A SIMPLE AND RAPID METHOD FOR SIMULTANEOUS
QUANTIFICATION OF DOXORUBICIN, EPIRUBICIN,
CYCLOPHOSPHAMIDE AND 5-FLUOROURACIL IN HUMAN
PLASMA BY LCMS/MS.
Gopinath Sambasivam*1, Deepak GopalShewade1, Biswajit Dubashi2, Rajan Sundaram1
1
Department of Pharmacology, JIPMER, Puducherry, India.
2
Department of Medical Oncology, JIPMER, Puducherry, India.
ABSTRACT
A reverse-phase HPLC method with detection by mass spectrometry is
described for the simultaneous determination of doxorubicin,
epirubicin, cyclophosphamide and 5 fluorouracil in human plasma. The
chromatographic separation was carried out on a Waters Xterra® C18
column (3.9mmx150 mm, 5μm particle size) with a mobile phase of
0.1% formic acid in water and methanol (30:70 v/v). The flow rate
was kept as 0.4ml/min with total run time of 7 minutes. A tandem
quadrupole MS was operated in multiple reaction monitoring (MRM)
in both positive and negative electrospray ionization (ESI) mode.
Doxorubicin epirubicin and cyclophosphamide were detected in positive mode and
5-Fluorouracil was detected well in negative ionisation mode. Protein precipitation method by
using 100% methanol was used to extract the drugs. To 200 μL of plasma along with internal
standard, 600 μL of 100% methanol was added then vortexed for 1 minute and centrifuged at 13,500 rpm for 5min. After centrifugation 100 μL of the supernatant layer was used for
analysis. The retention time of doxorubicin, epirubicin, 5-fluorouracil and cyclophosphamide
were 2.32min, 2.35 min, 2.43 min and 4.64 min respectively. Ifosamide was used as internal
standard and its retention time was 4.12 min. The developed method was validated in human
plasma with a lowest limit of quantification of 5 ng/mL for all the drugs. The method was
accurate with the intra-day and inter-day precision of 15% and the there was no endogenous
contamination of any of the analytes. This method was robust, precise and accurate for the
Volume 5, Issue 10, 747-757. Research Article ISSN 2277– 7105
*Corresponding Author
Gopinath Sambasivam
Department of
Pharmacology, JIPMER,
Puducherry, India.
Article Received on 26 July 2016,
Revised on 16 August 2016, Accepted on 06 Sep. 2016
determination of doxorubicin epirubicin cyclophosphamide and 5 fluorouracil in human
plasma using LC/MS/MS method.
KEYWORDS: Doxorubicin, Epirubicin, Cyclophosphamide 5-Fluorouracil, LC-MS/MS.
INTRODUCTION
Anthracyclines are one of the most active drugs prescribed for the treatment of breast cancer.
Doxorubicin (DOX), epirubicin and daunorubicin are the class of anthracyclines which
belongs to the tetracycline class of antibiotics. The anticancer activity of these drugs has been
mainly due to strong interactions with DNA in target cells. The cyclophosphamide, an
alkylating agent, is often administered with other antineoplastic agents like doxorubicin
epirubicin and 5-fluouracil in breast cancers. Anthracyclines and cyclophosphamide
combinations are used predominantly in the treatment of breast cancer.[1,2] These combination
regimens can be intolerable due to severe and life-threatening toxicities. Therefore maximum
clinical assessment is needed for the safer administration in different situations.[3]
5-Flourouracil is an anti-metabolite which is used for the treatment of various cancers such as
breast, lung and gastrointestinal tract cancers. Although toxicity is managed by treating the
symptoms and reducing the dose of anticancer drugs it results in under-dosing and reduced
efficacy. The optimal dose of anticancer drugs should have a maximal antitumor effect with
less toxicity. Clinical use of anticancer drugs may require therapeutic drug monitoring which
can be possible with analytical evaluation of these agents in biological fluids. A simple, rapid
and sensitive analytical method for estimation of these drugs is necessary for better
management of cancer patients. Various studies have reported the simultaneous plasma
determination of the anthracyclines and metabolites alone or combined with other drugs.[4-7]
Some studies have reported the environmental and biological monitoring of anticancer
drugs.[8, 9] Few studies described the determination of cyclophosphamide and 5-fluoroacil
with its metabolites in human plasma.[10-14] The sample preparation used in various studies
mostly involve liquid-liquid extraction (LLE) and solid phase extraction (SPE). Since HPLC
methods are labor intensive and have less sensitivity, LCMS method represents the better
choice for the analysis of antineoplastic drugs. LCMS methods have desired sensitivity and
less run time which enables for getting results within few hours. In the present study,
LCMS/MS method was developed to determine four drugs which are prescribed widely in
method for the simultaneous determination of doxorubicin, epirubicin, cyclophosphamide
and 5-Flurouracil in human plasma.
MATERIALS AND METHODS Chemicals and reagents
Pure drugs of doxorubicin, epirubicin ifosfamide, 5-Flurouracil and cyclophosphamide were
obtained from Sigma-Aldrich. Methanol (LC-MS chromasolv) and formic acid were also
purchased from Sigma-Aldrich (Missouri, USA). Milli-Q water (Millipore Corporation) was
used wherever required in this method. Ifosamide was selected as an internal standard as it
has similar in its physicochemical properties to that of cyclophosphamide which is one of the
drugs of interest for estimation.
LC-MS/MS conditions
An Alliance 2695 HPLC separation module (Waters USA) with PDA detector, cooled
autosampler and column oven was used. Chromatographic separation was achieved by using
Xterra MS C18 column (3.9mmx150 mm, 5μm; Waters, Milford, MA, USA). Column oven
temperature was maintained at 30°C and sample temperature at 20°C. The mobile phases
consisted of 0.1% formic acid-water (mobile phase A) and Methanol (mobile phase B). A
flow rate was 0.4 ml/min with 30% of Mobile Phase A and 70% of mobile phase B over 7
minutes. Total run time was 7 minutes. A tandem quadrupole MS was operated in multiple
reaction monitoring (MRM) in both positive and negative electrospray ionization (ESI)
modes for detection of all drugs. Data acquisition and processing were done by using
MassLynx 4.1 software. Quan Optimize software was used to optimize individually various
MS/MS parameters like cone energy collision energy, and retention time of all the drugs.
Solutions and standards
The stock solutions of doxorubicin, epirubicin, ifosfamide (IS) and cyclophosphamide were
prepared in methanol to yield a concentration of 1.0 mg/mL Preparation of working solutions
and calibration standards were done with 50% methanol in water. The working solutions for
calibration curve were prepared by serial dilutions of the stock solution. The calibration was
done over a range of 10ng/mL to 10μg/mL for doxorubicin and epirubicin, 10ng/mL to 50 μg/mL for cyclophosphamide and 5-flurouracil. Limit of detection (LOD) was observed up to
5ng/ml. The quality control (QC) working solutions were 10 ng/mL, 500 ng/mL, 10 μg/mL
methanol. Stock solutions were kept at -80 °C when not in use. Linear regression was used to
fit the calibration curves.
Sample preparation
The sample preparation from plasma was carried out using protein precipitation method by
using 100% methanol. 50 μL of internal standard working solution (100 ng/mL) was added to 200 μL of plasma and vortexed (30 s), followed by the addition of 600 μL of 100% methanol.
The whole solution was then vortexed for 1 minute and centrifuged at 13,500 rpm for 5min.
After centrifugation, 100 μL of the supernatant layer was transferred into vials for analysis and 20μL were injected into the system.
Method Validation
The bio-analytical method was validated according to the US FDA guidelines. Calibration
curves were done by using linear regression analysis of doxorubicin, epirubicin,
cyclophosphamide 5-flurouracil and ifosfamide (IS) versus concentration. The Intra-day and
inter-day variation, precision, and accuracy were assessed by analyzing QC samples. The QC
samples were also subjected for bench-top stability (27°C, 6 h, 12 h) and freeze/thaw stability
(-200°C,3 freeze/thaw cycles, 48 h) in plasma by comparing samples before and after the
stability tests.
RESULTS
The separation of four anticancer drugs by LC-MS/MS using ifosamide as an internal
standard was done. The mobile phases consisted of 0.1% formic acid-water (mobile phase A)
and methanol (mobile phase B). A flow rate of 0.4 ml/min with 30% of mobile. Phase A and
70% of mobile phase B with runtime of 7 minutes. The retention time of doxorubicin,
epirubicin, cyclophosphamide and ifosfamide (IS) in positive ion mode were 2.32min, 2.35
min, 4.64 and 4.12 min respectively.5-flurouracil retention time in negative ion mode was
2.43 min. A runtime of 7min was set to avoid carry-over effect of biological matrices. The
MRM transitions, cone voltage and collision energy of the analytes and IS are presented in
Table 1. A representative chromatograms of 100 ng of all four compounds after extraction in
Table 1: Multiple reaction monitoring of various parameters of drugs
Compound Formula/
Mass
Parent Ion m/z
Cone Voltage (Volt)
Daughter Ion m/z
Collision Energy
Ion Mode
Doxorubicin 543.17 544.27 16 397.05 12 ES+
Epirubicin 543.17 544.20 22 396.98 14 ES+
Cyclophosphamide 260.02 283.05 22 225.01 16 ES+
Ifosfamide 260.02 261.05 28 92.03 26 ES+
5-Flurouracil 130.01 128.98 24 42.10 12 ES-
Fig.1: Chromatogram of 100ng pure drug extracted in human plasma
Method validation Linearity and Sensitivity
The calibration curves were plotted by spiking blank human plasma over a wide range of
10ng/mL to 10μg/mL for doxorubicin and epirubicin. 10ng/mL to 50 μg/mL for
wide range. The lower limit of quantification (LLOQ) was 5 ng/mL for all the drugs which
defined the sensitivity of the method.
Precision and accuracy
The accuracy and precision were done for the QC samples at 10 ng/mL, 500 ng/mL,
10μg/mL and 50 μg/mL for doxorubicin, epirubicin, cyclophosphamide, and 5-flurouracil.
The accuracy of the present method was close to 100% over this range. The intra-day and
inter-day precision were within the limit of 15%.
Specificity and stability
The analytical run of blank samples with IS and without IS in plasma indicates there is no
cross interference of the drugs. The stability test was performed using four sets of QC
samples for benchtop stability and freeze-thaw stability. The first set of QC samples were
estimated immediately after preparation. The second set was kept at 27°C and was analyzed
after 6 hrs and 12 hrs for measuring bench top stability. The freeze-thaw stability was done
by keeping the fourth set of QC samples at -200°C and was analyzed at 24th hr and 48th hr.
The response deviation of the second set, third set and fourth set of samples were less than
10% from the first set of QC samples which indicated the stability of all these drugs in human
plasma.
DISCUSSION
The use of analytical methods for the simultaneous estimation of various drugs considerably
reduced the cost and time required for the analysis of drugs. HPLC methods with different
detectors were used for the estimation of doxorubicin, epirubicin cyclophosphamide and
5-flurouracil.[15-18] Most of the methods focused on individual drug estimation of these
commonly prescribed drugs. Sanson described the Simultaneous estimation methods of five
anticancer drugs using HPLC and liquid-liquid extraction method.[19] Ahmad described the
simultaneous determination of 5-flurouracil doxorubicin and cyclophosphamide by HPLC
with PDA detector.[20] Nevertheless, the mass spectrometry is an advanced detection method
which offers a lot of advantages over HPLC and more accessible to analysts as its cost
decreases. The detector is more superior in MS than UV because of its specificity and range,
and its detection limits are greater. Various studies have been carried out for the
determination of anthracyclines in LCMS.[21,22] Author wall described the method for the
estimation of epirubicin in LCMS by using the serum. A mixture of acetonitrile/ water/formic
method for the simultaneous determination of four anthracyclines and their metabolites by
LC-MS. The mobile phase was composed of 5 mM ammonium formate /acetonitrile (70:30
v/v) and the flow rate was 50 μL/min.[24]
Mahnik developed the method for the estimation of
anthracyclines in hospital effluents. The mobile phases were water/acetonitrile adjusted to pH
2.0. The flow rate was kept as 0.6 mL/min. Doxorubicin and Daunorubicin retention times
were 13.0 and 16.0 min.[25] In the present study, the mobile phases were 0.1% formic acid in
water and methanol (30:70) and flow rate was 0.4 ml/min. The retention time is 2.32 min and
2.35min for doxorubicin and epirubicin which is very much less than the other reported
studies. It offers advantages like faster estimation and the Limit of detection is 5 ng/ml which
shows the good sensitivity. Di Francesco described a method for the simultaneous estimation
of cyclophosphamide, doxorubicin, and doxorubicinol.[26] Solid-phase extraction was used for
sample extraction. Detection was achieved in positive, mixed reaction monitoring mode on a
triple quadrupole mass spectrometer. In the present study detection of cyclophosphamide
doxorubicin and epirubicin was achieved by positive MRM mode which is like in other
studies. In our study protein, precipitation method was used. It is a very convenient method
and less cumbersome than solid phase extraction. Few studies described the quantification
and validation of 5-flurouracil and its metabolites in dog and human plasma.[27-29] Author
usawanuwat described the monitoring of 5-fluorouracil cyclophosphamide and hydroxyl urea
in water samples.[30] Most of the studies described the estimation of 5-fluorouracil was
carried out in negative ionization mode in multiple reaction monitoring which is in agreement
with our study. The mobile phase of formic acid in water and methanol was suitable for
separation of the cyclophosphamide hydroxyl urea and 5 fluorouracil in waters sample.[30] In
the present study, the mobile phase of 0.1 formic acid in water and methanol was sufficient
enough for the separation of 5-fluorouracil cyclophosphamide as well as doxorubicin and
epirubicin. Calibration standard in our study was 10ng/mL to 10μg/mL for doxorubicin and
epirubicin. 10ng/mL to 50 μg/mL for cyclophosphamide and 5-flurouracil. The present
method was accurate with the intra-day and inter-day precision of 15% which is the accepted
criteria. The analytical run of blank samples with IS and without IS shown that there is no
cross interference of the drugs. The good background in the analytical run indicates that there
was no endogenous contamination by any of the analytes. It was indicative of the excellent
specificity of this current method for quantification of all four drugs used over a wide range.
Our method is robust precise and accurate for the estimation of doxorubicin epirubicin
CONCLUSION
In the current study, an LC/MS/MS method for the simultaneous estimation of three
chemically and structurally different anticancer drugs was developed. Protein precipitation
was used for the extraction of all drugs. This method is simple, fast and validated for the
estimation of doxorubicin, epirubicin, 5-flurouracil and cyclophosphamide in human plasma.
ACKNOWLEDGEMENT
We acknowledge the JIPMER and ICMR for funding the study.
CONFLICT OF INTEREST There is no conflict of interest.
REFERENCES
1. Nabholtz JM, Falkson C, Campos D. Szanto J. Docetaxel and doxorubicin compared with
doxorubicin and cyclophosphamide as first-line chemotherapy for metastatic breast
cancer: results of a randomized, multicentre, phase III trial. J Clin Oncol, 2003; 21(6):
968-75.
2. Biganzoli L, Cufer T, Bruning P. Coleman R. Doxorubicin and paclitaxel versus
doxorubicin and cyclophosphamide as first-line chemotherapy in metastatic breast cancer:
the European Organization for Research and Treatment of Cancer 10961 multi-center
phase III trial. J Clin Oncol, 2002; 20(14): 3114-21.
3. Egorin MJ, Forrest A, Belani CP. Ratain MJ.A limited sampling strategy for
cyclophosphamide pharmacokinetics. Cancer Res. 1989; 49(11): 3129-33.
4. Badea I, Lazar L, Moja D, Nicolescu D, Tudose A. A HPLC method for the simultaneous
determination of seven anthracyclines. J Pharm Biomed Anal. 2005; 39(1-2): 305-9.
5. Loadman PM, Calabrese CR. Separation methods for anthraquinone related anticancer
drugs. J Chromatogr B. 2001; 764(1-2): 193-206.
6. Chen C, Thoen KK, Uckun FM. High-performance liquid chromatographic methods for
the determination of topoisomerase II inhibitors. J Chromatogr B. 2001; 764(1 2): 81-119.
7. Larson RR, LMB, Khazaeli, Kenneth DH. Development of an HPLC method for
simultaneous analysis of Five Antineoplastic Agents. Appl Occup Environ Hyg. 2003;
18(2): 109-19.
8. Sottani C, Tranfo G, Bettinelli M, Faranda P. Trace determination of anthracyclines in
method for assessing exposure of hospital personnel. Rapid Commun Mass Spectrom.
2004; 18(20): 2426-36.
9. Gomez-Canela C, Ventura F, Caixach J. Occurrence of cytostatic compounds in hospital
effluents and wastewaters, determined by liquid chromatography coupled to
high-resolution mass spectrometry. Anal Bioanal Chem. 2014; 406(16): 3801-14.
10.Baumann F, Lorenz C, Jaehde U, Preiss R. Determination of cyclophosphamide and its
metabolites in human plasma by high-performance liquid chromatography–mass
spectrometry. J Chromatogr B Biomed Sci Appl. 1999; 729(1-2): 297–305.
11.De Jonge ME, van Dam SM, Hillebrand MJ Rosing H. 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. J Mass Spectrom. 2004; 39(3): 262–71.
12..Ciccolini J, Mercier C, Blachon MF, Favre R. A simple and rapid high-performance
liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and
possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD)
activity. J. Clin Pharm. Therap. 2004; 29(4): 307-15.
13.Compagnon E, Thiberville N, Moore C, Thudlez C. Simple high-performance liquid
chromatographic method for the quantitation of 5-fluorouracil in human plasma. J
Chromatogr B. 1996; 677(2): 380-83.
14.Nassim MA1, Shirazi FH, Cripps CM, Veerasinghan S. An HPLC method for the
measurement of 5-fluorouracil in human plasma with a low detection limit and a high
extraction yield. Inter J Mol Med, 2002; 10(4): 513-516.
15.Camaggi CM1, Comparsi R, Strocchi E, Testoni F, Pannuti F.HPLC analysis of
doxorubicin, epirubicin and fluorescent metabolites in biological fluids. Cancer
Chemother Pharmacol. 1988; 21(3): 216-20.
16.Mahdadi R, Lhermitte M, Lafitte JJ. Determination of Adriamycin and its fluorescent
metabolites in human plasma by HPLC. Biomed. Chromatogr. 1987; 2(1): 38-40.
17.Di Paolo A, Danesi R, Ciofi L, Vannozzi F. Improved analysis of 5-fluorouracil and
5,6-dihydro-5- fluorouracil by HPLC with diode array detection for determination of cellular
dihydropyrimidine dehydrogenase activity and pharmacokinetic profiling. Ther Drug
18.Burton LC, James CA. Rapid method for the determination of ifosfamide and
cyclophosphamide in plasma by high-performance liquid chromatography with
solid-phase extraction. J Chromatogr B Biomed Sci. 1988; 431(2): 450-54.
19.Sanson AL, Silva SCR, Martins MCG, Giusti-Paiva A. Liquid-liquid extraction combined
with high performance liquid chromatography-diode array-ultra-violet for simultaneous
determination of antineoplastic drugs in plasma. Braz J Pharm Sci. 2011; 47(2): 363-371.
20.Ahmad M, Usman M, Madni A. A fast and simple HPLC-UV method for simultaneous
determination of three anti-cancer agents in plasma of breast cancer patients and its
application to clinical pharmacokinetics. Afr J Pharm Pharmacol. 2011; 5(7): 915-22.
21.Arnold RD, Slack JE, Straubinger RM. Quantification of doxorubicin and metabolites in
rat plasma and small volume tissue samples by liquid chromatography/electrospray
tandem mass spectroscopy. J Chromatogr B. 2004; 808(2): 141-52.
22.Sottani C, Tranfo G, Bettinelli M, Faranda P. Trace determination of anthracyclines in
urine: A new high-performance liquid chromatography/tandem mass spectrometry
method for assessing exposure of hospital personnel. Rapid Commun Mass. 2004; 18(20):
2426-36.
23.Wall R, McMahon G, Crown J, Clynes M. Rapid and sensitive liquid chromatography–
tandem mass spectrometry for the quantitation of epirubicin and identification of
metabolites in biological samples. Talanta. 2007; 72(1): 145-54.
24.Lachatre F, Marquet P, Ragot S, Gaulier JM. Simultaneous determination of four
anthracyclines and three metabolites in human serum by liquid
chromatography-electrospray mass spectrometry. J Chromatogr B. 2000; 738(2): 281-91.
25.Mahnik SN, Rizovski B, Fuerhacker M, Mader RM. Development of an analytical
method for the determination of anthracyclines in hospital effluents. Chemosphere, 2006;
65(8): 1419-25.
26.Di Francesco R, Griggs JJ, Donnelly J, DiCenzo R. Simultaneous analysis of
cyclophosphamide, doxorubicin and doxorubicinol by liquid chromatography coupled to
tandem mass spectrometry. J Chromatogr B. 2007; 852(1-2): 545–53.
27.Vainchtein LD, Rosing H, Schellens JH, Beijnen JH. A new, validated HPLC-MS/MS
method for the simultaneous determination of the anti-cancer agent capecitabine and its
metabolites: 5'-deoxy-5- fluorocytidine, 5'-deoxy-5-fluorouridine, 5-fluorouracil and 5-
28.Wattanatorn W, McLeod HL, Cassidy J, Kendle KE. High performance liquid
chromatographic assay of 5-fluorouracil in human erythrocytes, plasma and whole blood.
J Chromatogr B. 1997; 692(1): 233-37.
29.Chu D1, Gu J, Liu W, Paul Fawcett J. Sensitive liquid chromatographic assay for the
simultaneous determination of 5-fluorouracil and its prodrug, tegafur, in beagle dog
plasma. J Chromatogr B. 2003; 795(2): 377-82.
30.Usawanuwat J, Narin B, Suwanna KB. Analysis of Three Anticancer Drugs
(5-Fluorouracil, Cyclophosphamide and Hydroxyurea) in Water Samples by HPLC-MS/MS.