Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 114118

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Journal of Pharmaceutical and Biomedical Analysis

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A validated HPLC method with uorescence detection for the glucuronides of

Combretastatin A1 in human plasma, and studies on their cistrans isomerisation
Michael R.L. Stratford , Lisa K. Folkes
University of Oxford, Gray Institute for Radiation Oncology and Biology, Department of Oncology, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK

a r t i c l e

i n f o

Article history:
Received 8 November 2011
Received in revised form
14 December 2011
Accepted 16 December 2011
Available online 24 December 2011
Keywords:
Plasma OXi4500 glucuronide
CA1P
cistrans isomerisation
HPLC
Fluorescence detection

a b s t r a c t
Two monoglucuronides (CA1G1 and CA1G2) of the catecholic cis-stilbene Combretastatin A1 (CA1,
OXi4500), have been identied in a clinical trial of the bisphosphate prodrug of OXi4500, OXi4503. A
validated assay for the two glucuronides in human plasma using HPLC with uorescence detection after
post-column photolysis is described. The assay was linear over the range 25 nM (CA1G1) or 50 nM (CA1G2)
5000 nM, R2 0.996. The intra-day precision for CA1G1 was better than 8.7% RSD (19.4% at the LLOQ),
and the inter-day precision was better than 5.5% RSD (7.6% at the LLOQ). The intra- and inter-day accuracies were better than 12.6% relative error (14.8% at the LLOQ) and 4.8% (5.4% at the LLOQ) respectively.
For CA1G2, the intra-day precision was better than 5.7% RSD (7.5% at the LLOQ), and the inter-day precision was better than 4.8% RSD (11.9% at the LLOQ). The intra- and inter-day accuracies were better than
10.1% relative error (12.6% at the LLOQ) and 2.2% (3.8% at the LLOQ) respectively. Recovery from plasma
was measured at three concentrations (125, 625 and 2500 nM). Mean recovery of CA1G1 was 94.5% and
ranged from 94.4 to 99.2%. Mean recovery of CA1G2 was 90.7%, range 8892%.
During the validation process, one of the isomers was unexpectedly found to be unstable. CA1G1,
substituted ortho to the stilbene, was relatively stable, but the meta-substituted CA1G2 readily converted
from the cis-stilbene conformation to the trans isomer. This was catalysed by acid and heavy metals,
and could be inhibited by antioxidants such as ascorbic acid. Isomerisation could also be induced by
one-electron oxidation processes such as horseradish peroxidase and azide radicals.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Combretastatin A1 phosphate (CA1P, OXi4503) is a bisphosphate pro-drug of the colchicine analogue Combretastatin A1 (CA1,
OXi4500, Fig. 1) which acts as a vascular disrupting agent (VDA) by
depolymerising tubulin in the endothelial cell, resulting in breakdown of tumour blood ow [1], and has recently completed a
Cancer Research UK-supported phase I trial as an anti-cancer agent.
The active drug, the catechol cis-CA1, was expected from previous
experience with the analogue Combretastatin A4 (CA4, Fig. 1) to be
subject to glucuronidation at one or other of the hydroxyl groups
[2] and this was found to be the case. Although the cis isomers of
the combretastatins are not naturally very uorescent, the trans
isomers are generally much more so, and this paper describes the
application of post-column photolytically induced isomerisation

Abbreviations: CA1(P), Combretastatin A1 (phosphate); CA1G1(2), CA1 glucuronide 1(2); CA4, Combretastatin A4; DTPA, diethylenetriaminepentaacetic acid;
DTT, dithiothreitol; IS, internal standard; PBS, phosphate buffered saline; TBA, tetrabutylammonium; VDA, vascular disrupting agent.
Corresponding author. Tel.: +44 1865 617343; fax: +44 1865 617334.
E-mail address: michael.stratford@oncology.ox.ac.uk (M.R.L. Stratford).
0731-7085/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jpba.2011.12.022

[3] to the development of a validated method for the determination

of the two glucuronide metabolites (CA1G1 and CA1G2) in human
plasma by HPLC with uorescence detection. During the validation process, we noted that CA1G2 was unexpectedly susceptible
to cis/trans isomerisation, and this was investigated in a number of
in vitro systems.
2. Materials and methods
2.1. Chemicals
Structures of the compounds used are shown in Fig. 1. CA1G1
and CA1G2 were from Quintiles, Kansas City, MO, USA. As
supplied, the position of the glucuronide substitution was not
given. The structures were assigned from the 1D-GOESY NMR
spectra obtained with solutions in DMSO-d6 , using a Bruker
Advance DPX400 spectrometer. CA4 was from Pharm-Eco Laboratories (Devens, MA, USA). Methanol and acetonitrile (HPLC grade)
and tetra-n-butylammonium hydrogen sulphate (TBAHSO4 , ECD
grade), were from Fisher (Loughborough, UK), ammonium formate (LCMS grade) and sodium ascorbate were from Fluka (Poole,
UK), dipotassium hydrogen orthophosphate (K2 HPO4 ) and ascorbic acid were from VWR (Lutterworth, UK) and formic acid, sodium

M.R.L. Stratford, L.K. Folkes / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 114118

115

inter-day precision and accuracy, analyte recovery, and analyte

stability.
2.4. Plasma sample preparation
All extraction procedures were carried out in reduced lighting.
To 50 L aliquots of plasma, 20 L IS, 40 L 0.1 M TBAHSO4 , and
1 mL methanol were added, with vortex mixing after each addition.
Samples were then kept at 4 C for 15 min, then spun at 20,000 g
for 10 min at 10 C. The supernatant was decanted into 4 mL amber
glass vials and taken to dryness in a heated centrifugal evaporator. The extracts were re-dissolved by the addition, with mixing, of
40 L methanol, followed by 60 L water, and transferred to amber
HPLC injection vials containing 200 L glass inserts. The injection
volume was 25 L.
2.5. Isomerisation studies
Aqueous solutions of the glucuronides (20 M, 300 L) were
prepared in either 10 mM HCl 1 mM ascorbic acid, or 10 mM
potassium phosphate, pH 7.0 20 M FeSO4 200 M DTPA. Samples were pipetted into plastic 300 L HPLC vials, placed in the
HPLC autosampler maintained at 30 C, and timed sequential 20 L
injections made from each vial.
2.6. HRP oxidation
Aqueous solutions of the glucuronides (200 M) were incubated
in PBS at 37 C with hydrogen peroxide (0.5 mM) and HRP (CA1G1,
4 g, CA1G2, 0.2 g), total volume 1 mL. Aliquots (50 L) were
taken at various times, added to 40 L acetonitrile, centrifuged and
analyzed by HPLC. Control incubations were without HRP.
2.7. Oxidation by azide radicals
Azide radical (N3 ) is a strong one-electron oxidant formed
through the reaction of hydroxyl radicals ( OH) with aqueous
sodium azide solutions. Radiolysis of water produces in particular
OH and hydrated electrons (e ). Through the following reacaq
tions, N3 is generated with yield of 0.56 M/Gy:
eaq + N2 O OH + OH + N2
OH

Fig. 1. Structures of the compounds studied.

azide, diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase (HRP) and dimethyl sulphoxide (DMSO) were from Sigma
(Poole, UK).
2.2. Stock solutions
For the isomerisation studies, CA1G1 and CA1G2 were prepared
in water at 1 mM; for the plasma validation studies, CA1G1 was
prepared in water and CA1G2 and CA4 (IS) in DMSO, all at 1 mM.
CA4 was diluted to 50 M in 30% DMSO and stored at 20 C, and
on a weekly basis, this stock was diluted to a working concentration
of 2.5 M in 30% methanol/water and stored at room temperature.
2.3. Validation methods
A full validation of the assay for human plasma was performed
following ICH guidelines [4] covering specicity, intra-day and

+ N3 N3 + OH

Solutions of CA1G1 and CA1G2 (20 M) were prepared in

sodium azide (5 mM) containing potassium phosphate buffer
(10 mM, pH 7.5) which had been previously deaerated with N2 .
Solutions were aliquoted into acid washed 4 mL resealable vials,
saturated with nitrous oxide and irradiated in a Caesium-137 GSR
D1 irradiator (Gamma-Service Medical GmbH, Leipzig, Germany)
at a dose rate of 1.5 Gy/min for 010 min. Products were analyzed
by HPLC.
2.8. HPLC
2.8.1. Validated plasma assay
The HPLC system consisted of a 2695 separations module and
a 474 uorescence detector tted with a 5 L ow cell, both from
Waters (Watford, UK). Chromatographic separation took place on
an ACE 3 m C18 column (150 mm 3 mm) (Hichrom, Reading, UK)
maintained at 35 C. Solvent A was 20% methanol, 5 mM KH2 PO4 ,
5 mM H3 PO4 , solvent B was methanolwater (75:25, v/v) and C,
acetonitrilewater (75:25, v/v). Initial conditions were 73% A, 27%
B, with a linear gradient to 45% A, 55% B over 9 min, then to 5% A,
55% B, 40% C over 5 min, and nally to 100% C over 0.5 min, held

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M.R.L. Stratford, L.K. Folkes / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 114118

Table 1
Recovery of CA1G1 and CA1G2 from human plasma.

CA1G1

CA1G2

Conc. (nM)

Recovery (%)

%RSD

2500
625
125
2500
625
125

95.8
94.4
99.2
92.8
88.0
91.4

7.3
3.8
6.1
8.8
3.7
2.3

for 2 min before returning to initial conditions. The ow rate was

0.5 mL/min and the run time was 23 min. A photolysis coil comprising of 75 cm 0.006 PTFE tubing wound round a mercury lamp
from a Waters 441 detector was placed between the column outlet and the uorescence detector [3]. The uorimeter wavelengths
used were excitation 320 nm, emission 390 nm. Data were acquired
using Waters Empower software.
2.8.2. Isomerisation and other product analysis studies
The HPLC system consisted of a 2695 separations module and
a 2996 diode array detector, both from Waters (Watford, UK).
Chromatographic separation took place on an ACE 3 m C18 column (125 mm 3 mm) maintained at 35 C. Solvent A was 20%
methanol, 5 mM KH2 PO4 , 5 mM H3 PO4 , solvent B was methanol,
the ow rate was 0.5 mL/min, and samples were either run with
a gradient from 4570% B over 4 min, run time 9 min, or isocratically at 45% B with a run time of 4.5 min. The cis and trans
isomers were separated under these conditions (retention times:
cis-CA1G1, 3.14 min; trans-CA1G1, 4.27 min; cis-CA1G2, 3.80 min;
trans-CA1G2, 4.18 min), and the areas of each peak at 300 nm
determined. Data were acquired using Waters Empower software.
Product identity after azide radical oxidation of CA1G1 was conrmed with a mass spectrometric detector (Waters ZQ) placed after
the 2996, using the same column, but with 10 mM formic acid as
the A solvent, gradient 3580% B over 5 min. The ow rate was
0.45 mL/min, and the solvent was split after the 2996 so that only
250 L went through the mass spectrometer. The detector was
operated in either positive (2.5 kV) or negative (2.1 kV) electrospray
mode, cone voltage 20 V.
3. Results
3.1. Validation of the plasma assay
3.1.1. Extraction and chromatography of CA1G1 and CA1G2 from
human plasma
Because the analysis of the glucuronides was normally performed on the same sample extract as that used for CA1P, the
TBAHSO4 included to improve recovery of CA1P [5] was used for this
validation; it may well be that it is not necessary to achieve good
recovery of the glucuronides, but this has not been formally investigated. Likewise, although considerable effort was made to achieve a
shorter analysis time, the requirement to resolve interfering peaks
in control plasma resulted in a gradient which was identical in
slope and organic composition to that used for CA1P, but run in
the absence of TBAHSO4 , and at a lower pH which enhances the
uorescence response of the glucuronides [3]. Recovery of the conjugates from human plasma was determined by comparing peak
areas with those of equivalent aqueous solutions, and is shown in
Table 1. Mean recoveries in excess of 90% for both metabolites were
achieved.
3.1.2. Specicity
Ten individual control plasma samples were extracted; none
showed signicant interferences where the analytes eluted. Fig. 2A
shows a typical chromatogram of a control plasma extract, and one

Fig. 2. Panel A: chromatograms of a control human plasma extract (dotted line)

and after spiking with 25 nM CA1G1 and 50 nM CA1G2. Panel B: chromatogram of a
human plasma extract 1.2 h after a dose of 14 mg/m2 OXi4503.

spiked with 25 and 50 nM CA1G1 and CA1G2. Neither CA1P (retention time 7.5 min) or CA1 (retention time 15.4 min) interfered. Also
shown is a chromatogram of a plasma extract from a patient 1.2 h
after a dose of 14 mg/m2 OXi4503 (Fig. 2B). CA1G1 was the major
glucuronide metabolite seen.
3.1.3. Linearity
Four calibration curves (CA1G1, 255000 nM; CA1G2,
505000 nM) were obtained for each analyte by plotting the
ratio of the peak area to the IS area, tted by linear regression
weighted to 1/concentration2 . The correlation coefcients in all
cases were 0.996, and the %RSD < 3%.
3.1.4. Precision and accuracy
Intra- and inter-day precision and accuracy (%RSD and %RE)
were calculated from replicate analyses of QC samples spiked with
ve concentrations of the analytes on three separate occasions
(Table 2). For CA1G1, precision at 25 nM was 19.4%, so this was
taken as the LLOQ. For CA1G2, accuracy at 25 nM was >20%, so
50 nM, where precision and accuracy were all <20%, was taken as
the LLOQ, consistent with the lower response for this compound
[3].
3.1.5. Plasma stability
QC samples were stored at room temperature for 4 h, at 80 C
for 12 months, and subjected to 3 freeze/thaw cycles. Extracted
QC samples were also stored at 20 C for 24 h before analysis. In
all cases, measurements were within 15%, indicating that both
CA1G1 and CA1G2 were stable under these conditions.
3.2. Isomerisation of CA1G2
Although CA1G1 solutions were very stable, during the validation process, we observed substantial variation in the amount of
the trans isomer in aqueous solutions of CA1G2. Fresh stocks were
invariably >98% cis-CA1G2 (the certicate of analysis quoted 99%),
but the subsequent degree of isomerisation was highly dependent

M.R.L. Stratford, L.K. Folkes / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 114118

117

Fig. 3. Panel A: isomerisation of CA1G2 in pH 7.0 10 mM phosphate buffer or 10 mM HCl 1 mM ascorbic acid. Closed symbols represent the cis isomer, open symbols trans.
Panel B: effect on the isomerisation of CA1G2 at pH 7.0 of 20 M FeII 200 M DTPA. Each data point is the mean of 4 points. Error bars are 1 standard deviation.

on the storage conditions. We therefore carried out some studies

to further characterise this phenomenon.
3.2.1. Role of pH and heavy metals
The isomerisation of CA1G2 was strongly pH dependent, proceeding more than an order of magnitude faster in 10 mM HCl than
at pH 7 (Fig. 3A). In this panel, the area percentage of each isomer at
300 nm is shown, with solid symbols the cis isomer, open symbols
the trans. Also shown is the complete protection provided by ascorbic acid (1 mM); similar results were seen with dithiothreitol and
dithionite. The role of heavy metals in the catalysis of isomerisation, and its inhibition by the chelator DTPA was also investigated.
As this chelator only functions efciently at neutral pH, the two
Table 2
Intra- and inter-assay precision and accuracy.

CA1G1
Intra-assay

CA1G1
Inter-assay

CA1G2
Intra-assay

CA1G2
Inter-assay

Nominal
concentration
(nM)

Calculated mean
concentration (nM)

Precision
(%RSD)

2500
625
125
50
25
2500
625
125
50
25
2500
625
125
50
25
2500
625
125
50
25

2539
620
127
55.2
28.5
2522
637
130
52.4
26.3
2638
639
121
54.6
32.1
2512
614
122
48.1
22.2

1.79
1.28
0.79
5.21
19.4
1.20
2.72
3.91
5.47
7.60
3.18
3.77
5.73
4.85
1.54
3.48
2.52
4.84
11.9
7.64

n = 3 for intra-assay, n = 9 for inter-assay.

Accuracy
(%RE)

glucuronides were incubated at pH 7.0 in 10 mM phosphate buffer

at 30 C in the presence of 20 M Fe2+ 200 M DTPA. The rate of
isomerisation of CA1G2 was accelerated by 20 M Fe2+ and almost
completely inhibited in the presence of DTPA (Fig. 3, panel B); in
parallel studies under both sets of conditions with CA1G1, no signicant isomerisation was seen.
3.2.2. Oxidation by HRP
cis-CA1G2, incubated with 0.2 g HRP, showed a rapid decrease
in concentration (initial slope 79 nmoles/min/g HRP), with a
concomitant increase in the trans-CA1G2. In contrast, cis-CA1G1
incubated with 20-fold more HRP showed only a very slow decrease
in peak area (0.82 nmoles/min/g HRP), and the trans isomer which
was present as a minor impurity (5%) paralleled this decrease.
3.2.3. Oxidation by azide radical
Oxidation of CA1G1 and CA1G2 by azide radicals showed a large
difference in the chemistry between the two isomers (Fig. 4). The
initial slope of the loss of CA1G1 was 85% of the initial radical yield.

1.57
.080
1.60
10.3
14.0
0.90
1.96
4.09
4.78
5.38
5.51
2.29
3.20
9.2
28.5
0.49
1.80
2.22
3.84
11.2
Fig. 4. Oxidation of CA1G1 and CA1G2 by azide radical.

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M.R.L. Stratford, L.K. Folkes / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 114118

Fig. 5. Reaction scheme for the azide radical one-electron induced isomerisation of CA1G2.

Mass spectrometric analysis showed that the main end product

was a dimer, M. Wt 1014.5 probably resulting from combination of
2 intermediate phenoxy radicals. No trans isomer was generated.
In contrast, loss of CA1G2 was 200% of the initial radical yield
suggesting that a radical chain reaction was occurring, with the
trans isomer being the only signicant product seen.
4. Discussion
The validated method for determining the two glucuronides
has been successfully applied to around 300 plasma samples from
patients in a clinical trial of CA1P with no signicant interferences
observed. All measured concentrations were within the validated
range, the highest concentration observed being 3.5 M CA1G1.
We had only very limited amounts of CA1G2, and no further supplies were available, and it was necessary to keep sufcient stocks of
the solid, which appeared to be stable over a period of several years,
to complete the validation and subsequent analysis of the clinical
trial samples. We were therefore only able to carry out limited studies to characterise the two positional isomers, but there were clear
differences between them. CA1G1 was very stable in aqueous solution, but CA1G2 exhibited acid and heavy metal-catalysed cistrans
isomerisation, and this was also observed using the one-electron
oxidants HRP and azide radical. Isomerisation of CA1G2 and not
CA1G1 after oxidation to an intermediate phenoxy radical can be
rationalised as indicated in Fig. 5. For cis-CA1G2, delocalisation of
the phenoxy radical on to the stilbene bond allows isomerisation
to the more energetically favoured trans isomer [6]. A chain reaction may occur following reaction of the latter with the parent cis
glucuronide, resulting in further loss of parent. For CA1G1, this
cannot occur as electron delocalisation onto the stilbene bond is
not possible. One electron oxidation of CA1G1 to a phenoxy radical intermediate appears to result in the formation of a dimer as
seen with other phenols such as tyrosine, where dityrosine is used
as a marker of oxidative stress [7,8]. Similar considerations apply
to the HRP data, where oxidation also proceeds through one electron steps, while the spontaneous rearrangement seen on storage
of aqueous solutions of CA1G2 is most likely due to trace amounts
of transition metals such as iron and copper causing Fenton-type
reactions which are inhibited by DTPA. This may explain the considerable variability observed in the rate of this isomerisation after
preparing solutions of CA1G2 depending on the extent of this trace

contamination. Radical-driven isomerisation is analogous to that

observed by Clarke et al. [9] with cis-AF-2, which has a double bond
conjugated with a nitrofuran and a furan aromatic ring. Formation
of the nitrofuran radical-anion led to high efciency isomerisation
to the trans form.
The data highlights the importance of rigorous checks on the
stability of analytes during the validation process. Because we had
reasonable supplies of CA1G1, we focussed our initial attention on
this isomer which proved very stable, and assumed that CA1G2
would behave in a similar fashion. We were fortunate that we had
specied that ascorbic acid, which as shown here inhibits the isomerisation of CA1G2, be added to the trial plasma samples in order
to prevent oxidation of the catechol CA1, enabling us to be condent
in the data generated for all three metabolites.
Acknowledgements
This work was supported by Cancer Research UK. We are grateful
to Peter Wardman for helpful discussions and to Mo Latif for the
NMR spectral analysis.
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