New RP HPLC method for the simultaneous estimation of terbutaline and theophylline in pharmaceutical dosage form

Ashok K A et al / Journal of Pharmacreations Vol-3(1) 2016 [40-63]
40
Pharmacreations | Vol.3 | Issue 1 | Jan- Mar- 2016
Journal Home page: www.pharmacreations.com
Research article Open Access
New RP HPLC method for the simultaneous estimation of terbutaline
and theophylline 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 Terbutaline and Theophylline dosage forms.
Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 20Mm
Phosphate buffer (KH2PO4) pH: 3.5 Acetonitrile (80:20v/v/v), with detection of 250 nm. Linearity was observed in
the range 1.25-3.75 µg /ml for Terbutaline (r2
=0.9975) and 50-150/ml for Theophylline (r2
=0.9994) 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.
Key words: Phosphate buffer (KH2PO4) pH: 3.5 Acetonitrile (80:20v/v/v), Terbutaline and Theophylline
INTRODUCTION
High Performance Liquid Chromatography is
the most widely used of all the analytical
separation techniques. The reasons for its
popularity are its sensitivity, ready adaptability to
quantitative determination, suitable for non-
volatile and thermally fragile species, wide
applicability to variety of substances such as
amino acids, carbohydrates, nucleic acids,
proteins, hydrocarbons, terpenoids, pesticides,
steroids, metal-organic species and inorganic
species. As high pressures (around 3000 psi) are
used for the separation of the analytes down the
column, it is often termed as High Pressure Liquid
Chromatography. 4, 5, 6
Types of HPLC
HPLC is classified into various types
Based on polarity of stationary and mobile
phase
Normal Phase Chromatography
Reverse Phase Chromatography
Based on the principle of separation
Adsorption Chromatography
Partition Chromatography
Ion Pair Chromatography
Size Exclusion Chromatography
Chiral Phase Chromatography
Journal of Pharmacreations

41
Based on elution technique
Isocratic Elution
Gradient Elution
Based on scale of operation
Analytical HPLC
Preparative HPLC
Based on the polarity of the stationary phase and
the mobile phase, it is of two types:
Normal Phase (NP) HPLC
In this type, the stationary phase is polar and
the mobile phase is non-polar, polar compounds
are retained for a longer periods because of more
affinity towards the stationary phase, hence non-
polar compounds travel faster and are eluted first.
Reverse Phase (RP) HPLC
In this type, the stationary phase is non-polar
and the mobile phase is polar, non-polar
compounds are retained for longer periods as they
have more affinity towards the stationary phase.
Hence, polar compounds travel faster and are
eluted first.3, 4,5,6
AIM AND PLAN OF WORK
Aim
To develop new RP HPLC method for the
simultaneous estimation of TERBUTALINE &
THEOPHYLLINE in pharmaceutical dosage form.
Plan of work
 Solubility determination of Terbutaline &
Theophylline in various solvents and buffers.
 Determine the absorption maxima of the drug in
UV–Visible region in different solvents/buffers
and selecting the solvents for HPLC method
development.
 Optimize the mobile phase and flow rates for
proper resolution and retention times.
 Validate the developed method as per ICH
guidelines.
METHODOLOGY
Mobile Phase
A mixture of 80 volumes of Phosphate buffer
pH 3.5:20volumes of Acetonitrile. The mobile
phase was sonicated for 10min to remove 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
TERBUTALINE
50 mg of Terbutaline was weighed and
transferred in to 500ml 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
ofTHEOPHYLLINE
50mg of Theophylline was weighed in to 500ml
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
Terbutaline
Soluble in methanol, sparingly soluble in DMSO,
insoluble in Water,.
Theophylline
Freely Soluble in Methanol. Slightly Soluble in
Water and DMF.
Wavelength determination
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 241 nm for Terbutaline and
Theophylline 278 and 250 nm for the combination.

42
Fig. 1: UV-VIS spectrum of terbutaline
Obeservation
λmax was found to be 241 nm for Terbutaline shown in the figure 1
Fig. 2: UV-VIS spectrum of Theophylline
Observation
λmax was found to be 278 nm for Theophylline shown in the figure 2
Fig. 3: UV-VIS spectrum of Terbutaline and Theophylline and the isosbestic point was 250 nm

43
Observation
The Isosbestic point was found to be 250nm for
Terbutaline and Theophylline in combination and
was shown in figure 3
METHOD DEVELOPMENT OF
TERBUTALINE & THEOPHYLLINE
Trial- 4
Preparation of mixed standard solution
weigh accurately 2.5mg of Terbutaline and 100
mg of Theophylline in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase From above stock
solution 2.5 µg/ml of Terbutaline and 100 µg/ml of
Theophylline is prepared by diluting 1ml to 10ml
with mobile phase. This solution is used for
recording chromatogram.
Fig. 4: Chromatogram of terbutaline and theophylline by using mobile phase.

44
Observation
The peaks showed more efficiency and more resolution. Hence this method was optimised.
Table 1: Optimized chromatographic conditions
Mobile phase Phosphate buffer (KH2PO4) pH: 3.5 Acetonitrile (80:20v/v/v),
pH 3
Column INERTSIL column,C18(150x4.6 ID) 5µm
Flow rate 1.0 ml/min
Column temperature Room temperature(20-25o
C)
Sample temperature Room temperature(20-25o
C)
Wavelength 250nm
Injection volume 20 µl
Run time 10 min
Retention time About 2.337 min for Terbutaline and 4.028min for Theophylline
ASSAY
Preparation of samples for Assay
Weigh accurately 2.5mg of Terbutaline and 100
mg of Theophylline in 100 ml of volumetric flask
and dissolve in 10ml of mobile phase and make up
the volume with mobile phase From above stock
solution 2.5 µg/ml of Terbutaline and 100 µg/ml of
Theophylline is prepared by diluting 1ml to 10ml
with mobile phase. This solution is used for
recording chromatogram.
Preparation of sample solution
5tablets (each tablet contains 2.5mg of
Terbutaline and 100mg of Theophylline) were
weighed and taken into a mortar and crushed to
fine powder and uniformly mixed. Tablet stock
solutions of Terbutaline (25μg/ml) and
Theophylline (1000μg/ml) were prepared by
dissolving weight equivalent to 2.5mg of
Terbutaline and 100 mg of Theophylline 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 2.5μg/ml of Terbutaline and 100
μg/ml of Theophylline was made by adding 1ml of
stock solution to 10 ml of mobile phase.
Fig. 5: Chromatogram of Assay standard preparation-1

45

46
Fig. 10: Chromatogram of Assay sample preparation-1

47
Table No.2: Assay Results
TERBUTALINE THEOPHYLLINE
Standard Area Sample Area Standard Area Sample Area
Injection-1 187.838 179.164 5678.797 5541.595

48
Injection-2 175.570 175.851 5436.955 5508.623
Injection-3 176.601 184.101 5494.221 5523.567
Injection-4 179.819 180.499 5575.961 5406.592
Injection-5 179.681 185.581 5483.936 5614.223
Average Area 179.902 181.039 5533.974 5518.92
Tablet average weight 150.75mg 150.75mg
Standard weight 2.5mg 100mg
Sample weight 150.75mg 150.75mg
Label amount 2.5mg 100mg
std. purity 99.2 99.3
Amount found in mg 2.495 99.03
Assay(%purity) 99.60 99.03
Observation
The amount of Terbutaline and Theophylline
present in the taken dosage form was found to be
99.60 and 99.03% respectively.
VALIDATIONS
Specificity by Direct comparison method
There is no interference of mobile phase, solvent
and placebo with the analyte peak and also the peak
purity of analyte peak which indicate that the method
is specific for the analysis of analytes in their dosage
form.
Preparation of samples for Assay
2.5 µg/ml of Terbutaline and 100 µg/ml of
Theophylline solution is prepared with mobile phase.
This solution is used for recording chromatogram.
Preparation of sample solution
5tablets (each tablet contains 2.5mg of Terbutaline
and 100mg of Theophylline) were weighed and
taken into a mortar and crushed to fine powder and
uniformly mixed. Tablet stock solutions of
Terbutaline (25μg/ml) and Theophylline (1000μg/ml)
were prepared by dissolving weight equivalent to
2.5mg of Terbutaline and 100 mg of Theophylline
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 2.5μg/ml of Terbutaline and 100 μg/ml
of Theophylline was made by adding 1ml of stock
solution to 10 ml of mobile phase.
Fig. 15: Blank chromatogram for specificity by using mobile phase

49
Fig. 16: Chromatogram for specificity of Terbutaline & Theophylline sample
Fig. 17: Chromatogram for Specificity of Terbutaline & Theophylline standard
Observation
It is observed from the above data, diluent or
excipient peaks are not interfering with the
Terbutaline & Theophylline peaks.
Linearity and range
Weigh accurately 2.5 mg of Terbutaline and
100 mg of Theophylline in 100 ml of volumetric
flask and dissolve in 10ml of mobile phase and
make up the volume with mobile phase. Further
take 1ml into 10ml volumetric flask and make upto
10ml with mobile phase.
Table 3: Linearity Preparations
Preparations
Volume from standard stock
transferred in ml
Volume made up in ml
(with mobile phase)
Concentration of solution(µg
/ml)
Terbutaline Theophylline

50
Fig. 18: Chromatogram of Terbutaline and Theophylline preparation-1
Preparation 1 0.0125 0.5 1.25 50
Preparation 2 0.0185 0.75 1.85 75
Preparation 3 0.025 1 2.5 100
Preparation 4 0.0315 1.25 3.15 125
Preparation 5 0.0375 1.5 3.75 150

51
Fig.21: Chromatogram of Terbutaline and Theophylline preparation-4
Fig. 22: Chromatogram of Terbutaline and Theophylline for preparation-5
Table 4: linearity of Terbutaline
S.No. Conc.(µg/ml ) Area
1 1.25 78.029
2 1.85 122.306
3 2.5 154.766
4 3.15 190.241
5 3.75 218.291
Table 9.3.8: linearity of THIOCOLCHICOSIDE
S.No. Conc.(µg/ml ) Area
1 50 2790.728
2 75 3866.934
3 100 5285.723
4 125 6121.454
5 150 6361.533

52
Fig. 23: Linearity graph of Terbutaline And Theophylline
Observation
The correlation coefficient for linear curve
obtained between concentration vs. Area for
standard preparations of Terbutaline and
Theophylline is 0.998 and 0.999. The relationship
between the concentration of Terbutaline and
Theophylline and area of Terbutaline and
Theophylline 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 75%, 100%, 125%. 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
75%, 100% & 125%.
Fig. 24: Chromatogram of 75%recovery (injection 1)
y = 27.805x + 20.597
R² = 0.9994
0
1000
2000
3000
4000
5000
6000
7000
8000
0 50 100 150 200
Area
Conc
Linearity of Theophylline
y = 55.753x + 13.343
R² = 0.994
0
50
100
150
200
250
0 1 2 3 4
Area
Conc
Linearity of Terbutaline

53
Fig. 25: Chromatogram of 75% recovery (injection 2)

54
Fig.29: Chromatogram of 100% recovery (injection 3)
Fig. 30: Chromatogram of 125% recovery(injection 1)

55
Acceptance criteria
The % recovery of Terbutaline and Theophylline should lie between 98% and 102%.
Table 5 : Recovery results for Terbutaline
Recovery
level
Accuracy Terbutaline Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
75% 1.87 92.288
95.500 1.86 99.01
99.49%
1.875 95.414
1.875 98.797
100% 2.5 166.024
166.230 2.47 98.982.5 165.944
2.5 166.723
125% 3.125 190.241

56
3.125 197.059 195.503 3.14 100.48
3.125 199.208
Table 6 : Recovery results for Theophylline
Observation
The percentage mean recovery of Terbutaline
and Theophylline is 99.49% and 99.12%
respectively.
PRECISION
Method precision
Prepared sample preparations of Terbutaline and
Theophylline as per test method and injected 6 times
in to the column.
Acceptance criteria
The % Relative standard deviation of Assay
preparations of Terbutaline and Theophylline should
be not more than 2.0%.
Fig. 33: Chromatogram of precision injection 1
Recovery
level
Accuracy Theophylline Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
75% 75 3190.245
3198.010 73.90 98.54
99.12%
75 3270.81
75 3132.976
100% 100 5293.994
5042.760 98.52 98.52100 4982.116
100 4852.17
125% 125
125
5941.213
5944.656 5944.582 125.40 100.32
125 5947.876

57

58
Table 7: Results for Method precision of Terbutaline and Theophylline
S.No. Rt Area S.No. Rt Area
1 1.923 182.723 1 3.423 5585.32
2 1.933 178.936 2 3.417 5418.603
3 1.897 178.962 3 3.353 5438.747
4 1.907 180.307 4 3.393 5499.970
5 1.943 181.926 5 3.457 5516.145
6 1.990 188.635 6 3.550 5645.393
avg 1.9322 181.915 avg 3.432 5517.363
stdev 0.0329 3.633 stdev 0.067 86.311
%RSD 1.70 2.00 %RSD 1.96 1.56

59
Observation
Test results for Terbutaline and Theophylline are
showing that the %RSD of Assay results are within
limits. The results were shown in table Table 7.
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.
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. 39: Chromatogram of Terbutaline and Theophylline Robustness (0.8 ml/min)
Fig. 40: Chromatogram of Terbutaline and Theophylline for Robustness (1.2 ml/min)

60
Fig. 41: Chromatogram of Terbutaline and Theophylline for Robustness (249nm)
Fig. 42: Chromatogram of Terbutaline and Theophylline for Robustness (250nm)
Fig. 43: Chromatogram of Terbutaline and Theophylline for Robustness (251nm

61
Table 8: Result of Robustness study
Parameter
Retention time(min) Tailing factor Retention time(min) Tailing factor
Flow
0.8ml/min
1.0 ml/min
1.2ml/min
2.817
2.32
2.022
1.73
1.732
1.72
4.860
4.035
3.487
1.585
1.586
1.57
Wavelength
249nm
250nm
251nm
2.367
2.320
2.367
1.722
1.732
1.741
4.080
4.035
4.082
1.571
1.586
1.585
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. 44: Chromatogram of Analyst 01 standard preparation

62
Fig. 45: Chromatogram of Analyst 01 sample preparation
Fig. 46: Chromatogram of Analyst 02 standard preparation
Fig. 47: Chromatogram of Analyst 02 sample preparation

63
Table 9: Results for Ruggedness
Terbutaline %Assay Theophylline %Assay
Analyst 01 97.99 Analyst 01 99.96
Analyst 02 98.37 Analyst 02 97.59
%RSD 0.27 %RSD 1.69
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 Terbutaline and Theophylline 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
industries, approved testing laboratories, bio-
pharmaceutical and bio-equivalence studies and in
clinical pharmacokinetic studies in near future.
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