Analytical Method Development and Stability Studies of Carvedilol

Master's Thesis, 2011

78 Pages, Grade: 8.0




2.1 Literature review

3.1Scope and objectives
3.2Plan of work

4.1UV-VIS method development
4.2RP-HPLC method development
4.3Forced degradation Studies

5.1UV-VIS Analysis
5.2RP-HPLC Analysis
5.3Forced degradation studies


7.1 References.

1. Introduction

1.1 Spectrophotometric methods

Spectrophotometric method are a large group of analytical methods that are based on atomic and molecular spectroscopy i.e. the integration between electromagnetic radiation and matter when electromagnetic radiation passes through a layer of analyte certain frequencies may be selectively removed by absorption, a process in which electromagnetic energy is transferred to the atom, ion or molecules composing the sample. Absorption promotes these particles from their normal ground state, to one or higher exited states.

The absorption of light by analytes by raising an electron or electrons to a higher level and other functional group. Every functional group in a molecule of substances is characterized by the absorption of light in a definite region of the spectra and this property is used for the identification of the substances in a drug. In addition to chromospheres, a molecule may contain one or more functional groups that themselves do not absorb in visible region being scanned , but can affect the behavior of the chromospheres that are conjugated with this group called auxochromes (eg. SH, NH2, OH) usually causes absorption by a chromophores at higher wavelength and at a higher value of absorptive then feature in the given chromophores. [1]

Importance of visible spectrophotometry in Pharmaceutical Analysis

Spectrophotometry is generally preferred especially by small scale industries as the cost of the equipment is less and the maintain problems are minimal. The method of analysis is based on measuring the absorption of monochromatic light by colored compound in the visible path of the spectrum .if the analytes are colorless compound they are converted into colored compounds by reaction with suitable compounds .in case majority of the compound are complex and complex legends. The later must be stable and have a constant composition and high color intensity. The photometric methods of analysis are based on the Bouger-Lambert-Beer’s law, which establishes the absorbance of a solution is directly proportional to the concentration of the analyte. The fundamental principle of operation of spectrophotometer covering UV region consists in that light of definite interval of wavelength passes through a cell with solvent and falls on to the photoelectric cell that transforms the radiant energy into electrical energy measured by a galvanometer. [2].

Important applications of spectrophotometer:

- Identification of many types of organic, inorganic molecules and ions.
- Quantitative determination of many biological, organic and inorganic species.
- Quantitative determination of mixtures of analyte.
- Monitoring and identification of chromatographic effluents.
- Determination of equilibrium constants.
- Determination of stoichiometry and chemical reactions.
- Monitoring of environmental and industrial process.
- Monitoring of reaction rates.
- Typical analysis times range from 2 to 30 min for sample.


This technique of ultra violet spectroscopy is one of most frequently employed method in pharmaceutical analysis. It involves the measurement of the amount of UV radiation (190-380 nm) or visible (380-800 nm) radiation absorbed by a substance in solution. Ultraviolet spectroscopy involves the promotion of electrons (σ, π, n electrons) from the ground state to higher energy state. It is useful to measure the number of conjugated double bonds and also aromatic conjugation with the various molecules.

The ultraviolet region of the electromagnetic spectrum is frequently subdivided into as follows:

- Far vacuum Ultraviolet region (10-200 nm).
- Near ultraviolet region (200-400 nm).
- Visible region (380-780 nm).

Diagram of an analytical instrument showing the stimulus and measurement of response.

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a) Simultaneous equation method

If a sample contains two absorbing drugs (X and Y) each of which absorbs at λ max of the others it may be possible to determine both drugs by the technique of simultaneous equation ( Vierodt’s method) provided that criteria apply.

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Information required is

1. The absorptivities of X at λ1 and λ2 and ax2, respectively
2. The absorptivities of Y at λ1 and λ2 and ay2, respectively
3. The absorbance’s if the diluted sample at λ1 and λ2, A1 and A2 respectively.

Let cx and cy be the concentrations of X and Y respectively in the diluted sample. Two equations are constructed based upon the fact that at λ1 and λ2

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Criteria for obtaining maximum precision, based upon the absorbance ratios, have been suggested (Glenn, 1960) that place limits on the relative concentrations of the components of the mixture. The criteria are the ratios

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It should lie outside the range 0.1-2.0 for the precise determination of X and Y respectively. These criteria are satisfied only when the λ max of the two components is reasonably dissimilar. An additional criterion is that the two components do not interact chemically, thereby negating the initial assumption that the total absorbance is equal to sum of the individual absorbance’s.

b) Derivative Spectrophotometric method

This method involves the conversion of the normal spectrum into first, second or higher derivative spectrum. The transformation that occurs in the derivative spectrum are understood by reference to a Gaussian band which represents an ideal absorption band.

The first derivative (D1) spectra is a plot of the ratio of change of absorbance with wavelength against wavelength, i.e. a plot of slope of the fundamental spectrum against wavelength or a plot of dA/dλ Vs λ. At λ2 and λ4, the maximum positive and maximum negative slope respectively in the D°. Spectrums correspond with maximum and minimum respectively in the D1 spectrum. The λmax at λ3 is a wavelength of zero slope and gives dA/dλ 0, i.e a cross-over point, in the DI spectrum .

The first order derivative spectrum of absorption band is characterized by a maximum, a minimum and a cross-over at a λmax of the absorption band. These spectral transformations confer two main advantages on derivative Spectrophotometry. Firstly an even order spectrum is of narrower spectral band width than its fundamental spectrum.

Derivative spectrum shows better resolution of overlapping bands than the fundamental spectrum and may permit the accurate determination of λmax of the individual bands. Secondly, derivative spectroscopy discriminates in favours of the substances of narrow spectral bandwidth against broad band width substances.

Beer–Lambert law

The law states that there is a logarithmic dependence between the transmission and transmissivity, T, of light through a substance and the product of the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e. the path length), ℓ. The absorption coefficient can, in turn, be written as a product of either a molar absorptive (extinction coefficient) of the absorber, ε, and the concentration c of absorbing species in the material, or an absorption cross section, σ, and the (number) density N' of absorbers.

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I 0 and I are the intensity of the incident light and the transmitted light.

σ is cross section of light absorption by a single particle.

N is the density (number per unit volume) of absorbing particles.

Correlation Coefficient

The Correlation Coefficient “r” (X, Y) is the most useful to express the relationship of the chosen scale. To obtain the Correlation Coefficient, the covariance is divided by the product of the standard deviation of X and Y

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Selectivity of the method

The determination of an analyte may be disturbed by matrix and interference effects. Some of the excipients and additives present in pharmaceutical formulation may sometimes interfere in the assay of the drug and in such instances appropriate separation procedure is to be adopted initially. The selectivity of the method the method is ascertained by studying the effect, of wide range of excipients and other additives usually present in the pharmaceutical formulation in optical condition. In the initial studies a fixed concentration of drug is determined several times by the optimum procedure in presence of suitable (1-1000 fold.) molar concentration of the foreign compound under investigation and its effects on the absorbance are be noticed .the the foreign compound considered to vbe non-interfering if at this concentration, it constantly produces an error less than 3% in the absorbance produce in pure solution.

Linearity and Sensitivity of the method

Knowledge of the sensitivity of the chromogen is important and the following terms are commonly employed for expressing sensitivity. Accordingly the Bouger-Lambert-Beer’s law,

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The absorbance (A) is proportional to the concentration (c) of absorbing species is absorptive ( ) and thickness of the medium (t) are constant. When ‘C’ is molar /liter, the constant is called the molar absorptive. The concentration is expressed in moles per unit volume, the molar absorptive (ε) is used in L-mol−1-cm−1.


High performance liquid chromatography is convenient separation technique used for wide types of samples, exceptional resolving power, and speed and nano molecular detection levels. It is presently used in pharmaceuticals research and development. [11]

1.3.1Modes of CHROMATOGRAPHY [ 12-29 ]

Modes of chromatography are defined essentially according to the nature of the interactions between the solute and the stationary phase, which may arise from hydrogen bonding, Vander walls forces, electrostatic forces or hydrophobic forces or basing on the size of the particles (e.g. Size exclusion chromatography).

Different modes of chromatography are as follows:

- Normal Phase Chromatography
- Reversed Phase Chromatography
- Reversed Phase – ion pair Chromatography
- Ion-Exchange Chromatography
- Ion Chromatography
- Affinity Chromatography
- Size Exclusion Chromatography

1) Normal Phase Chromatography

In normal-phase chromatography (NPC) the stationary phase is more polar than the mobile phase, the opposite of RPC. Usually, the mobile phase is a mixture of organic solvents without added water and the column packing is either an inorganic adsorbent (silica or occasionally alumina) or a polar bonded phase (cyano, diol, or amino) on a silica support. Regardless of the mobile or stationary phase used, sample retention in normal-phase chromatography increases as the polarity of the mobile phase decreases (the opposite of RPC).

Normal-phase chromatography has been used for separating both neutral and ionic compounds, but neutral samples predominate. Normal-phase chromatography for ionic samples can involve the use of water in the mobile phase, and the retention process is then somewhat complex. When ionic samples are separated by normal-phase chromatography, it is usually advisable to add triethylamine to the mobile phase for basic compounds and acetic or formic acid for acidic compounds. Neutral samples are often separated equally well by either reversed-phase chromatography or normal-phase chromatography, the main difference being a reversal of elution order for the two HPLC methods. In normal-phase chromatography, less polar compounds elute first, while more polar compounds leave the column last: this behavior can be contrasted with the opposite RPC behavior.

Application: Separation of nonionic, non-polar to medium polar substances.

2) Reverse Phase Chromatography

Reversed-phase chromatography (RPC) is the first choice for most regular samples. Reversed-phase chromatography typically more convenient and rugged than other forms of liquid chromatography and is more likely to result in a satisfactory final separation. High-performance RPC columns are efficient, stable, and reproducible. Detection often is easier in reversed-phase chromatography because of the solvents used. Finally, most workers have more experience with reversed-phase chromatography than with other HPLC methods.

Although many organic compounds have limited solubility in the mobile phase, this is not a practical limitation because only small amounts (nanograms or micrograms) of sample are usually injected. In those cases where sample solubility in reversed-phase chromatography mobile phases is exceptionally poor, normal-phase chromatography is a preferred alternative. Similarly, samples that are unstable in aqueous media can also be separated by normal phase chromatography using non-aqueous solvents.

Simple compounds are better retained by the reversed phase surface, the less water- soluble (i.e. the more non-polar) they are. The retention decreases in the following order: aliphatic > induced dipoles (i.e. CCl4) > permanent dipoles (e.g.CHCl3) > weak Lewis bases (ethers, aldehydes, ketones) > strong Lewis bases (amines) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids). Also the retention increases as the number of carbon atoms increases.


Ion-pair and reversed-phase HPLC share several features. The column and mobile phase used for these separations are generally similar, differing mainly in the addition of an ion-pair reagent to the mobile phase for ion-pair chromatography (IPC). For most applications that involve ionic samples, Reversed-phase chromatography separation should be explored first, before considering ion-pair chromatography. Ion-pair chromatography separations are more complicated to develop and are subject to additional experimental separation due to poor band spacing, ion-pair chromatography provides an important additional selectivity option. Thus ion-pair chromatography is a logical follow-up for reversed-phase chromatography separations that need improvement.


Ion exchange chromatography (IEC) was an important HPLC method. Its application for the separation of most sample types gradually diminished compared to other HPLC methods. Today it is used infrequently, except for certain “special” samples. These include mixtures of biological origin, inorganic salts, and some oreganos metallic’s.

Because of the similarity of ion-exchange and ion-pair HPLC retention, many separations that are possible using ion exchange chromatography can also be achieved using ion-pair chromatography. For the separation of typical small-molecule samples, ion-pair chromatography may have certain advantages like higher column efficiencies, easier control over selectivity and resolution, and more stable and reproducible columns.

Classification of Chromatographic Methods : -

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1.3.2 Elution TECHNIQUES [30]

Two types of elution techniques generally used. They are,

i) Isocratic elution
One particle solvent or mixture is pumped through the whole analysis.
ii) Gradient elution

For some determinations the solvent composition may be altered gradually gradient elution system can be classified as low pressure and high pressure system. In low pressure gradient elution system the eluent components are miner proportion varying with time at low pressure and the mixture is pumped in order to be delivered at high pressure to the column. In high pressure gradient elution system components or mixtures of fixed composition are each pumped by separate pump and then mixed at high pressure in a ratio varying with time.


Methods for analyzing drugs can be developed, provided one has knowledge about the nature of the sample, namely, its molecular weight, polarity, ionic character and the solubility parameter. An exact recipe for HPLC, however, cannot be provided because method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase. In general, one begins with reversed phase chromatography, when the compounds are hydrophilic in nature with many polar groups and are water soluble.

Elution of drug molecules can be altered by changing the polarity of the mobile phase. The elution strength of a mobile phase depends upon its polarity, the stronger the polarity, higher is the elution. Ionic samples (acidic or basic) can be separated, if they are present in un-dissociated form. Dissociation of ionic samples may be suppressed by proper selection of pH.

The pH of the mobile phase has to be selected in such a way that the compounds are not ionized. If the retention times are too short, the decrease of the organic phase concentration in the mobile phase can be in steps of 5 %. If the retention times are too long, an increase in 5 % steps of the organic phase concentration is needed.

Optimization can be started only after a reasonable chromatogram has been obtained. A reasonable chromatogram means that all the compounds are detected by more or less symmetrical peaks on the chromatogram. By a slight change of the mobile phase composition, the shifting of the peaks can be expected. From a few experimental measurements, the position of the peaks can be predicted within the range of investigated changes. An optimized chromatogram is the one in which all the peaks are symmetrical and are well separated in less run time.

The peak resolution can be increased by using a more efficient column (column with higher theoretical plate number, N), which can be achieved by using a column of smaller particle size, or a longer column. These factors, however, will increase the analysis time. Flow rate does not influence resolution, but it has a strong effect on the analysis time.

The parameters that are affected by the changes in chromatographic conditions are,

- Resolution (Rs),
- Capacity factor (k’),
- Selectivity (a),
- Column efficiency (N) and Peak asymmetry factor (As).

The presence of metabolites or more than one drug in a formulation usually demands a more sophisticated separation for their measurement especially, when two or more drugs are of similar physical and chemical nature. Chromatography is a separation technique that is based on differing affinities of a mixture of solutes between at least two phases. The result is a physical separation of the mixture into its various components.

The important factors, which are to be taken into account to obtain reliable quantitative analysis, are

1. Careful sampling and sample preparation.
2. Appropriate choice of the column.
3. Choice of the operating conditions to obtain the adequate resolution of the mixture.
4. Reliable performance of the recording and data handling systems.
5. Suitable integration/peak height measurement technique.
6. The mode of calculation best suited for the purpose.
7. Validation of the developed method.

1.4.1. Completing the HPLC Method Development

The final procedure should meet all the objectives that were defined at the beginning of method development. The method should also be robust in routine operation and usable by all laboratories and personnel for which it is intended.


Validation is defined as documented evidence which gives a high degree of confidence that a process, system, facility will consistently produce a product meeting its predetermined specifications and quality attributes .

Method Validation

Method validation is the process of proving that an analytical method is acceptable for its intended purpose. For pharmaceutical methods, guidelines from the United States Pharmacopoeia (USP), International Conference on Harmonization (ICH), World Health Organization (WHO) and the Food and Drug Administration (FDA) provide a framework for performing such validations.

Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Methods need to be validated or revalidated,

- Before their introduction into routine use
- Whenever the conditions change for which the method has been validated, e.g., instruments with different characteristics.
- Whenever the method is changed, and the change is outside the original scope of the method. The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceutical for human use has developed a consensus text on the validation of analytical procedures. The document includes definitions for eight validation characteristics.

The parameters as defined by the ICH and by other organizations

- Specificity
- Selectivity
- Precision
- Repeatability
- Intermediate precision
- Reproducibility
- Accuracy
- Linearity
- Robustness
- Ruggedness
- Range
- Limit of detection
- Limit of quantization

1.5.1. Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures.

An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and the assay. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure.

1.5.2. Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or on an accepted reference value and the value found. Assay

- Assay of Active Substance
- Assay of Medicinal products

Several methods are available to determine the accuracy.

a. Application of an analytical procedure to an analyte of known purity (e.g. reference material).
b. Comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure, the accuracy of which is stated and/or defined (independent procedure).
c. Application of the analytical procedure to synthetic mixtures of the product components to which known quantities of the substance to be analyzed have been added. Impurity (Quantification)

Accuracy should be assessed on sample (substance /products) spiked with known amounts of impurities. It should be clear how the individual or total impurities are to be determined.

E.g. Weight / Weight or area percent.

1.5.3. Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision of an analytical procedure is usually expressed at the variance, standard deviation or coefficient of variation of a series of measurements.

Validation of tests for assay and for quantitative determination of impurities includes an investigation of precision. Repeatability

Express the precision under the same operating conditions over a short interval of time. Repeatability is also termed as intra - assay precision. It should be assessed using a minimum of nine determinations covering the specified range for the procedure (e.g. three concentration/three replicates each) or a minimum of determinations at 100% of the test concentration. Intermediate Precision

The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. Reproducibility

Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility should be considered in case of the standardization of an analytical procedure, for instance inclusion of procedures in Pharmacopoeias.

1.5.4. Linearity

Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods. For example, calculation of a regression line by the method of least square. Therefore data from regression line itself may be helpful to provide mathematical estimates of the degree of linearity.

1.5.5. RANGE

Range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample including these concentrations for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

The following minimum specified ranges should be considered

- For the assay of an active substance or a finished product normally from 80-120 percent of the test concentration.
- For the content uniformity, covering a minimum of 70-130 percent of the test concentration.
- For dissolution testing, 20% over the specified range (e.g.), If the specifications for a controlled release product cover a region from 20% after 1 hour, upto 90% after 24 hours, the validated range would be 0-110% of label claim.
- For the determination of an impurity, the reporting level of an impurity to 120% of the specifications.


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Analytical Method Development and Stability Studies of Carvedilol
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This thesis was submitted in the year 2011 when I (Kishanta Kumar Pradhan) was lecturer at Royal College of Pharmacy and Helath Sciences, Berhampur, Odisha, India. The Project conducted under my guidance along with a person from industry. There after I have moved to Birla Institute of Technology, Mesra, Ranchi on 2012. I have been awarded with GOLD MEDAL being topper amongst all M.Pharm students by Governer of Odisha in the year 2008. I have also qualified GATE-2005. I have 20 publications in various national and international journals.
analytical, method, development, stability, studies, carvedilol
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M. Pharm., Ph. D. Kishanta Kumar Pradhan (Author)M. Pharm. Ranganadha Rao K (Author)M. SC., PhD P. Srinivasulu (Author), 2011, Analytical Method Development and Stability Studies of Carvedilol, Munich, GRIN Verlag,


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