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\begin{document}

\title{Do you care about molar activity?}



\author[1]{SRSTT Radiopharmaconnect}%
\affil[1]{Radiopharmaconnect}%


\vspace{-1em}



  
  \date{April 25, 2024}


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\subsubsection*{\texorpdfstring{by~\href{https://kclpure.kcl.ac.uk/portal/salvatore.bongarzone.html}{Salvatore
Bongarzone}~and~\href{https://radnuk.meduniwien.ac.at/en/home/science-research/radiopharmaceutical-sciences/medical-radiochemistry-and-biomarker-development/}{Verena
Pichler}}{by~Salvatore Bongarzone~and~Verena Pichler}}

{\label{466848}}

\par\null

\begin{quote}
\section*{\texorpdfstring{\emph{The term molar activity is not just a
concept for radiochemists and radiopharmacists but it should also be
taken into account by all disciplines in nuclear
medicine.}}{The term molar activity is not just a concept for radiochemists and radiopharmacists but it should also be taken into account by all disciplines in nuclear medicine.}}\label{the-term-molar-activity-is-not-just-a-concept-for-radiochemists-and-radiopharmacists-but-it-should-also-be-taken-into-account-by-all-disciplines-in-nuclear-medicine.}
\end{quote}

Here, we give an overview on the most important key principles on molar
activity (and specific activity) with a focus on \textsuperscript{11}C
and~\textsuperscript{18}F short-lived radionuclides.

~

\subsection*{\texorpdfstring{Definition of molar activity
(A\textsubscript{m}), specific activity (A\textsubscript{s}), apparent
A\textsubscript{m} and apparent
A\textsubscript{s}}{Definition of molar activity (Am), specific activity (As), apparent Am and apparent As}}

{\label{554901}}

When the radiotracer and its non-radioactive compound are present in the
formulation, we define:

\emph{Molar activity} (\textbf{A\textsubscript{m}})~and \emph{specific
activity}~(\textbf{A\textsubscript{s}}) as the measured activity per
mole and per gram of compound, respectively.

\emph{~}

If in the formulation non-radioactive impurities are also present, and
they might interact with the same biological target as the radioactive
compound we define:

\emph{~}

\emph{Apparent molar activity} (\emph{apparent}
\textbf{A\textsubscript{m}})~and \emph{apparent specific activity}
(\emph{apparent} \textbf{A\textsubscript{s}}) as the measured activity
per mole and grams, respectively, of all compounds (impurities and
non-radioactive compound). However, in most cases the main impurity is
the precursor.

~

\textbf{A\textsubscript{m}} \emph{} and \emph{apparent}
\textbf{A\textsubscript{m}} are expressed in Bq/mol (GBq/\selectlanguage{greek}µ\selectlanguage{english}mol).

\textbf{A\textsubscript{s}} \emph{} and \emph{apparent}
\textbf{A\textsubscript{s}} are expressed in Bq/g (GBq/\selectlanguage{greek}µ\selectlanguage{english}g).

~

Due to radioactive decay, the time of measurement of
\textbf{A\textsubscript{m}}, \textbf{A\textsubscript{s}},
\emph{apparent} \textbf{A\textsubscript{m}} and \emph{apparent}
\textbf{A\textsubscript{s}} must be always stated.

\subsection*{}

{\label{636368}}

\subsection*{Why is molar activity important? Is high molar activity
always the
goal?}

{\label{196261}}

There are different situations that will determine if high
\textbf{A\textsubscript{m}} (or \textbf{A\textsubscript{s}}) is a
critical factor or not for the study. Three categories can be
identified:

~

\textbf{High A\textsubscript{m} is critical} when studying:

\selectlanguage{ngerman}·~~~~~ bioactive or toxic molecules, as the quantity of injected
non-radioactive compound can produce undesired pharmacodynamic or
toxicological effects.

·~~~~~low-density receptors. The injection of an excess of the
non-radioactive compound can have an effect on the receptor occupancy
and, therefore, alter the apparent receptor characteristics. The
receptor occupancy with the non-radioactive compound should be kept
below 5\% in order to avoid pharmacological or pharmacodynamic effects.

·~~~~~ kinetic modelling parameters, as low
\textbf{\textbf{A\textsubscript{m}}} can lead to an unfavourable
signal-to-noise ratio within the image. \textbf{A\textsubscript{m}} can
have a substantial impact on the quantitative analysis and qualitative
interpretation of nuclear medicine images.

\par\null

\textbf{High A\textsubscript{m}} \textbf{is not critical} when studying:

·~~~~~ endogenous compounds normally found in high concentrations in the
body (e.g., {[}\textsuperscript{15}O{]}water,
{[}\textsuperscript{11}C{]}acetate, {[}\textsuperscript{11}C{]}glucose)

·~~~~~ processes in which the biological target or function is not
easily saturated (e.g. glucose metabolism, hypoxia, enzyme activity).
Nevertheless, the \textbf{A}\textsubscript{\textbf{m}} can dramatically
impact the image outcome, e.g. when the patient eats candies directly
before measuring the glucose metabolism.

\par\null

\textbf{Intermediate to low A\textsubscript{m}} is necessary when
studying:

·~~~~~ antibodies, due to liver depletion which reduces the biological
half-time of small amounts of antibodies in the bloodstream.

·~~~~~ some peptides or proteins, to optimize the biodistribution
properties in terms of reducing radiation burden in specific organs
(mostly emunctories) by low organ-to-tumour ratios.

\textbf{~}

\textbf{Table 1. Examples of radiotracers falling into the three
categories of A\textsubscript{m}.}

\textbf{~}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Low A\textsubscript{m}}{} & \textbf{Intermediate
A\textsubscript{m}}{} & \textbf{High
A\textsubscript{m}}{}\tabularnewline
{[}\textsuperscript{18}O{]}H\textsubscript{2}O &
{[}\textsuperscript{11}C{]}PIB &
{[}\textsuperscript{11}C{]}PHNO\tabularnewline
{[}\textsuperscript{11}C{]}Methionine & {[}\textsuperscript{11}C{]}DASB
& ~\tabularnewline
{[}\textsuperscript{18}F{]}FDOPA{} & {[}\textsuperscript{11}C{]}mHED &
~\tabularnewline
{[}\textsuperscript{68}Ga{]}PSMA\textbf{{}} & ~ & ~\tabularnewline
\bottomrule
\end{longtable}

\textbf{~}

~

\subsection*{What is the maximum theoretical molar
activity?}

{\label{868274}}

Theoretical \textbf{A\textsubscript{m}} \textsubscript{} values for PET
radionuclides are very high (e.g. 341.1 TBq/\selectlanguage{greek}µ\selectlanguage{english}mol for carbon-11, 63.3
TBq/\selectlanguage{greek}µ\selectlanguage{english}mol for fluorine-18) however these values are very far from those
obtained in laboratories (e.g. 10-5000 GBq/\selectlanguage{greek}µ\selectlanguage{english}mol for carbon-11, 10-1000
GBq/\selectlanguage{greek}µ\selectlanguage{english}mol for fluorine-18).

\textbf{~}

\textbf{Table 2. Half-life and maximum theoretical A\textsubscript{m} of
some PET radionuclides.}

\textbf{~}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Radionuclide}{} & \textbf{Half-life (minutes)}{} &
\selectlanguage{greek}\textbf{Maximum theoretical A\textsubscript{m} (TBq/µmol)}\selectlanguage{english}{} &
\textbf{Examples of radiotracers produced with high A\textsubscript{m}}
\selectlanguage{greek}\textbf{(TBq/µmol){}}\selectlanguage{english}\tabularnewline
\textbf{\textsuperscript{11}C} & ~~~~~~~ 20.4 & ~~~~~~~~~~~~~~~~~ 341.1
& 4.9 \selectlanguage{ngerman}± 2.4 ({[}\textsuperscript{11}C{]}Raclopride) at
EOS{}\tabularnewline
\textbf{\textsuperscript{18}F} & ~~~~~ 109.8 & ~~~~~~~~~~~~~~~~~~~ 63.3
& 4.4 ({[}\textsuperscript{18}F{]}DCFPyL) at EOS\tabularnewline
\textbf{\textsuperscript{15}O} & ~~~~~~~~~ 2.0 & ~~~~~~~~~~~~~~~ 3394.0
& ~\tabularnewline
\textbf{\textsuperscript{13}N} & ~~~~~~~ 10.0 & ~~~~~~~~~~~~~~~~~ 699.3
& ~\tabularnewline
\textbf{\textsuperscript{68}Ga} & ~~~~~~~ 68.0 & ~~~~~~~~~~~~~~~~~ 102.3
& ~\tabularnewline
\bottomrule
\end{longtable}

~

Low~\textbf{A\textsubscript{m}} might be due to a dilution process with
stable nuclides.~

\textbf{A\textsubscript{m}} is decreased by:

(i)~~~~~~~~~~~~ the generation of the radioactive precursor in the
cyclotron. Impurities potentially come from the target gas, the valves,
pressure regulators, seals, target body, target windows or even residues
from cleaning solvents.

(ii)~~~~~~~~~~ the transfer of the irradiated gas/liquid to the hot
cells. Long-time processes decrease~\textbf{A\textsubscript{m}}. Keeping
the lines pressurized with high purity gases and avoiding fluorine-rich
materials during the preparation of~\textsuperscript{18}F radiotracer
should help to maintain~\textbf{A\textsubscript{m}}.~

(iii)~~~~~~~~ the synthesis process. The use of high purity reagents and
precursors is the most important factor. Reagents should be prepared and
stored under adequate inert conditions to prevent contamination. The
synthesis unit should be also cleaned and dried thoroughly and kept
isolated from atmosphere/contaminants;

~

\textbf{A\textsubscript{m}} can be increased either by:

(i)~~~~~~~~~~~~ increasing beam time and/or current (up to a certain
level), loading/unloading the target before irradiation, discarding the
first 1-3 irradiations of the day and keeping the target under pressure
between runs.

(ii)~~~~~~~~~~ decreasing the amount of non-radioactive sources that
might potentially contaminate the reaction. Contaminants from the
atmosphere ( e.g. atmospheric CO\textsubscript{2} during the preparation
of~\textsuperscript{11}C radiotracer \emph{via}
{[}\textsuperscript{11}C{]}CO\textsubscript{2}), or undesired chemicals
released from tubes, etc. or the presence of impurities in reagents
could contribute to decrease~the~\textbf{A\textsubscript{m}}.~

~

\par\null

\subsection*{How to calculate the molar
activity.}

{\label{479778}}

The methods for measuring the \textbf{A\textsubscript{m}} will depend on
the physical state of the radioactive material. Gaseous and liquid
materials can often be analysed with the use of radiogas chromatography,
high pressure liquid chromatography (HPLC), ion chromatography or mass
spectrometry.

~

Here we give an example of a procedure to calculate
the~\textbf{A\textsubscript{m}} for a~\textsuperscript{11}C-radiotracer
using the HPLC system.

~

\textbf{What you need:} HPLC, analytical column, pipettes, falcon tubes,
\textsuperscript{12}C-reference compound, PET dose calibrator, a
solution of a \textsuperscript{11}C-radiotracer, laptop, solvents.

~

1)~~~ Develop an HPLC method to identify the reference compound using an
analytical HPLC column and define the:

\selectlanguage{ngerman}·~~~~~~ UV wavelength (measured at the maximal UV absorption);

·~~~~~~ mobile phase;

·~~~~~~ column temperature;

·~~~~~~ flow rate;

·~~~~~~ injection volume (e.g. 20 \selectlanguage{greek}µ\selectlanguage{english}L).

2)~~~ From a stock solution of the~\textsuperscript{12}C-reference
compound (e.g. 1 \selectlanguage{greek}µ\selectlanguage{english}M), prepare seven standard solutions in a non-volatile
solvent (e.g. 200 nM, 100 nM, 50 nM, 25 nM, 12.25 nM, 6.13 nM, 3.06 nM).
From the concentration of the standard solutions calculate the amount
(\selectlanguage{greek}µ\selectlanguage{english}mol) present in the HPLC injected volume (e.g. 20 \selectlanguage{greek}µ\selectlanguage{english}L). Inject the
standard solutions into the HPLC resulting in chromatograms with a
single peak - note: you should inject three times each standard
solution.~

3)~~~ From the UV detector chromatogram, determine the area under the
curve (AUC) of the peak corresponding to the compound (\textbf{Fig.
1A}). Enter these values and amount of reference compound (\selectlanguage{greek}µ\selectlanguage{english}mol) into a
computer spreadsheet. Create a calibration curve by plotting the AUCs as
a function of injected amount of reference compound (in \selectlanguage{greek}µ\selectlanguage{english}mol)
(\textbf{Fig. 1B}). Analyse the data by applying a linear regression
analysis (linear least squares fit). This yields the equation~\emph{y =
mx + y\textsubscript{0}}, where~\textbf{y} is the area,~\textbf{m}
represents the slope, and~\textbf{y\textsubscript{0}} is a constant that
describes the background. The amount of compound
(\textbf{x~}expressed\textbf{~}in \selectlanguage{greek}µ\selectlanguage{english}mol) present in a radioactive sample
may be calculated from this equation.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Blog-molar-Activity1/Blog-molar-Activity1}
\caption{{\textbf{A)} HPLC UV-chromatograms of standard concentrations of
a~\textbf{}reference compound. \textbf{B}) Calibration curve of AUC vs.
amount of reference compound (\selectlanguage{greek}µ\selectlanguage{english}mol).
{\label{683115}}%
}}
\end{center}
\end{figure}

\subsubsection*{}

{\label{674600}}

4)~~~ Synthesize and isolated the \textsuperscript{11}C-radiotracer.

5)~~~ Measure an exact volume of a solution containing the
\textsuperscript{11}C-radiotracer and amount of radioactivity via dose
calibrator and decay correct the activity at end of synthesis (EOS) (or
at end of delivery - EOB). Calculate the activity in GBq in the HPLC
injection volume (e.g. in 20 \selectlanguage{greek}µ\selectlanguage{english}L \textbf{activity\textsubscript{inj}}).

6)~~~ Inject a sample (20 \selectlanguage{greek}µ\selectlanguage{english}L) of a solution containing the
\textsuperscript{11}C-radiotracer into the HPLC using the same method
used to derive the calibration curve. Obtain the area from the
UV-chromatograms.

7)~~~ Using the appropriate calibration curve, this area is converted to
the number of moles of compound injected
(\textbf{amount\textsubscript{inj}}, in \selectlanguage{greek}µ\selectlanguage{english}mol).

8)~~~ Divide the \textbf{activity\textsubscript{inj}} by
\textbf{amount\textsubscript{inj}} to obtain the
\textbf{A}\textsubscript{\textbf{m}} \textbf{} in GBq/\selectlanguage{greek}µ\selectlanguage{english}mol (and stated
that it is corrected at EOS or EOB).

9)~~~ To obtain the \textbf{A}\textsubscript{\textbf{s}} \textbf{} in
GBq/\selectlanguage{greek}µ\selectlanguage{english}g, divide \textbf{A}\textsubscript{\textbf{m}} by the molecular
weight of the compound.

~

Note: The calibration curve has to be repeated at anytime if any of the
HPLC parameters are changed due to maintenance (e.g. change of UV lamp),
or a new HPLC column is used, or after a determined time period (e.g.
this is stated in your standard operating procedure).

~

\subsection*{Closing remarks and critical
aspects}

{\label{871270}}

The time-dependency of the molar activity makes it a necessity to
provide a time-cut-off for the applicability of a radiopharmaceutical to
avoid side-effects for the last patient. Besides, also the maximal
injectable volume in terms of potential unwanted pharmacodynamics or
side effects should be stated in the release criteria.

In most cases, only the \textbf{A}\textsubscript{\textbf{m}} is stated,
as the apparent \textbf{A}\textsubscript{\textbf{m}} is very difficult
to determine as precise information on impurities affecting the target
region must be available. Nevertheless, the apparent
\textbf{A\textsubscript{m}} is the more critical one in terms of
toxicology, interdependencies and signal-to-noise ratio. A starting
point is to include the residual amount of precursor in the calculation
of the maximal applicable dose and the time-cut-off. Interestingly, this
is already standard procedure for peptides, proteins and antibodies as
here the ratio of the activity per unlabelled compound is determined, as
the separation of precursor and radiopharmaceutical is not possible.
Therefore, the borders between the definitions of
\textbf{A}\textsubscript{\textbf{m}} and apparent
\textbf{A}\textsubscript{\textbf{m}} are fluid for these cases.

~

~

\subsection*{References}

{\label{915402}}

1.~~~~~~~~ Noguchi, J.; Zhang, M. R.; Yanamoto, K.; Nakao, R.; Suzuki,
K. In vitro binding of {[}\textsuperscript{11}C{]}raclopride with
ultrahigh specific activity in rat brain determined by homogenate assay
and autoradiography.~\emph{Nucl Med Biol~}\textbf{2008},~\emph{35},
19-27. (\href{https://www.ncbi.nlm.nih.gov/pubmed/18158939}{link})

2.~~~~~~~~ Ravert, H. T.; Holt, D. P.; Chen, Y.; Mease, R. C.; Fan, H.;
Pomper, M. G.; Dannals, R. F. An improved synthesis of the radiolabeled
prostate-specific membrane antigen inhibitor,
{[}\textsuperscript{18}F{]}DCFPyL.~\emph{J Labelled Comp
Radiopharm~}\textbf{2016},~\emph{59}, 439-50.
(\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516894/}{link})

3.~~~~~~~~ AGENCY, I. A. E. \emph{Cyclotron Produced Radionuclides:
Principles and Practice}. INTERNATIONAL ATOMIC ENERGY AGENCY: Vienna,
\textbf{2008}.~(link)

4.~~~~~~~~ G\selectlanguage{ngerman}ómez-Vallejo, V.; Gaja, V.; Koziorowski, J.; Llop, J.
Specific Activity of \textsuperscript{11}C-Labelled Radiotracers: A Big
Challenge for PET Chemists.
\textbf{2012}.~(\href{https://www-pub.iaea.org/books/iaeabooks/7849/Cyclotron-Produced-Radionuclides-Principles-and-Practice}{link})

5.~~~~~~~~ Pandit-Taskar, N.; O'Donoghue, J. A.; Morris, M. J.; Wills,
E. A.; Schwartz, L. H.; Gonen, M.; Scher, H. I.; Larson, S. M.; Divgi,
C. R. Antibody mass escalation study in patients with
castration-resistant prostate cancer using \textsuperscript{111}In-J591:
lesion detectability and dosimetric projections for 90Y
radioimmunotherapy. \emph{J Nucl Med} \textbf{2008}, \emph{49},
1066-74.~(\href{https://www.ncbi.nlm.nih.gov/pubmed/18552139}{link})

6.~~~~~~~~ Pichler, V.; Zenz, T.; Philippe, C.; Vraka, C.;
Berroteran-Infante, N.; Pfaff, S.; Nics, L.; Ozenil, M.; Langer, O.;
Willeit, M.; Traub-Weidinger, T.; Lanzenberger, R.; Mitterhauser, M.;
Hacker, M.; Wadsak, W. Molar activity - The keystone
in~\textsuperscript{11}C-radiochemistry: An explorative study using the
gas phase method.~\emph{Nucl Med Biol~}\textbf{2018},~\emph{67},
21-26.~(\href{https://www.ncbi.nlm.nih.gov/pubmed/30380463}{link})

7.~~~~~~~~ Coenen, H. H.; Gee, A. D.; Adam, M.; Antoni, G.; Cutler, C.
S.; Fujibayashi, Y.; Jeong, J. M.; Mach, R. H.; Mindt, T. L.; Pike, V.
W.; Windhorst, A. D. Consensus nomenclature rules for
radiopharmaceutical chemistry - Setting the record straight. \emph{Nucl
Med Biol} \textbf{2017}, \emph{55},
v-xi.~(\href{https://www.ncbi.nlm.nih.gov/pubmed/29074076}{link})

8.~~~~~~~~ Wurzer, A.; Pollmann, J.; Schmidt, A.; Reich, D.; Wester, H.
J.; Notni, J. Molar Activity of Ga-68 Labeled PSMA Inhibitor Conjugates
Determines PET Imaging Results. \emph{Mol Pharm} \textbf{2018},
\emph{15}, 4296-4302.

(\href{https://www.ncbi.nlm.nih.gov/pubmed/30011372}{link})

~

\subsection*{Authors:}

{\label{295770}}

\textbf{\href{https://kclpure.kcl.ac.uk/portal/salvatore.bongarzone.html}{Salvatore
Bongarzone}}

Salvatore has worked at King's College London (KCL) since Apr 2014 under
the supervision of Prof Antony Gee, originally as MRC postdoctoral
fellow (Apr 2014 - Dec 2017) and from December 2017 as translational
radiochemist in the Wellcome EPSRC Centre Medical Engineering KCL. His
active research focuses on developing carbon-11 and fluorine-18 PET
radiotracers. He received his Ph.D. in Physics and Chemistry of
Biological Systems (2011) from the International School of Advanced
Studies (SISSA/ISAS) Trieste. In November 2013, he completed a
postdoctoral research fellowship at the Institute of Research in
Biomedicine (IRB), Barcelona, Spain, cofunded by EU MarieCurie Actions.~

~

\textbf{\href{https://radnuk.meduniwien.ac.at/en/home/science-research/radiopharmaceutical-sciences/medical-radiochemistry-and-biomarker-development/}{Verena
Pichler}}

Verena finished her Ph.D. in Bioinorganic Chemistry at the University of
Vienna in 2013. Afterwards, she was research associate and lecturer at
the Institute of Biomedical Engineering at the University of Applied
Sciences in Vienna. Since August 2016, she works as postdoctoral fellow
and PET production manager at the Medical University of Vienna in the
working group of Prof. Marcus Hacker. Besides her research interest for
carbon-11 and fluorine-18 radiochemistry, she establishes new methods
for preclinical evaluation of PET tracers based on spheroid cultures.

\par\null

\selectlanguage{english}
\FloatBarrier
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