Development of RP-HPLC method
The simultaneous determination of KR and DQA was done with the development of the RP-HPLC method. The mobile phase was a mixture of components A and B wherein, A corresponded to 0.01 M potassium phosphate buffer of pH 3.2 and B had methanol as an organic phase. The gradient program was run and the varied composition with respect to time is represented in Table 1. The reversed-phase C18 X Bridge column (150×4.6 mm, 5µm) was used to get a sharp, narrow, and symmetrical shape. Better separation of KR and DQA was obtained with a flow rate of 1 mL/min and column oven temperature of 30˚C (Fig. 2).
Validation of RP-HPLC method
Specificity
Specificity was determined after comparison of the chromatograms of KR, blank DQAsomes without KR (placebo), and KR-loaded DQAsomes. KR in pure form and in the formulation showed similar retention time indicating no interference of other peaks. It has been represented in Fig. 3.
System suitability
The system suitability was evaluated after six replicate injections into the system for a solution of 100 µg/mL for both the analytes, KR and DQA. The %RSD value was found to be well within 2% for different parameters like peak area, tailing factor, and theoretical plates. Supplementary Table 1 (Table S1) represents system suitability parameters with average, standard deviation, and % RSD values.
Linearity
The linearity was established from 10 µg/mL to 250 µg/mL for both KR and DQA. The calibration curve was plotted for peak area against its respective concentration. The equation, Y= 30.155X-49.011 with r2 value of 0.9995 was obtained for KR while Y= 15.175X-37.774 with r2 value of 0.9995 was obtained for DQA which represented a good correlation (Supplementary Fig. S1). Supplementary Table 2 (Table S2) represents the linearity study of KR and DQA.
Accuracy
The accuracy was evaluated in terms of % recovery for 50 µg/mL, 100 µg/mL, and 150 µg/mL solutions for the analytes, KR and DQA. It was found that % the recovery was from 99% to 101% for KR and 98 to 100% for DQA. The results are incorporated into Table 2. The values were within the range of 98-102% and % RSD was also less than 2%.
Table 2. Accuracy study for analysis of KR and DQA
Analyte
|
Nominal concentration (µg/mL)
|
Found concentration* (µg/mL)
|
% Accuracy
|
%RSD
|
Kinetin Riboside
|
50
|
49.66 (±0.30)
|
99.31 (±0.60)
|
0.60
|
100
|
101.32 (±0.26)
|
101.32 (±0.26)
|
0.26
|
150
|
148.47 (±0.18)
|
98.98 (±0.12)
|
0.12
|
Dequalinium chloride
|
50
|
50.37 (±0.77)
|
100.74 (±1.54)
|
1.53
|
100
|
100.47 (±0.17)
|
100.47 (±0.17)
|
0.17
|
150
|
147.04 (±1.48)
|
98.03 (±1.21)
|
1.00
|
*Data expressed as mean (±SD); n=3.
Precision
The method showed acceptable precision study and % RSD was found to be less than 2% which is acceptable for precision study outlined by ICH guidelines (Table 3).
Table 3. (a) Precision study of KR (Day 1); (b) Precision study of DQA (Day 1); (c) Precision study of KR (Day 2); (d) Precision study of DQA (Day 2)
(a)
Sr.No.
|
Retention time (min)
|
Peak area
|
1
|
5.58
|
3172.76
|
2
|
5.58
|
3184.08
|
3
|
5.58
|
3190.96
|
4
|
5.59
|
3196.83
|
5
|
5.59
|
3223.00
|
6
|
5.58
|
3207.80
|
Mean
|
5.58
|
3195.90
|
SD
|
0.003
|
17.76
|
%RSD
|
0.05
|
0.55
|
(b)
Sr.No.
|
Retention time (min)
|
Peak area
|
1
|
6.99
|
1564.03
|
2
|
6.99
|
1568.09
|
3
|
6.99
|
1571.90
|
4
|
6.99
|
1573.20
|
5
|
6.99
|
1585.01
|
6
|
6.99
|
1580.58
|
Mean
|
6.99
|
1573.80
|
SD
|
0.001
|
7.79
|
%RSD
|
0.01
|
0.49
|
(c)
Sr.No.
|
Retention time (min)
|
Peak area
|
1
|
5.58
|
3157.97
|
2
|
5.58
|
3166.30
|
3
|
5.58
|
3184.40
|
4
|
5.58
|
3177.22
|
5
|
5.58
|
3182.07
|
6
|
5.58
|
3170.63
|
Mean
|
5.58
|
3173.10
|
SD
|
0.001
|
10.06
|
%RSD
|
0.01
|
0.32
|
(d)
Sr.No.
|
Retention time (min)
|
Peak area
|
1
|
6.99
|
1579.98
|
2
|
6.99
|
1569.20
|
3
|
6.99
|
1572.52
|
4
|
6.99
|
1568.13
|
5
|
6.99
|
1568.23
|
6
|
6.99
|
1562.64
|
Mean
|
6.99
|
1570.12
|
SD
|
0.001
|
5.79
|
%RSD
|
0.01
|
0.37
|
Limit of detection (LOD) and limit of quantitation (LOQ)
The LOD and LOQ were calculated by the Signal to Noise ratio (S/N) method. The limit of detection for KR and DQA was found to be 3.26 µg/mL and 3.03 µg/mL, respectively as it showed S/N ratio of 3.3:1 when compared to baseline. The limit of quantitation for KR and DQA was found to be 10.48 µg/mL and 10.12 µg/mL, respectively to get S/N ratio of 10:1 compared to baseline.
Robustness
The robustness was performed by doing small but deliberate changes in terms of flow rate (±0.2 mL/min), the temperature of column (±5˚C), wavelength of detection (±2 nm), and change in mobile phase composition (±2% organic phase change). The results of the robustness study are tabulated in Table 4.
Table 4. Robustness study
Sr. No.
|
Parameter
|
Analyte
|
Peak area*
|
Retention time* (Rt)
|
Tailing factor* (T)
|
- Change in flow rate
|
1.
|
0.8 mL/min
|
Kinetin Riboside
|
4013.75
|
6.58
|
1.45
|
Dequalinium chloride
|
1969.09
|
7.92
|
1.56
|
2.
|
1 mL/min
|
Kinetin Riboside
|
3173.10
|
5.58
|
1.39
|
Dequalinium chloride
|
1570.12
|
6.99
|
1.49
|
3.
|
1.2 mL/min
|
Kinetin Riboside
|
2690.18
|
5.05
|
1.39
|
Dequalinium chloride
|
1336.39
|
6.57
|
1.49
|
- Change in column temperature
|
1.
|
25˚C
|
Kinetin Riboside
|
3345.56
|
6.99
|
1.39
|
Dequalinium chloride
|
1573.9
|
7.00
|
1.50
|
2.
|
30˚C
|
Kinetin Riboside
|
3173.10
|
5.58
|
1.39
|
Dequalinium chloride
|
1570.12
|
6.99
|
1.49
|
3.
|
35˚C
|
Kinetin Riboside
|
2846.77
|
5.60
|
1.39
|
Dequalinium chloride
|
1522.90
|
7.03
|
1.59
|
- Change in wavelength
|
1.
|
267 nm
|
Kinetin Riboside
|
2846.77
|
5.60
|
1.39
|
Dequalinium chloride
|
1385.63
|
7.03
|
1.59
|
2.
|
269 nm
|
Kinetin Riboside
|
3173.10
|
5.58
|
1.39
|
Dequalinium chloride
|
1570.12
|
6.99
|
1.49
|
3.
|
271 nm
|
Kinetin Riboside
|
2861.72
|
5.60
|
1.39
|
Dequalinium chloride
|
1367.78
|
7.01
|
1.58
|
- Change in mobile phase (Buffer: Methanol) composition
|
1.
|
78:22
|
Kinetin Riboside
|
2859.29
|
4.87
|
1.56
|
Dequalinium chloride
|
1454.29
|
6.77
|
2.00
|
2.
|
80:20
|
Kinetin Riboside
|
3173.10
|
5.58
|
1.39
|
Dequalinium chloride
|
1570.12
|
6.99
|
1.49
|
3.
|
82:18
|
Kinetin Riboside
|
2858.05
|
4.91
|
1.64
|
Dequalinium chloride
|
1432.04
|
6.84
|
2.19
|
*Data expressed as mean; n=3
Forced degradation study
The forced degradation study was carried out under stress conditions including acidic, basic, neutral, oxidative, thermal, and photolytic conditions. The results showed that KR in its formulation showed one major degradation product (37% degradation) under acidic conditions, and % recovery was found to be 96.6%, whereas under basic conditions, 9% degradation was observed. Under photolytic conditions (UV light; 27h), 4 degradants were observed, amounting to around 59% degradation, and % recovery was determined to be 70%. No degradation products were observed under peroxide oxidation, neutral and thermal conditions. KR when subjected to degradation, underwent oxidative degradation (2.26% degradation), thermal degradation (3.33% degradation) and hydrolytic degradation (8% degradation), while the rest of the trend was similar to that observed with KR in the LR-loaded formulation. In the case of DQA, no degradants were observed either in the placebo or in KR-loaded formulations in any of the conditions except for basic and photolytic degradation. The detailed analysis of KR and DQA in the blank formulation and KR-loaded formulation under varied stress conditions is tabulated in Table 5. The overlay of chromatograms of blank, KR, blank formulation (placebo), and KR-loaded formulation in each of the stressed conditions are depicted in Supplementary Fig. S2 (a-f). Fig. S2 (i) represents chromatograms at 269 nm (Detection wavelength of KR), while Fig. S2 (ii) represents chromatograms at 310 nm (Detection wavelength of DQA).
Table 5. (a) Force degradation study of KR in formulation; (b) Force degradation study of KR (c) Force degradation study of DQA in placebo formulation (d) Force degradation study of DQA in formulation
(a)
Stress conditions
|
Treatment
|
% Assay
|
% Degradation
|
% Recovery
|
Unstressed
|
--
|
96.36
|
--
|
96.36
|
Acid
|
1 N HCl (60˚C, 48h)
|
56.20
|
36.95
|
96.67
|
Base
|
1 N NaOH (60˚C, 48h)
|
51.89
|
9.04
|
63.23
|
Peroxide Oxidation
|
Hydrogen peroxide (30%, RT, 7 days)
|
96.14
|
--
|
99.77
|
Temperature
|
Thermal (80˚C, 48h)
|
82.16
|
--
|
85.26
|
Hydrolysis
|
Water (RT, 24h)
|
92.85
|
--
|
96.36
|
Photolysis
|
UV light (27h)
|
8.18
|
59.26
|
69.99
|
(b)
Stress conditions
|
Treatment
|
% Assay
|
% Degradation
|
% Recovery
|
Unstressed
|
--
|
96.36
|
--
|
96.36
|
Acid
|
1 N HCl (60˚C, 48h)
|
87.08
|
5.22
|
95.79
|
Base
|
1 N NaOH (60˚C, 48h)
|
75.75
|
7.56
|
86.45
|
Peroxide Oxidation
|
Hydrogen peroxide (30%, RT, 7 days)
|
80.26
|
2.26
|
85.63
|
Temperature
|
Thermal (80˚C, 48h)
|
92.98
|
3.33
|
99.91
|
Hydrolysis
|
Water (RT, 24h)
|
87.43
|
7.99
|
99.03
|
Photolysis
|
UV light (27h)
|
54.54
|
33.31
|
91.17
|
(c)
Stress conditions
|
Treatment
|
% Assay
|
% Degradation
|
% Recovery
|
Unstressed
|
--
|
|
--
|
99.62
|
Acid
|
1 N HCl (60˚C, 48h)
|
92.89
|
--
|
93.24
|
Base
|
1 N NaOH (60˚C, 48h)
|
9.82
|
72.35
|
82.48
|
Peroxide Oxidation
|
Hydrogen peroxide (30%, RT, 7 days)
|
94.59
|
--
|
94.94
|
Temperature
|
Thermal (80˚C, 48h)
|
92.21
|
--
|
92.56
|
Hydrolysis
|
Water (RT, 24h)
|
83.51
|
--
|
83.83
|
Photolysis
|
UV light (27h)
|
89.41
|
--
|
89.75
|
(d)
Stress conditions
|
Treatment
|
% Assay
|
% Degradation
|
% Recovery
|
Unstressed
|
--
|
99.62
|
--
|
99.62
|
Acid
|
1 N HCl (60˚C, 48h)
|
92.89
|
--
|
93.24
|
Base
|
1 N NaOH (60˚C, 48h)
|
11.81
|
80.66
|
92.82
|
Peroxide Oxidation
|
Hydrogen peroxide (30%, RT, 7 days)
|
94.59
|
--
|
94.94
|
Temperature
|
Thermal (80˚C, 48h)
|
92.21
|
--
|
92.56
|
Hydrolysis
|
Water (RT, 24h)
|
83.51
|
--
|
83.83
|
Photolysis
|
UV light (27h)
|
16.59
|
82.79
|
99.76
|
Applications of validated RP-HPLC method
Formulation and characterization of KR-loaded DQAsomes
Initially, preliminary batches of DQAsomes were formulated to assess the impact of any formulations and process parameters on particle size and zeta potential. Based on preliminary investigation, it was found that probe sonication amplitude had the highest impact on particle size and zeta potential. Thus, 3 batches, namely, F1, F2, and F3, were formulated and subjected to different probe sonication amplitudes, viz., 30, 50, and 70A, respectively. The formulation and process parameters for 3 batches and their respective size of particles, polydispersity index and zeta potential were enlisted in Table 6. Out of three formulations, the F3 batch with a sonication amplitude of 70A had a particle size of 215.3±3.54 nm and a PDI 0.12±0.004. It can be seen that as probe sonication amplitude increased, particle size decreased along with PDI. The plot of particle size and PDI is shown in Fig. 4. The formulation F3 showed a zeta potential of +39.4 ±3.04 mV (Fig. 4).
Evaluation of % entrapment efficiency and drug loading of KR in formulated DQAsomes
The RP-HPLC method that has been validated, was used for the evaluation of entrapment efficiency and drug loading of KR in DQAsomes by the equations (Eqs. 4 and 5) stated earlier. The % entrapment efficiency of the formulation with a probe sonication amplitude of 70A was found to be 81.12±5.62% and %drug loading was found to be 45.23±4.83% (Table 6).
Table 6. Formulation and process parameters and Characterization of KR-loaded DQAsomes
Parameters
|
Formulations
|
|
F1
|
F2
|
F3
|
KR conc (mM)
|
15
|
15
|
15
|
DQA conc (mM)
|
15
|
15
|
15
|
Rotational speed (rpm)
|
100
|
100
|
100
|
Rotational temperature (˚C)
|
35
|
35
|
35
|
Methanol volume (mL)
|
10
|
10
|
10
|
HEPES volume(mL)
|
10
|
10
|
10
|
Probe sonication time (h)
|
1
|
1
|
1
|
Probe sonication amplitude (A)
|
30
|
50
|
70
|
Particle size (nm)
|
344.97±6.11
|
289.50±9.41
|
215.32±3.54
|
PDI
|
0.26±0.03
|
0.20±0.01
|
0.12±0.004
|
Zeta potential (mV)
|
24.47±1.50
|
29.2±4.20
|
39.4±3.04
|
% Entrapment efficiency
|
71.17±6.78
|
75.90±7.12
|
81.12±5.62
|
% loading capacity
|
35.30±3.61
|
43.29±7.54
|
45.23±4.83
|
In vitro drug release study of KR-loaded DQAsomes
In vitro drug release from DQAsomes was performed by dialysis bag membrane (MWCO 14 kDa) for 3 batches, namely, F1, F2 and F3 [31]. F3 showed initial burst release to a greater extent (25%) than rest 2 formulations in 15 minutes. This might be attributed to the release of unentrapped KR from DQAsomes. After 1 h, the F3 batch showed 38.6% release while F1 and F2 showed 21% and 36.6% cumulative KR release, respectively. F2 and F3 batch showed 40% and 45% cumulative KR release, respectively, till 24 h, which confirmed the sustained release of KR from DQAsomes. The overall in vitro drug release profile can be well correlated with particle size obtained for 3 batches, with F1 having a greater particle size and showing less drug release than F3, having a smaller particle size with the highest drug release. An In vitro cumulative drug release profile for 3 optimization batches of DQAsomes is depicted in Fig. 5.