3. Bioanalytical Method Development and Validation 3.1 ...shodhganga.inflibnet.ac.in/bitstream/10603/4436/12/12_chapter3.pdf · Bioanalytical Method Development and Validation 21

Chapter 3

Bioanalytical Method Development and Validation

21

3. Bioanalytical Method Development and Validation

3.1.Background

A sensitive, specific bioanalytical method is critical for a reliable pharmacokinetic

experiment. Chromatographic techniques, especially, high performance liquid

chromatography (HPLC) coupled with different detection systems is a preferred technique,

routinely employed in bioanalytical laboratories as compared to any other method of analysis

owing to their precision, accuracy, reliability and applicability to large-scale analysis. The

guidance document by USFDA systematically describes method development and validation

procedures required to demonstrate the reliability and reproducibility of the bioanalytical

method(CDER, 2001). According to the guidance document, the fundamental parameters of

validation include accuracy, precision, selectivity, sensitivity, reproducibility and stability.

The process of method validation involves laboratory investigations, documentation and

assessment of performance characteristics and their conformity to the acceptance criteria.

Though many validated methods have been reported, it is not necessary that all of them work

in a given laboratory. This occurs due to different instrument and assay conditions and other

environmental variables. Therefore, for a given laboratory, modification of the reported

method or development of a new method becomes essential. Most of the reported methods

are for human experiments and involve higher plasma volume. Our study was a non-clinical

one, which demanded a simple, economical, sensitive and specific bioanalytical method with

lesser plasma volume. Based on the physico-chemical properties of the drugs and the

available literature, we developed and validated two separate HPLC methods with UV

detection for the estimation of glibenclamide (GLI) and metformin (MET).

GLI is an oral hypoglycemic agent belonging to second-generation sulfonylureas. GLI lowers

the blood glucose by stimulating insulin release from pancreatic beta cells and may also

increase the insulin levels by reducing hepatic clearance of insulin. (Laurence, 2006). MET is

an anti-hyperglycemic drug belonging to the class of biguanides. It acts by reducing hepatic

glucose production and enhancing insulin action on muscle and fat tissue. MET brings down

fasting glucose and insulin levels, improves the lipid profile and causes weight loss in type-2

diabetic patients (Kirpichnikov et al., 2002, Laurence, 2006).

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3.2. Materials and Methods

3.2.1. Reference/Working Standards

Name of the Standard Supplier/Manufacturer

Glibenclamide Micro labs, India

Glimepiride Sigma Chemicals, USA

Metformin Marksans Pharma, India

Atenolol Sigma Chemicals, USA

3.2.2. Reagents, chemicals and Instruments

Chemical/Reagent Grade Manufacturer

Acetonitrile HPLC Labscan,Thailand

Dichloromethane HPLC Labscan,Thailand

Formic acid Suprapur Merck India Ltd, Mumbai

Methanol HPLC Labscan,Thailand

Methyl tertiary butyl ether HPLC Labscan,Thailand

n-Hexane AR S.D Fine Chemicals Ltd, Mumbai.

Potassium dihydrogen

orthophosphate

AR S.D Fine Chemicals Ltd, Mumbai.

Potassium hydroxide AR S.D Fine Chemicals Ltd, Mumbai.

Sodium Hydroxide AR S.D Fine Chemicals Ltd, Mumbai.

Water Milli-Q Millipore, USA

Instrument Brand/model Supplier/manufacturer

HPLC-System Prominence Shimadzu, Japan

Detector (PDA) SPD-M20A Shimadzu, Japan

Analytical column Betasil C18 Thermo Electron corporation

Lichrocart-CN Merck, Germany

Analytical balance Sartorius Sartorius Mechatronics, India

Centrifuge Heraeus Multifuge Thermo Electron corporation

Deep Freezer Carrier Carrier India

Water Purification System Milli-Q Millipore, USA

Multi-Pulse Vortexer Glass Col Terre Haute, USA

Micropipette Eppendorf Eppendorf, Germany

Nitrogen evaporator Turbovap LV Caliper Instruments,USA

Vortexer Spinix Spinix, India

Ultrasonic Cleaner UCB150 Spectral Lab, India

pH meter Orion 3 star Thermo Electron corporation

Hot air oven JRIC 7/A Osworld equipment, Mumbai

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23

3.2.3. Blank Plasma

The blank plasma for method development and validation was obtained from healthy animals

from Central Animal Research Facility, Manipal University, Manipal.

3.2.4. Method Development

Literature review was done to understand the physico-chemical properties of the analytes and

the validated methods reported so far. This was followed by method optimization procedures

involving effect of pH, stationary phase, solvent, mobile phase ratio, flow rate, internal

standard selection and optimization of extraction procedure.

3.2.5. Method development: Glibenclamide

3.2.5.1. Preparation of solutions and buffers

3.2.5.1.1. Mobile Phase [25mM Potassium dihydrogen orthophosphate buffer (pH 6.5)]

1701.125mg of Potassium dihydrogen orthophosphate was dissolved in 500 mL of water and

pH was adjusted to 6.50 with potassium hydroxide. The resulting solution was filtered

through 0.22µ millipore membrane filter using vacuum filtration system.

3.2.5.1.2. Rinsing and reconstitution 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.

3.2.5.1.3. Diluent [ methanol : water (50:50% v/v)]

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

well and sonicated.

3.2.5.1.4. Extraction Buffer (0.20% v/v formic acid in water )

400µL formic acid was mixed with 200 mL water and sonicated

3.2.5.2. Preparation of Stock solutions

3.2.5.2.1. Glibenclamide main stock (1.000 mg/mL)

10.000 mg of Glibenclamide was taken in 10 mL volumetric flask and 5 mL methanol was

added and sonicated to dissolve. The volume was made up to the mark with methanol.

Chapter 3


24

3.2.5.2.2. Glibenclamide intermediate stock (200.000 µg/mL)

2 mL of Glibenclamide main stock was taken in a 10 mL volumetric flask and made up to the

volume by diluent.

3.2.5.2.3. Internal standard (Glimepiride) stock solution (200.000µg/mL)

2.000 mg of Glimepride was taken in 10 mL volumetric flask and 5 mL methanol was added

and sonicated to dissolve well. The volume was made up to the mark with methanol.

3.2.5.2.4. ISTD Working standard solution (40.000 µg/mL)

Working standard solution was prepared by diluting 2mL of stock solution to 10 mL

volumetric flask with diluents. Then it was vortexed for 2 minutes to mix well.

3.2.5.2.5. Preparation of Calibration standards and Quality Control Samples

Table 1.1: CC Standards and QCs preparation of GLI

Standards

Intermediate

stock taken

(mL)

Final volume

(mL)

Spiking

stock conc.

(ng/mL)

Plasma conc.

(ng/mL)

STD-1 0.010 10 200.000 10.000

STD-2 0.020 10 400.000 20.000

STD-3 0.060 10 1200.000 60.000

STD-4 0.120 10 2400.000 120.000

STD-5 0.500 10 10000.000 500.000

STD-6 0.800 10 16000.000 800.000

STD-7 1.000 10 20000.000 1000.000

STD-8 1.200 10 24000.000 1200.000

LLOQC 0.010 10 200.000 10.000

LQC 0.030 10 600.000 30.000

MQC 0.600 10 12000.000 600.000

HQC 1.100 10 22000.000 1100.000

3.2.6. Method development: Metformin

3.2.6.1. Preparation of solutions and buffers

3.2.6.1.1. 50mM Sodium hydroxide

Sodium hydroxide (200mg) was weighed accurately, transferred to a glass bottle containing

100 mL of Milli-Q water and sonicated.

Chapter 3


25

3.2.6.1.2. Rinsing solution: [ 0.1% ammonia in { methanol : water (70:30%,v/v}]

1 mL of ammonia solution was transferred in to a glass bottle containing 999 mL of

methanol: water (70:30 v/v) and sonicated to mix well

3.2.6.1.3. Diluent [ methanol : water (40:60% v/v)]

40 mL of methanol was transferred into a glass bottle containing 60 mL of Milli-Q water,

mixed well and sonicated.

3.2.6.1.4. Reconstitution solution [methanol : water (50:50% v/v)]

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

mixed well and sonicated.

3.2.6.1.5. Buffer for mobile phase (25mM KH2PO4, pH 7.00)

1701.125mg of Potassium dihydrogen orthophosphate was dissolved in 500 mL of Milli-Q

water and pH was adjusted to 7 with potassium hydroxide. The resulting solution was filtered

through 0.22µ millipore membrane filter using vacuum filtration system

3.2.6.2. Preparation of Stock solutions

3.2.6.2.1. Metformin main stock (2.000 mg/mL)

20.000 mg of metformin was taken in 10 mL volumetric flask and 5 mL methanol was added

and sonicated to dissolve. The volume was made up to the mark with methanol.

3.2.6.2.2. Metformin intermediate stock (1000.000 µg/mL)

5 mL of metformin main stock was taken in to 10 mL volumetric flask and diluted up to the

mark with diluent.

3.2.6.2.3. Internal standard (atenolol) main stock solution (1.000 mg/mL)

25.000 mg of atenolol was taken in 25 mL volumetric flask and 10 mL methanol was added

and sonicated to dissolve well. The volume was made upto mark with methanol.

3.2.6.2.4. IS Working standard solution (100.000µg/mL)

Working standard solution was prepared by diluting 1 mL of main stock (IS) to 10 mL

volumetric flask with diluent. Then it was vortexed to mix well.

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26

3.2.6.2.5. Preparation of Calibration standards and Quality Control Samples

Table 1.2: CC Standards and QCs preparation of MET

Standards

Intermediate

stock taken

(mL)

Final

volume

(mL)

Spiking

stock

conc.(ng/mL)

Plasma conc.

(ng/mL)

STD-1 0.012 10 1200.000 60.000

STD-2 0.024 10 2400.000 120.000

STD-3 0.090 10 9000.000 450.000

STD-4 0.180 10 18000.000 900.000

STD-5 0.360 10 36000.000 1800.000

STD-6 0.720 10 72000.000 3600.000

STD-7 1.080 10 108000.000 5400.000

STD-8 1.440 10 144000.000 7200.000

STD-9 1.800 10 180000.000 9000.000

LLOQC 0.012 10 1200.000 60.000

LQC 0.036 10 3600.000 180.000

MQC 0.900 10 90000.000 4500.000

HQC 1.620 10 162000.000 8100.000

3.2.7. Optimization of extraction method

Different extraction techniques like, protein precipitation, liquid-liquid extraction and solid

phase extraction were tried. Optimization was done to minimize interferences at the retention

times of analyte and ISTD. All extraction trials were done at the MQC concentration. Trials

were taken to achieve consistent recovery.

3.2.7.1. Protein Precipitation Method

Various protein precipitation agents like acetonitrile, methanol, and 5% perchloric acid were

attempted.

3.2.7.2. Solid Phase Extraction Method

Different SPE trials were taken by altering buffering and washing procedures in order to

optimize extraction efficiency.

3.2.7.2.1. Liquid-Liquid Extraction Method

Methyl tertiary-butyl ether, n-Hexane, and ethyl acetate and methyl tertiary butyl ether in

combination with dichloromethane, methyl tertiary butyl ether in combination with n-Hexane

at different concentrations were attempted.

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3.2.8. Method validation

Validation experiments were conducted as per the USFDA guidelines. The parameters

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

accuracy, recovery, dilution integrity and stability experiments. Following acceptance criteria

was applied to qualify validation tests

Table 1.3: Acceptance criteria for bioanalytical method validation

Validation Test Acceptance Criteria

System Suitability Area ratio: %CV should be ≤ 5.0%

RT: %CV should be ≤ 2.0%

Carryover Check ≤ 20% of response of the mean extracted analyte at LLOQ.

≤5% of response of the mean extracted ISTD

Linearity r2 ≥ 0.9800

Sensitivity Mean % deviation from the nominal concentration should be within

± 20%

% CV of area ratio of LLOQ should be 20%.

Specificity/Selectivity No endogenous peak shall be present within 10% window of the

retention time of analyte and an internal standard

If any peak is present at the retention time of analyte, its response

should be ≤ 20% of response of an extracted Lower calibration

standard i.e. LLOQ standard

Accuracy and Precision The mean calculated concentration of the LLOQC must be within

80-120% of its mean % nominal concentration and for low, medium

and high quality control standards must be within 85-115% of their

nominal concentration

The %CV for LLOQC must be ≤ 20% and for low, medium and

high quality standards must be ≤ 15%

Recovery % CV across QC level must be ≤ 15%

Dilution integrity

% deviation from the nominal concentration should be within ± 15%

% CV of diluted samples must be ≤ 15%

Stock Solution Stability Mean % change must be within ± 10%

Plasma Stability The calculated concentration of the LQC and HQC must be within

85-115% of its nominal concentration

Mean % change must be within ± 15%

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3.3. Results and Discussion

3.3.1. Method Development

3.3.1.1. Optimized Chromatographic Conditions

Table 1.4: Final Chromatographic conditions

Conditions Glibenclamide Metformin

Chromatographic mode Reversed Phase Reversed Phase

Isocratic/gradient mode Isocratic Isocratic

Internal Standard Glimepiride Atenolol

Rinsing solution Methanol : Water

(50:50%, v/v)

Methanol : Water

(50:50%, v/v)

Tray temperature (°C) 5 5

Injection volume (µl) 30 50

Stationary Phase

Column

Length x i.d (mm)

Particle size (µ)

Thermo Betasil C18

150 x 4.6 mm i.d.,

5

Lichrocart-CN

250 x 4.0 mm i.d.

5

Mobile phase Acetonitrile: 25mM KH2PO4

at pH 6.50

(45:55%, v/v)

Acetonitrile: 25mM KH2PO4

at pH 7.00

(28:72 %v/v)

Column Temperature (°C) 40 40

Flow rate (ml/min) 0.6 0.7

Detection wavelength (nm) 228 236

Retention time (min)

Analyte

IS

8.20 (Glibenclamide)

9.20 (Glimepiride)

6.66 (Metformin)

7.69 (Atenolol)

Run time (min) 11 10

3.3.1.2. Optimized Extraction Procedure

Glibenclamide:

Various methods were tested for glibenclamide extraction. Protein precipitation and solid

phase extraction were ruled out because of poor recovery. Multiple trials were taken with the

organic solvents, Tertiary-butyl methyl ether (TBME), n-Hexane, ethyl acetate, TBME with

dichloromethane and TBME with n-Hexane. TBME+n-Hexane at different strengths were

Chapter 3


29

attempted. Both analyte and ISTD were extracted well by TBME: n-Hexane (90:10% v/v)

with no significant interferences.

0.100 mL of validation/study sample was taken in a microcentrifuge tube and 0.010 mL of IS

working solution was added except for blank, where 0.010 mL of diluent was added.

Thereafter, 0.100 mL of buffer (0.2% formic acid in water) was added and votexed to mix.

1.00 mL of extraction solvent (TBME:n-Hexane 90:10 v/v) was added and vortexed for five

minutes. The mixture was centrifuged at 3500 rpm for 05 minutes. The supernatant was

separated and dried in nitrogen evaporator at 40oC then reconstituted with 0.300 mL of

methanol:water (50:50 v/v), votexed to mix well and transferred in to auto sampler vial

containing inserts.

Metformin:

After taking trials with different methods of extraction, protein precipitation method was

optimized with 2% v/v acetic acid in acetonitrile as a precipitating agent for the extraction of

metformin from the plasma and following procedure was used.

0.100 mL of validation/study sample was taken in a micro centrifuge tube and 0.010 mL of IS

working solution was added except in blank plasma, where 0.010 mL of diluent was added in

place of IS working solution. 0.010 mL of 50 mM sodium hydroxide was added and mixed

on the vortexer. To that mixture, 0.75 mL of 2% acetic acid in acetonitrile was added and

centrifuged for 10 min at 10,000 r.p.m. at 4 oC. 0.200 mL of supernatant was transferred in to

auto sampler vial and 0.200 mL of reconstitution solution was added and injected in to

HPLC system.

3.3.1.3.Chromatography

Typical chromatograms obtained from blank sample, lower limit of quantification, upper

limit of quantification, low, medium, high quality control samples are represented in the

following figures. The retention times of glibenclamide and ISTD (glimepiride) were

approximately 8.2 and 9.2 respectively. The retention times of metformin and ISTD

(atenolol) were approximately 6.6 and 7.9 respectively. Representative chromatograms for

GLI are presented in Figure 1.1 to 1.8 and for MET in Figure 1.9 to 1.16

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Figure 1.1: Representative Chromatogram of blank solution (GLI study)

Figure 1.2: Representative Chromatogram of aqueous MQC equivalent (GLI study)

Figure 1.3: Representative Chromatogram of blank K2EDTA rat plasma (GLI study)

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Figure 1.4: Representative Chromatogram of LLOQ (GLI study)

Figure 1.5: Representative Chromatogram of ULOQ (GLI study)

Figure 1.6: Representative chromatogram of LQC (GLI study)

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Figure 1.7: Representative Chromatogram of MQC (GLI study)

Figure 1.8: Representative Chromatogram of HQC (GLI study)

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Figure 1.9: Representative Chromatogram of blank solution (MET study)

Figure 1.10: Representative Chromatogram of aqueous MQC equivalent (MET study)

Figure 1.11: Representative Chromatogram of blank K2EDTA rat plasma (MET study)

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Figure 1.12: Representative Chromatogram of LLOQ (MET study)

Figure 1.13: Representative Chromatogram of ULOQ (MET study)

Figure 1.14: Representative Chromatogram of LQC (MET study)

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Figure 1.15: Representative Chromatogram of MQC (MET study)

Figure 1.16: Representative Chromatogram of HQC (MET study)

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3.3.2. Bioanalytical Method validation

3.3.2.1.System suitability

System suitability test was performed to ensure that the complete testing system (including

instrument, reagents, column, analyte) is suitable for the intended application. System

suitability test was done separately before the start of every sequence. Five replicates of

system suitability solution were injected. Percent coefficient of variation (%CV) for the peak

area ratio and retention times of analyte & ISTD were calculated. The observed percentage

CV for peak response ratio was within the acceptance limits with a value of ≤0.74% for GLI

and ≤1.06% for MET. The observed RTs were within the acceptance limits with the values of

≤0.43% and ≤0.58% for GLI and MET respectively.

3.3.2.2.Calibration curve

The calibration curve was constructed based on reported Cmax of the analyte. Calibration

curve consisted of reconstitution solution, blank sample (matrix sample processed without

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

and non-zero samples (calibration standards). The calibration curves were linear over the

range of 10 to 1200 ng/mL for glibenclamide and 60 to 9000 ng/mL for metformin. The

coefficient of determination was ≥0.9960. The linearity parameters are presented in Table 1.5

and 1.10 for GLI and MET respectively. Typical calibration curves for GLI and MET are

presented in figure 1.17 and 1.18 respectively.

3.3.2.3.Specificity/Selectivity

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

presence of other components in the sample. For selectivity, blank plasma of six different lots

were taken and analyzed. Selectivity was evaluated by comparing extracted blank plasma

response with extracted LLOQ. No significant interference from the blank plasma was

observed at the retention times of both analyte and internal standards in both glibenclamide

and metformin study.

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Figure 1.17: Representative calibration curve of GLI in the range of 10.000 to 1200 ng/mL

Figure 1.18: Representative calibration curve of MET in the range of 60.000 to 9000 ng/mL

3.3.2.4.Sensitivity

The lower limit of quantification is the lowest concentration that can be determined with

acceptable accuracy and precision (20% variation) using a given method. This was performed

by injecting six different aliquots of extracted LLOQ concentration. Percentage deviation

y = 0.0014x - 0.0204

R² = 0.9966

0

0.6

1.2

1.8

2.4

0 200 400 600 800 1000 1200 1400

Calibrat ion Curve

y = 0.0002x + 0.0264

R² = 0.9979

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 2000 4000 6000 8000 10000

Calibrat ion Curve

Chapter 3


38

from the nominal concentration and percentage CV were calculated. The developed methods

were found to be sensitive with % CV of 15.80, Mean % concentration of 96.68 for

glibenclamide and %CV of 13.16%, Mean % Concentration of 107.04 for metformin.

3.3.2.5.Carryover

Carry over test was performed to confirm the absence carryover of analyte from the previous

injection. The experiment was performed at ULOQ level. The samples were injected in the

following order: blank reconstitution solution; aqueous equivalent ULOQ sample; blank

reconstitution solution; aqueous equivalent ULOQ sample; blank reconstitution solution;

extracted blank matrix sample; extracted ULOQ sample; extracted blank matrix sample;

extracted ULOQ sample; extracted blank matrix sample, followed by three LLOQs. No

significant carryover was observed in both GLI and MET studies.

3.3.2.6.Accuracy and precision

The accuracy of a method is the closeness of mean values obtained under prescribed

conditions. The precision of an analytical method is the closeness of individual measures

when different aliquots of same solution are analysed. Within-batch accuracy and precision

were assessed by analyzing one calibration curve and 4 sets of QC samples (6 replicates each

of the LLOQC, LQC MQC and HQC) in different batches. Between batch accuracy and

precision evaluation were also assessed by analyzing 4 batches on different days. The

accuracy and precision for all the batches at LLQC and LQC, MQC and HQC levels were

calculated. Mean percentage nominal concentration and coefficient of variation for all the

batches individually (intra) and collectively (inter) were found to be within acceptance limits

for both GLI and MET studies.

For glibenclamide, within batch accuracy was 95.46 to 105.6% and precision 15.80 to

19.76% at LLOQC, accuracy of 88.82 to 109.19 % and precision of 2.07 to 13.16% across

LQC, MQC and HQC.

For glibenclamide, between-batch accuracy was 98.44% and precision of 17.03% at LLOQC,

accuracy of 97.23 to 103.78% and precision of 6.18 to 9.46% across LQC, MQC and HQC.

The data is presented in the Table 1.6

For metformin, within batch accuracy was 93.04 to 117.10 % and precision 9.37% to 16.99%

at LLOQC, accuracy of 89.35 to 109.19 % and precision of 2.07 to 13.16% across LQC,

MQC and HQC.

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For metformin, between-batch accuracy was 98.44% and precision of 17.03% at LLOQC,

accuracy of 97.23 to 113.08% and precision of 1.94 to 9.55% across LQC, MQC and HQC.

The data is presented in the Table 1.11

3.3.2.7.Recovery

Recovery of an analyte was determined at low, medium and high quality control samples.

Analyte was spiked to the reconstituted solvent of the above blank samples to obtain the post

spiked LQC, MQC and HQC samples. Post spiked and extracted quality control samples

were analyzed and percentage recovery at each level was calculated by comparing the

response area of low, medium and high quality control levels and an internal standard. Mean

percentage recovery across all QC levels for GLI and MET is 93.45% and 73.26%

respectively. The mean percentage recovery for glimepiride (ISTD in GLI study) and atenolol

(ISTD in MET study) were 93.52% and 76.20% respectively. The summary statistics is

presented in Table 1.7 and 1.12 for GLI and MET respectively.

3.3.2.8.Dilution integrity

Dilution integrity was done in order to handle high sample concentrations exceeding ULOQ

or the possibility of insufficient sample volume in the study samples. A test for sample

dilution with blank matrix was performed. This was carried out by spiking 2xULOQ spiking

stock solutions in biological matrices to 1/2 and 1/4 dilute samples. Six aliquots each of

diluted samples and calibration standards were processed, extracted and analyzed.

Percentage CV and accuracy were calculated. The mean percentage nominal concentrations

and %CV were found to be within acceptance limits.

3.3.2.9.Stability

3.3.2.9.1. Stock solution stability

Short term and long term solution stabilities for the main stock and spiking stock solutions at

room temperature and at 2-8°C respectively were evaluated at MQC level. Five injections

each of comparison (freshly prepared MQC equivalent concentration) and stability samples

were performed. Mean percentage change was calculated by comparing the area of stability

and comparison samples. Both, main stock and spiking stocks of GLI and MET studies were

found to be stable at 2-8 oC for 9 days (long term) and for 8 hours at room temperature. The

results are presented in Table 1.8 and 1.13 for GLI and MET respectively.

3.3.2.9.2. Stability of drug in plasma

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40

The stability of the analytes in plasma were assessed at different conditions anticipated

during the pharmacokinetic studies like bench top stability, freeze thaw stability, in-injector

stability and long term stability at LQC and HQC levels.

3.3.2.9.3. Bench top stability

Bench top stability was evaluated to confirm that analyte degradation does not occur during

time of exposure to room temperature, which was based on the expected duration of the

samples maintained at room temperature during the study. Samples were prepared at low and

high quality control levels and kept at bench top at room temperature for a minimum of four

hours (stability samples). Later, fresh calibration standards and quality control samples

(comparison samples) were prepared, extracted and analyzed with the stability samples.

Mean percentage change was calculated.

The plasma samples were found to be stable at room temperature for 07 hours with mean%

change of 0.98% at LQC level and 6.67% at HQC levels for glibenclamide and 3.73% at

LQC, 1.76% at HQC for metformin.

3.3.2.9.4. Freeze and thaw stability

From the stage of collection to analysis, the samples undergo multiple cycles of freeze and

thaw, at least two times. Therefore, it is necessary to verify the validation samples to multiple

freeze-thaw cycles. The samples were exposed to four freeze thaw cycles. Stability samples

were analyzed against fresh calibration standards and quality control samples (comparison

samples). Mean % change was calculated and verified against acceptance criteria.

The plasma samples were found to be stable after repeating three cycles of freezing at -20oC

and thawing to room temperature with mean% change of 3.11% at LQC level and 5.26% at

HQC levels for glibenclamide and 0.56% at LQC and 8.21% at HQC for metformin

3.3.2.9.5. In-injector stability

Stability of processed samples in the instrument over the anticipated run time was assessed.

Stability samples were analyzed with fresh calibration standards along with low and high

quality control samples (comparison samples) and mean percentage change was calculated.

The plasma samples were found to be stable 4oC for 30 hours with mean% change of 5.14%

at LQC level and 5.83% at HQC levels for glibenclamide and 1.48% at LQC and 4.54% at

HQC for metformin

Chapter 3


41

3.3.2.9.6. Long term stability

To confirm analyte stability in the matrix during storage period, long-term stability studies

were conducted. Stability samples were stored at -20°C for 70 days. Long-term stability was

assessed by analyzing fresh calibration standards, low and high quality control samples

(comparison samples) with stability samples. Mean percentage change was calculated.

The plasma samples were found to be stable at -20oC for 70 days with mean% change of

3.83% at LQC level and 1.62 % at HQC levels for glibenclamide and 1.55% at LQC and

1.01% at HQC for metformin.

The stability data is presented in Table 1.9 and 1.14 for GLI and MET respectively.

3.4. Conclusion

Sensitive, specific and precise HPLC assays for the determination of glibenclamide and

metformin in rat plasma were developed and validated. The developed methods are suitable

for pharmacokinetic drug interaction studies in rodents.

Chapter 3


42

Table 1.5: Summary of calibration curve parameters of glibenclamide

Linearity Slope Intercept r2

1 0.001429379 -0.019494894 0.9996

2 0.001428497 -0.026486986 0.9995

3 0.001430286 -0.020494674 0.9966

4 0.001429299 -0.017495085 0.9996

Table 1.6: Between batch accuracy and precision of glibenclamide

QC Levels LLOQC

(10ng/mL)

LQC MQC HQC

(30 ng/mL) (600ng/mL) (1100ng/mL)

Batch-1

8.005 33.343 631.561 933.422

8.502 32.569 672.4 931.014

7.909 27.036 707.629 1053.373

11.935 28.572 659.422 1008.384

12.006 34.326 612.934 947.001

9.234 26.948 646.838 989.017

Batch-2

8.005 28.885 595.038 962.811

8.652 34.192 572.4 1096.594

12.326 33.375 597.629 982.373

11.492 30.572 583.598 1038.384

10.874 29.095 612.934 1226.001

12.034 31.572 606.838 1209.017

Batch-3

8.652 31.45 545.038 1162.811

7.997 28.392 572.475 1096.594

8.212 29.343 647.629 1230.429

11.326 27.569 615.598 1218.384

9.084 34.036 630.934 1056.034

12.002 38.572 628.838 990.017

Batch-4

10.234 30.326 685.543 1098.116

8.095 33.772 672.4 1062.174

9.671 29.343 597.629 1090.429

7.874 28.569 612.257 1069.575

10.134 27.936 610.425 1126.001

12.002 32.092 626.816 1090.017

n 24 24 24 24

Mean 9.844 30.912 622.7 1069.499

% CV 17.03 9.462 6.197 8.648

Mean %

nominal conc. 98.44 103.04 103.78 97.23

Chapter 3


43

Table 1.7: Recovery across QC levels (GLI study)

QC Levels Mean % Recovery

Glibenclamide

LQC 94.04

MQC 92.06

HQC 94.27

Mean 93.45

SD 1.21

CV 1.30

ISTD Glimepiride

93.52

n=6 for all QCs; n=18 for ISTD

Table 1.8: Stock solution stability (GLI Study)

Stability

Level

Glibenclamide Glimepiride

Mean % Change Mean % Change

Long term

(9 days at 2-8oC) Main Stock -4.539 -1.032

Spiking

stock -2.8603 -2.98

Short term

(08 hours at room

temperature)

Spiking

stock -2.921 -3.258

Table 1.9: Glibenclamide plasma stability

Stability Level

Fresh Sample Conc.

(ng/mL)

Stability sample Conc.

(ng/mL) Mean%

Change Mean %CV Mean %CV

Bench topa LQC 30.851 6.84 30.550 4.16 -0.98

HQC 1169.624 6.42 1091.585 2.40 -6.67

In-Injectorb LQC 30.851 6.84 29.267 6.73 -5.14

HQC 1169.624 6.42 1101.452 1.69 -5.83

Freeze thawc LQC 30.851 6.84 29.890 5.32 -3.11

HQC 1169.624 6.42 1108.053 8.92 -5.26

Long termd LQC 30.251 5.26 29.092 4.95 -3.83

HQC 1124.826 10.17 1106.653 10.69 -1.62 aStability in plasma at room temperature for 7hours bStability in plasma at 4oC in auto sampler for 30 hours cStability in plasma after 3rd freeze (-20oC) - thaw cycle dStability in plasma for 70 days at -20oC

n=6; Specified concentrations: LQC: 30.000 ng/mL; HQC:1100.000 ng/mL

Chapter 3


44

Table 1.10: Summary of calibration curve parameters of metformin

Linearity Slope Intercept r2

1 0.00173465 0.0085769 0.9945

2 0.00024236 0.0019262 0.9979

3 0.00175078 0.0267986 0.9976

4 0.00171293 0.00936981 0.9938

Table 1.11: Between batch accuracy and precision of metformin

QC levels LLOQC LQC MQC HQC

(60 ng/mL) (180 ng/mL) (4500 ng/mL) (8100 ng/mL)

Batch-1

54.75 172.271 4420.618 7774.503

58.127 189.724 4342.535 7781.07

55.232 165.217 4437.871 8065.91

54.658 172.747 4268.01 7722.569

46.82 182.975 4246.815 8206.582

65.341 206.873 4425.854 9198.712

Batch-2

71.102 181.395 5111.759 9016.458

48.794 207.44 4967.067 8950.74

66.515 199.876 4847.924 8891.085

68.939 185.483 4764.899 8667.464

53.869 199.96 4867.679 8463.503

49.612 187.454 5063.789 8958.234

Batch-3

64.515 188.24 4167.647 7681.264

50.444 218.204 4550.722 7456.511

59.108 201.134 4687.722 7469.813

66.335 196.096 4372.766 7351.797

73.078 186.171 4545.001 7916.367

71.843 198.349 4851.92 7436.367

Batch-4

72.15 192.362 3962.1 7084.241

58.428 202.969 4129.928 7292.116

78.617 200.941 4034.504 7289.145

71.939 238.1 3895.433 7995.818

69.86 180.067 3979.694 8104.048

70.567 206.855 4123.938 8122.56

n 24 24 24 24

Mean 62.527 194.204 4461.091 8037.370

% CV 14.76 8.16 8.19 7.88

Mean %

nominal conc. 104.21 107.89 99.14 99.23

Chapter 3


45

Table 1.12: Recovery across QC levels (MET study)

QC Levels Mean % Recovery

Metformin

LQC 71.69

MQC 70.21

HQC 77.88

Mean 73.26

SD 4.069

CV 5.55

ISTD Atenolol

76.20

n=6 for all QCs; n=18 for ISTD

Table 1.13: Stock solution stability (MET Study)

Stability

Level

Metformin Atenolol

Mean % Change Mean % Change

Long term

(9 days at 2-8oC) Main Stock -0.86 -1.032

Spiking

stock 2.15 1.85

Short term

(08 hours at room

temperature)

Spiking

stock 1.15 -4.75

Table 1.14: Metformin plasma stability

Stability Level

Fresh Sample Conc

(ng/mL)

Stability sample Conc

(ng/mL) Mean%

Change Mean %CV Mean %CV

Bench topa LQC 178.300 3.20 171.655 2.54 -3.73

HQC 8503.279 2.03 8353.754 2.69 -1.76

In-Injectorb LQC 178.300 3.20 180.930 1.49 -1.48

HQC 8503.279 2.03 8117.427 1.89 -4.54

Freeze thawc LQC 178.300 3.20 179.299 2.71 0.56

HQC 8503.279 2.03 7804.766 2.85 -8.21

Long termd LQC 183.633 3.01 180.780 1.96 -1.55

HQC 7923.612 2.72 8003.311 1.95 1.01 aStability in plasma at room temperature for 7hours bStability in plasma at 4oC in auto sampler for 30 hours cStability in plasma after 3rd freeze (-20oC) - thaw cycle dStability in plasma for 70 days at -20oC

n=5; Specified concentrations: LQC:180.000 ng/mL; HQC:8100.000 ng/mL

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