Chapter 2.

1
Simultaneous quantification of cyclophosphamide,
4-hydroxycyclophosphamide, N,N,N-triethylenethiophosphoramide (thiotepa) and N,N,N-triethylenephosphoramide (tepa) in human plasma by high-performance
liquid chromatography coupled with electrospray ionization
tandem mass spectrometry (LC-MS/MS)
Milly E de Jonge, Selma M van Dam, Michel JX Hillebrand, Hilde Rosing,
Alwin DR Huitema, Sjoerd Rodenhuis, Jos H Beijnen

Abstract
The alkylating agents cyclophosphamide (CP) and N,N,N-triethylenethiophosphoramide
(thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens
can be complicated by the occurrence of severe and sometimes life-threatening toxicities,
pharmacokinetically guided administration of these compounds, to reduce variability in exposure,
may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we
have developed and validated an assay, using liquid chromatography coupled with electrospray
tandem mass spectrometry (LC-MS/MS), for the routine quantification of CP, thiotepa, and their
respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N,N,N-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is
derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as
4OHCP-SCZ.
Sample pretreatment consisted of protein precipitation with a mixture of methanol and
acetronitrile using 100 l plasma. Chromatographic separation was performed on an Zorbax
Extend C18 column (150 x 2.1 mm ID, particle size 5 m), with a quick gradient using 1 mM
ammoniumhydroxide in water and acetonitrile, at a flow rate of 0.4 ml/min. The analytical run time
was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and
multiple reaction monitoring was used for drug quantification. The method was validated over a
concentration range of 200 to 40,000 ng/ml for CP, 50 to 5,000 ng/ml for 4OHCP-SCZ and 5 to
2,500 ng/ml for thiotepa and tepa, using 100 l of human plasma. These dynamic concentration
ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an
internal standard. The coefficients of variation were less than 12% for both intra-day and inter-day
precisions for each compound. Mean accuracies were also between the designated limits (15%).
This robust and rapid LC-MS/MS assay is now successfully applied for routine therapeutic drug
monitoring of CP, thiotepa and their metabolites in our hospital.

J Mass Spectrom 2004; 39: 262-271

Chapter 2.1

Introduction
High-dose chemotherapy with the alkylating agents cyclophosphamide (CP), carboplatin
and thiotepa (CTC) is widely used in the treatment of advanced or metastatic breast,
ovarian and testis tumors [1-6]. Because of the haematological stem cell support in this
high-dose setting, the doses of the individual compounds can be increased up to 10-fold
since bone marrow toxicity is no longer dose-limiting. However, high-dose CTC regimens
can be complicated by the occurrence of severe and sometimes life-threatening nonmarrow toxicities such as mucositis, veno-occlusive disease, oto- and cardiotoxicity [1-6].
The large interindividual variability of these toxicities may be due to interindividual
pharmacokinetic variation of the compounds involved. Studies have shown that
exposures (expressed as area under the plasma concentration versus time curve, AUC)
of the compounds and their metabolites are related to toxicity [7-13]. Therefore,
therapeutic drug monitoring (TDM) may be an important tool to minimize toxicity in the
CTC regimen. To make routine and fast TDM in the CTC setting feasible, a fast and
robust analytical method is required quantifying the necessary compounds. With this
method, analytical results should become available within a few hours to make dose
adaptations during a chemotherapy course possible. Since for carboplatin a simple
method of analysis is available [14], it was intended to develop a method for
simultaneously quantifying CP, thiotepa and their relevant metabolites.
A
Cl
Cl

H
O N
CH 2 CH 2
N P
CH 2 CH 2
O

B
Cl
Cl

H
O N
CH 2 CH 2
N P
CH 2 CH 2
O

OH

N P N

N P N

Figure 1. Chemical structures of A) cyclophosphamide, B) 4-hydroxycyclophosphamide, C)

thiotepa and D) tepa.

CP is an oxazaphosphorine prodrug requiring activation by the cytochrome P450

enzyme system (CYP), to form its pharmacologically active metabolite
4-hydroxycyclophosphamide (4OHCP) (Figure 1A and B). 4OHCP can enter target cells
where it is metabolised to form the final cytotoxic agent phosphoramide mustard. Since
the 4OHCP level in plasma is an accurate determinant of the alkylating activity of CP [15],

84

Bioanalysis of cyclophosphamide, thiotepa and metabolites

it is necessary to quantify both CP and 4OHCP for TDM purposes. 4OHCP is a very
unstable compound, with a plasma half-life of only a few minutes [16]. Accurate
quantification of 4OHCP therefore requires instantaneous derivatization of the compound
at the bedside to form a stable derivative which can subsequently be measured. Several
methods for stabilizing 4OHCP have been described [17]. The method using stabilization
with the derivatizing agent semicarbazide (SCZ), resulting in formation of 4OHCP-SCZ
(Figure 2) [18,19], is robust, easy to perform and therefore feasible in clinical practice.
The ethylenimine thiotepa is rapidly metabolized by oxidative desulfuration mediated
by CYP to yield its metabolite tepa (Figure 1C and D). Thiotepa and tepa have a similar
alkylating activity [20,21] and therefore it is important to quantify both compounds for
TDM purposes.
An assay for the simultaneous determination of CP, 4OHCP, thiotepa and tepa has
not yet been developed. In our laboratory, a gas chromatographic assay was developed
for the simultaneous quantification of CP, thiotepa and tepa in plasma [22]. However,
sample pretreatment consisted of a labour-intensive liquid/liquid extraction, and in
practice the assay appeared not to be very robust. We have also developed an assay for
the determination of 4OHCP in plasma (quantified as 4OHCP-SCZ, Figure 2), using highperformance liquid chromatography (HPLC) with UV detection [18]. This assay, however,
requires 1000 l of plasma per sample, has a lengthy sample pretreatment procedure
and a run time of 20 min. To reduce the turn-around time of the samples, a combined
assay with a simple sample pretreatment procedure and a short run time is necessary.
This can be achieved by using tandem mass spectrometric (MS/MS) detection with
multiple reaction monitoring (MRM). Because of the high selectivity and specificity of this
technique, it permits the use of short run times and minimal sample-clean up procedures.

H
Cl

CH 2 CH 2

Cl

CH 2 CH 2

OH

N
O

N P
O

4-hydroxycyclophosphamide
H
N
H 2N

NH 2

Cl

CH 2 CH 2

Cl

CH 2 CH 2

H2

NH 2
N N

N P
O

semicarbazone derivative of
4-hydroxycyclophosphamide

semicarbazide

Figure 2. Reaction of 4-hydroxycyclophosphamide with semicarbazide resulting in the formation

of the semicarbazone derivative of 4-hydroxycyclophosphamide.

85

Chapter 2.1

Assays quantifying CP and/or 4OHCP using HPLC with MS detection have been
described [23-25]. Sottani et al [24] described the quantification of CP in urine using
LC-MS/MS applying liquid/liquid extraction with ethylacetate as sample pretreatment.
Baumann et al [23] described a LC-MS method, using single-ion monitoring, for the
simultaneous determination of CP, 4OHCP and three other CP metabolites in 450 l
human plasma. Methylhydroxylamide was used for derivatizing 4OHCP and a solidphase extraction was used for sample pretreatment. Sadagopan et al [25] described an
LC-MS/MS assay simultaneous determining CP and 4OHCP in mouse plasma.
Methylhydroxylamide was used for derivatizing 4OHCP and protein precipitation followed
by evaporation and reconstitution of the supernatant was used for sample pretreatment.
To our knowledge, quantification of thiotepa and tepa using LC-MS/MS has not been
described.
In this paper, we present the development and validation of a rapid, sensitive and
specific method for the simultaneous quantification of CP, 4OHCP, thiotepa, and tepa in
human plasma using HPLC coupled with electrospray ionization (ESI)MS/MS. The
combination of a uniform, simple and above all fast sample pretreatment with a
chromatographic system that provides a run time of 10 min allows high sample
throughput and has proven very useful for routine TDM. The validation of the method was
performed based on the most recent international guidelines for bioanalytical validation
[26].

Experimental
Chemicals
CP, 4-hydroperoxycyclophosphamide (4OOHCP), and all other CP metabolites used for
selectivity and specificity tests were a generous gift from Dr. Niemeyer, ASTA Medica,
Frankfurt, Germany (purity >95%). Thiotepa (Ledertepa) was obtained from AHP
Pharma (Hoofddorp, The Netherlands). Tepa was synthesized at the Faculty of
Chemistry, University of Utrecht, according to the method described by Craig and
Jackson [27] (purity >98%). The internal standard (IS) hexamethylphosphoramide (HMP)
(Figure 3) originated from Sigma (Zwijndrecht, The Netherlands). Acetonitrile and
methanol were HPLC-grade reagents and were obtained from Biosolve BV
(Valkenswaard, The Netherlands). Potassium dihydrogenphosphate (suprapure grade)
was from Merck (Darmstadt, Germany). SCZ hydrochloride (analytical reagent grade)
was purchased from Acros (Geel, Belgium). A 2 M solution of SCZ in 50 mM potassium
phosphate buffer (pH 7.4) was prepared (stored at 4C for a maximum of 6 months).
Distilled water was used throughout the analysis and all other chemicals used were of
analytical grade and used without further purification. Drug free human plasma originated
from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service
(Amsterdam, the Netherlands).
86

Bioanalysis of cyclophosphamide, thiotepa and metabolites

H3C
H3C

H 3 C CH 3
N
N P N
O

CH 3
CH 3

Figure 3. Chemical structure of the internal standard hexamethylphosphoramide.

Chromatographic and mass spectrometric conditions

An Agilent Technologies, (Palo Alto, CA, USA) HPLC system was used, consisting of a
Model 1100 Series pump and cooled autosampler (10C). Separation was carried out on
a Zorbax Extend C18 column (150 x 2.1 mm ID, particle size 5 m; Agilent Technologies)
protected with an Agilent Extend C18 narrow-bore guard column (12.5 x 2.1 mm ID,
particle size 5 m; Agilent Technologies). A stepwise gradient was used to elute the
compounds from the column. At time zero a mixture of 96% eluent A (1 mM ammonium
hydroxide in water) and 4% eluent B (100% acetonitrile) was flushed through the column.
After 2 min, the fraction of acetonitrile was increased to 25% during 1 min. This mobile
phase composition was maintained for 3 min. Subsequently, during 0.1 min, the mobile
phase composition was set back at 96% eluent A and helt there for the final 4 min of the
run. The flow-rate was 0.4 ml/min. The column outlet was connected directly to the
electrospray sample inlet (Sciex, Thornhill, ON, Canada). The source temperature was
set at 400C. Ions were created at atmospheric pressure and were transferred to an API
3000 triple-quadrupole mass spectrometer (Sciex). The curtain gas (1.1 ml/min) and the
collision-induced dissociation (CID) gas (342 * 1015 molecules/cm2) consisted of nitrogen
(grade 5.0) and the nebulizer and turbo gases (1.6 l/min and 7.0 l/min, respectively) were
zero air. The electrospray source was operated in the positive ion mode. The
electrospray voltage was +2.5 kV and the dwell time was 50 ms with a 5 ms pause
between scans. Q1 and Q3 were operating at unit mass resolution.
MRM was used for drug quantification. Precursor ions of analytes and IS were
determined from spectra obtained during the infusion of standard solutions using an
infusion pump connected directly to the electrospray source. As a result of the very soft
ionization, provided by the electrospray ion source, only singly charged molecular ions
were observed. Each of the precursor ions was subjected to CID to determine the product
ions. The transitions of the protonated precursor/product ion pairs that were used for
recording the selected-ion mass chromatograms are listed in Table 1. Data were
processed by Analyst software (Applied Biosystems/ MDS Sciex, Analyst software
version 1.2).
Sample collection, pretreatment and processing
Whole blood samples were collected in patients receiving high-dose CTC chemotherapy,
including CP (6,000 or 4,000 mg/m2 in 4 days), carboplatin (1,600 or 1,067 mg/m2 in 4

87

Chapter 2.1

days) and thiotepa (480 or 320 mg/m2 in 4 days) [2-6]. Samples were collected in heparin
tubes and immediately placed on ice. Plasma was immediately obtained by centrifuging
the whole blood sample at 3,500 g for 3 min at 4 C. A 500 l volume of plasma was
added to a polypropylene tube containing 50 l of SCZ solution. Samples were whirlmixed for 10 s, placed in a water-bath at 35 C for 2 h and then stored at -70 C. For
analysis, to 100 l of this solution 25 l of the IS solution was added (containing
approximately 100 ng/ml HMP in ethanol). After whirl-mixing for 10 s, 300 l of the protein
precipitation reagent methanol-acetonitrile (1:1, v/v) was added. The mixture was vortex
mixed for 30 s, and automatically mixed (Labinco, Breda, The Netherlands) for 5 min.
Subsequently, the samples were centrifuged at 7,000 g for 15 min. A volume of 50 l of
supernatant was diluted with 400 l of a 1 mM ammonium hydroxide solution in water and
whirl-mixed for 10 s. From this mixture, a volume of 10 l was injected on to the analytical
column.
Preparation of stock solutions, working solutions and plasma standards
Two fresh stock solutions of CP, thiotepa and tepa were prepared independently in
ethanol, at concentrations of approximately 5 mg/ml (CP) and 500 g/ml (thiotepa and
tepa). Stock solutions of 4OHCP were prepared by dissolving 4OOHCP in water, which
decomposes after dissolution into hydrogen peroxide and 4OHCP, resulting in a final
concentration of approximately 1 mg/ml. One solution was used to spike the plasma
calibration samples and the other was used to prepare the quality control (QC) samples.
Stock solutions were diluted further with water and the solutions of each analyte were
added together to obtain working solutions. After spiking 1950 l of drug-free human
plasma with 50 l of these working solutions, the following concentrations in plasma were
obtained: 200, 400, 1,000, 4,000, 10,000, 20,000 and 40,000 ng/ml for CP, 50, 100, 250,
500, 1,000, 2,500, 5,000 ng/ml for 4OHCP and 5, 10, 50, 250, 500, 1,000 and 2,500
ng/ml for thiotepa and tepa. The choice for these calibration ranges was based on the
concentrations achieved in patients in daily practice of TDM. QC samples at low, medium
and high concentration levels were prepared in a similar way. To all of these mixtures 200
l of SCZ solution was added. The samples were whirl-mixed for 10 s, placed in a waterbath at 35 C for 2 h and subsequently stored at -70 C until analysis. After thawing, 100
l volumes of each calibration sample were processed as described for the blood
samples.
Validation procedures
The validation of the assay was based on the FDA guidelines for Bioanalytical Method
Validation [26].
Linearity
Calibration standards were prepared and analysed in duplicate in three independent runs.
Calibration curves (area ratio to the IS versus nominal analyte concentration) were fitted
88

Bioanalysis of cyclophosphamide, thiotepa and metabolites

by least-squares linear regression without weighting and using 1/x and 1/x2
(x=concentration) as weighting factors. In order to establish the best weighting factor,
back-calculated concentrations were determined. The model with the lowest total bias
and the most constant bias across the concentration range was considered to be the best
fit. To assess linearity, deviations of the mean calculated concentrations over three runs
should be within 15% from nominal concentrations for the non-zero calibration
standards. At the lower limited of quantitation (LLQ) level a deviation of 20% was
permitted.
Accuracy and precision
Inter-assay accuracy and intra- and inter-assay precisions of the method were
determined by assaying five replicates of each of the QC samples with analyte
concentrations around the LLQ, and in the low, medium and high concentration ranges in
three separate analytical runs.
Inter-assay accuracy was determined as the percentage difference between the mean
concentration after three analytical runs and the nominal concentration. Accuracy should
be within 15% except at the LLQ concentration, where it should be less than 20%.
The coefficient of variation (CV) was used as a measure for intra- and inter-assay
precision. Precision should not exceed 15% CV except for the LLQ where it should not
exceed 20% CV.
To validate the accuracy and precision of the analysis of samples originally above the
upper limit of quantification, an extra QC sample containing analyte concentrations two
times higher than the concentrations of the highest QC sample, was diluted twofold with
drug-free human plasma prior to analysis.
Recovery
The absolute recovery of the analytes after protein precipitation was determined by
comparing the analytical results for plasma QC samples at the three concentration levels
to a corresponding set of spiked plasma extracts (containing 100% of the theoretical
concentration). Three replicates were analysed at each concentration level. Recovery
should be consistent, precise and reproducible.
To determine the amount of ion suppression, the analytical results for spiked plasma
extracts were compared with those for a corresponding set of diluted working solutions (in
triplicate, at three concentration levels).
Selectivity and specificity
Out of six batches control human plasma, double blank samples (no analyte, no IS),
blank samples (no analyte, with IS) and LLQ samples were prepared, processed and
analysed to determine whether endogenous plasma constituents interfered with the
assay. Interference may occur when co-eluting endogenous compounds produce ions at
the same m/z values that are used to monitor the analyte and IS.

89

Chapter 2.1

To investigate the potential interference of other metabolites of CP and co-medication

with the quantification of the analytes, the metabolites and co-medicated drugs were
added to LLQ samples at therapeutic relevant concentrations. The samples were then
processed and assayed according to the described method. The following metabolites
were tested: Carboxyphosphamide (400 g/ml), 2-dechloroethylcyclophosphamide (175
g/ml), ketophosphamide (450 g/ml) and phosphoramide mustard (70 g/ml). The
following drugs were tested: Aciclovir (100 g/ml), amphotericine B (20 g/ml), caffeine
(1000 g/ml), carboplatin (1000 g/ml), ciprofloxacine (15 g/ml), dexamethasone (200
g/ml), fluconazole (60 g/ml), granisetrone (150 g/ml), itraconazole (15 g/ml),
lorazepam (5 g/ml), mesna (150 g/ml), methoclopramine (1,5 g/ml), morphine (1000
g/ml), ondansetrone (150 g/ml), paracetamol (1000 g/ml), ranitidine (5 g/ml),
roxithromycine (150 g/ml) and temazepam (15 g/ml). Areas of peaks co-eluting with the
analyte peaks should not exceed 20% of the area at the LLQ level. At the IS retention
time the interference should not exceed 5% of the IS peak area.
Stability
The stability of the analytes was investigated under various conditions including the
stability in plasma, in the supernatant and in the final extract. The analytes were
considered to be stable when 80-120% of the initial concentration was found.
Stability of freshly prepared QC samples, containing thiotepa, tepa, CP and
4OHCP-SCZ at a low, medium and high concentration level, was assessed after three
freeze (-70 C)-thaw cycles. The concentrations of the analytes were related to the initial
concentration as determined for the samples that were freshly prepared and processed
immediately. The long-term stability of thiotepa, tepa, CP and 4OHCP-SCZ in plasma at
-70 C was studied by re-analysing previously measured patient samples at different
concentrations.
The processed sample stability of all analytes in the supernatant after storage for 10
days at 4 C was assessed at two different concentration levels. Also, the stability in the
final extract was studied after 10 days storage at 4 C at two different concentration
levels. The measured concentrations of the analytes in the stored processed samples
were related to the measured concentrations of the same QC samples immediately after
processing.

Results and discussion

Mass spectrometry
The most sensitive mass transitions for MRM analysis are depicted in Table 1.
Representative chromatograms of control human plasma and a QC sample are
presented in Figure 4. In Figure 5, tandem mass spectra of the four compounds are
shown. For CP, the most abundant fragment in the product ion mass spectrum was seen
90

Bioanalysis of cyclophosphamide, thiotepa and metabolites

at m/z ratio of 140. This product ion is a result of cleavage of the nitrogen-phosphorus
bond connecting the ring structure with the amine, resulting in the loss of
di(2-chloroethyl)amine. For 4OHCP-SCZ, the most abundant fragment at m/z 221 was
probably the result of cleavage of the carbon-oxygen group in the middle of the molecule
resulting in the loss of phosphoramide mustard. Detected product ions for thiotepa and
tepa are both the result of loss of azacyclopropane (m/z 43) resulting in products with m/z
147 and 131, respectively. Formation of the product ion of HMP at m/z 135 is based on
the same mechanism with concomitant loss of dimethylamine (m/z 45). The MS/MS
settings were adjusted to maximize the response of each of the precursor-product ion
combinations.
Table 1. Retention times, capacity factors (k) and monitored transitions of the analytes and
internal standard.
Compound

Retention time
(min)

Precursor (m/z)

Product (m/z)

CP
9.2
8.2
261
140
4OHCP-SCZ
7.2
6.2
334
221
TT
7.7
6.7
190
147
T
2.4
1.4
174
131
HMP
7.0
6.0
180
135
CP= cyclophosphamide; 4OHCP-SCZ= semicarbazone derivative of 4-hydroxycyclophosphamide;
TT= thiotepa; T= tepa; HMP= hexamethylphosphoramide.

Sample pretreatment
During the development of the sample pretreatment procedure we focussed on nonlabor-intensive methods to accelerate sample processing. Moreover, because of the
potential volatility of thiotepa, but especially tepa, a heating or evaporating step was not
preferred in sample pretreatment. A protein precipitation method was then validated using
a mixture of organic solutions (methanol-acetonitrile (1:1, v/v)) as precipitation reagent.
The kinetics of the derivatization procedure of 4OHCP in plasma were investigated in
more detail. Applying a 2M SCZ solution for the reaction [18], we found that the
derivatization reaction was only completed after 5 h at room temperature (20 C) or after
1-2 h at 35 C at all low, medium (Figure 6) and high concentration levels of 4OHCP.
Cooling the mixture to 4 C during derivatization slows down the reaction dramatically
and heating the mixture more than 35 C does not increase the rate of the derivatization
reaction (data not shown). The heating process at 35 C appeared not to influence the
amount of measured thiotepa, tepa and CP in the mixture. Therefore, it was concluded
that the most optimal derivatization conditions were at 35 C for 2 h, which is feasible in
clinical TDM practice.

91

Chapter 2.1

Intensity (cps)

1000

TIC double blank

800
600
400
200
0
0

10

Time (min)

Intensity (cps)

100000

XIC cyclophosphamide (m/z 261)

80000
60000
40000
20000
0
0

10

Time (min)
Intensity (cps)

1500

XIC 4-hydroxycyclophosphamide-semicarbazide (m/z 334)

1200

900
600
300
0
0

10

Time (min)
Intensity (cps)

50000

XIC thiotepa (m/z 190)

40000

30000
20000
10000
0
0

10

Time (min)

Intensity (cps)

25000

XIC tepa (m/z 174)

20000
15000
10000
5000
0
0

10

Intensity (cps)

Time (min)
30000
25000
20000
15000
10000
5000
0

XIC hexamethylphosphoramide (m/z 180)

10

Time (min)

Figure 4. Total ion-chromatogram (TIC: A) of a processed control plasma sample and the
extracted single ion chromatograms (XIC: B to F) of a processed quality control sample at the
medium concentration level. Cyclophosphamide (B, 4.03 g/ml); semicarbazone derivative of
4-hydroxycyclophosphamide (C, 1.01 ng/ml); thiotepa (D, 265 ng/ml); tepa (E, 251 ng/ml);
hexamethylphosphoramide (F, 25.0 ng/ml).

92

Bioanalysis of cyclophosphamide, thiotepa and metabolites

350000

Intensity (cps)

m/z=140

H
N
N P
Cl CH2 CH2
O

300000

Cl CH2 CH2

250000
200000

m/z=140

150000

m/z=261

100000
50000
0
0

50

100

150

200

250

300

m/z

12000000

Intensity (cps)

10000000

H2
N
N P
Cl CH2 CH2
O
Cl CH2 CH2

8000000
6000000

N N

m/z=221
NH2

m/z=221

4000000
2000000

m/z 334

0
0

50

100

150

200

250

300

350

400

m/z

1200000

Intensity (cps)

m/z=147

1000000

800000

m/z=190

600000

m/z=147

400000
200000
0
0

20

40

60

80

100

120

140

160

180

200

m/z
3500000

D
Intensity (cps)

m/z=174

3000000

2500000

2000000

m/z=131

1500000
1000000

m/z=131

500000
0
0

20

40

60

80

100

120

140

160

180

200

m/z

Figure 5. MS/MS spectra of cyclophosphamide (A, m/z 261) semicarbazone derivative of

4-hydroxycyclophosphamide (B, m/z 334) thiotepa (C, m/z 190) and tepa (D, m/z 174).

93

Chapter 2.1

4OHCP-SCZ (%)

100
80
60
40
20
0
0

Time (h)

Figure 6. Percentage of the formation of the semicarbazone derivative of 4-hydroxycyclophosphamide (4OHCP-SCZ) versus time in a quality control sample at medium concentration level at
20 C (!) and 35 C (!) (taking the maximum measured value of 4OHCP-SCZ as 100%).

Chromatography
HPLC separation of the four analytes was performed under basic conditions using an
eluent composed of aqueous 1 mM ammonium hydroxide and acetonitrile together with a
column containing a base-stable stationary phase (the pH of the aqueous component is
approximately 10). This approach was chosen since it resulted in excellent
chromatography for thiotepa and tepa compared to neutral or acidic conditions.
Moreover, thiotepa and tepa appeared to be stable at pH 10 [28-30]. The most
hydrophilic compound, tepa, could be retained on the stationary phase when the aqueous
content of the mixture was high in the first 2 min of the run (96% ammonium hydroxide
and 4% acetonitrile). Thereafter, the fraction of acetonitrile was increased to 25% and in
the final 4 min of the run the initial eluent composition was again used (column
conditioning period). This chromatographic system resulted in adequate and reproducible
retention for tepa to separate it from endogenous components. All other analytes were
eluted from the column in the column conditioning period due to the extended lag-time of
the HPLC system used. The run time could be limited to 10 min (Figure 4).
Validation procedures
The assay was linear over the validated concentration ranges of 200-40,000 ng/ml for
CP, 50-5,000 ng/ml for 4OHCP and 5-2,500 ng/ml for thiotepa and tepa. The best fit for
the calibration curves was obtained by using a weighting factor of 1/concentration2 for CP
and tepa and 1/concentration for 4OHCP and thiotepa. The deviations from the nominal
concentration were <15% for all analytes at all concentrations. The correlation
coefficients were at least 0.994. Unfortunately, stable isotopically labelled ISs for the
analytes were not available during development of the assay. Based on its structural
94

Bioanalysis of cyclophosphamide, thiotepa and metabolites

similarities with thiotepa and tepa, HMP was chosen as IS. HMP also proved useful as IS
for the quantification of CP and 4OHCP. Its structure is shown in Figure 3.
The intra- and inter-assay performance data are presented in Table 2. Accuracies
were within 3.9% for the LLQ and within 12.4% for the other concentrations. The interassay precision, expressed as CV, was <11.2% for all concentrations tested. The mean
intra-assay precision did not exceed 10.0%. Samples originally above the upper limit of
quantitation (ULQ) could be quantified with acceptable accuracy and precision after
dilution with drug-free human plasma. Measured accuracies ranged from 1.2% to 14.9 %.
The protein precipitation recovery data for the analytes and IS are listed in Table 3. It
was not possible to determine the recovery of 4OHCP-SCZ, since a pure reference
standard for 4OHCP-SCZ was not available. Recovery values of the other analytes were
between 89 and 105%. The degree of ion suppression for all analytes (again with the
exception of 4OHCP-SCZ) was between 0 and 12% (measured during three runs).

Table 2. Intra- and inter-assay performance data of the analytes at four concentration levels in
three analytical runs.
Compounda

CP

4OHCP-SCZ

TT

Nominal

Measured

concentration
(ng/ml)

concentration
(ng/ml)

Inter-assay
accuracy

Intra-assay

Inter-assay

(%)b

precision

precision

(%)

(%)

201.50
402.90
4,029.00
20,147.00

193.9
399.7
4,317.0
20,920.0

-3.74
-0.79
7.15
3.84

6.32
2.73
2.75
1.94

9.79
3.64
3.22
2.13

55.28
110.60

53.15
102.65

-3.86
-7.16

9.71
6.24

11.21
8.18

552.80
2,764.00

511.50
2,568.00

-7.47
-7.09

5.18
4.70

5.75
5.30

5.110
10.220
255.500
1,022.000

5.05
10.47
277.00
1,138.00

-1.14
2.43
8.41
11.35

7.72
4.30
3.00
2.23

8.28
6.56
3.77
2.96

5.013
4.821
-3.81
10.00
10.030
9.448
-5.76
6.10
250.700
278.400
11.07
2.86
1,003.000
1,126.000
12.35
1.84
a
CP= cyclophosphamide; 4OHCP-SCZ= semicarbazone derivative of 4-hydroxycyclophosphamide;
TT= thiotepa; T= tepa.
b
Accuracy: [(measured concentration nominal concentration) / nominal concentration] * 100%.

10.10
6.28
3.34
2.22

95

Chapter 2.1

Selectivity and specificity

MRM chromatograms of six batches of control human plasma contained no endogenous
peaks co-eluting with any of the analytes. Figure 4A shows a representative TIC of a
control human plasma sample. LLQ samples, prepared in these six batches of human
plasma, could be quantified within the required 20% deviation from the nominal
concentration (data not shown). No chromatographic interferences were found from the
CP metabolites and co-medicated drugs tested in control human plasma; LLQ samples
spiked with the tested drugs and CP metabolites could be quantified within the required
20% deviation.
Stability
Thiotepa, tepa, CP and 4OHCP-SCZ appeared to be stable after three freeze-thaw
cycles (Table 4). Moreover, the stability of the analytes in the supernatant and final
extract at 4 C is guaranteed for at least ten days (Table 4). The long-term stability of
thiotepa, tepa, CP and 4OHCP-SCZ in plasma was established in previously analysed
patient samples stored at -70 C that were 2, 5 and 7 months old. All measured
concentrations were within 80-120% limits (data not shown). More stability data for the
four compounds at various conditions have been published previously by our group
[18,22].

Table 3. Mean protein precipitation recovery and CV% for the analytes and internal standard after
three workups.
Compound

CP

Nominal
concentration
(ng/ml)

Recovery

CV

(%)

(%)

402.90
100.00
0.741
4,029.00
92.00
8.570
20,147.00
97.00
4.980
TT
10.22
96.00
8.370
255.50
86.00
13.800
1,022.00
97.00
11.800
T
10.03
105.00
6.450
250.70
95.00
4.940
1,003.00
95.00
2.260
HMP
100.00
99.00
4.200
CP= cyclophosphamide; TT= thiotepa; T= tepa; HMP= hexamethylphosphoramide.

96

Bioanalysis of cyclophosphamide, thiotepa and metabolites

Table 4. Stability of the analytes.

Compound
CP

4OHCP-SCZ

TT

Matrix

Conditions

Final extract

4 C, 10 days

Supernatant

4 C, 10 days

Plasma

3 freeze (-70 C)thaw cycles

Final extract

4 C, 10 days

Supernatant

4 C, 10 days

Plasma

3 freeze (-70 C)thaw cycles

Final extract

4 C, 10 days

Supernatant

4 C, 10 days

Plasma

3 freeze (-70 C)thaw cycles

Final extract

4 C, 10 days

Supernatant

4 C, 10 days

Plasma

3 freeze (-70 C)thaw cycles

Nominal conc.
(ng/ml)
402.90
20,150.00
402.90
20,150.00
402.90
4,029.00
20,150.00
110.60
2,764.00
110.60
2,764.00
110.60

Deviation
(%)
-0.520
5.150
-0.074
8.140
0.099

CV
No. of
(%)
replicates
0.129
3
4.050
3
1.860
3
3.190
3
5.840
3

5.310 3.970
4.570 0.725
8.250 10.400
-10.700 4.190
10.100 8.010
2.480 4.960
1.000 6.600

3
3
3
3
3
3
3

552.80
2,764.00
10.22
1,022.00
10.22
1,022.00
10.22

-9.850
-8.100
-1.200
-7.950
-6.910
-12.900
-4.570

5.120
4.150
1.050
7.090
6.800
1.890
2.210

3
3
3
3
3
3
3

255.50
1,022.00
10.03
1,003.00
10.03
1,003.00
10.03

3.330
9.260
3.500
7.960
10.800
9.240
-5.770

3.470
0.517
4.660
1.530
6.800
3.530
6.410

3
3
3
3
3
3
3

250.70
8.250 1.850
1,003.00
11.700 0.893
CP= cyclophosphamide; 4OHCP-SCZ= semicarbazone derivative of 4-hydroxycyclophosphamide;
TT= thiotepa; T= tepa.

3
3

Conclusion
A validated assay is described for the simultaneous quantification of CP, thiotepa, and
their respective metabolites 4OHCP and tepa in human plasma using LC-MS/MS,
requiring only 100 L plasma per sample. The unstable metabolite 4OHCP is converted
into the stable 4OHCP-SCZ derivative, using a single-step derivatization procedure. This
sensitive and selective LC-MS/MS method allowed minimal matrix interference, and
therefore the use of a simple sample clean-up procedure. Accuracies and precisions

97

Chapter 2.1

were well within the predefined limits. The relatively short run time allows high-throughput
analysis.
The method described is routinely applied in our hospital to monitor patients receiving
the high-dose CTC chemotherapy regimen. Analytical results are available within a short
time-window, making dose adaptations during a chemotherapy course possible. The
validated ranges have proved to be suitable for routine TDM measurements (Figure 7).
So far, approximately 500 samples have been analysed using the described method.

100000

Concentration (ng/ml)

10000
1000
100
10
0

10

15

20

Time (h)

25

Concentration (ng/ml)

10000

1000

100

10
0

Time (h)

Figure 7. Plasma concentrations of A) cyclophosphamide (!) and 4-hydroxycyclophos-phamide

(") and B) thiotepa (!) and tepa (") in a patient treated with a 1-h infusion of CP (1000 mg/m2)
followed by a 1-h infusion of carboplatin (265 mg/m2) and a 30 min infusion of thiotepa (40
mg/m2).

98

Bioanalysis of cyclophosphamide, thiotepa and metabolites

Acknowledgements
This work was supported with a grant from the Dutch Cancer Society (project NKI 20012420).

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Hussein AM, Petros WP, Ross M, et al. A phase I/II study of high-dose cyclophosphamide, cisplatin, and
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Colby C, Koziol S, McAfee SL, et al. High-dose carboplatin and regimen-related toxicity following autologous
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Chapter 2.2
Sorption of thiotepa to polyurethane catheter causes falsely
elevated plasma levels
Milly E de Jonge, Ron AA Matht, Selma M van Dam, Sjoerd Rodenhuis, Jos H Beijnen

Abstract
Central venous access catheters are commonly used in clinical oncology. The double lumen
variant is applied in pharmacokinetic studies for simultaneous administration and blood sampling
when frequent blood collections are necessary. Occlusion of one lumen, a common complication,
necessitates the investigator to perform blood sampling through the administration lumen after
interrupting the infusion. Plasma concentrations measured in this sample can be influenced by
sorption of the previously infused compound to the catheter lumen. In this study, the quality of
cyclophosphamide, thiotepa, and carboplatin plasma concentrations is investigated when
sampling is performed through the administration lumen.

Ther Drug Monit 2003; 25: 261-263

Chapter 2.2

Introduction
The use of semipermanent central venous access devices has become a common
practice in clinical oncology, in particular when high-dose chemotherapy regimens are
used. Surgically implanted central venous catheters are used for administration of
chemotherapy, reinfusion of hematopoietic cells, and intensive supportive care
management, including frequent blood sampling and infusion of antibiotics, analgesics,
anti-emetics, blood products, and parenteral nutrition [1]. Prolonged venous access may
improve comfort and quality of life of cancer patients by circumventing the need of
frequent peripheral venous punctures.
In pharmacokinetic studies, multilumen catheters are usually applied, allowing blood
sampling during and after administration of the chemotherapy without interrupting the
infusion. Catheter occlusion is a complication that frequently occurs, precluding blood
sampling through the plugged lumen. Formation of clots inside the catheter is probably
the result of the interaction between the catheter material and the physiologic
mechanisms of blood clotting [2]. When lumen occlusion occurs during pharmacokinetic
studies, the investigator is forced to collect blood samples from a lumen that is not
blocked. Most of the times this will be the same lumen through which administration is
realized. When the infused drug interacts with the material of the lumen, measured
plasma concentrations of the previously infused drug may be falsely elevated.
Cyclophosphamide, carboplatin, and thiotepa are chemotherapeutic agents that are
frequently used in high-dose combination regimens in the treatment of patients with
cancer. For drug concentration monitoring, blood samples are drawn during and after
administration of the respective compounds. Concentrations measured in the plasma
provide information for treatment optimization and, therefore, need to be of high quality.
The goal of this study was to investigate if and to what extent cyclophosphamide,
carboplatin, and thiotepa exhibit sorption to the catheter and how this interaction
contaminates the collected plasma samples.

Patients and methods

Patients had a double-lumen polyurethane Arrow-Howes Central Venous Catheter with
Blue FlexTip (Arrow International Inc., Reading, PA) located in the right subclavian vein.
The infusion was connected to the lumen with the end most downstream. Whole blood
samples were collected from the proximal opening [3].
Three patients were treated for 4 consecutive days with cyclophosphamide (1,000 or
1,500 mg/m2/day in 500 ml 0.9% NaCl) during 1 h, followed by carboplatin (265 or 400
mg/m2/day in 500 ml glucose 5%) during 1 h, and thiotepa (40 or 60 mg/m2/day in 100 ml

102

Sorption of thiotepa to catheter

0.9% NaCl) in two 30-min infusions every 12 h. After the end of the thiotepa infusion,
other supportive fluids were slowly infused.
For therapeutic drug monitoring, blood samples were taken immediately after end of
cyclophosphamide, carboplatin, and thiotepa infusions and 30 min after end of infusion of
each compound. Samples were simultaneously drawn from both lumina: first from the
proximal ending lumen not used for the infusions (collection lumen), and second from the
distal ending lumen used for administration of the chemotherapy (administration lumen).
Sampling through the administration lumen was carried out after removing the drugcontaining fluid from the lumen by rinsing the lumen with 5 ml saline solution. After
withdrawing and discarding 3 ml of blood, the actual sample of 5 ml whole blood is
withdrawn using a 5-ml vacuum heparinized tubing. Collection of 5 ml whole blood from
the collection lumen (control sample) was performed likewise after withdrawing and
discarding 3 ml of blood. After sampling, the lumen was flushed with 5 ml of saline
solution.
After collection, the samples were immediately placed on ice. Plasma was
subsequently separated by centrifuging the sample at 3,500 rpm for 3 min at 4C. Plasma
ultrafiltrate, for carboplatin determination, was prepared immediately using the Amicon
micropartition system with a YMT-14 membrane (Millipore Corporation, Bedford, MA).
Ultrafiltrate was prepared by transferring 0.5 ml plasma in the micropartition system and
centrifuging the system at 2,500 rpm for 15 min. Plasma and ultrafiltrate were stored at
-70 C until analysis. Samples were analyzed within 1 week after collection. Analyses of
thiotepa and cyclophosphamide were performed using a GC-NPD method [4], and
carboplatin was determined using AAS [5]. Within-day and between-day precision and
accuracy were less than 10% for both analysis methods.
The patients participated in clinical studies that were approved by the Committee on
the Medical Ethics of the Netherlands Cancer Institute. Written informed consent to
participate in the pharmacokinetic study was obtained from all patients.

Results
Plasma concentrations of cyclophosphamide, carboplatin, and thiotepa in samples
collected immediately after end of their respective infusions are presented in Table 1.
Concentrations are classified according to the lumen of collection. For thiotepa, plasma
concentrations observed in the sample collected from the administration lumen just after
end of infusion were approximately 100% higher when compared with concentrations in
the samples taken from the collection lumen. However, the cyclophosphamide and
carboplatin concentrations were similar after sampling through both administration and
collection lumen. In the samples taken 30 min after the end of infusion, during which the
lumen has been constantly perfused with supporting fluids, thiotepa concentrations in
samples taken from both lumina were similar (data not shown).

103

Chapter 2.2

Other sampling procedures for accurately measuring thiotepa from the administration
lumen immediately after end of thiotepa infusion have been investigated. Rinsing the
lumen with 5 ml saline followed by withdrawing and discarding 10 or 20 ml whole blood
(in stead of 3 ml), still resulted in thiotepa plasma concentrations that were 35% to 50%
higher in samples drawn from the administration lumen compared with those drawn from
the collection lumen.
Table 1. Concentrations of thiotepa, cyclophosphamide, and carboplatin at the end of infusion
after blood sampling through both collection and administration lumen.
Patient

Thiotepa (ng/ml)
Cyclophosphamide (g/ml)
a
b
a
b
I
1472
2551
49.09
50.62
II
1086
2107
35.06
34.55
III
919.4
1917
45.48
44.90
a= collection lumen; b= administration lumen

Carboplatin (M)
a
b
65.51
61.99
55.07
54.10
90.12
83.97

Discussion and conclusion

Catheter occlusion, a result of thrombus formation, is a frequently seen problem in
semipermanent central venous catheters, preventing blood sampling through the plugged
lumen and necessitating urokinase instillation [2,6]. Even with effective flushing and the
use of a heparin lock in the lumen that is not frequently used, catheter occlusion cannot
always be prevented. With persistent occlusive problems, the catheter has to be removed
[2].
Pharmacokinetic sampling from the lumen through which administration of the drug is
realized is possible after interruption of the infusion. According to our results, this can
sometimes be complicated by falsely elevated plasma levels of the drug. Thiotepa
appears to interact with the polyurethane catheter, leading to absorption onto the surface
of the lumen. Because the interaction is reversible, thiotepa is slowly released from the
lumen. In subsequent collected blood samples, thiotepa concentrations are elevated.
Cyclophosphamide and carboplatin have apparently minor affinity to the catheter.
Cyclosporin A [7-11], tacrolimus [12], and isosorbide dinitrate [13-15] are examples of
drugs that also have been shown to exhibit sorption to infusion systems. The extent of
sorption appears to depend on the lipophilicity of the infused drug, its vehicle [12,13], and
the material of the catheter [8,12-15]. For the highly lipophilic drugs cyclosporin A and
tacrolimus, concentrations in plasma samples, taken from the catheter previously used
for their administration, were significantly greater than concentrations obtained
peripherally. Sorption of cyclosporin A and tacrolimus persisted for several days [7-12],
depending on catheter material [8,12] and continued use of the catheter for administration
of other fluids [9-11].

104

Sorption of thiotepa to catheter

Thiotepa sorption to the polyurethane catheter is not as extensive as reported for

cyclosporin A and tacrolimus [8,11,12]. The latter compounds are accordingly much more
lipophilic than thiotepa, necessitating the use of the vehicles Cremophor EL and
Cremophor HC, respectively. Thiotepa sorption to the catheter appeared to be completely
neutralized after 30-min infusion of fluids through the lumen. However, applied rinsing
methods after end of thiotepa infusion appeared not to eliminate thiotepa from the lumen
completely.
Based on these findings, we recommend that a catheter lumen used for infusion of
thiotepa should not be used to monitor thiotepa plasma concentrations during and
immediately after end of infusion. This is an important finding needing recognition by
researchers since otherwise clinical interventions will be based on falsely elevated
plasma levels. It is likely that contamination of drug-monitoring samples by sorption of the
respective drug to the catheter is relevant for many other, particularly lipophilic, drugs.
Investigators should be aware of this and should avoid the need of sampling through the
same lumen by which the drug is administered. Alternatives, if possible, could be
sampling from a peripheral vein or the initial use of a triple-lumen catheter. However,
preventing lumen occlusion to occur is, off course, the best strategy.

Acknowledgements
This work was supported with a grant from the Dutch Cancer Society (project NKI 20012420).

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