Simultaneous Determination of Paracetamol and its ......high performance liquid chromatography...
Transcript of Simultaneous Determination of Paracetamol and its ......high performance liquid chromatography...
1232 2, Issue.39, Vol.2740-, ISSN: 2051Pharmaceutical AnalysisInternational Journal of
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Simultaneous Determination of Paracetamol and its
Metabolites in Rat Serum by HPLC Method and its
Application Supplement-Drug Pharmacokinetic
Interaction Bayan Alkhawaja
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Tawfiq Arafat
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Eyad Mallah
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Nidal Qinna
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Naser Idkaidek
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Wael Abu Dayyih
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Hamza Alhroub
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan Adnan Badwan
Jordan Pharmaceutical Manufacturing Company.
Corresponding Authors E-mall: [email protected], [email protected]
ABSTRACT
In this report, we describe a reliable HPLC method for
simultaneous estimation of paracetamol and its
metabolites in rat serum. In addition, we investigate the
impact of coadminstration of glucosamine, a dietary
supplement used in osteoarthritis, on paracetamol and its
metabolites. Accordingly, HPLC method with detection at
245nm was developed. Paracetamol and its metabolites
were quantitated within a 10 minutes run time. The
method was linear over the range of 0.5-100 µg/ml for
paracetamol (P), paracetamol glucuronide (PG) and
paracetamol sulphate (PS) as well as 0.05–10 µg/mL for
paracetamol mercapturate (PM) and paracetamol cysteine
(PC) in rat serum. The intra-day CV% for P, PG, PS, PM
and PC in serum were less than 3.577, 3.303, 3.184, 3.276
and 3.142 % , respectively. While the inter-day values
were less than 2.953, 3.096, 2.755, 3.521, 3.100 %,
respectively. Co-administration of glucosamine impact
were evaluated. Accordingly, paracetamol maximum
serum concentration (Cmax) and area under the curve
(AUClast) 34.65 µg/ml and 64.08µg*h/ml at 60 mg/kg
dose of paracetamol were reduced but not significantly
with co-administration of glucosamine 31.15 µg/ml and
50.43µg*h/ml. However, PG Cmax was significantly
elevated 40.96 & 70.54 µg/ml without and with
glucosamine, respectively. On the other hand, Cmax and
AUC last of toxic metabolites PM and PC were reduced,
this reduction was significant for PC. These results
indicate that both drugs could be used together without
dose adjustment and this combination may provide a
potential measure to mitigate paracetamol hepatotoxicty
since it recently acquires universal attention.
Keywords -Paracetamol metabolism, Paracetamol
hepatotoxicity, Glucosamine, Osteoarthritis, HPLC.
1. INTRODUCTION
Paracetamol (P) is considered one of the most widely
administrated analgesic and antipyretic substance
worldwide. Despite the wide popularity of paracetamol, its
precise mechanism of action is still not completely
understood[1].
In recent years, concerns about paracetamol associated
hepatotoxicity have been raised. From 1998 to 2003,
paracetamol was the primary cause of acute liver failure
(ALF) in the United States, and about 48% of
paracetamol-related were inadvertent overdose [2].More
recently, about 46% of ALF cases in USA were due to
paracetamol according to acute liver failure study group
(ALFSG) [3].
Paracetamol toxicity is closely related to its metabolism
which is somehow complex. Paracetamol metabolized via
both phase 1 and 2 thus it could be involved in many drug-
drug interaction[4]. Majority of paracetamol conjugated
with glucuronide (PG) and sulfate (PS) 55% and 35%,
respectively. Metabolism via phase 1 will result in toxic
metabolite formation namely, N-acetyl-p-benzoquinone
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imine (NAPQI) which leads to hepatotoxicty. Toxicity of
paracetamol occurs beyond the therapeutic dose when the
level of NAPQI exceed the detoxifying capacity of
glutathione (figure1) [5].
NAPQI will be readily detoxified and conjugated with
glutathione to be further degraded to paracetamol
mercapurate and paracetamol cysteine (PM & PC)[6].
A reliable method to detect paracetamol and its
metabolites of oxidation and conjugation pathway is
required in toxicity and interactions studies. In this regard,
high performance liquid chromatography (HPLC) with
ultraviolet (UV) detection has been widely used for the
quantification of paracetamol and its metabolites.
However, most of previous methods were only directed to
detect paracetamol major metabolites (PG and PS). Hence
could not be use for toxicity purposes [7]. Moreover, LC-
MS was also used for quantification of paracetamol and its
metabolites [8,9,10]. However, most of these methods are
only directed to detect paracetamol major metabolites only
or required long run time. In order to understand the
systemic metabolism of paracetamol comprehensive
information on the whole paracetamol metabolic pathways
is in demand.
Figure 1.Metabolism pathways of paracetamol.
Drug interaction is one of the eight drug related problems
that interferes with the optimum or desired therapeutic
effect of the drug owing to the presence of other drug,
food or other factors [11,12]. Beside drugs, Supplemental
products, may also cause interactions when combined with
conventional drugs and thus require deep monitoring [13].
Glucosamine (GluN) is used as dietary supplement in
Osteoarthritis patient (OA). The Osteoarthritis Research
Society International (OARSI) has recommended GluN as
a symptomatic treatment and structure-modifying agent
for knee OA. It could be used in combination with other
supplements such as chondroitin or alone in the form of
GlcN hydrochloride or GlcN sulphate[14].Referring to the
2007 National Health Interview Survey, which included
questions on natural products used by Americans, the most
used products were fish oil/omega 3/DHA (37.4 percent)
followed GluN (19.9 percent) [15]. OA is not curable,
management are directed to relief symptoms, analgesics
such as paracetamol and Non-steroidal anti-inflammatory
drugs and GluN supplement are widely used [16].
Paracetamol is widely recommended as intermitted or
continued therapy in OA patient [17] and thus it's possible
to be used concomitantly with GluN. Since the
metabolism of paracetamol is very complex and involve
both phase one and phase two of metabolism [18].
In addition, pharmacokinetic studies in rats showed that
GluN undergoes extensive first-pass metabolism which
resulted in low bioavailability [19]. There is a possibility
of interaction between them.
Herein, we presented a reliable, rapid and simple HPLC
method for the quantification of paracetamol and its all
metabolites in rat serum using a simple extraction
paracetamol PS
PG
NAPQI
PC PM
GSH
CYP
55%
35%
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procedure and short run time. This analytical method was
applied to detect the potential metabolic interaction
between paracetamol and glucosamine in rats.
2. METHODS
2.1 Instrumentation
The Dionex HPLC (Dionex HPLC Ultimate 3000) system
used consisted of a LPG-3400SD pump, a WPS-3000(RS)
autosampler, a VWD-3x00(RS) detector and TCC-
3x00(RS) column compartment. The chromatographic
system was controlled by computer system (Microsoft
Windows XP Professional 5.1, Build 2600).
2.2 Chemicals and reagents
Paracetamol metabolites were of analytical purity grade;
PG, PS, PM and PC were purchased from Toronto
Research Chemicals Inc. Canada (Batch#: 1-ACH-16-1, 1-
SXG-144-1, 13-XJZ-43-1, 13-XJZ-72-1, respectively).
Paracetamol was obtained from Joswe medical (Batch
#1210661). Cefadroxil was obtained from United
Pharmaceuticals (Batch # R09392).Glucosamine HCL was
kindly donated by JPM. Acetonitrile, Water and Methanol
of were of HPLC gradient grade and purchased from Fisher
scientific Inc. Other chemicals were all of analytical grade.
2.3 Chromatographic conditions
A mobile phase consisting of water: acetonitrile:
triethylamine (92.95:7.00:0.05,v/v) with pH of 2.75 (
adjusted with phosphoric acid) was circulated through a
reversed-phase Thermo scientific column (BDS
HYPERSIL C18) with particle size of 5 µm and
dimensions of 150mm×4.6mm. The flow rate was 1ml min-
1 with injection volume of 20 µl. Absorption was measured
at 245 nm wavelength that was optimum for P, PG, PS, PC,
PM and IS. The retention times for P, PG, PS, PC,PM and
IS were approximately 4.32, 2.55, 3.45, 3.06, 8.69 and 7.19
minutes, respectively.
2.4 Preparation of calibration standards
(STD) and quality control samples
Stock solutions of P, PG, PS, PC and PM were prepared in
methanol to get concentrations (5000, 5000, 5000, 500 and
500) µg/ml, respectively. Working solutions A were
prepared by taking 30 µl from each stock solution to get
working solution, another dilution was done to get working
solution B by taking 300 µl. In order to get the first three
spiked levels, calculated volumes were taken from serial
solution A and spiked in 1.5ml of serum. The rest spiked
levels were prepared by spiking calculated volumes from
working solution B into 1.5 ml of serum. The obtained STD
concentrations were: 0.5, 2, 5, 10, 25, 50 and 100 µg/ml for
P, PG and PS in serum and were: 0.05, 0.2, 0.5, 1, 2.5, 5
and 10µg/ml for PC and PM in serum. Quality control (QC)
samples were prepared using the same preparation
procedure as the calibration samples. The serum
concentrations of QC samples were 1.5, 40 and 80 µg/mL
for paracetamol and its major metabolites (PG and PS) and
0.15, 4 and 8 µg/mL for minor metabolites (PC and PM).
2.5 Study design
The study protocol was approved by ethical committee of
the High Research Council, faculty of pharmacy and
medical science, University of Petra, Amman, Jordan.
Adult female Sprague Dawley (140-240g) rats were
weighed and randomized into groups. Each group contains
an average of 8 rats. All rats fasted 24hr before experiment
day and given either 15mg/kg or 60mg/kg oral gavage
dose of paracetamol. However, in order to investigate the
impact of GluN on paracetamol and its metabolites,
another two groups were pre-administered with GlucN in
drinking water for 2 days and half an hour before
paracetamol was given, a booster dose of GlucN either 12
mg/kg or 50 mg/kg was administrated.
2.6 Analysis of serum samples
Blood samples were collected from the rates tails at (0.00,
0.25, 0.50, 1.00, 2.00, 3.00, 4.00, 6.00) hr into an Eppendorf,
centrifuged at 4000 rpm for 10 minutes. Supernatant
collected and kept in deep freezer at -200C till analysis. 75 µl
of samples were transferred to an Eppendorf tube, 75 µl of
the working solution of internal standard (IS) (50 µg/ml
cefadroxil in 5% PCA) was added, after vortexing for
1minute, the samples were centrifuged for 10 minutes at
14000 rpm. The clear supernatant was transfer to a flat
bottom insert and 20 µl was injected into the HPLC
column.
2.7 Analytical method validation
2.7.1 Accuracy and Precision
The intra-day precision and accuracy were evaluated by
analyzing six replicates of the QC samples (low, mid,
high) and lower limit of quantization (LLOQ) samples on
a single day. The inter-day precision and accuracy were
determined by analyzing three runs of QC samples and
LLOQ samples on three different days. The accuracy (%)
was calculated by dividing a measured mean concentration
over the nominal analyte concentration. Precision was
presented as CV%. The acceptable limits of accuracy and
precision should be below 15% except at the LLOQ, for
which accuracy and precision should be below 20% [20].
2.7.2 Linearity
Linearity was determined by a series of six injections to a
seven calibration concentration levels for each analyte
(paracetamol and its metabolites. Peak areas of the
calibration standards were plotted in the Y-axis against the
nominal standard concentration, and the linearity of the
plotted curve was evaluated through the value of the
correlation coefficient (R2) which should be more than
0.98[20].
2.7.3 Stability
Stability of the analyte in the rat serum was evaluated
using low and high QC samples were analyzed
immediately after preparation and after the applied storage
conditions that are to be evaluated.
conditions were:
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Autosampler stability
freeze-thaw stability (after 3 cycles)
Short term stability at room temperature (24 h)
The mean concentration should be within ±15% of the
nominal concentration [20].
2.7.4 Absolute recovery
Absolute recovery was measured at concentrations of (1.5,
40, 80) μg/ml for P, PG and PS and concentration of (0.15,
4, 8) μg/ml for PM and PC. Serum samples (n=3) at each
concentration were extracted and injected. Three samples
of the same amount of compound in solution were directly
injected.
The absolute recovery was calculated by comparing the
AUCs for serum extracted samples at the three mentioned
concentration with un-extracted samples those represent
100% recovery.
Recovery of the analytes need not to be 100% but the
recovery of the analytes and IS should be consistent and
reproducible [20].
2.8. Data analysis
Pharmacokinetics parameters were calculated by non-
compartmental analysis (NCA) model using Winnonlin
software V 5.2.Parameters were estimated as follows:
Area under the curve to 6 hr (AUClast), area under the
curve to infinity (AUC inf), maximum concentration of
drug in serum (Cmax), time to achieve Cmax (Tmax) and
half life (t0.5).
The differences between the major pharmacokinetic
parameters (AUClast and Cmax) were assessed by
calculating the 90% confidence interval for the geometric
means ratio of the test (coadminstration of GluN) over
reference (control group). Lack of Drug-Drug interaction
(no effect) is claimed if the ratio of the means and 90% CI
of the ratio of the geometric means were contained within
the interval of 80.00-125.00%.
The statistical significance of difference in mean of a
normally distributed variable, like Cmax and AUClast
between 2 groups was assessed using the independent
samples Student’s t-test using Excel programme. In the
case of Tmax, t-test was performed without Ln-
transformation. P value <0.05 is considered significant.
3. RESULTS
3.1 Interference and retention time
Using the defined chromatographic method paracetamol,
PG, PS, PC, PM and IS were separated within 10 minutes
run time. Figure 2 shows representative chromatograms
from a serum sample containing the internal standard
50μg/ml only (A) and a serum sample containing
Paracetamol 5 μg/ml, PG 5 μg/ml, PS 5 μg/ml, PC 0.5
μg/ml , PM 0.5 μg/ml and internal standard 50 μg/ml (B).
Figure 2.(A) Chromatograms of serum containing IS (50µg/ml) (B) spiked rat serum with paracetamol (5µg/ml),
PG (5µg/ml), PS (5µg/ml), PM (0.5µg/ml), PC(0.5µg/ml) and IS, eluted at 4.32, 2.55, 3.45, 8.69, 3.06 and 7.19
minutes, respectively.
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3.2 Validation results
3.2.1 Accuracy and Precision
Over the range of concentrations of each analyte, the intra-
day accuracy % were (91.33-112.50), (91.28-108.00),
(89.64-106.00), (86.33-114.00) and (89.33-113.00) for P,
PG, PS, PM and PC, respectively. Meantime, the inter-day
accuracies were (95.42-105.05 %), (95.55-101.72%),
(94.05-100.46 %), (91.65-110.83%) and (92.61-107.06%)
for P, PG, PS, PM and PC, respectively (table 1).
The overall results showed that intra-day CV% for P, PG,
PS, PM and PC in serum were less than 3.577, 3.303,
3.184, 3.276 and 3.142 %, respectively. While the
corresponding inter-day values were less than 2.953,
3.096, 2.755, 3.521, 3.100 %, respectively (table 1).
3.2.2 Linearity
The calibration curves for paracetamol and its four
metabolites were analyzed by least-squares linear
regression with weighing factor (1/x2). The correlation
coefficient (R2) for the calibration curves of all analytes
were more than 0.99.
Table 2 represents the calibration curve data for
paracetamol and its metabolites in rat serum.
Table1. Intra- and inter-day precision and accuracy for the determination of APAP and its metabolites in rat serum
compound concentration
(µg/ml)
Day one Day two Day three Inter-day
Accuracy CV% Accuracy CV% Accuracy CV% Accuracy CV%
paracetamol 0.5
104.80 0.77 105.35 1.63 105.00 3.58 105.05 2.18
1.5
96.78 1.78 96.18 1.84 95.67 1.67 96.21 1.73
40
95.38 3.15 95.26 3.15 95.61 3.11 95.42 2.95
80
101.38 0.70 101.46 0.69 101.57 0.58 101.47 0.62
PG 0.5
101.07 0.79 102.42 1.35 101.67 3.15 101.72 1.99
1.5
100.17 1.81 97.08 1.90 96.53 1.83 97.93 2.42
40
95.53 3.28 95.49 3.30 95.63 3.30 95.55 3.10
80
100.42 0.74 100.46 0.73 100.59 0.66 100.49 0.67
PS 0.5
99.37 1.66 101.80 1.72 100.20 3.18 100.46 2.39
1.5
96.12 1.73 95.72 1.87 94.55 1.92 95.46 1.87
40
94.83 2.79 94.40 2.76 92.92 2.78 94.05 2.76
80
100.43 0.68 99.68 0.70 97.61 0.85 99.24 1.42
PM 0.05
110.33 1.48 111.50 1.68 110.67 1.95 110.83 1.67
0.15
95.39 1.60 90.28 2.34 89.28 1.88 91.65 3.52
0.4
93.75 3.16 92.68 3.21 92.33 3.28 92.92 3.09
0.8
99.75 0.73 99.78 0.68 100.56 0.64 100.03 0.75
PC 0.05
103.17 0.73 109.33 2.06 108.67 1.71 107.06 3.06
0.15
93.56 2.00 92.72 1.45 91.56 1.28 92.61 1.76
0.4
95.30 3.31 95.37 3.31 95.51 3.27 95.39 3.10
0.8
100.98 0.75 101.03 0.77 101.10 0.84 101.04 0.74
Table 2.Calibration curve data of paracetamol and its metabolites in rat serum
Compound Concentration
range (µg/ml) slope Intercept R2 P value
Paracetamol 0.5-100 0.0847 -0.01961 0.999 <0.001
PG 0.5-100 0.0494 0.00907 0.999 <0.001
PS 0.5-100 0.0637 -0.00638 0.999 <0.001
PM 0.05-10 0.0538 -0.00459 0.999 <0.001
PC 0.05-10 0.04034 -0.00154 0.999 <0.001
3.2.3 Stability
3.2.3.1 Autosampler stability
Regarding autosampler stability, all results were within the
ICH accepted range where the accuracy % doesn’t exceed
15% for both QC low and QC high.
3.2.3.2 Short term stability at room temperature or
processing temperature
All the analytes (paracetamol and its four metabolites)
were stable after 24 hr at room temperature (RT) since the
accuracy results were within the accepted criteria i.e.,
within 85%-115%.
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3.2.3.3 Freeze and thaw stability
The QC samples were frozen in the freezer at the intended
temperature and then thawed at room or processing
temperature. After complete thawing, samples were
refrozen again applying the same conditions. At each
cycle, samples were frozen for at least 12 hours before
they were thawed. The accuracy for QC low and high for
paracetamol and its four metabolites after 3 cycles were
within the accepted range (85-115%).
3.2.4 Absolute recovery (extraction coefficient)
Table 3 shows absolute recovery for both analytes and IS.
Over the concentration range outlined above, paracetamol
and the four metabolites have good recovery range
between (83.32-96.16%) as well as the IS showed good
recovery range between (93.35-94.41%) which
comparable to that of the analytes. The results were
reproducible too.
Table 3.Absolute recovery of paracetamol, PG, PS, PM and PC
Concentration
Mean Serum
(AUC)
Mean
Solution
(AUC)
Absolute
Recovery
paracetamol
1.5 µg/ml (QC Low) 0.756 0.884 85.55
40.0 µg/ml (QC Mid) 21.471 24.74 86.79
80.0 µg/ml (QC High) 45.237 50.638 89.33
PG
1.5 µg/ml (QC Low) 0.449 0.475 94.59
40 µg/ml (QC Mid) 12.835 13.781 93.14
80 µg/ml (QC High) 26.671 27.735 96.16
PS
1.5µg/ml (QC Low) 0.488 0.585 83.32
40.0 µg/ml (QC Mid) 14.552 17.24 84.41
80.0 µg/ml (QC High) 30.7 36.532 84.04
PM
0.15 µg/ml (QC Low) 0.048 0.054 88.8
4.00µg/ml (QC Mid) 1.407 1.61 87.36
8.00 µg/ml (QC High) 2.953 3.251 90.84
PC
0.15 µg/ml (QC Low) 0.035 0.038 91.31
4.00 µg/ml (QC Mid) 1.006 1.115 90.16
8.00µg/ml (QC High) 2.143 2.259 94.86
IS
(QC Low) 6.439 6.82 94.41
(QC Mid) 6.669 7.143 93.35
(QC High) 7.116 7.543 94.33
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3.3. Estimation the impact of pre-
administration of GluN on paracetamol and
its metabolites pharmacokinetic parameters.
The pharmacokinetic parameters of paracetamol and its
metabolites at dose 15 and 60 mg/kg are illustrated in
tables 4 and 5 respectively.
At the lower dose of paracetamol, the impact of GluN
(both doses) on the pharmacokinetics parameters of
paracetamol and its metabolites were not significant.
3.3.1 Effect of GluN on paracretamol and PG.
At the lower dose of GluN, no alteration in serum levels of
paracetamol. AUClast and Cmax values without and with
GluN were (11.57 ± 0.46, 11.48 ± 0.33, 8.03 ± 0.78, 8.07 ±
0.92, respectively). In addition, 90% CI ratio of the means
of both AUClast and Cmax were within the pre-specified
range. It also appears that GluN has no clinically
significant effects on the pharmacokinetics parameters
(Cmax, AUC and Tmax) of paracetamol at 60 mg/kg dose.
However trend of reduction of paracetamol and elevation
of PG was observed at the higher dose of GluN 50mg/kg.
Serum concentration-time profiles of paracetamol and PG
at dose 15mg/kg with and without GluN are shown in
figure3.
At 60mg/kg dose of paracetamol, significant increase in
AUClast and Cmax of PG at 50mg/kg booster dose of
GluN was observed (table 6, figure 4). Moreover, the 90%
CI of means ratio were not completely contained in (80-
125%) i.e. higher than 125% for both Cmax and AUClast
(table7).
3.3.2 Effect of GluN on PM and PC.
On the other hand, coadministration of GluN resulted in
decrease levels of both Cmax and AUClast of PM at both
doses of paracetamol (15 and 60 mg/kg) which provide a
beneficial effect in mitigating paracetamol associated
toxicity (figure 5).Ratio of means and 90% CI of the ratio
of means were estimated for major parameters (Cmax and
AUClast). CI were not completely contained in (80-125%)
i.e. ratio of the means were lower than 80% for both Cmax
and AUClast at both doses 15 and 60mg/kg when given
with GluN 12mg/kg (table 7). Moreover, there was
significant reduction of AUClast for 60mg/kg dose of
paracetamol when combine with GluN (12mg/kg) (P value
= 0.03) (table 6).
Finally, the last studied metabolite was PC, the second
toxic metabolite. Pharmacokinetic parameters are shown
in(table 5). Serum levels of PC were very low, lower than
our detection limits. Thus only data of 60mg/kg was
utilized.
Promising results were obtained from co-administration of
GluN as both AUC and Cmax levels were reduced
compared with paracetamol alone (figure 5).
Both AUClast and Cmax were reduced Significantly
(p<0.01) in dose dependent manner of GluN (table 6).
Noteworthy, all 90% CI ratio of the means were not within
the (80-125%) range i.e. below 80% (table 7).
Table 4.Pharmacokinetic parameters of paracetamol and its metabolites at dose 15 mg/kg.
Paracetamol PG PS PM
PK
parameters
no of
groups mean SD
no of
groups mean SD
no of
groups mean SD
no of
groups Mean SD
Cmax (µg/ml)
6
8.03 1.92 6 9.28 1.82
6
21.65 2.38 6 0.75 0.38
Tmax (h)** 0.25 (0.25-
0.25) 6 1.00
(0.5-
2) 1.17
(0.50-
2.00) 6 2.25
(0.50-
6.00)
AUClast
(µg*h/ml) 11.57 1.12 6 26.40 5.64 66.36 11.51 6 2.03 0.72
AUCINF 13.29 1.35 3* 27.87 3.24 94.28 11.22 3* 2.46 0.08
AUMClast
(µg*h2/ml)
17.77 3.35 6 59.27 16.93 153.66 27.84 3* 8.98 3.9
AUMCINF 34.09 8.75 3* 114.36 41.03 507.22 251.42 6 5.72 2.4
MRTlast (h) 1.53 0.22 3* 4.03 1.01 2.31 0.12 6 2.83 0.39
MRTINF 2.55 0.5 6 2.23 0.19 5.26 2.29 6 3.63 1.47
HL_Lambda_z
(h) 2.41 0.17 3* 3.07 1.07 3.74 1.92 6 2.06 1.44
* no clear elimination were estimated so these parameters could not be calculated.
** data presented as mean (range)
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Table 5. Pharmacokinetic parameters of paracetamol and its four metabolites at dose 60 mg/kg.
paracetamol PG PS PM PC
PK
parameter
s
no of
groups mean SD
no of
groups mean SD
no of
groups mean SD
no of
groups mean SD
no of
groups mean SD
Cmax
(µg/ml)
5
34.65 6.4
5
40.96 4.87
57.53 27.76 5 3.14 1.37 5 0.69 0.12
Tmax (h) 0.35
(0.25
-
0.50)
1.00 (1-1) 1.4 (1.00-
2.00) 5 2.6
(2.00
-
4.00)
5 1 (1.00-
1.00)
AUClast
(µg*h/ml) 64.08 20.77 120.15 13.67 185.59 47.8 5 6.53 0.81 5 1.78 0.33
AUCINF 67.78 20.37 144.97 11.32 282.39 84.21 2* 7.78 0.11 5 1.84 0.34
AUMClast
(µg*h2/ml)
95.14 35.23 270.91 31.60 473.05 108.79 5 17.76 1.71 5 3.61 0.85
AUMCINF 126.7
6 36.38 522.25
159.8
9
1724.0
3
1098.6
5 5 29.97 4.03 5 4.11 0.98
MRTlast
(h) 1.47 0.09 2.26 0.15 2.56 0.13 5 2.76 0.5 5 2.02 0.21
MRTINF 1.89 0.31 3.58 0.97 5.75 2.43 2* 3.86 0.57 5 2.22 0.3
HL_Lamb
da_z (h) 1.64 0.38 2.46 0.86 3.89 1.86 2* 1.77 0.05 5 1.12 0.19
*No clear elimination were estimated so these parameters could not be calculated.
**Data presented as mean (range)
5
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Figure 3.Mean plasma paracetamol and PG concentration–time profiles at 15 mg/kg with and without GluN 50mg/kg (n=4 and 6 groups, respectively). Results are
shown as (mean +SEM).
Figure 4.Mean plasma paracetamol and PG concentration–time profiles at 60 mg/kg with and without GluN 50mg/kg (n=4 and 5 groups, respectively). Results are
shown as (mean +SEM).
-10
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5 6 7
Pla
sma
con
cetr
atio
n (
µg/
ml)
Time (hr) paracetamol 60mg/kg alone paracetamol 60mg/kg+ GluN booster dose 50mg/kg
PG 60mg/kg PG 60 mg/kg + GLuN booster dose 50mg/kg
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Figure5. Impact of coadminstration of GluN on PM AUClast and Cmax. Data presented as (mean + SEM). * significant decrease in AUClast with coadministration of GluN
compared with the control group (P<0.05).
Figure 6.Impact of GluN on PC (paracetamol cysteine).Data are shown as (mean, +SEM) for (PC60 mg/kg ) and (PC 60 mg/kg+ GluN booster dose 12mg/kg) and as (mean, -
SEM) for (PC 60mg/kg+ GluN booster dose 50mg/kg GluN).
6.53
4.96
6.28
3.14
1.57
3.27
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
paracetamol 60mg/kg paracetamol 60mg/kg+ GluNbooster dose 12mg/kg
paracetamol 60mg/kg+ GluNbooster dose 50mg/kg
PM AUC last PM Cmax
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 2 3 4 5 6 7
Pla
sma
con
cen
trat
ion
(u
g/m
l)
Time (h)
PC 60 mg/kg PC 60mg/kg + GluN booster dose 12mg/kg PC 60mg/kg + GluN booster dose 50mg/kg
*
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Table 6.Summary of major pharmacokinetic parameters for paracetamol and its metabolites in rats plasma after a single dose paracetamol alone compared with paracetamol
in combination with GluN.
paracetamol PG PS PM PC
PK
parameter Treatment
no of
groups mean SEM mean SEM mean SEM mean SEM mean SEM
AUClast
paracetamol 15mg/kg 6 11.57 0.46 26.40 2.30 66.36 4.70 2.03 0.30
15mg/kg+12mg/kg GluN 5 11.48 0.33 27.15 2.35 65.33 7.16 1.45 0.19
15mg/kg+50mg/kg GluN 4 8.97 0.27 30.16 5.17 63.49 5.81 1.43 0.04
paracetamol 60mg/kg 5 64.08 9.29 120.15 6.12 185.59 21.38 6.53 0.36 1.78 0.15
60mg/kg+12mg/kg GluN 3 54.91 0.48 142.86 12.18 217.22 20.01 4.96* 0.42 1.28* 0.07
60mg/kg+50mg/kg GluN 4 50.43 2.56 148.56* 10.72 204.12 13.07 6.28 1.13 0.61* 0.11
Cmax
paracetamol 15mg/kg 6 8.03 0.78 9.28 0.74 21.65 0.97 0.75 0.16
15mg/kg+12mg/kg GluN 5 8.07 0.92 10.02 0.83 21.65 2.18 0.44 0.07
15mg/kg+50mg/kg GluN 4 6.49 0.42 12.37 1.71 22.70 1.52 0.57 0.02
paracetamol 60mg/kg 5 34.65 2.86 40.96 2.18 57.53 12.41 3.14 0.61 0.69 0.06
60mg/kg+12mg/kg GluN 3 29.26 0.31 46.12 1.60 58.08 6.99 1.57 0.48 0.43* 0.02
60mg/kg+50mg/kg GluN 4 31.15 1.87 70.54* 8.97 83.47 12.62 3.27 0.58 0.33* 0.06
Tmax**
paracetamol 15mg/kg 6 0.25 0.25-
0.25 1.00
0.5-
2.00 1.17
0.5-
2.00 2.25
0.5-
6.00
15mg/kg+12mg/kg GluN 5 0.25 0.25-
0.25 0.85
0.25-
1.00 0.75
0.25-
1.00 2.40
1.00-
4.00
15mg/kg+50mg/kg GluN 4 0.25 0.25-
0.25 1.25
1.00-
1.00 0.69
0.25-
1.00 1.75
1.00-
3.00
paracetamol 60mg/kg 5 0.35 0.25-
0.50 1.00
1.00-
1.00 1.40
1.00-
2.00 2.60
2.00-
4.00 1.00
1.00-
1.00
60mg/kg+12mg/kg GluN 3 0.25 0.25-
0.25 1.67
1.00-
3.00 1.67
1.00-
3.00 5.00
3.00-
6.00 1.00
1.00-
1.00
60mg/kg+50mg/kg GluN 4 0.25 0.25-
0.25 1.50
1.00-
2.00 1.50
1.00-
2.00 3.75
1.00-
6.00 1.00
1.00-
1.00
*Statistically significant (p<0.05).
**Data presented as mean (range)
'
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Table 7. The 90% CI of the geometric mean ratios of primary pharmacokinetc parameters (AUClast and Cmax) for paracetamol and its four metabolites.
Paracetamol PG PS PM PC
PK
parameter Treatment
Ratio
%*
90%
CI
Ratio
%*
90% CI Ratio
%* 90% CI
Ratio
%*
90%
CI
Ratio
%*
90% CI
AUClast
paracetamol
15mg/kg+ 12mg/kg
GluN 99.46
(86.00-
115.03)
103.11 (84.05-
126.5)
97.19
(78.56-
120.25) 72.92≠
(55.61-
95.61)
paracetamol
15mg/kg+ 50mg/kg
GluN 77.69≠
(66.53-
90.72)
112.09 (90.15-
139.39)
95.78
(76.34-
120.18) 74.25≠
(55.62-
99.12)
paracetamol
60mg/kg +12mg/kg
GluN 88.65
(74.39-
105.65)
118.68 (92.75-
151.86)
118.68
(92.75-
151.86) 75.82≠
(54.68-
105.12) 72.62≠
(51.28-
102.85)
paracetamol
60mg/kg+ 50mg/kg
GluN 81.10
(69.04-
95.28)
123.39 (98.38-
154.75)
123.39
(98.38-
154.75) 82.68
(61.24-
111.62) 32.98≠
(23.96-
45.40)
Cmax
paracetamol
15mg/kg+ 12mg/kg
GluN 100.08
(82.88-
120.87)
108.33 (87.61-
133.95)
98.45
(74.63-
129.87) 63.72≠
(39.53-
102.71)
paracetamol
15mg/kg+ 50mg/kg
GluN 82.02
(67.08-
100.29)
132.18≠ (105.41-
165.76)
104.68
(77.92-
140.64) 86.21
(51.83-
143.41)
paracetamol
60mg/kg+ 12mg/kg
GluN 75.79≠
(61.54-
93.33)
113.12 (87.56-
146.13)
113.12
(87.56-
146.13) 49.67≠
(27.93-
88.33) 62.98≠
(44.38-
89.37)
paracetamol
60mg/kg+ 50mg/kg
GluN 75.79≠
(62.59-
91.76)
168.97≠ (133.56-
213.78)
168.97≠
(133.56-
213.78) 115.9
(68.3-
196.68) 46.36≠
(33.61-
63.93)
* Ratio were calculated assuming paracetamol alone (control groups) is reference.
≠ value is not contained within the (80-125 %)
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4. DISCUSSION
Herein we reported a simple, rapid and reliable HPLC
method using UV detector at wavelength 245 nm to
separate and detect paracetamol and its four conjugated
metabolites, glucuronide, sulfate, cysteine and
mercapturate in serum samples in less than 10 minutes run
time. This method is characterized by using of 75µl serum
samples. Sample preparation was a single step PCA (1:1)
precipitation without any further processing. Accordingly,
the overall analysis of the paracetamol metabolites of both
pathways; conjugation and oxidation, will be very useful
to get pharmacological and toxicological information
about this drug.
In order to demonstrate the reliability of our method for
the determination of paracetamol in rat serum, a full
method validation according to EMA guidelines was
carried out. As illustrated in the result part, the present
assay provides reasonable accuracy, precision, linearity,
stability and recovery for paracetamol and its metabolites
over the concentration range tested since all of the results
were within the acceptance criteria of validation
guidelines.
This assay was applied to determine the impact of GluN
on plasma concentrations of paracetamol and its
metabolites. Paracetmol could to be used concomitantly
with GluN in OA and other cases. In this regard, almost all
the conducted studies deal with the efficacy issues of these
agents i.e. paracetamol and GluN in treatment of OA [21].
Moreover, most clinical trials were conducted to compare
between drugs used in OA and their effectiveness and
limitation [21].
To the best of our Knowledge, our study considered the
first study that addresses the quantification of paracetamol
metabolites following its interaction with GluN.
Plasma conc-time profiles for paracetamol were
constructed at both doses by giving paracetamol to
previously fasted female rats (24 hr before the dose)
(figure 3 and 4).
Surprisingly, plasma conc-time profiles for paracetamol at
both doses dose not altered significantly when GluN was
pre-administered to rats for two days in drinking water
(12mg/ml). Pharmacokinetics parameters were reduced
not significantly, and possible drug-drug interaction could
not be concluded (table 6). Thus, it was speculated that
higher dose of GluN would show a clear effect. Again no
significant alteration in pharmacokinetics parameters was
observed (table 6).
The bioequivalence EMA guidance was used to
investigate a potential drug–drug interaction between
GluN and paracetamol. Geometric mean ratios and 90%
confidence intervals (90% CI) were estimated for
maximum plasma concentration (Cmax) and area under
the plasma concentration–time curve (AUClast). No drug-
drug interaction is concluded if the results were within the
acceptance range (80-125%) [22,23].
Mean ratios were within the pre-specified range for
AUClast and Cmax at 12mg/kg booster dose of GluN.
These findings suggest that both paracetamol and GluN
could be administrated together without a significant effect
on pharmacokinetics of paracetamol and thus dose
adjustment for paracetamol would not be necessary if the
two drugs are co-administered.
Paracetamol is extensively metabolized and very small
percent of a therapeutic dose is excreted unchanged in the
urine. The major metabolites of paracetamol are the
glucuronide (PG) sulphate (PS) conjugates. These major
conjugates being more water-soluble than the paracetamol,
are eliminated from the liver and blood mainly via urine
(both) and a little via bile (PG). About 30% and 55% of
administered paracetamol is excreted in urine as PS and
PG, respectively. However, important differences in
conjugation pathways between species are found. Rats
excrete PS more than PG [6,24,25].
Plasma profile and major pharmacokinetic parameters of
PG and PS are summarized in tables (4 and 5). In this
regard, both metabolites mainly PS showed higher plasma
levels than that of paracetamol and that behavior were also
observed in the previous studies [24, 26].
Effect of co-administration of GluN on paracetamol major
metabolites was evaluated. Accordingly, GluN pre-
administration resulted in an increase PG maximum
plasma concentration (Cmax) and area under the plasma
concentration-time curve (AUC) significantly but did not
affect time to reach the maximum plasma concentration
(Tmax) (table 6). Elevation of PG plasma level would
explain the slight reduction of paracetamol plasma level.
This increase was significant at 60mg/kg dose of
paracetamol but not at 15mg/kg dose. A possible
explanation for this finding that at higher dose, metabolic
pathways would be saturated and thus any alteration in
these pathways would be more obviously revealed.
90% CI means ratio for both AUClast and Cmax of PG
metabolite were calculated. Accordingly, at higher dose of
GluN these ratio were not contained within the pre
specified range of bioequivalence i.e. above the higher
limit of the ratio of 80-125% and this implies that PG
plasma levels were elevated when GluN was concomitant
administrated with paracetamol.
Coadminstration of GluN.HCL with paracetamol showed
no significant effect on PS pharmacokinetic parameters at
both doses of GluN. Accordingly, alteration in this
metabolic pathway could not be concluded.
CYP450 involved in paracetamol metabolism. Oxidation
of paracetamol to NAPQI is mostly mediated by CYP2E1
isoform, CYP1A2 and CYP3A4 are also involved [27].
NAPQI detoxifed by glutathion to be further proccessed to
PC and PM and then cleared out the body safely.However,
the monitoring of NAPQI in plasma is not possible
because it is further rapidly metabolized to the glutathione
conjugate[28]. Moreover, NAPQI is not stable and readily
hydrolyzes to 1,4-benzoquinone or hydroquinone [29].
Therefore, monitering PM and PC levels confirm the
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formation of NAPQI. In this regard, the most interesting
finding was the reduction of both AUClast and Cmax of
paracetamol toxic metabolites, namely PM and PC with
GluN coadminstration. PM both AUClast and Cmax were
reduced by coadministration of GluN at both doses of
paracetamol 15 mg/kg and 60 mg/kg. However, this
reduction was not significant. On the other hand, PC levels
were significantly reduced. Both AUClast and Cmax were
reduced in dose dependant manner with coadminstration
of GluN.
The most obvious finding emerged from these results that
coadminstration of GluN may provide toxicity reduction.
Hence, this combination provides a potential good
candidate for mitigation of hepatotoxicty associated with
paracetamol.
5. CONCLUSION
In conclusion, we have reported a rapid and reliable HPLC
method using UV detector for the simultaneously
determination of paracetamol and its four metabolites
namely, PG, PS, PM and PC in rat serum with significant
advantages over other methods used for detection of the
five compounds in biological fluids.
Moreover, this study offers some insights into the
usefulness of combination of both GluN and paracetamol
in terms of paracretamol toxicity. Further work needs to
address these findings. This work may provide a starting
point to unique formulation contain both paracetamol and
GluN with high efficacy and low side effects.
ACKNOWLEDGEMENTS
The authors are very grateful for the Jordanian
Pharmaceutical Manufacturing Co. (JPM), University
of Petra and "Jordan Center for Pharmaceutical
Research" for all their supports to complete this work.
They also thank Mr. Mohamad Al Bayed guanine help
and contribution to this work.
REFERENCES
[1]. Graham, G.G., Davies, M.J., Day, R.O.,
Mohamudally, A., Scott, K.F., 2013. The modern
pharmacology of paracetamol: therapeutic actions,
mechanism of action, metabolism, toxicity and
recent pharmacological findings.
Inflammopharmacology 21, 201–32.
doi:10.1007/s10787-013-0172-x
[2]. Larson, A.M., Polson, J., Fontana, R.J., Davern,
T.J., Lalani, E., Hynan, L.S., Reisch, J.S., Schiødt,
F. V, Ostapowicz, G., Shakil, a O., Lee, W.M.,
2005. Acetaminophen-induced acute liver failure:
results of a United States multicenter, prospective
study. Hepatology 42, 1364–72.
doi:10.1002/hep.20948
[3]. Additional Research Areas -FDA, 2012. Drug-
Induced Liver Toxicity.
[4]. Ward, B., Alexander-Williams, J.M., 1999.
Paracetamol revisited: A review of the
pharmacokinetics and pharmacodynamics. Acute
Pain 2, 139–149. doi:10.1016/S1366-
0071(99)80006-0
[5]. Bertolini, A., Ferrari, A., Ottani, A., Guerzoni, S.,
Tacchi, R., Leone, S., 2006. Paracetamol: new
vistas of an old drug. CNS Drug Rev. 12, 250–75.
doi:10.1111/j.1527-3458.2006.00250.x
[6]. Bessems, J.G., Vermeulen, N.P., 2001. Paracetamol
(acetaminophen)-induced toxicity: molecular and
biochemical mechanisms, analogues and protective
approaches. Crit. Rev. Toxicol. 31, 55–138.
doi:10.1080/20014091111677
[7]. Vertzoni, M.., Archontaki, H.., Galanopoulou, P.,
2003. Development and optimization of a reversed-
phase high-performance liquid chromatographic
method for the determination of acetaminophen and
its major metabolites in rabbit plasma and urine
after a toxic dose. J. Pharm. Biomed. Anal. 32,
487–493. doi:10.1016/S0731-7085(03)00246-2
[8]. Gicquel, T., Aubert, J., Lepage, S., Fromenty, B.,
Morel, I., 2013. Quantitative analysis of
acetaminophen and its primary metabolites in small
plasma volumes by liquid chromatography-tandem
mass spectrometry. J. Anal. Toxicol. 37, 110–6.
doi:10.1093/jat/bks139
[9]. Hewavitharana, a K., Lee, S., Dawson, P. a,
Markovich, D., Shaw, P.N., 2008. Development of
an HPLC-MS/MS method for the selective
determination of paracetamol metabolites in mouse
urine. Anal. Biochem. doi:10.1016/j.ab.2007.11.011
[10]. Tan, Q., Zhu, R., Li, H., Wang, F., Yan, M., Dai, L.,
2012. Simultaneous quantitative determination of
paracetamol and its glucuronide conjugate in human
plasma and urine by liquid chromatography coupled
to electrospray tandem mass spectrometry:
application to a clinical pharmacokinetic study. J.
Chromatogr. B. Analyt. Technol. Biomed. Life Sci.
893-894, 162–7.
doi:10.1016/j.jchromb.2012.02.027
[11]. Ruths, S., Viktil, K.K., Blix, H.S., 2007.
[Classification of drug-related problems]. Tidsskr.
den Nor. lægeforening Tidsskr. Prakt. Med. ny
række 127, 3073–6.
[12]. Palleria, C., Di Paolo, A., Giofrè, C., Caglioti, C.,
Leuzzi, G., Siniscalchi, A., De Sarro, G., Gallelli,
L., 2013. Pharmacokinetic drug-drug interaction
and their implication in clinical management. J.
Res. Med. Sci. 18, 601–610.
[13]. ( National Center for Complementary and
Alternative Medicine, n.d. Using Dietary
Supplements Wisely | NCCAM [WWW
Document].
1246 2, Issue.39, Vol.2740-, ISSN: 2051Pharmaceutical AnalysisInternational Journal of
© RECENT SCIENCE PUBLICATIONS ARCHIVES | December 2014|$25.00 | 27704062|
*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*
[14]. Chan, K.O.W., Ng, G.Y.F., 2011. A review on the
effects of glucosamine for knee osteoarthritis based
on human and animal studies. Hong Kong
Physiother. J. 29, 42–52.
doi:10.1016/j.hkpj.2011.06.004
[15]. Barnes, P.M., Bloom, B., Nahin, R.L., 2008.
Complementary and alternative medicine use
among adults and children: United States, 2007.
Natl. Health Stat. Report. 1–23.
[16]. Laba, T.-L., Brien, J., Fransen, M., Jan, S., 2013.
Patient preferences for adherence to treatment for
osteoarthritis: the MEdication Decisions in
Osteoarthritis Study (MEDOS). BMC
Musculoskelet. Disord. 14, 160. doi:10.1186/1471-
2474-14-160
[17]. Hochberg, M.C., Altman, R.D., April, K.T.,
Benkhalti, M., Guyatt, G., McGowan, J., Towheed,
T., Welch, V., Wells, G., Tugwell, P., 2012.
American College of Rheumatology 2012
recommendations for the use of nonpharmacologic
and pharmacologic therapies in osteoarthritis of the
hand, hip, and knee. Arthritis Care Res. (Hoboken).
64, 465–474. doi:10.1002/acr.21596
[18]. Jaeschke, H., Bajt, M.L., 2006. Intracellular
signaling mechanisms of acetaminophen-induced
liver cell death. Toxicol. Sci. 89, 31–41.
doi:10.1093/toxsci/kfi336
[19]. Du Souich, P., 2014. Absorption, distribution and
mechanism of action of SYSADOAS. Pharmacol.
Ther. doi:10.1016/j.pharmthera.2014.01.002
[20]. EMA, 2012. European Medicines Agency - News
and Events - European Medicines Agency updates
guideline on drug interactions [WWW Document].
[21]. Chou, R., McDonagh, M.S., Nakamoto, E., and
Griffin, J., 2011. Analgesics for Osteoarthritis.
Agency for Healthcare Research and Quality (US).
[22]. Nunes, T., Sicard, E., Almeida, L., Falcão, A.,
Rocha, J.-F., Brunet, J.-S., Lefebvre, M., Soares-da-
Silva, P., 2010. Pharmacokinetic interaction study
between eslicarbazepine acetate and topiramate in
healthy subjects. Curr. Med. Res. Opin. 26, 1355–
62. doi:10.1185/03007991003740861
[23]. EMEA, 2010. Guidlineon the Investigationof
Bioequevelence 1, 1–27.
[24]. Ishii, M., Kanayama, M., Esumi, H., Ogawara, K.-
I., Kimura, T., Higaki, K., 2002. Pharmacokinetic
analysis of factors determining elimination
pathways for sulfate and glucuronide metabolites of
drugs. I: studies by in vivo constant infusion.
Xenobiotica. 32, 441–50.
doi:10.1080/00498250210123094
[25]. Prescott, L.F., 1980. Kinetics and metabolism of
paracetamol and phenacetin. Br. J. Clin. Pharmacol.
10 Suppl 2, 291S–298S.
[26]. Brunner, L.J., Bai, S., 1999. Simple and rapid assay
for acetaminophen and conjugated metabolites in
low-volume serum samples. J. Chromatogr. B.
Biomed. Sci. Appl. 732, 323–9.
[27]. Tan, S.C., New, L.S., Chan, E.C.Y., 2008.
Prevention of acetaminophen (APAP)-induced
hepatotoxicity by leflunomide via inhibition of
APAP biotransformation to N-acetyl-p-
benzoquinone imine. Toxicol. Lett. 180, 174–81.
doi:10.1016/j.toxlet.2008.06.001
[28]. Albano, E., Rundgren, M., Harvison, P., Nelson, S.,
Moldeus, P., 1985. Mechanisms of N-acetyl-p-
benzoquinone imine cytotoxicity. Mol. Pharmacol.
28, 306–311.
[29]. Bedner, M., MacCrehan, W. a, 2006.
Transformation of acetaminophen by chlorination
produces the toxicants 1,4-benzoquinone and N-
acetyl-p-benzoquinone imine. Environ. Sci.
Technol. 40, 516–22.