Recent applications of a single quadrupole mass spectrometer in 11C, 18F and radiometal chemistry

Article abstract of DOI:10.1016/j.jfluchem.2018.02.009

Mass spectrometry (MS) has longstanding applications in radiochemistry laboratories, stemming from carbon-dating. However, research on the development of radiotracers for molecular imaging with either positron emission tomography (PET) or single photon emission computed tomography has yet to take full advantage of MS. This inertia has been attributed to the relatively low concentrations of radiopharmaceutical formulations and lack of access to the required MS equipment due to the high costs for purchase and maintenance of specialized MS systems. To date, single quadrupole (SQ)-MS coupled to liquid chromatography (LC) systems is the main form of MS that has been used in radiochemistry laboratories. These LC–MS systems are primarily used for assessing the chemical purity of radiolabeling precursor or standard molecules but also have applications in the determination of metabolites. Herein, we highlight personal experiences using a compact SQ-MS in our PET radiochemistry laboratories, to monitor the small amounts of carrier observed in most radiotracer preparations, even at high molar activities. The use of a SQ-MS in the observation of the low mass associated with non-radioactive species which are formed along with the radiotracer from the trace amounts of carrier found is demonstrated. Herein, we describe a pre-concentration system to detect dilute radiopharmaceutical formulations and metabolite analyses by SQ-MS. Selected examples where SQ-MS was critical for optimization of radiochemical reactions and for unequivocal characterization of radiotracers are showcased. We also illustrate examples where SQ-MS can be applied in identification of radiometal complexes and development of a new purification methodology for Pd-catalyzed radiofluorination reactions, shedding light on the identity of metal complexes present in the labelling solution.

Full text of DOI:10.1016/j.jfluchem.2018.02.009

Accepted Manuscript
Title: Recent applications of a single quadrupole mass
spectrometer in 11C, 18F and radiometal chemistry
Authors: Thomas L. Collier, Kenneth Dahl, Nickeisha A.
Stephenson, Jason P. Holland, Adam Riley, Steven H. Liang,
Neil Vasdev
PII:
S0022-1139(17)30516-X
DOI:
Reference:
https://doi.org/10.1016/j.jfluchem.2018.02.009
FLUOR 9123
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FLUOR
Received date:
Revised date:
Accepted date:
30-11-2017
17-2-2018
17-2-2018
Please cite this article as: Collier TL, Dahl K, Stephenson NA, Holland JP,
Riley A, Liang SH, Vasdev N, Recent applications of a single quadrupole mass
spectrometer in 11C, 18F and radiometal chemistry, Journal of Fluorine Chemistry
(2010), https://doi.org/10.1016/j.jfluchem.2018.02.009
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Recent applications of a single quadrupole mass
spectrometer in 11C, 18F and radiometal chemistry
Thomas L. Collier1,2,* Kenneth Dahl,1 Nickeisha A. Stephenson,1,† Jason P. Holland,1,‡ Adam Riley,1
Steven H. Liang,1 and Neil Vasdev1,
ł
1Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital (MGH) &
Department of Radiology, Harvard Medical School, Boston, Massachusetts, USA
2Advion, Inc., Ithaca, New York, USA
Present addresses:
†
Department of Chemistry, University of the West Indies, Mona, Kingston, Jamaica
‡
University of Zurich, Department of Chemistry, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland
ł Azrieli Centre for Neuro-Radiochemistry, Centre for Addiction and Mental Health & Department of
Psychiatry, University of Toronto, Canada
*Corresponding Author:
T.L. Collier, Ph.D.; Tel: +1-201-921-7510; collierl@advion.com
1

Graphical abstract
1. Abstract
Mass spectrometry (MS) has longstanding applications in radiochemistry laboratories, stemming
from carbon-dating. However, research on the development of radiotracers for molecular imaging
with either positron emission tomography (PET) or single photon emission computed tomography
has yet to take full advantage of MS. This inertia has been attributed to the relatively low
concentrations of radiopharmaceutical formulations and lack of access to the required MS equipment
due to the high costs for purchase and maintenance of specialized MS systems. To date, single
quadrupole (SQ)-MS coupled to liquid chromatography (LC) systems is the main form of MS that
has been used in radiochemistry laboratories. These LC/MS systems are primarily used for assessing
the chemical purity of radiolabeling precursor or standard molecules but also have applications in the
determination of metabolites. Herein, we highlight personal experiences using a compact SQ-MS in
2

our PET radiochemistry laboratories, to monitor the small amounts of carrier observed in most
radiotracer preparations, even at high molar activities. The use of a SQ-MS in the observation of the
low mass associated with non-radioactive species which are formed along with the radiotracer from
the trace amounts of carrier found is demonstrated. Herein, we describe a pre-concentration system
to detect dilute radiopharmaceutical formulations and metabolite analyses by SQ-MS. Selected
examples where SQ-MS was critical for optimization of radiochemical reactions and for unequivocal
characterization of radiotracers are showcased. We also illustrate examples where SQ-MS can be
applied in identification of radiometal complexes and development of a new purification
methodology for Pd-catalyzed radiofluorination reactions, shedding light on the identity of metal
complexes present in the labelling solution.
2. Introduction
Mass spectrometry (MS) is among the most well-established structural characterization methods used
in the field of chemistry.[1-6] MS has an established role in radiochemistry, particularly in
radiocarbon dating.[7] For instance, the introduction of accelerator mass spectrometry (AMS)
technology as a means of measuring natural levels of 14C has been described as the "Third
Radiocarbon Revolution."[8] Elsewhere in radiochemistry, single quadrupole (SQ)-MS has become
the most widely used MS method. In combination with liquid chromatography (LC) systems, LC/MS
has become commonplace for standard quality control analysis of radioactive precursors and
standards. LC/MS is also used increasingly for detection of metabolites/radiometabolites to assess
radiochemical stability in vitro and ex vivo, as well as to identify potential radiotracers for
neuroimaging.[9-12] Whereas radiochemistry with 3H and 14C have benefitted from the advances of
MS, radiochemistry for molecular imaging by positron emission tomography (PET) or single photon
emission computed tomography (SPECT) has yet to take full advantage of MS technology. The
historically high cost of MS purchase, maintenance and operation of this specialized instrumentation
3

are likely key factors which have held back MS from more widespread use in PET or SPECT
radiochemistry laboratories. Furthermore, the relatively low concentrations (vide infra) of
radiopharmaceutical formulations, and the regulatory restrictions around radioactivity in a central
core MS laboratory, have also impeded access of this technology to PET and SPECT chemists.
Our radiopharmaceutical programs focus on the development of novel radiochemical
reactions and on the characterization of new PET radiotracers for medical imaging in drug discovery
and clinical research.[13] The majority of our clinical research employs two radionuclides: carbon-
11 (11C; t1/2 = 20.4 min) or fluorine-18 (18F; t1/2 = 109.7 min). Unfortunately, the sensitivity of SQ-
MS is not sufficient to detect the radiolabelled species themselves in typical clinical-grade
radiopharmaceutical formulations. For example, clinical-grade 18F-labelled PET radiotracers have
typical specific activities around 5 Ci/mol with administered radioactive doses in humans around 10
to 20 mCi.[14] For an average human male (~75 kg) this administered dose of radiotracer equates to
around ~2 to 4 nmol (ca. 25 to 50 pmol/kg body weight). Given the theoretical maximum specific
activity of fluorine-18 (1712.9 Ci/mol), typical 18F-radiotracers have an isotopic dilution factor of
19
around ~350 (equivalent to approximately one radioactive atom for every 350 non-radioactive F
atoms; ~0.3%). Hence, the number of moles of radioactive species present in a standard human
injection is around 6 to 12 pmol. For carbon-11 radiotracers, even smaller quantities of radiolabelled
material are produced in clinical productions. For example, the number of atoms contained in 10 mCi
of 11C is ~6.5 x 1011 atoms, equivalent to 1.08 x 10-12 moles.[15] Using a typical radioactive
concentration of 10 mCi/mL, a 5 µL injection used for standard LC/MS analysis represents only ~5
femtomoles of the 11C-labeled radiotracer. These calculations illustrate that the vast majority of PET
radiotracers observe the tracer principle,[16] wherein the chemical dose of administered PET
radiotracers is so small that the agents have no effect on biochemical processes in vivo. Although a
SQ-MS cannot determine the amount of radioisotope in a sample, MS has found applications in PET
radiochemistry,[17-19] specifically for radiometabolite analysis.[20]
4

Herein, we showcase applications of a compact SQ-MS in our laboratories. Specifically, we
describe the use of SQ-MS for: 1) coupling with a novel pre-concentration system to detect dilute
radiopharmaceutical formulations and application of this system in metabolite analyses; 2)
11
18
optimization of radiochemical reactions and simultaneous characterization of C- and F-labeled
89
radiotracers; 3) purification and identification of radiometal complexes with Zr and Pd-catalyzed
radiofluorination reactions.
3.
Integration of a Pre-concentration System to a SQ-MS
For practical applications of SQ-MS in PET radiochemistry, it is necessary to increase the sample
concentration. The major benefit of a pre-concentration step in PET radiotracer analysis is the ability
to concentrate the non-radioactive (cold) mass associated with the radioactive species. Concentrating
the sample also helps to eliminate (or significantly reduce) interferences, such as salts (trifluoroacetic
acid, phosphate etc.) or high concentrations of solvents such as dimethyl sulfoxide (DMSO),
acetonitrile or other organic solvents that may affect the chromatography directly or cause
suppression of the mass spectrometry signal. Pre-concentration systems have been used in the
determination of metals and ions since the late 1970’s.[21] Initially these systems found their major
use in groundwater analysis and generally consist of an injector system where the injection loop has
been replaced by an adsorbent.[2, 22-24] However, other modifications including the replacement of
the injection loop with an electrochemical cell for the pre-concentration of divalent metals[25] were
reported. The basic trapping system replaces the injection loop with a trapping cartridge and the rest
of the system remains as standard for the chromatography system. The sample may require dilution
using water to allow the desired materials to be trapped and can be washed with water or a suitable
eluting solvent to remove salts, ion pairs etc. that may interfere with the chromatography or to
eliminate / reduce materials that are not well retained but are present in high concentration. From a
radiochemical perspective, these pre-concentration systems are analogous to solid-phase strong
cation exchange methods that are used to trap 68Ga3+ ions and purify the radionuclide from non-
5

radioactive impurities (Zn2+, Fe3+ etc.) and radioactive materials (68Ge) that are leached during
standard 68Ga-generator elution protocols.
A
B
Sample in
Waste
Sample in
Waste
Trapping cartridge
Trapping cartridge
To Column and
Detector
From HPLC Pump
To Column and
Detector
From HPLC Pump
C
Trapping cartridge
Figure 1. A: In this position the trapping cartridge is cleaned for trapping by washing with ethanol
and then water. The sample is then applied to the trapping cartridge to perform the concentration of
the materials. If necessary, the sample may require dilution or adjustment of the pH to affect the
retention of the material on the trapping cartridge. The trapping cartridge is then washed with water
or other solution to remove interfering materials. At the same time, the analytical system is
equilibrated to minimize the analysis time. B: In this position, the trapping cartridge is moved into
the flow path with the analytical column to perform the analytical analysis. C: The trapping cartridge
(Valco Instruments Co. Inc, Houston, TX, Finger-tight cartridge assembly #SFECH412) can be
packed by the user with any standard solid phase extraction or resin packing.
An example of the utility of the pre-concentration system is shown in Figure 2, where for
comparison approximately 0.8 pmol (5 µL of a 0.16 nmol /mL solution) of the Kallikrein inhibitor
peptide (Ac-Pro-Phe-Arg-Ser-Val-Gln-NH2) was injected onto the LC/MS system. The protonated
molecular ion peak, [M+H]+, can be observed in the mass spectrum at an m/z 774.6 (monoisotopic
MW = 773.4); however, there is considerable noise in the spectrum. The additional noise in the mass
spectra makes it difficult to determine which of the peaks are attributed to the desired compound. It
is possible to concentrate the sample by evaporation, assuming the sample is stable to the process.
6

Figure 2. Mass spectrum obtained from a 5 µL injection of the Kallikrein inhibitor peptide (Ac-Pro-
Phe-Arg-Ser-Val-Gln-NH2) solution (0.16 nmol /mL) on to the LC/MS system. The intensity of the
[M+H]+ peak is similar to the other noise peaks.
The use of the pre-concentration system allows the user to increase the amount of mass
injected on to the LC-SQ-MS system with minimal manipulations and with minimal losses. Figure 3
shows the effect of concentrating a 1 mL sample of the 0.16 nmol/mL solution of the same Kallikrein
Inhibitor peptide (Ac-Pro-Phe-Arg-Ser-Val-Gln-NH2) observed in Figure 2. In this case,
concentrating the sample increased the amount of peptide to be injected on to the LC/MS system to
~160 pmol. The ability to wash the trapping cartridge, after the trapping of the compound of interest,
reduces the salts and solvent present while not increasing the volume of the sample injected to the
analytical column. The mass spectrum observed in Figure 3 is absent of other interfering peaks and
the signal to noise is extremely high. Thus, pre-concentration greatly improved the signal-to-noise
ratio and overall quality of the spectrum obtained.
7

Figure 3. Mass spectrum obtained from 1 mL of the Kallikrein Inhibitor peptide (Ac-Pro-Phe-Arg-
Ser-Val-Gln-NH2) solution (0.16 nmol /mL), concentrated on the trapping cartridge containing a
hydrophilic modified styrene polymer solid phase extraction material (HLB). The cartridge was then
washed with water (5 mL, LC/MS grade) to remove contaminants such as saline and was then injected
on to the LC/MS system. The protonated molecular ion, [M+H]+, is the only peak seen in the mass
spectrum.
4.0 Metabolite Analysis
Given the importance of assessing the metabolism of a radiopharmaceutical in vivo for quantitative
analysis it is not surprising that LC/MS has found applications in PET imaging studies.[20] A basic
outline of the methodology for LC/MS based metabolomics was described by Zhou et al.[26] With
access to a high sensitivity triple quadrupole MS, Shetty et al[27] determined that it is possible to
observe 11C-labeled species and their respective 12C ([M+1]) isotopologues/carriers for four PET
radiotracers. While the sensitivity of the SQ cannot observe the radioisotopically labelled species, it
is still possible to obtain data by observing the small amount of “carrier” material or metabolites when
present. Typically, when performing metabolite analysis on a new radiotracer, the most common
types of information obtained are simply to what extent metabolites are observed and whether these
metabolites are more or less lipophilic that the parent radiotracer. Detailed knowledge of the chemical
composition and structure of radiometabolites is often lacking. A common method for the analysis of
metabolites is a column-switching approach reported by Hilton et al.[28] Column-switching HPLC
has been used extensively in the analysis of drugs and their metabolites in plasma.[29, 30] The
8

materials trapped on the capture column are then eluted directly onto an analytical chromatographic
column, without loss of materials during the transfer. In the paper by Hilton sample sizes of up to 4
mL of plasma were used. Recently, we reported an automated version of this column-switching HPLC
system.[31, 32]
An alternative pre-concentration methodology for dilute radiotracer samples, including
plasma, takes advantage of thin layer chromatography (TLC) to separate the intact radiotracer[33, 34]
from metabolites. The benefit of using TLC is two-fold: first, larger amounts of material can be
spotted on to the TLC plate (up to 250 µL of plasma) without the need to remove all proteins from
the sample; second, by extending the time that the TLC plate is exposed to the phosphorimager plate
of a Cyclone Plus Storage Phosphor System (PerkinElmer, Inc., Waltham, MA USA) or using an
AR-2000 (Eckert & Ziegler Radiopharma Inc., Hopkinton MA, USA) the entire TLC lane can be
imaged over a set period of time or can be set to continue to collect data until a sufficient number of
counts are detected.
N
N N
N
N N
O
O
N
O
N
H
N
O
N
H
1) TBAOH
2) 11CH3I
11CH3
O
OH
Cl
Cl
[5-(3-Chloro-4-[11C]methoxyphenyl)-1,3-oxazol-4-yl][3-(1H-
1,2,4-triazol-1-yl)propylamino]formaldehyde
[5-(3-Chloro-4-hydroxyphenyl)-1,3-oxazol-4-yl][3-(1H-
1,2,4-triazol-1-yl)propylamino]formaldehyde
[11C]PF-367
Figure 4. Radiochemical synthesis of the glycogen synthase kinase 3 (GSK-3) radiotracer, [11C]PF-
367
Figure 4 shows the radiochemical methylation reaction of the phenolic precursor to form the
GSK-3 radiotracer [11C]PF-367. TLC metabolite analysis was performed on a 1 mL plasma sample
obtained at various times post-injection of the glycogen synthase kinase 3 (GSK-3) radiotracer,
[11C]PF-367 (Figure 4, monoisotopic molecular weight 361.1) to which 135µg/ kg of “cold” PF-367
had been administered.[35] Analysis of the TLC plate by MS-SQ was performed by automatically
9

extracting the soluble materials from the TLC plate at a spacing interval of ~4 mm, and thirty to
thirty-five separate spots on each TLC plate were analyzed (Figure 5).
Figure 5. TLC/MS of the plasma sample, [11C]PF-367. The arrows indicate the positions that
were sampled by the TLC/MS system. The silica is sometimes removed during the process and this
is indicated by the dark oval. For the 1 minute post injection plasma sample, the blue arrows indicate
the position where the proteins, di and tri-glycerides were observed. The green arrow indicates the
position where the parent compound, [11C]PF-367 was observed.
In the case of PF-367, the most intense peaks, observed were for the [M+Na]+, and were seen
at m/z 384.1 for the parent compound + sodium ion and m/z 370.1 for the precursor + sodium ion
(Figure 6).
Figure 6. Mass spectra of compounds identified in the TLC analysis of the plasma sample obtained
at 1 minute post injection A: Spectrum obtained from material at an Rf of 0 - 0.1 (Blue arrows in
Figure 5) and indicates the presence of proteins, triglycerides and diglycerides. C: Spectrum from
peak at Rf = 0.9 (Green arrow in Figure 5) indicates the presence of the intact PF-367.
To highlight the use of the trapping system method (vide supra) for the determination of metabolites,
the plasma sample obtained at 10 minutes post-injection of the glycogen synthase kinase 3 (GSK-3)
radiotracer, [11C]PF-367 (Figure 4) to which 135µg/ kg of “cold” PF-367 had been administered.[35]
In this case, ~100 µL of plasma was injected onto the trapping cartridge followed by washing with 5
mL of 1% acetonitrile / water to remove salts. The material trapped was analyzed by LC/MS and the
resulting total ion chromatogram (TIC) is shown in Figure 7. The chromatogram was then extracted
10

to obtain the intensity of the protonated parent compound ([M+H]+ = 362.1) and the protonated
precursor ([M+H]+ = 348.1). The XIC is a means of obtaining a chromatogram without the other
masses which are not related to the ions being investigated. The metabolism of a compound may be
predicted by using the MetaPrint2D-React metabolic product predictor, which can be found on the
Center for Molecular Informatics website (University of Cambridge, http://www-
metaprint2d.ch.cam.ac.uk/metaprint2d-react/). In the case of PF-367, the major predicted metabolite
coincides with the observed demethylated phenol, production of which is attributed to metabolism by
P-450 enzymes in vivo.[36]
Figure 7. Plasma metabolite sample 10 minutes post-injection of the glycogen synthase kinase 3
(GSK-3) radiotracer, [11C]PF-367 (Figure 4) to which 135µg/ kg of “cold” PF-367 had been
administered. A: Total ion chromatogram of PF-367 plasma sample. B: Extracted ion chromatogram
(XIC) of the protonated primary expected metabolite m/z 348.1 (demethylated phenol). C: Extracted
ion chromatogram (XIC) of the protonated parent compound of [M+H]+ m/z 362.1.
5. Analysis of PET reactions to optimize reaction conditions
The many advantages of using MS during the optimization process for producing radiotracers are
illustrated by our [18F]fluoride displacement and [11C]CO2 fixation reactions. During our
radiochemical development phase, we obtain mass spectra on the major ions, and especially the
chemical impurities formed from the non-radioactive reagents to allow for the optimization of the
11

reaction and the minimization of potential co-eluting impurities, thereby facilitating our purification
steps. For example, mass spectrometry was critical in our two-step microfluidic radiosynthesis of the
high affinity partial agonist of the serotonin 1A receptor, [18F]FEMPT (pKi = 9. 79; Ki = 0.16
nM).[37]
As depicted in Figure 8, initially, high concentrations of the diethyl tosylate resulted in low
radiochemical incorporation yields of the radiofluorinated product, however the formation of the
intermediate [18F]fluoroethyltosylate was high (~55-60%). The LC/MS of this reaction mixture
showed large amounts of the tosylate, hydroxyl and the elimination product in the reaction mixture.
By reducing the concentration of ethyl ditosylate and hence; reducing the competition of the precursor
for either [18F]fluoroethyltosylate or diethyltosyltate, the radiochemical incorporation yield was
increased and the chemical impurities were reduced. This allowed for significantly simpler separation
conditions to be used and allowed for higher chemical and radiochemical purity.
To determine the products being formed during the radiosynthesis the final formulation were analyzed
by LC/MS using 1 mL of the final product solution trapped on the concentration system as described
in section 3 (vide supra). The direct HPLC injection of 5 L of reaction mixture revealed no
observable UV signal. However, when 1 mL of the solution of the purified radiotracer was injected
via the trapping system, the UV traces indicates the presence of a number of species with retention
times close to the desired product (Figure 8).
12

TBA+
O
HO
O
-O
N
N
O
O
TBAOH
N N
N N
N
N
N
N
Desmethyl-MPT
Reactor 1
[18F]TEAF, DMSO
18F
OTs
TsO
+
OTs
TsO
N
100 mm X 2 m reactor
OTs
170oC, 50 mL/min
Reactor 2
O
O
18F
N
O
N N
N
[18F]FEMPT
TBA+
O
-O
O
O
N
O
N
HO
O
N N
N N
N
N
N
N
18F
OTs
OTs
Desmethyl-MPT TBA salt
+
HydroxyEMPT
250 mm X 1m
150-200oC
50 mL/min
O
O
TsO
N
TsO
O
N N
N
N
TosylEMPT
O
O
N
O
N N
N
N
EthyleneMPT
Figure 8. Radiosynthesis of [18F]FEMPT. In the first microfluidic reactor two products are found at
the output of the first reactor, The desired [18F]fluoroethyltosylate and the starting reagent
ethylditosylate. The output of the first reactor is directly fed into the second reactor. Using high
concentrations of the ethylditosylate in the first reactor, the major product was the TosylEMPT and
hydroxylEMPT. However by minimizing the diethyltosylate in the first reactor, the major products
were the desired [18F]FEMPT, and small amounts of the hydroxylEMPT, tosylEMPT and
ethyleneMPT.
13

Figure 9. Analysis of the final formulated product by LC/MS, using direct injection of 5 L (blue
line) and using the trapping system to improve the sensitivity (Red line). Displayed is the UV
spectrum observed at 254 nm for both methodologies. Column: 3 µm, 4.6 x 150 mm C18,
Phenomenex, Luna. Solvent CH3CN : 0.1% formic acid, Flow rate= 1mL/min, Gradient from 20%
CH3CN to 95% CH3CN at 10 minutes, hold for 2 minutes at 95% CH3CN. Insets are the structures
identified using the MS data. Mass spectra obtained using an Expression-L Compact Mass
Spectrometer (Advion Inc., USA), APCI ion source operating in positive ion mode and corona
discharge of 5 μA, m/z scan range: 200-500.
A number of the observed impurities were able to be identified using LC/MS and the major impurities
observed were the expected elimination product and the hydroxyl product. However, all of these
materials were below the mass seen for the desired FEMPT and the combined signals were used in
the molar activity calculation. Overall, [18F]FEMPT was obtained in ~7% isolated radiochemical
yield and in >98% radiochemical and chemical purity. The molar activity of the final product was
determined to be >148 GBq /µmol (>4 Ci/ µmol).
A large number of [11C]CO2 fixation reactions,[38-42] which can now be performed “in-
11
loop” to form C-labelled carbamates, oxazolidinones and ureas (Figure 10) were studied using
LC/MS to determine the products formed during the reaction by monitoring the small mass of non-
radioactive species formed.[43] Sufficient non-radiolabeled mass of material is produced even at high
molar activities (≥1 Ci /µmol) to obtain MS data on the species formed during the radiosynthesis. It
14

is noteworthy that for these reactions, the intermediates and products formed during the reactions are
readily ionized using an electrospray ionization (ESI) source yielding high signal-to-noise ratios.
O
11 C
O
N
O
11
A
B
11
[11C]CO2
O- +BEMP
POCl3
N
MeOH
NH2
N
O
H
H
BEMP, DMF
O
11 C
O
11
N
O
11
[11C]CO2
POCl3
O- +BEMP
Benzylamine
N
NH2
BEMP, DMF
N
N
H
H
H
Figure 10. A: Radiosynthesis of the desired product, methyl N-benzylcarbamate using [11C]CO2
fixation methodology to form the intermediate [11C]isocyanate, which then reacts with methanol to
produce the carbamate. B: By-product of the radiosynthesis where the symmetrical urea, 1,3-
dibenzylurea, is formed by reaction of the intermediate [11C]isocyanate reacts with another molecule
of benzylamine.
Interestingly, the major 11C-labelled product that was obtained had a different retention time
than the standard molecule, methyl N-benzylcarbamate. The results of the radioactive-LC/MS
analysis are shown in Figure 11. It should be noted that the radioactive species, observed at retention
times of ~7.8 and 8.1 minutes were not observed using SQ-MS, but the small amount of non-
radioactive mass (always present during the radiosynthesis) was seen. The mass spectrum of the major
peak at a retention time of ~7.8 min had a major ion at m/z 241.1, which did is not the desired
carbamate, but corresponds to the protonated molecular ion, [M+H]+ of the symmetrical urea, N,N′-
dibenzylurea (Figure 11, inset A). The minor radioactive peak, at the retention time of ~8.1 min, had
a major ion that did correspond to the protonated molecular ion [M+H]+ ion of the desired carbamate,
methyl N-benzylcarbamate (Figure 11, inset B)
15

300
250
200
150
100
50
Radiation Detector
A
O
O
B
N
N
H
N
O
H
H
155
158
161
164
167
170
230
233
236
239
242
245
0
0
1
2
3
4
5
6
7
8
9
Figure 11. Radio-HPLC showing the radiation detector output for products of the [11C]CO2 fixation
with benzylamine and methanol. Inset A: Mass spectrum obtained from the major radiation peak,
(Red arrow) which does not correspond to the desired methyl N-benzylcarbamate, but to the [M+H]+
of N,N’-dibenzylurea, m/z 241.1. Inset B: Mass spectrum obtained from the minor radioactive peak
at a retention time of ~8.1' (Blue arrow), which has the correct [M+H]+ ion for the desired carbamate,
m/z 166.1
By knowing the identity of the major impurity (N,N′-dibenzylurea) observed in the radio-
HPLC of the initial reaction attempted, it was possible to modify the reaction conditions to minimize
the formation of the symmetrical urea (Figure 12). Judicious control of the reaction conditions
allowed chemoselective synthesis of the desired 11C-carbamate as the major product.[43]
Figure 12. With insight from MS, the reaction conditions were altered to synthesize the desired
carbamate as the major product, and the symmetrical urea was significantly suppressed.
16

In the past, to obtain positive and negative ion scans it was necessary to perform two different
analyses. With improvements in electronics and mass spectrometer design, most commercial SQ-MS
systems now can rapidly change the parameters of the ion source and mass spectrometer. The typical
use of this switching ability is to obtain both positive and negative ions at the same time. However,
it is also possible to use the same polarity but to alter the ion source characteristics, such as source
voltage, etc. This can be used to either induce or to minimize fragmentation during the analysis, which
effectively occurs simultaneously, and both molecular ion and fragmentation data can be obtained in
the same analysis. An example of this is illustrated in Figure 13, where the standard ion source
settings and ion source settings with the source voltage raised 30V above the standard settings. With
the standard ion source settings, only the protonated molecular ion is observed for the Kallikrein
Inhibitor peptide (Ac-Pro-Phe-Arg-Ser-Val-Gln-NH2). The mass spectrum obtained using the ion
source setting with the higher source voltage, it can easily be seen that there has been increased
fragmentation and this information can be used to determine additional structural information.
Figure 13. A: Mass spectrum which shows only the protonated molecular ion obtained during LC-
SQ-MS run using the standard ion source parameter which minimize fragmentation, Inset: Expanded
plot showing molecular ion region; B: During the same run as above, the ion source voltage was
increase by 30V to induce fragmentation and as observed in the mass spectrum, there is further
information which can aid in the elucidation of structure.
17

6. Purification and identification of metal complexes
The development of radiotracers based on radioactive metal ion complexes is becoming increasingly
important for both nuclear imaging and molecularly targeted radiotherapy. Often, coordination
chemistry is employed to attach radiometal ions to biological targeting vectors such as peptides,
proteins, antibodies, and more elaborate systems based on nanoparticles.[44, 45] Coordination of the
metal ion relies on derivatizing the vector with a bifunctional chelate (BFC). The BFC is chosen to
present an optimum set of donor atoms to facilitate stable and selective metal ion complexation, as
well as offer a reactive handle to allow covalent functionalization of the vector molecule. Ideally, the
metal ion complex should be thermodynamically, kinetically and metabolically stable to prevent
unwanted escape of the metal ion in vivo. In addition, the conjugation chemistry is often chosen to
minimize the rate of degradation or loss of the metal ion complex from the vector. Nevertheless, for
most metal ion BFC systems, a certain degree of degradation or loss of the radiometal ion is
commonly observed. For example, Cu2+ ions can be coordinated by various chelates including the
macrocycles DOTA, NOTA, NODGA and CB-TE2A.[46]
Dramatic differences in the
biodistribution profiles have been observed for systems that employ these different chelates with high
liver uptake often attributed to demetallation of the Cu2+ ion in vivo. Understanding the nature of the
metal ion complexes, and potential degradation products is crucial for future design of new metal ion
chelates.
Increasing interest in the use of 89Zr-radiolabeled antibodies for immuno-PET imaging
provides a suitable case-study. Zirconium-89 is a positron-emitting radionuclide with a has a half-life
of 78.4 hours and exists as the Zr4+ ion, and is readily coordinated by the hexadentate, tris-
hydroxamate ligand, desferrioxamine B. Density functional theory (DFO) calculations have predicted
that, due to the increased size of the first coordination sphere, Zr4+ ions coordinated by DFO have the
potential to increase their coordination number from between 6 and 8 donor atoms.[47] No crystal
structures of the predicted [Zr(DFO)(H2O)2]+ are available but MS data confirm the DFT predictions
18

which suggest that the water molecules are loosely bound and kinetically labile (Figure 12).
While most human applications of 89Zr-radiolabeled antibodies have found that the 89Zr-DFO
complex remains stable in vivo, concern still exists about the potential loss of 89Zr ions from the
89
chelate and associated problems for radiation dosimetry. In mouse models Zr-labeled antibodies
typically show accumulation of the radioactivity in bone that reaches ca. 5 – 10% ID/g of administered
dose.[47, 48] Interestingly, bone accumulation of 89Zr is typically found to be higher in animal models
that have tumour models which are positive for the target antigen which points toward specific
intratumoral metabolism as the source of the bone seeking 89Zr-species. Metabolic degradation of the
bifunctional linker and potential demetallation are two possible explanations for the bone tropism.
However, at present no studies have confirmed the chemical nature of the 89Zr-species that is released
from the 89Zr-DFO-antibody constructs. Studies have shown that 89Zr ions administered as either the
tetraoxalate complex, [89Zr(C2O4)4]4- or as the ZrCl4 species dissolved in a phosphate buffer show
high accumulation of radioactivity in the bone.[49] It is likely that detailed MS studies will be
required to solve these outstanding questions about zirconium metabolism.
Figure 14. A: Structure of the zirconium desferrioxamine complex. Note: loosely associated solvent
water molecules are not observed in the MS. The monoisotopic molecular weight of [Zr(DFO)]+,
C25H45N6O8Zr, is 647.2; B: the predicted spectrum of [Zr(DFO)]+; C: total ion chromatogram from
the flow injection analysis (FIA) of the [Zr(DFO)]+complex. D: the spectrum obtained by FIA
matches the predicted intensities based on isotopic abundance.
19

A further example is the development of a new purification method for a palladium(IV) fluoride
complex. Hooker & Ritter et al. disclosed the radiosynthesis and utility of a novel Pd(IV)18F complex
that was able to undergo fluorination reactions with palladium aryl complexes to yield 18F-aryl
fluorides.[50] This discovery was important to the field of PET radiochemistry with [18F]fluoride, as
now the ability to carry out “pseudo-electrophilic” fluorination reactions without the need for [18F]F2
gas and its derivatives (e.g. [18F]Selectfluor™) meant that readily available and high specific activity
18
[18F]fluoride could be used for F-labelling of non-activated aromatic rings. The purification of
[18F]Pd(IV)F requires the use of a polypyridine resin (JandaJel™-polypyridine)( Figure 15).
However, this resin is no longer commercially available and a new method was needed to purify the
complex.
Me
2
2
OTf
1) [18F]fluoride
18F
OTf
N
18-cr-6, KHCO3,
acetone, rt, 10 min
N
N
N
N
N
N
N
N
Pd
N
Pd
N
N
N
N
B
2) Filtration with
JandaJel™- polypyridine
N
B
N N
N N
[18F]Pd(IV)fluoride
complex
Pd(IV)Picoline
complex
Figure 15. Radiosynthesis of [18F]Pd(IV)F using JandaJel™-polypyridine for purification of the
Pd(IV)F complex from the Pd(IVpicoline precursor.
20

Since the fluorinated species was the desired product to be isolated from the reaction mixture, we
postulated that the Pd(IV)F complex could be purified using HPLC, however the stability of this
complex towards HPLC conditions was unknown. We used LC/MS to test the viability of HPLC
purification (Figure 16). The complex was initially injected onto the LC/MS using acetonitrile and
formic acid as the eluent. Data from the MS showed that under these conditions the Pd(IV)F complex
was not stable as the loss of a pyrazole from the tetrpyrazolborate ligand was observed Figure 14.
This result suggested that acids should be avoided in the purification step and that the ideal HPLC
solvent system should be carried out at neutral or near neutral pH. When the HPLC purification was
repeated using acetonitrile and potassium triflate (0.1M) the MS showed that the major product was
indeed the Pd(IV)F complex.
Figure 16. Upper two traces: initial purification methodology tested and resulted in the loss of one
of the pyrazole groups. Lower 2 traces: Final purification methodology which resulted in the major
product formed during the purification as the desired complex.
21

Isolation of the complex from the HPLC and subsequent analysis by MS demonstrated that HPLC
purification was a viable method to purify Pd(IV)F. Further investigation of the observed isotope
pattern of the MS showed that it matched well with the predicted isotope pattern of the complex
(Figure 17).
OTf
A
F
N
N
Pd
N
N
N
B
N
N
N N
B
B
C
576 578 580 582 584 586 588 590
Figure 17. A: Pd(IV)F complex; B: the predicted isotope patterns and C: the pattern observed in the
mass spectrometer.
7. Conclusion
Herein we have described a pre-concentration system integrated to an SQ-MS to detect dilute
radiopharmaceutical formulations and metabolites. We also show examples of how SQ-MS was used
for optimization of radiochemical reactions, identification of radiometal complexes and development
of a new purification methodology for radiofluorination reactions. The examples of SQ-MS in our
PET radiochemistry laboratories presented herein hopes to inspire the radiochemistry community as
well as researchers involved in organic synthesis, organofluorine chemistry, organometallic
22

chemistry, preclinical and clinical translation to make more use of this readily available
instrumentation in both basic and applied science.
Acknowledgements:
We thank Ellen Osborn and Asia Knight for their contributions to our early mass spectrometry
studies. JPH thanks the University of Zurich, the Swiss National Science Foundation (SNSF
Professorship PP00P2_163683), and the European Research Council (ERC-StG-2015, NanoSCAN –
676904) for financial support. N.V. thanks Advion, the US Department of Energy for an R&D Isotope
Program Project grant (DE-SC0015556), and the National Institute on Ageing of the NIH
(R01AG054473) for funding. S.H.L. is a recipient of an NIH career development award (DA038000)
and an Early Career Award in Chemistry of Drug Abuse and Addiction (ECHEM, DA043507) from
the National Institute on Drug Abuse.
23

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