Results and Discussion Bundelkhand University, Jhansi

CHAPTER 3: RESULTS AND DISCUSSION

3.1 ANALYTICAL SECTION:

3.1.1 HPLC method development for Pure Erythromycin:

The linearity of the response of the drug was verified from 1 to 10 µg/ml
concentrations. The calibration graphs were obtained by plotting the response versus
the concentration. The calibration curve was found to be linear in the aforementioned
concentrations. The correlation coefficient (r2) of determination was 0.9999 which
indicates that the method is accurate.

Table 3.1: Linearity of Erythromycin for HPLC method development

Sr. Concentration (µg/ml) Area

No.
1 1.004 68125
2 2.009 137259
3 3.013 208252
4 4.018 275685
5 5.022 345621
6 6.026 414865
7 7.031 485023
8 8.035 555210
9 9.040 625125
10 10.044 691242
Slope 69271
Intercept -1752
Correlation co-efficient 0.99999

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Table 3.2: Optical and Regression Characteristics of the proposed HPLC method
for Erythromycin

Parameter Erythromycin

Absorption maxima 210 nm

Linearity Range (µg/ml) 1-10

Correlation co-efficient 0.9999

Table 3.3: Peak Results for Erythromycin WS

Sr. Name RT Area % Area USP Plate USP Tailing

no. Count Factor
1 Erythromycin 4.993 315374 100.0 11281 1.05
WS

Table 3.4: Peak Results for Erythromycin formulation Samples A and B

Sr. Name RT Area Height Purity Purity Purity

No. Angle threshold flag
1 Erythromycin 4.994 315453 44214 0.173 0.357 No
Erytop Cream
2 Erythromycin 4.993 314169 43984 0.181 0.307 No
Acnet Cream

Method precision was evaluated by carrying out the independent assays for both
Erythromycin formulations. Each sample of known concentration was injected thrice
for every formulation. The relative standard deviation for each formulation was then
calculated. These precision assay RSD values indicates the method is reproducible

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Table 3.5: HPLC Method Precision Values

Formulation Assay Value of Erythromycin as RSD

% of Labeled Amount
99.98
Erytop Cream 99.76 0.281
99.33
99.54
99.68
Acnet Cream 0.301
99.93
99.67

The recovery values performed at 50%, 100% and 150% levels were found to range
between 99.2 % to 100.3 %. Accuracy of the method was studied by recovery
experiments using one topical formulation. The percentage recovery values indicates
that there no interference from the excipients present in the formulation that
developed method is found to be sensitive, accurate, precise and most reproducible.

Table 3.6: Recovery Study Values by HPLC

Formulation Percentage Recovery

Erytop Cream 100.30
Acnet Cream 99.90

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3.1.2 Development of HPLC Method for Erythromycin Dermal Formulations

3.1.2.1 Specificity / Purity Plots:

Specificity of method was verified by injecting Erythromycin working standard and

Erythromycin sample solutions in the chromatographic system. No interference was
found at the retention time of Erythromycin. The purity plots data reveals that
Erythromycin peak passes the peak purity criteria. Hence, it can be concluded that the
method is specific for assay of Erythromycin formulations.

Table 3.7: Peak Purity Results for Erythromycin Formulation Samples A and B

Sr. Name RT Area Height Purity Purity Purity

No. Angle threshold flag
1 Erythromycin 4.994 315453 44214 0.173 0.357 No
Erytop Cream
2 Erythromycin 4.993 314169 43984 0.181 0.307 No
Acnet Cream

3.1.2.2 System Precision:

Assay of method precision was evaluated by injecting six replicates of the standard
solution into HPLC system. The observed RSD was 0.36% which is less than 2.00, a
mandatory need for the data to be precise as per the USP. Hence, based on the system
precision data it can be concluded that the system is precise.

Table 3.8: Determination of Precision for HPLC system validation

Sr. No. Percentage assay value

for Precision
1 99.43
2 99.64
3 99.60
4 99.08
5 99.20
6 100.12
Mean 99.50
RSD 0.36

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3.1.2.3 Method Precision:

Assay of method precision (intra-day precision) was evaluated by carrying out six
independent assays of test samples of A and B formulations. The intermediate
precision (inter-day precision) of the method was also evaluated using two different
analysts, systems and different days in the same laboratory. The relative standard
deviation (RSD) and assay values obtained by two analysts were 0.38 & 99.50, 0.27
& 99.60, for A formulation and 0.50 & 98.21, 0.49 & 98.23 for B formulation
respectively. These values indicate good reproducibility of the method for both
formulations.

Table 3.9: Determination of Precision for HPLC method validation

Sample Assay of Erythromycin as % of labeled amount
number A formulation B formulation

Analyst-II
Analyst-I (Intra- Analyst-II (Inter- Analyst-I (Intra-
(Inter-day
day precision) day precision) day precision)
precision)
1 99.43 99.73 98.26 98.44
2 99.62 99.20 98.12 98.23
3 99.50 99.88 98.22 98.22
4 99.18 99.57 98.15 98.23
5 99.22 100.00 98.28 98.44
6 100.10 99.23 98.22 98.43
Mean 99.50 99.60 98.21 98.23
RSD 0.38 0.27 0.50 0.49

3.1.2.4 Accuracy (Recovery test)

Accuracy of the method was studied by recovery experiments. The recovery was
performed at three levels, 50, 100% and 150% of Erythromycin standard
concentration. The recovery samples were prepared by before mentioned procedure.
Three samples of each marketed formulation (A and B) were prepared for each
recovery level. The solutions were then analyzed, and the percentage recoveries were
calculated from the calibration curve. The result of analysis the recovery studies
presented in Table 3.9 and 3.10. The percentage recovery values indicates that there

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no interference from the excipients present in the formulation that developed method
is found to be sensitive, accurate, precise and most reproducible.

Table 3.10: Recovery Studies for HPLC method validation

Formulation Level %Recovery %RSD*

50% 99.20 0.2834
A 100% 99.90 0.3050
150% 99.60 0.3491
50% 99.40 0.2801
B 100% 99.50 0.3101
150% 99.70 0.3280
* RSD of six observations

Table 3.11: Analysis of Formulation for HPLC method validation

Formulation Amount % label %RSD*

Labeled Found claim
0.9860 %
A 1% w/w 98.60 0.2223
w/w
0.9921 %
B 1% w/w 99.21 0.3432
w/w
0.9883 %
C 1% w/w 98.83 0.3131
w/w
* RSD of six observations

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3.2 IN VITRO PENETRATION OF ERYTHROMYCIN FORMULATION:

Two Erythromycin formulations were studied for their permeation through the
cadaver skin. The amount of drug permeated through the skin preparation was
estimated by HPLC method and the results are represented in Table 3.11.

In, in vitro experiment for the penetration study of Erythromycin using the human
cadaver skin, the graph plotted against the amount of Erythromycin penetrated with
respect to time for each formulation reveals that, the Erythromycin penetrates steadily
by following the zero order kinetics.

Table 3.12: Amount of Erythromycin Penetrated through the In vitro Skin

Preparation
Time in Amount in µg of
Erythromycin Penetrated
Minutes
through the In vitro Skin
Preparation
Formulations Formulations
A B
Erytop Acnet Cream
Cream
30 11.184 13.66
60 16.822 16.77
90 21.853 20.12

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3.3 IN VIVO DPK OF ERYTHROMYCIN FORMULATION:

Fifteen (15) male volunteers were screened out of that twelve (12) were considered
eligible as per protocol. All the twelve subjects successfully completed period of the
nstudy. Samples from all the male subjects who completed both the periods of the
study were analyzed. The Stratum Corneum samples were used for pharmacokinetic
analysis of Erythromycin.

The subjects were examined within 21 days prior to their first administration of study
medication and assessed for their eligibility to participate. No clinically relevant
abnormalities in physical examinations were reported in subjects who were included
in the study. Post study well being was performed by the physical examination by the
physician.

Vital signs and physical examinations showed no marked changes throughout the
study. All the other subjects who participated in the study were declared healthy at the
post-study examination, except those subjects who failed to follow-up for further post
study examination. All subjects enrolled in the study underwent safety assessments
until the completion of the study.

To the principal investigator’s knowledge, all subjects refrained from using any
prescription and over the counter medications, for two and one weeks respectively,
before the first administration of study medication and for the duration of the study,
with the exception of the study medication taken on clinic days.

No moderate or serious adverse events (AEs) were reported to the investigators.

Potential recall bias of AEs in this study was not likely because only one dose of each
formulation was administered during each treatment; subjects were under medical
surveillance in the clinical unit.

Only one kind of adverse event was reported during the entire clinical study and it
was observed in two volunteers. Subject DPK 14 and DPK 07 had mild laceration on
the right forearm on the dosing day of period I. The nature of the adverse event was
“mild” and resolved on the same day without any concomitant medication. This
adverse event, more than being associated with the formulations, was associated with

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the ‘tape stripping method’ of DPK analysis. Hence, the relationship of the adverse
event to study medication was assumed to be “unlikely” but it could be due to the
‘tape stripping method’ employed for DPK analysis.

Twelve subjects were enrolled in the comparison between two formulations of

Erythromycin (mean age, 25.16 years). The bioequivalence values of the test drug A
were Cmax of 23.767±2.398 µg/ml, tmax of 1.75 ±0.261 h, AUC0-t of 100.507± 10.455
h. µg/ml, AUC0-∞ of 178.286± 22.859 h. µg/ml; of the test drug B Cmax of 24.084±
2.216 µg/ml, tmax of 1.75 ±0.261 h, AUC0-t of 100.586±11.15 h. µg/ml, AUC0-∞ of
179.887± 21.553 h. µg/ml.

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Table 3.13: Occurrence of ADRs

Subject Period Adverse Treatment Intensity Relationship

event to study
medication
Laceration
DPK 14
Period I on right C Mild Unlikely
SAR
forearm
Laceration
DPK 07
Period I on left C Mild Unlikely
VSC
forearm

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Table 3.14: Subject wise concentration data for Formulation A (Erytop Cream)
in µg/cm2

Amount of Erythromycin absorbed in µg/cm2 in time in hours

Subject
0 0.5 1.0 1.5 2.0 3.0 4.0 6.0
Initials

SDS 0 10.12 15.75 24.22 21.22 18.14 16.55 10.66

VPJ 0 13.46 18.25 22.23 25.23 22.12 19.15 15.23

SAA 0 12.50 18.84 20.53 19.23 16.20 13.04 12.32

MDM 0 17.16 22.24 26.24 25.10 22.45 21.23 20.79

ACP 0 14.23 15.64 19.48 19.32 16.44 10.97 9.23

VSC 0 15.00 18.24 26.12 19.35 17.25 14.78 13.86

NAL 0 16.25 18.45 22.56 25.96 24.63 19.36 17.23

BPH 0 14.10 15.83 18.45 22.00 20.56 19.12 17.02

RMD 0 12.25 15.00 16.42 21.13 17.05 15.09 12.65

CKA 0 14.56 18.21 25.98 24.24 21.36 18.05 15.23

YKT 0 8.00 12.96 22.23 24.56 21.32 19.85 16.85

SAR 0 10.56 12.78 17.76 23.73 21.16 19.12 14.43

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Table 3.15: Subject wise concentration data for Formulation B (Acnet Cream) in

Amount of Erythromycin absorbed in µg/cm2 in time in hours

Subject
Initials
0 0.5 1.0 1.5 2.0 3.0 4.0 6.0

SDS
0 11.85 14.56 23.96 21.56 17.56 16.55 11.23

VPJ
0 13.56 16.85 21.12 26.42 21.96 18.98 16.56

SAA
0 12.50 17.99 20.13 18.98 15.62 12.05 12.34

MDM
0 17.66 21.965 26.25 25.45 20.36 22.56 20.98

ACP
0 15.32 17.53 20.56 18.32 16.23 11.25 9.635

VSC
0 14.86 17.85 25.96 18.96 17.25 15.85 10.86

NAL
0 17.00 19.84 24.28 26.60 23.54 18.52 18.88

BPH
0 14.26 16.56 17.32 23.01 19.98 20.10 16.56

RMD
0 12.26 15.86 16.89 22.35 16.08 14.32 11.45

CKA
0 15.20 17.29 24.96 24.56 20.87 19.25 14.62

YKT
0 8.45 11.95 21.86 25.01 21.33 19.45 17.99

SAR
0 11.01 13.02 17.45 23.78 21.31 19.56 15.20

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GRAPHS:

30 Combined graph for Subject 01

25

Conc in µg/cm2
20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.11: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 01

30 Combined graph for Subject 02

25
Conc in µg/cm2

20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.12: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 02

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25 Combined graph for Subject 03

20

Conc in µg/cm2
15

10 Erytop Cream
Acnet Cream
5

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.13: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 03

30 Combined graph for Subject 04

25
Conc in µg/cm2

20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.14: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 04

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25 Combined graph for Subject 05

20

Conc in µg/cm2
15

10 Erytop Cream
Acnet Cream
5

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.15: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 05

30 Combined graph for Subject 06

25
Conc in µg/cm2

20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.16: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 06

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30 Combined graph for Subject 07

25

Conc in µg/cm2
20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.17: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 07

25 Combined graph for Subject 08

20
Conc in µg/cm2

15

10 Erytop Cream
Acnet Cream
5

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.18: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 08

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25 Combined graph for Subject 09

20

Conc in µg/cm2
15

10 Erytop Cream
Acnet Cream
5

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.19: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 09

30 Combined graph for Subject 10

25
Conc in µg/cm2

20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.20: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 10

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30 Combined graph for Subject 11

25

Conc in µg/cm2
20

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.21: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 11

Combined graph for Subject 12

25

20
Conc in µg/cm2

15
Erytop Cream
10 Acnet Cream

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.22: Combined DPK Time Vs Concentration Profile of Formulations A

and B for Subject 12

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Results and Discussion Bundelkhand University, Jhansi

Mean Combined graph for All Subjects

25

20
Conc in µg/cm2

15
Erytop Cream
Acnet Cream
10

0
1 2 3 4 5 6 7 8
Time in Hours

Figure 3.23: Mean Combined DPK Time Vs Concentration Profile of

Formulations A and B for all Subjects

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3.4 IN VITRO – IN VIVO CORRELATION:

The In vitro – In vivo correlation was established using the data obtained from the in
vitro and in vivo experiments carried out. The results are represented by the following
line chart in which the In vivo results are plotted on X-axes and the In vitro results are
plotted on the Y-axes. The results of both the formulations are plotted on the
overlapping plot for the comparison. It was observed that, for each formulation the
correlation could be represented as straight line equation. The characteristics of these
straight lines representing the correlation is summarized in Table 3.15

The linear In vitro - In vivo correlation observed for both Erythromycin Topical
Formulations, reveals the fact that, the penetration and the dermal absorption of
Erythromycin from these formulations are relative phenomenon and indicative of the
results and predictions for each other, even though, the experimental design. Time
duration for the In vitro experiments using the cadaver skin is more time consuming
than the In vivo DPK absorption experiments. This is because in DPK experiments,
multiple skin sampling for the same sampling time is possible and more importantly,
the results are real time and not the predication from the results obtained using the
dead tissue. These results again strengthen the original idea that the DPK studies by
skin stripping experiments are accurate, more reliable, easily reproducible, easy to set
up, and are essentially the next needed incorporations in the regulatory guidelines for
the Bioequivalence studies for the dermal products.

Table 3.16: The In vitro In vivo correlation for Erythromycin Topical

formulations

The In vitro In vivo correlation for

Erythromycin Topical formulations
Straight Line Formulations Formulations
A B
Characteristics
Erytop Acnet Cream
Cream
r2 1 0.999
Slope 3.338 2.547
Intercept -0.098 6.563

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3.5 STATISTICAL RESULTS

Table 3.17: Statistical Data for Formulation A and B

Formulation Cmax Tmax AUClast AUCINF_obs

Erytop Number of Subjects 12 12 12 12
Cream A Mean 23.767 1.75 100.507 178.286
SD 2.398 0.261 10.455 22.859
Min 19.48 1.5 84.32 145.56
Median 24.39 1.75 100.9 178.33
Max 26.24 2 118.25 219.47
CV% 10.1 14.9 10.4 12.8
Geometric Mean 23.651 1.732 100.009 176.955
Acnet Number of Subjects 12 12 12 12
Cream B Mean 24.084 1.75 100.586 179.887
SD 2.216 0.261 11.15 21.553
Min 20.13 1.5 83.58 143.6
Median 24.46 1.75 103.1 186.69
Max 26.61 2 118.45 212.43
CV% 9.2 14.9 11.1 12
Geometric Mean 23.986 1.732 100.011 178.642

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Table 3.18: Pharmacokinetic Comparison between the formulations A and B

Dependent Test FormRef RefGeoLSM TestGeoLSM Ratio[%Ref] CI_90_Lower CI_90_Upper

Ln(AUCINF_obs) A B 178.64 176.96 99.06 87.73 111.85

Ln(AUClast) A B 100.01 100.01 100 93.03 107.48

Ln(Cmax) A B 23.99 23.65 98.60 92.24 105.40

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