UPLC-UV Method

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SIMULTANEOUS DETERMINATION OF
ASPIRIN AND ITS METABOLITE FROM
HUMAN PLASMA BY UPLC-UV DETECTION:
APPLICATION TO PHARMACOKINETIC
STUDY
Chinmoy Ghosh
a
, Anita Upadhayay
a
, Ajay Singh
a
, Saumya
Bahadur
a
, Priya Jain
a
& Bhaswat S. Chakraborty
a
a
Cadila Pharmaceuticals Limited, Gujarat, India
Available online: 18 Nov 2011
To cite this article: Chinmoy Ghosh, Anita Upadhayay, Ajay Singh, Saumya Bahadur, Priya Jain &
Bhaswat S. Chakraborty (2011): SIMULTANEOUS DETERMINATION OF ASPIRIN AND ITS METABOLITE FROM
HUMAN PLASMA BY UPLC-UV DETECTION: APPLICATION TO PHARMACOKINETIC STUDY, Journal of Liquid
Chromatography & Related Technologies, 34:19, 2326-2338
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SIMULTANEOUS DETERMINATION OF ASPIRIN AND ITS
METABOLITE FROM HUMAN PLASMA BY UPLC-UV DETECTION:
APPLICATION TO PHARMACOKINETIC STUDY
Chinmoy Ghosh, Anita Upadhayay, Ajay Singh, Saumya Bahadur,
Priya Jain, and Bhaswat S. Chakraborty
Cadila Pharmaceuticals Limited, Gujarat, India
& A rapid and sensitive liquid chromatography method with UV detection for simultaneous
measurement of aspirin (ASA) and salicylic acid (SA) was developed and validated completely
in human plasma. ASA, SA, and Benzoic acid (BA) as internal standard were extracted via pro-
tein precipitation with Perchloric acid. An isocratic elution with binary mode was used to separate
interference peaks using a C18 Acquity column with only three minutes of analysis time. The lin-
earity range was 15 to 6000 n g mLÀ1
. Calibration functions, LOQ, stability, intra- and inter-day
reproducibility, and accuracy were estimated. The inter- and intra-day % CV for ASA and SA are
Æ15% and the percentage change of all stability samples with comparison samples were within
10%. The in vitro conversion of ASA to SA was also studied and it was controlled to keep at a
minimum (<10%). With respect to other published methods this method is most sensitive in UV
detection, as well as its sensitivity and throughput is comparable or even better than published
LC-MS=MS methods. This method was successfully applied to a single dose Bioequivalence (BE)
study of following administration of 81 mg enteric coated Aspirin tablets.
Keywords aspirin, Benzoic acid, Bioequivalence=protein precipitation, salicylic acid,
UPLC-UV
INTRODUCTION
Aspirin, also known as acetylsalicylic acid (ASA)[1]
(CAS number:
50-78-2; Figure 1), is often used as an analgesic to relieve minor aches
and pains, as an antipyretic to reduce fever, and as an anti-inflammatory
medication. Aspirin also has an antiplatelet or ‘‘anti-clotting’’ effect and
is used in low doses to prevent long-term heart attacks, strokes and blood
clot formation in people at high risk.[2]
Furthermore, it has been
Address correspondence to Chinmoy Ghosh, Research Scientist, Contract Research Organization,
Cadila Pharmaceuticals Limited, 1389, Trasad Road, Dholka-387 810, Ahmedabad, Gujarat, India.
E-mail: chinmoy_ghosh@yahoo.com
Journal of Liquid Chromatography & Related Technologies, 34:2326–2338, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2011.589092
Downloadedby[ChinmoyGhosh]at20:3122November2011

established that low doses of aspirin may be given immediately after a heart
attack to reduce the risk of another heart attack or of the death of cardiac
tissue.[3,4]
The main undesirable side effects of aspirin are gastrointestinal
ulcers, stomach bleeding, and tinnitus, especially in higher doses. In chil-
dren under 19 years of age, aspirin is no longer used to control flu-like
symptoms or the symptoms of chickenpox, due to the risk of Reye’s
syndrome.[5]
Salicylic acid (SA)[6]
(CAS number: 69-72-7; Figure 2), is a colorless
crystalline organic acid and is widely used in organic synthesis and func-
tions as a plant hormone. It is derived from the metabolism of salicin. In
addition to being a compound that is chemically similar to but not identical
to the active component of aspirin (acetylsalicylic acid), it is probably best
known for its use in anti-acne treatments. It is poorly soluble in water
(0.2 g=100 ml H2O at 20
C).[7]
Several LC methods with UV detection[8–16]
have been reported for
estimation of ASA alone or with its metabolite, that is, SA. Although the
other reported LC-UV methods were not sensitive enough, the reported
lower limit of quantitation (LLOQ) for simultaneous determination of
both ASA and SA is around 100 ng mLÀ1
with long analysis time. On the
other hand, there were some LC-MS=MS methods[17–19]
that are compara-
ble with this LC-UV method; as in the present method, the limit of quanti-
fication (LLOQ) is 15 ng mLÀ1
for both ASA and SA, which is sufficient
with low doses of ASA with only 3 min. of analysis time. In the present
method, a simple precipitation method was used as the extraction tech-
nique instead of other complex extraction techniques reported in other
FIGURE 2 SA (Salicylic Acid).
FIGURE 1 ASA (Acetyl Salicylic Acid).
Determination of Aspirin and Its Major Metabolite by UPLC-UV 2327

methods.[12,13]
In the present method, benzoic acid (BA) was used as inter-
nal standard (IS), whereas some other method used isotope labeled IS.[14]
More over there is no UPLC method available, so it is the only UPLC-UV
method. The novelty of this method is that, its sensitivity and throughput
is equivalent or better to reported LC-MS=MS methods.[17–19]
Additionally,
this manuscript also studies the degradation of aspirin in human plasma. As
a result, this present method can be very useful for those laboratories where
costly instruments such as LC-MS=MS or GC-MS are not available; however,
by applying this method, the equivalent sensitivity and throughput can be
achieved.
The aim of the present research work was to develop an accurate and
sensitive UPLC-UV method with a dynamic linearity range that can cover
the plasma concentrations following a single oral dose of aspirin. The
method has been validated by evaluating the precision, accuracy, and other
validation parameters for human plasma samples, as mentioned in regulat-
ory guidelines.[20]
EXPERIMENTAL
Chemicals and Reagents
ASA and the IS were procured from the Cadila Pharmaceuticals Ltd.,
Ahmedabad. SA was obtained from the Chemisynth Laboratory Limited,
Mumbai, India. Potassium dihydrogen phosphate, Ortho phosphoric Acid
(OPA), and Hydrochloric acid used during analysis were procured from
S.D. Fine Chemicals Ltd., Ankleshwar, Gujarat, India and were of AR grade.
Perchloric Acid (about 70% Purity) was procured from Merck Pharmaceu-
ticals Limited, Mumbai, India. Water used was collected from water purifi-
cation systems (Milli Q, Milli Pore, USA) installed in our Laboratory where
this study was conducted. Methanol and acetonitrile were of HPLC grade
and were supplied by J. T. Baker, USA. Fresh frozen Human Plasma
(K2-EDTA as anticoagulant) was used during validation, which was supplied
by Prathma Blood Centre, Ahmedabad, India. Plasma was stored at
À70 Æ 5
C.
Apparatus and Software
Waters Acquity Ultra Performance Liquid Chromatography (UPLC) sys-
tem with UV-PDA detector (Waters Corporation, USA) for detection and
quantitation of samples was utilized. Chromatographic acquisition and
integration were performed using Empower 2 software developed and inte-
grated with the UPLC by Waters Corporation, USA. Other instruments
2328 C. Ghosh et al.

used were Analytical Balance (Mettler Toledo, Switzerland), Refrigerated
Centrifuge (Heraeus Multifuge 3S–Rþ, Thermo Electron Corporation,
Germany), Vortexer (Remi, Mumbai, India), Deep freezer (Sanyo, Japan),
and Refrigerator (Samsung, Ahmedabad, India).
Standards and Working Solutions
A standard stock solution of ASA containing 1 mg mLÀ1
was prepared
by dissolving pure compound in acetonitrile, where separate stock standard
solutions of SA and BA (each 1 mg mLÀ1
) were prepared by dissolving pure
compounds in methanol. Intermediate and working solutions of ASA were
prepared from corresponding stock solutions by diluting with acetonitrile,
whereas intermediate and working solutions of SA and BA were prepared
from corresponding stock solutions by diluting with water:methanol
(50:50 v=v). Calibration standards were established between 15 to
6000 ng mLÀ1
of ASA and SA both, using nine concentration levels. Quality
control standards at three different levels low (45 ng mLÀ1
), medium
(2480 ng mLÀ1
), and high (5000 ng mLÀ1
) were also prepared. All these
stock solutions, calibration standards, and quality control samples were
stored at 4 Æ 2
C. These solutions were found to be stable and used for
the complete method validation.
Chromatographic Conditions
Chromatographic separation was performed on an Acquity UPLC BEH
C18 column (100 Â 2.1 mm i.d., particle size 1.7 mm). The mobile phase
used was a mixture of Potassium dihydrogen phosphate (pH: 2.1 Æ 0.05,
20 mM) in Milli-Q water:methanol:acetonitrile (70:15:15, v=v=v). The flow
rate was 0.350 mL minÀ1
. Total analysis time of single injection was three
min. Injection volume was 30 mL. Auto sampler rinsing volume was
400 mL. Strong Needle Wash was a mixture of acetonitrile:water at a ratio
of 50:50 v=v. Weak needle wash was 100% Milli-Q water.
Sample Preparation
Protein precipitation extraction technique was used to extract the ASA
and SA from human plasma samples. Hydrochloric acid (1M) was used for
providing an acidic medium to prevent conversion of ASA to SA. Perchloric
acid was used as extracting solvent. Plasma samples (500 mL) were trans-
ferred to a centrifuge tube for analysis. An amount of 20 mL of IS
(20 mg mLÀ1
) followed by 50 mL of 1.0 M hydrochloric acid was added into

it and the samples were vortexed for 15 sec. Perchloric acid solution
(200 mL of 20% v=v) was added to precipitate the plasma samples and vor-
texed for 1 min and centrifuged at 4500 rpm for 5 min at 5
C. The super-
natant was centrifuged at 10000 rpm for 5 min at 5
C and the whole
supernatant was injected onto UPLC.
Degradation Study for Bioanalytical Purpose
It has been reported[21]
that ASA converts to SA by hydrolysis over time
under normal conditions. Therefore, tests were performed to monitor per-
centage degradation of ASA into SA in different conditions of pH (1 M
HCl, 0.1 M HCl, 1% Formic Acid, 1% Acetic Acid), Base (1 M KOH), and
without addition of any acid or base at different time intervals.
Method Validation
The validation parameters were specifically, linearity, sensitivity,
accuracy, precision, and hemolysis effects of the assay and the recovery
and stability in human plasma, according to the US Food and Drug Admin-
istration (FDA) guidance for the validation of bioanalytical methods.[20]
Selectivity was studied by comparing the chromatograms of six different
lots of plasma obtained from six subjects, with the plasma samples having
been spiked with SA, ASA, and the IS. Calibration curves were prepared
by assaying standard plasma samples at SA and ASA concentrations, ran-
ging from 15 to 6000 ng=mL for both SA and ASA.
The linearity of each method matched calibration curve was deter-
mined by plotting the peak area ratio (y) of SA or ASA to the IS versus
the nominal concentration (x) of SA or ASA, respectively. The calibration
curves were constructed by weighing (1=x2
) least-squares linear regression.
The LLOQ for SA or ASA in human plasma was defined as the lowest
concentration giving acceptable accuracy (80–120%), and sufficient
precision (within 20%); this was verified by analysis of six replicates.
Intra- and inter-day accuracy and precision for this method were deter-
mined at three different concentration levels on three different days, and
on each day, six replicates were analyzed with independently prepared cali-
bration curves. The percentage accuracy was express as (mean observed
concentration)=(nominal concentration) Â 100, and the precision was the
relative standard deviation [RSD (%)].
For recovery calculation, the peak areas obtained by direct injection of
solvent (or neat) standard solutions spiked after extraction into plasma
extracts as A and the peak areas for solvent (or neat) standard solutions
spiked before plasma extraction as B, the extraction recovery value can

be calculated as follows:
Extraction recovery ð%Þ ¼ B=A Â 100
The stability of SA or ASA in human plasma was assessed by analyzing six
replicate samples at LQC and HQC levels for SA and ASA, respectively,
under six conditions: after long term storage of 6 months at À70
C; after
four freeze-thaw cycles; after sample preparation for 6 hr on bench top
and after 49 hr within the auto sampler. The concentrations obtained were
compared with the nominal values of the QC samples.
Clinical Protocol
The Independent Medical Ethics Committee (IEC) of Cadila Contract
Research Organization, Ahmedabad, Gujarat, India, approved the bio-
equivalence study protocol and bioanalytical method presented in this
paper. The study was a randomized, open-label, two-treatment, two-period,
two-sequence, two-way crossover study, during which subjects were adminis-
tered two different formulations of a single dose of Aspirin 81 mg
tablet along with 240 mL of drinking water. Dose were administered after
an overnight fasting for at least 10 hr in each period with at least 14 d of
the washout period between each administration. Volunteers were healthy,
adult males from India. In each period, a total of 16 blood samples were
collected including a predose sample prior to drug administration and
after drug administration at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0,
8.0, 10.0, 12.0, 14.0, and 16.0 hr. These samples were assayed by the
developed and validated UPLC method described in this paper.
RESULTS AND DISCUSSIONS
Method Development
Method development was started with UPLC BEH C8 column
(50 Â 2.1 mm i.d., particle size 1.7 mm), but there was almost no resolution
between SA and ASA with poor chromatography. As a result, the column
length was increased to 100 mm keeping all other specifications the same,
but poor chromatography was observed for both SA and ASA with little res-
olution between SA and ASA. Therefore, BEH C18 column (50 Â 2.1 mm
i.d., particle size 1.7 mm), was tried, which showed good chromatography
for SA, ASA, and BA with comparatively better resolution. Initially, potass-
ium dihydrogen phosphate (pH: 2.1 Æ 0.05, 20 mM) in Milli-Q water:aceto-
nitrile (75:25, v=v) was tried as the mobile phase. Later, to improve the

resolution between the peaks, methanol was introduced along with acetoni-
trile as the organic phase. Finally, the mobile phase used was a mixture of
potassium dihydrogen phosphate (pH: 2.1 Æ 0.05, 20 mM) in Milli-Q water:-
methanol:acetonitrile (70:15:15, v=v=v). The flow rate was 0.350 mL minÀ1
.
Total analysis time of a single injection was three min. Injection volume was
30 mL. Wave length was selected from the available literatures.
Method Validation
The method was validated in terms of linearity, specificity, LLOQ, recov-
ery, accuracy, precision, dilution integrity, hemolysis effect, and stability stu-
dies (e.g., short term stock solution stability, freeze thaw stability, bench top
stability, and auto sampler stability). The accuracy and precision determi-
nation were carried out in three different d with six replicates of LLOQ,
low, medium, and high quality control samples.
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 of the assay ranged from 15 to
6000 ng mLÀ1
(r 0.9900). In aqueous solution linearity test, all calibration
standards showed an accuracy within 85–115%, except LLOQ where it was
between 80–120%.
Specificity and Selectivity
Six different lots of plasma were analyzed to ensure that no endogenous
interference with the retention time chosen for ASA, SA, and the IS. Six
LLOQ level samples along with the plasma blank from the respective
plasma lot were prepared from six different lots of plasma and analyzed.
In all six plasma blanks, the response at the retention time of ASA and
SA was less than 20% of LLOQ response and at the retention time of IS,
the response was less than 5% of mean IS response in LLOQ. Figure 3
shows a typical chromatogram of plasma blank and Figure 4 represents
the chromatogram at LLOQ.
Accuracy and Precision
For the validation of the assay, QC samples were prepared with three
concentration levels of low, medium, and high. Six replicates of each QC
samples and LLOQ level were analyzed together with a set of calibration

standards. The accuracy of each sample preparation was determined by
injection of calibration samples and LLOQ, LQC, MQC, and HQC samples
in six replicate for 3 d. The obtained accuracy and precision (inter- and
intra-day) are presented in Table 1 for ASA and SA. The results showed that
the analytical method is accurate, as the accuracy is within the acceptance
limits of Æ20% of the theoretical value at LLOQ and Æ15% at all other con-
centration levels. The precision around the mean value was always within
15% at any of the nominal concentration studied.
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 mean recovery of ASA was 58.11% and the CV (%) of mean recovery
FIGURE 4 Representative chromatogram of LLOQ (15.0 ng mLÀ1
).
FIGURE 3 Representative chromatogram of plasma blank.

of at all three QCs was 1.98, whereas the mean recovery of SA was 70.64. CV
(%) of mean recovery of at all three QCs was 6.09. Recovery of the IS was
73.22%.
Hemolysis Effects
To determine hemolysis effects on the estimation of plasma concen-
tration, six hemolyzed plasma blank and QC samples were prepared in
hemolyzed plasma with two concentration levels of low and high. Six repli-
cates of each QC samples were analyzed together with a set of calibration
standards prepared in normal plasma. The accuracy of each sample prep-
aration was determined by injection of calibration samples and two QC
samples in six replicates. The average % accuracy of LQC and HQC level
was 104.61 and 100.75, respectively, for ASA and % accuracy of LQC and
HQC level was 98.32 and 101.71, respectively, for SA. The CV (%) of
LQC was 5.86 and for HQC was 9.27 for ASA and CV (%) of LQC was
10.11 and for HQC was 9.51 for SA.
Stability Studies
The stability of ASA, SA, and IS were investigated in the stock and work-
ing solutions, in plasma during storage, during processing (i.e., bench top
TABLE 1 Inter and Intra-day Accuracy and Precision of ASA and SA
ASA SA
QC Levels
Mean
Accuracy
Mean
Precision (% CV)
Mean
Accuracy
Mean
Precision (% CV)
Day 1 LLOQ 98.73 4.66 104.63 3.40
LQC 91.44 9.14 97.08 2.27
MQC 95.33 1.25 112.13 1.59
HQC 101.53 4.33 113.59 2.30
Day 2 LLOQ 103.83 4.05 111.25 1.20
LQC 93.75 2.03 92.51 1.50
MQC 98.21 3.24 103.52 4.75
HQC 99.63 10.24 110.49 3.33
Day 3 LLOQ 100.33 12.75 98.79 3.84
LQC 100.79 4.11 85.69 2.55
MQC 93.71 3.93 100.74 3.65
HQC 100.93 2.91 108.05 1.32
Inter day LLOQ 100.96 7.94 104.88 5.71
LQC 95.42 7.09 92.12 5.48
MQC 95.75 3.47 105.46 5.76
HQC 100.69 6.24 110.71 3.13
Each mean and % CV of intra-day accuracy and precision represent six observations (n ¼ 6). The
inter-day accuracy and precision are averages and % CV of three intra-day observations.

stability), after four freeze-thaw cycles, and in the final extract (i.e., auto
sampler stability). Stability samples were compared with freshly processed
calibration standards and QC samples for comparison. Analyte, metabolite,
and IS were considered stable when the change of concentration was Æ10%
with respect to initial concentration. Summary of stability data is presented
in Table 2 for ASA and SA.
Calibration Curve Parameter
The summaries of calibration curve parameters were as follows.
The mean y-intercepts and slope for ASA were À0.0086 (range: À0.0149
to À0.0032) and 0.00033 (range: 0.00027 to 0.00037) respectively. The
mean correlation coefficient, r was 0.9968 (range: 0.9942 to 0.9987).
For SA, the mean y-intercepts and slope were À0.0050 (Range: À0.0201
to À0.0224) and 0.00063 (range: 0.000497 to 0.00072), respectively. The
mean correlation coefficient, r was 0.9994 (range: 0.9935 to 0.9978).
Degradation Study
Summary of data after conversion of ASA to SA is summarized in
Figure 5. The maximum conversion was observed in basic condition which
is suitable for the studies where only ASA is to be analyzed in terms of SA.
The minimum conversion is obtained under acidic conditions. Hence, this
condition was selected for simultaneous estimation of ASA and SA. Among
all the trials it has been observed that after six hr almost 70% of ASA was
converted to SA in 1 M potassium hydroxide solution at room temperature,
whereas approximately 10% of ASA is converted to SA in 1 M hydrochloric
acid solution at room temperature after six hr.
TABLE 2 Summary of Stability Data of ASA
ASA SA
Experiment
Name QC Level
Mean
Accuracy
Mean
Precision
(%CV)
Percent
Change
Mean
Accuracy
Mean
Precision
(%CV)
Percent
Change
Stability
Duration
Bench top LQC 94.88 8.30 1.21 94.03 4.13 9.73 06 hr
HQC 94.46 1.09 À5.19 99.06 4.65 À8.32
Freeze thaw LQC 91.44 4.74 4.61 95.72 12.77 4.02 4 cycles
HQC 102.75 1.50 À1.90 106.80 0.73 À3.96
Auto sampler LQC 97.54 13.88 4.04 93.70 6.36 9.35 60 hr
HQC 95.34 1.48 À4.30 110.10 0.91 1.89
Each mean accuracy, % CV and % change of each stability represents six observations (n ¼ 6) of
corresponding QC levels.

Pharmacokinetic Study
The sensitivity and specificity of the assay were found to be sufficient for
accurately characterizing the plasma pharmacokinetics of SA and ASA in
healthy volunteers. Profile of the mean plasma concentration and time
are shown in Figure 6. Maximum plasma concentration (Cmax of SA is
800 Æ 100 ng=mL and ASA is 5500 Æ 500 ng=mL) was achieved at 2.50 hr
for SA and 6.00 hr for ASA. The higher sensitivity of this method compared
with the currently existing methods in literature facilitates the quantitation
of SA and ASA at lower concentration. Figure 7 represents the chromato-
gram of real sample.
FIGURE 5 Representative graph of % conversion of ASA to SA.
FIGURE 6 Pharmacokinetic profile of plasma concentration and time of single volunteer.

CONCLUSION
A simple, sensitive, selective, precise, and accurate UPLC-UV method
for the simultaneous determination of ASA and its metabolite, SA, in
human plasma, over a range of 15–6000 ng=mL for ASA and SA, was
developed and validated. This is the only method for ASA and SA, which
was developed and validated using UPLC with UV detection. This method
requires only 0.500 mL of biological samples, owing to simple sample prep-
aration and short run time (3 min), it allows high sample throughput. The
method was successfully applied to a single dose 81 mg enteric coated tablet
Bio equivalence study of ASA and its major metabolite, SA.
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