A Novel Bioanalytical Method Of Stability

Indicating Samidorphan And Olanzapine By Using

Rp-Hplc

S.Marakatham1, Dr.P.Shanmugapandiyan2*

1Research Scholar, Department of Pharmacy, PRIST UNIVERSITY Deemed to be university, Thanjavur, Tamilnadu,
India
2Research Supervisor, Sathyabama School of pharmacy, Sathyabama Deemed to be university, Chennai, Tamilnadu,

India

Correspondence Author: P.SHANMUGAPANDIYAN

Email: shanmugapandiyan@gmail.com
DOI: 10.47750/pnr.2022.13.S07.751

A simple method was develop for the simultaneous estimation method for Samidorphan and Olanzapine in plasma using the
Dolutegravir as internal standard by RP-HPLC technique. The conditions used are stationary phase Inertsil 250 4.6mm, 5m,
Mobile phase is 0.01N Ammonium acetate Buffer and Acetonitrile 60:40 and flow rate was found to be 1.0ml/min, detection wave
length was 228 nm, temperature was set to 30oC and diluent was mobile phase Retention time of Samidorphan and Olanzapine
were found to be 2.909 min and 3.408min. % CV of the Samidorphan and Olanzapine was found to be 0.42% and 0.52%. %
Recovery was obtained as 99.96% and 101.47%. The linearity concentration is in the range of 2.750-275.000 for Samidorphan and
4.750-475.000 for Olanzapine and linearity is (r2 = 0.999). Further, the method was done as per the ICH guidelines and found to
be well within the acceptable range.

Key words: Samidorphan, Olanzapine, RP-HPLC

INTRODUCTION

Samidron is a 17-cyclopropylmethyl-4,14-dihydroxy-6-oxomorphinan-3-carboxamide. Olanzapine is a 2-methyl-4-4-

methyl-1-piperazinyl-10h-thien benzodiazepine. These drug are used for the management of schizophrenia and bi
polar disorders in patients

Materials:

Samidorphan and Olanzapine, Distilled water, Acetonitrile, Ammonium Acetate buffer,

Triethylamine,Orthophosphoric acid All the above materials are from Rankem

Methods:

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Buffer: 0.01N Ammonium Acetate

0.77g of of Ammonium Acetate was weighed in a 1L of Standard flask thawed in 900ml of water added and
degasses to sonicate and finally made up to 1000ml with water then add 1ml of Triethylamine and P H is adjusted to
3.0 with dil. Orthophosphoric acid solution.

Preparation of Samidorphan Stock solution (0.11 mg/ml):

0.11mg/ml of drug was prepared by dissolving 11mg in 100ml of diluent.

Preparation of Olanzapine Stock solution (0.19 mg/ml):

0.19 mg/ml of drug was prepared by dissolving 19mg in 100ml of diluent

Preparation of Samidorphan Spiking Solutions (2.75ng/ml to 275 ng/ml):

The above samidorphan stock solution 0.01ml, 0.02ml, 0.03ml, 0.2ml, 0.5ml, 0.6ml, 0.8ml and 1.0 ml was pipette
out and transfer to 8 individual flask of 10 ml standard flask and dissolved to the mark with diluent to produce 110
ng/ml, 220 ng/ml, 330 ng/ml, 2200 ng/ml, 5500 ng/ml, 6600 ng/ml, 8800 ng/ml and 11000 ng/ml.

Calibration standards (CS) and quality control (QC) samples were done by spiking blank plasma with working stock
dilutions of drug to produce 2.75 ng/ml, 5.5 ng/ml, 8.25 ng/ml, 55 ng/ ml, 137.5 ng/ ml, 165ng/ ml, 220ng/ ml and
275 ng/ ml.

Preparation of Olanzapine Spiking Solutions (4.75 ng/ ml to 475 ng/ ml):

From the above stock solution 0.01ml, 0.02ml, 0.03ml, 0.2ml, 0.5ml, 0.6ml, 0.8ml and 1.0 ml was pipette and transfer
to 8 individual flask of10 ml Standard flask and dissolved to the mark with diluent to produce 190 ng/ml, 380 ng/ml,
570 ng/ml, 3800 ng/ml, 9500 ng/ml, 11400 ng/ml, 15200 ng/ml and 19000ng/ml.

Calibration standards (CS) and quality control (QC) samples were done by spiking blank plasma with working stock
dilutions of drug to produce 4.75 ng/ml, 9.5 ng/ml, 14.25 ng/ml, 95 ng/ml, 237.5 ng/ml, 285 ng/ml, 380 ng/ml and
475 ng/ml.

Preparation of internal standard Solution (675 ng/ml):

10 mg of Dolutegravir in 10 ml conical flask and mixed with diluent. From stock add 1 ml of solution into 10 ml
Standard flask and mixed with diluent, from this solution take 0.3ml of solution into 10ml Standard flask and mixed
with diluent to produce 3000 ng/ml solutions.

Finally internal standard sample was prepared by spiking blank plasma to produce 675ng/ml.

Validation: METHOD VALIDATION

System suitability of Samidorphan and Olanzapine

Drug ISTD Area

Drug Name File Name Drug Area ISTD Area
RT (min) RT (min) Ratio
5.234 3.595 0.19683
MEAN 6.631 3.625 0.50444

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 0.0221 0.0347 0.001367
SD 0.0344 0.0143 0.011593
0.42 0.96 0.69
%CV 0.52 0.39 2.30

Table no 1: System Suitability of Samidorphan and Olanzapine

Matrix factor evalution of Samidorphan and Olanzapine

Batch ID Date
HQC LQC
Standard Concentration (µg/ml)
220.000 8.250
380.000 14.250
S. No. Plasma Lot No.
187.000-253.000
7.013-9.488
323.000-437.000
12.113-16.388

Calculated Concentration (µg/ml)

n 18 18
215.9681 8.1357
Mean
381.7575 14.2163
3.57836 0.15681
SD
4.63303 0.36203
1.66 1.93
% CV
1.21 2.55
98.17 98.61
% Mean Accuracy
100.46 99.76
No. of QC Failed 0 0

Table no 2: Matrix factor evaluation of Samidorphan and Olanzapine

2. Linearity of Samidorphan and Olanzapine

Linearity of Samidorphan
Batch ID STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8

Standard Concentration (ng/mL)

2.750 5.500 8.250 55.000 137.500 165.00022 220.000 275.000

0.000
Standard Concentration Range (ng/mL)

2.200- 4.675- 7.013- 46.750- 116.875- 140.250- 187.000- 233.750-

3.300 6.325 9.488 63.750 158.125 189.750 253.000 316.250

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 Back Calculated Concentration (ng/mL)

P&A1 2.792 5.386 8.240 54.690 134.750 159.312 216.045 272.429

P&A2 2.781 5.414 8.181 54.711 136.153 164.011 212.501 272.150

P&A3 2.678 5.384 8.257 52.727 134.756 162.670 218.447 276.452

n 3 3 3 3 3 3 3 3

Mean 2.7503 5.3947 8.2260 54.0427 135.219 161.9777 215.6643 273.6770

7
SD 0.0628 0.07677 0.03989 1.13945 0.80830 2.41247 2.99122 2.40727
8
%CV 2.29 0.37 0.48 2.11 0.60 1.49 1.39 0.88

% Mean 100.01 98.08 99.71 98.26 98.34 98.17 98.03 99.52

Accuracy

Table no 3: Linearity of Samidorphan

Linearity of Olanzapine
Batch ID STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8

Standard Concentration (ng/mL)

4.750 9.500 14.25 95.000 237.500 285.000 380.000 475.000

0
Standard Concentration Range (ng/mL)

3.800- 8.075- 12.11 80.75- 201.87- 242.250- 323.000- 408.750-

5.700 10.925 3- 109.25 109.25 327.750 437.000 546.250
16.38
8
Back Calculated Concentration (ng/mL)

P&A1 4.83 9.56 14.63 95.66 236.77 286.84 380.21 475.56

0
P&A2 4.82 9.49 14.31 94.36 239.89 285.07 379.20 473.70
0
P&A3 4.86 9.49 13.98 94.71 231.17 285.25 380.36 476.79
0
n 3 3 3 3 3 3 3 3

Mean 4.8376 9.5133 14.30 94.9100 235.943 285.3200 379.9233 475.3500

67 3
SD 0.0208 0.04047 0.325 0.67268 4.41839 0.97411 0.63090 1.55567
2 07
%CV 0.43 0.42 2.27 0.71 1.87 0.34 0.17 0.33

% Mean 101.82 100.14 100.4 99.91 99.34 100.25 99.98 100.07

Accuracy 0

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Table no 4: Linearity of Olanzapine

Fig no 1: calibration curve of Samidorphan

0.0300
0.0250 y = 0.0116x + 0.0001
R² = 0.9999
0.0200
0.0150 Series1
0.0100 Linear (Series1)
0.0050
0.0000
0 0.5 1 1.5 2 2.5 3

Fig no 1: calibration curve of Olanzapine

1
y = 0.0016x + 0.0016
0.8 R² = 0.9992
0.6
Series1
0.4
Linear (Series1)
0.2
0
0 100 200 300 400 500

3.Precision&Accuracy (intra-day runs of Samidorphan and Olanzapine)

HQC MQC1 LQC LLOQ QC

Standard Concentration (µg/mL)

220 137.500 8.250 2.750

380 237.500 14.250 4.750

Batch ID Date Nominal Concentration Range (µg/mL)

187.000-253.000 116.875-158.125 7.013-9.488 2.200-3.300

323.000-437.000 201.875-273.125 12.113-16.388 3.800-5.700

Back Calculated Concentration (µg/mL)

n 6 6 6 6

216.0887 134.7637 8.1468 2.7480

Mean
380.0500 237.9117 14.2200 4.8333

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 2.83324 1.37909 0.21096 0.05969
SD
0.98801 1.37149 0.64817 0.17614
1.31 1.02 2.59 2.17
%CV
0.26 0.58 4.56 3.64
98.22 98.01 98.75 99.93
% Mean Accuracy
100.01 100.17 99.79 101.75

inter Batch Precision and Accuracy

n 18 18 18 18

216.0402 135.0419 8.1536 2.7489

Mean
380.1672 238.1989 14.3072 4.8200
2.21548 1.21511 0.14300 0.04444
SD
0.61811 1.30376 0.48711 0.14046
1.03 0.90 1.75 1.62
%CV
0.16 0.55 3.40 2.91
98.20 98.21 98.83 99.96
% Mean Accuracy
100.04 100.29 100.40 101.47

Table no 4: precision data for intra-day runs of Samidorphan and Olanzapine

4.Recovery

Recovery of Samidorphan and Olanzapine

Batch ID
HQC MQC1 LQC
S. No. Un extracted Extracted Un extracted Extracted Un extracted Extracted
Report Report Report Report Report Report
n 6 6 6 6 6 6
145225 141948 90636 90092 5472 5431
Mean
285798 282178 173602 175377 10169 10174
2693.95 1305.82 783.27 404.06 40.95 31.66
SD
2251.13 1450.77 3118.55 2376.43 35.26 25.90
1.86 0.92 0.86 0.45 0.75 0.58
% CV
0.79 0.51 1.80 1.36 0.35 0.25
97.74 99.40 99.26
% Mean Recovery
98.73 101.02 100.05
Overall % Mean 98.801
Recovery 99.934
0.9185
Overall SD
1.1488
0.93
Overall % CV
1.15

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Table no 5: Recovery of Dolutegravi

Recovery - Internal standard

Acquisition Batch ID
Un extracted Area
S.No. Extracted Area Ratio
Ratio
n 6 6
Mean 468166.7 461813.0
SD 4430.31 2344.72
% CV 0.95 0.51 0.25
% Mean Recovery 98.64

Table no 6: Recovery of Lumacaftor (IS)

5. Stabilities

Long term stock solution stability at Zero Samidorphan and Olanzapine

Batch ID

HQC LQC
Standard Concentration (µg/mL)
220.000 8.250
Replicate No. 380.000 14.250
Standard Concentration Range (µg/mL)
187.000-253.000 7.013-9.488
323.000-437.000 12.113-16.388
Calculated Concentration (µg/mL)
n 6 6
216.1487 8.1423
Mean
380.5250 14.3217
2.83580 0.11637
SD
1.17716 0.21414
1.31 1.43
% CV
0.31 1.50
98.25 98.69
% Mean Accuracy
100.14 100.50

Table no 7: Long term stock solution stability at Zero Samidorphan and Olanzapine

stability of matrix samples at -28 ± 5 C for 37 days (Samidorphan and Olanzapine)

Date
HQC LQC
Standard Concentration (ng/mL)

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220.000 220.000 8.250 8.250

380.000 380.000 14.250 14.250

Standard Concentration Range (ng/mL)
(187.000-253.000) (187.000-253.000) (7.013-9.488) (7.013-9.488)

(323.000-437.000) (323.000-437.000) (12.113-16.388) (12.113-16.388)

Calculated Concentration (ng/mL)
Assessment Samples Stability Samples Assessment Samples Stability Samples
6 6 6 6
216.1033 216.2867 8.1409 8.1523

381.3967 379.8567 14.5367 14.2550

2.33142 2.65299 0.16862 0.13572\0.27472

0.90139 1.31527 0.28374

1.08 1.23 2.07 1.66

0.24 0.35 1.95 1.93

98.23 98.31 98.68 98.82

100.37 99.96 102.01 100.04

100.08 100.14

99.60 98.06

SUMMARY AND CONCLUSION

Parameters SAMIDORPHAN OLANZAPINE

LIMIT
Linearity 2.750- 4.750-475.000ng/ml
Range(ng/ml) 275.000ng/ml R2< 1
Regression coefficient 0.999 0.999
Slope 0.0116 0.0001

Intercept 0.0001 0.001

Regression equation y = 0.0116x + y = 0.001x + 0.001
(Y=mx+c) 0.0001
Specificity Specific Specific No interfering of any
peak
System precision %CV 1.31 0.26 NMT 15.0%

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 Method precision %CV 1.03 0.16 NMT 15.0%

Accuracy %recovery 99.96% 101.47% 80-120%

Table no 10: summary for Samidorphan and Olanzapine

Conclusion

A simple, accurate method was developed for the estimation of the Samidorphan and Olanzapinein plasma using the
Dolutegravi as internal standard. Retention time of Samidorphan and Olanzapinewere found to be2.909 min and
3.408min. %CV of Samidorphan and Olanzapinethe was found tobe0.42% and 0.52%. % Recovery was obtained
as99.96% and 101.47%. The linearity concentration is in the range of 2.750-275.000 for Samidorphan and 4.750-
475.000 for Olanzapine and linearity is (r2 = 0.999) and stability studies were performed and its values within the
ICH limits. Further, the stated method was done as per ICH guidelines and within the suitable range.

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