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International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.38, Issue.2 1134

© RECENT SCIENCE PUBLICATIONS ARCHIVES| September 2013|$25.00 | 27702833 |

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Development and Validation of a Stability–

Indicating HPLC Method for Determination of

Bupivacaine in Human Plasma

Wael Abu Dayyih Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences;

University of Petra, Amman-Jordan Enas Alkhader

Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences;

University of Petra, Amman-Jordan Eyad Mallah


University of Petra, Amman-Jordan Tawfiq Arafat


University of Petra, Amman-Jordan Corresponding Author Email: [email protected]

ABSTRACT

The purpose of the research described herein was to

develop and validate a stability indicating HPLC method

for determination of Bupivacaine in human plasma .A

simple, rapid, accurate, and precise gradient reversed-

phase (RP-HPLC) method has been developed for the

determination of Bupivacaine in human plasma. BDS

Hypersil C18 Column (150mm x 4.6mm, 5μm, Thermo

Electron Corp. using 66.5 % water, 33.5 % Acetonitrile, 1

ml/L Triethylamine, pH = 2.5,as mobile phase at a flow rate

of 1.0 ml/min. UV Detector λ = 202 nm. at column

temperature (40°C).

Retention times dependent upon HPLC conditions which,

3.1 – 3.3 minutes Bisoprolol as (Internal Standard) and 3.5 –

3.7 minutes for the Bupivacaine the correlation coefficient

was 0.99930which represent a linearity strength of this

method . The proposed method was further applied to the

determination of Bupivacaine in human plasma, with good

percent recovery.

The accuracy and the precision of the method were

validated on intraday and interday basis in accordance

with ICH guidelines

1. INTRODUCTION

Bupivacaine is a local anesthetic which has various uses.

Bupivacaine often is administered by epidural injection

before total hip arthroplasty[1-6,11]Bupivacaine

hydrochloride monohydrate is that is freely soluble in 95%

ethanol, soluble in water, and slightly soluble in

chloroform or ether [7-11].Bupivacaine hydrochloride is

chemically designated as 2- piperidinecarboxamide, 1-

butyl-N-(2,6- dimethylphenyl)-, monohydrochloride,

monohydrate [7,8]with a molecular mass of 342.9 and has

the structure is shown in figure 1 [12]:

Figure 1: Bupivacaine Chemical Structure.

Few Jordanian hospitals use Bupivacaine combined with

Fentanyl, most of them use single dose of Bupivacaine

HCl (Macraine ®) in a dose of 1.0-1.25 mg/ml.

There are few and variety reported methods for the

determination of Bupivacaine levels in human plasma

using HPLC [13]. A HPLC equipped with a variable

wavelength UV detector [14]. A coupled-column HPLC

system, consists of one reversed-phase and one ion-

exchange column. [15].A Rp- HPLC method has been

developed and validated for the quantification of

Bupivacaine in human plasma. Bupivacaine and the

internal standard, lidocaine,. [16]. Another pharmaceutical

methods of analysis had been obtained for

biopharmaceutical studies and application to seven other

local anesthetics or by gas chromatography,using alkyldiol

silica precolumn and determination of bubivacaine with

another anesthesia[17-25]. However, these methods all

have their own weakness or disadvantages like the

sensitivity, low recovery, long retention time and the using

of phosphate buffer which have negative effects on HPLC

instrument on the long run.

As shown there are few and variety analytical methods

reported for the determination of Bupivacaine in human

plasma and this work will concentrate on developing a

new HPLC-UV method that is rapid, simple, selective, and

reproducible to overcome the disadvantages found in the

previous methods.




2. EXPERIMENTAL PART

2.1 Chemicals and Reagents

- Bupivacaine hydrochloride was obtained from JCPR,

- Bisoprolol as internal standard was obtained from Tabouk

Pharma

- The blank plasma was collected from Blood Bank

(harvested from donors), Islamic Hospital (Amman, Jordan).

- Methanol HPLC grade(Fischer, USA).

- Acetonitrile HPLC grade (Fischer, USA).

- Phosphoric acid 85%. (Fischer, USA).

- Quality deionized water, HPLC grade-JCPR

- Triethylamine(TEA). (ACROS, Geel – Belgium).

2.2 Instrumentation

- A Dionex HPLC auto-sampler system composed of a

constant solvent delivery system (P580), a 100 µL fixed

volume injector (Rheodyne 7125), UV Detector

(UVD340S), λ = 202 nm, Autosampler (ASI-100),

Computer System, MicroSoft Windows 2000, Hypersil

Thermo Electron Corporation, BDS C-18 Column (150 mm

x 4.6 mm, 5μm).

- Balance: Sartorious, model ME2355.

- Centrifuges: Eppendorf centrifuge model 5417 C and 5702

R.

- Vortex Mixer: EKA, manual.

2.3 Chromatographic Conditions

Chromatographic conditions in this experiment are

illustrated in table 1:

Table 1: Chromatographic conditions of Bupivacaine analysis

Retention times dependent upon HPLC conditions.

2.4 Standard, quality control and buffer

samples

Stock solutions of Bupivacaine were prepared by dissolving

the accurately weighed reference compound in methanol to

give a final concentration of 500 µg/ml. The stock solution

of I.S, bisoprolol figure .2 was prepared in methanol to get

1000 µg/ml and then diluted to 10 µg/ml

Figure 2: Bisoprolol Chemical Structure

For preparation of standard curve or quality control , the

standard working solution (500 µg/ml) were used ,spiked

plasma samples were prepared by using Bupivacaine stock

solution . Samples of the standard curve in plasma were

prepared using seven concentrations, not including zero and

these concentrations are:10, 50, 250, 500, 1000, 2000 and

4000 ng/ml, and kept at -40 oC, standard samples were given

daily together with the quality control samples.

Preparation of QC samples ,spiked plasma samples were

prepared using Bupivacaine solution (stock solution) which

contains 500 μg/ml of Bupivacaine Table 2 shows data of

preparation of Quality Control samples in plasma[26,27].

Buffer is used to enhance the baseline, 1.0 M NaOH and 1.0

M Na2CO3 were studied and compared with water.

Tab.2. Quality control samples preparation procedure

Solution

No.

Stock

Conc.

(µg/ ml)

Volume

Taken

from Stock

(µl)

Total

Volume

(ml)

Working

solution

Conc.

(µg/ ml)

QC ID

Volume

taken from

W.S.

(µl)

Total

Volume

(ml)

Final

Conc.

(ng/ ml)

S11 500 30 10 1.5 QC

Low 1000 50 30

S12 500 1500 10 75 QC Mid 1000 50 1500

S13 500 3000 10 150 QC

High 1000 50 3000

Column BDS Hypersil C18 Column (150mm x 4.6mm, 5μm, Thermo Electron Corp.

Solvent System (Mobile

phase)

66.5 % water, 33.5 % Acetonitrile, 1 ml/L Triethylamine, pH = 2.5 adjust with

H3PO4

Detection UV Detector λ = 202 nm.

Injection Volume 50 micro litters

Retention Times*

Bisoprolol

Bupivacaine

3.1 – 3.3 minutes (Internal Standard)

3.5 – 3.7 minutes

Flow Rate 1.0 ml/min.

Oven Temperature 40oC




2.5 Validation Tests 2.5.1 Linearity and calibration curve

Standards for linearity at five calibration curves consisting

of the validation, was performed on three separate days,

with seven standard calibration level lines (not including

zero) on each day. Each day of validation included plasma

samples representing blank, Zero, standard calibration

curve, 10 replicates of Quality Control (QC) samples

(Q.C. Low, Q.C. Mid, Q.C. High). The validation

parameters have not to exceed the limits by the Guidance

for Industry [26,27].

2.5.2. Accuracy

Measured using ten replicates for each of QC levels; low,

mid and high for Bupivacaine, peak areas of replicates were

analyzed daily and the concentrations were back-calculated

by employing the regression equation established on the

same day.

2.5.3. Precision

Was measured by the intra-day and inter-day percent

coefficient of variation. Precision was estimated from QC

samples and standard curve levels

2.5.4. Intra-day Accuracy and Precision

Intra-day accuracy and precision was measured by

analyzing ten replicates for each of QC level (Low, Mid

and High) in the batch for Bupivacaine.

2.5.5. Inter-day Accuracy and Precision

Ten replicates for each of QC levels (Low, Mid and high)

in the batch of Bupivacaine were analyzed to measure the

inter-day accuracy and precision in the batch for

Bupivacaine, the above was applied for 3 successive days.

2.5.6 Recovery

Tested by preparing triplicates from each QC level of

plasma samples and triplicates from each QC level

prepared in the mobile phase (out of extraction procedure)

2.5.7 Specifity

Examined by analyzing blank and standard zero samples

of the biological matrix (plasma) and it was obtained from

six different sources. Each of the blank and zero standards

were tested for interference.

2.5.8 Selectivity

Done by the analysis of six different most common drugs

that most people might take and it was done to make sure

there is no significant interference at Bupivacaine and

Bisoprolol retention time.

For selectivity common drugs were injected to check

interferences at drug or internal standard retention time e.g

(caffiene, acetylsalicylic acid, acetaminophen, ibuprofen,

nicotine, mefanimic acid and sulfamethoxazole).

2.5.9 Stability

Plasma samples containing QC Low, QC Mid and QC

High concentrations were prepared in order to determine

the analyte's stability under the analytical conditions as

indicated. The stability of analyte was determined in terms

of, short term stability, room temperature (Bench top)

stability post-operative stability (Auto-Sampler Stability),

freeze and thaw stability and long term stability.

The stability of stock solution of Bupivacaine and IS were

evaluated at room temperature for at least 24 hours.

Samples then refrigerated and the stability of the

refrigerated stock solutions was determined after 14 days,

then the stability were tested by comparing the instrument

response with that of freshly prepared solutions

3. RESULTS AND DISCUSSION

3.1 Internal Standard

Internal standard chosen should match the

chromatographic properties[28], recovery and ionization

properties of the analyte, bisoprolol and propanolol were

found to match these criteria, upon running them after

applying the extraction procedure both suggested I.S

showed no interfering but propanolol retention time was

4.5 min while bisoprolol retention time was in the range of

3.1-3.3 min so bisoprolol was found to match the required

criteria and also serve our purpose of method

development, therefore it was chosen as an IS.

Bisoprolol did not alter or deteriorate the performance of

the proposed method.

3.2 Sample Preparation and Method of

Extraction Procedure

According to the sample preparation and method of

extraction development mentioned, 5.0 ml of tert-butyl-

methyl-ether was the chosen solvent as it has the best

intensity, 1.0 M sodium carbonate was chosen as buffer as

it has the best baseline, with 200 µl of formic acid.

A 50 µl of bisoprolol (10.0 µg/ml) was added to 0.25 ml of

plasma in test tube, then 50 µl of 1.0 M sodium carbonate

was added, the mixture was vortex-mixed, then added 5.0 ml

of tert-butyl-methyl-ether, vortex for 2.0 minutes followed

by centrifugation for 6.0 minutes at 4400 rpm. After 6 min of

centrifugation, the lower layer was carefully transferred to

350 µl flat bottom insert and 50 μl of this sample was

injected on BDS Hypersil C18 column. Bupivacaine and the

internal standard were separated from endogenous

substances.

3.3 Separation and Chromatography

The chromatographic conditions were optimized through

several test and error trials to achieve symmetric peaks

shapes for the analyte and I.S, as well as shortest run time.

The good results were obtained with a mobile phase

consists of water/ acetonitrile (66.5/33.5% v/v), 0.1%

triethylamine and pH of 2.5 adjusted with phosphoric acid

and oven temperature set at 40 oC. Focused on the short

run time to assure high throughput, with good peak shapes

and high sensitivity. The retention times of Bupivacaine

and IS were 3.1-3.3 and 3.5-3.7, respectively. Figure 3




shows plasma blank chromatogram, and no endogenous

interfering peaks with comparison to LLOQ

chromatogram, which is represented in figure 4 with a

concentration of 10 ng/ml. Zero concentration of plasma

sample showing in its chromatogram in figure 5, also a

clean area in the place of Bupivacaine and I.S peaks in this

chromatogram we could see the I.S peak. A good

chromatographic separation can be indicated by Upper

Limit of Quantitation (ULOQ) level of standard

calibration curve that appears in figure 6, its specified

amount is 4000 ng/ml for Bupivacaine, theoretical plates

in HPLC chromatogram for ULOQ of Bupivacaine is

8948, asymmetry value is 1.04 and resolution value is

2.84.

Figure 3: HPLC Chromatogram of Plasma Blank.

Figure 4: HPLC Chromatogram of LLOQ of Bupivacaine (10 ng/ ml).

Figure 5: HPLC Chromatogram of Zero Bupivacaine Concentration.




Figure 6: HPLC Chromatogram of Bupivacaine ULOQ (4000 ng/ ml)

3.4 Validation Day One, Two and Three,

linearity, Accuracy and Precision Data

Table 12 represents validation of day one for standard

calibration curve.

Intra-day accuracy data derived from standard calibration

curve back calculation, and, figure 7 shows the plot of

calibration curve levels against their analytical response

and regression linear equation.

Day 1 validation results showed an accuracy range of

98.61-102.11% while the accepted criteria is 85.00-

115.00% except for the LLOQ which is 80.00-120.00%,

this work's day 1 validation results passed the required

criteria in terms of accuracy.

The coefficient of determination (R2) should be equal or

more than 0.98 to be within the accepted criteria while in

figure 9 we could find it equals 0.999930 which represents

the strength of the correlation, the correlation coefficient

of standard calibration curve was consistently greater than

0.99 during the course of the validation.

Table 12: Standard calibration curve of validation day one, intraday accuracy data derived from standard calibration

curve back calculation.

Raw data of the standard curve with regards to Correlation, Slope, R2 and intercept for Day I are illustrated in table 13:

Table 13: Raw data of the standard curve with regards to correlation, slope, R2 and intercept for Day I

Correlation Slope R2 Intercept

0.999965 0.000618 0.999930 -0.001274

Theoretical conc.

ng/ml AUC of Drug AUC of I.S AUC Ratio Measured Conc. Accuracy %

10.000 0.038 7.718 0.005 10.059 100.590

50.000 0.225 7.698 0.029 49.338 98.680

250.000 1.224 7.818 0.157 255.266 102.110

500.000 2.317 7.581 0.306 496.175 99.240

1000.000 4.698 7.719 0.609 986.086 98.610

2000.000 9.529 7.642 1.247 2018.103 100.910

4000.000 18.822 7.622 2.469 3994.973 99.870




ng/ml

AU

C R

atio

ng/ml

Figure 7: The plot of calibration curve levels against their analytical response and regression linear equation in validation

Day I.

Table 14 ,15 and figure8 represents validation of day two for standard calibration curve.

Table 14: Standard calibration curve of validation day two, intraday accuracy data derived from standard calibration


Theoretical conc. ng/ml Drug

Area

IS

Area Ratio

Measured

Conc. Accuracy

10.000 0.035 6.937 0.005 10.181 101.810

50.000 0.209 6.829 0.031 50.893 101.790

250.000 1.103 7.106 0.155 249.017 99.610

500.000 2.175 7.170 0.303 484.272 96.850

1000.000 4.292 7.036 0.610 971.579 97.160

2000.000 8.644 6.651 1.300 2067.426 103.370

4000.000 17.688 7.072 2.501 3976.632 99.420

Table 15: Raw data of the standard curve with regards to correlation, slope, R2 and intercept for day II


0.999749 0.000629 0.999498 -0.001426


day II.

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00 4500.00

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 1000.00 2000.00 3000.00 4000.00 5000.00

AU

C R

atio

Y = 0.000629X - 0.001426

(R2= 0.999498 )




ng/ ml

In the same way table 16, 17 and figure 9 represents validation of day three for standard calibration curve.

Table 16: Standard calibration curve of validation day three, intraday accuracy data derived from standard calibration


Theoretical conc.

ng/ml

Drug

Area IS Area Ratio

Measured

Conc. Accuracy

10.000 0.041 7.412 0.006 9.200 92.000

50.000 0.232 7.621 0.030 51.649 103.300

250.000 1.138 7.392 0.154 261.776 104.710

500.000 2.248 7.511 0.299 509.088 101.820

1000.000 4.385 7.329 0.598 1018.126 101.810

2000.000 8.366 7.517 1.113 1894.110 94.710

4000.000 17.767 7.437 2.389 4066.051 101.650

Table 17: Raw data of the standard curve with regards to correlation, Slope, R2 and intercept for day III


0.999471 0.000588 0.998942 0.000150


day III.

Table 18, 19 and figure 10 represents calibration curve Number 4. Intra-day accuracy data derived from standard

calibration curve back calculation, and figure 10 shows the plot of calibration curve levels against their analytical

response and regression linear equation.

Table 18: Standard calibration curve number 4, intraday accuracy data derived from standard calibration curve back

calculation

Theoretical conc.

ng/ml

Drug

Area IS Area Ratio

Measured

Conc. Accuracy

10.000 0.040 7.757 0.005 9.918 99.180

50.000 0.237 7.766 0.031 50.692 101.380

250.000 1.235 7.886 0.157 253.188 101.280

500.000 2.363 7.683 0.308 495.723 99.140

1000.000 4.783 7.831 0.611 982.682 98.270

2000.000 9.746 7.786 1.252 2012.181 100.610

4000.000 19.330 7.755 2.493 4005.616 100.140

Table 19: Raw data of the standard curve number 4 with regards to correlation, slope, R2 and intercept


0.999968 0.000623 0.999936 -0.001028

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 1000.00 2000.00 3000.00 4000.00 5000.00

AU

C R

atio

Y = 0.000588X + 0.000150

(R2= 0.998942 )




AU

C R

atio

AU

C R

atio

ng/ ml

Figure 10: The plot of calibration curve number 4 levels against their analytical response and regression linear equation.

Table 20,21 and figure 11 represent Calibration curve Number 5. Intra-day accuracy data derived from standard

calibration curve back calculation, and figure 11 shows the plot of calibration curve levels against their analytical

response and regression linear equation.

Table 20: Standard calibration curve number 5, intraday accuracy data derived from standard calibration curve back

calculation

Theoretical conc.

ng/ml

Drug

Area IS Area Ratio Measured Conc. Accuracy

10.000 0.043 6.805 0.006 10.087 100.870

50.000 0.215 6.683 0.032 51.579 103.160

250.000 1.076 6.964 0.154 248.599 99.440

500.000 2.104 7.030 0.299 481.905 96.380

1000.000 4.165 6.908 0.603 970.865 97.090

2000.000 8.426 6.538 1.289 2075.543 103.780

4000.000 17.192 6.972 2.466 3971.422 99.290

Table 21: Raw data of the standard curve number 5 with regards to correlation, slope, R2 and intercept


0.999689 0.000621 0.999378 0.000094

Figure 11: The plot of calibration curve number 5 levels against their analytical response and regression linear equation.

Table 22 shows linearity and linear working range of Bupivacaine data based on back calculated area ratio derived from

standard calibration curve.

Table 23 shows linearity and linear working range of Bupivacaine data based on normalized concentration derived from

standard calibration curves.

Table 24 shows linearity and linear working range of Bupivacaine data based on calculated concentration derived from

standard calibration curves while figure 12 shows the plot of linearity of five calibration curves levels against their

analytical response and regression linear equation.

Table 25 shows the results of raw data for five calibration curves with regards to correlation, slope, R2 and intercept

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 1000.00 2000.00 3000.00 4000.00 5000.00

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 1000.00 2000.00 3000.00 4000.00 5000.00

Y = 0.000623X - 0.001028

(R2= 0.999936)

ng/ ml

Y = 0.000621X + 0.000094

(R2= 0.999378 )




Table 22: linearity and linear working range of Bupivacaine data based on back calculated area ratio derived from

standard calibration curve.

Calibration

Curve #

AUC Ratio for Standard Point

10.000 50.000 250.000 500.000 1000.000 2000.000 4000.000

1 0.005 0.029 0.157 0.306 0.609 1.247 2.469

2 0.005 0.031 0.155 0.303 0.610 1.300 2.501

3 0.006 0.030 0.154 0.299 0.598 1.113 2.389

4 0.005 0.031 0.157 0.308 0.611 1.252 2.493

5 0.006 0.032 0.154 0.299 0.603 1.289 2.466

Mean 0.005 0.031 0.155 0.303 0.606 1.240 2.464

STD 0.001 0.001 0.001 0.004 0.005 0.075 0.044

CV% 10.904 3.339 0.780 1.233 0.883 6.016 1.798

Min 0.005 0.029 0.154 0.299 0.598 1.113 2.389

Max 0.006 0.032 0.157 0.308 0.611 1.300 2.501

Table 23: linearity and linear working range of Bupivacaine data based on normalized concentration derived from

standard calibration curves

Calibration

Curve #

Normalized Concentration

10.000 50.000 250.000 500.000 1000.000 2000.000 4000.000

1 1.006 0.987 1.021 0.992 0.986 1.009 0.999

2 1.018 1.018 0.996 0.969 0.972 1.034 0.994

3 0.920 1.033 1.047 1.018 1.018 0.947 1.017

4 0.992 1.014 1.013 0.991 0.983 1.006 1.001

5 1.009 1.032 0.994 0.964 0.971 1.038 0.993

Mean 0.989 1.017 1.014 0.987 0.986 1.007 1.001

STD 0.040 0.019 0.022 0.022 0.019 0.036 0.009

CV% 4.010 1.835 2.122 2.208 1.951 3.602 0.946

Min 0.920 0.987 0.994 0.964 0.971 0.947 0.993

Max 1.018 1.033 1.047 1.018 1.018 1.038 1.017

Table 24: linearity and linear working range of Bupivacaine data based on calculated concentration derived from

standard calibration curves.

Calibration

Curve #

Concentration for each Standard Point

10.000 50.000 250.000 500.000 1000.000 2000.000 4000.000

1 10.059 49.338 255.266 496.175 986.086 2018.103 3994.973

2 10.181 50.893 249.017 484.272 971.579 2067.426 3976.632

3 9.200 51.649 261.776 509.088 1018.126 1894.110 4066.051

4 9.918 50.692 253.188 495.723 982.682 2012.181 4005.616

5 10.087 51.579 248.599 481.905 970.865 2075.543 3971.422

Mean 9.889 50.830 253.569 493.433 985.868 2013.473 4002.939

STD 0.397 0.933 5.381 10.893 19.236 72.516 37.879

CV% 4.010 1.835 2.122 2.208 1.951 3.602 0.946

Accuracy % 98.890 101.660 101.428 98.687 98.587 100.674 100.073

Min 9.200 49.338 248.599 481.905 970.865 1894.110 3971.422

Max 10.181 51.649 261.776 509.088 1018.126 2075.543 4066.051

Raw data for five calibration curves with regards to Correlation, Slope, R2 and intercept are illustrated in table 25.

Table 25: Raw data for five calibration curves with regards to correlation, slope, R2 and intercept


0.999982 0.000617 0.999964 -0.001874




Figure 12: plot of linearity of five calibration curves levels against their analytical response and regression linear

equation.

Table 26 illustrates Inter-day accuracy and precision for the quality control samples of Bupivacaine in the three days of

validation.

Table 26: Inter-day accuracy and Precision for the quality control Samples of Bupivacaine in the three days of validation

30.000 1500.000 3000.000

Day

One

Day

Tow

Day

Three Day One Day Tow

Day

Three Day One Day Tow

Day

Three

Mea

sure

d C

on

cen

tra

tio

n

31.675 30.155 32.504 1503.266 1478.174 1530.652 2854.949 2870.699 3085.664

32.258 30.336 30.528 1468.852 1510.895 1553.222 2861.015 2920.147 3059.512

30.407 32.037 31.399 1479.798 1441.865 1518.744 2866.204 2814.624 3040.301

28.746 32.520 29.964 1485.290 1430.252 1545.274 2854.340 3032.196 3069.725

29.863 30.965 30.925 1470.600 1504.582 1547.417 2937.956 2950.737 3045.970

31.476 30.680 30.992 1498.451 1478.893 1532.803 2856.972 2878.036 3084.505

33.038 29.670 31.131 1470.191 1508.241 1550.991 2865.546 2916.419 3086.361

31.383 31.709 31.154 1478.167 1439.374 1591.342 2868.273 2826.634 3041.608

30.179 32.592 30.043 1485.380 1428.365 1582.858 2852.653 3036.325 3066.321

28.909 30.255 29.753 1474.112 1537.454 1588.412 2937.817 2951.032 3041.811

Mean 30.908 1503.797 2952.478

STD 1.088 45.104 93.873

CV% 3.520 2.999 3.179

Accuracy

% 103.027 100.253 98.416

y = 0.000617x - 0.001874 R² = 0.999964

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0.00 1000.00 2000.00 3000.00 4000.00 5000.00

AU

C R

atio

ng/ ml




4. CONCLUSION

A sensitive HPLC-UV method for quantitative

determination of Bupivacaine in human plasma was

validated in terms of linearity, sensitivity, accuracy,

precision, recovery, specifity, selectivity, dilution integrity

and stability according to the United States Food and Drug

Administration and ICH-guidelines

The application of this method is to study the feasibility of

applying this method for the determination of Bupivacaine

in pregnant women during labor.

ACKNOWLEDGMENT

The authors wish to express our sincere thanks to the

University of Petra and Jordan Centre for Pharmaceutical

Research JCPR for their generous support to this work

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Anaesth., 2012, 6(4), 336-340.

[2]. Abouleish E.I., 1987, Epinephrine Improves the

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Cesarean Section. Anesthesia & Analgesia

Journal, Vol. 66, No. 5:395-400

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