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




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%




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:




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.




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%.




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




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)




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




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




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

*




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)

'




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 %)




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




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.

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simultaneous determination of paracetamol and its ......high performance liquid chromatography...

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