A simple and selective LC method is described for the determination of Sulbactum and Ceftriaxone tablet dosage forms. Chromatographic separation was achieved on a C18 column using mobile phase consisting of a mixture of mixture of 60 volumes of 20mM Phosphate buffer pH 3.5: 40 volumes of Acetonitrile (60:40 v/v) with detection of 210 nm. Linearity was observed in the range 30-70 µg /ml for Sulbactum (r2 =0.9998) and 60-140µg /ml for Ceftriaxone (r2 =0.9983) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.

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Available online at www.icjpir.com ISSN: 2349-5448
Intercontinental journal of pharmaceutical
Investigations and Research
ICJPIR |Volume 3 | Issue 1 | Jan – Mar- 2016 Research Article
New RP HPLC method for the simultaneous estimation of sulbactum and
ceftriaxone in pharmaceutical dosage form
M. Sambasiva Rao, A. Sunil Kumar Reddy, A. Ashok Kumar
Professor & HOD OF Vijaya college of pharmacy, Munaganur (village), Hayathnagar (Mandal),
Ranga redy (District), Pin-501511
Corresponding Author: A. Ashok Kumar
Email: ashok576@gmail.com
ABSTRACT
A simple and selective LC method is described for the determination of Sulbactum and Ceftriaxone tablet
dosage forms. Chromatographic separation was achieved on a C18 column using mobile phase consisting of a
mixture of mixture of 60 volumes of 20mM Phosphate buffer pH 3.5: 40 volumes of Acetonitrile (60:40 v/v)
with detection of 210 nm. Linearity was observed in the range 30-70 µg /ml for Sulbactum (r2
=0.9998) and 60-
140µg /ml for Ceftriaxone (r2
=0.9983) for the amount of drugs estimated by the proposed methods was in good
agreement with the label claim. The proposed methods were validated. The accuracy of the methods was
assessed by recovery studies at three different levels. Recovery experiments indicated the absence of
interference from commonly encountered pharmaceutical additives. The method was found to be precise as
indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the
methods and can be used for routine analysis of pharmaceutical dosage form.
Keywords: C18 column, Sulbactum, Ceftriaxone and %RSD
INTRODUCTION
High performance liquid chromatography
Chromatography is the method of separation
that finds applications in all branches of science. It
was first invented by Russian Botanist Mikhail
Twsett. This technique was used separate various
plant pigments like chlorophylls and xanthophylls
by passing solutions of these compounds through a
glass column packed with finely divided calcium
carbonate. The separated species appeared as
colored bands on the column hence the name of the
process (Greek chroma meaning “color” and
graphein meaning “writing”).3
Chromatography is defined as a non- destructive
procedure for resolving multi-component mixture of
trace, minor, or major constituents into its individual
fractions. In chromatography, the sample is dissolved
in the mobile phase which may be a gas, liquid, or a
supercritical fluid. The principle involved in HPLC is
that when a mixture containing different compounds

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is introduced into the mobile phase and allowed to
flow over a stationary phase, the individual
compounds travel at different speeds and get
separated based on the relative affinities to the
stationary phase and the mobile phase. The
compounds are separated based on the polarity of the
stationary phase and the mobile phase.
Chromatography is of various types based on the
physical state of the stationary and the mobile phase:
Solid - Liquid type
The stationary phase is a solid and the mobile
phase is a liquid. Ex: Thin layer chromatography,
High performance liquid chromatography (HPLC).
Liquid - Liquid type
The stationary phase is a liquid and the mobile
phase is also a liquid. Ex: Paper Chromatography,
HPLC.
Liquid - Gas Type
The stationary phase is a liquid and the mobile
phase is a gas. Ex: Gas chromatography.
METHODOLOGY
Mobile Phase
A mixture of 60 volumes of 20mM Phosphate
buffer pH 3.5:40volumes of Acetonitrile. The
mobile phase was sonicated for 10min to remove
gases.
Preparation of Phosphate buffer (20mM)
2.721 gm. of potassium di hydrogen phosphate
(KH2PO4) was weighed and dissolved in 100ml of
water and volume was made up to 1000ml with
water. Adjust the pH to 3.5 using ortho phosphoric
acid. The buffer was filtered through 0.45µ filters
to remove all fine particles and gases.
Determination of Working Wavelength
(λmax)
In simultaneous estimation of two drugs
isobestic wavelength is used. Isobestic point is the
wavelength where the molar absorptivity is the
same for two substances that are interconvertible.
So this wavelength is used in simultaneous
estimation to estimate both drugs accurately.
Preparation of standard stock solution of
CEFTRIAXONE
25 mg of Ceftriaxonewas weighed and
transferred in to 250ml volumetric flask and
dissolved in methanol and then make up to the
mark with methanol and prepare 10 µg /ml of
solution by diluting 1ml to 10ml with methanol.
Preparation of standard stock solution of
SULBACTAM
25mg of Sulbactam was weighed in to 250ml
volumetric flask and dissolved in Methanol and
then dilute up to The mark with methanol and
prepare 10 µg /ml of solution by diluting 1ml to
10ml with methanol.
RESULTS AND DISCUSSION
Solubility Studies
These studies are carried out at 25 0
C
Ceftriaxone
Sparingly soluble in methanol, Freely Soluble in
Water
Sulbactam
It is freely soluble in water, sparingly soluble in
methanol, very slightly soluble in ethanol
Determination of wavelength
The wavelength of maximum absorption (λmax)
of the drug, 10 μg/ml solution of the drugs in
methanol were scanned using UV-Visible
spectrophotometer within the wavelength region of
200–400 nm against methanol as blank. The
resulting spectra are shown in the fig. no. 1, 2 and 3
and the absorption curve shows characteristic
absorption maxima at 240 nm for Ceftriaxone, 208
nm for Sulbactamand210 nm for the combination.

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Fig. 1: UV-VIS spectrum of CEFTRIAXONE
Observation
λmax was found to be 240 nm for CEFTRIAXONEshown in the figure 1
Fig. 2: UV-VIS spectrum of SULBACTAM
Observation
λmaxwas found to be 208 nm for SULBACTAM shown in the figure 2
Fig. 3: UV-VIS spectrum of CeftriaxonE and Sulbactam and the isosbestic point was 210 nm

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Observation
The Isosbestic point was found to be 210nm for
Ceftriaxone and Sulbactam in combination and was
shown in figure 3
TRIAL-7: (OPTIMISED)
Chromatographic conditions
Mobile phase: KH2PO4: ACN
Ratio : 60:40
Column : Inertsil ODS, C18 (250×4.6× 5µ)
Wavelength : 210 nm
Flow rate : 1ml/min
pH : 3.5
Preparation of mixed standard stock solution
Weigh accurately 10 mg of Ceftriaxone and 5
mg of Sulbactam in 100 ml of volumetric flask and
dissolve in 10ml of mobile phase and make up the
volume with mobile phase. The above stock
solution contains 100 µg/ml of Ceftriaxone and 50
µg/ml of Sulbactam. This solution is used for
recording chromatogram.
Fig. 4: Chromatogram of Ceftriaxone and Sulbactam by using mobile phase
Table1: Result for Ceftriaxone and Sulbactam by using mobile phase
S.No. Name Rt (min) Peak Area Asymmetry Efficiency Resolution
1 SULBACTAM 2.753 331.377 1.500 3086 -
2 CEFTRIAXONE 4.190 3608.736 2.065 3646 6.038
Observation
The peak response was good for both
Ceftriaxone and Sulbactam and the efficiency was
good for both Ceftriaxone and Sulbactam, and the
retention time was also satisfactory for Ceftriaxone
and Sulbactam.The details are given in the table 1
and figure 4, hence it was taken for optimization.
Mobile phase KH2PO4: ACN
pH 3.5
Column Inertsil ODS, C18 (250×4.6× 5µ)
Flow rate 1.0 ml/min
Column temperature Room temperature(20-25o
C)
Sample temperature Room temperature(20-25o
C)
Wavelength 210 nm
Injection volume 20 µl
Run time 6.0 min
Retention time About 4.190min for SULBACTAM and 2.753min for CEFTRIAXONE

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ASSAY
Preparation of samples for Assay
Standard sample
Weigh accurately 10 mg of Ceftriaxone and 5
mg of Sulbactam in 100 ml of volumetric flask and
dissolve in 10ml of mobile phase and make up the
volume with mobile phase. The above stock
solution contains 100 µg/ml of Ceftriaxone and 50
µg/ml of Sulbactam. This solution is used for
recording chromatogram.
Tablet sample
10tablets (each tablet contains 500 mg of
Sulbactamand 1000mg of Ceftriaxone) were
weighed and taken into a mortar uniformly mixed.
Test stock solutions of Sulbactam (50μg/ml) and
CeftriaxonE(100μg/ml) were prepared by
dissolving weight equivalent to 500 mg of
Sulbactam and 1000 mg of Ceftriaxone and
dissolved in sufficient mobile phase. After that
filtered the solution using 0.45-micron syringe
filter and Sonicated for 5 min and dilute to 100ml
with mobile phase. Further dilutions are prepared in
5 replicates of 50μg/ml of Sulbactamand 100μg/ml
of Ceftriaxonewas made by adding 1 ml of stock
solution to 10 ml of mobile phase.
Fig. 5: Chromatogram of Assay standard preparation-1 Fig. 6: Chromatogram of Assay standard preparation-
2
Fig. 7: Chromatogram of Assay standard preparation-3 Fig. 8: Chromatogram of Assay standard
preparation-4

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Fig. 9: Chromatogram of Assay standard preparation-5
Fig.10: Chromatogram of Assay sample
preparation-1
Fig. 11: Chromatogram of Assay sample
preparation-2
Fig. 12: Chromatogram of Assay sample
preparation-3
Fig. 13: Chromatogram of Assay sample
preparation-4
Fig. 14: Chromatogram of Assay sample
preparation-5
Table No.2: Assay Results
SULBACTAM CEFTRIAXONE
Standard Area Sample Area Standard Area Sample Area
Injection-1 351.533 337.476 3426.737 3390.215

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Injection-2 364.739 335.597 3399.205 3403.940
Injection-3 347.31 351.238 3405.464 3464.683
Injection-4 348.496 352.701 3383.819 3436.878
Injection-5 347.226 366.228 3406.047 3406.121
Average Area 354.527 348.648 3404.254 3420.367
Tablet average weight 1500.2 1500.2
Standard weight 50 100
Sample weight 149.2 149.2
Label amount 500 1000
std. purity 99.6 99.7
Amount found in mg 492.43 1007.22
Assay(%purity) 98.49 100.72
Observation
The amount of Sulbactam and Ceftriaxone present in
the taken dosage form was found to be 99.49 % and
100.72% respectively.
VALIDATIONS
System suitability
Standard solutions were prepared as per the test
method and injected into the chromatographic
system. The system suitability parameters like
theoretical plates, resolution and asymmetric factor
were evaluated.
Table 3: Results for system suitability of SULBACTAM
Injection Retention time (min) Peak area Theoretical plates (TP) Tailing factor (TF)
1 2.983 358.060 3267 1.462
2 2.967 360.049 4136 1.522
3 2.943 354.753 3088 1.538
4 2.947 355.992 3283 1.385
5 2.953 359.264 4136 1.522
6 2.947 349.258 3086 1.500
Mean 2.9567 356.229 - -
SD 0.0154 3.949 - -
%RSD 0.52 1.11 - -
Table 4: Results for system suitability of CEFTRIAXONE
Injection Retention time (min) Peak area Theoretical plates Tailing factor Resolution
1 4.457 3515.269 3625 2.097 5.940
2 4.443 3513.118 3614 2.129 6.248
3 4.423 3484.753 3688 1.969 6.021
4 4.413 3501.992 3893 2.032 6.263
5 4.387 3453.219 3614 2.129 6.248
6 4.307 3483.064 3646 2.065 6.038
Mean 4.405 3491.903 - - -
SD 0.054 23.339 - - -
%RSD 1.22 0.67 - - -

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Observation
The % RSD for the retention times and peak
area of Sulbactam and Ceftriaxone were found to
be less than 2%. The plate count and tailing factor
results were found to be satisfactory and are found
to be within the limit.
Specificity by Direct comparison method
Preparation of samples for Assay
Standard sample
100 µg/ml of Ceftriaxone and 50 µg/ml of
Sulbactam solution was prepared. This solution is
used for recording chromatogram.
Tablet sample
Test stock solutions of Sulbactam (50μg/ml)
and Ceftriaxone (100μg/ml) were prepared by
dissolving weight equivalent to 500 mg of
Sulbactam and 1000 mg of Ceftriaxone and
dissolved in sufficient mobile phase.. Further
dilutions are prepared in 5 replicates of 50μg/ml of
Sulbactam and 100 μg/ml of Ceftriaxone was made
by adding 1 ml of stock solution to 10 ml of mobile
phase.
Fig.15: Blank chromatogram for specificity by using mobile phase
Fig. 16: Chromatogram for specificity of Sulbactam and Ceftriaxone standard.
Fig. 17: Chromatogram for Specificity of Sulbactam and Ceftriaxone sample

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Observation
It is observed from the above data, diluent or
excipient peaks are not interfering with the
Sulbactam and Ceftriaxone peaks.
Linearity and range
Preparation of mixed standard stock solution
100 µg/ml of Ceftriaxone and 50 µg/ml of
Sulbactam solution was prepared. This solution is
used for recording chromatogram. This solution is
used for recording chromatogram and further
dilutions were given in the table No 5.
Table 5: Linearity Preparations
Preparations
Volume from standard
stock transferred in ml
Volume made up in ml
(with mobile phase)
Concentration of solution(µg /ml)
SULBACTAM CEFTRIAXONE
Preparation 1 0.6 10 30 60
Preparation 2 0.8 10 40 80
Preparation 3 1.0 10 50 100
Preparation 4 1.2 10 60 120
Preparation 5 1.4 10 70 140
Fig. 18: Chromatogram of Sulbactam and
Ceftriaxonepreparation-1
Fig. 19: Chromatogram of Sulbactam and
Ceftriaxonepreparation-2
Fig. 20: Chromatogram of Sulbactam and
Ceftriaxone preparation-3
Fig.21: Chromatogram of Sulbactam and
Ceftriaxonepreparation-4

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Fig. 22: Chromatogram of Sulbactam and Ceftriaxone for preparation-5
Table 6: linearity of Sulbactam AND Ceftriaxone
S.No. Conc.(µg/ml ) Area
1 30 211.018
2 40 269.576
3 50 329.564
4 60 378.706
5 70 436.719
S.No. Conc.(µg/ml ) Area
1 60 2124.986
2 80 2866.660
3 100 3508.071
4 120 4106.525
5 140 4872.434
Fig. 23: Linearity graph of Sulbactam Fig. 24: Linearity graph of Ceftriaxone
Observation
The correlation coefficient for linear curve
obtained between concentration vs. Area for
standard preparations of Sulbactam and Ceftriaxone
is 0.9998 and 0.9983. The relationship between the
concentration of Sulbactam and Ceftriaxone and
area of Sulbactam and Ceftriaxone is linear in the
range examined since all points lie in a straight line
and the correlation coefficient is well within limits.
ACCURACY
Accuracy of the method was determined by Recovery
studies. To the formulation (pre analyzed sample), the
reference standards of the drugs were added at the
level of 80%, 100%, 120%. The recovery studies were
carried out three times and the percentage recovery
and percentage mean recovery were calculated for
drug is shown in table. To check the accuracy of the
method, recovery studies were carried out by addition
of standard drug solution to pre-analyzed sample
solution at three different levels 80%, 100%,120%.
Linearity of Ceftriaxone
y = 33.674x + 128.35
R2
= 0.9983
0
1000
2000
3000
4000
5000
6000
0 50 100 150
Conc
Area
y = 27.905x + 3.7902
R² = 0.9998
0
100
200
300
400
500
600
0 5 10 15 20
Area
Conc
Linearity of Sulbactam

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Fig. 25: Chromatogram of 80% recovery (injection 1)
Fig. 26: Chromatogram of 80% recovery (injection 2)
Fig. 27: Chromatogram of 80% recovery (injection 3)
Fig. 28: Chromatogram of 100% recovery (injection 1)

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Fig. 29: Chromatogram of 100% recovery(injection 2)
Fig. 30: Chromatogram of 100% recovery (injection 3)
Fig. 31: Chromatogram of 120% recovery(injection 1)
Fig. 32: Chromatogram of 120% recovery (injection 2)

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Fig. 33: Chromatogram of 120% recovery(injection 3)
Acceptance criteria
The % recovery of Sulbactam and Ceftriaxone should lie between 98% and 102%.
Table 7 : Recovery results for Sulbactam
Table 8 : Recovery results for Ceftriaxone
Recovery
level
Accuracy SULBACTAM Average%
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 50 338.031 335.910 49.83 99.65
99.30%
50 341.651
50 328.047
100% 60 392.018 388.703 58.97 98.29
60 397.207
60 376.884
120% 70 435.176 441.674 69.98 99.97
70 445.499
70 444.347
Recovery
level
Accuracy CEFTRIAXONE Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 100 3664.032 3628.807 101.27 101.27
101.48%
100 3636.35
100 3586.04
100% 120 4437.275 4260.968 121.46 101.22
120 4286.931
120 4058.699
120% 140 4857.176 4885.320 142.76 101.97
140 4913.236
140 4885.547

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Observation
The percentagemean recovery of Sulbactam and
Ceftriaxone is99.30% and 101.48% respectively.
PRECISION
Method precision
Prepared sample preparations of Sulbactam and
Ceftriaxone as per test method and injected 6 times in
to the column.
Acceptance criteria
The % Relative standard deviation of Assay
preparations of sulbactam and Ceftriaxone should be
not more than 2.0%.
Fig. 34: Chromatogram of precision injection 1
Fig. 35: Chromatogram of precision injection 2
Fig. 36: Chromatogram of precision injection 3

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Fig. 37: Chromatogram of precision injection 4
Fig. 38: Chromatogram of precision injection 5
Fig. 39: Chromatogram of precision injection 6
Table 9: Results for Method precision of Sulbactam and Ceftriaxone.
SULBACTAM CEFTRIAXONE
S.No. Rt Area S.No. Rt Area
1 2.983 358.060 1 4.457 3515.269
2 2.967 360.049 2 4.443 3513.118
3 2.943 354.753 3 4.423 3484.753
4 2.947 355.992 4 4.413 3501.992
5 2.953 359.264 5 4.387 3453.219
6 2.947 349.258 6 4.307 3483.064
Avg 2.9567 356.229 Avg 4.405 3491.903
Stdev 0.0154 3.949 Stdev 0.054 23.339
%RSD 0.52 1.11 %RSD 1.22 0.67

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Observation
Test results for Sulbactam and Ceftriaxone are
showing that the %RSD of Assay results are within
limits. The results were shown in table Table9
Limit of Detection
Where, σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte.
Fig. 40: Calibration graphs of Sulbactam and Ceftriaxone
Table No 10.: Results for calibration graph
S.No. SULBACTAM CEFTRIAXONE
Concentration µg/ml Peak Area Concentration µg/ml Peak Area
1 30 211.018 60 2124.986
2 40 269.576 80 2866.660
3 50 329.564 100 3508.071
4 60 378.706 120 4106.525
5 70 436.719 140 4872.434
S.D. 4.0 110 31.623 1090
Slope 27.9 34.44
Observation
The LOD for this method was found to be
0.47µg/ml & area 13.05for SULBACTAM and 3.03
µg/ml & area 104.49 for CEFTRIAXONE.
Limit of Quantification
Where,
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte. The LOQ for this method was
found to be 1.42 µg/ml & area 39.54 for
SULBACTAMand 9.18 µg/ml & area 316.63 for
CEFTRIAXONE.
ROBUSTNESS
Chromatographic conditions variation
To demonstrate the robustness of the method,
prepared solution as per test method and injected at
different variable conditions like using different
conditions like Temperature and wavelength.

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System suitability parameters were compared with
that of method precision.
Acceptance criteria
The system suitability should pass as per the
test method at variable conditions.
Fig. 41: Chromatogram of Sulbactam and Ceftriaxone Robustness (Flow:0.8 ml/min)
Fig. 42: Chromatogram of sulbactam and Ceftriaxone robustness (Flow: 1.2 ml/min)
Fig. 43: Chromatogram of Sulbactam and Ceftriaxone for Robustness (208nm)

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Fig. 44: Chromatogram of Sulbactam and Ceftriaxone for Robustness (212nm)
Table 11: Result of Robustness study
Parameter
SULBACTAM CEFTRIAXONE
Retention time(min) Tailing factor Retention time(min) Tailing factor
Flow
0.8ml/min
1.0ml/min
1.2ml/min
4.110
2.833
3.203
1.579
1.462
1.714
6.293
4.263
3.203
2.087
2.097
1.714
Wavelength
208nm
210nm
212nm
2.803
2.833
2.793
1.444
1.462
1.481
4.257
4.263
4.253
1.800
2.097
1.882
Observation
From the observation it was found that the
system suitability parameters were within limit at
all variable conditions.
Ruggedness
The ruggedness of the method was studied by
the determining the analyst to analyst variation by
performing the Assay by two different analysts
Acceptance criteria
The % Relative standard deviation of Assay values
between two analysts should be not more than 2.0%.
Fig. 45: Chromatogram of Analyst 01 standard preparation

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Fig. 46: Chromatogram of Analyst 01 sample preparation
Fig. 47: Chromatogram of Analyst 02 standard preparation
Fig. 48: Chromatogram of Analyst 02 sample preparation
Table 12: Results for Ruggedness
SULBACTAM %Assay CEFTRIAXONE %Assay
Analyst 01 97.42 Analyst 01 99.48
Analyst 02 101.41 Analyst 02 100.73
%RSD 0.72% %RSD 0.78%

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Observation
From the observation the %RSD between two
analysts Assay values not greater than 2.0%, hence
the method was rugged.
CONCLUSION
From the above experimental results and
parameters it was concluded that, this newly
developed method for the simultaneous estimation of
SULBACTAM and CEFTRIAXONE was found to be
simple, precise, accurate and high resolution and
shorter retention time makes this method more
acceptable and cost effective and it can be effectively
applied for routine analysis in research institutions,
quality control department in meant in industries,
approved testing laboratories, bio-pharmaceutical and
bio-equivalence studies and in clinical
pharmacokinetic studies in near future.
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http://wolfson.huji.ac.il/purification/PDF/IonExchange/AMERSHAM_iIEXandChromatofocManual.pdf