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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 |
*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*
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
Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences;
University of Petra, Amman-Jordan Tawfiq Arafat
Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences;
University of Petra, Amman-Jordan Corresponding Author Email: wabudayyih@uop.edu.jo
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.
International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.38, Issue.2 1135
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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
International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.38, Issue.2 1136
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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
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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.
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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
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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
curve back calculation.
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
Correlation Slope R2 Intercept
0.999749 0.000629 0.999498 -0.001426
Figure 8: The plot of calibration curve levels against their analytical response and regression linear equation in validation
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 )
International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.38, Issue.2 1140
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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
curve back calculation.
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
Correlation Slope R2 Intercept
0.999471 0.000588 0.998942 0.000150
Figure 9: The plot of calibration curve levels against their analytical response and regression linear equation in validation
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
Correlation Slope R2 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 )
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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
Correlation Slope R2 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 )
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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
Correlation Slope R2 Intercept
0.999982 0.000617 0.999964 -0.001874
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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
International Journal of Pharmaceutical Analysis, ISSN: 2051-2740, Vol.38, Issue.2 1144
© RECENT SCIENCE PUBLICATIONS ARCHIVES| September 2013|$25.00 | 27702833 |
*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*
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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