The Pareto Chart Analysis

Categories: Chemistry Materials Physics Scientific method

Analysis, Pages 7 (1501 words)

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Figure 2 Represents the Pareto Chart, Normal Plot, Normal probability plot, Versus Fit, Versus order, contour plot and Surface plot of Zeta potential (ZP)

Determination of Entrapment Efficiency (EE)

The entrapment efficiency was found to be in the range of 40.5% to 28.5%. The response curves and the contour plots explained the effect of various independent variables on entrapment efficiency. The F-value of 75.46 presented that the model was significant at p < 0.0001. The R2 value, adjusted R2 and Predicted R2 values were 0.99, 0.98 and 0.89 respectively. The difference between adjusted R2 value and predicted R2value was less than 0.20 which suggested the close agreement between the both, percentage EE was influenced by the total amount of lipids and concentration of surfactant positively i.e., increase in the total amount of lipids and concentration of surfactant increased the entrapment efficiency of the drug). Percentage EE depends on A, C and C2 (equation no.5). Escalation of homogenization speed had nonsignificant effect on decrease in EE. The main challenge of this experiment was to maximize the amount of drug entrapped in the nanostructured lipid carrier and deliver the hydrophilic drug moiety to CNS.

The model summary and related ANOVA details are given in table 5 and Pareto chart, residual plots, contour plots and response surface curve is given in figure 3

%EE = 37.3 + 0.0269 A - 0.00213 B + 4.65 C - 0.000108 A*A - 0.000000 B*B - 3.23 C*C+ 0.000008 A*B - 0.00550 A*C + 0.000475 B*C (5)

Table 5 ANOVA of the % entrapment efficiency of the experimental design

Model summary

S R-sq R-sq(adj) R-sq(pred)

0.504810 0.99 0.97 0.89

Analysis of variance

Source Degree of freedom Sum of Square Mean Square F-Value P-Value

Model 9 173.062 19.229 75.46 0.000

Linear 3 156.562 52.187 204.79 0.000

C 1 2.000 2.000 7.85 0.038

B 1 2.311 2.311 9.07 0.030

A 1 152.251 152.251 597.45 0.000

Square 3 6.294 2.098 8.23 0.022

C*C 1 2.413 2.413 9.47 0.028

B*B 1 0.124 0.124 0.49 0.516

A*A 1 4.333 4.333 17.00 0.009

2-Way Interaction 3 10.205 3.402 13.35 0.008

C*B 1 0.902 0.902 3.54 0.119

C*A 1 0.303 0.303 1.19 0.326

B*A 1 9.000 9.000 35.32 0.002

Error 5 1.274 0.255

Lack-of-Fit 3 1.148 0.383 6.04 0.145

Pure Error 2 0.127 0.063

Total 14 174.336

A= Total amount of lipid, B= homogenisation speed, C= Conc. of surfactant

Figure 3 Represents the Pareto Chart,Normal Plot, Normal probability plot, Versus Fit, Versus order, contour plot and Surface plot of % Entrapment Efficiency (%EE)

Table 6 Observed and predicted response of dependent variables like particle size, Zeta potential and entrapment efficiency

Formulation Code Particle Size Zeta Potential % Entrapment efficiency

Observed value Predicted value Observed value Predicted value Observed value Predicted value

1 189.8 182.225 -24.4 -24.67

32.3 32.5750

2 178.0 175.625 -27.9 -28.20 38.3 38.3000

3 139.8 142.175 -26.5 -26.20

28.5 28.5000

4 168.7 176.275 -28.6 -28.32

40.5 40.2250

5 180.0 178.575 -25.3 -25.21

30.6 30.1375

6 177.5 178.375 -26.8 -26.68

39.6 39.4125

7 93.9 98.625 -24.8 -24.91 29.5 29.6875

8 124.9 126.325 -29.0 -29.08

37.4 37.8625

9 198.7 207.700 -26.2 -26.11 36.5 36.6875

10 186.0 185.050 -25.7 -26.0875

34.2 34.6625

11 137.8 138.750 -26.4 -26.31 35.2 34.7375

12 124.0 122.000 -28.1 -27.98

34.8 34.6125

13 145.0 144.667 -28.5 -28.53 36.4 36.1667

14 147.0 144.667 -28.7 -28.53 36.2 36.1667

15 142.0 144.667 -28.4 -28.53 35.9 36.1667

Responses Optimization: % Entrapment Efficiency, Zeta potential, Particle size

In analysis of experiment data, it was found that the experimental model selected, was significant and for all responses, the adjusted R2 value and predicted R2 value had good agreement among them. In this study, the focus was to increase the permeability of the hydrophilic drug through CNS, so the drug was entrapped in nanostructure lipid carriers. But the challenge was to entrap the hydrophilic drug in the lipid moiety and to enhance the stability of the NLCs. Hence the %(EE) entrapment efficiency and zeta potential were given 2 times more importance than Particle size. The drug ceftriaxone being negatively charged the zeta potential had negative sign and stability of the prepared NLCs depends on the numerical value values of zeta potential. Minitab 19 software was used to optimize the experimental responses, the goal had to be set at minimum values for particle size and zeta potential (zeta potential values set with negative signs) and that for entrapment efficiency set at maximum value for stability. Minitab helped to find the optimal desirability of each individual response at different goals fixed according to the requirement of the design and it also gives the optimal composite desirability of model. Composite desirability was 0.8551 which suggested that the model selected was the best to obtain the desired response and it was found that if the independent variables were fixed at concentration of surfactant at 2%, homogenization speed at 12868.69 rpm and total amount of lipids at 400 mg the response and individual desirability of % EE was found to be 38.68 and 0.83, respectively. The individual desirability values of the predicted zeta potential (-29.69mV) and particle size (129.05nm) of the suggested optimized formation it's desirability was l and was 129.05nm and desirability was 0.66. It is known that the value of desirability ranges between 0 and 1. When this value approaches 1 it suggests that the setting would give optimal result for all responses.()

Table 7 Response optimization for Composite desirability optimal of model and individual desirability optimal of ZP, EE, PS.

Parameters

Response Goal Lower Target Upper Weight Importance

ZP Minimum -29.0 -24.4 1 2

EE Maximum 28.5 40.5 1 2

PS Minimum 93.9 198.7 1 1

Solution

Solution A B C ZP

Fit EE

Fit PS

Fit Composite

Desirability

1 400 12868.7 2 -29.2570 38.4523 129.049 0.855085

Multiple Response Prediction

Variable Setting

A 400

B 12868.7

C 2

Response Fit SE Fit 95% CI 95% PI

ZP -29.257 0.279 (-29.975, -28.540) (-30.322, -28.192)

EE 38.452 0.460 (37.270, 39.635) (36.697, 40.208)

PS 129.05 5.68 (114.44, 143.66) (107.35, 150.75)

ZP= Zeta Potential, PS= Particle Size, EE= entrapment efficiency

Figure 4 Graph of composite desirability optimal of model and individual desirability of responses like Zeta potential, Particle size and Entrapment efficiency

(Zeta potential actually is the maximum value but as the charge of ceftriaxone sodium is negative and we are getting the mathematical model so to get the appropriate result ZP have been fitted negative)

Selection of optimized NLC

It is evident from response optimization that the main independent factors are the amount of drug and surfactant concentration which effects the responses. Therefore F8 was selected as the optimised formulation. It was further lyophilized, was subjected to various evaluation.

DSC Analysis

The DSC analysis showed the endothermic peak of pure lipid glyceryl monostearate at 68.230C, representing the melting peak of the solid lipid.()For the pure drug ceftriaxone it was found to have peak at 132.700C. The peak value of glyceryl monostearate decreases to 57.810C when the drug and the liquid lipid were added to it and in the admixture another melting peaks was also noted at 131.390C. In FZD NLC it was found that the peak of drug was almost missing revealing that the drug has fully dispersed in the lipid. The decrease in the melting points of FZD NLC of ceftriaxone compared to that of solid lipid suggests that particles size has been reduced to nanometric size as attributed to the Kelvin effect given by Thomson equation and the results of DSC and XRD suggest the formation of imperfection in crystal structure of NLC.

Figure 5 DSC thermograms of solid lipid, drug, physical admixture of drug, solid lipid and liquid lipid

X-ray diffraction

The study of the crystalline orientation of drug (Ceftriaxone sodium), solid lipid Glyceryl monostearate, physical admixture of drug, solid lipid and liquid lipid and Freeze dried NLC of optimised formulation was done by X ray diffraction technique. Pure Ceftriaxone sodium exhibited large number of discrete sharp and robust peaks at the 2? values given in table ---- which exhibiting the high crystallinity structure of drug (Degree of crystallinity was found 83%). The FWHM values suggest the crystal size and the stress-strain gathered in the material during the process of size reduction. The value of FWHM is inversely related to the particle size and sharper XRD peaks give small value of FMHM; for pure drug in crystalline form FMHM has value less than 1, as it has been mentioned in the table 1.The value of FWHM to corresponding 2 theta is less than 1 indicates drugs crystalline nature and also suggests that the drug has not been subjected to any stress. The intensity and the number of peak of solid lipid was less and the corresponding FWHM value shows that its amorphous nature character predominates. The number of peaks and intensity further decreases for physical admixture of drug ,solid lipid and liquid lipid. But the XRD pattern of optimized freeze dried NLC shows that there were less number and widened peak related to the pure drug and the solid lipid revealing that the crystalline structure has been converted into amorphous form, means that in NLC formulation ceftriaxone was molecularly solubilized (dispersed) forming the lipid matrix and the FWHM value (1.95 and 2.88 ) and wide peak of freeze dried NLC gives evidence that the system was subjected to stress to reduce the particle size suggesting structural disorder.

Table 8 XRD data for drug, solid lipid, physical admixture of drug, solid lipid and liquid lipid and Freeze dried NLC of optimized formulation

Drug Solid lipid Physical admixture of drug and lipids Freeze dried NLC

2-theta FWHM(deg) 2-theta FWHM(deg) 2-theta FWHM(deg) 2-theta FWHM(deg)

11.036(10) 0.225(16) 19.061(12) 0.45(2) 18.996(18) 0.28(4) 19.12 1.95

11.52(2) 0.21(4) 19.555(13) 0.477(14) 19.435(17) 0.63(7) 23.71 2.88

12.440(13) 0.201(17) 20.68(8) 3.76(19) 20.35(5) 2.1(4)

18.32(3) 0.34(3) 22.910(19) 0.35(2) 23.56(4) 1.97(12)

18.807(16) 0.268(16) 23.927(19) 1.53(4)

19.901(11) 0.64(7)

21.116(10) 0.287(8)

22.681(9) 0.295(7)

23.699(12) 0.292(10)

25.09(2) 0.29(2)

26.66(4) 0.28(4)

28.152(9) 0.65(3)

29.36(6) 0.48(6)

30.55(7) 0.45(6)

33.76(3) 0.34(2)

FWHM -full width half maximum of XRD peak

Figure 6 showing XRD of drug, solid lipid, physical admixture of drug, solid lipid and liquid lipid and Freeze dried NLC of optimized formulation

Conclusion

NLC formulation containing ceftriaxone sodium were formulated by hot emulsification technique and optimised using Box-Behken design. The effects of different independent variables ( amount of drug, homogenisation speed and concentration of surfactant ) on various dependent variables which attributes to the quality of nanocarriers in terms of particle size,entrapment efficiency and zeta potential were studied.From the results it is evident that NLCs of Ceftriaxone sodium can be anticipated as the reliable formulation for the administration of the same and also gives a novel approach to fabricate the lipid nanocarriers for advancement of hydrophilic drug delivery.