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.
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 875
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).
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 876
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
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 877
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:
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 878
(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.
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 879
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).
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 880
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.
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 881
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. (0.1 µg/mL) 0.095 (Quantity recovered); 0.450 0.094 (Quantity recovered); 1.01
Spiked Conc. (0.2 µg/mL) 0.189 (Quantity recovered); 0.367 0.191 (Quantity recovered); 0.956
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
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 882
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.
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 883
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®
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 884
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)
Muhammad Hassan et al., J.Chem.Soc.Pak., Vol. 41, No. 05, 2019 885
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.
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