Appendix II- Bioanalytical Method Development and...

Appendix II- Bioanalytical Method Development and Validation

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A2. Bioanalytical method development

1. Optimization of chromatographic conditions

Method development and optimization of chromatographic parameters is of utmost important for

validating a method in biological matrix (Plasma).

1.1 Selection of wavelength

A stock solution of 1.0 mg/ml of Silybin was prepared in methanol. Solution was diluted to

10μg/ml and scanned between 200-800nm using PDA detector. The obtained absorption

maximum (λ max) for Silybin was 286nm and used as working wavelength for Silybin.

1.2 Selection of chromatographic method

Silybin (CAS No. - 65666-07-1) is available as yellow color powder. Its molecular weight is

482.44 Da, molecular formula C25H22O10, log value 2.4, melting point 230 - 233 C. It is soluble

in methanol and insoluble in water and oil. It is a mixture of flavonoids extracted from seeds of

the Silybum marianum. It primarily consists of silybin and its isomers viz., silicristin and

silidianin. The RP-HPLC was selected for the initial separation because of its simplicity,

suitability, reproducibility, ruggedness, and wide acceptability.

Figure AII.1. Structure of Silybin A and Silybin B


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Table AII.1. Physical properties of silybin

Name Silybin

IUPAC name

(2R,3S)-3,5,7-Trihydroxy-2-[(2R)-2-(4-hydroxy-3-

methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-

benzodioxin-6-yl]-2,3-dihydro-4H-chromen-4-one

Physical state yellowish powder

Melting point 230 - 233 C

Formula C25H22O10

Mol. wt. 482.44 Da

Solubility Soluble in methanol and insoluble in water and oil

LogP/Hydrophobicity 2.4

pKa 6.418

2. Chromatographic conditions

HPLC : Shimadzu- SPD-M20A

Compound : Silybin (sigma-s0292-10g) Batch no.-107k0762

Column : RP-C18 (Merck® 100 RP-18 endcapped (5µm))

Chromatographic mode : Reverse phase

Run time : 27.0 min

Detection wavelength : 286nm

Flow rate : 1 ml/min

Injection volume : 20 µL


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3. Mobile phase selection

The following mobile phases were used and the chromatograms were recorded.

Trial (1) ACN : Water (50:50%v/v), pH 3.5 adjusted with glacial acetic acid

Comment: poor resolution and poor recovery

Trial (2) ACN : Phosphate buffer (40:60%v/v), pH 3.5 adjusted with glacial acetic acid

Comment: Poor resolution

Trial (3) ACN : Phosphate buffer (20:80%v/v) pH3.5

Comment: poor resolution

Trial (4) ACN : Phosphate buffer (70:30%v/v), pH 3.5

Comment: poor resolution

Trial (5) Methanol : Water (40:60%v/v), pH 3.5 adjusted with glacial acetic acid

Comment: Poor resolution

Trial (6) Methanol : Water (50:50%v/v), pH 3.5 adjusted with glacial acetic acid

Comment: Better resolution and good recovery

From the above trials, trial 6 was found to be better for elution of various component of Silybin.

The retention time of Silybin A and Silybin B was 17.7 and 19.8 min respectively in the same

condition.


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4. PREPARATION OF SOLUTIONS

Rinsing solution [ Methanol : Water (50:50%v/v)]

250 mL of methanol was added into a glass bottle containing 250 mL of water and the

resulting mixture was mixed well and sonicated for 10 minutes.

Buffer (0.002%v/v Acetic acid)

999.80mL milli-Q water and 20 µl glacial acetic acid was mixed and sonicated.

Diluent [ Methanol : Water (50:50%v/v) pH3.5]

100 mL of methanol was transferred into a glass bottle containing 100 mL of water,

mixed well and sonicated.

5. Selection of internal standard (ISTD)

Selection of internal standard (ISTD) was based on structural, physico-chemical and

pharmacological similarity of drug and ISTD. Various standard drugs were chromatographed

viz., rutin, quercetin and alpha napthol. Among these α- napthol was well resolved. Hence it was

selected as internal standard (ISTD) for the estimation of Silybin from plasma.

6. PREPARATION OF STOCK SOLUTION

Main Stock solution of 1mg/mL was prepared. From that intermediate solution of

2µg/mL was prepared and was used for the preparation of calibration curve.

Calibration Curve

A standard plot was made using the following concentrations:- 15, 30, 60, 120, 180, 200 and

320ng/mL of Silybin.

Internal Standard Working Solution (250ng/mL) (IS/WS)

α-Napthol of concentration 250ng/mL was prepared and used as internal standard.

7. OPTIMIZATION OF EXTRACTION TECHNIQUE

Various extraction procedures were tried to extract analyte and internal standard from plasma

concentrating on better and consistent recovery without interfering with the quantification of

analyte and ISTD. Solid phase extraction, liquid liquid extraction and protein precipitation

methods were tried. Out of these three methods, protein precipitation was found to be economic,


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simple and gave good recovery. So it was selected for further validation. Various protein

precipitants like acetonitrile, methanol, 1:1 ratio of methanol and acetonitrile were used. All

these precipitants were also tried in cold condition. 1:1 ratio of methanol and acetonitrile gave

the best recovery in cold condition among all tried precipitant.

Sample Extraction Procedure (Protein Precipitation Method)

To 95μL of plasma, 5μL of analyte and 5μL of internal standard working solution was added.

300mL of 1:1 ratio of methanol and acetonitrile (extraction solution) was added and vortexed for

5min and after that sample was centrifuged at 14000rpm. Supernatant was separated and 40μL

was injected into the HPLC system.


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BIOANALYTICAL METHOD VALIDATION

Bioanalytical method for silybin was validated as per USFDA guidelines. The parameters

evaluated were: system suitability, carryover, specificity, sensitivity, linearity, precision and

accuracy, recovery and stability. The acceptance criteria for these parameters are summarized in

table AII.2.

Table AII.2. Method validation acceptance criteria for following parameters

Parameters Acceptance criteria

System suitability eak area ratio: CV ≤ 5.0 .

etention times: CV ≤ 2.0

Carryover ≤20 of response of mean extracted LLOQ Area

response at retention time of analyte

≤5 of response of mean extracted IST .

Linearity r2≥0.9800

Specificity If any peak is present at RT of analyte its response

should be ≤20 of extracted LLOQ.

Sensitivity % deviation from nominal concentration should be

within ±20%.

CV of calculated concentration should be ≤ 20 .

Precision and Accuracy The calculated concentration at each QC level should

be within 85-115% of their nominal concentration

except for LLOQC where it must be within 80-

120%.

The precision determined at each concentration level

should not exceed 15% of the coefficient of variation

(%CV) except for the LLOQ, where it should not

exceed 20% of the %CV.

Recovery Recovery across QC level should be ±15%

Stock solution stability Mean % change should be ±10%

Stability in plasma Mean % change should be ±15%


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Preparation of calibration curve standard and Quality control samples:- Aqueous dilution of

Silybin was prepared by taking respective volume of stock solution as mentioned in table AII.3

and diluted with diluents to get the desired concentration.

Table AII.3. Calibration curve and quality control sample dilution

SILYBIN SPIKING STOCK PREPARATION

Weight taken (mg)

Silybin α-Napthol

2µg/mL 250ng

Level

Vol. of

Intermediate stock

taken (mL)

Final vol

(mL)

Spiking stock Conc.

(ng/mL)

Plasma Conc

(ng/mL)*

STD-1 0.075

10.00 300.000 15.000

STD-2 0.150

10.00 600.000 30.000

STD-3 0.300

10.00 1200.000 60.000

STD-4 0.600

10.00 2400.000 120.000

STD-5 0.900

10.00 3600.000 180.000

STD-6 1.000

10.00 4000.000 200.000

STD-7 1.600

10.00 6400.000 320.000

LLOQC 0.075

10.00 300.000 15.000

LQC 0.225

10.00 900.000 45.000

MQC 0.800

10.00 3200.000 160.000

HQC 1.450

10.00 5800.000 290.000

* Percentage of spiking = 5.0%v/v


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8.1. Bulk spiking

Preparation of stock solution of silybin

A stock solution of Silybin was prepared for bulk spiking of calibration standards and quality

control samples for the method validation. The aqueous spiking stock solution of Silybin for

seven calibration standard concentrations ranging from 15.000 to 320.00 ng/mL and for LLOQC,

LQC, MQC and HQC of concentrations 15.000 ng/mL, 45.00 ng/mL, 160.00 ng/mL and 290.00

ng/mL was prepared in diluent. The working stock solution of α-napthol (ISTD) was prepared in

same diluents at concentration of 250 ng/mL. All seven standards and quality control samples

were bulk spiked in plasma and stored at -70 ºC. Aliquots of these stock solution were also

stored under refrigeration at 2-8ºC for determination of stock solution stability.

8.2. Preparation of plasma calibration curve, quality control samples, system suitability

sample

The drug free plasma lots stored at -70ºC were withdrawn and allowed to thaw at room

temperature and were then vortexed adequately before use.

8.2.1. Preparation of blank sample:

To 95 μL of drug free plasma, 5 μL of diluents were added and sample was extracted as per

sample extraction procedure.

8.2.2 Preparation of Zero sample:

To 95 μL of drug plasma, 5 μL of diluents and 5 μL of Alpha naphthol (250ng/mL) were added

and sample was extracted as per sample extraction procedure.

8.2.3 Preparation of calibration standards:

The plasma concentration ranges of Silybin are 15, 30, 60, 120, 180, 200 and 320 (ng/mL).

These were prepared by adding 5 μL of ST -1 to STD- respectively and 5 μL of Alpha

naphthol (250 ng/mL) to 95 μL of plasma.


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8.2.4. Preparation of system suitability:

Five microliters of aqueous MQC spiking stock solution of Silybin, 5 µL of working stock

solution of alpha napthol (ISTD) and 300 µL of reconstitution solution (RS) was taken into a vial

containing 350 µL insert and the auto sampler was programmed to inject 40 µL into the HPLC

system.

8. Result:

9.1. System suitability

System suitability test was done separately before start of every new sequence. Five injections of

above system suitability solution were injected in HPLC. Percentage CV was found to be less

than 1.29 for peak area which was within the acceptance criteria. (Table AII.4)

Table AII.4. System suitability

S. No. Area ratio of Silybin to ISTD

Batch-1 Batch-2 Batch-3 Batch-4

1 0.933 0.924 0.932 0.931

2 0.912 0.942 0.935 0.925

3 0.941 0.934 0.941 0.941

4 0.942 0.937 0.94 0.944

5 0.932 0.938 0.935 0.935

Mean 0.932 0.935 0.9366 0.9352

S.D. 0.012 0.007 0.004 0.008

% CV 1.29 0.73 0.40 0.82

9.2. Specificity / selectivity:

Specificity of a method is the ability of method to differentiate and quantify the analyte in the

presence of other components in the sample. For selectivity, at least six independent lots of


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blank samples of the appropriate biological matrix was analysed.

There was no interference of endogenous material in the quantification of Silybin and IS (α-

Napthol) (Table AII.5)

Table AII.5. Screening and selectivity of silybin in six different lots of Wistar rat plasma

Sr.

No.

Blank

Plasma

ID

Interference

at Rt of

analyte

LLOQC

area

% of

LLOQC

Interference at

Rt of ISTD ISTD area

%

ISTD

1 BP-01 0.00 3766 0.00 0.00 192004 0.00

2 BP-02 0.00 3876 0.00 0.00 202900 0.00

3 BP-03 0.00 3789 0.00 0.00 197288 0.00

4 BP-04 0.00 3827 0.00 0.00 196700 0.00

5 BP-05 0.00 3749 0.00 0.00 197217 0.00

6 BP-06 0.00 3687 0.00 0.00 197501 0.00

Mean 3782 197268

S.D. 65.237 3458.584

% CV 1.72 1.75


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Figure AII.2. Representative Chromatogram of LLOQ in pooled Wistar rat plasma

Figure AII.3. Representative Chromatogram of blank Wistar rat plasma

Figure AII.4. Representative Chromatogram of zero sample in Wistar rat plasma


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9.3. Carry over check:

Carry over test was performed to find out that there is no carryover of analyte from the previous

injection. The experiment was performed at ULOQ level.

There was no significant carry over effect observed in blank sample . Results are shown in table-

AII.6.

Table AII.6. Carry over check for silybin

S.

No. Sample Name

Area % area of LLOQ

Silybin α- napthol Silybin α- napthol

1 Blank-RS 0 0 NA NA

2 AQ-ULOQ 55526 185478 NA NA

3 Blank-RS 0 0 0 0

4 AQ-ULOQ 56864 197563 NA NA

5 Blank-RS 0 0 0 0

6 EXT-Blank plasma 0 0 0 0

7 EXT-ULOQ 55896 204765 NA NA


9 EXT-ULOQ 54859 205755 NA NA


11 LLOQ-1 3876 192004

12 LLOQ-2 3789 202900

13 LLOQ-3 3827 197288

Mean 3831 197397


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9.4. Sensitivity

Sensitivity test is performed for establishing the reproducibility and accuracy of bioanalytical

method at lowest limit of quantification. Percentage CV determination at LLOQ level was found

to be 5.024% for silybin. (Table AII.7)

Table AII.7. Sensitivity for silybin

Sr. No. LLOQ (ng/mL) Specified conc. (15ng/mL)

Calculated concentration % Nominal concentration

1 14.779 98.527

2 16.319 108.793

3 14.543 96.951

4 16.136 107.571

5 15.615 104.099

6 16.294 108.628

N 6

Mean calc. conc. 15.614

S.D. 0.784

% CV 5.024

Mean % Nominal

concentration

104.095

9.5. Linearity / calibration (standard) curve

A calibration (standard) curve is the relationship between instrument response and known

concentrations of the analyte. (Table AII.8 and Table AII.9)


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A calibration curve should consist of:

A blank sample (matrix sample processed without internal standard)

A zero sample (matrix sample processed with internal standard), and

Six to eight non-zero samples covering the expected range, including LLOQ.

Calibration curves were found to be consistently accurate and precise over 15-320 ng/mL.

Analyte concentration at each calibration level was back calculated from the calibration

curves.

The calibration curve meets the acceptance criteria of r2 ≥ 0.98. ( Table AII.8)

Table AII.8. Summary of calibration curve parameters of silybin

Linearity Slope Intercept r2

1 0.00090 0.00750 0.9993

2 0.00086 0.00851 0.9997

3 0.00091 0.00830 0.9993

4 0.00079 0.00762 0.9992


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Table AII.9. Back-calculated concentrations of calibration curve standards of silybin in Wistar rat plasma

Linearity

Conc. (ng/mL)

STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7

Sr. No. 15.000 30.000 60.000 120.000 180.000 200.000 320.000

1 12.842 26.583 60.456 119.752 173.041 191.190 304.272

2 13.453 27.703 66.446 125.816 160.906 185.721 326.792

3 14.186 27.143 68.244 129.455 165.760 190.643 306.524

4 15.287 27.703 66.267 127.636 173.648 187.909 305.961

Mean 13.942 27.283 65.353 125.665 168.339 188.866 310.888

S.D. 1.05 0.54 3.38 4.21 6.12 2.54 10.65

% CV 7.543 1.965 5.179 3.352 3.633 1.345 3.424

% Accuracy 92.95 90.94 108.92 104.72 93.52 94.43 97.15


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9.6. PRECISION AND ACCURACY (P&A)

The accuracy of an analytical method describes the closeness of mean test results obtained by

the method to the true value (concentration) of the analyte. It is determined by replicate analysis

of samples containing known amounts of the analyte. Precision and accuracy was checked at

quality control samples eg. LLOQC, LQC, MQC & HQC.

9.6.1. Intra-batch or within-batch precision & accuracy

% CV & % nominal concentration for QC samples i.e. LLOQC, LQC, MQC and HQC

samples of Silybin for all four precision and accuracy batches were calculated.

% Accuracy for LLOQC was 93.51 and ranging from 98.08 to102.657 for LQC, MQC

and HQC. % Precision for LLOQC was 9.30 for LQC, MQC and HQC it ranges from

2.38 to 6.03%.

9.6.2. Inter-batch or between-batch precision & accuracy

The inter-day precision and accuracy of the day were estimated from the assay of QC

samples of different days. (Table AII.10)

Global mean % accuracy for quality control samples i.e. LLOQC, LQC, MQC and HQC

samples were 89.31, 98.29, 98.47 and 102.29 respectively, which are within acceptance

criteria of 85-115% for LQC, MQC and HQC samples and 80-120% for LLOQC.

% global precision for quality control samples i.e. LLOQC, LQC, MQC and HQC

samples were 8.74, 3.81, 3.23 and 6.60 respectively, which are within acceptance criteria

of ≤ 15 for LQC, MQC and HQC samples and of ≤ 20 for LLOQC.


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Table AII.10. Inter- day Precision & Accuracy batch for silybin

Specified conc.

(ng/mL)

LLOQC

(15ng/mL)

LQC

(45ng/mL)

MQC

(160ng/mL)

HQC

(290ng/mL)

Batch-1

1 16.295 42.619 156.578 279.006

2 14.359 47.053 164.030 299.486

3 12.318 43.499 157.765 303.932

4 13.945 44.055 153.201 312.550

5 13.709 42.276 156.790 273.367

6 13.538 45.321 154.609 317.899

Batch-2

7 13.916 42.619 156.578 264.804

8 10.970 47.053 164.030 299.486

9 12.318 43.499 157.765 303.932

10 13.945 44.055 152.717 312.550

11 13.709 42.276 156.790 273.367

12 12.460 45.321 154.609 317.899

Batch-3

13 15.021 43.745 160.555 259.312

14 11.523 47.618 166.308 302.163

15 12.318 44.006 160.031 308.543

16 14.508 44.448 150.494 313.127

17 14.288 42.389 153.444 273.367

18 13.007 45.491 149.093 321.321

Batch-4

19 13.915 42.410 160.210 273.229

20 13.719 46.888 165.772 313.002

21 12.318 43.481 159.432 297.439

22 13.302 43.919 150.893 313.127

23 12.448 42.184 153.201 273.367

24 13.658 45.327 166.446 313.072

Number 24 24 24 24

Mean 13.396 44.231 157.556 296.639

SD 1.171 1.687 5.096 19.592

%CV 8.74 3.81 3.23 6.60

Mean % nominal

conc. 89.31 98.29 98.47 102.29


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9.7. EXTRACTION RECOVERY:

Recovery pertains to the extraction efficiency of an analytical method within the limits of

variability. Recovery is calculated by comparing detector response obtained from extracted QC

sample to that of true concentration of pure authentic standard.

Recovery of silybin at LQC, MQC and HQC level were 102.72%, 98.03% and 98.78%

respectively. Mean recovery of α-napthol was found to be 88.44% . Recovery of analyte and

ISTD was found to be consistent, precise and reproducible.

9.8. STABILITY STUDIES:

Drug stability in a biological fluid depends on the storage conditions, chemical properties of the

drug, matrix, and container system. This study evaluated the stability of the analyte during

sample collection and handling, after long-term (frozen at the intended storage temperature) and

short-term (bench top, room temperature) storage, and after going through freeze and thaw

cycles and the analytical process. Normally, as an example, the following stability tests should

be evaluated:

Freeze and thaw stability of the analyte in the matrix (plasma) from freezer storage

conditions to room temperature.

Bench top stability of the analyte in matrix (plasma) at room temperature.

Long term stability of the analyte in matrix (plasma) stored in the freezer for long time

In-injector/ auto sampler stability of the processed sample at injector or auto sampler

temperature.

Process stability: Process stability was conducted with six replicates of LQC and HQC

samples. For this, samples were kept for 2 hours before vortexing at room temperature

and 2 hours after reconstitution at room temperature to ensure the stability of analytes and

ISTD during sample preparation.

Main stock stability was performed at MQC level. Main stock was stable for 6 days at 2-8°C

with mean % change of 0.57%.


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The stability tests performed for silybin in plasma were bench top stability, process stability, in-

injector stability, freeze and thaw stability and long term stability. Result of stability shows that

silybin was stable for all the above stabilities performed. (Table AII.11)

Table AII.11. Stability study

QC level Stability Type Mean SD %CV %Accuracy % Change

LQC Bench top 45.450 2.770 6.094 101.001 2.67

Process for 4hr 45.488 1.476 3.246 101.085 2.76

In-injector 44.976 0.627 1.394 99.946 1.60

3rd

Freeze/Thaw 45.301 1.522 3.359 100.668 2.33

15 days at -70°C 45.724 2.117 4.631 101.608 3.29

HQC Bench top 301.542 16.309 5.409 103.980 1.29

Process for 4hr 305.450 11.224 3.675 105.328 2.60

In-injector 303.436 12.813 4.223 104.633 1.92

3rd

Freeze/Thaw 292.288 7.684 2.629 100.789 -1.82

15 days at -70°C 303.606 10.569 3.481 104.692 1.98

9.9. RUGGEDNESS

Ruggedness of the method was checked using different lots of column and reagents. One

precision and accuracy (P&A) batch was carried out with different column of same type

(different lot) and one P&A batch was carried out with reagents of different manufacturer.

For column and solvent ruggedness, different lot of columns and solvents of different

manufacturers were used and a linearity followed by precision and accuracy was performed for


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quality control samples at LLOQC, LQC, MQC and HQC levels and % Accuracy were 97.18,

101.38, 99.30 and 98.82% respectively for Silybin in column ruggedness. % Accuracy for

quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 97.73, 101.53, 98.24

and 98.07% respectively in solvent ruggedness. % CV for quality control samples i.e. LLOQC,

LQC, MQC and HQC samples were 6.78, 4.20, 4.40 and 4.60% respectively for Silybin in

column ruggedness. % CV for quality control samples i.e. LLOQC, LQC, MQC and HQC

samples were 6.16, 3.29, 6.45 and 6.21% respectively for Silybin in solvent ruggedness. All the

values were within acceptance criteria of 85-115% for LQC, MQC and HQC samples and of 80-

120% for LLOQC.

Appendix II- Bioanalytical Method Development and...

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