Analysis of novel melphalan hydrolysis products formed under isolated lung perfusion conditions using liquid chromatography/tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6515

Rationale: Melphalan is a widely used cytotoxic agent in cancer treatments. This phenylalanine analog has been shown an effective drug in the treatment of breast cancer, multiple myeloma and melanoma of the extremities. A good knowledge of the drug's degradation and metabolism are crucial for understanding its activity during cancer treatments. Methods: The formation of hydrolysis products of melphalan is studied using ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS). Aqueous melphalan solutions were incubated at elevated temperatures and analyzed by UPLC/MS/MS. Two previously described hydrolysis products, mono- and dihydroxymelphalan (MOH and DOH), were formed in vitro and could be characterized during MS/MS and high-resolution experiments. Results: Novel compounds with m/z values >500 Da were discovered. Comparison of the fragmentation patterns of these new molecules with those of MOH and DOH show great similarities. The higher masses are explained by the presence of two or more melphalan units. In total, more than 15 new hydrolysis products were found. Experiments were set up to study the formation and the chemical structures of these molecules. Conclusions: The hydrolysis of melphalan is studied in the scope of a phase II clinical trial (isolated lung perfusion, ILuP). Patient samples were screened for the presence of all documented and novel melphalan hydrolysis products. This study reports the formation of a new class of oligomeric compounds in both in vivo and in vitro samples. Copyright

Full text of DOI:10.1002/rcm.6515

Research Article
Received: 22 November 2012
Revised: 9 January 2013
Accepted: 11 January 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 835–841
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6515
Analysis of novel melphalan hydrolysis products formed under
isolated lung perfusion conditions using liquid chromatography/
tandem mass spectrometry
Jasper Boschmans1, Ernst de Bruijn2, Paul Van Schil3 and Filip Lemière1
*
1University of Antwerp, Chemistry, Center for Proteomics, Groenenborgerlaan 171 V.417b, Antwerp, BE 2020 Belgium
2University of Leuven, Lab Experimental Oncology, UZ Gasthuisberg, CDG Building, Herestraat 49, Leuven, BE 3000 Belgium
3Antwerp University Hospital, Thoracic and Vascular Surgery, Wilrijkstraat 10, Antwerp, BE 2650 Belgium
RATIONALE: Melphalan is a widely used cytotoxic agent in cancer treatments. This phenylalanine analog has been
shown an effective drug in the treatment of breast cancer, multiple myeloma and melanoma of the extremities. A good
knowledge of the drug’s degradation and metabolism are crucial for understanding its activity during cancer treatments.
METHODS: The formation of hydrolysis products of melphalan is studied using ultra-performance liquid chromatography
(UPLC) tandem mass spectrometry (MS/MS). Aqueous melphalan solutions were incubated at elevated temperatures and
analyzed by UPLC/MS/MS. Two previously described hydrolysis products, mono- and dihydroxymelphalan (MOH and
DOH), were formed in vitro and could be characterized during MS/MS and high-resolution experiments.
RESULTS: Novel compounds with m/z values >500 Da were discovered. Comparison of the fragmentation patterns of
these new molecules with those of MOH and DOH show great similarities. The higher masses are explained by the
presence of two or more melphalan units. In total, more than 15 new hydrolysis products were found. Experiments were
set up to study the formation and the chemical structures of these molecules.
CONCLUSIONS: The hydrolysis of melphalan is studied in the scope of a phase II clinical trial (isolated lung perfusion,
ILuP). Patient samples were screened for the presence of all documented and novel melphalan hydrolysis products.
This study reports the formation of a new class of oligomeric compounds in both in vivo and in vitro samples.
Copyright © 2013 John Wiley & Sons, Ltd.
Melphalan (Mel; Fig. 1) is a widely used chemotherapeutic
agent in cancer treatments. This phenylalanine analog has
been shown an effective drug in the treatment of breast
cancer, multiple myeloma, bladder cancer and melanoma of
the extremities.[1] Nitrogen mustards like Mel, cyclophospha-
mide and chlorambucil are alkylating agents because they
can form covalent bonds with the nucleobases of DNA,
thereby adding an alkyl group on the DNA strand and
forming a DNA adduct (Fig. 1).[2–4] The dose of orally or
intravenously administrated Mel is limited by bone marrow
suppression. A solution to overcome this toxicity is to apply
Mel locoregionally. The Department of Thoracic and Vascular
Surgery of the University Hospital Antwerp (UZA) started
investigating the use of Mel during an isolated lung perfusion
(ILuP).[5–8] To treat pulmonary (micro)metastases, the lungs
are isolated from the rest of the blood circulation and per-
fused with a high dose of Mel.
(MOH), which further hydrolyzes to dihydroxymelphalan
(DOH; Fig. 1). Using liquid chromatography/tandem mass
spectrometry (LC/MS/MS) we here identified multiple earlier
unknown Mel hydrolysis products.
EXPERIMENTAL
Chemicals and reagents
Melphalan (Mel; min. 95%) was purchased from Sigma-
Aldrich (Bornem, Belgium). Acetonitrile (ACN; HPLC grade
for gradient analysis) and formic acid (FA; 99+ %) were
obtained from Acros (Geel, Belgium). Reversed osmosis (RO)
water was prepared using a Silex water filtering system from
Eurowater (Nazareth-Eke, Belgium). Ethanol (EtOH) was
obtained from Carlo Erba Reagenti (Rodano, Italy).
It is known that Mel hydrolyzes spontaneously in aqueous
solutions (e.g. blood, perfusion fluid).[9–12] Earlier studies
report two hydrolysis products: monohydroxymelphalan
Samples
Reactivity and oligomerization samples
A 5 mM stock solution of Mel was prepared using RO water.
Because of the low solubility of Mel in water the solution was
vortexed and sonicated to improve solubility. This dilution was
incubated at 37 C for 3 h using a gas chromatography (GC)
oven (HP, type 5890) or Thermomixer (Eppendorf, type 5436)
* Correspondence to: F. Lemière, University of Antwerp,
Chemistry, Center for Proteomics, Groenenborgerlaan 171
V.417b, Antwerp, BE 2020 Belgium.
ꢀ
E-mail: filip.lemiere@ua.ac.be
Rapid Commun. Mass Spectrom. 2013, 27, 835–841
Copyright © 2013 John Wiley & Sons, Ltd.

J. Boschmans et al.
Figure 1. Nucleophilic substitution on melphalan (m/z 305) and formation of
hydrolysis products MOH (m/z 287), DOH (m/z 269) or DNA adducts.
depending on the sample volume. The solution was filtered
Xevo TQ MS
prior to MS analysis using a 0.2 mm Nylon filter (4 mm HPLC
syringe filter; Alltech, Lokeren, Belgium). Samples used for
product ion analysis were concentrated 10 times.
Full scan MS spectra, product ion spectra and MRM ana-
lyses were performed on a Xevo TQ mass spectrometer
(in vivo samples).
Electrospray experiments in positive ion mode (ES+) were
performed on a Xevo TQ mass spectrometer (Waters, Milford,
MA, USA). Capillary voltage: 1 kV. Cone voltage: 27ꢀ V.
ILuP samples
ꢀ
During the isolated lung perfusion (ILuP) the blood circula-
tion of the treated lung is isolated from the general circulation
and flushed with a perfusion fluid containing melphalan.
This perfusion fluid contains melphalan, Plasma-Lyte A
(Baxter; Lessines, Belgium), Voluven (6 %, Fresenius Kabi;
Schelle, Belgium), heparin and blood. Before and during the
ILuP samples are taken from the perfusion fluid. All samples
are immediately snap-frozen and stored at À80 ꢀC. Preceding
our analyses, samples had already been thawed once for
LC-UV quantitation of Mel.
Source temperature: 150 C, desolvation temperature: 500 C.
Resolution settings in MS/MS mode were 2.9 for both LM 1
and LM 2. For HM 1 and HM 2 resolution was set to 15.
Cone voltage and collision energy were optimized for
each of the 12 compounds (Table 1). QuanLynx software
(Waters, Milford, MA, USA) was used to integrate the areas
of all peaks recorded in the multiple reaction monitoring
(MRM) method.
Q-Tof II
Accurate mass data were acquired on a Q-Tof II mass
spectrometer (Micromass, Manchester, UK), equipped with
a chip-based NanoMate source (Advion, Ithaca, NY, USA).
The NanoMate was connected with the nanoAcquity pump
using an LC-coupler. A spray voltage of 1.7 kV was applied.
Sample preparation
ILuP samples
A volume of 50 mL of the perfusion fluid sample was mixed
with 900 mL ice-cold ethanol (EtOH) and cooled at À20 ꢀC
for 60 min. Afterwards theꢀ sample was centrifuged for
15 min at 13 500 rpm and 10 C (Eppendorf centrifuge, type
5415 R). Samples were evaporated to remove all EtOH and
reconstituted in 50 mL H2O. Samples were centrifuged for
4 min at 14 000 rpm (Eppendorf miniSpin plus) in order to
remove small particles, since usage of a filter was not possible
due to small sample volumes.
ꢀ
Cone voltage: 20 V. Source temperature: 80 C. Desolvation
temperature: 60 ꢀC.
TOF MS scans were performed with the collision energy at
5 eV. A mass range for studying dimers and trimers: m/z 100
to 900. Mass ranges and MS profile settings were adjusted
when studying tetramers. Scan time: 0.9 s, inter-scan delay:
0.050 s.
Collision energies for product ion scans (MS/MS): 25 to
50 eV. The mass range for dimers and trimers: m/z 50–600 or
m/z 50–900, for tetramers: m/z 100–1200. Scan time: 0.9 s,
inter-scan delay: 0.050 s.
LC-MS conditions
Acquity and nanoAcquity UPLC
A binary gradient was developed on a Waters Acquity LC
system (Milford, MA, USA). Guard column: 2.1 mm  5 mm
1.8 mm Vanguard HSS T3. Analytical column: 2.1 mm  100 mm
1.8 mm Acquity HSS T3 (both Waters, Milford, MA, USA).
Column temperature: 30 ꢀC. Gradient method used both acid-
ified (0.1% formic acid; FA) H2O (A) and ACN (B) for analysis
as well as wash purposes. The gradient had a run time of
4 min, starting with 0.89 min of on-column focusing. Going
from 99% A to 60% B in 3.20 min, the gradient is followed by
0.20 min of washing (99% B) and 0.30 min of equilibration at
initial conditions. Injection: 5 mL full loop (overfill factor 1).
A similar method was developed on a nanoAcquity UPLC
system, for coupling with the Q-Tof II mass spectrometer.
Trapping column: 180 mm  20 mm 5 mm Symmetry C18.
Analytical column: 100 mm  100 mm 1.7 mm Acquity BEH130
C18 column (both Waters, Milford, MA, USA).
RESULTS AND DISCUSSION
Formation of hydrolysis products and oligomers
Separation of a 5 mM incubated solution of Mel results
in a complex chromatogram (Fig. 2). Based on existing
literature only three compounds are expected: Mel,
MOH and DOH. These are detected at retention time (tR)
1.72 min, 2.99 min and 3.77 min and their identity is con-
firmed by relative elution order in RP chromatography
and their MS spectra. All other compounds present were
initially unknown.
Both MOH and DOH are formed by reaction of Mel with
H2O: one or two Cl atoms are substituted for a hydroxyl
group. In this reaction H2O is the nucleophile; however, other
molecules in solution can act as a nucleophiles. Potential
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Melphalan hydrolysis products formed under ILuP conditions
Table 1. Optimized cone voltages and collision energies for a total of 12 compounds as used in the developed MRM method
Exact mass Chemical formula Retention time
Name
DOH
MOH
Mel
Transitions
Cone voltage Collision energy
+
269.1501
287.1162
305.0824
501.2712
519.2819
519.2374
537.2036
537.2480
537.2480
555.2141
555.2141
573.1802
C13H21O4N2Cl0+
C13H20O3N2Cl1+
C13H19O2N2Cl2+
C26H37O6N4Cl0+
C26H39O7N4Cl0+
C26H38O5N4Cl1+
C26H35O4N4Cl2+
C26H38O6N4Cl1+
C26H38O6N4Cl1+
C26H37O5N4Cl2+
C26H37O5N4Cl2+
C26H36O4N4Cl3
1.72
2.99
3.77
2.69
2.54
3.05
3.38
3.00
2.92
3.49
3.39
3.80
269 > 176; 287 > 150
287 > 228; 287 > 150
305 > 246; 305 > 194
25
25
20
35
35
30
35
30
30
25
30
35
30; 30
25; 30
35; 35
25; 30
25; 30
30; 35
35; 35
25; 30
25; 30
25; 30
25; 30
25; 30
MOH-eDOH 501 > 251; 501 > 234
MOH-DOH
Mel-eDOH
Mel-eMOH
519 > 251; 519 > 234
519 > 269; 519 > 252
537 > 252; 537 > 168
MOH-MOH 537 > 251; 537 > 234
Mel-DOH
MOH-Mel
Mel-MOH
Mel-Mel
537 > 269; 537 > 252
555 > 251; 555 > 234
555 > 269; 555 > 252
573 > 269; 573 > 252
nucleophiles are organic solvents used during reaction (e.g.
alcohols), carboxylate or other anions or Mel itself. Reaction
between two Mel molecules results in dimeric structures with
masses >500 Da and MS signals and isotope patterns charac-
teristic for their chlorine content.
Apart from signals for MOH and DOH, additional peaks at
other retention times were observed in the RICs. Analysis of
the MS/MS data reveals that these peaks originate from Mel
dimers. As far as we know, dimerization or oligomerization
has never been described in the literature before.
Dimeric structures can be formed between an electrophile
(Mel or MOH) and a nucleophile (Mel, MOH or DOH).
Potential nucleophilic sites present in Mel, MOH and DOH
are the carboxyl and amine groups of the phenylalanine
moiety and the hydroxyl group on the mustard side of a
(partially) hydroxylated Mel molecule (MOH or DOH). All
of these compounds have a characteristic isotope pattern
and exact mass. Potential dimeric structures, together with
their chemical formulas, exact masses and isotope patterns
are summarized in Fig. 3 and Table 2. This list compromises
a total of 15 dimers including several isomers, resulting in
only four unique masses.
Identification of melphalan dimers, isomers and isobars
RICs for m/z 573 and 537 contain multiple peaks. Peaks
arising from [2M+H]+ clusters of Mel, MOH and DOH are
also present in these RICs but these are easily identified
as such. Multiple chromatographic peaks are also observed
in RICs for the predicted dimers with mass-to-charge ratios
m/z 555 and 519. The presence of multiple chromatographic
peaks, identical accurate masses and matching isotope
patterns indicate that for some dimers multiple isomers
are formed.
Figure 2. UPLC/MS analysis (TIC) of a melphalan incubation sample (3 h, 37 ꢀC). Apart from
melphalan and its hydrolysis products, MOH and DOH, numerous other compounds are
present. The polar DOH elutes early in the gradient resulting in a poor peak shape.
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J. Boschmans et al.
Figure 3. In aqueous solution melphalan oligomerizes and forms dimers. Possible
structures are classified by their bond type: ethers, esters and amines. Further hydrolysis
of these compounds originates in more products.
When assigning a chemical structure to the detected
dimers, we need to identify the bond between two Mel
molecules. Four possible bond types are suggested: an ether,
pyrazine, amine or ester bond (see Fig. 3). A dimer with an
ether linkage (a) has a structure with two free phenylalanine
groups and (one or two) unreacted chlorine atoms. The
hydroxyl group of MOH or DOH acts as the nucleophile that
binds to the electrophilic Mel or MOH. A pyrazine bond (b) is
formed when the NH2 group of the amino acid of a first
molecule (Mel, MOH or DOH) reacts with both chloro ethyl
groups of a Mel molecule. An ester (or amine; c or d)
bond is formed when the carboxyl (or amine) group acts
as the nucleophile.
two fragmentation patterns: 269/252 for the unmodified
mustard (ÀCl) and 251/234 for the hydrolyzed mustard
(ÀOH). The accurate mass data of the product ions is in
agreement with the elemental composition of these proposed
product ions.
Knowing the structural possibilities (i.e. bond types) of a
Mel dimer, tentative chemical structures can be assigned to
all observed compounds and their isomers.
m/z 573
A single chromatographic peak is found for the product ions
of m/z 573. The isotope pattern and accurate mass of m/z
+
573.1793 (tR = 3.80 min; C26H36O4N4Cl3 , calc. 573.1802 Da)
Melphalan-related compounds are characterized by their
typical fragmentation in which the bond between Mel and
its reaction partner is broken adjacent to Mel’s ethyl group.[13]
Depending whether the second ethyl group of the mustard is
unmodified (ÀCl) or hydrolyzed (ÀOH) this fragment is
found at m/z 269 or 251. Further loss of ammonia yields a
product ion at m/z 252 and 234, respectively. This results in
reveal three chlorine atoms in its structure. In a Mel dimer
with an ether or pyrazine linkage, a maximum of only two
chlorine atoms can be present excluding these bond types
for m/z 573. Only the formation of an amine or ester bond
between two Mel molecules yields a dimer with three chlor-
ine atoms (m/z 573) as found at tR = 3.80 min. The product
ion spectrum shows a signal at m/z 269 and one at m/z 252
Table 2. Dimeric structures: chemical formulas, exact masses and isotope patterns
Exact mass
519.2819
Chemical formula
Name(s)
Bond type
Retention time (s)
+
C26H39O7N4Cl0
MOH-MOH
MOH-DOH
MOH-DOH
Mel-MOH
Mel-DOH
Mel-DOH
Mel-Mel
Ether
Ester
Amine
Ether
Ester
Amine
Ether
Ester
–
2.54
2.54
–
+
537.2480
555.2141
573.1802
C26H38O6N4Cl1
MOH-MOH
MOH-MOH
2.92
2.92
–
3.00
3.00
+
C26H37O5N4Cl2
Mel-MOH
Mel-MOH
Mel-Mel
MOH-Mel
MOH-Mel
3.39
3.39
3.80
3.80
3.49
3.49
Amine
Ester
Amine
+
C26H36O4N4Cl3
Mel-Mel
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Melphalan hydrolysis products formed under ILuP conditions
typical for a structure with a Mel residue with a remaining
Cl atom. Based on our current data, ester or amine linkage
cannot be distinguished.
The elemental composition as derived from its accu-
rate mass, including the presence of two Cl atoms for the
m/z 537.2036 compound (tR = 3.38 min), implies that this
structure is the result of an elimination of water from the
m/z 555.2141 dimer (tR = 3.49 min; C26H37O5N4Cl+2). Further
hydrolysis of this compound results in a dimer at m/z 519.2411
(C26H36O5N4Cl1+, calc. 519.2374 Da) and m/z 501.2724
(C26H37O6N4Cl0+, calc. 501.2713 Da). These compounds are
indeed detected at tR = 3.05 and 2.69 min, respectively.
m/z 555
Two peaks are observed in the RIC for m/z 555 (tR = 3.39 and
3.49 min). The mass difference with the previous compound
(18 Da) and the difference in their isotope pattern (one Cl
atom) can be explained by the hydrolysis of the chlorine
atom of m/z 573. Hydrolysis can take place for any of the
chlorines present (see Fig. 4): substitution for -OH of -ClA
or -ClB leads to identical dimers due to free rotation of the
mustard part (C–N bond). Substitution of ClC leads to an
isomeric molecule with identical exact mass but with differ-
ent retention time.
m/z 519
Plotting the RIC for m/z 519 shows the presence of two
chromatographic peaks at tR = 2.54 and 3.05 min. MS spectra
for these peaks reveal that both compounds have isobaric
masses. The isotope patterns reveal that the compound at
tR = 2.54 min contains no chlorine atoms, whereas the other
structure (tR = 3.05 min) contains a single chlorine atom.
The product ion spectrum for the dimer at tR = 2.54 min
(m/z 519.2827, C26H39O7N4Cl0+, calc. 519.2819 Da) shows
the 251/234 fragmentation pattern, confirming the absence
of a chlorine atom in the part of the ion that is split off
and analyzed.
MS spectra at both retention times show an ion with a dou-
+
ble chlorine isotope pattern at m/z 555.2120 (C26H37O5N4Cl2 ,
calc. 555.2141 Da), indicating that these compounds are
isomers. The product ion spectrum for each chromatographic
peak shows the presence of the 269/252 (tR = 3.49 min; –OHC
position) or the 251/234 (tR = 3.39 min; –OHA or –OHB
position) fragmentation pattern, identifying the position of
the hydrolysis.
The presence of the Cl atom in the product ion pattern
(269/252) for the ion present at tR = 3.05 min reveals that both
chlorines of the second Mel moiety were hydrolyzed followed
by elimination of H2O resulting in the elemental composition
+
m/z 537
as detected for this product (m/z 519.2411, C26H36O5N4Cl1 ,
In the RIC for m/z 537, three chromatographic peaks are
found. For each compound the product ion spectrum is
recorded. The compounds eluting at tR = 2.92 and 3.00 min
calc. 519.2374 Da).
Higher-order oligomers
+
(m/z 537.2469; C26H38O6N4Cl1 , calc. 537.2480 Da) show a
Observation and identification of the Mel dimers in our
samples led us to the assumption that higher-order oligomers
of Mel can also be formed. Theoretical structures, their masses
and isotope patterns were calculated and spectra and RICs
were searched for the presence of these compounds.
Spectra show clear but low-abundance signals in RICs
for predicted masses at m/z 751.4000, 769.3691, 769.4136,
1001.5348 and 1019.5009 (data not shown). Cluster ions do
not appear to contribute to these signals.
Chromatographic data shows that our method is not
optimized for the separation of these low-abundance trimers
and tetramers resulting in peak broadening and overlapping
chromatographic peaks.
single chlorine isotope pattern whereas the peak at tR = 3.38
min (m/z 537.1995; C26H35O4N4Cl2 , calc. 537.2036 Da) con-
+
tains two chlorine atoms. The presence of the chlorine atoms
is confirmed by the accurate mass data.
The product ion spectrum of the peak at tR = 2.92 min
shows the presence of the m/z 269/252 fragment ions (Fig. 4).
The 251/234 fragments are observed for the compound with
identical accurate mass eluting at tR = 3.00 min.
The compound at tR = 3.38 min also contains the 269/252
fragments. When comparing all three data sets, addi-
tional fragment ions (with low intensities) are observed at
tR = 3.38 min. These additional fragments are formed after
the loss of e.g. 28 Da, 45 Da or 101 Da from the [M+H]+ ion.
We were not yet able to assign all of these signals to specific
fragment ions.
Multiply charged ions
In the MS spectra of the dimers, intense signals were
observed at half the m/z value of the analyzed compounds.
Closer investigation showed that for dimeric compounds,
[M+2H]2+ ions are observed while in the spectra of trimeric
compounds additional [M+3H]3+ ions are observed as well.
The number of Mel subunits determines the maximum
charge state.
MRM analysis of ILuP samples
An MRM method was developed on the Xevo TQ MS detector
and included Mel, MOH, DOH and all dimers (see Table 1).
Four perfusate samples acquired at different time points for
each of two treated patients were analyzed: before the start of
the ILuP (ILuP blank) and 5 min, 15 min and 30 min after the
start of ILuP. For patient 2, an extra sample, i.e. arterial blood,
Figure 4. Independent of whether the melphalan dimers are
linked through an ester or amine bond, substitution can take
place on the three unreacted Cl atoms (ClA, ClB or ClC). Frag-
mentation of the ion results in an m/z 269 ion which, after
additional loss of NH3, leads to an m/z 252 ion. Substitution
on ClC by –OH gives a 251/234 fragmentation pattern.
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J. Boschmans et al.
Figure 5. MRM analysis of an ILuP sample showing the presence in an in vivo sample of MOH, DOH and two of the
newly discovered dimeric compounds. Other dimeric compounds are present (data not shown).
was available that was acquired 60 min after injection of Mel
(or 30 min after end of ILuP). The samples were analyzed in
triplicate, separated by blanks (H2O) to prevent and check
for carryover.
detected as well. The dimers differ in the number of remain-
ing Cl atoms after elimination of H2O or the hydrolysis of
the chlorine atom.
Samples originating from two patients treated with an
isolated lung perfusion were analyzed. In accordance with
in vitro experiments (data not shown), ILuP samples show
higher signals for all monomeric hydrolysis products com-
pared to the signals for all dimeric products.
Analysis of the perfusate samples acquired before the start
of the perfusion (ILuP blanks) shows no signal for Mel, MOH
or DOH, nor for the analyzed dimers as expected, because no
Mel has been introduced in the circuit at this point. Samples
acquired after the start of ILuP show signals for the mono-
meric hydrolysis compounds and at lower signal intensities
the newly characterized dimeric compounds (see Fig. 5).
The arterial blood sample for patient 2 allowed to check
for a leakage of Mel into the system after rinsing of the lung
and decannulation (restoring of the normal blood circuit of
the lung). Mel, MOH and DOH are detected in the blood
samples (S/N >5). The dimeric compounds are below the
detection limit.
The presence of Mel and its hydrolysis products in the
arterial blood can be explained by a minor leakage of
the perfusion fluid into the system. Even though after ILuP
the lung is rinsed with fresh perfusion fluid, some remaining
melphalan can enter the blood stream after decannulation of
the pulmonary artery.
Different patients show different profiles of Mel hydrolysis
during the ILuP treatment.
The availability of an arterial blood sample acquired
post-perfusion allowed us to investigate the leakage of
melphalan into the blood stream. Data shows that there
are low but clear signals visible for melphalan, MOH
and DOH (S/N >5).
Using the described LC/MS/MS method we were able to
confirm the presence of mono- and dimeric hydrolysis pro-
ducts of melphalan in in vivo perfusate samples of individual
patients. The effect of these hydrolysis products on the treat-
ment of lung metastases during an isolated lung perfusion
with melphalan is not yet known.
Interpretation of the differences and potential applica-
tion of this information requires further development of
the analytical method and patient follow-up in a larger
study.
CONCLUSIONS
Acknowledgements
It is known that aqueous solutions of the nitrogen mustard
and chemotherapeutic agent melphalan contain the (mono-
meric) hydrolysis products MOH and DOH. Based on LC/
MS/MS data, we report the existence of a new class of
hydrolysis products that were identified as oligomers of
melphalan. Dimers were characterized based on accurate
mass, isotope pattern and product ion spectra. Low-abun-
dance signals that are attributed to trimeric structures were
We would like to thank the Hercules Foundation for finan-
cial support; Professor P. Van Schil, M.D., for providing us
information about and giving us the opportunity to attend
the ILuPs; the surgeons from the Department of Thoracic
and Vascular Surgery (UZA) for providing us with the
patient samples; and Gunther Guetens and Gert De Boeck
for helping us with the perfusion fluid sample preparation.
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Melphalan hydrolysis products formed under ILuP conditions
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