Simultaneous Determination of Etoposide and Paclitaxel in ... · [email protected]* (Received on 24th May 2018, accepted in revised form 21st December 2018) Summary: A simple,

Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 874

Simultaneous Determination of Etoposide and Paclitaxel in Biological and

Pharmaceutical Samples by RP-HPLC; Method Development, Validation and

Applications of the method for Evaluation of Polymeric Nanoparticles

Muhammad Hassan, Zafar Iqbal, Fazli Nasir, Ismail Khan, Fahim Ullah Khan, Farhad Ullah,

Saifullah Khan and Nabeela Niaz

Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan. [email protected]*

(Received on 24th May 2018, accepted in revised form 21st December 2018)

Summary: A simple, rapid and sensitive RP-HPLC-UV method was developed for quantification of

paclitaxel (PTX) and etoposide (ETO) in biological and pharmaceutical samples. Optimization of

experimental conditions were performed and standard guidelines were used for the validation of the

method. Analytes were separated on Pruospher® Star RP- 18e (250mm × 4.6mm, 5µm) column

using ACN and TFA (0.025%) as mobile phase in the ratio of (60:40V/V) with a flow rate of 1

mL/min and detector set at 235 nm. Protein precipitation method was applied for the extraction of

analytes from biological samples. The method was applied to in-vitro and in-vivo evaluation of

polymeric nanoparticles of etoposide. Solvent evaporation technique was used for the preparation of

polymeric nanoparticles (NPs) using polymer PLGA (75:25) and poloxamer as surfactant. The

linearity of the method is in the range of 14-500 ng / mL for paclitaxel and 12-1000 ng/mL for

etoposide. The LLOD were 5 and 6 ng/mL while the LLOQ were 12 and 14 ng/mL for etoposide and

paclitaxel, respectively. The developed method was precise and its intra and inter day co-efficient of

variance was below 1%. The method was used for in-vitro and in-vivo evaluation of PLGA

polymeric nanoparticles of etoposide. In-vitro evaluation included determination of drug content and

drug release while in-vivo evaluation consisted of pharmacokinetic evaluation.

Key words; RP-HPLC-UV, Etoposide, Paclitaxel, PLGA nanoparticles, Pharmacokinetics.

Introduction

Etoposide (ETP) is a derivative of

podophyllotoxin (Fig. 1A) that inhibits

topoisomerase-II and/or induces direct breakage of DNA [1-2]. It is used in the treatment of small cell

lung cancer, testicular tumor, Kaposi’s sarcoma,

lymphoma and leukemia [1-3].

It has been reported in many studies that

using etoposide for the treatment of various ailments

may also leads to secondary leukemia due to which it

is necessary to optimize its drug regimen and to avoid

the potential risks associated with its use and to

achieve maximum therapeutic benefits [4-6]. The

unwanted affects produced by ETP includes nausea

and vomiting, alopecia and bone marrow suppression (myelosuppression) [7]. Paclitaxel (PTX) is a natural

diterpenoid extracted from the bark of the Pacific

yew. The structural formula of PTX is given in (Fig.

1. B). Paclitaxel has the ability to stabilize and

protect microtubules from disassembly, and interfere

with the breakdown of microtubules during cell

proliferation. PTX has been extensively used to treat

different kind of tumors (ovarian, breast, non-small

cell lung, and prostate tumors) [8-10]. In order to get

optimum results, it is mandatory to monitor the PK

parameters of any drug after IV administration.

A number of analytical methods have been

reported for the quantification of etoposide and PTX

in human plasma and pharmaceutical preparations, individually. No method has been reported for

simultaneous determination of the two drugs. More

over the reported methods have various limitations

such as a first comprehensive review regarding the

liquid chromatographic methods for the

determination of ETP was published in 2001 [11].

Later on, it was also determined in physiological

fluids on liquid chromatography coupled with

different detectors such as ultraviolet, fluorescence,

electrochemical/electron captured (ECD), MS and

ELISA [11-13]. However the methods already

developed have used complex extraction procedures [14-16], complicated gradient systems, [16] and

longer analysis time [16-17] which made them

unsuitable for routine analysis. So an HPLC linked

with UV method was developed for the analysis of

etoposide in biological matrices and dosage forms.

The method was found to be, rapid, inexpensive, and

accurate as compared to other reported methods [11,

18-23].HPLC–UV is a widely applied method for

quantification of PTX and other taxanes in biological

matrices [24-43]. Most assays quantify PTX and

taxanes in human, although some assays have been designed for quantification of taxanes in serum [30-

*To whom all correspondence should be addressed.


31, 39], tissue [28, 44], urine [25-26, 32, 35, 44-46]

or feces [44]. LC-MS techniques are now widely

used for the analysis of PTX [47-49]. Selectivity and

sensitivity of the method is very high, often used for

the determination of PTX and other taxanes in biological matrices. The HPLC-UV methods reported

till now for PTX and other taxanes have some major

problems such as; detection at a 227 nm because at

this low wavelength potential interference of other

endogenous compounds may occur, lack of

sensitivity, large samples volumes to be injected and

the longer analysis times [27-28, 38], while LC/MS

methods are very expensive.

The method which is developed in this

research study is first method which has the ability to

determined three (02) different anticancer drugs i.e. etoposide and paclitaxel in a shortest possible time in

human plasma and dosage form. The internal

standard (sorafenib) used in this method also belongs

to anticancer group. Due to sensitivity, accuracy and

cost effectiveness this method is the most suitable

method to be used for the determination of the above

mentioned anticancer drugs. Standard guidelines

were considered for the validation of this RP- HPLC-

UV method.

Experimental

Chemicals and reagents

Etoposide (≥ 99.9%), Sorafenib (≥ 99.9 %),

and Paclitaxel (≥ 99.9%) were procured from Qilu

Antibiotic Pharmaceutical Co Ltd China. HPLC

grade ACN, Methanol, Di-chloromethane, Tri-

flouroacetic acid (TFA) and poloxamer 407 were

procured from sigma Aldrich (Germany). Polylactic

Co-glycolic acid (PLGA, 75:25) was purchased from

Evonik Germany. Distilled water was prepared by

Millipore (Milford, USA) distillation apparatus.

Instrumentation and Chromatographic Conditions

Perkin Elmer HPLC system (Norwalk,

USA) consisted of a pump (series 200), on-line

vacuum degasser (series 200), auto sampler (series

200), column oven (series 200), linked by a network

chromatography interface (NCI) 900 with a UV/VIS

detector (series 200) was used in this study. Perkin

Elmer Total Chrom Workstation Software (version

6.3.1) was used to quantify the data. The separation

of ETP and PTX was performed using Pruospher® STAR RP- 18e (250mm × 4.6mm, 5µm) column at a

wavelength of 235 nm. Various chromatographic

columns like ACE 5 C18 (150 mm x 4.6 mm, 5 µm),

Discovery HS C18 column (250 mm x 4.6 mm, 5

µm), Symmetry C8 column (150 mm × 3.9 mm, 5

µm) and Symmetry C8 (250 mm × 4.6 mm, 5 µm)

were also tried with pre-column guard cartridge

protected by a Perkin Elmer C18 (30 mm × 4.6 mm,

10µm; Norwalk, USA) pre-column guard cartridge.

The samples were analysed while maintaining the

temperature of the column oven at 25 ºC using trifluroacetic acid (0.025%) and ACN in (40:60 V/V)

ratio in isocratic mode. The samples were pumped at

1.0 mL/ min. Sorafenib was an IS during the process

of determination of etoposide and paclitaxel.

Autosampler was used for injecting the analytes in to

the analytical system.

Fig. 1: Chemical structure of Etoposide (A), and Paclitaxel (B).


Preparation of Standard Solutions

Acetonitrile was used for the preparation of

stock solution of the etoposide, paclitaxel and

sorafenib (IS). The concentration of stock solution was 0.1 mg/mL and was kept at - 20ºC. Dilutions

were prepared from the stock solutions on need basis

using mobile phase as a solvent.

Sample Preparation

Extraction Procedure

Extraction of analytes from plasma was

performed with different organic solvents which

include diethyl ether, acetonitrile, dichloromethane

and ethyl acetate. Dicholoro methane and diethyl ether were also used in 1:1 mixture for the purpose of

extraction of analytes. Among all these solvents the

best organic solvent for the purpose of extraction was

considered on the basis of maximum recovery of the

analytes (etoposide, paclitaxel and internal standard

sorafenib).

Spiked Plasma Samples

Blood samples were collected in Heparin

glass tubes and centrifuged at 8000 rpm for 5 min to isolate plasma. Plasma sample was spiked with

paclitaxel, etoposide and IS. Protein was precipitated

with acetonitrile and volume was made up to 1 mL

with mobile phase according to the scheme shown in

Table-1. The samples which were reconstituted were

then injected, in volume ranging from 10 to 50 µl, in

to the system with the help of an autosampler.

Table-1: Scheme for extraction of analytes from

plasma

Rabbits and Mice Plasma Samples

Paclitaxel was injected to the marginal ear

vein of the rabbit and Etoposide was injected in to the

tail vein of mice. Heparin containing glass tubes were

used for the collection of blood samples from both

the animals at predetermined time. The blood was

centrifuged to isolate plasma and protein

precipitation was carried out with ACN (3 times volume of plasma) and then volume was made up to

1 mL with mobile phase.

Optimizations of Experimental Parameters

Different chromatographic conditions such

as stationary phase (column), mobile phase

composition, flow rate, Lambda max (λ) and column

oven temperature were optimized.

Various analytical columns such as

Pruospher® STAR RP- 18e (250mm × 4.6mm, 5µm), Discovery HS C18 column (250 mm x 4.6 mm, 5

µm), ACE 5 C18 (150 mm x 4.6 mm, 5 µm), CNW

Athena C18-WP (100 mm x 4.6 mm, 5 µm),

Symmetry C8 column (150 mm × 3.9 mm, 5 µm) and

Symmetry C8 (250 mm × 4.6 mm, 5 µm) were tried

with pre-column guard cartridge RP18 (30 × 4.6 mm,

10 µm; Norwalk, USA) were tried to separate

etoposide and paclitaxel. Suitable column was

selected based on peak characteristics such as peak

shape and better resolution.

Different solvents like methanol, ACN,

tetrahydrofuran (THF, 0.05% and 0.1%) and different

buffers which includes phosphate buffer in the range

of 10–100 mM and 50 mM NaH2PO4 in different

compositions were used in isocratic as well as in

gradient mode for the analysis of these anticancer

drugs. Different compositions of mobile phase were

used during analysis but the composition which

produced higher sensitivity, shortest possible run

time and best peak resolution was considered.

Mobile phase flow rate was evaluated in the range of 0.8-1.5 mL/min and its effect on resolution

and peak characteristics were studied. The flow rate

showing greater sensitivity and good resolution was

selected.

The analysis was performed at various

column oven temperature i.e. from 25 to 45 ºC to

determine the effect of temperature on the analytes.

This study was performed at various

wavelengths (λmax) ranges i.e. from 235 to 255 nm and that λmax (235 nm) was selected which produced

best peak resolutions and sensitivity.

Various compounds like sorafenib,

Tenifovir, Atenolol and piroxicam were analyzed to


be used as an IS. The one showing better recovery

and instrumental response was selected as internal

standard.

Method Validation

The method was validated according to USP

and ICH guidelines [50].

Specificity

The specificity of analytical method was

determined by analyzing the analytes (etoposide,

paclitaxel and sorafenib) in liquid phase, blank

plasma and 1:1 mixture (having 1µg/mL of analytes

and IS) and plasma samples spiked with 1µg/mL each of analyte and sorafenib (IS).

Accuracy

Percent (%) recovery technique was used to

assess the method accuracy. The percent recovery of

the analytes was determined at three concentrations

levels (0.1, 0.2, 0.4 ug/mL) by spiking the plasma

(200µl) with appropriate concentration of each

analyte and IS. Percent recovery was calculated

according to the following equation:

(1)

where

X = response ratio of the analyte with reference to IS

in the spiked plasma samples, Y = response ratio of

analyte with reference to internal standard in the

mobile phase control plasma; and Z = response ratio

of the analyte with reference to IS in control plasma

(1:1 mixture). Percent recovery was calculated in

triplicate and results were presented in mean ± SD.

Linearity

The method linearity was determined by

plotting construction curves at seven (07)

concentration points of each analyte in the mobile

phase and plasma. The response ratio and

concentration of each analytes were plotted against

each other to obtain the calibration curve using a

linear least square regression. Slope (m), intercept

(b), and correlation coefficient (r) of the curve were calculated from the regression equation using

Microsoft (MS) Excel 2013.

Precision

Precision study was determined on the basis

of repeatability and intermediate precision (inter-day

and intra-day) studies. Plasma samples (n=6) were repeatedly injected into HPLC system to determined

the repeatability of the method and results were

calculated.

The intra-day study was performed by

analyzing plasma samples for 24 hrs at an interval of

8 hrs. Inter-day study was carried out by analyzing

samples on daily basis (at 24 hrs interval) for seven

days. At each sampling point, analysis was performed

in triplicate and mean was taken. The recovered

amounts were calculated in the form of concentration

by the following equation:

(2)

where X and Y are peak areas of the analyte in

plasma samples and 1:1 mixture, respectively; A and

B are peak areas of the internal standard in 1:1

mixture and plasma samples, respectively; CS is the

concentration of analyte in the 1:1 mixture; and CD is

the dilution factor.

Sensitivity

The sensitivity of the method was evaluated

by quantifying the limit of detection (LOD) and limit

of quantification (LOQ) for each analyte. The limit of

detection (LOD) of the analyte is the concentration at

which signal-to-noise ratio (S/N) is three while a

signal to noise ratio of ten was taken as limit of

quantification (LOQ). Various dilutions of both the

analytes were prepared and their instrumental response was evaluated.

Robustness

The robustness/ruggedness of the reported

method was assessed by bringing small deliberate

changes in the various experimental conditions, like

mobile phase composition, column oven temperature

(± 5ºC), and flow rate of mobile phase (0.2 mL/min)

and their effect on peak characteristics was evaluated.

Stability

Stability studies of samples were conducted

at room temperature (18–23oC), 4 ºC and freezing

temperature (-20oC) for one week. Samples were

analyzed on daily basis in triplicate and % stability

was calculated by the following equation:


(3)

where Qt is stability of analyte at time t, and Q0 is

stability at initial time [51]. Fresh samples (Day-1)

were taken as standard and other results were

compared with it.

Formulation Development

Etoposide loaded polymeric (PLGA)

nanoparticles were prepared by oil in water single

emulsion solvent evaporation method. High pressure

sonication was performed during this process. PLGA

(15 mg) and drug (1, 2, 3, 4, 5 mg) were dissolved in

3 mL of acetonitrile (ACN). This solution was then

added drop wise to the stabilizer aqueous solution

(Poloxamer- 407, 0.025% and 0.05% solution) with

the help of a syringe with continuous stirring. After

complete addition of organic phase to aqueous phase,

the emulsion was then sonicated with the help of

probe sonicator for 2 minutes and the formed dispersion was placed on a magnetic stirrer for the

complete removal/evaporation of organic phase. The

formed nanosuspension was then centrifuged at

14000 rpm for 25 minutes at 5 ºC to collect the

formed nanoparticles. The NPs were then washed

thrice with double distal water and lyophilized for

further investigations. Process parameters are given

in Table-5.

Application of the Method

The proposed method was applied to the

evaluation of in-vitro drug release and

pharmacokinetic (PK) study in animal models. This

research study was performed as per the guideline,

set forth for this purpose by the World Medical

Associations, Declaration of Helsinki-ethical principles for medical research involving animal

subjects. Prior approval for conducting this study has

also been obtained from the Department of

Pharmacy, University of Peshawar departmental

“Committee for Research Ethics”. Pharmacokinetic

parameters were determined for optimal formulations

of polymeric nanoparticles and compared with the

conventional preparations of etoposide.

For in-vitro drug release, a weighed quantity

of etoposide loaded polymeric nanoparticles was

suspended in phosphate buffer saline (pH 7.4) and

aliquot (1 mL) was taken in dialysis bag. PBS (100

mL) was used as dissolution media and agitated at 60

± 2 rpm in a shaking water bath. Samples (1 mL)

were collected at designated times interval (0.25,

0.5,1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168

hrs) and replaced with same volume of dissolution

media. The samples were then analyzed by HPLC-

UV to determine the release of the drug.

Etoposide loaded PLGA nano formulations

were introduced into tail vein of the mice with the

help of fine needle (27 G) and blood samples (0.2–

0.4 mL) were collected at specified time intervals in

heparinized tubes. Centrifugation was performed at

10,000 rpm for 5 min at 0 ºC in order to separate the

plasma. ACN was used for the de-proteination of

plasma samples and it was prepared according to the

extraction procedure given in section 2.4.1.

Compartmental models were used to

determine different pharmacokinetic (PK)

parameters. These PK parameters includes Time of

Peak concentration (Tmax), Mean Residence time

(MRT), Peak plasma concentration (Cmax), Half-life

(t½), Clearance, AUC max, Area under the curve

(AUC) and AUMC. The PK data was analyzed using

MS Excel 2013, Graph Pad Prism 5 and PK-

Summit® pharmacokinetic software.

Results

The method developed here is rapid, robust,

and easy to automate for the determination of ETP

and PTX in plasma and standard samples using

sorafenib as an IS. Different experimental parameters

and chromatographic conditions were optimized

according to the standard guideline [50].

Optimization of Experimental Conditions

For selection of optimum experimental

conditions all the parameters were optimized within

the specified range. Perfect resolution and peak shape

were obtained with Pruospher ® Star RP- 18e among

the different columns used for this purpose.

Broadening of peaks and over lapping were observed with other columns for both analytes and IS. Various

combinations of organic solvents and trifluroacetic

acid (Methanol: Water (50:50, v/v) Acetonitrile:

Water (pH 3, adjusted with phosphoric acid) (35:65),

ACN: Phosphate Buffer (pH 3) (45:55), ACN: TFA

(0.05%) (50:50, V/V, acetonitrile: trifluroacetic acid

(0.025%), (65:35, V/V)) were tried to get the

optimum mobile phase. Best results in terms of peak

shape and area were obtained with acetonitrile:

trifluroacetic acid (0.025%), (65:35, V/V). With

increase in ratio of ACN in mobile phase, retention time of all the compounds decreased that resulted in

shorter analysis time as shown in Fig. 2. Increase in

the ratio of TFA caused decreasing in peak area and

increase in tailing factor for both the anticancer drugs

(analytes). Best results were obtained at 35:65V/V

ratios of tri-fluroacetic acid and acetonitrile,

respectively, and was used for further analysis.


Fig. 2: A. Effect of mobile phase (ACN: TFA 0.025%) combination on peak shapes and retention times

A=50:50, B=55:45, C=60:40, D=65:35 and E=70:30 v/v. B: Effect of flow rate on retention times and

peak shape, Where A=0.5 mL/min, B=0.8 mL/min, C=1 mL/min, D=1.2 mL/min, E=1.5 mL/min.

Retention time, peak shape and peak area

are greatly affected by the mobile phase flow rate. It

has been noted that increase in the flow rate of

mobile phase leads to the decrease in the retention

time of all the analytes and lessen analysis time as

shown in the Fig. 2. Best peak resolution of the

analytes was determined at increased flow rate but

sensitivity of the method decreased greatly. Best

possible results were determined at flow rate of 1.0

mL/min which was considered as suitable flow rate for the analysis of analytes.

Column oven temperature is an important

parameter and it has a significant effect on the

chromatogram. Its impact on the analytes was studied

in different ranges i.e. from 25 – 45oC. Sensitivity

was observed as high at decrease temperature (25oC)

and vice versa. Both peak shape and peak height

were enhanced with reduction in temperature. Best

peak shape and resolution were obtained at 25oC (Fig.

3).


Fig. 3: A. Effect of column oven temperature on peak shape, Peak area and retention time, 1;25ºC, 2;30ºC, 3;35ºC, 4;40ºC, 5;45ºC. B. Effect of detector wavelength on peak shape and peak height 1; 235 nm, 2;

240 nm, 3; 245 nm, 4; 250 nm, 5; 255nm.

Wave length of maximum absorbance of

ETP and PTX is 286 nm and 227 nm, respectively.

For selection of working wave length of the detector,

solutions of both the analytes were scanned

separately using double beam UV spectrophotometer.

Scan spectra of both the analytes were overlapped

and wave length at which both the analytes exhibited

maximum absorbance was chosen as working wave

length. On the basis of the scan results, 235 nm was

selected as working λ max for the analysis of

Etoposide and paclitaxel.

Best resolution and sensitivity of the peaks

at 235 nm (Fig. 3) confirmed suitability of the

detector wavelength and it was selected as optimum

detector wavelength for further analysis.


Different drugs like sorafenib, Tenifovir,

Atenolol and piroxicam were tried to be used as an

IS. After evaluating all these sorafenib showed better

recovery and resolution and was considered as an

internal standard.

Preparation of Samples

Stock solutions of the analytes and internal

standard were prepared on the basis of their

solubility. Stock solutions of Etoposide, Paclitaxel

and IS (Sorafenib) were prepared using acetonitirle as

a solvent. Concentrations of all stock solutions were

0.1 mg/mL. As per the standard practice the working

solutions were prepared from the previously prepared

stock solution on daily basis with the help of the

mobile phase.

Method Validation

This method of analysis was validated as per

the standard guidelines, laid down for this purpose in

terms of selectivity, linearity, sensitivity, recovery,

precision and robustness.

Specificity and Selectivity

This RP-HPLC UV method of analysis was

highly selective and specific due to the reason that

peaks of ETO, PTX and IS were completely resolved. Separation of peaks was confirmed by using blank

plasma, plasma spiked with ETP, PTX and IS as

shown in Fig. 4. A.

Linearity

Calibration curve of the standard mixtures

and spiked plasma samples were used to determine

the linearity of this method. The Calibration curves

were constructed at seven (07) different

concentration levels in the range of 12–1000 ng/mL

and 14–500 ng/mL for ETO and PTX, respectively, for standard mixture and spiked plasma. Within the

mentioned concentration range, the method was quite

linear. The regression equation and correlation co-

efficient values are presented in Table-3. The overlay

chromatograms of plasma sample spiked with

Etoposide, paclitaxel and sorafenib (IS) are presented

in Fig. 4.

Table-2: Recovery and Precision of the method.

Parameter Analytes

Paclitaxel Mean; %RSD Etoposide Mean; %RSD Recovery

Spiked Conc. (0.4 µg/mL) (97.59); 1.151 (97.79); 0.552

Spiked Conc. (0.2 µg/mL) (98.04); 0.916 (98.17) ; 0.560

Spiked Conc. (0.1 µg/mL) (n = 5) (97.23); 1.208 (98.12); 0.352

Precision

Injection repeatability

Spiked Conc. (0.20 µg/mL) 2621 (Peak area); 0.916 2130 (Peak area); 0.560

Spiked Conc. (0.40 µg/mL) (n = 5) 7.67 (Retention time); 1.044 3.18(Retention time); 0.790

Analysis repeatability

Spiked conc. (0.4 µg/mL) (n = 5) 0.385 (Quantity recovered); 0.189 0.384(Quantity recovered);0.335

Intermediate precision

Intraday reproducibility

Spiked Conc. (0.1 µg/mL) 0.092Quantity recovered); 0.137 0.094 (Quantity recovered); 0.914

Spiked Conc. (0.2 µg/mL) 0.196 (Quantity recovered); 0.295 0.196 (Quantity recovered); 0.898

Spiked Conc. (0.4 µg/mL) (n = 5) 0.384 (Quantity recovered); 0.189 0.385(Quantity recovered); 0.304

Inter day reproducibility



Spiked Conc. (0.4 µg/mL) (n = 5) 0.388 (Quantity recovered); 0.251 0.383 (Quantity recovered); 0.870

Table-3: Calibration Range, Linearity, and Sensitivity of the Method.

Parameters Analytes

Etoposide Paclitaxel

Calibration range (ng/mL) 12- 1000 14-500

Linearity

Standard mixture

Regression equation y = 0.010x + 1.342 y = 0.019x + 0.544

Correlation co efficient 0.999 0.998

Spiked plasma samples

Regression equation y = 0.018x+0.592 y = 0.013x + 0.517

Correlation co efficient 0.999 0.998

Sensitivity

Limit of detection (LOD) ng/mL 5 6

Lower limit of quantification (LLOQ) ng/mL 12 14


Fig. 4: A. Chromatograms of spiked samples of Paclitaxel, Etoposide and IS. B. Overlay chromatograms of

plasma sample spiked with Etoposide, Paclitaxel and Sorafenib (IS).

Accuracy of the Method

Accuracy of this suggested method was

determined by % recovery from plasma. It was determined at 03 concentration levels (0.1µg/mL,

0.2µg/mL and 0.4µg/mL) of both analytes as shown

in Table-2.

Precision of the Method

Precision of the method was evaluated in

terms of injection and analysis repeatability.

Intermediate precision was determined by of intra-

day and inter-day study. Results of analysis

repeatability, inter day precision and intraday

precision are presented in Table-2. There is complete

harmony among repeated injections, repeated

analysis, inter day and intraday study.

Stability of Solutions

Stability study was conducted at room

temperature (18–23oC), 4 ºC and freezing

temperature (-20oC) for one week. All the samples were stable at 4ºC and –20oC for one

week. At room temperature both the analytes

(etoposide and paclitaxel) degraded to a

significant level which showed that all the samples, to be analysed, must be stored at 4ºC so

as to avoid any chances of instability and to get

maximum results. Sensitivity (LOD and LOQ)

Limit of detection (LOD) and limit of

quantification ( LOQ) of etoposide and paclitaxel are

mentioned in Table-3. These values proved that the

said method of analysis is more sensitive then the

already published methods for these two compounds.

The respective chromatograms of LOD and LOQ of

ETO and PTX are presented in Fig. 5 A.


Fig. 5: A. (A) Limit of detection (LOD) and (B) limit of quantification (LOQ) of Paclitaxel and Etoposide. B.

Pharmacokinetics studies of Etoposide and Paclitaxel.

Formulation Evaluation

Two nano-formulations i.e., EP 3 and EP 7 in

all these formulations have the required particle size

(177 ± 1.5nm, 136 ± 1.52 nm), zeta potential (-10 ±

0.30, -11 ± 1.46), PDI (0.3 ± 0.02, 0.221 ± 0.09),

entrapment efficiency (96 %, 88%), drug loading (2.94

mg, 1.98 mg) respectively with round morphology.

These nanoformulations showed good in-vitro and

pharmacokinetic profile (Fig. 5 and 6) as well as in-vitro

release as compared to the previous results.

Application of the Method

In the present study several polymeric

nanoformulations of anticancer drug etoposide were

prepared which the help of a polymer (PLGA) and

stabilizing agents (poloxamer-407) which were also

evaluated by in-vitro and in-vivo means and this method

was an integral part of this research project. This

method was successfully applied for the in-vitro and in-

vivo evaluation (Fig. 6, Table 4 and 6) of nano-

pharmaceutical formulations (optimized) of etoposide in

animal model (mice) as well as for the conventional

dosage forms in animals and human plasma. Different

in-vitro release techniques are usually employed, during

the process of formulation development to select a

suitable formulation that can provide adequate

therapeutic activity. Structural characteristics of the

polymeric material can easily be understood on the basis

of in-vitro studies and drug release behavior from it. The method developed during this research project was

successfully applied for in-vitro evaluation and

pharmacokinetic determination of etoposide in animal

model after I/V administration (Fig. 6, Table-4 and 6) to

Balb C mice. The drug content of each sample was

determined with the help of RP-HPLC-UV and PK

parameters were determined while applying

pharmacokinetics software PK Summit®


Table-4: Plasma Pharmacokinetic Parameters of drugs after I/V Administration. Plasma Pharmacokinetic Parameter after intravenous

administration of Paclitaxel solution in Rabbits (n=05) Plasma Pharmacokinetic Parameter after intravenous administration of

Etoposide solution in Mices (n=05) Parameters Value Parameters Value

A (mg/l) 6.4 A (mg/l) 18.5

α (1/hr) 0.954 α (1/hr) 1.501

B (mg/l) 2.749 B (mg/l) 18.5

β (1/hr) 2.196 β (1/hr) 3.162

Vc (l/kg) 2.6 Vc (l/kg) 2.1

t1/2α (hr) 5.355 t1/2α (hr) 1.705

t1/2β (hr) 4.038 t1/2β (hr) 1.026

K21 (1/hr) 0.138 K21 (1/hr) 0.152

K10 (1/hr) 0.161 K10 (1/hr) 0.112

K12 (1/hr) 0.21 K12 (1/hr) 0.08

AUC [(mg/l).hr] 6.6 AUC [(mg/l).hr] 5.7

CLs[(mg/kg)/h/(mg/l)] 0.44 CLs[(mg/kg)/h/(mg/l)] 1.821

MRT (hr) 5.3 MRT (hr) 4.2

Table-5: Process parameters for Etoposide loaded polymeric nanoparticles. Code Drug PLGA Poloxamer-407 (20 mL) Sonication speed

(%)

Mean size

(nm)

PDI Zeta potential

(mV)

Encapsulation efficiency

(%)

EP 1 1 mg 15 mg 0.25% 99 % 121±3.60 0.16±0.02 -11±1.15 82

EP 2 2 mg 15 mg 0.25% 99 % 134±1.15 0.14±0.03 -10±0.55 55

EP 3 3 mg 15 mg 0.25% 99 % 177±1.5 0.3±0.02 -10±0.30 96

EP 4 4 mg 15 mg 0.25% 99 % 178±4.0 0.10±0.05 -6±0.7 31

EP 5 5 mg 15 mg 0.25% 99 % 212±2.51 0.09±0.02 -3±1.5 38

EP 6 1 mg 15 mg 0.5% 99 % 182±1.5 0.08±0.03 -12±0.5 72

EP 7 2 mg 15 mg 0.5% 99 % 136±1.52 0.22±0.09 -11±1.46 88

EP 8 3 mg 15 mg 0.5% 99 % 162±1.15 0.11±0.02 -9±0.11 82

EP 9 4 mg 15 mg 0.5% 99 % 160±1.15 0.33±0.02 -6±0.57 44

EP 10 5 mg 15 mg 0.5% 99 % 197±1.52 0.10±0.04 -4±0.5 28

Table-6: Plasma Pharmacokinetic Parameters after Intravenous

Administration of Etoposide Nano-formulations in Mice (n=05)

Parameters Plasma

EP 3 EP 7

E Half-life (hr) 55.49 57.32

Cmax (obs)µg/mL 65.10 67.89

AUC(0-t) (obs area)µg-hr/mL 1469.28 1563.07

AUMC∞ (area)µg-hr*hr/mL 42900 44776

MRT (expo)hr 122.40 126.30

Vd (obs area)mL 184.16 217.57

CL (area)mL/hr 2.89 2.60

Fig. 6: In-Vivo Pharmacokinetics and In-Vitro Release profile of Etoposide EP 3 and EP 7.

Table-7: Various mobile phase combinations used in this research study for the determination of analytes. S.No Aqueous Phase(%V) Organic Phase (%V)


1 Water (50%) Methanol (50%)

2 Water pH 3 adjusted with phosphoric acid (35%) Acetonitrile (65%)

3 Phosphate Buffer pH 3 (55%) Acetonitrile (45%)

4 Water TFA 0.005% (50%) Acetonitrile (50%)

5 Water TFA 0.025% (40%) Acetonitrile (60%)

Conclusion

The present method of analysis was

developed and validated as per the standard

international guidelines. Several experimental

parameters were optimized for determination of

sorafenib and paclitaxel in spiked human plasma and

in plasma of Rabbits/Mice. The extraction procedure

for biological samples was simple as mobile phase was used for this purpose. All the analytes were well

separated from each other and mobile phase was

simple and easy to prepare. The method will be

applied for in-vitro and in-vivo analysis of polymeric

nanoparticles with slight modification.

Acknowledgement

We are thankful to Directorate of Science

and Technology (DoST) and University of Peshawar

for providing research facilities to carry out the study.

References

1. M. L. Slevin, The clinical pharmacology of

etoposide, Cancer., 67, 319 (1991).

2. K. Hande, Etoposide: four decades of

development of a topoisomerase II inhibitor,

Eur. J. Cancer., 34, 1514 (1998).

3. T. Stumpp, E. Sasso‐Cerri, E. Freymüller and

S. M. Miraglia, Apoptosis and testicular

alterations in albino rats treated with etoposide

during the prepubertal phase, Anat Rec A Discov

Mol Cell Evol Biol., 279, 611 (2004).

4. C. Kollmannsberger, J. Beyer, J. Droz, A. Harstrick, J. Hartmann, P. Biron, A. Flechon, P.

Schöffski, M. Kuczyk and H. Schmoll,

Secondary leukemia following high cumulative

doses of etoposide in patients treated for

advanced germ cell tumors, J. Clin. Oncol., 16,

3386 (1998).

5. S. RamaChandran and H. Ariffin, Secondary

acute myeloid leukemia after etoposide therapy

for haemophagocytic lymphohistiocytosis,

Pediatr. Blood Cancer., 53, 488 (2009).

6. S. Ezoe, Secondary leukemia associated with the anti-cancer agent, etoposide, a topoisomerase II

inhibitor, Int. J. Environ. Res. Publ. Health., 9,

2444 (2012).

7. D. E. Thurston, Chemistry and pharmacology of

anticancer drugs, CRC press., (2006).

8. D. G. Kingston, The chemistry of taxol,

Pharmacol. Ther., 52, 1 (1991).

9. S. G. Arbuck, M. Christian, J. Fisherman, L.

Cazenave, G. Sarosy, M. Suffness, J. Adams, R.

Canetta, K. Cole and M. Friedman, Clinical

development of Taxol, J Natl Cancer Inst

Monogr., 15, 11 (1993).

10. E. K. Rowinsky, N. Onetto, R. M. Canetta and S.

G. Arbuck. Taxol: the first of the taxanes, an important new class of antitumor agents, Semi

oncol., 19, 646 (1992).

11. C.-L. Chen, K. K. Thoen and F. M. Uckun,

High-performance liquid chromatographic

methods for the determination of topoisomerase

II inhibitors, Journal of Chromatography B:

Biomedical Sciences and Applications., 764, 81

(2001).

12. T.-H. Tsai, Analytical approaches for traditional

Chinese medicines exhibiting antineoplastic

activity, J Chromatogr B Biomed Sci Appl., 764, 27 (2001).

13. S. Pang, N. Zheng, C. A. Felix, J. Scavuzzo, R.

Boston and I. A. Blair, Simultaneous

determination of etoposide and its catechol

metabolite in the plasma of pediatric patients by

liquid chromatography/tandem mass

spectrometry, J. Mass Spectrom., 36, 771 (2001).

14. H. Chen, S. Shi, M. Zhao, L. Zhang, H. He and

X. Tang, A lyophilized etoposide submicron

emulsion with a high drug loading for

intravenous injection: preparation, evaluation,

and pharmacokinetics in rats, Drug Dev. Ind. Pharm., 36, 1444 (2010).

15. A. Sistla, D. J. Smith, N. L. Kobrinsky and K.

Kumar, Pharmacokinetics and tissue distribution

of liposomal etoposide in rats, Drug deliv., 16,

423 (2009).

16. M. Krogh-Madsen, S. H. Hansen and P. H.

Honoré, Simultaneous determination of cytosine

arabinoside, daunorubicin and etoposide in

human plasma, J. chromatogr B., 878, 1967,

(2010).

17. T. Zhang, Q. Shen and W. Pan, LC Determination of the intestinal absorption of

etoposide in vitro and in rat plasma,

Chromatographia., 71, 993 (2010).

18. R. Strife, L. Jardine and M. Colvin, Analysis of

the anticancer drugs etoposide (VP 16-213) and

teniposide (VM 26) by high-performance liquid

chromatography with fluorescence detection,


J Chromatogr B Biomed Sci Appl., 224, 168

(1981).

19. B. Floor, A. Klein, N. Muhammad and D. Ross,

Stability‐indicating liquid chromatographic

determination of etoposide and benzyl alcohol in

injectable formulations, J. Pharm. Sci., 74, 197

(1985). 20. M. Van Opstal, J. Blauw, J. Holthuis, W. Van

Bennekom and A. Bult, On-line electrochemical

derivatization combined with diode-array

detection in flow-injection analysis: Rapid

Determination of Etoposide and Teniposide in

Blood Plasma, Anal. Chem. Acta., 202, 35

(1987).

21. P. Canal, C. Michel, R. Bugat, G. Soula and M.

Carton, Quantification of teniposide in human

serum by high-performance liquid

chromatography with electrochemical detection, J Chromatogr B Biomed Sci Appl., 375, 451

(1986).

22. T. Littlewood, A. Hutchings, D. Bentley and B.

Spragg, High-performance liquid

chromatographic determination of etoposide in

plasma using electrochemical detection,


(1984).

23. G. Duncan, R. Farmen, H. Movahhed and K.

Pittman, High-performance liquid

chromatographic method for the determination of

etoposide in plasma using electrochemical detection, J Chromatogr B Biomed Sci Appl.,

380, 357 (1986).

24. M. Suno, T. Ono, S. Iida, N. Umetsu, K.-i.

Ohtaki, T. Yamada, T. Awaya, M. Satomi, Y.

Tasaki and K. Shimizu, Improved high-

performance liquid chromatographic detection of

paclitaxel in patient's plasma using solid-phase

extraction, and semi-micro-bore C 18 separation

and UV detection, J. chromatogr B., 860, 141

(2007).

25. J. Rizzo, C. Riley, D. von Hoff, J. Kuhn, J. Phillips and T. Brown, Analysis of anticancer

drugs in biological fluids: determination of taxol

with application to clinical pharmacokinetics, J

Pharm Biomed Anal., 8, 159 (1990).

26. R. Mader, B. Rizovski and G. Steger, On-line

solid-phase extraction and determination of

paclitaxel in human plasma, J. chromatogr B,

769., 357 (2002).

27. S.-H. Lee, S. D. Yoo and K.-H. Lee, Rapid and

sensitive determination of paclitaxel in mouse

plasma by high-performance liquid

chromatography, J Chromatogr B Biomed Sci Appl., 724, 357 (1999).

28. S. C. Kim, J. Yu, J. W. Lee, E.-S. Park and S.-C.

Chi, Sensitive HPLC method for quantitation of

paclitaxel (Genexol®) in biological samples with

application to preclinical pharmacokinetics and

biodistribution, J Pharm Biomed Anal., 39, 170 (2005).

29. A. El-Yazigi and A. Yusuf, Expedient liquid

chromatographic assay for paclitaxel in plasma

after its administration to cancer patients, Ther

Drug Monit., 17, 511 (1995).

30. M. Andreeva, P. D. Niedmann, L. Binder, V. W.

Armstrong, H. Meden, M. Binder and M.

Oellerich, A simple and reliable reverse-phase

high-performance liquid chromatographic

procedure for determination of paclitaxel (taxol)

in human serum, Ther Drug Monit., 19, 327

(1997). 31. F. Coudoré, N. Authier, D. Guillaume, A. Beal,

E. Duroux and J. Fialip, High-performance

liquid chromatographic determination of

paclitaxel in rat serum: application to a

toxicokinetic study, J Chromatogr B Biomed Sci

Appl., 721, 317 (1999).

32. M. Huizing, H. Rosing, F. Koopman, A. Keung,

H. Pinedo and J. Beijnen, High-performance

liquid chromatographic procedures for the

quantitative determination of paclitaxel (Taxol)

in human urine, J Chromatogr B Biomed Sci Appl., 664, 373 (1995).

33. S. Vasantha Kumar, S. Srinath and R. N. Saha, A

Simple and Rapid 3D View Method for Selective

and Sensitive Determination of Paclitaxel in

Micro Volume Rat Plasma by LC-Diode Array

UV and Its Application to a Pharmacokinetic

Study, J Chromatogr Sci,, 50, 259 (2012).

34. J. Leslie, J. Kujawa, N. Eddington, M. Egorin

and J. Eiseman, Stability problems with taxol in

mouse plasma during analysis by liquid

chromatography, J Pharm Biomed Anal., 11,

1349 (1993). 35. S. M. Longnecker, R. C. Donehower, A. E.

Cates, T.-L. Chen, R. B. Brundrett, L. B.

Grochow, D. S. Ettinger and M. Colvin, High-

performance liquid chromatographic assay for

taxol in human plasma and urine and

pharmacokinetics in a phase I trial, Cancer Treat

Rep., 71, 53 (1987).

36. N. Martin, J. Catalin, M. Blachon and A.

Durand, Assay of paclitaxel (Taxol) in plasma

and urine by high-performance liquid

chromatography, J Chromatogr B Biomed Sci Appl., 709, 281 (1998).

37. D. Song and J. L.-S. Au, Isocratic high-

performance liquid chromatographic assay of

taxol in biological fluids and tissues using


automated column switching, J Chromatogr B

Biomed Sci Appl., 663,3 37 (1995).

38. L. Wang, P. Ho, H. Lee, H. Vaddi, Y. Chan and

C. S. Yung, Quantitation of paclitaxel in micro-

sample rat plasma by a sensitive reversed-phase HPLC assay, J Pharm Biomed Anal., 31, 283

(2003).

39. H. Yonemoto, S. Ogino, M. N. Nakashima, M.

Wada and K. Nakashima, Determination of

paclitaxel in human and rat blood samples after

administration of low dose paclitaxel by

HPLC‐UV detection, Biomed. Chromatogr.,

21, 310 (2007).

40. Y. Wei, Z. Xue, Y. Ye, P. Wang, Y. Huang and

L. Zhao, Pharmacokinetic and tissue distribution

of paclitaxel in rabbits assayed by LC‐UV after

intravenous administration of its novel liposomal

formulation, Biomed. Chromatogr., 28, 204

(2014).

41. S. Bermingham, R. O'Connor, F. Regan and G. P. McMahon, Simultaneous determination of

anthracyclines and taxanes in human serum

using online sample extraction coupled to high

performance liquid chromatography with UV

detection, J. Sep. Sci., 33, 1571 (2010).

42. L. Z. López, A. A. Pastor, J. M. A. Beitia, J. A.

Velilla and J. G. Deiró, Determination of

docetaxel and Paclitaxel in human plasma by

high-performance liquid chromatography:

validation and application to clinical

pharmacokinetic studies, Ther Drug Monit., 28,

199 (2006). 43. A. Andersen, D. J. Warren, P. F. Brunsvig, S.

Aamdal, G. B. Kristensen and H. Olsen, High

sensitivity assays for docetaxel and paclitaxel in

plasma using solid-phase extraction and high-

performance liquid chromatography with UV

detection, BMC Pharmacol Toxicol., 6, 2 (2006).

44. A. Sparreboom, O. van Tellingen, W. J. Noojien

and J. H. Beijnen, Determination of paclitaxel

and metabolites in mouse plasma, tissues, urine

and faeces by semi-automated reversed-phase

high-performance liquid chromatography,


(1995).

45. M. B. Garg and S. P. Ackland, Simple and

sensitive high-performance liquid

chromatography method for the determination of docetaxel in human plasma or urine,


(2000).

46. L. Yan, J. YanYan, C. MinChun, Y. Jing, S.

Ying, L. ChengTao, G. Jie, L. CaiYang, Z.

ZhenXing and W. AiDong, High-performance

liquid chromatographic analysis of felotaxel, a

novel anti-cancer drug, in rat plasma and in

human plasma and urine, J Chromatogr Sci., 51,

292 (2013).

47. R. A. Parise, R. K. Ramanathan, W. C. Zamboni

and M. J. Egorin, Sensitive liquid chromatography–mass spectrometry assay for

quantitation of docetaxel and paclitaxel in human

plasma, J. chromatogr B, 783, 231 (2003).

48. P. Guo, J. Ma, S. Li and J. M. Gallo,

Determination of paclitaxel in mouse plasma and

brain tissue by liquid chromatography–mass

spectrometry, J. chromatogr B., 798, 79 (2003).

49. L. D. Vainchtein, B. Thijssen, E. Stokvis, H.

Rosing, J. H. Schellens and J. H. Beijnen, A

simple and sensitive assay for the quantitative

analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem

mass spectrometry, Biomed. Chromatogr., 20,

139 (2006).

50. N. Epshtein, Validation of HPLC techniques for

pharmaceutical analysis, Pharm Chem J., 38,

212 (2004).

51. A. Khan, M. I. Khan, Z. Iqbal, Y. Shah, L.

Ahmad and D. G. Watson, An optimized and

validated RP-HPLC/UV detection method for

simultaneous determination of all-trans-Retinol

(Vitamin A) and α-Tocopherol (Vitamin E) in

human serum: Comparison of different particulate reversed-phase HPLC columns, J.

chromatogr B., 878, 2339 (2010)

Simultaneous Determination of Etoposide and Paclitaxel in ... · [email protected]* (Received on 24th May 2018, accepted in revised form 21st December 2018) Summary: A simple,

Documents