RESEARCH ARTICLE
LC–MS/MS method development for quantification of
doxorubicin and its metabolite 13‐hydroxy doxorubicin in mice
biological matrices: Application to a pharmaco‐delivery study
Q1 Serena Mazzucchelli
1
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Alessandro Ravelli
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Fausto Gigli
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Mauro Minoli
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Fabio Corsi
1
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Pierangela Ciuffreda
1
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Roberta Ottria
1
1
Dipartimento di Scienze Biomediche e
Cliniche “Luigi Sacco”, Università degli Studi di
Milano, Italy
2
Dipartimento di Scienze Biomediche,
Chirurgiche ed Odontoiatriche, Sezione di
Tossicologia Forense, Università degli Studi di
Milano, Italy
Correspondence
Roberta Ottria, Università degli Studi di
Milano‐Dipartimento di Scienze Biomediche e
Cliniche “Luigi Sacco,” Via G.B. Grassi 74,
20157 Milano, Italy.
Email: roberta.ottria@guest.unimi.it
Abstract
This study describes the development of simple, rapid and sensitive liquid chromatography tan-
dem mass spectrometry method for the simultaneous analysis of doxorubicin and its major
metabolite, doxorubicinol, in mouse plasma, urine and tissues. The calibration curves were linear
over the range 5–250 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in plasma and
tumor, over the range 25–500 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in
liver and kidney, and over the range 25–1000 ng/mL for doxorubicin and doxorubicinol in urine.
The study was validated, using quality control samples prepared in all different matrices, for accu-
racy, precision, linearity, selectivity, lower limit of quantification and recovery in accordance with
the US Food & Drug Administration guidelines. The method was successfully applied in determin-
ing the pharmaco‐distribution of doxorubicin and doxorubicinol after intravenously administra-
tion in tumor‐bearing mice of drug, free or nano‐formulated in ferritin nanoparticles or in
liposomes. Obtained results demonstrate an effective different distribution and doxorubicin pro-
tection against metabolism linked to nano‐formulation. This method, thanks to its validation in
plasma and urine, could be a powerful tool for pharmaceutical research and therapeutic drug
monitoring, which is a clinical approach currently used in the optimization of oncologic
treatments.
KEYWORDS
doxorubicin, doxorubicinol, LC–MS/MS, nano‐formulated drug, quantification
1
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INTRODUCTION
Doxorubicin (DOX), an anthracycline glycoside antibiotic, is an excep-
tionally good antineoplastic agent and is widely used in the treatment
of various cancers, including lung, ovarian and breast cancer and malig-
nant lymphoma (Duggan & Keating, 2011
Q2 ). However, long‐term clinical
use is limited due to the development of a progressive dose‐dependent
cardiomyopathy that irreversibly evolves toward congestive heart fail-
ure (Ho, Fan, Jou, Wu, & Sun, 2012). The current thinking is that DOX
is toxic per se but gains further cardiotoxicity after one‐electron reduc-
tion with reactive oxygen species overproduction or two‐electron
reduction with conversion to a secondary alcohol metabolite
doxorubicinol (DOXol). It became clear that is essential to quantify this
toxic metabolite of DOX in as much biomatrices as possible to study its
distribution in the organism after drug administration to understand
better the side effect mechanisms linked to DOX treatment. Further-
more, the antitumor activity of the drug was noticeably enhanced
when it was nano‐formulated. Indeed, DOX has been found to be more
effective in mice when loaded in nano‐drug delivery systems such as
polymeric nanoparticles, liposomes and bionanoparticles. Moreover
nano‐formulation protects DOX from undesired metabolism reducing
the formation of toxic derivatives as DOXol (Lianga et al., 2014; Park
et al., 2009). Actually, it is well‐known that nano‐formulation improves
drug bioavailability, delivery and accumulation to the tumor site. At the
Abbreviations used: CS, calibration standard; %CV, percentage coefficient of
variance; DAU, daunorubicin hydrochloride; DOX, doxorubicin; DOXol,
doxorubicinol; HQC, high quality control; LLE, liquid–liquid extraction; LOD,
limits of detection; LOQ, limits of quantification; LQC, low quality control;
MQC, medium quality control; QC, quality control; %RSE, percentage relative
standard error.
Received: 17 June 2016 Revised: 28 September 2016 Accepted: 4 October 2016
DOI 10.1002/bmc.3863
Biomedical Chromatography 2016; 1–10 Copyright © 2016 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc 1
Journal Code Article ID Dispatch: 02.11.16 CE:
B M C 3 8 6 3 No. of Pages: 10 ME:
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same time, the tumor targeting of the drug implies the reduction of
organ‐sensitive toxicity because of the better tissue biodistribution.
Moreover, slow drug release from a storage structure help to enhance
the therapeutic index and reduce side effects (Rao et al., 2015).
Although the mechanisms by which targeted drugs are more efficient
is becoming increasingly clear, only few details about less toxicity asso-
ciated to nanoparticle‐loaded DOX than the free drug are available.
Information about the biodistribution of DOX delivered by nanoparti-
cles and, in particular, that of its cardiotoxic metabolite DOXol should
lead to a better understanding of the mechanisms related to reduced
nano‐formulated drug toxicity. Various analytical methods in which
detection limits, adequate to analyze plasma or serum from patients
receiving conventional chemotherapeutic treatments, have been
reported. The reported methods have mostly used HPLC coupled with
fluorescence (Zhou & Chowbay, 2002
Q3 ), electrochemical (Ricciarello
et al., 1998) and chemiluminescence detection (Ahmed et al., 2009).
Moreover, apart from some published LC–MS/MS‐based methods
with validated quantifications of DOX and/or DOXol in some biologi-
cal matrices (Sottani, Poggi, Melchiorre, Montagna, & Minoia, 2013),
human plasma (Ibsen et al., 2013), tumors from mice (Liu, Yang, Liu,
& Jiang, 2008), rat plasma (Lachâtrea et al., 2000), human serum, less
attention was paid so far for analysis of DOX and its 13‐hydroxy
metabolite in mouse tissue samples suitable to study the tissue distri-
bution profile of nanoparticle‐delivered DOX (Arnold, Slack, &
Straubinger, 2004; Cao & Bae, 2012
Q4 ; Park et al., 2006). In the present
study, a simple, fast and inexpensive HPLC method with MS–MS
detection has been developed and validated for quantification of
DOX and DOXol in mice biomatrices to obtain a powerful tool for drug
distribution evaluation in pharmaceutical research. The method was
applied to investigate in BALB/c tumor‐bearing mice the bioavailability
and biodistribution of DOX, differently formulated, and its reduced
metabolite, DOXol. We aimed to study the contribution of different
kinds of nano‐formulation to improve DOX bioavailability and
biodistribution in a murine in vivo tumor model. Moreover, the method
can also be applied in therapeutic drug monitoring, a clinical approach
used in the optimization of oncologic treatments.
2
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MATERIALS AND METHODS
2.1
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Materials
DOX hydrochloride and the internal standard daunorubicin hydrochlo-
ride (DAU) were purchased from Sigma (St. Louis, MO, USA). DOXol
trifluoroacetate salt was obtained from AlsaChim (Bioparc, Illkirch,
France). The HPLC grade solvents were purchased from Sigma.
2.2
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Control plasma, urine and mouse tissues
collection
Control human plasma and urine samples used for purification and
extraction studies and for validation experiments were obtained from
healthy volunteers. Blood was collected into a vial containing K
+
‐
EDTA, which was immediately centrifuged. Aliquots of 15 mL of
pooled plasma were stored at −80°C. Human urine, obtained from
volunteer colleagues, was collected after a circadian cycle and aliquots
of 15 mL of pooled urine were stored at −80°C.
Kidney and liver tissues used for purification and extraction
studies and for validation experiments were obtained from healthy
BALB/c mice.
The tumor tissue samples used in this study have been obtained
from an orthotopic model of murine breast cancer. The tumors were
generated by injecting in to the mammary fat pad of 8‐week‐old
BALB/c females 1 × 10
5
4T1‐Luc cells (Bioware‐Ultra 4 T1‐Luc2 cell
line; PerkinElmer
Q5). 4 T1‐Luc is a murine cell line stably transfected with
luciferase, which generates a very aggressive breast cancer. The
tumors were allowed to grow for 10 days, at which time they reached
a size of approximately 0.8 cm
3
. Mice were killed and organs were
explanted, weighted, transferred in a polypropylene plastic Eppendorf
tubes, immediately frozen by liquid nitrogen immersion and stored at
−80°C. Before extraction, whole organs were homogenized in water
(10% w/v) with potter
Q6(Glas‐Col homogenizer) and divided in aliquots
of 200 μL.
2.3
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Preparation of standard solutions, calibration
standards and quali ty control samples
Stock solutions of DOX and DOXol were separately prepared in meth-
anol at a concentration of 1 mg/mL from powder. Even DAU (internal
standard) stock solution was prepared in methanol from powder at
concentration of 1 mg/mL. Three working solutions containing the
mixture of DOX and DOXol at concentrations of 10 μg/mL, 1 μg/mL
and 100 ng/mL, were prepared in methanol mixing and diluting first
stock solutions at 1 mg/mL. Similarly, the DAU working solution was
prepared in methanol at a concentration of 100 ng/mL by diluting
the first stock solution. Aliquots of first stock solutions and second
stock solutions were stored at −80°C while the aliquot in use was
stored at −20°C.
Calibration standard (CS) samples were prepared in plasma, liver,
kidney and tumor tissue homogenates (0.2 mL of homogenate 10%
w/v in water) by adding different volumes of the second stock solu-
tions of mixed DOX and DOXol to reach final concentrations of 1.25,
2.5, 5, 10, 25, 50, 100, 250 and 500 ng/mL. Each solution was spiked
with DAU internal standard solution (DAU 1 μg/mL, and 100 ng/mL
final concentration). CS for DOX and DOXol quantification in urine
samples were prepared in 0.1 mL of human urine by adding different
volumes of the second stock solutions of mixed DOX and DOXol to
reach final concentrations of 25, 50, 100, 250, 500, 750 and
1000 ng/mL. Each solution was spiked with DAU as previously
described for other biomatrices.
Quality control (QC) samples were prepared in plasma, liver, kid-
ney and tumor tissue homogenates (0.2 mL of homogenate 10% w/v
in water). Each sample was spiked with different volumes of the sec-
ond stock solutions of mixed DOX and DOXol at low (LQC), medium
(MQC) and high (HQC) concentration levels (5, 25 and 100 ng/mL for
DOX and 1.25, 5 and 25 ng/mL for DOXol) and with the second stock
solution of DAU. QC samples for validation in urine (0.1 mL) were pre-
pared spiking different volumes of second stock solutions of mixed
DOX and DOXol at LQC, MQC and HQC concentration levels (50,
250 and 750 ng/mL) and a second stock solution of DAU.
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Pooled plasma or urine used for validation experiments were
prepared combining 30 different samples derived from healthy
volunteers.
All CS and QC samples
Q7 were extracted as described below and
immediately injected or stored at −80°C until the injection.
Liquid–liquid extraction (LLE) methods commonly used for drug
extractions from human plasma or tissue have been assessed. LLE
were performed in 15 mL glass tubes. Different aqueous solutions
in combination with different organic phases have been tested.
Aqueous solutions were sodium borate solution (pH 9), acetate
buffer (pH 5), KOH 1
M and H
2
SO
4
1mM whereas the organic solu-
tions were acetonitrile/methyl alcohol 70: 30, chloroform/isopropyl
alcohol 50: 50, hexane/ethyl acetate 50: 50, hexane/ethyl acetate
90: 10, chloroform/heptane/isopropyl alcohol 50: 33: 17, dichloro-
methane/isopropyl alcohol 80: 20 and chloroform/acetone 50: 50.
In addition, homogenization of solid tissues directly in acetonitrile/
methyl alcohol 70: 30 has been tested. Comparing all the extrac-
tion methods that were tested, the combination of H
2
SO
4
and
chloroform/isopropyl alcohol, as the organic phase, gave better
extraction yields. Consequently, CS and real samples were
extracted in the following way: 50 μL of plasma, or 25 μL of urine
or 200 μL of tissue homogenates (10% in water w/v), spiked with
DAU, diluted to 1 mL with H
2
SO
4
1mM and extracted with chloro-
form/isopropyl alcohol 50: 50. After organic phase evaporation, the
residual was dissolved in 50 μL of the initial mobile phase (water/
acetonitrile, 95: 5 v/v) and 20 μL were injected in to the HPLC
for analysis.
2.4
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HPLC–MS/MS analysis
The LC system was composed by a Dionex Ultimate 3000 Rapid Sep-
aration LC system (DionexThermo Fischer, Rodano Milanese, Italy).
Mass analyses were performed on a ABSciex 4000 Q‐trap LC–MS/
MS system (AB Sciex, Foster City, CA, USA). Ionization of analytes
was performed using electrospray ionization in a positive mode; the
ion source temperature was 550°C, ion spray voltage was 5500 V
and declustering potential was 62 V for DOX, DOXol and DAU. Direct
infusion and flow injection analysis of DOX, DOXol and DAU made it
possible to optimize the MS parameters for fragmentation in a multiple
reaction monitoring mode.
Separation of the analytes was carried out on a Phenomenex
Gemini C18 column (150 mm × 2 mm ID 3) at a flow rate of
0.350 mL/min. Mobile phase A was ammonium formate 10 m
M, daily
prepared by means of a Milli‐Q Synthesis A10 System (Millipore,
Billerica, MA, USA), containing 0.1% v/v formic acid and mobile phase
B was acetonitrile. Several gradients of mobile phase A and B have
been tested for the chromatographic separation and the following
gradient has been selected: 0.0–1.0 min 5% B; 1.0–3.0 min to 90% B;
3.0–5.0 min to 95% B; 5.0–6.0 min 95% B; 6.0–6.1 min to 5% B; and
6.1–8.5 min 5% B. The retention times obtained in a total run of
8.5 min, comprising re‐equilibration at 5% B, are listed in Table
T1 1. A
representative HPLC–MS/MS analysis of a mouse plasma sample is
reported in Figure
F1 1. Quantifications were performed using Multiquant
1.2.1 software by AB Sciex.
2.5
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Validation
The analytical method was validated to meet the acceptance criteria of
the US Food & Drug Administration guidelines
Q8(US Department of
Health and Human Services, Food and Drug Administration, Center
for Drug Evaluation and Research, CDER, 2001). Important parameters
such as linearity, accuracy, precision, sensitivity (limits of detection
[LODs] and limits of quantification [LOQs]), specificity, recovery, sta-
bility and influence of matrix effects were determined using plasma,
urine and tissue samples.
2.6
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Selectivity, carry‐over and sensitivity
To exclude any interference or false positive response derived from
extractive procedure, reagents or disposable, blank water was
extracted according to the method
Q9and analyzed in triplicate.
The carry‐over was evaluated by analyzing a solvent sample
(water/acetonitrile 95: 5 v/v) just after the highest CS. The signal–
noise ratio of the eventual DOX or DOXol peak in the solvent sample
was <2.5.
Sensitivity of the method was expressed by LOD and LOQ calcu-
lated on calibration curves prepared in plasma, urine, liver, kidney and
tumor tissue. LOD and LOQ are expressed respectively as 3.3 and 10
times the ratio between the standard deviation of the response and
the slope of the calibration curve (equations 1 and 2). The LOD and
LOQ values calculated for all biomatrices are reported in Table
T22.
LOD ¼ 3:3×
SDav slope
Av slope
(1)
LOQ ¼ 10 ×
SDav slope
Av slope
(2)
2.7
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Linearity, precision and accuracy
The linearity response of analytes was assessed on the five different
biomatrices over their respective calibration range from three batches
of analytical runs. Different calibration ranges for DOX and DOXol and
for different biomatrices have been chosen in relation to
TABLE 1 Multiple reaction monitoring transitions (m/z values), CE
(eV) and RT (min) used to identify and quantify analytes
Compound Mass Precursor Product CE RT
Doxorubicin 543.52 544.2 397.5
a
19 4.49
361.5
a
24
355.5 24
130.0 38
Doxorubicinol 545.54 546.2 399.5
a
21 4.43
363.5
a
35
130.0 30
Daunorubicin
hydrochloride
527.52 528.2 363.5
a
21 4.54
321.5 21
CE, collision energy; RT, retention times.
a
Product ions used for quantification.
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concentrations expected in real samples. CS were prepared in plasma,
urine and tissues and extracted in triplicate as described. Twenty
microliters of eluates were injected and analyzed. A linear model was
used to describe the relationship between analyte concentration and
instrument response (analyte peak area/internal standard peak area)
and determination and variation coefficients (r
2
and CV) were calcu-
lated (Table
T3 3).
Precision and accuracy were determined by QC analyses at LQC,
MQC and HQC concentrations over three batch runs. For each QC,
analysis was performed in six replicates on each day. Precision was cal-
culated using equation 3 and is denoted by percentage coefficient of
variance (%CV). Accuracy was calculated using equation 4, where nom-
inal means theoretical amounts, and is denoted by a percentage rela-
tive standard error (%RSE). The accuracy and precision were required
to be within ±15% RSE of the nominal concentration and ≤15% CV
(Table
T44).
%CV ¼
SD
Mean
×100 (3)
%RSE ¼
Mean−nono min al
nono min al
×100 (4)
2.8
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Recovery and matrix effect
To evaluate absolute recovery two sets of samples were prepared in
plasma, urine and tissue samples. The pre‐extraction spiked QC
FIGURE 1 Q17HPLC‐MS/MS spectra of a real sample of mouse plasma: chromatographic separation and MS/MS analysis of DOX, DOXol and DAU.
DAU, daunorubicin hydrochloride; DOX, doxorubicin; DOXol, doxorubicinol; MRM, multiple reaction monitoring (mode)
TABLE 2 Limit of the assay: LOD (expressed in ng/mL) and LOQ (expressed in ng/mL). Recovery expressed as percentage and matrix effect
expressed as percentage of ion suppression
DOX DOXol
Matrix effect Recovery Matrix effect Recovery
LOD LOQ LQC HQC LQC MQC HQC LOD LOQ LQC HQC LQC MQC HQC
Plasma 0.04 0.15 3 8 75 96 65 0.24 0.82 12 25 59 68 64
Liver 0.12 0.42 37 21 60 93 88 0.30 1.02 13 30 47 66 72
Kidney 0.43 1.48 25 38 73 86 90 0.32 1.05 32 28 66 73 85
Tumor 0.52 1.73 37 23 58 63 70 0.35 1.17 19 26 63 62 78
Urine 0.025 0.08 1 3 82 68 83 0.09 0.32 12 10 70 72 82
DOX, doxorubicin; DOXol, doxorubicinol; HQC, high quality control; LOD, limit of detection; LOQ, limit of quantification; LQC, low quality control; MQC,
medium quality control.
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samples (test samples) were prepared as described at low (DOX 5 ng/
mL; DOXol 1.25 ng/mL), medium (DOX 25 ng/mL; DOXol 5 ng/mL)
and high (DOX 100 ng/mL; DOXol 25 ng/mL) concentration levels for
all matrices except for urine where low, medium and high concentra-
tions were 50, 250 and 750 ng/mL for both DOX and DOXol. For
the post‐extraction spiked samples (reference samples), aliquots of
blank matrices (plasma/urine/liver/kidney/tumor tissue) were proc-
essed using the extraction method to yield post‐extraction superna-
tant. Pooled aliquots of post‐extraction supernatant were then
spiked using DOX and DOXol stock solutions to yield post‐extraction
samples containing DOX and DOXol at 5–25 and 100 ng/mL and
1.25–5 and 25 ng/mL respectively
Q10(50, 250 and 750 ng/mL for both
DOX and DOXol for urine). All samples were analyzed sixfold and ana-
lyte recovery was determined at each concentration level using the
equation 5 where the ratio of the analyte peak areas of the test and
reference samples were expressed as a percentage recovery (%RE).
The average %RE was determined and the calculated precision (CV)
did not exceed 15%.
RE ¼
Peak area of test sample
Peak area of reference sample
×100 (5)
The presence of suppression or enhancement of the analytical sig-
nal was investigated using the post‐extraction spike method. Three
samples of pooled plasma, urines, liver, kidney and tumor tissue and
blank water were extracted following the proposed method. The stan-
dards were added to 50 μL of eluate at two concentration levels LQC
and HQC (5–250 ng/mL for different matrices and 25–750 ng/mL for
urine for DOX, and 1.25–25 ng/mL for different matrices and
25–750 ng/mL for urine for DOXol). Mean peak areas of standards
spiked in eluate from water (A
w
) and from biomatrices (A
p
) obtained
for each concentration were used for calculations (equation 6) and
results are reported as the ion suppression percentage (Table 2).
Matrix effect% ¼ 1−
A
p
A
w
×100 (6)
2.9
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Sample stability
Stock solutions stability was established by quantification of samples
from dilution of two stock solutions stored at −80°C for 1 month and
at room temperature for 6 h. Long‐term storage freeze/thaw and
bench‐top stabilities were determined at LQC and HQC. Long‐term
storage stability in processed biomatrix was tested up to 40 days upon
storage at −80°C. Bench‐top stability was evaluated from samples kept
at room temperature for 15 h before extraction. Freeze/thaw stability
was tested over five cycles of freezing and thawing.
2.10
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Application to real samples
Tumor‐bearing BALB/c mice were anesthetized and injected into the
lateral tail vein with DOX, free or encapsulated in ferritin nanoparticles
(HFer‐DOX) or in liposomes (CAELYX) (1.24 mg kg
−1
; n = 24 mice/
group). One, 2, 24 and 48 h after injection mice were killed (n = 6 mice
TABLE 3 Linearity (n = 5) of the analytical method. Calibration curves built in biomatrices according to the formula y = mx + b (m = slope and b = intercept)
m ±SD b ±SD r
2
± SD CV* m ±SD b ±SD r
2
± SD CV*
DOX 25–500 ng/mL DOXol 1.25–25 ng/mL
Liver 0.0108 ± 0.0002 –0.0822 ± 0.052 0.9975 ± 0.0016 11.3 0.0069 ± 0.000086 0.0051 ± 0.0004 0.9948 ± 0.0027 14.8
Kidney 0.0067 ± 0.00003 0.0224 ± 0.011 0.9986 ± 0.0025 8.5 0.0085 ± 0.00018 0.0118 ± 0.00028 0.9965 ± 0.0010 14.9
DOX 5–250 ng/mL DOXol 1.25–25 ng/mL
Plasma 0.037 ± 0.000003 0.0037 ± 0.0012 0.9958 ± 0.0012 6.3 0.0021 ± 0.0001 –0.001 ± 0.00007 0.9969 ± 0.0015 14.7
Tumor 0.0058 ± 0.000026 0.0187 ± 0.015 0.9984 ± 0.0018 11.3 0.0064 ± 0.000047 0.0053 ± 0.00019 0.9981 ± 0.003 11.5
DOX 25–1000 ng/mL DOXol 25–1000 ng/mL
Urine 0.0053 ± 0.0003 0.0199 ± 0.028 0.9985 ± 0.002 8.1 0.0046 ± 0.00007 0.0011 ± 0.0028 0.9947 ± 0.0014 10.8
CV, coefficient of variance; DOX, doxorubicin; DOXol, doxorubicinol.
*CV expressed as percentage.
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