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A SIMPLE AND RAPID METHOD FOR SIMULTANEOUS QUANTIFICATION OF DOXORUBICIN, EPIRUBICIN, CYCLOPHOSPHAMIDE AND 5 FLUOROURACIL IN HUMAN PLASMA BY LCMS/MS

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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

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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

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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

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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

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[image:5.596.61.522.85.606.2]

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

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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

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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

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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.

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Figure

Table 1: Multiple reaction monitoring of various parameters of drugs

References

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