Carbon-based nanomaterials have received a lot of interest in recent years. Carbon quantum dots (CQDts), novel zero-dimensional nanomaterials based on carbon, are well-known for their small size and comparatively strong fluorescence characteristics. CQDts not only inherit the excellent optical properties of traditional semiconductor quantum dots but also overcome their shortcomings in terms of cytotoxicity, environmental danger, and biohazard by having superior water solubility, chemical stability, and photobleaching resistance. It has caused tremendous anxiety among researchers in a number of fields, including biology, chemical sensing, nanomedicine, and photo electrocatalysis [37].
CQDts were created from ascorbic acid as a carbon source. It was dissolved in water and subjected to hydrothermal carbonization. Alcohol (polyethelene glycol) added in excess formed the ligand and capped the CQDts, producing yellow emitters. The ligand groups on the surface of the CQDts were visible in the FT-IR spectrum (Figure S1). The peak at 3383 cm-1 denotes the presence of the O-H group, while the peaks at 2943 and 1655 cm− 1 denote the stretching vibrations of the C-H and C = O, respectively, and the peak at 1086 cm− 1 denotes the symmetric and asymmetric vibration of the C-O-C [38]. Because of the hydroxyl and carbonyl groups, CQDts has a strong water solubility.
The aqueous excitation-emission spectra are presented in Fig. 2 at wavelengths of 325 nm and 524 nm, respectively. The average diameter of the spherical CQDts shape in the transmission electron microscopy (TEM) image was 10 nm (Figure S2).
3.1. Optimization of Experimental Factors:
To examine all potential combinations of the variables and their influence on various responses, the procedure optimization was conducted using a two-level full factorial design. Factorial design has distinct advantages over the conventional optimization method, including the use of fewer experiments, shorter operation times, and the ability to generate data that can be statistically analyzed to reveal important details about the interactions between experimental parameters [39]. A total of 18 experiments were conducted to determine the effects of time, buffer volume, buffer pH, and reagent volume on the ΔF for the three drugs. The factors and ranges selected for consideration were based on previous univariate studies which show best quenching when the range of volume of reagent was 0.2 to 0.5 mL (A), pH of the buffer was 4 to 6 (B) and volume of buffer was 3 to 6 mL (C). After addition of the reagent solution to the drugs, different time intervals were studied from zero to 15 min (D).
Half-normal plots and Pareto charts generated by the factorial design are shown in Figure S3 demonstrating that the volume of the reagent (A) had a significant effect on the ΔF of each of the three drugs. Additionally, the volume of the reagent with the buffer's pH (AB) had a significant effect on the ΔF of only nimodipine and nifedipine, whereas the volume of the buffer (C) had a significant effect on the ΔF of lercanidipine alone.
In order to investigate of these independent factors and their interactions effects the responses, an approximated Fisher Statistical Test for Variance Analysis (ANOVA) model [40] was employed to analyze the responses and used to determine the significance of the independent factors.
The equation for the four-factor experimental design could be presented as follows:
γ = β0 + β1A + β2B + β3C + β4D + β2AB + β2AC + β2BC + β2BD + β2A2 + β2B2 + β2C2 + β2D2 :
where:
γ stands for response, β stands for regression coefficients, and A, B, C and D stand for quantum dot reagent, buffer pH, volume of buffer and time of incubation, respectively.
The interaction between different factors was confirmed by the interaction and main effect plots (Figure S4) which showed that there are interactions between the factors affecting the method and these can’t be shown using univariate optimization.
The composite desirability of a response is determined by the Minitab response optimizer. The value of (D), which ranges from zero to one, indicates if the responses fall within accepted limits. One implies that the condition attained is ideal, hence its value should be one or near to one. Zero is unacceptable since it shows that many of the responses are outside of their acceptable ranges. It provided the response optimizer (Fig. 3) which show that the optimum conditions to achieve the highest ΔF when the volume of reagent was 0.5, pH was 6, volume of buffer was 6 mL and the incubation time was 15 min.
3.2. Analytical Performance of the proposed method
The suggested technique was approved according to ICH Q2 recommendations [41]. For each of the three drugs, the fluorescence intensity quenched linearly in the range of 0.5–20.0 µg/mL. Linear regression analysis of the data are summarized in Table 1, and represented by the following equations:
∆F = 38.41 + 23.38C (r = 0.9999) for lercanidipine
∆F = 24.22 + 18.58C (r = 0.9999) for nimodipine
∆F = 117.37 + 21.14C (r = 0.9999) for nifedipine
Where ∆F is the quenching in the fluorescence intensity, ∆F = (the native fluorescence of quantum dot solution (Fº) - fluorescence of the reaction product (F)), C is the concentration of the drug (µg/mL) and r is the correlation coefficient.
The limits of quantitation (LOQ) were calculated following ICH Q2 Recommendations [41[ and were found to be 0.33 for lercanidipine, 0.30 for nimodipine and 0.37 µg/mL for nifedipine. LOQ was calculated from the following equation ]41[:
LOQ = 10 Sa / slope
The limits of detection (LOD) were calculated accordingly and were found to be 0.11 for lercanidipine, 0.10 for nimodipine and 0.12 µg/mL for nifedipine. LOD was calculated from the following equation ]41[.
LOD = 3.3 Sa / slope
The proposed methods were tested for linearity, specificity, accuracy and precision (Table 1).
Parameter
|
Lercanidipine
|
Nimodipine
|
Nifedipine
|
Table 1
Performance data of the studied drugs by the proposed methods
concentration range(µg/ mL ).
|
0.5–20.0
|
0.5–20.0
|
0.5–20.0
|
-LOD (µg/mL ).
|
0.11
|
0.10
|
0.12
|
-LOQ (µg/ mL).
|
0.33
|
0.30
|
0.37
|
-Correlation coefficient ( r).
|
0.9999
|
0.9999
|
0.9999
|
-Slope
|
23.38
|
18.58
|
21.14
|
-Intercept
|
38.14
|
24.22
|
117.73
|
-Sy/x
|
1.1097
|
0.8131
|
1.215
|
-Sa
|
0.7718
|
0.541
|
0.845
|
-Sb
|
0.0675
|
0.0462
|
0.074
|
-% Error
|
0.445
|
0.100
|
0.29
|
-%RSD
|
1.090
|
0.246
|
0.712
|
-No.of Experiments.
|
6
|
6
|
6
|
-Mean found (%) ± SD.
|
100.04 ± 1.09
|
99.95 ± 0.25
|
99.93 ± 0.71
|
N.B.
-
-Sy/x =standard deviation of the residuals.
-
-Sa = standard deviation of the intercept of regression line.
-
-Sb = standard deviation of the slope of regression line
-
-% Error = RSD% / √ n.
For examining precision, three concentrations of each drug (5,10,15 µg/mL) in its pure form on three separate occasions were tested, and the results are displayed in (Table 2). The drugs were repeatedly studied in pure form using different concentrations summarized in (Table 2) over a period of three days in order to achieve intermediate precision (also known as ruggedness). Moreover, the consistency of the difference in fluorescence intensity (ΔF) with the slight changes in the experimental parameters proved the method’s robustness. These parameters included pH 6 ± 0.2, volume of buffer 6 ± 0.2 and volume of quantum dot reagent 0.5 ± 0.02. These minimal changes that may take place during the experimental operation doesn't greatly affect the decrease in the fluorescence intensity (∆F).
Table 2
Precision data of the proposed methods for determination of the studied drugs in pure form
Drug
|
Conc.
(µg/mL)
|
Intra-day
|
Inter-day
|
Mean ± S. D
|
%RSD
|
% error
|
Mean ± S. D
|
%RSD
|
% error
|
Lercanidipine
|
5
|
99.17 ± 0.99
|
1.00
|
0.58
|
99.35 ± 1.03
|
1.04
|
0.60
|
10
|
99.95 ± 0.42
|
0.42
|
0.24
|
99.59 ± 0.91
|
0.92
|
0.24
|
15
|
99.97 ± 0.60
|
0.60
|
0.34
|
99.78 ± 0.73
|
0.73
|
0.42
|
Nimodipine
|
5
|
99.67 ± 0.54
|
0.54
|
0.31
|
99.81 ± 0.37
|
0.37
|
0.21
|
10
|
99.98 ± 0.59
|
0.59
|
0.34
|
99.65 ± 1.13
|
1.14
|
0.66
|
15
|
99.55 ± 0.94
|
0.95
|
0.55
|
99.75 ± 0.66
|
0.66
|
0.38
|
Nifedipine
|
5
|
99.68 ± 0.61
|
0.61
|
0.78
|
99.4 ± 0.8
|
0.83
|
0.44
|
10
|
100.4 ± 0.9
|
0.92
|
0.53
|
99.8 ± 0.5
|
0.51
|
0.29
|
15
|
99.6 ± 0.6
|
0.74
|
0.43
|
100.25 ± 0.35
|
0.36
|
0.21
|
The proposed and comparison methods for each of the three drugs did not show significant difference when it came to accuracy and precision, according to statistical analysis of the results using the Student's t-test and variance ratio F-test [42].
The comparison techniques used spectrophotometric measurements of the three drugs' absorbances in their solutions at wavelengths of 332 nm, 238.5 nm, and 387 nm for lercanidipine, nimodipine, and nifedipine, respectively [5, 13, 25].
The proposed methods are simple, fast, sensitive and have wider range over the comparison method
Pharmaceutical Applications
The proposed method was effectively utilized in the determination of each of the three drugs in their dosage forms Caredipine®, Nimodipine® and Epilat Retard® tablets.
By observing any interference caused by common tablet excipients such as talc, lactose, starch, avisil, gelatin, and magnesium stearate, the method's specificity was examined. The acceptable % recoveries of the examined concentrations showed that these excipients didn't interfere with the suggested approach. The results for the analysis of tablets and in pure form using the suggested method are shown in (Table 3). Comparison with the spectrophotometric approach previously reported, showing good accuracy and precision.
Table 3: Application of the proposed method and reference method for determination of the studied drugs in commercial tablets
Compound
|
Proposed Method
|
Comparison methods [5,13,25]
|
Amount taken
(µg/mL)
|
Amount found
(µg/mL)
|
% Found
|
% Found
|
Caredipine
|
5
|
4.920
|
98.40
|
100.92
|
10
|
9.898
|
98.98
|
99.09
|
15
|
14.932
|
99.55
|
100.31
|
Mean
|
98.98
|
100.11
|
± S.D.
|
0.575
|
0.93
|
t-test
|
1.81(2.78)
|
F-test
|
2.62(19.00)
|
Nimodipine
|
5
|
4.991
|
99.83
|
100.79
|
10
|
9.857
|
98.57
|
99.28
|
15
|
15.054
|
100.36
|
100.29
|
Mean
|
99.59
|
100.12
|
± S.D.
|
0.919
|
0.77
|
t-test
|
0. 83(2.78)
|
F-test
|
1.42(19.00)
|
Epilat retard
|
5
|
5.0100
|
100.21
|
100.47
|
10
|
9.9240
|
99.24
|
98.89
|
15
|
15.0400
|
100.27
|
99.93
|
Mean
|
99.91
|
99.76
|
± S.D.
|
0.578
|
0.80
|
t-test
|
0.27(2.78)
|
F-test
|
1.92(19.00)
|
Compound
|
Proposed Method
|
Comparison methods [5,13,25]
|
Amount taken
(µg/mL)
|
Amount found
(µg/mL)
|
% Found
|
% Found
|
Caredipine
|
5
|
4.920
|
98.40
|
100.92
|
10
|
9.898
|
98.98
|
99.09
|
15
|
14.932
|
99.55
|
100.31
|
Mean
|
98.98
|
100.11
|
± S.D.
|
0.575
|
0.93
|
t-test
|
1.81(2.78)
|
F-test
|
2.62(19.00)
|
Nimodipine
|
5
|
4.991
|
99.83
|
100.79
|
10
|
9.857
|
98.57
|
99.28
|
15
|
15.054
|
100.36
|
100.29
|
Mean
|
99.59
|
100.12
|
± S.D.
|
0.919
|
0.77
|
t-test
|
0. 83(2.78)
|
F-test
|
1.42(19.00)
|
Epilat retard
|
5
|
5.0100
|
100.21
|
100.47
|
10
|
9.9240
|
99.24
|
98.89
|
15
|
15.0400
|
100.27
|
99.93
|
Mean
|
99.91
|
99.76
|
± S.D.
|
0.578
|
0.80
|
t-test
|
0.27(2.78)
|
F-test
|
1.92(19.00)
|
Values between parentheses are the tabulated t and F values respectively, at p = 0.05. [42]
Study of Interference:
the possible interference from other species that might be present was examined to assess the analytical method's selectivity. Taking into account that the target samples to be analyzed are CCBs, the main compounds that may interfere in the determination are other antihypertensive drugs as atenolol [6], captopril, atorvastatin and valsartan. Other interfering agent were introduced to examine the selectivity these agents including ascorbic acid, uric acid, magnesium sulphate and copper sulphate.
If the signal variation was less than 5%, we assumed that no interference had occurred. In general, the tolerance to the presence of foreign species is much higher than the concentration at which these compounds are often present with the analytes in pharmaceuticals (Table 4). Satisfactory tolerances were obtained due to the selectivity of the CQDts system towards the studied drugs, observing no interaction with other antihypertensive drugs and interfering agents.
If the signal variation was less than 5%, we assumed that no interference had occurred. In general, the tolerance to the presence of foreign species is much higher than the concentration at which these compounds are often present with the analytes in pharmaceuticals (Table 4). Satisfactory tolerances were obtained due to the selectivity of the CQDs system towards the studied drugs, observing no interaction with other antihypertensive drugs and interfering agents.
Interfering substance
|
Tolerance limit
|
Table 4
Study of interference of excipients:
Atenolol
|
11.50
|
Captopril
|
20.00
|
Atorvastatin
|
13.33
|
Valsartan
|
6.67
|
Ascorbic acid
|
16.67
|
Uric acid
|
18.57
|
Magnesium sulphate
|
80
|
Copper sulphate
|
53.33
|
The tolerance limit is the maximum concentration in µg/mL of the interfering substance that caused a relative error (RE) less than 5% for the determination of the studied drugs. |
Mechanism of Quenching:
The studied drugs were added to the synthesized CQDts, which exhibited fluorescence that was significantly quenched. The excitation and emission fluorescence spectra of CQDts at 325 and 528 nm, which were noticeably sensitive to each drug and gradually declined with increasing drugs concentration, are shown in (Figure 4). Fluorescence is known to be quenched through a variety of mechanisms, including fluorescence resonance energy transfer (FRET), inner filter effect (IFE), dynamic quenching, and static quenching [43].
where a is blank of CQDTs and from b to g is the reaction product with different concentrations (0.5–20 µg/mL). a- Lercanidipine b- Nimodipine c- Nifedipine.
For each of the three drugs, the CQDts excitation spectra and UV absorbance spectra overlapped (Fig. 5), suggesting the possibility of IFE. In order to fully investigate the quenching mechanism, Eq. 1 was used in conjunction with correction of the fluorescence intensity of the CQDts upon addition of increasing concentrations of the quencher (lercanidipine, nimodipine, nifedipine).
Fcorr = Fobs X 10 (Aex+Aem)/2 (1)
Fcorr is the corrected fluorescence intensity after eliminating IFE from Fobs, where Fobs is the observed fluorescence intensity. The terms Aex and Aem relate to the quencher's absorbance at the fluorophore's (CQDts) excitation and emission wavelengths, respectively. The suppressed efficiency (% E) for the measured and corrected fluorescence intensity was then calculated using Eq. 2.:
%E = \(\left[1-\frac{F}{F^\circ }\right]\times 100\) (2)
Plotting % E of the observed and corrected fluorescence intensities of CQDts against the molar concentration of each of the three drugs indicated that IFE was the mechanism of quenching for NIM in this study (Fig. 6), while LER and NIF were found to have different mechanisms, requiring the use of Stern Volmer Eq. (3).
the Stern-Volmer plots were constructed by plotting Fo/F versus [M] according to Stern-Volmer equation [43]:
Fo /F = 1 + Ksv [M] (3)
{KSV is the Stern-Volmer quenching constant and [M] is the molar concentration of each of LER and NIF}.
The formation of ground-state complexes provides static quenching, whereas the interaction of quencher molecules with the excited fluorophore results in dynamic quenching. These mechanisms can be found by examining the relationship between temperature and quenching rate constant.
When the temperature rises during dynamic quenching, the quencher and fluorescent molecules are tempted to disperse and collide, increasing the quenching rate constant. The quencher/fluorophore ground-state complex becomes less stable at higher temperatures during static quenching, which results in a decrease in the quenching rate constant.
In this research, the effects of fluorescence quenching at 298, 308, and 318 K were examined. The experimental data were analyzed using the Stern-Volmer equation, and Ksv values were determined from the slope of the plots of F0/F versus [Q] (Figure S5). The results revealed a direct correlation between Ksv values and temperature (Table 5), demonstrating that reaction of lercanidipine and nifedipine with CQDts was IFE and dynamic interactions dominate the control of the fluorescence quenching process.
Table 5
A summary of the Stern-Volmer parameters for the reaction of lercanidipine and nifedipine with CQDts.
Drug
|
Temperature
(°k)
|
Stern-Volmer quenching constant (Ksv)
|
Correlation coefficient (r)
|
Lercanidipine
|
298
|
0.018
|
0.9914
|
308
|
0.0364
|
0.9984
|
318
|
0.0478
|
0.9992
|
Nifedipine
|
298
|
0.0414
|
0.987
|
308
|
0.116
|
0.9981
|
318
|
0.1796
|
0.9989
|