Clinical Chemistry 43: 615-618, 1997;
(Clinical Chemistry. 1997;43:615-618.)
© 1997 American Association for Clinical Chemistry, Inc.
Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction
Rita Paronia,
Cinzia Arcelloni,
Elena De Vecchi,
Isabella Fermo,
Davide Mauri and
Renzo Colombo
Laboratory of Chromatography and Separative Techniques and 1 Department of Urology, I.R.C.C.S. H San Raffaele, via Olgettina 60, 20132 Milano, Italy.
a Address correspondence to this author at: Lab. HPLC, H San Raffaele, Via Olgettina 60, 20132 Milano, Italy. Fax +39 2 26432640.
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Abstract
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The aim of this study was to set up a method for quantification of
plasma mitomycin C (MMC) concentrations during intravesical
chemotherapy delivered in the presence of local bladder hyperthermia
(HT). In comparison with existing methods, this assay, characterized by
relative simplicity and efficiency, resulted in the facilitation of
performance with nondedicated instrumentation or nonspecialized staff.
Purification from plasma matrix was carried out by solid-phase
extraction under vacuum. The purified drug was then collected directly
into the vials of the HPLC autosampler. Chromatographic analysis was
performed on a reversed-phase C18 column with
water:acetonitrile (85:15 by vol) as the mobile phase and the UV
detector set at 365 nm. The use of porfiromycin as internal standard
provided a method with good within-day precision (CV 6.0% at 5 µg/L,
n = 6), linearity (0.5–50 µg/L), and specificity. The lower
limit of detection (
0.5 µg/L) proved to be suitable for plasma
pharmacokinetics monitoring in two tested patients treated with MMC+HT
for superficial bladder cancer.
Key Words: indexing terms: plasma assay • bladder hyperthermia • intravesical chemotherapy • porfiromycin • pharmacokinetics
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Introduction
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Mitomycin C [aziridine(2',3':3,4) pyrrole (1, 2-
)
indol-4,
7-dione-6-amino-1,1a,2,8,8a,8b-exahydro-8-(hydroxymethyl) 8a-metoxy-5-methylcarbamate]
(MMC) is an antimicrobial and anticancer agent that was isolated in
1958 from the fermentation fluid culture medium of Streptomyces
caespitosus (1).1
Since then, several clinical trials involving MMC
as a single agent or in combination with other chemotherapeutic drugs
and with different dosage schedules have been reported
(2). The most significant MMC toxicity in humans is
myelosuppression, widely confirmed to be a delayed and dose-related
effect (3). Anemia, hepatotoxicity, and renal failure,
although unusual, have also been well documented. One of the most
peculiar MMC administration routes is intravesical instillation as a
preventive therapy for the recurrence and stage progression of
superficial bladder cancer after transurethral resection. Intravesical
administration has resulted in extremely low serum concentrations, thus
with virtually no systemic side effects
(4)(5). Several investigations
(6) have clearly evidenced a synergistic interaction
between local increase of tumor temperature and anticancer agents'
tumoral cell killing capacity. In our Institute, a new system
(SBTS-101®) designed to simultaneously deliver local
bladder hyperthermia (HT) and intravesical chemotherapy (ICT) has been
recently developed and clinically tested on recurrent superficial
transitional cell carcinoma of the bladder (STCCB)
(7)(8). Preliminary results (9)
were suggestive of a better effectiveness of microwave-induced HT
combined with local chemotherapy in respect to chemotherapy alone.
However, the question is whether temperature enhancement of bladder
mucosa could potentially affect the systemic absorption of the
cytostatic drug, rendering important the monitoring of plasma
concentration–time profile during ICT+HT treatment.
To quantify MMC in biological fluids, a wide number of different
analytical methods have been applied on the basis of microbiological
(10), immunological (11), and chromatographic
(HPLC) procedures (12)(13)(14)(15)(16)(17). None of the aforementioned
methods, however, completely presented the characteristics of
sensitivity (<0.5 µg/L), precision, rapidity, and simplicity ideally
required. Our aim was to develop a low-cost HPLC assay for MMC
determination in serum that could be easily transferred to the routine
laboratory and, therefore, also carried out by nonspecialized personnel
and without dedicated or unusual instrumentation.
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Materials and Methods
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chemicals
The organic solvents (HPLC grade) were obtained from J.T. Baker
(Deventer, The Netherlands). Bidistilled water was used throughout the
study.
calibration solutions and calibrators
As calibrator MMC, Mitomycin-C® (injectable powder at
4% purity in NaCl) from Kyowa Hakko Kogyo (Tokyo, Japan) was used. As
internal standard, porfiromycin (PFM) was synthesized in Japan (>99%
pure) and kindly donated by Kyowa Hakko Kogyo. MMC and PFM stock
solutions were prepared at 1.72 mmol/L (0.5 g/L) and 2.87 mmol/L (1
g/L) in methanol, respectively. These solutions were stored at
-20 °C for up to 1 month without notable degradation. MMC working
solution was 3.44 and 1.72 µmol/L (1 and 0.5 mg/L) in water, whereas
PFM was 2.87 µmol/L (1 mg/L) in 2 mol/L PBS (potassium phosphate
buffer), pH 7.4. New working solutions were freshly prepared for
dilution of the stocks at each analytical series. For unknown sample
measurement, calibrators in plasma were prepared at three different MMC
concentrations (1, 5, 20 µg/L) and treated as described below in the
sample extraction procedure.
apparatus
Chromatographic analysis was carried out on a C18
column (Ultrasphere 150 x 4.6 mm, 5 µm; Beckman, Palo Alto, CA)
connected to a Kontron chromatographic system (Kontron, Zurich,
Switzerland) equipped with two pumps (Model 420), an autosampler (Model
460), a double-beam UV detector (Model 430) set at 365 nm, and a Data
System 450. The mobile phase of water:acetonitrile (85:15 by vol) was
delivered at 1.0 mL/min.
sample preparation and storage
As a preliminary study, we evaluated MMC systemic absorption in
two patients with STCCB in which MMC instillation was associated with
microwave-induced local HT (homogeneous heating of bladder mucosa
within a medium temperature of 42.5 °C–46.5 °C). A total amount
of 40 mg of MMC was administered in 60 min but, to avoid the drug
degradation throughout the entire procedure, the dose was delivered
with two 30-min instillations of 20 mg/50 mL bidistilled water. The
procedure followed in this study was in accordance with the ethical
standards of our Institution's responsible committee.
Blood samples were taken into heparinized tubes before and every 15 min
during the 60-min instillation. As soon as possible, plasma was
obtained and stored at -20 °C until analysis took place.
sample extraction procedure
After thawing and avoiding any exposure to light, plasma samples
(1 mL) were added to the internal standard PFM (50 µL = 50 ng)
and diluted 1:1 (by vol) with water. For solid-phase extraction, 1-mL
Supelclean extraction tubes filled with 100 mg of LC-18 end-capped
sorbent were used (Supelco, Bellefonte, PA). The disposable tubes were
connected to the Visiprep Solid Phase Extraction Vacuum Manifolds
(Supelco) and, after vacuum application with a water pump, preactivated
by washing with 2 mL of methanol followed by 2 mL of water. The vacuum
was then disconnected to prevent the columns from drying, the diluted
plasma samples were loaded, and the vacuum was applied again, not
exceeding the flow rate of ~1 mL/min. The C18 columns
were washed with water (2 x 1-mL aliquots), followed by
water:methanol 90:10 (1 mL) and water:methanol 70:30 (0.2 mL) mixtures.
Columns were completely dried by flushing air for 1–3 min; then the
vacuum was disconnected and the 12-mL polypropylene tubes used to
collect washings were discharged and replaced with the 0.5-mL
polypropylene microvials of the HPLC autosampler. MMC was eluted under
vacuum with 2 x 200-µL fractions of water:methanol 50:50. The
vials were directly positioned into the sample tray of the autosampler
and 100–200 µL of the samples injected into the HPLC column.
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Results
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chromatographic analysis
Under the conditions described in Materials and
Methods, the MMC and PFM showed a Rt of 4.9
and 7.2 min, respectively, with a total analysis time of 10 min. In
Fig. 1
A, the chromatogram of a blank plasma sample enriched with MMC
(20 µg/L) and PFM (50 µg/L) before solid-phase extraction is shown,
whereas in Fig. 1B
and C
the same control plasma was supplemented with
2.5 and 0.5 µg/L MMC, respectively. In Fig. 1D
, the analysis of a
serum sample taken from a patient after a 60-min instillation with MMC
and homogeneous heating of the bladder walls at 46.5 °C is shown.
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Figure 1. Chromatographic separation of plasma samples purified by
solid-phase extraction as described in Materials and
Methods.
(A) Control plasma (1 mL) supplemented with 20 ng of MMC
and 50 ng of PFM; 100 µL/400 µL eluted from the C18
cartridges injected  5 ng of MMC. (B) Control plasma
supplemented with 2.5 ng of MMC; 100 µL/400 µL injected 0.6 ng.
(C) Control plasma (1 mL) supplemented with 0.5 ng of
MMC. The sample eluted from the C18 disposable cartridge
was reduced by nitrogen evaporation for a few minutes to ~250–300
µL and 200 µL was injected into the HPLC column. (D)
Plasma taken 30 min after ICT+HT treatment (heating at 46.5 °C) from
a patient with superficial bladder cancer. The MMC peak corresponds to
5.4 µg/L. Arrows indicate MMC
(Rt = 4.9 min) and PFM
(Rt = 7.2 min), respectively.
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We found that the length of the chromatographic separation could be
properly reduced to only 3 min (1.9 and 2.7 min
Rt of MMC and PFM, respectively) simply by
replacing the 15-cm C18 column with a 3 cm x 3
mm x 3 µm C18 column (Supelco) (data not shown).
Because of the rapidity of the preanalytical phase, however, we
considered the analysis length of 10 min acceptable for our purpose,
and we preferred the use of the 15-cm column, thus reducing the
possibility of chromatographic interferences and improving the
specificity of the separation.
analytical variables
The use of PFM as the internal standard ensured good
reproducibility of the entire procedure. The extraction efficiency and
reproducibility of MMC and PFM from the C18 columns were
assessed in triplicate on plasma samples supplemented with 50 µg/L of
the internal standard PFM and with MMC (50, 20, and 5 µg/L). MMC
recovery was 98% ± 4.0%, 92% ± 6.0%, and 89% ± 7.1% at the
three concentrations tested, respectively, whereas PFM showed a mean
recovery of 85.0% ± 6.1% (n = 9). The interference study was
carried out with caffeine, theophylline, amphotericin B, epirubicin,
ampicillin, amikacin, cefazolin, and ceftriaxone. None of these
commonly administered drugs was detected under the analytical
conditions used for MMC assay. The intraday precision of the method
(CV) calculated on the MMC/PFM area ratio gave 5.8% (n = 6) and
6.0% (n = 6) at 10 and 5 µg/L, respectively. The
reproducibility of the method calculated within 1 year showed a CV of
10.7% and 9.5% for the 10 and 5 µg/L concentrations (n = 10),
respectively. Linearity of the assay was assessed by supplementing
human control plasma in the 0.5–50 µg/L concentration range. Linear
regression of the area ratio (y) vs MMC plasma concentration
expressed in µg/L (x) gave the equation y
= 0.0108(±0.0001)x + 0.005(±0.003), r =
0.999, Sy|x =
0.0072. By injecting 0.5 µg/L directly after solid-phase extraction,
the MMC signal was still evaluable with a peak area of 0.011 mVmin. To
achieve a better response at this concentration, avoiding larger
injection volumes, we reduced the methanol fraction of the sample by
subjecting it to nitrogen evaporation for few minutes before HPLC
injection, thus potentially improving the detection limit (Fig. 1C
).
Control plasma showed no peaks at the Rt of the
two drugs.
mitomycin c pharmacokinetics
The plasma pharmacokinetics concentration profiles recorded in two
patients when an intravesical dose of MMC (20 mg/50 mL replaced after a
30-min instillation) was given in the presence of adjuvant local HT are
shown in Fig. 2
. In both patients, the curves reached the maximum peak of MMC
plasma concentration between 45 and 60 min of instillation, with
concentrations from 1.2 to 5.9 µg/L in the first patient and a
slightly more increased systemic absorption in the second one (plasma
concentration 8.1–13.3 µg/L).
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Figure 2. Plasma pharmacokinetics absorption of MMC after ICT+HT.
The bladder cavity was filled for 60 min with MMC (20 mg/50 mL of
distilled water). After 30 min the bladder was emptied and the solution
replaced with a fresh one. The curves refer to two
different patients.
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Discussion
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The method set up for MMC quantification was suitable for
monitoring the kinetics of absorption in plasma during ICT associated
with local HT.
The chosen chromatographic conditions were the same as those described
by Tjaden et al. (16), but without buffering the mobile
phase because we were interested in MMC determination only. Those
authors obtained a fully automated analysis with a continuous-flow
system and a valve-switching technique coupled to a 100 x 3 mm
C8 HPLC column. Although feasible for a routine working
load, this assay procedure needed dedicated instrumentation with
nonstandard chromatographic apparatus and >2 mL of plasma to reach a
MMC sensitivity of <1 µg/L.
Den Hartigh et al. (12) used a liquid–liquid extraction
with chloroform:2-propanol (1:1 by vol), whereas Dalton et al.
(17) used ethyl acetate alone. Despite the very high
solvent:sample ratio used by these authors (1:10 by vol), and the very
critical step of the solvent drying up under reduced pressure or
nitrogen and thus causing MMC decomposition and affecting the recovery
rate, this kind of procedure appeared unsuitable for a clinical
chemistry laboratory where air exhausters and (or) equipment for
solvent evaporation are not always easily available. Moreover, the
chromatographic separation of the ethyl acetate extract shown by Dalton
et al. (17), although referring to only 1 ng of MMC
injected, appears contaminated and full of potentially interfering
peaks. The use of solid-phase extraction for MMC purification from
serum has already been reported (13)(14).
However, Tjaden et al. (13) used homemade Amberlite XAD-2
columns requiring high washing and elution volumes, with a drying step
of the purified methanol extract (6 mL) before HPLC analysis.
The main feature of our proposed assay consists of the solid-phase
extraction under vacuum of 1 mL of sample with commercially available
100-mg disposable columns, which assures a reproducible performance,
avoids the use of conspicuous extraction volumes, and allows the direct
injection of the sample eluted in the last purification step. The
chromatographic analysis does not require a dedicated apparatus
(16) or an unusual detector (13), so MMC
analysis can be easily scheduled on the same HPLC apparatus used for
other routine assays (drugs or metabolites). In addition, the manual
preanalytical purification can be fully automated with ASPEC (Gilson,
Villiers Le Bel, France) equipment for automated column extraction
directly linked to the HPLC system. The manual handling of the
purification procedure allows the simultaneous cleanup of 12 samples
every 15 min, with the use of <3 mL of solvent and a quite low cost
analysis (<$1 including the column and the extraction eluents).
The pharmacokinetics curves reported here are only preliminary results
of a larger trial in which the effects of local HT when associated with
ICT will be thoroughly investigated. Moreover, any eventual relation
between temperature delivered during the hyperthermic treatment and the
systemic absorption of the drug is presently under careful examination.
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Acknowledgments
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This work was supported in part by a grant of C.N.R. and in part by
a grant of Associazione Italo-Brasiliana per la Diagnosi e la Terapia
delle Malattie dell'Apparato Urinario.
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Footnotes
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1 Nonstandard abbreviations: MMC, mitomycin C; HT, hyperthermia; ICT, intravesical chemotherapy; STCCB, superficial transitional cell carcinoma of the bladder; and PFM, porfiromycin. 
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Abstract
Introduction
