EPA DHA jlcrt

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SIMULTANEOUS DETERMINATION
OF DOCOSAHEXAENOIC ACID AND
EICOSAPENTAENOIC ACID BY LC-ESI-MS/
MS FROM HUMAN PLASMA
a a a a
Chinmoy Ghosh , Vijay Jha , Chinmay Patra , Ramesh Ahir &
a
Bhaswat Chakraborty
a
Bio-Analytical Department, Cadila Pharmaceuticals Limited,
Dholka, Gujarat, India

Accepted author version posted online: 10 Apr 2012. Version of
record first published: 07 Aug 2012

To cite this article: Chinmoy Ghosh, Vijay Jha, Chinmay Patra, Ramesh Ahir & Bhaswat Chakraborty
(2012): SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACID
BY LC-ESI-MS/MS FROM HUMAN PLASMA, Journal of Liquid Chromatography & Related Technologies,
35:13, 1812-1825

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Journal of Liquid Chromatography & Related Technologies, 35:1812–1825, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2011.627603

SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC
ACID AND EICOSAPENTAENOIC ACID BY LC-ESI-MS/MS
FROM HUMAN PLASMA

Chinmoy Ghosh, Vijay Jha, Chinmay Patra, Ramesh Ahir, and
Bhaswat Chakraborty
Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012

Bio-Analytical Department, Cadila Pharmaceuticals Limited, Dholka, Gujarat, India

& A sensitive and rapid method based on liquid chromatography=tandem mass spectrometry
(LC=MS=MS) with simple single step protein precipitation has been developed and validated for
the quantitative determination of docosahexaenoic acid (DHA) and eicosapentaenoic acid
(EPA) in human plasma. After addition of internal standard to human plasma, samples were
extracted by simple protein precipitation using acetonitrile as the precipitating agent. The extracts
were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring
(MRM) mode. This method for the simultaneous determination of DHA and EPA is accurate
and reproducible, with limits of quantitation of 50.00 ng=mL in plasma. The standard calibration
curves for both DHA and EPA are linear (r > 0.99) over the concentration ranges 50.00–
7498.50 ng=mL in human plasma, respectively. The intra- and inter-day precision over the con-
centration range for DHA and EPA are less than 10.16 and 6.72 (relative standard deviation,
%RSD), and accuracy is between 91.17–104.74% and 95.81–108.33%, respectively.

Keywords docosahexaenoic acid, eicosapentaenoic acid, LC-MS=MS, matrix effects,
poly unsaturated fatty acid, protein precipitation

INTRODUCTION
The x-3 fatty acids found in fish oil, eicosapentaenoic acid (EPA; 20:5
n-3) [(5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5, 8, 11, 14, 17-pentenoic acid] and
docosahexaenoic acid (DHA; 22:6 n-3) [(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-
docosa-4,7,10,13,16,19-hexaenoic acid], are essential for growth and devel-
opment, and may also play an important role in the prevention and treat-
ment of cardiovascular disease, inflammatory diseases, and cancer.[1,2]
Several species of marine fish offer rich dietary sources of polyunsaturated
fatty acids (PUFA), for example EPA and DHA, but these foods are not

Address correspondence to Mr. Chinmoy Ghosh, Research Scientist, Cadila Pharmaceuticals
Limited, 1389, Trasad Road, Dholka, Gujarat, India. E-mail: chinmoy_ghosh@yahoo.com

Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1813

regularly included in the western diet. For the majority of the population,
the alternative dietary source of long chain of x-3 fatty acids might be the
precursor, a-linolenic acid (ALA; 18:3 n-3). Previous reports suggested that
increased intake of ALA, similar to intake of EPA and DHA, may have ben-
eficial effects in health and in the control of chronic diseases.[1]
Very few bioavailability studies have been undertaken for EPA and DHA;
among them this is the only method that was developed by using LC-MS=MS.
Methods currently used for the analysis of mono- and poly-hydroxy fatty acids
include gas chromatography (GC)[3–5] and HPLC with a chemiluminescence
labeling method.[6] Among all these published methods, there is only one
method available for determination of EPA and DHA from human plasma,[5]
and the rest are either from fish,[3] perilla oil,[4] or human serum.[6]
In contrast to all reported methods for simultaneous determination of

EPA and DHA, the current method is the simplest, fastest and most sensitive
one. The other reported methods have very long extraction procedures[3–6]
with comparatively high LLOQ value. The extraction method adopted by
Chauke et al.[3] used mechanical homogenization followed by liquid-liquid
extraction (LLE) and methyl esterification for GC analysis. In another method
reported by Kurowska et al.,[4] several intermediate steps were performed that
included liquid-liquid extraction (LLE) followed by hydrolyzed incubation,
precipitation, again incubation, and finally LLE followed by GC analysis.
Similarly, Rusca et al.[5] performed the LLE, followed by de-esterification,
methyl esterification, and again LLE before injecting into GC. LLE followed
by chemiluminescence labeling reaction was reported by Hidetaka et al.[6]
Whereas, the present manuscript describes a sensitive, simple, single step pro-
tein precipitation technique that achieves a LLOQ value of 50 ng=mL. More-
over, this is the fastest method with respect to any other reported methods,[3–6]
with only 2.25 min of analysis time and a very wide range of assay linearity, over
the concentration range of 50.00–7498.50 ng=mL. LC-MS=MS combines the
resolving power of liquid chromatography with the detection specificity of
MS=MS and overcomes the limitations of the conventionally used approaches,
thus providing the means of a rapid, versatile, and sensitive methodology over
the GC methods, which is not widely used for bioanalysis nowadays. Moreover,
no methods were reported with as short an analysis time, as simple an extrac-
tion technique, or as wide a linearity range. As a result, this developed method
is worthwhile for bioanalysis of EPA and DHA by LC-MS=MS.

EXPERIMENTAL
Chemicals and Reagents
DHA (98% purity) and EPA (99% purity) were obtained from the Sigma
Aldrich, MO, USA and Nevirapin (internal standard, IS, 100.3% purity) was

1814 C. Ghosh et al.

purchased from Sequent Scientific Limited, New Mangalore, India.
Methanol and acetonitrile (J.T. Baker, IL, USA) was of HPLC-grade, and
other chemicals used were of analytical grade. Water used for the prep-
aration of the mobile phase and other solutions was collected from Milli
QPS (Millipore, NY, USA). Human K2EDTA Plasma, lipemic, and hemolyzed
plasma were used during validation and study sample analysis was supplied
by the clinical unit of Cadila Pharmaceuticals Limited, Ahmedabad, India.
Plasma was stored at À30 Æ 5 C before sample preparation and analysis.
Plasma was obtained by centrifugation of blood treated with the anticoagu-
lant K2EDTA and stored at À30 C until analysis.

Instrumentation

The HPLC system with an auto sampler was a Shimadzu LC-20AD
(Shimadzu, Tokyo, Japan) coupled with Applied Biosystem Sciex (MDS
Sciex, ON, Canada) API 4000 Tandem mass spectrometry. The auto sam-
pler was SIL-HTC from Shimadzu, Tokyo, Japan. The solvent delivery mod-
ule was LC-20AD from Shimadzu, Tokyo, Japan. The chromatographic
integration was performed by Analyst software 1.4.2 (Applied Biosystems,
ON, Canada).

Standards and Quality Control Samples
Stock solutions of DHA and EPA were prepared by dissolving the accu-
rately weighed reference compounds in methanol to give a final concen-
tration of 1000 mg=mL of both. The solution was then diluted with diluent
(methanol:water 70=30, v=v) to achieve mixed standard working solutions
at concentrations of 50.00, 100.00, 499.90, 1999.60, 3499.30, 4999.00,
6198.75, and 7498.50 ng=mL for both DHA and EPA. Stock solution of IS
was prepared in methanol at the concentration of 1000 mg=mL and diluted
to 30 mg=mL with diluent. Structural formulae of DHA, EPA, and IS are
shown in Figure 1. All solutions were stored at À30 C and were brought
to room temperature before use.
For the preparation of standard curves or quality control samples, the
pre-mixed standard working solutions (10 mL) were used to spike blank
plasma samples (190 mL), both in pre study validation and during the analy-
sis of samples from the pharmacokinetic study.

Sample Preparation
To a 0.2 mL aliquot of plasma sample, 20 mL of internal standard
(30 mg=mL) was added. The samples were briefly mixed and 1 mL of


FIGURE 1 Structure of (A) docosahexaenoic acid, (B) eicosapentaenoic acid, and (C) nevirapine
(internal standard).

acetonitrile was added. The mixture was vortex-mixed for approximately
1 min and placed on a centrifuge machine. After centrifugation at
4500 rpm for 5 min, the upper organic layer was removed and transferred
into HPLC vial and was injected onto the LC–MS=MS system for analysis.

Chromatographic Conditions
Chromatography was performed on a Kromasil-100 C18 analytical
column (50 mm Â 4.6 mm i.d., 5 m, Eka Chemicals, Bohus, Sweden). The
column was maintained at 30 C. The compounds were eluted isocratically
at a flow rate of 0.45 mL=min. The mobile phase consisted of 0.1% v=v
ammonia solution:acetonitrile:methanol (10:81:09, v=v=v).

Mass Spectrometric Conditions
The mass spectrometer was operated in the negative ion detection
mode. Nitrogen was used as the nebulizing, turbo spray, and curtain gas,
with the optimum values set at 25, 30, and 25 psi, respectively. The tempera-
ture of the vaporizer was set at 400 C and ESI needle voltage was adjusted
to À4500 V. The declustering potential was set at À80, À85, and À80 V for
DHA, EPA, and IS, respectively. Identification was performed using mul-
tiple reaction monitoring (MRM) of the transitions of m=z 327.30 ! m=z
283.20 for DHA, m=z 301.30 ! m=z 257.20 for EPA and m=z 265.00 ! m=z
182.20 for nevirapine (IS), respectively, with a dwell time of 200 ms per
transition. For collision-induced dissociation (CID), nitrogen was used as


the collision gas at a pressure of 8 psi. The collision energy was À20 V,
À22 V, and À35 V for DHA, EPA, and IS, respectively.

Method Validation
EPA and DHA, are the endogenous compounds, so the back-calculated
concentration and accuracy were calculated by using a validated Microsoft
Excel sheet, which was validated by SAS software. Four zero standard sam-
ples were injected with each calibration curve to calculate the average area
ratio of the endogenous components. The calculated average area ratio was
subtracted from the area ratio of each sample to get the actual area ratio of
the components, which were further used to back calculate the concen-
tration and accuracy of calibration standards and quality control samples

using the validated Excel.
The sample concentrations were calculated using weighted (1=x2) least
squares linear regression. To evaluate linearity, plasma calibration curves
were prepared and were assayed on three separate days. Accuracy and
precision were also assessed by determining QC samples at three concen-
tration levels on three different validation days. The accuracy was expressed
by, (observed concentration=spiked concentration) Â 100% and the pre-
cision by relative standard deviation (RSD). The extraction recoveries of
DHA and EPA at three QC levels were evaluated by comparing peak areas
of analytes obtained from plasma samples with the analytes spiked before
extraction to those spiked after the extraction. The matrix effect was car-
ried out by extracting low and high quality control samples in triplicate
from six different plasma sources. The hemolysis effect and lipemic effect
experiments were carried out at two different concentration levels.
The stability of DHA and EPA in the diluents was assessed by placing QC
samples under ambient conditions for at least 6 hr. The freeze–thaw stability
of DHA and EPA was also assessed by analyzing QC samples undergoing four
freeze (À20 C) and thaw (room temperature) cycles. Similarly stability of
DHA and EPA was assessed inside the auto sampler and in plasma at room
temperature. Subsequently, the DHA and EPA concentrations were
measured and compared with freshly prepared samples, respectively.

Clinical Protocol
The pharmacokinetic study protocol presented in this paper was
approved by the Independent Medical Ethics Committee of Cadila
Contract Research Organization, Ahmedabad, Gujarat, India. In both per-
iods, the subject was administered a single dose of omega-3 fatty acid cap-
sule along with 200 mL of drinking water after an overnight fasting of at


least 10 hr in each period. The test capsule contains 30 mg DHA and
250 mg EPA, whereas the reference capsule contains 250 mg DHA and
350 mg EPA. The administered subject was a healthy, adult, male, human
volunteer of Indian origin. In each period, a total of 17 blood samples were
collected including four pre-dose sample prior to drug administration. The
blood samples were immediately centrifuged at 2000 rpm for 10 min at 4 C,
and the plasma samples were stored at À30 C until LC-MS=MS analysis.

RESULTS AND DISCUSSION
Optimization of Chromatographic Condition
The successful analysis of the analytes in biological fluids using HPLC-MS=

MS relies on the optimization of chromatographic conditions, sample prep-
aration techniques, chromatographic separation, post column detection,
and so forth.[7–10] Thus, for better selectivity and sensitivity, different types
of columns and mobile phases were used. Length of the column varied from
50 mm to 150 mm, and the particle size varied from 3.5 m to 5 m. Columns of
different types of stationary phase such as C8 and C18 were used which showed
some remarkable effect on peak shape. Finally, Kromasil-100 C18 analytical
column (50 mm Â 4.6 mm i.d., 5 m, particle size) was selected for analysis.
The influence of buffer molarities, pH, and types of organic modifier on
the signal intensities was also studied. Initially, 90% acetonitrile: 10% of 0.1%
ammonia solution (v=v) at a flow rate of 0.500 mL=min was tried but it led to
improper resolution between the other endogenous peaks present in the
extracted plasma samples. Therefore, methanol was introduced in a small
quantity, that is, 9% v=v by decreasing the equal volume of acetonitrile.
Then, based on the peak intensity and peak resolution, 0.1% ammonia sol-
ution (v=v), acetonitrile and methanol (10:81:09, v=v) as the mobile phase at
a flow rate of 0.450 mL=min were selected for further studies.
In sample extraction technique, protein precipitation was adopted.
Initially, plasma samples were precipitated by using methanol as the preci-
pitating agent, but it extracts more endogenous compounds that interfere
with the main peak. Then, acetonitrile was used as the precipitating agent
which produced a better result. Initially, a conventional 2:1(v=v) ratio of
acetonitrile to plasma was used, but due to high extraction recovery, the
responses of the analytes caused the saturation of the detector; as a result,
to make a diluted sample, 5:1 (v=v) ratio was selected.

Method Validation
The validation parameters were linearity, sensitivity, accuracy, precision,
matrix effects of the assay, and recovery and stability in human plasma,


according to the U.S. Food and Drug Administration (FDA) guidance for
the validation of Bioanalytical methods.[11]

Linearity
Linearity of calibration standards was assessed by subjecting the spiked
concentrations and the respective peak areas using 1=X2 linear least-squares
regression analysis. Linearity ranged from 50.00 to 7498.50 ng=mL for DHA
and EPA both (r 0.990). In aqueous solution, accuracy of all calibration
standards was within 85–115%, except LLOQ where it was 80–120%.

Specificity and Selectivity
As DHA and EPA are endogenous components, the specificity experiment

was performed for IS only. Six different lots of normal plasma and one lot of
lipemic plasma and hemolyzed plasma were analyzed to ensure that no
endogenous interference took place with the mass transitions of the IS. LLOQ
level samples along with plasma blank from the respective plasma lot were pre-
pared and analyzed. In each plasma blank, the response at the retention time
of IS was 5% of the IS response in the respective LLOQ. Figure 2A and 2B
represents the plasma blank of DHA and EPA, respectively, with IS.

Accuracy and Precision
For the validation of the assay, QC samples were prepared at three con-
centration levels of low, medium, and high. Six replicates of each QC sam-
ples were analyzed together with a set of calibration standard. The accuracy
of each sample preparation was determined by injection of calibration sam-
ples and three QC samples in six replicates for 3 d. Obtained accuracy and
precision (inter- and intra-day) are presented in Table 1 for EPA and DHA.
The results show that the analytical method is accurate, as the accuracy is
within the acceptable limits of 100 Æ 20% of the theoretical value at LLOQ
and 100 Æ 15% at all other concentration levels. The precision around the
mean value was never greater than 15% at any of the concentration studied.

Limit of Quantitation
Process and inject six LLOQ and six ULOQ samples along with cali-
bration standards in the same range used for calculation of precision and
accuracy.
For DHA, the %CV at LLOQ level was 7.14 and at ULOQ level was 3.69.
The average %accuracy at LLOQ level was102.02 and at ULOQ level was
101.32. For EPA the %CV at LLOQ level was 4.98 and at ULOQ level was
3.74. The average %accuracy at LLOQ level was100.34 and at ULOQ level
was 99.30.


FIGURE 2 A) Representative chromatogram of plasma blank of DHA and IS and B) Representative
chromatogram of plasma blank of EPA and IS. (Color figure available online.)

Recovery Study
A recovery study was performed by comparing processed QC samples of
three different levels in six replicate with aqueous samples of same level.
The recovery of DHA at low quality control (LQC) level was 58.64%,


TABLE 1 Inter-Day and Intra-Day Accuracy and Precision

DHA EPA

Days QC Level Accuracy Precision Accuracy Precision

Day 1 LQC 92.35 7.97 98.42 5.25
MQC 108.10 2.05 104.61 4.02
HQC 94.35 4.21 96.84 5.03
Day 2 LQC 91.45 2.36 94.81 5.79
MQC 99.69 3.68 99.52 2.50
HQC 97.27 3.95 97.94 4.47
Day 3 LQC 97.71 5.77 100.05 0.92
MQC 104.74 0.93 108.33 1.00
HQC 97.07 2.41 99.35 4.02

medium quality control (MQC) level was 47.96%, and for high quality
control (HQC) level was 49.60%. The mean recovery of DHA was
52.07%. % coefficients of variation (%CV) of mean recovery of all three
QCs were 11.04.
The recovery of EPA at low quality control (LQC) level was 46.35%,
medium quality control (MQC) level was 41.25%, and for high quality con-
trol (HQC) level was 43.88%. The mean recovery of EPA was 43.83%. %CV
of mean recovery of all three QCs was 5.82. Recovery of internal standard
was 42.02%.

Matrix Effects
Matrix effect evaluated by eighteen LQC and eighteen HQC samples;
three each from six different plasma lots were processed and analyzed.
For DHA the average % accuracy for all LQC level was 97.83 and %CV of
all LQC samples was 3.14 and the average % accuracy for all HQC level
was 100.56 and %CV of all HQC samples was 2.06. Whereas, for EPA, the
average % accuracy for all LQC level was 108.93 and %CV of all LQC sam-
ples was 2.06 and the average % accuracy for all HQC level was 99.82 and
%CV of all HQC samples was 2.05.

Hemolysis Effects
To determine hemolysis effects, six QC samples were prepared in hemo-
lyzed plasma with three concentration levels of low, medium, and high. Six
replicates of each QC samples were analyzed together with a set of cali-
bration standard prepared in normal plasma. The accuracy of each sample
preparation was determined by injection of calibration samples and three
QC samples in six replicate. For DHA the average % accuracy for LQC level
was 99.45, for MQC level was 113.32, and for HQC level was 108.83. The
%CV of LQC was 7.32, for MQC was 3.72, and for HQC was 3.69.


TABLE 2 Summary of Stability Data of DHA

Mean Precision Mean Percent Stability
Stability QC Level (%CV) Accuracy Change Duration

Bench top LQC 4.85 85.72 À9.18 8 Hr
HQC 4.09 102.88 5.98
Freeze thaw LQC 5.76 92.70 1.04 4 Cycles
HQC 2.01 90.09 2.98
Auto sampler LQC 7.28 97.50 À0.57 24 Hr
HQC 1.86 97.27 À0.47

For EPA the average % accuracy for LQC level was 105.31, for MQC
level was 111.02, and for HQC level was 103.91. The %CV of LQC was
4.85, for MQC was 1.50, and for HQC was 2.02.

Lipemic Effects
To determine lipemic effects, six QC samples were prepared in lipe-
mic plasma with three concentration levels of low, medium, and high.
Six replicates of each QC samples were analyzed together with a set of
calibration standard prepared in normal plasma. The accuracy of each
sample preparation was determined by injection of calibration samples
and three QC samples in six replicate. For DHA the average % accuracy
for LQC level was 98.73, for MQC level was 106.72, and for HQC level
was 105.67. The %CV of LQC was 9.03, for MQC was 2.49, and for
HQC was 1.97.
For EPA the average % accuracy for LQC level was 93.17, for MQC level
was 107.34, and for HQC level was 103.80. The %CV of LQC was 3.11, for
MQC was 2.65, and for HQC was 2.53.

Stability Studies
The stability of DHA and EPA were investigated in the stock and working
solutions, in plasma during storage, during processing, after four freeze–
thaw cycles, and in the final extract. Stability samples were compared with

TABLE 3 Summary of Stability Data of EPA

Mean Precision Mean Percent Stability
Stability QC Level (%CV) Accuracy Change Duration

Bench top LQC 1.42 100.36 0.32 8 Hr
HQC 3.54 106.41 7.11
Freeze thaw LQC 2.98 98.30 2.36 4 Cycles
HQC 1.57 91.33 3.26
Auto sampler LQC 4.79 110.39 1.47 24 Hr
HQC 2.31 95.74 À0.82


freshly processed calibration standards and QC samples. Analytes were
considered stable when the change of concentration is Æ10% with respect
to their initial concentration.
The %CV of DHA at LQC and HQC levels for, bench top stability, auto
sampler stability, and freeze-thaw stability were 4.85, 7.82, and 5.76 and

FIGURE 3 (A) Representative chromatogram of real sample of DHA and (B) Representative chroma-
togram of real sample of EPA. (Color figure available online.)


4.09, 1.86, and 2.01, respectively, whereas, the %CV of PHA at LQC and
HQC levels were 1.42, 4.79, and 2.98 and 3.54, 2.31, and 1.57, respectively.
Summary of stability data are presented in Tables 2 and 3 for DHA and
EPA, respectively.

Calibration Curve Parameters
The summary of calibration curve parameters was as follows. For DHA
the mean slope and y-intercepts were 0.00573 (Range: 0.0004 to 0.0007)
and 0.051282 (Range: À0.0984 to 0.2982), respectively. The mean corre-
lation coefficient, r was 0.9985 (Range: 0.9965 to 0.9999).

FIGURE 4 (A) Plasma concentration-time curves for DHA (n ¼ 1) and (B) Plasma concentration-time
curves for EPA (n ¼ 1). (Color figure available online.)


For EPA the mean slope and y-intercepts were 0.000618 (Range: 0.0005
to 0.0008) and 0.0143 (Range: À0.0629 to 0.0743), respectively. The mean
correlation coefficient, r was 0.9984 (Range: 0.9962 to 0.9999).

APPLICATION
The validated method was applied to determine the concentration
time profile, following single dose oral administration of capsule in
healthy human volunteer. This developed method can also be applied
for estimation of EPA and DHA from urine, serum, and other biological
matrices to some extent. This method will be a valuable tool during in vitro
study of EPA and DHA. The method can also be helpful for routine
analysis of EPA and DHA in quality control departments. The chromato-

grams obtained from the analysis of real samples are presented in
Figure 3A and 3B for DHA and EPA, respectively. After LC-MS=MS analy-
sis, the plasma concentration of DHA and EPA were measured. Figure 4A
and 4B shows the plasma concentration-time curves for DHA and EPA,
respectively.

CONCLUSION
A LC=MS=MS method with simple protein precipitation has been
developed for the simultaneous quantitative determination of DHA and
EPA in human plasma. The present method affords the rapid, sensitivity,
accuracy, and precision necessary for fast quantitative measurements in
pharmacokinetic studies and therapeutic monitoring of DHA and EPA.

REFERENCES
1. Simopoulos, A. P. Omega-3 Fatty Acids in Health and Disease and in Growth and Development.
Am. J. Clin. Nutr. 1991, 54, 438–463.
2. Drevon, C. A. Marine Oils and Their Effects. Nutr. Rev. 1992, 50, 38–45.
3. Chauke, E.; Chkrowska, E.; Thaela-Chimuka, M. J.; Chimuka, L.; Nsengimana, H.; Tutu, H. Fatty
Acids Composition in South African Freshwater Fish as Indicators of Food Quality. Water SA.
2008, 34, 119–126.
4. Kurowska, E. M.; Dresser, G. K.; Deutsch, L.; Vachon, D.; Khalil, W. Bioavailability of Omega-3 Essen-
tial Fatty Acids from Perilla Seed Oil. Prostaglandins Leukot Essent Fatty Acids. 2003, 68, 207–212.
5. Rusca, A.; Stefano, A. F. D. D.; Doig, M. V.; Scars, C.; Perucca, E. Relative Bioavailability and Phar-
macokinetics of Two Oral Formulations of Docosahexaenoic Acid=Eicosapentaenoic Acid After
Multiple-Dose Administration in Healthy Volunteers. Eur. J. Clin. Pharmacol. 2009, 65, 503–510.
6. Hidetaka, Y.; Azuma, Y.; Maeda, N.; Kawasaki, H. High-Performance Liquid Chromatographic
Determination of Eicosapentanoic Acid in Serum by a Chemiluminescence Labeling Method. Chem.
Pharm. Bull. 1998, 36, 1905–1908.
7. Ghosh, C.; Jha, V.; Ahir, R.; Shah, S.; Shinde, C. P.; Chakraborty, B. A Rapid and Most Sensitive
Liquid Chromatography=Tandem Mass Spectrometry Method for Simultaneous Determination of


Alverine and Its Major Metabolite, Para Hydroxy Alverine, in Human Plasma: Application to a
Pharmacokinetic and Bioequivalence Study. Drug Test. Anal. 2010, 2, 284–291.
8. Ghosh, C.; Singh, R. P.; Inamdar, S.; Mote, M.; Chakraborty, B. Sensitive, Selective, Precise and Accu-
rate LC–MS Method for Determination of Clonidine in Human Plasma. Chromatographia 2009, 69,
1227–1232.
9. Ghosh, C.; Shinde, C. P.; Chakraborty, B. S. Ionization Polarity as a Cause of Matrix Effects, its
Removal and Estimation in ESI-LC-MS=MS Bio-analysis. J. Anal. Bioanal. Tech. 2010, 1, 106.
10. Ghosh, C.; Gaur, S.; Shinde, C. P.; Chakraborty, B. A Systematic Approach to Overcome the Matrix
Effect During LC-ESI-MS=MS Analysis by Different Sample Extraction Techniques. J. Bioequiv.
Availab. 2011, 3, 122–127.
11. Guidance for Industry, Bioanalytical Method Validation (2001). Food and Drug Administration, Center
for Drug Evaluation and Research (CDER), Dholka, India 2001.

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