Development and Validation of A LC/MS/MS Method For The Determination of Duloxetine in Human Plasma and Its Application To Pharmacokinetic Study

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ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry
http://www.ejchem.net 2012, 9(2), 899-911

Development and Validation of a LC/MS/MS Method


for the Determination of Duloxetine in Human Plasma
and its Application to Pharmacokinetic Study
*
D. CHANDRAPAL REDDY1, 2, A. T. BAPUJI1, V. SURYANARAYANA RAO3, V.
HIMABINDU 2, D. RAMA RAJU1, SYED SYEDBA1 and H. L. V. RAVIKIRAN1

1
Department of Clinical Pharmacology
APL-Research Centre, Hyderabad 500090, AP, India
2
Institute of Science and Technology
JNT University, Kukatpally, Hyderabad 500085, AP, India
3
Department of Chemistry SKD University
Anantapur 515 055, AP, India
dcpreddy007@yahoo.co.in

Received 8 August 2011; Accepted 4 October 2011

Abstract: A selective, high sensitive and high throughput liquid


chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been
developed and validated for the chromatographic separation and quantitation of
duloxetine in human EDTA plasma using fluoxetine (IS) as an internal standard.
Analyte and IS were extracted from human plasma by liquid-liquid extraction
using MTBE-n Hexane (80:20).The eluted samples were chromatographed on
X-terra RP8 (50 mm4.6 mm, 5 µm particle size) column by using mixture of
30 mM ammonium formate (pH-5.00.05) and acetonitrile as an isocratic mobile
phase at a flow rate of 0.40 mL/min and analyzed by mass spectrometer in the
multiple reaction monitoring (MRM) using the respective m/z 298.08→154.0 for
duloxetine and 310.02→148.07 for IS. The linearity of the response/
concentration curve was established in human plasma over the concentration
range 0.100-100.017 ng/mL. The lower detection limit (LOD,S/N>3) was 0.04
ng/mL and the lower limit of quantization (LOQ,S/N>10) was 0.100 ng/mL.
This LC-MS/MS method was validated with Intra-batch and Inter-batch
precision of 5.21-7.02.The Intra-batch and Inter-batch accuracy was 97.14-
103.50 respectively. Recovery of duloxetine in human plasma is 80.31% and
ISTD recovery is 81.09%. The main pharmacokinetic parameters were Tmax (hr)
= (7.251.581), Cmax (ng/mL) (44.594 0t, = (984.702526.502)
and AUC0, (1027.147

Keywords: Duloxetine, UPLC-MS/MS, Human plasma, Extraction efficiency, Bioequivalence study,


and pharmacokinetic.
900 D. C. REDDY et al.

Introduction
Duloxetine HCl (DLX) (Figure 1) is chemically, 2(+)-(S)-N-methyl-(gamma)-(1-naphthyloxy)-2
thiophenepropylamine hydrochloride1. Duloxetine hydrochloride is a newer selective
serotonin and norepinephrine reuptake inhibitor (SSNRI) used for major depressive
disorders2-3. The empirical formula is C18H19NOS.HCl and having a molecular weight of
333.88. It is used for the treatment of naturopathic pain associated with peripheral
neuropathy especially diabetic polyneuropathy for which it is first-line and as an add-on
treatment in stress urinary incontinence instead of surgery4-5, also indicated for the
management of fibromyalgia6-7. It restores the balance of neurotransmitters in the brain like
serotonin and norepinehrine8. Moreover it is also being used in the treatment of peripheral
neuropathy caused by certain anti cancer drugs9.

Figure 1. Chemical structure for duloxetine HCl.

In the references few bioanalytical methods are reported for the determination of
duloxetine in human plasma by LC-MS/MS10, HPLC analysis of duloxetine in human
plasma with SPE11, capillary electrophories with laser-induced fluorescence detection12, in
blood using HPLC with spectrometric detection and column switching13 and LC-MS (SIM
mode)14.
The following are the advantages of the proposed method over those reported earlier: (1).
Greater sensitivity is achieved (0.100 ng/mL) even with low plasma volumes and method is
well suited for pharmacokinetic analysis. (2). Employing a single-step liquid-liquid
extraction procedure minimizes the chances of errors, saves considerable time and simplifies
the sample preparation procedure. (3). Because of the use of less plasma volume (0.300 mL),
the volume of the sample to be collected for time point from subjects during the study is
reduced significantly-this allows inclusion of additional points (4). The rapid sample
analysis turnaround time of 3.00 minutes makes it an attractive procedure in high-throughput
bioanalysis of duloxetine in human plasma. The chromatographic conditions were optimized
and the results of validation in terms of Specificity, linearity, precision, accuracy, extraction
efficiency, dilution integrity, and Stabilities were provided. The devised method was used in
duloxetine clinical study, which was conducted in accord with USFDA guidelines. Typical
bioavailability including AUC0t (the area under plasma concentration-time curve) and Cmax
(the maximum plasma concentration) AUC0, (Area under the concentration time-curves
from time zero to infinity) parameters were compared.

Experimental
Reference standards of Duloxetine (Potency (w/w 99.5%) and Fluoxetine (99.9%) were
procured from Aurobindo pharma Ltd. (Hyderabad, India). Methanol and Acetonitrile were
of HPLC Grade purchased from J.T. Baker (Philipsburg, USA). Analytical-grade
Ammonium formate was purchased from sdfine chemicals (Mumbai, India.), tert-butyl
Development and Validation of a LC/MS/MS Method for the Determination 901

methyl ether and n-Hexane were of HPLC Grade purchased from Merck specialties (Mumbai,
India) and Formic acid (AR Grade) was purchased from (RFCL Chemicals New Delhi,
India). Polypropylene vials (Torsens products Pvt Ltd Kolkata, India.) Water used for the
LC-MS/MS analysis was prepared using a Milli Q water purification system procured from
Millipore (Bangalore, India). Human plasma was procured from Cauvery Diagnostics and
blood bank Hyderabad, India).and was stored at -20°C until use.
Liquid Chromatographic Conditions
A waters Acquity UPLC system (Milford, MA, USA) consisting of binary solvent manager,
sample manager and column manager was used for setting the reverse-phase liquid
chromatographic conditions. The separation of Duloxetine and Fluoxetine (IS) was
performed on X-terra RP8 (50mm×4.6mm (length inner diameter), with 5 µm particle size)
and was maintained at 35°C in column oven. The mobile phase consists of 30mM
Ammonium formate (pH 5.000.05) and acetonitrile in 10:90 (v/v) ratio. For isocratic
elution, the flow rate of the mobile phase was kept at 0.40 mL/min. The total
chromatographic run time was 3.0 min. The sample manager temperature was maintained at
10°C and the pressure of the system was 1200 psi.
Mass Spectrometric Conditions
Ionization and detection of analyte and IS was carried out on a triple quadrupole mass
spectrometer. WATERS, Quattro Micro (Milford, MA, USA) equipped with electro spray
ionization and operating in positive ion mode. Quantization was performed using multiple reaction
monitoring (MRM) mode to monitor Parent → Product ion (m/z) transitions for Duloxetine
298.08 → 154.0 and 310.02 → 148.07 for IS respectively. (Figure 2 shows Figure 2 (A). Product
ion mass spectra of Duloxetine (m/z 298.08→154.0, scan range 80-320 amu) and Figure 2 (B).
Product ion mass spectra of Fluoxetine (m/z 310.12→148.07 amu), scan range 50-350 amu).

Figure 2. (A). Product ion mass spectra of Duloxetine (m/z 298.08→154.0, scan range
80-320 amu).
902 D. C. REDDY et al.

Figure 2. (B). Product ion mass spectra of Fluoxetine (m/z 310.12→148.07 amu), scan
range 50-350 amu.

The source dependent parameters maintained for Duloxetine and Fluoxetine were
capillary; 3.50 kV; extractor; 0.0V; RF lens; 0.2V; source temperature: 100°C; desolvation
temperature: 400°C; cone gas flow; 80±10 L/h desolvation gas flow: 800±10 L/h. The
optimum values for compound dependent parameters (MRM file parameters) like cone
voltage and collision energy set were 10 V and 6.0 eV for the analyte and 15.0 V and
18.0 eV for IS respectively. The dwell time easy set at 500 ms. Mass Lynx software version
4.1 was used to control all parameters of UPLC and MS.
Standard Stock, Calibration Standards and Quality Control Sample Preparation
The standard stock solution of 1 mg/mL of duloxetine and fluoxetine was prepared by
dissolving requisite amount in methanol. Calibration standards and quality control (QC)
samples were prepared by spiking (2% total volume of blank plasma) blank plasma with stock
solution. Calibration curve standards were made at 0.100, 0.200, 0.500, 10.002 and 25004.
50.009, 80.014 and 100.017. ng/mL respectively while quality control samples were prepared
at four levels, viz. 77.752 ng/mL (HQC, high quality control), 42.064 (MQC, middle quality
control), 0.300 ng/mL (LQC low quality control), 0.100 ng/mL (LLOQQC lowest level quality
control). Stock solution (1 mg/mL) of the internal standard was prepared by dissolving 10 mg
of Fluoxetine in 10 mL of methanol. An aliquot of 50.0 µL of this solution was further diluted
to 10.0 mL in the same diluent to obtain solution of 5.000 µg/mL. All the solutions (standard
stock, calibration standards and quality control samples) were store at 2-8°C until use.
Protocol for Sample Preparation
Prior to analysis, all frozen subjects samples, calibration standards and quality control samples
were thawed and allowed to equilibrate at room temperature. To an aliquot of 300 µL of spiked
plasma sample, 50 µL internal standard was added and vortexed for 20 s. Further, 100 µL of
50 mM Ammonium formate was added and vortexed 20 s. To these samples, 2.5 mL of
Development and Validation of a LC/MS/MS Method for the Determination 903

extraction solvent (MTBE : n Hexane 20:80, v/v) was added and samples were extracted on
extractor at 32 × g for 10 10 min. centrifugation of the samples was done at 3200 × g, for
10 min at 10°C. 2.0 mL supernant was separated and evaporated to dryness under nitrogen at
50°C ± 0.5°C for 15 psi and 15 min. The dried samples were reconstituted with 300 µL of
mobile phase and 20 µL was injection in the chromatographic system.
Method Validation
The method validation was performed as per USFDA guidelines 15. System suitability
experiment was performed by injecting six consecutive injections using aqueous standard
mixture of Duloxetine and internal standard at the start of each batch during the method
validation. The carryover effect of the auto sampler was evaluated by injecting a sequence of
injections solutions of aqueous standard, Mobile phase, standard blank, extracted standard
equivalent to highest standard in the calibration range. As per the acceptance criteria, the
response in blank should not be greater than 20% of LLOQ response 16.
The linearity of the method was determined by analysis of five linear curves containing
eight non-zero concentration. The ratio of area response for drug and IS was used for
regression analysis. Each calibration curve was analyzed individually by using least square
weighed (1/x2) linear regression. The lowest standard on the calibration curve was accepted
as the lower limit of quantitation (LLOQ), if the analyte response was at least five times
more than that of the drug free (blank) extracted plasma. The deviation of than that of drug
free (blank) extracted plasma. The deviation of standards other than LLOQ from nominal
concentration should not to be more than ±15.0%.
The selectivity of the method towards endogenous plasma matrix components was
assessed in twelve batches (7 normal of K2 EDTA plasma, 2 haemolysed, 2 lipidemic and
and 1 heparinised) of blank human plasma. This was done to estimate the extent to which
endogenous plasma components contribute towards interference at the retention time of
analytes and IS. The cross talk of MRM for analytes and IS was checked using highest
standard on calibration curve and working solution of IS.
For determining the intra-day accuracy and precision, replicate analysis of plasma
samples of Duloxetine was performed on the same day. The run consisted of a calibration
curve and six replicates of LLOQ, LQC, MQC and HQC samples. The inter-day accuracy
and precision were assessed by analysis of three precision and accuracy batches on three
consecutive validation days. The precision of the method was determined by calculating the
percent coefficient of variation (%CV) for each level. The deviation at each concentration
level from the nominal concentration was expected to be within ±15.0% except LLOQ, for
which it should be within ±20.0%.
The relative recovery, matrix effect and process efficiency were assessed as
recommended by Matuszewski et al.17. All three parameters were evaluated at Std-1, Std-3,
Std-5, Std-6 and Std-8 levels in six replicates. Relative recovery (RE) was calculated by
comparing the mean area response of extracted samples (spiked before extraction) to that of
unextracted samples (spiked after extraction) at each CC level. The recovery of IS was
similarly estimated. Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions. The overall ‘process efficiency’ (%PE) was calculated by comparing the mean
area response of extracted samples (spiked before extraction) to that with mean area of neat
standard solutions at each CC level. The assessment of relative matrix effect was based on
direct comparison of the MS/MS responses (peak areas) of the analytes spiked into extracts
originating from different lots of plasma. The variability in these responses, expressed as
%CV was considered as the measure of relative matrix effect.
904 D. C. REDDY et al.

Stability experiments were carried out to examine the analyte stability in stock solutions
and in plasma samples under different conditions. Short term stability at room temperature
and long term stability of spiked solution stored at – 70ºC was assessed by comparing the
area response of stability sample of analyte and IS with the area response of sample prepared
from fresh stock solutions. The solutions were considered stable if the deviation from
nominal value was within ±10%. Autosampler, wet extract stability, bench top stability, dry
extract stability and freeze- thaw stability were performed at LQC and HQC, using six
replicates at each level. The samples were considered stable if the deviation from the mean
calculated concentration of freshly thawed quality control samples was within ±15%.
To authenticate the ruggedness of the proposed method, it was done on two precision
and accuracy batches. The first batch was analysed by different analysts while the second
batch was analysed on different column and different LC-MS/MS. Dilution integrity
experiment was conducted by diluting the stock solution prepared as spiked standard at
concentration of 200.034 ng/mL for Duloxetine. The precision and accuracy for dilution
integrity standards at 1/5th and 1/10th determined by analyzing the samples against
calibration curve standards.
Study Design
A pharmacokinetic study was conducted on 12 healthy, adult, male, human subjects under
fating conditions. (n = 12) following oral administration of 60 mg delayed release capsules.
Each volunteer was judged to be in good health through medical history, physical
examination and routine laboratory tests. Written consent was taken from all the volunteers
after informing them about the objectives and possible risks involved in the study. An
independent ethics committee constituted as per Indian council of Medical Research (ICMR)
approved the study protocol. The study was conducted strictly in accordance with guidelines
laid down by international conference on Harmonization and USFDA18. A single oral dose
of 60 mg drug was given to the volunteers with 240 mL of water. Blood samples were
collected at 0.0 (pre-dose), 1.00, 2.00, 3.00, 4.00, 4.50, 5.00, 5.50, 6.00, 6.50, 7.00, 7.50,
8.00, 10.00, 12.00, 16.00, 20.00, 24.00, 36.00, 48.00 and 72.00 h after oral administration of
the dose for test formulation in labeled K2 EDTA- vaccuettes. Plasma was separated by
centrifugation(3200 × g, 10ºC, 10 min) and kept frozen at – 70ºC until analysis. During
study, volunteers had a standard diet while water intake was free.

Results and Discussion


Method Development
Chromatographic resolution of Duloxetine and IS was initiated under isocratic conditions to
obtain adequate response, sharp peak shape and a short analysis time. Thus, separation was
tried using various combinations of methanol/acetonitrile, acidic buffers and additives like
formic acid on different reversed-phase columns with 5µm particle size viz. Chromolith,
Hypersil, X-terra, Kromasil, Intertsil and Grace ACE Cyano (150 mm and 250 mm × 4.6 mm),
Chromolith RP-18 (50 mm × 4.6 mm), Kromasil (50 mm and 100 mm × 4.6 mm) and
Gemini C-18 (50 mm × 4.6 mm) to find the optimal column that produced the best
sensitivity, efficiency and peak shape. The analytes showed poor separation and
reproducibility for proposed linear range except for x-terra RP-8 column that offered superior
peak shape, baseline separation, desired linearity and reproducibility. The mobile phase
consisting of 30 mM ammonium formate adjusted the pH 5.000.05 with formic acid and
methanol (30:70, v/v) ratio and having 30 mM ammonium formate pH around 5.0-5.5 were
found most suitable for eluting Duloxetine and IS at 1.50 and 1.48 min respectively. Also,
Development and Validation of a LC/MS/MS Method for the Determination 905

the reproducibility of retention times for the analytes, expressed as %CV was ≤2% for 100
injections on the same column.

The inherent selectivity of MS/MS detection was also expected to be beneficial in


developing a selective and sensitive method. The present study was conducted using ESI as
the ionization source as it gave high intensity for drug and IS as they have similar sites for
protonation. Initially, the extraction of Duloxetine and IS was carried out via protein
precipitation with common solvents like acetonitrile, methanol and acetone, but the
sensitivity and reproducibility were poor, in all the solvents with frequent clogging of the
column, which required online flushing of the column. Liquid-liquid extraction technique
was also tested to isolate the drugs from plasma using diethyl ether, dichromethane, ethyl
acetate, methyl tert butyl ether and isopropyl alcohol (alone and in combination) as
extracting solvents. However, the recovery was inconsistent with some ion suppression
(greater than 15% CV) in most of these solvent systems. Further, use of 100 µL of 50mM
ammonium formate to extraction in methyl tert butyl ether: n Hexane (80:20, v/v) gave
consistent recoveries for the analytes, especially at the LLOQ level with minimum matrix
interference. A general internal standard was used to minimize any analytical variation due
to solvent evaporation, integrity of the column and ionization efficiency of analytes.
Fluoxetine was used as an internal standard (IS) in the present study, which had similar
chromatographic behavior and was quantitatively extracted with the proposed extraction
procedure. Also, there was no effect of IS on analyte recovery, sensitivity or ion
suppression.

System Suitability and Auto Sampler Carryover


Throughout the method validation, the % CV of system suitability was observed below 4.0
at the retention time of duloxetine and the IS. Carryover evaluation was performed in each
analytical run so as to ensure that it does not affect the accuracy and the precision of the
proposed method. There was negligible carryover (≤4% of the LLOQ response) observed
during auto sampler carryover experiment, No enhancement in the response was observed in
double blank after subsequent injection of highest calibration standard (aqueous and
extracted) at the retention time of analytes and IS.

Linearity and Lower Limit of Quantification (LLOQ)


The calibration curves were linear over the concentration range of 0.100.100.017 ng/mL
with correlation coefficient r2 ≥ 0.9963 for duloxetine respectively. The equations for means
(n=5) of five calibration curves for duloxetine. The standard deviation value for slope,
intercept observed were 0.9921, 0.9952, 0.9951, 0.9997, 0.9996 and 0.00008, 0.00007:
0.0005, 0.0006: 0.0005 and 0.0005 for duloxetine respectively. The accuracy and precision
(%CV) observed for the calibration curve standards ranged from 91.14 to 104.59% and 0.82
to 11.54% respectively. The lowest concentration (LLOQ) in the standard curve for both the
isomers was measured at a signal-to-noise ratio (S/N) of ≥ 100. Figure 3 shows the
representative LC-MS/MS chromatograms of (A) calibration curve of duloxetine, (B)
Double blank plasma with out IS, (C) Blank plasma with IS and (D) Duloxetine and
fluoxetine at LLOQ.
906 D. C. REDDY et al.

Residual
Response

Figure 3. (a) Calibration curve of Duloxetine.

Figure 3. (b) Double blank plasma (without IS).

Figure 3. (c) Blank plasma with IS.


Development and Validation of a LC/MS/MS Method for the Determination 907

Figure 3. (d) Duloxetine and Fluoxetine at LLOQ (m/z) 298.08154.0.

Selectivity, Accuracy and Precision


To establish the selectivity of the method for interference due to endogenous plasma
components from haemolysed, lipidemic, heparinised and K2 EDTA blank plasmas, the %
change in the area ratio (analyte/IS) at LLOQ level was within 4-8%, while the precision
(%CV) in their measurement varied from 2.1 to 5.6%, representative MRM ion
chromatograms extracted (A) blank human plasma (double blank), (B) blank plasma
fortified with IS (m/z 310.02→148.07), duloxetine at LLOQ (m/z 298.08→154.0) the
selectivity of the method. The extraction procedure together with mass detection gave very
good selectivity for the analysis of both the drug and IS in the blank plasma. No endogenous
interferences were found at the retention times of analytes and IS.
The intra- and inter batch precision and accuracy were established from validation runs
performed at HQC, MQC, LQC and LLOQ QC levels. The intra- and inter batch precision
ranged from 0.74 to 9.76% for duloxetine.The accuracy values were within 97.14-104.49%
for both the analytes in intra- and inter batches. The precision and accuracy values for intra-
and inter day experiments in plasma are shown in Table 1(A).

Table 1. (A). Comparison of intra- and inter-batch precision and accuracy for Duloxetine.
Intrabatch
Nominal Mean
QC ID
concentration, ng/mL n concentration % CV % Accuracy
observed, ng/mL
LLOQQC 0.100 0.104 7.70 103.50
LQC 0.300 0.307 5.56 102.29
6
MQC 42.060 41.255 3.79 98.08
HQC 77.750 81.240 5.03 104.49
Intrabatch
Nominal Mean
QC ID
concentration, ng/mL n concentration % CV % Accuracy
observed, ng/mL
LLOQQC 0.100 0.103 6.85 103.25
LQC 0.300 0.308 7.02 102.59
24
MQC 42.060 40.863 5.21 97.14
HQC 77.750 79.684 5.52 102.48
908 D. C. REDDY et al.

(B). Stability of Duloxetine under various conditions (n=6).


Nominal Mean calculated % Mean
Storage conditions
Concentration, ng/mL conc. (ng/mL) ± SD accuracy
Bench top stability
(After 5.67 h at ~ at 25°C
LQC 0.300 0.298 ± 0.0090 99.28
HQC 77.750 75.382 ± 2.6005 96.95
Freeze thaw stability
(3 Cycles)
LQC 0.300 0.298 ± 0.0060 99.39
HQC 77.750 79.717 ± 4.9832 102.53
Dry extract stability
(25.82 h below 10°C)
LQC 0.300 0.299 ± 0.0087 99.67
HQC 77.750 83.442 ± 5.2259 107.32
Wet extract stability
(26.43 h below 10°C)
LQC 0.300 0.309 ± 0.0136 102.94
HQC 77.750 76.943 ± 3.4201 98.96
Auto sampler stability
(21.88 h 10°C)
LQC 0.300 0.297 ± 0.0112 98.89
HQC 77.750 73.886 ± 4.3496 95.00
Long term stability in plasma
at -70°C
(23.66 days at -70°C)
0.300 0.298 ± 0.0060 99.39
LQC 77.750 71.398 ± 5.4829 91.83
HQC

Recovery and Stability Results


The relative recovery, absolute matrix effect and process efficiency data at LQC, MQC and
HQC levels is presented. The recovery for drug and IS in human plasma was 80.31% and
81.09%. Further, the extent of matrix effect in different lots of plasma (spiked after
extraction) was within the acceptable limit as evident from the precision (%CV) values in
Table 2.
Stock solutions for short term stability of Duloxetine and IS were stable at room
temperature for minimum period of about 6 h and between 2 and 8°C for about 7 days.
Duloxetine in control human plasma (bench top) at room temperature was stable at least for
5.67 h at ambient temperature and for minimum of three freeze and thaw cycles. Auto
sampler stability of the spiked quality control samples maintained at 10°C was maintained
up to 21.88 h. Long-term stability of the spiked quality control samples stored at -70°C was
determined up to 23.66 days. The accuracy values for different stability experiment in
plasma are shown in Table 1(B).
Development and Validation of a LC/MS/MS Method for the Determination 909

Table 2. Absolute matrix effect, relative recovery and process efficiency for Mirtazapine.
Analyte Aa Bb Cc Absolute Relative Process
ISTD (%CV) (%CV) (%CV) matrix recovery efficiency
effect (%RE) e (%PE) f
(%ME) d
STD 1 179 188 109 94.23 85.40 80.47
Mirtazapine (8.31) (3.91) (4.32) 90.41 84.34 76.25
Zolpidem 42068 37907 40711
(0.49) (1.35) (1.32)
STD 3 567 591 460 104.38 77.77 81.18
Mirtazapine (3.23) (4.13) (1.22) 103.33 75.68 76.20
Zolpidem 52367 54113 40951
(1.31) (1.56) (1.80)
STD 5 23157 22868 18300 98.75 80.02 79.02
Mirtazapine (1.74) (1.69) (0.96) 97.91 83.67 81.93
Zolpidem 50420 49366 41307
(1.94) (1.65) (0.68)
STD 6 43730 43102 34517 98.57 80.08 78.93
Mirtazapine (1.09) (1.24) (1.01) 99.34 80.29 79.76
Zolpidem 47700 47385 38045
(1.53) (1.56) (1.07)
STD 8 77701 78734 63689 101.33 80.89 81.97
Mirtazapine (1.46) (1.94) (0.80) 102.28 87.30 89.29
Zolpidem 42800 43777 38217
(1.61) (2.23) (5.33)
a Mean area response of six replicate samples prepared in Mobile phase (neat samples), b Mean area
response of six replicate samples prepared by spiking in post extracted blank, c Mean area response of
six replicate samples prepared by spiking in plasma before extraction, d %Matrix effect: Post extracted
mean response/Aqueous (Neat) mean response x 100, e %Recovery: Extracted mean response / Post
extracted mean response x 100, f %Process efficiency: Extracted mean response / Aqueous Mean
response x 100.

Ruggedness and Dilution Integrity


The results of ruggedness study for Duloxetine was well within the acceptance limit of 15%
in Precision and 85.0-115.0. % in mean accuracy. The precision and accuracy values for
both experiment at LLOQ, LQC, MQC and HQC levels for Duloxetine ranged from 3.4 to
7.6% and 97.1 to 108.5% respectively.
The dilution integrity experiment was performed with an aim to validate the dilution test
to be carried out on higher analyte concentration above the upper limit of quantification
(ULOQ), which maybe encountered during real subject sample analysis. The precision and
accuracy values for 1/5th and 1/10th dilution ranged from 3.26 to 3.34% and 98.2 to 109.7 for
Duloxetine.
Application of the Method in Healthy Human Subjects
The validated method was successfully applied for the assay of Duloxetine in healthy male
Indian volunteers in the age group of 18-45 years. Figure 4 shows the plasma concentration
vs. time profile of Duloxetine human subjects under fating condition. The method was
sensitive enough to monitor the Duloxetine plasma concentration up to 72 h. Approximately
520 samples including the calibration and QC samples were within the acceptable limits.
910 D. C. REDDY et al.

The pharmacokinetic parameters viz. Cmax, Tmax, AUC0t and AUC0 were
calculated for Duloxetine in test formulations. (Figure 4). Shows the data of Mean plasma
concentration-time profile of Duloxetine Hydrochloride 60 mg Delayed Release Capsules
formulation to 12 healthy volunteers.
Linear mean plot of plasma Duloxetine Concentration Vs Time under Fasting conditions

45

40
Mean concentration, ng/mL

35

30

25

20

15

10

0
0 6 12 18 24 30 36 42 48 54 60 66 72
Time,(hr)
Time h

Parameter Duloxetine
Cmax, ng/mL 44.59418.599
Tmax (h) 7.251.581
AUC0t 984.702526.502
AUC0 1027.147572.790
Figure 4. Mean plasma concentration-time profile of Duloxetine Hydrochloride 60 mg
Delayed Release Capsules formulation to 8 healthy volunteers.

Conclusion
The proposed method successfully demonstrates chromatographic separation of duloxetine
from human plasma with high resolution. The bioanalytical methodology for their
simultaneous determination is highly specific, rugged and rapid for therapeutic drug
monitoring. The method involved a simple and specific sample preparation by liquid-liquid
extraction followed by isocratic separation in 3.0 min. The overall analysis time is proving
compared to other reported procedures for duloxetine.
Acknowledgment
The authors gratefully acknowledge Dr. A.T. Bapuji and Aurobindo Pharma Ltd Hyderabad,
India for providing necessary facilities for carrying out this study.

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