Microsoft word - reddy et al pdf.doc
Research In Pharmaceutical Biotechnology Vol. 2(1), pp. 001-006, February, 2010
Available online at http://www.academicjournals.org/RPB
Validation and stability indicating RP-HPLC method for
the determination of tadalafil API in pharmaceutical
B. Prasanna Reddy1*, K. Amarnadh Reddy2 and M. S. Reddy3
1Department of Quality control, Nosch Labs Pvt Ltd, Hyderabad-500072, A.P, India.
2 Department of AR and D, Aurigene Discovery Technologies Ltd, Bangalore, India.
3Department of Plant Pathology and Entomology, Auburn University, USA.
The present study describes the development and subsequent of a stability indicating RP-HPLC method
for the analysis of tadalafil. The samples separated on an Inertsil C18, (5
µ , 150 mm x 4.6 mm i.d) by
isocratic run using acetonitrile and phosphate buffer as mobile phase), with a flow rate of 0.8 ml/min,
and the determination wavelength was 260 nm for analysis of tadalafil. The described method was
linear within range of 70 - 130 µg/ml (r2 = 0.999). The precision, ruggedness and robustness values were
also within the prescribed limits (< 1% for system precision and < 2% for other parameters). Tadalafil
was exposed to acidic, basic, oxidative and thermal stress conditions and the stressed samples were
analyzed by the proposed method. Chromatographic peak purity results indicated the absence of co-
eluting peaks with the main peak of tadalafil, which demonstrated the specificity of assay method for
estimation of tadalafil in presence of degradation products. The proposed method can be used for
routine analysis of tadalafil in quality control laboratories.
RP-HPLC, tadalafil, validation, stability indicating assay, forced degradation.
1), is a phosphodiesterase type 5 inhibitor used in the
Chemical and reagents
management of erectile dysfunction. It is not official y
included in any of the pharmacopoeias. It is listed in the
Tadalafil standard and API were provided from Smilax laboratories
Merck Index (Budavari et al., 2001) and Martindle and
limited. HPLC grade sodium dihydrogen phosphate (NaH2PO4),
complete drug reference (Sean et al., 2002). There are
disodium hydrogen phosphate (Na2HPO4), acetonitrile, hydrogen
peroxide and sodium hydroxide were procured from Merck Ltd.
several (Cheng et al., 2005) methods for determination of
High pure water was prepared by using Mil ipore Mil i Q plus
tadalafil such as HPLC-EIMS (Zhu et al., 2005) and
capil ary electrophoresis methods (Aboul-Enein, 2005)
and by HPLC (Aboul, 1994). The present work was
designed to develop a simple, precise and rapid
HPLC instrumentation and conditions
analytical LC procedure, which would serve as stability
indicating assay method for analysis of tadalafil active
A high performance liquid chromatograph system, with LC solutions
data handling system, with an auto sampler was used for the
analysis. The data was recorded using LC 2010 solutions software.
The samples separation was performed on a Shimadzu Inertsil C
(5 µ, 150 mm x 4.6 mm, Japan) with the mobile phase consisting of
acetonitrile and phosphate buffer (pH 7.0) with a ratio of 60: 40 (v/v)
*Corresponding author. E-mail: firstname.lastname@example.org. Tel:
at ambient temperature. The flow rate was kept at 0.8 ml/min and
the determination wavelength was 262 nm.
Structure of Tadalafil.
Standard and sample preparation
Robustness of method was investigated by varying the
chromatographic conditions such as change of flow rate (± 10%),
organic content in mobile phase (± 2%), wavelength of detection (±
Mix 700 ml of acetonitrile to the buffer, the mobile phase was
5%) and pH of buffer in mobile phase (± 0.2%). Robustness of the
sonicated for 15 min and then it was filtered through 0.45 µm
developed method was indicated by the overal %RSD between the
Limit of detection (LOD)
The standard was dissolved with mobile phase to 0.5 mg/ml. The
The detection limit is determined by the analysis of samples with
test samples were dissolved with mobile phase. With the optimized
the known concentrations of analyte and by establishing the
chromatographic conditions, a steady baseline was recorded, the
minimum level at which the analyte can be reliably detected. The
standard solution was injected and the chromatogram was
recorded. This procedure was repeated for the sample solution.
Forced degradation studies
Limit of quantitation (LOQ)
Tadalafil was al owed to hydrolyze in different strengths of base
The quantitation limit is determined by the analysis of sample of
(0.005 N and 0.05 N NaOH), acid (0.05 N, 0.5 N and 1 N HCl) and
known concentration of analyte and by establishing the minimum
hydrogen peroxide (30%, 10, 3 and 1%). Tadalafil was also studied
level at which the analyte can be quantified with acceptable
for its thermal degradated at 80, 100, 120°C for 1 h respectively. An
accurately weighed 50 mg of tadalafil API was dissolved in 1 ml of
respective base (NaOH), acid (HCl) or hydrogen peroxide and kept
for specified period after which the volume was made up to 50 ml
with water: acetonitrile (70:30, v/v). Five mil iliters of the above
solution were diluted with water: acetonitrile (70:30, v/v) to get a
where S: Slope of the calibration curve, : Average standard
concentration of 100 ppm. Blank was also treated in same way.
Linearity was determined by injecting different concentration of
sample solutions (50 - 150 µg/ml). For system precision, standard
solution (100 µg) was injected to six replicates injections to check
%RSD (relative standard deviation) and for method precision six
time samples were prepared and each of those were injected in
In order to separate tadalafil API, phosphate buffer-
duplicate. Mean of al of these values gives to assay value.
To establish the within-day (intra-assay) and between-day (inter-
acetonitrile mixtures were used as the mobile phase.
assay) accuracy and precision of the method, tadalafil was assayed
Satisfactory resolution was obtained using the mobile
on one day and three separate days. Intra-assay and inter-assay
phase system of acetonitrile/phosphate buffer (70:30, v/v,
pH 7.0) at a flow rate of 0.8 ml/min with UV detection at
Chromatogram of Tadalafil API.
Results of force degradation studies of Tadalafil.
Alkaline degradation (0.005 N NaOH, 1 hr)
Alkaline degradation (0.05 N NaOH, 2 hr)
Oxidative degradation (30 % H2O2, 80°C for 10 min)
Thermal degradation (Solid sample, 80°C, 1 hr)
Thermal degradation (Solid sample, 100°C, 1 hr)
Thermal degradation (Solid sample, 120°C, 1 hr)
260 nm. Under these conditions tadalafil with the
stable under the applied stress conditions like heat, acid
retention time of tadalafil was 2.88 min. Figure 2 showed
and alkaline and oxidative degradation states.
a typical chromatogram obtained under these conditions
Forced degradation studies
The calibration curve showed good linearity in the range
of 50 - 150 µg/ml, for tadalafil API with correlation co-
During the study it was observed that upon treatment of
efficient (R2) of 0.9998 (Figure 4). A typical calibration
tadalafil with different strengths of base (0.005 N and
curve has the regression equation of y = 23646x
0.05 N NaOH), acid (0.05 N, 0.5 N and 1 N HCl) and
hydrogen peroxide (30, 10, 3 and 1%) the degradation
was observed only with the higher strengths (0.05 N
NaOH), acid (1 N HCl) and hydrogen peroxide (30%),
where as with the lower strengths of alkali (0.005 N
NaOH), acid (0.05 N and 0.05 N HCl) and hydrogen
The results of system precision (% RSD = 0.26), method
peroxide (1, 3 and 10%) no degradation was observed
precision (% RSD = 0.10) are found within the prescribed
(Table 1).Further it is important to note that from the
limit of ICH guidelines (% RSD < 1%, and % RSD < 2 %
chromatograms (Figure 3a to c), it is evident that
respectively in case of system precision and method
although the degraded peaks are observed. The tadalafil
Chromatograms of (a) Base hydrolyzed-degraded sample (b) Thermal degraded sample and (c) Acid hydrolyzed
Intra-assay and Inter-assay
The intra and inter-day variation of the method was
Influence of smal changes in chromatographic conditions
carried out and the high values of mean assay and low
such as change in flow rate (± 10%), organic content in
values of standard deviation and %RSD (%RSD < 2%)
mobile phase (± 2%), wavelength of detection (± 5%) and
within a day and day to day variations for tadalafil
pH of buffer in mobile phase (± 0.2%) studied to
revealed that the proposed method is precise in (Table 2
determine the robustness of the method are also in favor
(Table 4, %RSD < 2%) of the developed RP-HPLC
Linearity curve for Tadalafil.
Intra-assay precision data of proposed RP-HPLC method
Mean (% w/w)
Inter-assay precision data of proposed RP-HPLC method.
Mean (% w/w)
The influence of changes in chromatographic
parameters on RP-HPLC analysis of Tadalafil API
Change in parameter
method for the analysis of tadalafil API.
Aboul-Enein HY, Ali I (2005). Talanta 65(1): 276-280.
Aboul-Enein HY (1994). Chromatographia 60(3-4): 187-191.
LOD and LOQ
Abdul-Fattah AM, Bhargava HN (2002). A new high performance liquid
Chromatography (HPLC) method for the analysis of halofantrine (HF)
The minimum concentration level at which the analyte
in Pharmaceuticals. J. Pharm. Biomed. Anal.
can be reliably detected (LOD) and quantified (LOQ)
Avarez-Lueje A, Pujol S, Squel a JA, Nunez Vergara LJ (2003). A
Selective HPLC method for determination of lercanidipine in tablets.
were found to be 0.05 and 0.5 µg/ml, respectively.
Bebawy L (2003). I. Spectrophotometric and HPLC determination of
linezolid in the Presence of its alkaline-induced degradation products
Specificity and stability in analytical solution
and in pharmaceutical Tablets. Anal. Lett. 36: 1147-1161.
Budavari S, Eds (2001): In, The Merck Index, 13th Edn, Merck & Co.,
The results of specificity indicated that the peak was pure
Cheng CL, Chou CH (2005). J. Chromatogr. B Analyt. Technol. Biomed.
in presence of degraded sample. It is important to
mention here that the tadalafil API was stable in solution
ICH (1996). Validation of Analytical Procedures: Methodology, in:
Proceeding of the International Conference on Harmonization,
The results of linearity, precision, inter and intra-
ICH (2002).Guideline on Analytical method Validation, in: Proceeding
assays, method robustness, LOD, LOQ and specificity
of International Convention on quality for the pharmaceutical industry,
and stability in analytical solution established the
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validation of the developed RP-HPLC assay for the
Proceeding of the International conference on Harmonization,
Lambropouls J, Spanos GA, Lozaridis (1999). V.N. Method
development and Validation for the HPLC assay (potency and
related substances) for 20 mg paroxetine tablets. J. Pharm. Biomed.
Sean C, Sweetman Eds. (2002). In., Martindale, The Complete Drug
The present study is the first time report on stability
Reference, 34th Edn., The Pharmaceutical Press: London p. 875.
indicating assay of tadalafil in presence of degradation
The United States Pharmacopoeia” 26th ed. (2003). US Pharmacopoeia
products by HPLC. In this method isocratic elution
Zhu X, Xiao S, Chen B, Zhang F, Yao S, Wan Z, Yang D, Han H (2005).
method is selected for the analysis of tadalafil API
J. Chromatogr A., eb25;1066(1-2): 89-95.
because it gave better base line separation and peak
width, which is suitable for the routine analysis of
tadalafil. The developed method was validated as per
ICH guidelines (ICH, 1996) and its updated international
Stability testing forms an important part of the process
of drug product development. The purpose of stability
testing is to provide evidence on how the quality of a drug
substance varies with time under the influence of a
variety of environmental factors such as temperature,
humidity and light, and enables recommendation of
storage conditions, retest periods, and shelf life to be
established (Avarez-Lueje et al., 2003; Abdul-Fattah et
al., 2002; Lambropouls et al., 1999; Bebawy, 2003). The
assay of tadalafil API (Active Pharmaceutical Ingredient)
in stability test sample needs to be determined using
stability indicating method, as recommended by the
International Conference on Harmonization (ICH)
Below you find a summary of my findings on calculations with dimensional decibels. Although this should be quite straightforward and commonly known, I was not ableto find a concise calculation rule anywhere on the web. As such, I decided to try andwrite one myself. At the end, in ‘Examples’, you find my consequent opinion on a measure for phasenoise, the carrier-to-noise-density-ratio. If y
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