A Simple
Ultraviolet Spectrophotometric Method for the Estimation of Docetaxel
in Bulk Drug and Formulation
Mahawar Sheetal*
Department of Pharmacy, Shri Jagdish Prasad Jhabarmal Tibrewala University, Vidyanagari, Jhunjhunu,
Rajasthan, India
*Corresponding Author E-mail: sheetal.mpharmndds@gmail.com
The present investigation aimed at development and validation
of an analytical method for the estimation of Docetaxel
based on Ultra-violet Spectrophotometry. The reported methods of analysis
include the chromatographic methods such as HPLC (1-2), RP-HPLC (3-4), LC-MS/MS
(18) and HPLC/MS in plasma (5-7). An ion-pair extractive spectrophotometric
method has been reported in the literature for the determination of a docetaxel (8). Chromatographic methods are complex,
tedious, time consuming and are not suitable for routine analysis. However,
Ultraviolet spectrophotometric methods which are simple and fast are ideal for
routine analysis. Docetaxel exhibits a sharp peak at
229nm when scanned in the Ultraviolet region. Thus, it was selected as the
analytical wave-length. The developed method was found to be simple, sensitive,
accurate, precise and specific as evidenced by non-interference from the excipients present in the marketed injection. Linearity was
observed in the range of 5μg/ml to 45μg/ml and Accuracy was found
between 99.36 to 101.75%. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.174μ/ml &
0.194μ/ml respectively.
KEYWORDS: Docetaxel,
Validation, UV spectroscopic method
INTRODUCTION:
Docetaxel is a semi-synthetic drug, belonging to taxoid family, derived from precursor extracted from the
renewable needle biomass of the European yew tree, Taxus
baccata. Docetaxel, which
is chemically (2R,3S)-N-carboxy-3-phenylisoserine,N-tert-butyl ester,
13-ester with
5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one
4-acetate 2-benzoate, trihydrate, an anti neoplastic agent most commonly used in the treatment of
breast, ovarian, prostate, and non-small cell lung cancer (9). The molecular
empirical formula for Docetaxel is C43H53NO14.3H2O
(10). The structure of Docetaxel is shown in Figure
1. It is highly lipophilic and practically insoluble
in water, soluble in ethanol, methanol,
chloroform, acetone, acetonitrile (10).
It
is available in the market as an injection to be administered intravenously. Docetaxel is official in USP. The methods of estimation of Docetaxel in bulk drug and the formulation include the
chromatographic methods using UV detector (9). Other reported methods of
analysis are reverse phase (3-4) and ion pair (5) HPLC methods. So far, no
Ultraviolet spectrophotometric method for estimation of Docetaxel
is reported. Ultraviolet spectrophotometric methods are simpler and faster
compared to the chromatographic methods. Docetaxel in
acetonitrile exhibits a sharp peak at 229 nm when
scanned in the UV region between 200-400 nm and hence it was selected as the
analytical wave-length. Thus, the aim of the present investigation was to
develop a reliable spectrophotometric procedure for estimation of docetaxel in bulk and marketed formulation.
Docetaxel was obtained as a gift sample from RPG Life Sciences,
Mumbai, India. Acetonitrile AR Grade was obtained
from Fischer
Scientific, Mumbai, India. Docetaxel injection
IP (20 mg) (marketed formulation) of docetaxel was
purchased from a local retail pharmacy.
Figure
1 Structure of Docetaxel
Equipment
Digital analytical balance
(Shimadzu SCS, Switzerland), Spectrophotometric measurements were made on a
Shimadzu 1700 double beam UV-VIS spectrophotometer with a fix slit width of 1nm
coupled with computer loaded with Shimadzu UV PC software (UV probe) version
2.31.
Methods
Determination λmax
The
absorption maximum (λmax) of docetaxel
was determined by scanning 10 µg/ml solution against Acetonitrile
as reagent blank in spectrum mode between 200 to 400nm.
Preparation of Stock
Solution
Primary
stock solution of docetaxel in acetonitrile
(100µg/ml) was prepared by dissolving 5mg docetaxel
in 50ml acetonitrile. The primary stock solution was
stored at 2-8şC.
Preparation of
Calibration Curve in Acetonitrile
Appropriate
aliquots of the stock solution of docetaxel
(100µg/ml) were transferred to 10ml volumetric flasks and were diluted up to
the mark with acetonitrile. The absorbance of all the
prepared solutions (5, 10, 15, 20, 25, 30, 35, 40 and 45 µg/ml) was then
measured at the absorbance maxima, 229nm against the reagent blank (acetonitrile). The readings were recorded in triplicate.
Mean value (n=3) along with the standard deviation (SD) are recorded in Table
1. The regressed values of absorption were plotted graphically against the
concentrations, as shown in Figure 2. Stability of the solutions of docetaxel in acetonitrile was
ascertained by observing the changes in the absorbance of the solution at the
analytical wavelength, over a period of 24 hours, at room temperature. The
readings were recorded in triplicate.
Analytical Method Validation
Linearity
The linearity of an analytical method is its ability to
elicit, test results that are directly or a well defined mathematical
transformation proportional to the concentration of analytes
in samples within a given range (11). Beer’s law states that absorbance is
proportional to the concentration of the absorbing species. A calibration curve is prepared by
plotting a dependent variable (absorbance Y) as a function of an independent
variable (concentration X). This relation if found with a series of
measurements, which in practice, is often a linear one.
Y= mX +C
Where, m is the slope of the line and C is the
intercept on the Y axis (11).
Linearity of an analytical method for docetaxel in acetonitrile was
established by the regression coefficient.
Accuracy
Accuracy of an analytical
method is the closeness of the test results obtained by that method to true
value (12). Accuracy, sometimes also referred to as recovery is an indicator of
the trueness of test measurements. To determine the accuracy of the method
three quality control samples (10μg/ml, 20μg/ml and 30μg/ml) for
acetonitrile were used. The samples chosen were such
to represent the entire range of the standard curve i.e
lower, middle and higher concentrations of the range. Accuracy was calculated
by analysis of 3 replicate samples for the above described methods. The
observed concentrations of the drug were then back calculated using the
equation of standard calibration curve and compared with actual concentrations
(13-14). The accuracy results for the UV method are listed in table 3. The
contents of the vial of the marketed formulation containing 20 mg of Docetaxel were transferred to 100 ml volumetric flask and
were dissolved in the acetonitrile. A measured volume
of this solution was mixed with standard docetaxel
solution and diluted suitably to get the total docetaxel
concentrations of 10, 20 and 30 μg/ml
respectively. The absorbance of the solutions was measured at 229 nm using acetonitrile as blank. The study was performed in three
replicates and the mean was found at each concentration level.
Precision
Precision
of an analytical method is the degree of agreement among the individual test
results when the procedure is applied repeatedly to multiple scanning of
homogenous sample (12). Precision may be measure of either degree of
reproducibility or of repeatability of the analytical method under normal
operating conditions. The precision of an analytical method is usually
expressed as the standard deviation or confidence limit. The standard deviation
is calculated from the following formula (11).
SD = [
-1]1/2
Where
X = an individual measurement in the set, x=arithmetic mean of the set
n=
total number of replicated measurement taken in set
Precision
between different samples can be compared with RSD as follows:
% RSD = [SD/Mean]*100
For
current assay validation, within day precision and day to day precision were
used. Repeatability was determined
by using different levels of drug concentrations (5-45µg/ml) for acetonitrile prepared from independent stock solutions and
analyzed. Inter-day and intra-day variation was studied to determine precision
of the analytical methods. Different levels of drug concentrations in
triplicates were prepared three different times in a day and studied for
intra-day variation. Same procedure was
followed for three different days to study inter-day variation. The percent relative standard deviation
(%RSD) of the predicted concentrations from the regression equation was taken
as precision (13-14). The precision results for the UV method are listed in
table 4.
Limit of Detection and Limit of
Quantification:
The
Limit of Detection (LoD) is a quantitative parameter.
It is the lowest concentration of the analyte in a
sample that can be detected with acceptable precision and accuracy under stated
experimental conditions (12). It is expressed as the concentration of the analyte in the sample. The limit is expressed in terms of
µg/ml, ng/ml, pg/ml. etc. LoD
values are specific for a particular set of experimental conditions. Anything
that changes the sensitivity of a method, including instrument, sample
preparation etc. will change the detection limits.
Limit
of Quantification (LoQ) is the lowest concentration
of the analyte in a sample that may be measured in a
sample matrix such as impurities in bulk drug substances and degradation
products in finished pharmaceuticals. The value of LoQ
is almost 10 times higher than that of the blank (11). The LOD and LOQ of docetaxel were determined using calibration standards. LOD
and LOQ were calculated as 3.3 σ/S and 10 σ/S,
respectively, where S is the slope of the calibration curve and σ
is the standard deviation of y-intercept of regression equation (n=9)
(13-14).
RESULTS AND DISCUSSION:
Determination of λmax
Based
on the spectrophotometric scanning of docetaxel
(10µg/ml), the maxima was obtained at 229nm in acetonitrile, hence chosen as the analytical wavelength.
Calibration Curve of Docetaxel
Table 1 shows the mean absorbance values along with the
standard deviation of docetaxel in acetonitrile. The high correlation coefficient in the acetonitrile indicated that absorbance and concentration of
the drug was linearly related. Beer’s law was found to be obeyed in the range
of 5 to 45µg/ml in acetonitrile.
|
Table 1: Absorbance of Docetaxel in Acetonitrile
|
|
Concentration
(µg/ml)
|
Absorbance
± S.D.*
|
Absorbance ± S.D.*
(after 24 hours)
|
|
5
|
0.113
± 0.003
|
0.110 ± 0.013
|
|
10
|
0.221
± 0.002
|
0.208 ± 0.018
|
|
15
|
0.324
± 0.013
|
0.306 ± 0.020
|
|
20
|
0.433
± 0.023
|
0.416 ± 0.020
|
|
25
|
0.530
± 0.019
|
0.508 ± 0.015
|
|
30
|
0.645
± 0.009
|
0.597 ± 0.012
|
|
35
|
0.749
± 0.008
|
0.709 ± 0.009
|
|
40
|
0.851
± 0.011
|
0.799 ± 0.010
|
|
45
|
0.960
± 0.016
|
0.895 ± 0.020
|
|
*Mean ± SD (n =3)
|
Figure 2 Calibration curve of docetaxel
Analytical method validation
Linearity of the Assay
The linearity of the assay was determined by plotting
the standard calibration curves for the concentration range 5-45µg/ml at 229nm
in acetonitrile for 3 consecutive days as shown in table 2.
|
Table 2 Linearity of developed
analytical method
|
|
Solvent
|
λ max (nm)
|
Range
|
Regression Equation
|
Correlation Coefficient (R2)
|
|
Acetonitrile
|
229
|
5-45 µg/ml
|
y= 0.019x + 0.009
|
0.999
|
Accuracy
The
excellent mean % Accuracy values, close to 100%, and their low standard
deviation values (SD < 1.0) represent high accuracy of the analytical
methods (13-14).
The mean % accuracy for
lower (10µg/ml), intermediate (20µg/ml), and higher concentrations (30µg/ml),
were found to be 99.36 (0.568), 101.75 (0.548) and 99.76 (0.268), respectively
in acetonitrile. Thus, the accuracy of the developed
method of docetaxel in acetonitrile
was found between 99.36 to 101.75%.
|
Table 3 Accuracy of the
developed method in Acetonitrile
|
|
Concentration (µg/ml)
|
Calculated concentration
(µg/ml) (± SD)*
|
% Accuracy
(± SD)*
|
|
10
|
9.936 ± 0.046
|
99.36 ± 0.568
|
|
20
|
20.351 ± 0.089
|
101.75 ± 0.548
|
|
30
|
29.930 ± 0.066
|
99.76 ± 0.268
|
*Mean
± SD (n =3)
Above mentioned revealed that any small change in the
drug concentration in the solutions could be accurately determined by the
proposed analytical methods. The results suggest that the methods were very
accurate.
Precision
Precision was determined by
studying the repeatability and intermediate precision. The intra- and inter-
day precision of the method are recorded in the Table 4.
In precision study, %RSD
values were not more than 2.0% in all the cases. RSD values found for the
analytical methods were well within the acceptable range indicating that these
methods have excellent precision. Precision of the method to estimate docetaxel in presence of the excipients
present in the injection was ascertained by performing the assay of the
commercially available injection. Briefly, the contents of the vial of the
marketed formulation were dissolved in 100 ml acetonitrile.
Further, 1.0 ml of the solution was diluted to 100 ml with acetonitrile
and absorbance was measured at 229 nm using acetonitrile
as blank. The amount of docetaxel present in the
formulation was calculated by using the equation generated by the linearity
studies as shown in table 5. The above procedure was repeated six times and the
standard deviation was calculated for determining intraday precision. The same
procedure was repeated six times each on two separate days to determine the
interday precision.
LOD and LOQ
The
LOD and LOQ of docetaxel were determined using
calibration standards. LOD and LOQ were calculated as 3.3 σ/S and
10 σ/S, respectively, where S is the slope of the
calibration curve and σ is the standard deviation of y-intercept
of regression equation (n=9).
|
Table 4 Intra day and Inter
day precision for docetaxel determination in Acetonitrile
|
|
Conc. (µg/ml)
|
Intra day Absorbance
|
Inter day Absorbance
|
|
Mean ± SD*
|
% RSD
|
Mean ± SD*
|
% RSD
|
|
05
|
0.112±0.0008
|
0.729
|
0.112±0.0008
|
0.729
|
|
10
|
0.204±0.0036
|
1.767
|
0.202±0.0040
|
1.722
|
|
15
|
0.310±0.0035
|
1.134
|
0.302±0.0075
|
1.325
|
|
20
|
0.413±0.0025
|
0.609
|
0.409±0.0065
|
1.837
|
|
25
|
0.508±0.0055
|
1.085
|
0.501±0.0072
|
1.298
|
|
30
|
0.595±0.0044
|
0.733
|
0.589±0.0040
|
1.224
|
|
35
|
0.710±0.0050
|
0.709
|
0.705±0.0056
|
0.574
|
|
40
|
0.795±0.0069
|
0.871
|
0.793±0.0055
|
0.702
|
|
45
|
0.896±0.0056
|
0.621
|
0.889±0.0051
|
0.577
|
*Mean ± SD (n =3)
|
Table 5. Results
of the intra day and inter day precision
|
|
Set No.
|
Assay (% of labeled
amount)
|
|
Day 1
|
Day 2
|
Day 3
|
|
1
|
99.78
|
99.45
|
98.23
|
|
2
|
99.86
|
99.56
|
99.41
|
|
3
|
99.75
|
99.25
|
99.23
|
|
4
|
99.35
|
98.31
|
99.64
|
|
5
|
100.32
|
100.54
|
98.67
|
|
6
|
101.33
|
98.54
|
100.25
|
|
Mean
|
100.06
|
99.27
|
99.23
|
|
Standard deviation
|
0.69
|
0.80
|
0.71
|
CONCLUSION:
The UV-visible spectrum
obtained by scanning the 10μg/ml of docetaxel
recorded between 200 nm to 400 nm. It was observed that docetaxel
shows the characteristic peak at 229nm and thus it was selected as the
analytical wavelength. Analytical method for docetaxel
was developed and validated by UV spectrophotometrically in acetonitrile.
The high correlation coefficient in the above solvent indicated that absorbance
and concentration of the drug was linearly related. Beer’s law was found to be
obeyed in the range of 5 to 45µg/ml for docetaxel in acetonitrile.
There is negligible
difference in the absorbance values of the fresh and the stored solutions
indicating that docetaxel is stable over the period
of analysis. The accuracy of the developed method of docetaxel
in acetonitrile was found to close to 100%, between
98.00 to 102.00%. In precision study, %RSD values were not more than 2.0% in
all the cases. RSD values found for the analytical methods were well within the
acceptable range indicating that these methods have excellent precision. The LoD and LoQ for the assay of Docetaxel using Acetonitrile is
0.174 and 0.194 respectively indicating that the method is sensitive. It is
also selective as evidenced by the noninterference of the excipients
used in the formulation of the injection. The developed spectrophotometric
methods for determination of Docetaxel are simple,
specific, accurate, precise, rapid and economical which indicates its adequacy
for routine pharmaceutical analysis. It is concluded that the developed
spectrophotometric method can be successfully utilized for the routine
estimation of Docetaxel in bulk as well as
formulation.
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