Development and Validation of A LC/MS/MS Method For The Determination of Duloxetine in Human Plasma and Its Application To Pharmacokinetic Study
Development and Validation of A LC/MS/MS Method For The Determination of Duloxetine in Human Plasma and Its Application To Pharmacokinetic Study
Development and Validation of A LC/MS/MS Method For The Determination of Duloxetine in Human Plasma and Its Application To Pharmacokinetic Study
E-Journal of Chemistry
http://www.ejchem.net 2012, 9(2), 899-911
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
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.
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 AUC0t (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 5.000.05) 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.
the reproducibility of retention times for the analytes, expressed as %CV was ≤2% for 100
injections on the same column.
Residual
Response
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.
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.
The pharmacokinetic parameters viz. Cmax, Tmax, AUC0t 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 44.59418.599
Tmax (h) 7.251.581
AUC0t 984.702526.502
AUC0 1027.147572.790
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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