1. INTRODUCTION:

Duloxetine (DUX) is a potent selective serotonin and norepinephrine re-uptake inhibitor used to treat major depressive episodes, pain and stress urinary incontinence in human. DUX is extensively metabolized in liver by CYP1A2 and CYP2D6 isozymes [1]. The bioavailability of DUX after oral administration was ranged from 32% to 80% and a delay of peak concentration from 6 to 10 hours in presence of food [2,3]. DUX was found to be highly variable and results in variability in the blood concentrations [4]. After oral administration of DUX C_maxwas achieved at 6 hours and the elimination half-life was 12 hours [5]. To support pharmacokinetic, toxicokinetic, bioavailability and bioequivalence studies, it is critical to have a highly sensitive and selective bioanalytical method for the quantitative determination of analyte (s) in the biological matrix. Most accurate and reliable results are obtained by employing a well-characterized and fully validated bioanalytical methods for the determination of the analytes (s) [6].

Various analytical methods have been reported for the determination of DUX in biological matrix. DUX determination in the biological matrix using high performance chromatography (HPLC) [7-11], gas chromatography [12] and liquid chromatography coupled with mass spectroscopy (LC-MS) were reported [5,13,14,15,16,17,18].

HPLC methods for the determination of DUX is reported with the sensitivity of 5ng/mL [7], Ulu ST reported HPLC method with the sensitivity of 0.5ng/mL in neat solution and it is reported that HPLC method with the sensitivity of 2ng/mL using 1mL of plasma and it is reported that HPLC with the sensitivity of 2ng/mL using derivatization procedure [9,10,19] and it is reported that HPLC method with 2ng/mL using 0.450ml of plasma [11]. Gas chromatographic method is reported with the sensitivity of 1µg/mL using 0.5 mL of plasma volume with the run time of more than 7 mins [12]. LCMS/MS methods reported having the sensitivity of 0. 1ng.mL using 0.300mL of plasma [15] and reported with 0.8ng/mL sensitivity using 0.200mL of plasma [16].

As DUX shows large inter-individual variability [2,3]. it is necessary to develop a highly sensitive and selective analytical method to quantify DUX in human matrix. In the current research work we have developed a highly sensitive and rugged bioanalytical method using HPLC-MS/MS and the method is validated as per the USFDA guidelines [20]. The developed method is successfully employed for the determination of DUX in human plasma to support a clinical pharmacokinetic study. The range selected will fit to analyze the pharmacokinetic samples obtained from 30mg or 60mg formulations.

2. MATERIALS AND METHODS:

2.1 Chemicals and materials:

Analytical reference standard of DUX (99.95% purity, Lot# SJ104) and the internal standard (ISTD) Atomoxetine hydrochloride (99.9% purity) EP standard were procured from sigma Aldrich, India. The structure of DUX and Atomoxetine are presented in Figure 1.

Duloxetine

Atomoxetine

Figure1: Chemical Structure of Duloxetine and Atomoxetine

AR grade Ammonium trifluoroacetate, ammonium hydroxide were procured from Merck specialities Pvt Ltd, Mumbai India, HPLC grade Methanol, n-hexane, formic acid were procured from ACI Labscan, Thailand. Milli-Q water was prepared by Milli-Q Water purification system (USA) and used during the entire analysis period. Human blank plasma, with K₃EDTA as an anticoagulant used for method development and validation purpose. Blank plasma was procured from Supratech voluntary blood bank, Ahmedabad.

2.2 Instrumentation and chromatographic conditions:

The chromatography was carried out using Luna^® 5µm C8(2) 100 Å, LC column 100 x 4.6 mm, with the mobile phase comprising of Milli q water (0.05% formic acid and 0.1% ammonium trifluroacetate solution and Methanol (23:77%v/v), isocratic elution at 0.5mL/min flow rate. The column oven and auto sampler tray temperature were maintained at 35°C and 10°C respectively, throughout the analysis. The total chromatographic run time was about 2.2minutes. LC-MS/MS analysis was performed using Shimadzu HPLC (Shimadzu corporation, Japan) and MDS SCIEX API-4000 (Toronto, Canada) Mass spectrophotometer, equipped with electro spray ionization (turbo spray) ion source. The analysis was carried out with positive ionization mode and the ions were monitored using multiple reaction monitoring (MRM) mode. ESI source dependent parameters were set as follows; Curtain gas (CUR): 20, Ion spray Voltage (IS) 5000.00, Temperature (TEM); 450.00, Nebulizer gas (GS1);50, Heater Gas (GS2): 50, Interface heater (ihe): on and Collision gas: 10.00. The MRM transitions for DUX and Atomoxetine (ISTD) were 298.00 → 154.00 and 256.00 → 148.00 respectively. All chromatograms were acquired using analyst software.

2.3 Preparation of analyte stock solutions, calibrations and quality control (QC) standards preparation:

The standard analyte stock solution of 0.1mg/mL was prepared by dissolving 2.5mg of DUX in 25.0mL of methanol. Intermediate stock solution at the concentration of 2000ng/mL was prepared from the main stock using diluent (Water: Methanol 50:50%v/v) and used for the spiking stock solution preparation. Internal standard stock solution of 0.100µg/mL was prepared in methanol and working solution of 200ng/mL was prepared in diluent form the main stock solution. The concentration of the solution was calculated based on the potency and the amount of the analytes weighed. The spiking stock solutions were prepared in the diluent at the concentrations of 2.00, 4.00, 8.00, 20.00, 40.00, 100.00, 200.00, 500.00, 1200.00, 1600.00 and 2000.00 ng/mL for calibration standards (CS) and 2.00, 10.00, 120.00 and 1500.00ng/mL for quality control (QC) samples.

2.4 Plasma samples preparation procedure for method validation and study samples:

Method validation or study sample samples were withdrawn from the deep freezer and thawed at the room temperature. 0.200mL of the plasma sample was aliquoted into the prelabelled tubes and 30µL of ISTD working solution was spiked (except for blank samples) and vortexed for 20 seconds. To this 100µL of extraction buffer was added (0.1N sodium hydroxide in water) and vortexed for 20 seconds, followed by the addition of 2.5mL of n-hexane. Vortexed using vibramax for 5 minutes at 2500rpm. Centrifuged for 5 minutes for 4000 rpm at 5±4°C for 10 minutes. 2.5mL of supernatant was transferred into pre-labeled glass tubes and evaporated to dryness under the stream of nitrogen at the samples to dryness under nitrogen at 40°C. The dried samples were reconstituted with 200µL of mobile phase and vortexed to mix. Then the samples were transferred into auto sampler vials and injected into HPLC system for analysis.

2.5 Bioanalytical method validation:

The analytical method was validated as per the procedure drafted in guidance for industry Bioanalytical method validation by US Food and Drug Administration, 2013 [20]. Full method validation was performed, and the parameters studied were selectivity, sensitivity, accuracy and precision, recovery, calibration curve, reproducibility, matrix effect and stability studies (both analyte stability in solution and matrix). Freeze and stability experiment were performed at storing the samples at -20±5˚C during each cycle. The long-term stock solution stability was performed at 4±5˚C and plasma storage stability was performed at -20±5˚C. System suitability was checked every day of the validation to check the instrument performance. Auto sampler carryover check experiment was performed to rule out the any instrument error.

The CS and QC were performed in plasma prepared by 2% spiking. A total of ten calibration standards in plasma were prepared at the concentration of 0.10, 0.20, 0.40, 1.00, 2.00, 5.00, 10.00, 25.00, 60.00, 80.00 and 100.00ng/mL. A total of 4 quality controls samples in plasma were prepared at the concentration of 0.100(LLQC), 0.50(LQC), 50.00(MQC) and 75.00(HQC)ng/mL. Required quantity of aliquots of CS and QC were prepared and stored in deep freezer at -20±5°C until completion of the validation.

The concentration of the QC samples is calculated using linear regression equation (y = mx + b) with 1/x² weighting factor by calculating peak area ratio. Matrix effect was tested in 6 different lots of plasma including the heparinized plasma, lipemic and haemolysed plasma. Accuracy and precision (inter and intra day) was assed by analyzing the quality control samples at four different concentrations and four replicate samples at each level. Short term and long-term stability of analyte in plasma was performed by preparing the calibration standards by fresh spiking and the stability QC samples analysed against fresh calibration curve. The solution into the plasma and stability QC samples and Freeze thaw (FT) stability were tested at two different levels of QC samples at -20±5°C. Wet Extract (WE) stability (after reconstitution step of the extraction procedure) and Dry Extract (DE) Stability (before reconstitution in the extraction step) were performed at two different QC levels. Bench Top (BT) stability was performed at room temperature at two different QC levels. Auto sampler stability was performed at four different QC levels, to asses the stability of processed samples in the auto sampler conditions for expected maximum time of the analytical batch run. Recovery of the analytes was carried out at four different levels and ISTD recovery at the working standard level. Specificity/selectivity at LLOQ and precision and accuracy experiment at LQC and HQC level were performed with the anticipated concomitant medications used during the study to treat the common adverse events. Concentration equivalent to C_max of the concomitant medication concentration in plasma was spiked into the experiment samples. To assess the vulnerability of the method for the changes that occur during the routine analytical procedures, ruggedness of the method was tested by performing the experiments with different analytical column of the same make but of different batch number and different analysts short-term and long-term stock solution stability were performed at LLOQ and ULOQ levels. Long term (LT) stability of the analyte (DUX) in plasma was performed at two levels for the period covers entire period of the study samples storage period. Dilution integrity (DI) experiments were performed at ½ and ¹/₁₀dilution to handle low plasma volumes for one-time analysis of the clinical study samples. Stability of the analyte in blood at room temperature and ice-bath were determined to study the impact of the procedures happening between the blood collection from the study subjects till start of centrifuge procedures. The acceptance criteria of the results were set as per the USFDA bioanalytical method validation guidelines [20]. Accuracy of the calibration standards and the QC samples should be within 85-115% and for LLOQ calibration standard and LLOQC accuracy should be within 80-120% of the nominal concentration. At least 75% of non-zero (except blank and zero sample) standards should meet the criteria and including LLOQ. The precision of back calculated concentration should be within 20% of the nominal concentration at LLOQ level and within 15% at other levels including other CS and QC samples. For stock solution stability mean % change should be within 90.00 to 110.00% and %CV should be within 15.00%.

2.6 Clinical pharmacokinetic study design:

An open label, randomized, balanced, three-treatment, three-period, two-sequence, crossover, single-dose comparative oral bioavailability study of Duloxetine delayed release capsules 60mg of test and reference product was planned. A clinical study protocol was prepared and approved by the ethics committee before conduction of the study. The study subject’s selection was based on laboratory evaluations during screening, medical history, clinical examination, Chest X-ray, ECG recordings, urine screen for drugs of abuse and screen for alcohol breath test in each period. Informed consent was obtained from each subject prior to study participation. After overnight fasting of at least 10 hours, each subject administered one capsules orally with about 240mL of water in the sitting posture as per the randomization schedule. A total of sixteen (16) blood samples were collected from the fore arm vein at the time intervals of 0.00 (Prior to dosing) 1.00, 2.00, 3.00, 4.00, 6.00, 7.00, 8.00, 10.00,12.00, 16.00, 24.00, 36.00, 48.00, 72.00 and 96.00 hour (after dosing) in each period. Blood samples were collected in K₃EDTA vacutainers. The samples were centrifuged at 3000 rpm for 15 minutes at 4°C, plasma was separated into prelabelled polypropylene vials and stored -20 ± 10°C until analysis. A washout period of 07 days was maintained between each period of the study period.

2.7 Ethical considerations:

The study was commenced only after obtaining an approval for the study protocol from the Institutional Ethics committee and written informed consent from each subject. The study procedures were explained to the subjects in their respective native languages. The study was conducted as per the Good Clinical Practices, Declaration of Helsinki, and applicable requirements of Principles of Good Laboratory Practices.

2.8 Safety:

2.9 Pharmacokinetic parameters:

Using the concentration time profiles of DUX, the pharmacokinetic parameters of maximum plasma concentration (C_max), area under the concentration–time curve (AUC) AUC_0-t and AUC_0-∞, AUC_0-t/AUC_0-∞, time taken to achieve maximum level (T_max), first-order terminal elimination rate constant Kel and half-life (t½) were calculated. Pharmacokinetic parameters evaluation was carried out by using by Phoenix® WinNonlin Version 5.3 (Pharsight Corporation, USA). PK and statistical analyses were performed using SAS® Version 9.2. (SAS, USA).

3. RESULTS AND DISCUSSIONS:

3.1 Method development:

LC conditions were optimized to achieve good ionization efficiency and short runtime. The mobile phase was selected based on Pka of the analytes, to assist for maximum ionization. Mass detector parameters were optimized for the better response by fine tuning the Declustering potential (DP), Entrance potential (EP), Collision energy (CE). The positive ion spectrum showed a protonated molecular ion of [M+H] at m/z 298 and at m/z 256 for Duloxetine (DUX) and Atomoxetine (ATX) respectively. Internal standard of similar chemical structure was selected. Compound (for both analyte and internal standard) dependent parameters were; Declustering potential (DP): 75.00, Entrance potential (EP):10, Collision energy (CE):28.00 and 32.00, Collision cell exit potential (CXP):13.00 and Dwell time (milli seconds):400.00. Most abundant ion transitions were obtained at 298.00 → 154.00 and 256.00 → 148.00 DUX and ATX respectively. The mobile phase with different buffer concentrations of 10mM, 5mM and 2mM ammonium acetate/ammonium format (acidified with 0.1% formic acid) with Methanol and Acetonitrile at the compositions of 30:70% v/v, 20:80%v/v, 15:85%v/v and 10:90% v/v (Aqueous: Organic) were checked during method development. Mobile phase composition of Milli q water (0.05% formic acid and 0.1% ammonium trifluroacetate solution and Methanol (23:77%v/v) shown good instrument response, reproducibility of the analyte and internal standard area with the flow rate of 0.5mL per minute.

Many of the literatures presented the automated sample extraction techniques, despite of having advantages, many of the academic or research organizations cannot afford the cost the instrument and also requires special skill set. Complete automation may not be possible due to either different make of the instruments or lack of coordination between the instrument suppliers. Biostudies for regulatory submission will face technical challenges during the method development and incase instrument related mechanical errors happens during the sample preparation and noticed after the analysis, then it will be very difficult task to justify the event. We adopted manual procedure and every step of the extraction procedure is evident, can be reproduced in any lab. We focused on utilizing limited number of extraction solvents and minimum quantity. Best recovery results were obtained by using 2.5mL of: n-hexane as an extraction solvent. Luna^® 5 µm C8(2) 100 Å, LC Column 100 x 4.6 mm chromatography column was selected based on the column efficiency, fast column stabilization, peak shape, peak symmetry and low back pressure. The retention time of DUX and ATX was 1.20 and 1.17 minutes respectively with the analytical run time of 2.2 minutes. Carryover was eliminated by optimization of the auto sampler rinsing volume to 500µL using Methanol: 0.1% formic acid in water (50:50%v/v).

3.2 Method validation:

3.2.1 System suitability and carryover:

System suitability of all analytical runs were compiled and the precision of analyte RT, ISTD RT and area ratio were found to be ≤1.05%. The analyte carryover from ULOQ concentration was found to be ≤ 8.45% upon comparison with the area of LLOQ area and ≤ 1.56 for internal standard.

3.2.2 Specificity and selectivity:

Specificity was tested in ten lots of plasma (Including lipemic and Hemolyzed). The response (area) at the retention time of the analyte and internal standard was compared with that of area of LLOQ and internal standard sample. The area was found to be less than 20% at the RT of analyte and less than 5% for internal standard. The selectivity was tested at LLOQ level and accuracy and precision was found to be 108% and 5.12% respectively. The signal to ratio at LLOQ level was found to be ≥ 87.2%.

3.2.3 Linearity:

The weighting factor of 1/x² was given the good curve fit and used in the analysis. Calibration curve was constructed over the concentration range of 0.100 to 100.00ng/mL. The data of 6 analytical runs were compiled and mean slope and coefficient of determination were found to be 0.0540 and 0.9978 respectively. The calibrations curves were found to be linear over the calibration range with coefficient of determination (r²) ≥ 0.9967. The accuracy and precision of the back calculated concentrations were found to be ≤ 104.80% and ≤ 6.45% respectively. The precision (%CV) and accuracy results of back calculated concentration are presented in Table 1.

3.2.4 Matrix effect:

Matrix effect was determined by spiking the known concentration of the analytes at two different concentrations at LQC (0.50ng/mL) and HQC (75.00ng/mL) level. The mean bias at LQC land HQC level was in the range of -0.3 and 4.8% and -3.70 to 1.67 respectively. The precision was found to be ≤ 2.07 and 1.09 % analyte for LQC and HQC and 0.34% for ISTD respectively.

3.2.5 Inter and Intra run precision and accuracy:

Intra-run and inter-run precision and accuracy was determined at four different QC levels and the results were compiled from three different batches on two different days. The inter-run accuracy was found to be between 96.88 to 104.00% at LQC, MQC and HQC levels and 107% at LLOQC level. The precision was found to be ≤ 8.09% across LQC, MQC and HQC levels and 14.54 at LLOQC level. The intra-run accuracy was ranged from 96.11-106.14% across the LQC, MQC and HQC level and ≤ 109.00 at LLOQC level. The precision was found to be ≤ 8.90 % across the LQC, MQC and HQC level and ≤ 8.33% at LLOQC level.

3.2.6 Stability of analyte and Internal standard stock solutions:

The stock solution stability was established at room temperature and 4±5˚C. Main stock solution (in methanol) of analyte (100.00µg/mL) and internal standard were found to be stable at room temperature for 08 hours and 25 days at 4±5˚C. Working stock solution of DUX at 1000ng/mL and internal standard at 200ng/mL were found to be stable at room temperature for 06 hours and 25 days at 4±5˚C.

3.2.7 Recovery:

Recovery of the analyte was found to be 86.22%, 84.26% and 85.11% at LQC, MQC and HQC respectively. The precision was found to be ≤1.83% across the QC levels. The recovery of ISTD was found to be 92.67% and precision was 5.97%.

3.2.8 Dilution integrity (DI):

DI experiment was carried out at 2×ULOQ (2×100ng/mL) concentration with 1/10 and 1/2 dilution. The accuracy and precision were found to be 102.4% and 99.56% and 2.93% and 3.34% at 1/10(ng/mL) and 1/2 (ng/mL) dilutions respectively.

3.2.9 Stability of analyte in plasma:

Stability of the analyte in plasma was determined at different conditions at three different concentrations. DUX was found to be stable in bench top for 10 hours at room temperature; Wet extract of the analyte was stable at 4±5˚C for 48 hours after reconstituting with the reconstitution solution; Dry extract was stable at-20±5˚C for 60 hours. DUX was stable after five freeze thaw cycles -20±5˚C. Long term stability was established for 50 days at -20±5˚C.

3.2.10 Ruggedness:

Ruggedness of the experiment was checked by performing three precision and accuracy batches by two different analysts, different column of same make and different batch number and different instrument at three different concentrations. The accuracy was ranged between 97.80 to 110.18% at LQC, MQC and HQC and 98.00 to 110% at LLOQC with the precision ≤ 8.99 across the QC levels and ≤7.89 at LLOQC.

3.2.11 Re-injection reproducibility:

Re-injection reproducibility was tested by re-injecting the precision and accuracy batch with calibration standards after 60 hours at auto sampler temperature of 5±3˚C. The mean percentage change at LQC, MQC and HQC levels was found to be -2.00, -0.80, -4.75 respectively, with accuracy was ranged between 95.25 to 106.40 and precision was ≤ 4.98%. The mean percentage change at LLOQC was 2.30% accuracy and precision were 102.32% and 4.20% respectively.

3.2.12 Analytical batch run stability:

Batch size experiment was performed at 160 samples per batch. The accuracy and precision were found to be 94.56 to 103.33% and ≤4.18% at LQC, MQC, and HQC respectively. The accuracy and precision at LLOQC were found to be 109% and ≤ 8.38% respectively.

3.2.13 Whole blood stability:

Whole blood stability was established by comparing the area ratio between the fresh sample and stability sample. DUX in whole blood was found to be stable for 30 minutes at room temperature and 1.5 hours in wet ice bath.

3.3 Bioequivalence study design and ethical principles:

The validated analytical method was used for the analysis of bio study samples. Eighteen healthy male volunteers of aged between 18 and 44 years (both inclusive) with the BMI of 18.55 -24.88 were enrolled in the study. The study was conducted as per the Good Clinical Practices, Declaration of Helsinki and applicable requirements of Principles of Good Laboratory Practices under fasting conditions. During the entire study period, there were no major protocol deviations.

3.4 Safety:

Safety and tolerability of both DUX formulations for the enrolled subjects were assessed throughout the study by monitoring adverse events (AEs), standard clinical laboratory tests (clinical biochemistry, urinalysis, and hematology), physical examinations, vital signs, and 12-lead electrocardiograms (ECGs). Post-study safety follow-up was also carried out. The administered test and reference formulations of DUX were well tolerated by the subjects. All AEs were resolved. No significant or serious AEs occurred during the study that required medication(s).

3.5 Pharmacokinetic and statistical analysis:

The results of pharmacokinetic parameters are presented in Table 2.

Table 2: Pharmacokinetic parameters of DUX in 18 healthy subjects after oral administration of test and reference products

PK parameters (Arithmetic mean ± SD)	DUX Test (T)	DUX Reference ( R )
C_max(ng/mL)	44.1306 ± 20.8312	40.19465 ± 19.5290
AUC_0–t (ng h/mL)	922.1160 ± 653.9306	897.21205 ± 617.9416
AUC_0–∞ (ng h/mL)	942.1636 ± 680.8890	914.1859 ± 642.8547
T_max (h)	6.5 (3.00 - 8.00)	12 (2-20)
Kel	0.0588 ± 0.0156	0.0603 ± 0.0149
t½ (h)	12.751 ± 4.084	12.2435 ± 3.1605

The results confirm that the test product was bioequivalent to that of reference product. The data was assessed by employing average, population and individual bioequivalence approaches. The results are presented in Table 3.

Table 3: Bioequivalence summary by applying ABE, PBE and IBE approaches

ABE evaluation
variances		Test/reference values for log-transformed		90% Confidence interval
C_max (ng/mL)		102.99		91.44 – 116.00
AUC_0–t (ng h/mL)		112.55		100.80 – 125.66
AUC_0–∞ (ng h/mL)		113.00		101.17 – 126.23
Pass or fail ABE				Fail
PBE evaluation
variances	Linearized point estimate		95% upper confidence bound
C_max (ng/mL)	108.37		0.16 (Constant scale)
AUC_0–t (ng h/mL)	100.20		0.32 (Constant scale)
AUC_0–∞ (ng h/mL)	100.43		0.33 (Constant scale)
Pass or fail PBE			Fail
IBE evaluation
variances	Linearized point estimate		95% upper confidence bound
C_max (ng/mL)	108.37		−0.01 (Constant scale)
AUC_0–t (ng h/mL)	100.20		−0.02 (Constant scale)
AUC_0–∞ (ng h/mL)	100.43		−0.02 (Constant scale)
Pass or fail IBE			Pass

The mean plasma concentration is presented in Figure 2.

Figure 2: Mean plasma concentration of verses time profile of DUX Test and Reference products

4. CONCLUSION:

We have developed a highly sensitive and selective bioanalytical method for the determination of DUX at the concentration of 0.10ng/mL in human plasma using LC-MS/MS. The reported methods are either requires long runtime or reported with highly sophisticated instruments. Since Duloxetine is available in two different strengths, it is necessary to develop the bioanalytical method which fits the concentration of the analyte in the plasma. The calibration curve was found to be linear over the concentration range of 0.1 to 100.00ng/ mL with the coefficient of determination (r²) ≥ 0.9967. Simple liquid- liquid extraction technique was adopted to extract the samples form the plasma. All validation parameters sensitivity, selectivity, carryover, inter and intra run precision and accuracy, bench top, freeze thaw stability for five cycles at -20±5˚C, dry extract, wet extract, long term stability (50days) at -20±5˚C were found to be within the acceptable limits as per the USFDA bioanalytical method validation. The method was successfully employed to evaluate the bioequivalence between test and reference formulation of Duloxetine DR capsule. The method proves to be highly rugged and can be employed for the determination of DUX in human plasma, where very low concentration quantification is desired.

5. ACKNOWLEDGEMENT:

The authors are grateful to the School of Advanced Sciences, Vellore Institute of Technology (VIT), Vellore, India, for supplying the essential facilities and support for the making of this research article. The authors extend their gratitude to Strides Arcolab Limited (SAL), India and Jeevan Scientific Technology Limited, India for all their technical assistance and resource supports.

6. FUNDING:

Supported by Strides Arcolab Limited (SAL), India and Jeevan Scientific Technology Limited, Hyderabad, Telangana.

7. CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

8. ETHICAL APPROVAL:

The study was approved by Samkshema independent Ethics Committee. The principles outlined in the declaration of Helsinki were adhered to while performing the study.

9. INFORMED CONSENT:

Informed consent was obtained separately from all study subjects enrolled in the study.

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Received on 09.09.2019 Modified on 12.10.2019

Research J. Pharm. and Tech 2020; 13(5):2117-2124.

DOI: 10.5958/0974-360X.2020.00381.9

Cal. Std.	1	2	3	4	5	6	7	8	9	10	11
Actual Conc. (ng/mL)	0.100	0.200	0.400	1.000	2.000	5.000	10.00	25.00	60.00	80.00	100.00
Calculated Conc. (ng/mL)	0.101	0.204	0.399	0.998	2.025	5.100	10.00	26.20	59.50	80.20	98.00
STD DEV	0.005	0.010	0.137	0.203	0.5	0.120	0.189	0.4	2.56	4.00	3.56
%CV	4.9	3,98	2.78	4.89	3.47	2.89	1.09	2.2	3.28	6.45	3.78
Mean % Nominal conc. (ng/mL)	101	102	99.75	99.8	101.25	102	100	104.8	99.17	100.25	98