanol attachment ions, e.g. with ampicillin the positive ion FAB spectrum in addition to an intense [MH]+ showed an equally intense ion at m/z 382 corresponding The
ion at m/z 350
to [M+methanoL+H]+.
exact nature of these methanol adduct ions is not clear and
further work
to determine their genesis is in hand.
REFERENCES
1. 2. 3.
3-L. Cower, C. Beaugrand and C. Sallot, Biomedical Mass Spectrometry, 8 (1981), 36-40. M. Barber, R.S. Bordoli, R.D. Sedgwick, A-N. Tyler, B.N. Green, V.C. Parr and 3.L. Gower, Biomedical Mass Spectrometry, 9 (19821, 11-17. V.C. Parr, E3.N. Green, R-H. Bateman and 3-C. Bill, presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, May, 24-29, lY81.
431
FAST ATOM BOMBARDMENT MASS SPECTROMETRY OF B-LACTAM ANTIBIOTICS
3.L.GOWER
Beecham Pharmaceuticals,
Chemotherapeutic
Research Centre, Brockham Park,
Surrey RH3 7A3, England.
ABSTRACT The advantages and disadvantages of Fast Atom Bombardment mass spectrometry (FAB-MS) are described with reference to penicillins, cephalosporins and clavA comparison of FAB spectra with Field Desorption (FD) and Desulanic acid. orption Chemical Ionization (DCI) spectra is given together with brief comments on mass measurement of ions in FAB mass spectra, paper chromatography FAB-MS and the use of metastable scanning techniques with FAB-MS.
INTRODUCTION UnLil
recently
the
mass spectrometric analysis of penicillinsand
other 8-
lactam antibiotics as their free acids or alkali metal salts depended on derivatization to form either methyl or trimethylsilyl esters. ins such
as benzyl penicillin
the derivatization
With simple penicill-
procedure proved effective
a single ester was formed that was amenable to gas chromatography. complicated
need
With more
examples such as ampicillin or amoxycillin problems with mixtures
of products and the inability to gas chromatograph the
and
these products have led to
to find alternative methods of mass spectrometric analysis.
DCI-MS (ref.1) and FAB-MS
(ref.2) have both shown useful molecular weight and
structural information on penicillins and cephalosporins. spectra showing mqlecular weight information. MS of penicillins
Recently
and cephalosporins
FD-MS also gives
Since the original work on FAB-
a more detailed study has been
carried out
and some of the results are presented here.
FAB-MS With
of
B-LACTAM ANTIBIOTICS
free
acids e.g. ampicillin, the positive ion spectra show strong proton-
ated molecular ions CM]' often with relatively intense dimeric species [2M+H]+. Fragment ions are usually intense but obviously dependent on the side-chain structure, dominating ment
The negative ion spectra are often more intense with the molecular
ion observed in both
ion region. penicillins
A particularly and
common
[M-H]-
and intense
Q
frag-
cephalosporins _involves cleavage
across both rings to give
0020-7381/83/0000-0000/$03.00
ions
1983 Elsevier Scientific Publishing Company
432
I’i
R-C-NH
The FAB mass spectra of 8-lactam antibiotics as their alkali metal salts are usually, as might be expected, more intense than the free acid spectra with cationized species [M+X]' and [ZM+Xj+dominating the positive ion spectra. The negative ion spectra show intense ions for the two ring cleavage structure
mentioned above and for the carboxylate anion [M-Xl-and dimeric ion [2M-XI-. Losses of small molecules i.e. C02, CH3COOH are also common.
Molecular weight
determination of unknown penicillins has been aided by cation exchange using excess alkali metal ions in the glycerol e.g. Li+. The positive ion and negative ion FAB mass spectra of potassium clavulanate
(I> in general show the same characteristics
as the other 8-lactam antibiotics.
The positive ion FAB mass spectrum is dominated by the cationized molecular ion [M+K]+ at m/z 276 and dimeric species [ZM+K]+ at m/z 513.
The trimeric species
[3M+K]+ at m/z 750 and tetrameric species at m/z 987 [4M+K]+ are also intense. Glycerol adduct ions e.g., [M+K+glycerbl]+ at m/z 36Band
[2M+K+glycerol]+ at
m/z 605 are present.
Several significant fragment ions are seen in the mass
range up to m/z 250.
Comparison of the spectra with the spectra of the sodium
and lithium salts and MIKES studies of the spectra of the potassium salt are currently being carried out in order to assign structures to these ions. The base peak in the negative ion spectrum is the anion [M-K]- at m/z 198 with-an intense ion for the dimeric species [2M-K]- at m/z 435. molecular ion [M-H]- is present at m/z 236.
The pseudo
A major fragment ion at m/z 136
probably represents loss of carbon dioxide and water from the anion [M-K]-.
COMPARISON OF FAB, DCI, AND FD MASS SPECTRA OF AMPICILLIN
(II)
The positive ion FD mass spectrum of ampicillin is very simple consisting of the protonated molecular ion [MH]+ at m/z 350.
At high emitter currents a
few Low intensity fragment ions were detected but were very transitory. emitter loading produced very few spectra.
Each
Both positive and negative ion FAB
433
mass spectra are relatively simple with the negative ions spectrum being dominated by the two ring cleavage ion at m/z 207 and the @i-H] -ion at m/z 348. Loss of CQOH from this pseudomolecular
ion gave the other major fragment ion.
positive ion spectrum is more complex
The
with
side-chain fragment ions at
m/z 91 [PhCH2]*, and m/z 106 [PhCHNH2]+, together with the thiazolidine cleavage ion at m/z 160 dominating the low mass region of the spectrum and the protonated molecular ion [MH]+ at m/z 350 being
the major high mass ion.
The ammonia positive ion desorption CI spectra is complex with the protonated molecule ion and the ammonia adduct ion at m/z 350 and 367 respectively being observed.
Several intense fragment ions are observed several of which must
arise from complex rearrangements as previously described, (ref.1). In contrast, the methane negative ion desorption
CI spectrum is relatively
simple
with
molecular anion [Ml- at m/z 349 and several other minor fragment ions. base
a
The
peak in the spectra is the thiazolidine anion at m/z 159. The information provided by the three ionization methods is complementary
with FDMS being of most use with complex mixtures and DC1 in those problems where the
fragmentation
information is of value in the structural determination
of unknowns. The FAB mass spectra are the most useful with molecular weight and useful fragmentation information.
The fact that the ionization method produces long
lived species enabling mass measurement and metastable analysis to be carried out is of great importance. Work
on a more detailed comparison between the FD,
FAB,
DC1
and laser des-
orption mass spectra of penicillins will be published elsewhere.
ACCURATE MASS MEASUREMENT AND METASTABLE ANALYSIS Using peak matching on a VG-ZAB mass spectrometer the protonated and cationized molecular ions of a series polymer
of penicillins
have
been measured.
ions and ions from standard penicillins were used
Glycerol
as reference ions and
results with accuracies of 10 p.p.m. were routinely obtained. There were regions in the mass range where no suitable reference ion could be found using standard penicillins but a set of quaternary ammonium salts has been
434
built up to cover these gaps, The ed
stable
on FAB mass
phalosporins,
ion currents resulting from FAB enable MIKES spectra.
MIKES
scans
on
the
CMHl+
and
scans
[MH]-
to
be
obtain-
ions from two ce-
cephaloglycine and cephalexin have been reported previously
(ref
3).
Metastable
scanning methods are of particular importance In deciding the or-
igin of fragment ions especially when
there is the possibility that an ion may
have come from a protanated molecule on [M+H]+,
a cationized ion e.g. [M+K]+
or a glycerol attachment ion e.g. [M+K+glycerol]+.
PAPER
CHROMATOGRAPHY/FAB-MS
Recent
work has concentrated on paper
ed by FAB-MS.
chromatography
of penicillins follow-
Using Whatman No 1 paper and a butanol:ethanol:water
solvent
system a simple mixture of ampicillin and amoxycillin was chromatographed. spots were detected and each was cut from turn on the FAB probe. matrix
A
mixture of
the
methanol
Two
paper chromatogram and placed in and
glycerol was used as the
and both positive and negative FAB spectra were obtained on both spots
confirming the presence of ampicillin and amoxycillin.
The spectra were comp-
licated by what appears to be methanol attachment ions, e.g. with ampicillin the positive ion FAB spectrum in addition to an intense [MH]+ showed an equally intense ion at m/z 382 corresponding The
ion at m/z 350
to [M+methanoL+H]+.
exact nature of these methanol adduct ions is not clear and
further work
to determine their genesis is in hand.
REFERENCES
1. 2. 3.
3-L. Cower, C. Beaugrand and C. Sallot, Biomedical Mass Spectrometry, 8 (1981), 36-40. M. Barber, R.S. Bordoli, R.D. Sedgwick, A-N. Tyler, B.N. Green, V.C. Parr and 3.L. Gower, Biomedical Mass Spectrometry, 9 (19821, 11-17. V.C. Parr, E3.N. Green, R-H. Bateman and 3-C. Bill, presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, May, 24-29, lY81.