Simultaneous spectrophotometric
determination of Tipiracil and Trifluridine
Rangisetty Spandana Yasaswini, Mukthinuthalapati
Mathrusri Annapurna*, Sistla Mounica Pratyusha
Department of Pharmaceutical Analysis,
GITAM Institute of Pharmacy,
GITAM (Deemed to be) University,
Visakhapatnam-530045, India
*Corresponding Author E-mail:
mannapurna.mukthinuthalapati@gitam.edu
ABSTRACT:
Tipiracil and Trifluridine are anticancer drugs used for third or
fourth-line treatment of metastatic colorectal cancer. Trifluridine is an
active antiviral drug used as ophthalmic solutions for the treatment of primary
keratoconjunctivitis and recurrent epithelial keratitis due to herpes simplex
virus type 1 and 2. It’s antiviral action is produced by incorporating in to
viral DNA during replication, that results in defective proteins formation and
increased mutation rate. Tipiracil is approved for the treatment of
unresectable advanced or recurrent colorectal cancer in the form of combination
drug trifluridine and tipiracil. Tipiracil is a thymidine phosphorylase
inhibitor that inhibits degradation of trifluridine, thus increasing systemic
exposure to trifluridine. In the present study three new spectrophotometric
methods such as Dual wavelength method (Method A), Graphical absorbance ratio
method (Method B) and First derivative method (Method C) were proposed for the
simultaneous determination of Tipiracil and Trifluridine and phosphate buffer (pH
7.0). Linearity was observed over the concentration range1-80 µg/ml in all the
three methods for borate buffer pH 9.0; phosphate buffer pH 7.0 and both the
methods were validated as per ICH guidelines. These methods can be successfully
applied for the simultaneous determination of Tipiracil and Trifluridine in
tablets.
KEYWORDS: Spectrophotometric
Determination, Tipiracil, Trifluridine
INTRODUCTION:
Trifluridine is an active anti-viral agent
(Figure 1A) which is used ophthalmic solutions for the treatment of primary
keratoconjunctivitis1 and recurrent epithelial keratitis due to
herpes simplex virus type 1 and 2 and Tipiracil (Figure 1B) is used as an
anti-cancer drug when given in combination with Trifluridine. Tipiracil is a
thymidine phosphorylase inhibitor2-4 that inhibits degradation of
Trifluridine, thus increasing systemic exposure to Trifluridine. Few methods of
HPLC were developed for simultaneous determination of Trifluridine and
Tipiracil5-11. At present the authors have proposed three different spectrophotometric methods for the
simultaneous determination of Trifluridine and Tipiracil and the methods
were validated12.
Figure 1A:
Chemical structure of Trifluridine
Figure 1B:
Chemical structure of Tipiracil
MATERIALS AND METHODS:
Model No. UV-1800 double beam UV-VIS spectrophotometer (Shimadzu)
with quartz cells was used for the entire study and all the sample
solutions were scanned 200-400 nm. Stock solutions of Trifluridine and
Tipiracil were separately prepared in methanol. Using phosphate buffer pH 7.0
and borate buffer pH 9.0 a series of dilutions were made for the present study.
Method validation
From the stock solution a series of Trifluridine and Tipiracil were prepared
and scanned against their reagent blank i.e. phosphate buffer pH 7.0 and borate
buffer pH 9.0. At the selected wavelength the absorbance was taken and their
absorptivity values were calculated for Method A and Method B and the
individual concentrations of both drugs were determined, by substituting these
absorptivity values in the equations. In Method C the concentration of each
drug was calculated at the zero crossing point (ZCP) of the other drug (minima
observed) from the first derivative spectra and vice versa. A calibration curve
was drawn by taking the concentration of the drug solution on the x- axis and
the corresponding absorbance values were taken on the y-axis for Method A and
Method B and for Method C the derivative absorbance was taken.
The intra-day (same day) and inter-day (three different day)
precision studies were performed at three different concentration levels and the %RSD was calculated. Accuracy
studies were carried out by spiking the formulation solution with the pure drug
(50%, 100%, and 150%) and the % recovery was calculated for all the three
methods A, B and C.
Assay of formulations (Tablets)
Twenty tablets of two brands containing
both Trifluridine and Tipiracil were manufactured in the local lab (20 mg
Trifluridine + 8.9 mg Tipiracil) & (15 mg Trifluridine + 6.14 mg Tipiracil)
and extracted with methanol. The stock solution of two brands were filtered and
diluted with phosphate
buffer pH 7.0, borate buffer pH 9.0 solution as per the
requirement for Method A, B and C.
RESULTS AND DISCUSSION:
Three new spectrophotometric
methods - Dual wavelength method (Simultaneous equation method (Method A),
Graphical absorbance ratio method (Q – Analysis) (Method B) and First
derivative (Method C) spectrophotometric methods were proposed for the
estimation of Trifluridine and Tipiracil combined dosage forms (Tablets) in
phosphate buffer pH 7.0 and borate buffer pH 9.0. The literature review was given in Table
1.
Table 1: Literature review
|
Method
|
Mobile phase
(v/v) / Reagent
|
λmax
(nm)
|
Linearity
(µg/mL)
|
Ref.
|
|
HPLC
|
Methanol:
Water (65:35)
|
220
|
75–375 (TRF)
& 15–75 (TPL)
|
5
|
|
HPLC
|
15% NaClO4
buffer (pH 4.5 v/v), 85% Methanol.
|
260
|
0.5-4.0 (TRF)
& 0.2 -1.6 (TPL)
|
6
|
|
HPLC
|
Acetonitrile:
Water: Methanol (60:20:20)
|
230
|
66.6-330
(TRF) & 10-50 (TPL)
|
7
|
|
HPLC
|
Methanol and
Water (55:45)
|
292
|
3-9 (TRF) & 6-22 (TPL)
|
8
|
|
HPLC
|
potassium
dihydrogen phosphate buffer and acetonitrile (30:70) (pH 2.5)
|
240
|
20mg
(TRF) & 9mg (TPL)
|
9
|
|
HPLC
|
phosphate buffer
and methanol(30:70) (pH 3.0)
|
240
|
25-125 (TRF) & 15-75 (TPL)
|
10
|
|
HPLC
|
orthophosphoric
acid: acetonitrile (50:50)
|
292
|
1.02-15.30 (TRF) & 0.41-6.15 (TPL)
|
11
|
|
Spectroph
otometry
|
Phosphate buffer
pH 7.0 &
Borate buffer pH
9.0.
|
261.24
301.87
|
1-80 (TRF &
TPL)
|
Present method
|
Method validation
Dual wavelength method (Method A)
In Method A (Dual wavelength method) the absorption maxima of both
Trifluridine and Tipiracil were selected. Trifluridine has shown absorption
maxima (λmax) at 261.24 nm and that of Tipiracil at 301.87 nm.
From the absorbance recorded, the absorptivity values were calculated (Table 2)
at selected wavelength of their individual spectra and substituted in the
simultaneous equations for both the drugs. The linear regression equations for
borate buffer pH 9.0 are found to be y=0.0259x-0.0022 (0.9999), y=0.0004x+5E-05 (0.9999), y=0.0034x+0.0001
(0.9998), y=0.0375x+0.0033 (0.9997) for Trifluridine
at 261.24 nm, 301.87 nm and Tipiracil at 261.24nm, 301.87 nm respectively. The
linear regression equations for phosphate buffer pH 9.0 are found to be y =
0.0327x - 0.0044 (0.9999), y = 0.0026x - 0.0002 (0.9999), y = 0.0081x
+ 0.0012 (0.9999), y=0.0373x+0.0038 (0.9999) for Trifluridine at 261.24 nm,
301.87 nm; Tipiracil at 261.24 nm and 301.87 nm respectively. The overlay
spectrum of Trifluridine and Tipiracil were shown in Figure 2.
A1 and A2 represent the absorbance of the
formulation solution at 261.24 nm and 301.87 nm respectively; CTRF
and CTPL are the concentrations of Trifluridine and Tipiracil (g/100
ml) and the details of the equations were given in Table 3.
Table 2: Linearity of Trifluridine and Tipiracil (Method A)
|
Borate buffer
|
|
Trifluridine
|
Tipiracil
|
|
Conc. (µg/ml)
|
Absorbance at 261.24nm
|
€ 261.24
nm
|
Absorbance at 301.87 nm
|
€
301.87 nm
|
Conc. (µg/ml)
|
Absorbance at 261.24 nm
|
€ 261.24 nm
|
Absorbance at 301.87 nm
|
€ 301.87 nm
|
|
10
|
0.259
|
259.01
|
0.0036
|
3.6
|
10
|
0.0349
|
34.9
|
0.379
|
379.83
|
|
20
|
0.519
|
259.05
|
0.0072
|
3.6
|
20
|
0.069
|
34.5
|
0.749
|
374.5
|
|
30
|
0.772
|
257.33
|
0.011
|
3.66
|
30
|
0.103
|
34.33
|
1.131
|
377
|
|
40
|
1.035
|
258.75
|
0.0145
|
3.625
|
40
|
0.138
|
34.5
|
1.512
|
378
|
|
50
|
1.291
|
258.2
|
0.018
|
3.6
|
50
|
0.170
|
34
|
1.861
|
372.25
|
|
60
|
1.541
|
256.8
|
0.022
|
3.667
|
60
|
0.209
|
34.83
|
2.279
|
379.8
|
|
70
|
1.811
|
258.7
|
0.0253
|
3.614
|
70
|
0.239
|
34.14
|
2.65
|
378.5
|
|
80
|
2.061
|
257.62
|
0.029
|
3.625
|
80
|
0.276
|
34.15
|
2.978
|
372.2
|
|
Phosphate buffer pH 7.0
|
|
Trifluridine
|
Tipiracil
|
|
Conc. (µg/ml)
|
Absorbance at 261.72 nm
|
€
261.72 nm
|
Absorbance at
301.62 nm
|
€ 301.62 nm
|
Conc. (µg/ml)
|
Absorbance at 261.72 nm
|
€
261.72 nm
|
Absorbance at 301.62 nm
|
€
301.62 nm
|
|
10
|
0.321
|
321.06
|
0.026
|
26.81
|
10
|
0.081
|
81.21
|
0.374
|
374.12
|
|
20
|
0.645
|
322.84
|
0.053
|
26.74
|
20
|
0.163
|
81.52
|
0.758
|
379.06
|
|
30
|
0.975
|
325.31
|
0.079
|
26.53
|
30
|
0.245
|
81.94
|
1.132
|
377.65
|
|
40
|
1.296
|
324
|
0.105
|
26.42
|
40
|
0.327
|
81.75
|
1.494
|
373.52
|
|
50
|
1.648
|
329.6
|
0.132
|
26.45
|
50
|
0.407
|
81.41
|
1.851
|
370.21
|
|
60
|
1.959
|
326.5
|
0.157
|
26.33
|
60
|
0.489
|
81.66
|
2.268
|
378.01
|
|
70
|
2.264
|
323.42
|
0.184
|
26.28
|
70
|
2.613
|
373.28
|
2.613
|
373.28
|
|
80
|
2.621
|
327.62
|
0.212
|
26.50
|
80
|
2.987
|
373.37
|
2.987
|
373.37
|
Table 3: Details of the Equations
|
Borate buffer pH 9.0
|
Phosphate buffer pH 7.0
|
|
A1= Absorbance of the sample at iso-absorptive point
279.12 nm
|
A1= Absorbance of the sample at iso-absorptive point
259.03 nm
|
|
A2 = Absorbance of the sample at wavelength 261.72
nm.
|
A2 = Absorbance of the sample at wavelength 261.72
nm.
|
|
ax1 = Mean absorptivity of Trifluridine at 279.12 nm
|
ax1 = Mean absorptivity of Trifluridine at 259.03 nm.
|
|
ay1 = Mean absorptivity of Tipiracil at 261.72 nm
|
ay1 = Mean absorptivity of Tipiracil at 261.72 nm
|
|
Qm = Absorbance of formulation solution at 279.12 nm
/ Absorbance of formulation solution at 261.72 nm
|
Qm = Absorbance of formulation solution at 259.03 nm
/ Absorbance of formulation solution at 261.72 nm
|
|
Qx = Absorptivity of Trifluridine at 261.72nm /
Absorptivity of Trifluridine at 279.12 nm = 3.569
|
Qx = Absorptivity of Trifluridine at 261.72 nm /
Absorptivity of Trifluridine at 259.03 nm = 0.9401
|
|
Qy = Absorptivity of Tipiracil at 261.72 nm /
Absorptivity of Tipiracil at 279.12 nm = 0.2106
|
Qy = Absorptivity of Tipiracil at 261.72 nm /
Absorptivity of Tipiracil at 259.03 nm = 1.079
|
At 261.24 nm, A1 = 258.37 CTRF +
376.51 CTPL (Borate buffer pH 9.0)
A1 = 325.04 CTRF +
374.90 CTPL (Phosphate buffer pH 7.0).
At 301.87 nm, A2 = 3.621 CTRF +
34.418 CTPL (Borate buffer pH 9.0)
A2 = 26.50 CTRF +
81.57 CTPL (Phosphate buffer pH 7.0)
Figure 2: Overlay absorption spectrum of
Trifluridine and Tipiracil
Graphical absorbance ratio method (Method B)
In Method B the isosbestic point
i.e. 279.12 nm (borate buffer); 259.03 nm (phosphate buffer) and the absorption
maxima of one of the drugs (i.e. λmax Trifluridine = 261.72nm)
(borate and phosphate buffer) were chosen. The absorptivity (Ɛ) values
obtained at the selected wavelengths (Table 4) were substituted in the equation
given below.
Cx 
Cx = Concentration of Trifluridine
Cy 
Cy =Concentration of Tipiracil
The linear regression equations for borate buffer pH 9.0 are found
to be y = 0.0072x - 8E-05(0.9999), y = 0.0259x - 0.0022(0.9999), y = 0.0163x -
0.0011 (0.9999), y=0.0375x+0.0033(0.9997) for Trifluridine at 259.03nm, 261.72 and
Tipiracil at 259.03nm, 261.72 nm respectively. The linear regression equations
for phosphate buffer pH 9.0 are found to be y = 0.0347x - 0.0015 (0.9999), y = 0.0327x - 0.0044 (0.9999), y = 0.0076x + 0.0001 (0.9999), y =
0.0081x + 0.0012 (0.9999) for Trifluridine at 259.03nm, 261.72 and Tipiracil at
259.03nm, 261.72 nm respectively.
Beer-Lambert’s law was obeyed over the concentration range
1-80 µg/ml (borate buffer pH 9.0) and (phosphate buffer pH 7.0) in both Method A and Method
B for Trifluridine and Tipiracil respectively.
Calibration curves were drawn by taking the concentration on the x-axis and the
corresponding absorbance values on the y axis for Method B (Figure 3). The % RSD in precision and
accuracy studies for the simultaneous determination of Trifluridine and
Tipiracil in Methods A and B was found to be less than 2.0 indicating that
methods are precise and accurate.
Table 4:
Linearity of Trifluridine and Tipiracil (Method B)
|
Borate buffer
|
|
Trifluridine
|
Tipiracil
|
|
Conc. (µg/ml)
|
Absorbance at
279.12nm
|
€ 279.12
nm
|
Absorbance at
261.24nm
|
€ 261.249nm
|
Conc. (µg/ml)
|
Absorbance at
279.12 nm
|
€ 279.12nm
|
Absorbance at
261.24 nm
|
€ 261.24nm
|
|
10
|
0.072
|
72
|
0.259
|
259.01
|
10
|
0.163
|
163
|
0.0349
|
34.9
|
|
20
|
0.145
|
72.5
|
0.519
|
259.05
|
20
|
0.327
|
163.5
|
0.069
|
34.5
|
|
30
|
0.218
|
72.66
|
0.772
|
257.33
|
30
|
0.491
|
163.66
|
0.103
|
34.33
|
|
40
|
0.289
|
72.25
|
1.035
|
258.75
|
40
|
0.641
|
163.25
|
0.138
|
34.5
|
|
50
|
0.362
|
72.4
|
1.291
|
258.2
|
50
|
0.818
|
163.6
|
0.170
|
34
|
|
60
|
0.435
|
72.5
|
1.541
|
256.8
|
60
|
0.979
|
163.16
|
0.209
|
34.83
|
|
70
|
0.502
|
72
|
1.811
|
258.7
|
70
|
1.144
|
163.42
|
0.239
|
34.14
|
|
80
|
0.582
|
72.75
|
2.061
|
257.6
|
80
|
1.306
|
163.25
|
0.276
|
34.15
|
|
Phosphate
buffer pH 7.0
|
|
Trifluridine
|
Tipiracil
|
|
Conc. (µg/ml)
|
Absorbance
at 259.03 nm
|
€
259.03 nm
|
Absorbance at
261.72 nm
|
€
261.72 nm
|
Conc. (µg/ml)
|
Absorbance
at 259.03 nm
|
€
259.03 nm
|
Absorbance at
261.72 nm
|
€
261.72 nm
|
|
10
|
0.345
|
345.20
|
0.321
|
321.06
|
10
|
0.321
|
321.06
|
s
|
81.21
|
|
20
|
0.685
|
342.65
|
0.645
|
322.84
|
20
|
0.645
|
322.84
|
0.163
|
81.52
|
|
30
|
0.938
|
343.91
|
0.975
|
325.31
|
30
|
0.975
|
325.31
|
0.245
|
81.94
|
|
40
|
1.387
|
346.75
|
1.296
|
324
|
40
|
1.296
|
324
|
0.327
|
81.75
|
|
50
|
1.719
|
343.83
|
1.648
|
329.6
|
50
|
1.648
|
329.6
|
0.407
|
81.41
|
|
60
|
2.096
|
349.36
|
1.959
|
326.5
|
60
|
1.959
|
326.5
|
0.489
|
81.66
|
|
70
|
2.437
|
349.19
|
2.264
|
323.42
|
70
|
2.264
|
323.42
|
2.613
|
373.28
|
|
80
|
2.767
|
345.87
|
2.621
|
327.62
|
80
|
2.621
|
327.62
|
2.987
|
373.37
|
Figure 3: Calibration
curves of Trifluridine and Tipiracil (Method B)
Figure 4:
First order derivative absorption spectra of Trifluridine (TRF) and Tipiracil
(TPL)
Table 5: Linearity of Trifluridine and Tipiracil (Method C)
|
Borate buffer
|
Phosphate buffer pH 7.0
|
|
Conc. (µg/ml)
|
Trifluridine
(Zero crossing point of Tipiracil 253.61 nm)
|
Tipiracil
(Zero crossing point of Trifluridine 232.53 nm)
|
Trifluridine
(Zero crossing point of Tipiracil 253.13 nm)
|
Tipiracil
(Zero crossing point of Trifluridine 261.95 nm)
|
|
Absorbance
|
Absorbance
|
Absorbance
|
Absorbance
|
|
10
|
0.231
|
0.319
|
0.010
|
0.0003
|
|
20
|
0.461
|
0.589
|
0.020
|
0.0007
|
|
30
|
0.678
|
0.881
|
0.029
|
0.010
|
|
40
|
0.912
|
1.156
|
0.039
|
0.014
|
|
50
|
1.14
|
1.435
|
0.048
|
0.017
|
|
60
|
1.37
|
1.751
|
0.058
|
0.021
|
|
70
|
1.59
|
2.022
|
0.068
|
0.024
|
|
80
|
1.82
|
2.296
|
0.078
|
0.028
|
Table 6: Assay of Trifluridine and Tipiracil tablets
|
Brand
|
Drug
|
Label
claim (mg)
|
*Amount found
(mg)
|
*% Recovery
|
|
Method A
|
Method B
|
Method C
|
Method A
|
Method B
|
Method C
|
|
I
|
Trifluridine
Tipiracil
|
20
8.9
|
19.95
8.85
|
19.97
8.87
|
19.93
8.84
|
99.75
99.43
|
99.85
99.66
|
99.65
99.32
|
|
II
|
Trifluridine
Tipiracil
|
20
8.9
|
19.94
8.83
|
19.90
8.86
|
19.96
8.87
|
99.70
99.21
|
99.50
99.55
|
99.80
99.66
|
First derivative method (Method C)
In first derivative method, with the help of in built software the
individual zero order absorption spectra obtained for Trifluridine and
Tipiracil were transformed in to first order derivative spectra (Figure 4).
Trifluridine has showm zero crossing point (ZCP) at 261.95 and Tipiracil has
shown zero crossing point (ZCP) at 253.13 respectively. Trifluridine was
estimated at 253.61 nm (selected ZCP of Tipiracil); Tipiracil was estimated at
232.53 nm (selected ZCP of Trifluridine) for borate buffer pH 9.0 and
Trifluridine was estimated at 253.13 nm (selected ZCP of Tipiracil); Tipiracil
was estimated at 261.95 nm (selected ZCP of Trifluridine) for phosphate buffer
pH 7.0 (Table 5).
Beer-Lambert’s law was obeyed over the concentration range
1-80 µg/ml in Method C for Trifluridine and Tipiracil respectively for both the buffers.
Calibration curves were drawn by taking the concentration on the x-axis and the
corresponding derivative absorbance values on the y axis. The % RSD in
precision and accuracy studies of the simultaneous determination of
Teneligliptin and Metformin in three methods A, B and C was found to be less
than 2.0 indicating that the three methods are precise and accurate.
Assay
of formulations (Tablets):
The percentage purity was found to be 99.50-99.85 and
99.21-99.63 for Trifluridine and Tipiracil respectively (Table 6) in Method A, Method B and Method
C. The three methods can be successfully applied for the simultaneous
determination of Trifluridine and Tipiracil.
CONCLUSION:
The three spectrophotometric methods are simple, economical,
precise and accurate for the simultaneous determination of Trifluridine and
Tipiracil in pharmaceutical formulations (Tablets).
ACKNOWLEDGEMENT:
The authors are grateful to
M/s GITAM (Deemed to be) University, Visakhapatnam for providing the research
facilities and Biophore pharmaceuticals for providing the gift samples. There
is no conflict of interest.
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