Table S1: In-vitro drug release studies (Franz diffusion cell) with the transdermal patches.
Time(h) / Percent in vitro release of duloxetine HCl for formulations
A1 / A2 / A3 / A4 / B1 / B2 / B3 / B4 / C1 / C2 / C3 / C4
0.5 / 6.36 / 6.03 / 5.59 / 5.26 / 6.69 / 6.25 / 5.37 / 5.15 / 7.02 / 6.36 / 4.93 / 4.93
1.0 / 12.91 / 8.35 / 13.22 / 8.22 / 10.33 / 8.06 / 7.36 / 9.19 / 13.56 / 10.82 / 12.78 / 6.50
2.0 / 22.12 / 21.53 / 19.21 / 16.58 / 15.37 / 12.63 / 18.86 / 15.52 / 30.44 / 15.08 / 19.04 / 9.77
3.0 / 30.77 / 28.58 / 25.20 / 21.51 / 29.55 / 16.44 / 26.50 / 24.96 / 35.73 / 24.05 / 27.23 / 13.43
4.0 / 39.57 / 33.52 / 31.51 / 32.45 / 37.18 / 31.36 / 30.88 / 30.68 / 41.03 / 35.49 / 34.68 / 29.43
6.0 / 45.43 / 42.38 / 38.12 / 38.05 / 46.28 / 37.87 / 37.46 / 36.33 / 49.19 / 40.94 / 40.57 / 34.18
8.0 / 56.05 / 50.28 / 50.03 / 46.19 / 53.24 / 45.98 / 43.87 / 41.09 / 55.50 / 52.02 / 48.05 / 42.12
10 / 63.55 / 56.72 / 57.07 / 53.53 / 62.33 / 52.80 / 49.21 / 50.79 / 65.70 / 59.99 / 57.89 / 50.36
12 / 70.31 / 63.77 / 63.92 / 59.38 / 68.80 / 58.90 / 55.34 / 56.67 / 72.84 / 67.01 / 64.25 / 56.30
14 / 76.94 / 72.84 / 72.61 / 65.52 / 75.30 / 68.19 / 63.02 / 62.91 / 80.03 / 74.83 / 72.02 / 64.31
16 / 84.40 / 79.60 / 79.77 / 72.98 / 82.50 / 75.03 / 72.03 / 72.60 / 88.32 / 83.46 / 79.11 / 70.65
24 / 94.01 / 89.84 / 88.60 / 81.95 / 91.82 / 83.18 / 80.77 / 83.11 / 95.59 / 90.78 / 87.33 / 81.89
Table S2: Ex-vivo drug release studies (Franz diffusion cell) with the transdermal patches
Time(h) / Percent ex vivo release of duloxetine HCl for formulations
A1 / A2 / A3 / A4 / B1 / B2 / B3 / B4 / C1 / C2 / C3 / C4
0.5 / 4.82 / 4.28 / 4.71 / 4.17 / 5.70 / 5.04 / 4.71 / 3.73 / 5.26 / 4.71 / 4.49 / 4.49
1 / 7.60 / 6.63 / 7.58 / 6.39 / 7.42 / 6.76 / 6.05 / 5.11 / 8.22 / 7.91 / 7.22 / 6.45
2 / 12.57 / 11.13 / 11.68 / 11.62 / 11.57 / 9.39 / 7.69 / 6.63 / 11.53 / 10.63 / 9.28 / 8.07
3 / 20.71 / 18.18 / 14.94 / 14.05 / 22.37 / 14.49 / 11.33 / 8.38 / 19.24 / 17.34 / 15.90 / 14.53
4 / 24.96 / 21.76 / 19.25 / 17.92 / 26.58 / 21.87 / 16.50 / 12.59 / 26.33 / 23.39 / 19.89 / 20.45
6 / 30.10 / 27.16 / 23.85 / 22.47 / 30.65 / 26.32 / 22.51 / 17.32 / 34.05 / 29.06 / 26.95 / 26.14
8 / 34.83 / 31.16 / 26.75 / 27.08 / 34.84 / 29.84 / 26.54 / 22.03 / 41.74 / 38.93 / 36.30 / 34.21
10 / 40.21 / 35.84 / 32.43 / 30.62 / 39.71 / 35.08 / 31.89 / 26.85 / 49.15 / 45.37 / 42.61 / 39.92
12 / 47.42 / 41.98 / 38.82 / 34.60 / 46.67 / 40.44 / 36.10 / 31.06 / 58.22 / 53.78 / 52.67 / 45.82
14 / 55.39 / 49.09 / 44.42 / 39.72 / 53.82 / 47.32 / 40.79 / 35.93 / 66.30 / 62.03 / 60.35 / 53.74
16 / 61.21 / 55.31 / 49.56 / 44.96 / 59.14 / 53.51 / 45.01 / 40.66 / 73.44 / 69.21 / 67.30 / 60.47
24 / 73.74 / 65.67 / 59.72 / 54.66 / 69.06 / 62.74 / 58.33 / 53.61 / 81.63 / 77.27 / 74.53 / 68.90
Fig. S1. Percent in vitro release at various time intervals for formulations A1-A4, B1-B4, C1-C4.
Fig.S2. Percent ex-vivorelease at various time intervals for formulationsA1-A4, B1-B4,C1-C4.
VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR DULOXETINE
Figure. UV scan of duloxetine
The stock solution was serially diluted (1 to 100 µg/mL) with phosphate buffer (pH 7.4), and the absorbance values were measured in triplicate at 289 nm (λmaxof the drug) against phosphate buffer (pH 7.4) as blank.
Construction of calibration curve
A standard plot was obtained by using the absorbance readings of all the dilutions.
Spectrophotometric data for construction of standard plot of duloxetine
Sr. No. / conc. ( mcg /mL) / conc. ( g/100 mL) / Absorbance1 / 1 / 0.0001 / 0.016
2 / 2.5 / 0.00025 / 0.034
3 / 5.0 / 0.0005 / 0.095
4 / 10 / 0.001 / 0.181
5 / 20 / 0.002 / 0.376
6 / 30 / 0.003 / 0.562
7 / 40 / 0.004 / 0.754
8 / 50 / 0.005 / 1.157
9 / 60 / 0.006 / 1.165
10 / 70 / 0.007 / 1.351
11 / 80 / 0.008 / 1.544
12 / 90 / 0.009 / 1.737
13 / 100 / 0.01 / 1.927
Figure: Standard plot of duloxetine (conc. range 0.0001-.01 g/100mL)
Validation of the analytical procedure
The analytical procedure employed for the quantification of duloxetine by UV spectrophotometry was validated according to ICH guidelines. The method was validated with respect to linearity and range, accuracy and precision, limit of detection (LOD) and limit of quantification (LOQ), selectivity and robustness. The developed method was validated for the pure drug as well as marketed formulation of Delok 30 (duloxetine 30 mg).
- Calibration curve
A calibration curve was plotted by taking absorbance values of six separate series of solutions in the range (5-40 mcg/mL).
Figure:Calibration curve of duloxetine hydrochloride (5-40 mcg/mL)
Tablefor Calibration data
Sr. No. / Conc. (mcg/mL) / *Mean absorbance at 289 nm ± S.D / % RSD#1 / 5 / 0.095 ± 0.0008 / 0.9382
2 / 10 / 0.181 ± 0.0016 / 0.9244
3 / 20 / 0.376 ± 0.0020 / 0.5426
4 / 30 / 0.562 ± 0.0023 / 0.4120
5 / 40 / 0.7538 ± 0.0032 / 0.4312
*Calculated as mean of measurements in triplicate.
# Calculated as = standard deviation/ data set × 100
- Linearity and range
Six separate series of solutions of the drug (5-40 mcg/mL in 7.4 phosphate buffer) were prepared from the stock solution and analyzed. Excellent linearity was indicated by high r2 value (0.999) in the whole range (5-40 mcg/mL).
- Accuracy
Accuracy was evaluated by recovery studies which were carried out by standard addition method with the pure drug as well as by using marketed preparation. The percentage recoveries were then calculated. The corresponding data given in Tables 19 and 20 shows standard addition method both with pure drug and with marketed formulation respectively. The percentage recovery was found to be within acceptable range of 95-105 % was indicating good accuracy of the method.
Table: Results of recovery studies of pure drug duloxetine by standard addition method
Initial concentration (mcg/ml) / Volume (ml) / Volume added (ml) / Amount added (mcg) / Total amount (mcg) / Amount found (mcg) / Recovery % (± SD) / *RSD %5 / 5.0 / 1.0 / 5 / 30 / 29.01 / 96.70 ± 0.61 / 0.6321
10 / 5.0 / 1.0 / 5 / 55 / 53.78 / 97.79 ± 0.88 / 0.9020
20 / 5.0 / 1.0 / 5 / 105 / 101.00 / 96.15 ± 0.52 / 0.5446
30 / 5.0 / 1.0 / 5 / 155 / 151.61 / 97.81 ± 0.77 / 0.7931
40 / 5.0 / 1.0 / 5 / 205 / 202.11 / 98.59 ± 49 / 0.5051
50 / 5.0 / 1.0 / 5 / 255 / 247.64 / 97.11 ± 1.19 / 1.2324
*Calculated as relative standard deviation between recovery values in triplicate.
Analysis of marketed preparation and assessment of accuracy.
Powder weight equivalent to 15 mg of duloxetine was taken and dissolved in 100 mL of phosphate buffer to make a solution A (150 mcg/mL). The solution was analyzed with suitable dilution at 289 nm. The percentage recovery was 99.05 % (amount recovered = 14.85mg). The amount per capsule was found to be 29.71 mg. Now for the assessment of accuracy, three equal volumes (20 ml each) of the solution A were taken and by adding appropriate volumes of a previously prepared 400 mcg/mL pure drug solution (solution B) their drug concentration was increased to 225 mcg/mL, 300 mcg/mL and 375 mcg/mL (i.e. A+50%, A+100% and A+150% respectively). The above three solutions were analyzed with the spectrophotometrically at 289 nm and their percent recovery, in comparison with the recovery of solution A was utilized for determination of accuracy.
Table: Results for analysis of marketed formulation and assessment of accuracy
Formulation / Volume of solution A added (mL) / Volume of solution B added (mL) / Final conc. (mcg/mL) / Total amount (mcg) / Amount recovered (mcg) / Percentage recovery / Drug amount found per capsule / Percentage recovery compared to solution ADelok 30 / 14.0 / 6.0 / 225 / 4.5 / 4.42 / 98.26 / 29.48 ± 0.27 / 99.2
Delok 30 / 8.0 / 12.0 / 300 / 6.0 / 5.88 / 98.05 / 29.41 ± 0.36 / 98.99
Delok 30 / 2.0 / 18.0 / 375 / 7.5 / 7.33 / 97.78 / 29.33 ± 0.43 / 97.71
Mean / 98.03 / 29.41 ± 0.35 / 98.97
- Precision:
Precision was investigated by analyzing different concentrations of duloxetine (5-50 mcg/mL) in independent replicates on the same day (intra-day precision) and on three consecutive days (inter-day precision). The data is represented as relative standard deviation (RSD) and results have been shown in Table 21. Intra- day and Inter- day analysis have shown low relative standard deviation (RSD) values which indicated the good precision of the method.
Table 21. Precision of the proposed methods for the analysis of duloxetine
Duloxetine (mcg/ml) / Intraday, n=6 / Inter-day, n=6*Mean ± SD / #RSD % / *Mean ± SD / #RSD %
5 / 0.095 ± 0.0005 / 0.6056 / 0.0956 ± 0.0005 / 0.6035
10 / 0.181 ± 0.0005 / 0.3183 / 0.182 ± 0.0015 / 0.8362
20 / 0.378 ± 0.0017 / 0.4582 / 0.380 ± 0.0028 / 0.7590
30 / 0.562 ± 0.0020 / 0.3699 / 0.565 ± 0.0030 / 0.5403
40 / 0.757 ± 0.0043 / 0.57581 / 0.761 ± 0.0066 / 0.8745
*Calculated as mean of measurements in hexaplicate.
#Calculated as the relative standard deviation between measurement values 1, 2, 3, 4, 5 and 6.
- Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ of the method were established using the following formulae as per ICH definitions.
LOD = 3.3 σ ∕s … (16)
LOQ = 10 σ ∕s …(17)
Where; σ - Mean standard deviation of blank determination
s - Slope of the standard curve
- Molar absorptivity
A concentration vs time plot was obtained with concentration in (moles/liter) on the X – axis and absorbance value on the Y – axis was plotted. The value of molar absorptivity was assessed from theslope of the straight line plot.
.
Figure: Plot for the determination molar absorptivity
- Sandell’s sensitivity
Sandell’s sensitivity was calculated by dividing molecular weight by molar absorptivity.
- Robustness
Repeatability is based on the results of the method operating over short interval of time under same conditions. Robustness was examined by evaluating the small variations in different experimental conditions such as heating temperatures (± 2° C), working wavelengths and concentration of reagents (Tables 22, 23 and 24)
Robustness data for the developed method.
Table: Robustness at different temperatures.
Serial no. / Temperature / Concentration (mcg/mL) / *Mean absorbance ± SD / Percent recovery / #Robustness1 / Room temperature (26 °C) / 20 / 0.370
± 0005 / 98.11 / 0.405
2 / At temperature (28 °C) / 20 / 0.368
± 0015 / 97.75
3 / At temperature (23 °C) / 20 / 0.372
± 0005 / 98.55
*Calculated as mean of measurements in triplicate.
#Calculated as the relative standard deviation between recovery values 1, 2 and 3.
Table: Effect of change in concentration of reagent by 2%
Serial no. / Test / Concentration (mcg/mL) / *Mean absorbance ± SD / Percent recovery / #Robustness1 / Normal buffer / 20 / 0.370
± 0005 / 98.11 / 0.27
2 / Potassium phosphate (-2%) / 20 / 0.369
± 0020 / 97.75
3 / Disodium hydrogen orthophosphate (-2%) / 20 / 0.371
± 0005 / 98.28
*Calculated as mean of measurements in triplicate.
#Calculated as the relative standard deviation between recovery values 1, 2 and 3.
Table: Effect of change in wavelength.
Serial no. / Wavelenth / Concentration (mcg/mL) / *Mean absorbance ± SD / Percent recovery / #Robustness1 / 289 / 20 / 0.370
± 0005 / 98.11 / 1.10
2 / 291 / 20 / 0.362
± 0005 / 95.99
3 / 287 / 20 / 0.365
± 0005 / 96.78
*Calculated as mean of measurements in triplicate.
#Calculated as the relative standard deviation between recovery values 1, 2 and 3.
Table: Optical Characteristics and regression equation of duloxetine hydrochloride
Validation parameter(in phosphate buffer 7.4) / Results
Absorption maximum / 289 nm
Regression equation (y= mx+c) / y = 188.9x - 0.003
Slope / 188.9
Intercept / 0.003
Beer’s law limit (μg/mL) / 5-50 µg/ml
Molar Absorptivity (L/mol.cm) / 6292
Sandell’s sensitivity (μg/cm2/0.001 A.U) / 0.0529
Coefficient of correlation(r) / 0.999
Limit of detection (LOD) / 0.41 mcg/ml
Limit of quantification (LOQ) / 1.24 mcg/Ml
Accuracy / 99.02 ± 0.35
Precision / Intraday = 0.46
Inter-day = 0.72
Robustness / Less than 1.2%
The proposed UV method for the quantification of duloxetine in bulk and formulation (capsule dosage form) is simple, accurate, precise, specific and highly sensitive. The method is economical rapid and does not require any sophisticated apparatus in contrast to chromatographic methods. Hence, the proposed method can be successfully used for routine quality control analysis of pure drug and marketed formulations containing duloxetine.