ANALYTICAL TECHNIQUES IN PHARMACEUTICLES

BIR Nitish1, BHATTI Roshni2

1,2

Deparment of Chemical Engineering, Dr. B R Ambedkar National Institute of Technology

Jalandhar-144001, Punjab

ABSTRACT
The development of the pharmaceuticals has revolutionized the medical science. These improvised
pharmaceuticals would serve their purpose only if they are free from all the contaminants and are
used in appropriate quantity. In order to make drugs serve their purpose effectively, various chemical
and instrumental methods were developed at regular intervals which are involved in the estimation of
drugs. It is important to detect and quantitate the pharmaceuticals as it may develop impurities at
various stages of their development, storage and transportation. For this various analytical methods
plays an important role. This paper depicts the role of various analytical methods in determing the
quality of the drugs which includes titrimetric, spectroscopic, chromatographic, electrophoretic, and
electrochemical and their corresponding methods.
Keywords: Pharmaceuticals, Analytical techniques, Drugs

1. INTRODUCTION
Under the aegis of pharmacology and clinical sciences, and driven by chemistry, pharmaceutical
research in the past has played a vital role in the development of variety of pharmaceutical drugs. The
contribution of various fields of science including chemistry, pharmacology, biochemistry and
microbiology has set a standard in the discovery of drugs where new drugs are being generated by
the exchange of ideas between chemists and biologists and not just by the imagination of chemist
alone. Analytical techniques play a great role ranging from various stages of drug development to
marketing and post marketing areas. The role of these techniques can also be seen in the
understanding of physical and chemical stability of drugs, impact on the selection and design of the
dosage form, assessing the stability of the drug molecules, quantitation of the impurities and
identification of these impurities with the help of various analytical techniques.
This paper aimed at highlighting the role of various analytical techniques and methods in the analysis
of pharmaceutical drugs.
2. Analytical techniques
2.1. Titrimetric techniques
Titrimetric method of analysis was originated somewhere in the middle of the 18th century. In this
method, a solution of a suitable reagent of exactly known concentration is treated with the substance
being determined. This reagent is added to the substance until the amount added is equivalent to the
amount of substance to be determined. The change in colour of an auxiliary reagent (the indicator) is
usually the point of equivalence (end point), but may also be detected by electrical means
such as potentiometric, titrimetry, conductometric, etc. Various advantages of this method include the
likes of saving time and labour, high precision and most importantly, there is no need of using
reference standards. In the past these methods have been used for the determination of gabapentin
[1], albendozole [2] and Sparfloxacin [3] on commercial scale. It is also being used in the estimation of
degradation products of the pharmaceuticals [4].

2.2. Spectroscopic techniques

2.2.1. Spectrophotometer
One of the important groups of methods which find an important place in the field of pharmacopoeias
are spectrophotometric methods based on natural UV absorption and chemical reactions [5].
Spectrophotometry is basically the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength. The advantages of these methods include low
labour and time consumption and excellent precision.
The use of UVVisible spectrophotometry in the field of pharmaceutical analysis has rapidly increased
over the past few years [6], [7]. The colorimetric methods are usually based on the following aspects:
Complex-formation reaction
Oxidation-reduction process
Catalytic effect
It is important to mention that colorimetric methods are regularly used for the determination of bulk
materials. For example, the blue tetrazolium assay is used for the determination of corticosteroid drug
formulations [8], [9]. Several approaches using spectrophotometry for determination of active
pharmaceutical ingredients in bulk drug and formulations have been reported and details of these
methods are recorded in Table 1.
Derivative spectroscopy uses first or upper derivatives of absorbance with respect to wavelength for
qualitative investigation and estimation. In late 1950s, the concept of derivative spectroscopy was
introduced as it was having some advantages. However, in spite of certain advantages, the technique
received little consideration mainly due to the complexity of generating derivative spectra using early
UVVisible spectrophotometers. It was only in late 1970s that the introduction of microcomputers
made it generally convincing to use mathematical methods to generate derivative spectra quickly,
easily and reproducibly. This significantly increased the use of the derivative technique. The
applications of derivative method are not confined to only UV spectrophotometry but are also seen in
infrared spectroscopy [10], atomic absorption, fluorescence spectrometry [11], and fluorimetry [12].
The use of derivative spectrometry is more advantageous whenever quantitative study of normal
spectra is problematic. Some of the disadvantages associated with this method includes the
differential degrades the signal-to-noise ratio, so that some form of smoothing is required in
conjunction with differentiation [13].
Table 1 Quantitative analysis of drugs in pharmaceutical formulations by UVvisible spectrophotometric
procedures
Reagent used

Name of drug

max

m-Cresol
p-Chloranilic acid

Acetaminophen
Quetiapine fumarate
Milrinone
Flunarizine dihydrochloride
Aripiprazole
Diltiazem HCl
Rasagiline
Rasagiline mesylate
Zidovudine
Rasagiline mesylate
Pregabalin
Lisinopril

640
520
519
380
400
750
414
414
520
414
402.6
530

Iodine
Sodium metavanadate
Bromothymol blue
Bromocresol green
Chloramin T
Bromophenol blue
Ninhydrin
Ascorbic acid

2.2.2. Near infrared spectroscopy (NIRS)

Near infrared spectroscopy (NIRS) is critical method which provides multi component analysis of
almost any matrix. Recently, NIR spectroscopy has been widely used in pharmaceutical industry for
testing of raw materials, controlling product quality and process monitoring. Due to its various
advantages like easy sample preparation without any requirement of pre-treatments, the probability of
separating the sample measurement position by use of fibre optic probes, and obtaining physical and
chemical sample parameters from one single spectrum, NIRS is being used on a large scale and is
usually preferred over other analytical techniques. The major pharmacopoeias have generally
adopted NIR techniques. The collaboration of NIR spectroscopy with multivariate data analysis opens
many interesting methods in pharmaceutical analysis, both qualitatively and quantitatively. A plethora
of publications have been reported the quantitative analysis by NIRS for the measurements of active
ingredient in tablets [14]. Apart from this, many review articles have also been published highlighting
various applications of the NIRS in analysis of pharmaceutical [15], [16].
2.2.3. Nuclear magnetic resonance spectroscopy (NMR)
NMR was first reported in 1996 for the analysis of pharmaceuticals and since then NMR has been
progressing promptly. Over the last few years, a variety of state-of-the art approaches have been
presented and found a widespread application in both pharmaceutical and academic research.
Various applications of NMR includes its use in quantitative analysis in order to determine the impurity
of the drug [17], characterization of the composition of the drug products and in quantitation of drugs
in pharmaceutical formulations and biological fluids [18].
2.3 Chromatographic Techniques
2.3.1 Thin Layer Chromatography
In this method, a solid phase, the adsorbent, is coated onto a solid support as a thin layer usually on a
glass, plastic, or aluminium support. Several factors determine the efficiency of this type of
chromatographic separation. First of all the adsorbent should show extreme selectivity toward the
substances being separated so that the dissimilarities in the rate of elution are large. For the
separation of any given mixture, some adsorbents may be strongly or weakly adsorbing. A number of
chromatographic adsorbents in the order of adsorptive power are highlighted in Table 2. Due to
distinctive advantages such as minimal sample clean-up, flexibility in sample distinction, wide choice
of mobile phases, high sample loading capacity and low cost, TLC has become a popular technique
for the analysis of a wide variety of organic and inorganic materials. TLC is a powerful tool for
screening unknown materials in bulk drugs [19]. TLC has been popularly used for the determination of
some steroids [20], noscapine [21] and pioglitazone [22] and also for the determination of various
impurities in pharmaceuticals [23]. TLC plays a vital role in the early stages of drug development
when information about the impurities is inadequate.

Table 2. Chromatographic adsorbents

Most strong adsorbent

Alumina
Charcoal
Florisil

Al 2O3
C
MgO/SiO 2 (anhydrous)

Least strong adsorbent

Silica gel

SiO 2

Source: http://www.chem.wisc.edu/courses/342/Fall2004/TLC.pdf.

2.3.2 High-performance liquid chromatography (HPLC)

HPLC is an advanced form of chromatographic technique which plays an important and critical role in
the field of pharmaceutical analysis. It was first used for the analysis of bulk drug material materials
(United States Pharmacopoeia, 1980). It is being used for both qualitative and quantitative analysis
i.e., testing of products and detection of raw ingredient used to make them. Moreover, the importance
of HPLC uses in these fields falls under the stringent regulations established by the U.S. Food and
Drug Administration (FDA) and this make all pharmaceutical companies to analyse the quality of their
products with the help of HPLC before allowing them to sell it in the global market.
HPLC method has excellent specificity and precision but this high specificity, accuracy and precision
are attainable only if wide-ranging system suitability tests are carried out before the HPLC analysis
and this will ultimately going to increase the expense to be paid.
In HPLC, all the components are completely detected only if the choice of detection approach is
perfect for a given material. UV detector is one of the widely used detectors in HPLC which is capable
of monitoring several wavelengths concurrently with the help of multiple wavelength scanning
programs.
The utilisation of refractive index detector to analyze the content of volgibose in pharmaceutical
formulations was reported by Lakshmi and Rajesh [24]. The electrochemical detector responds to the
substances that are either oxidizable or reducible and the electrical output results from an electron
flow triggered by the chemical reaction that takes place at the surface of the electrode. This detector
was applied recently to analyze the content of glutathione in human prostate cancer cells and lung
adenocarcinoma cells [25]. One of the most sensitive detectors among the LC detectors is
fluorescence detector. Typically its sensitivity is 101000 times higher than that of the UV detector for
strong UV absorbing materials used as an advantage in the measurement of specific fluorescent
species in samples. One of the most important applications of fluorescence is the estimation of
pharmaceuticals [26]. Over a certain period of time most workers used the reversed- phase mode with
UV absorbance detection whenever appropriate, because this provided the best available reliability,
analysis time, repeatability and sensitivity. Several drugs have been assayed in pharmaceutical
formulations [27] and in biological fluids [28] using HPLC. Thus, HPLC provides a major service in
answering many questions posed by the pharmaceutical industry.
However, it also bears some limitations including high price of columns, solvents and a lack of long
term reproducibility due to the proprietary nature of column packing. Liquid chromatography combined
with mass spectrometry (LCMS) is considered as one of the most important techniques of the last
decade of 20th century [29]. It became the method-of-choice for analytical support in many stages of
quality control and assurance within the pharmaceutical industry [30]. Recently HPLC-MS has been
used for assay of drugs [31]. In addition to the application of HPLC in analyzing the drugs, it is also
used in the determination of impurities present in pharmaceuticals [32] and degradation products [33].
2.3.3. Gas chromatography
Another chromatographic technique is gas chromatography, which is a powerful separation technique
for detection of volatile organic compounds. Combining separation and on-line detection allows
accurate quantitative determination of complex mixtures, including traces of compounds down to parts
per trillions in some specific cases. Gas liquid chromatography commands a substantial role in the
analysis of pharmaceutical product [34]. The creation of high-molecular mass products such as
polypeptides, or thermally unstable antibiotics confines the scope of this technique. Its main constraint
rests in the comparative non-volatility of the drug substances therefore, derivatization is virtually
compulsory.

Recently, gas chromatography has been used for the determination of drugs such as cocaine [35],
isotretinion [36], and also used in the determination of residual solvents in betamethasone valerate
[37]. Gas chromatography is also an important used for the analysis of impurities of pharmaceutical
[38].
2.4. Electrochemical methods
Electrochemical techniques have been used on a large scale in the analysis of drugs and
pharmaceuticals since its inception. The use of sophisticated instruments is one of the reasons that
researchers are very much interested in electrochemical techniques. Table 3 shows the application of
various electrochemical modes in the analysis of drugs and pharmaceuticals.
The quantification of pharmaceuticals can be done with the wide number of electro analytical methods
available in market. Determination of imipramine, trimipramine and desipramine was made possible
with the development of amberlite XAD-2 and titanium dioxide nanoparticles. Cyclic voltammetry,
adsorptive stripping differential pulse voltammetry and electrochemical impedence spectroscopy has
been used for the investigation of electrochemical behaviour of these drugs [39].The capsaicin
modified carbon nanotube modified basal-plane pyrolitic graphite electrode or p-chloranil modified
carbon paste electrodes have been developed for the determination of benzocaine and lidocaine. The
electrochemically initiated formation of capsaicin-benzocaine adduct causes a linear decrease in the
voltammetric signal corresponding to capsaicin which correlates to the added concentration of
benzocaine [40]. A copper (II) complex and silver nanoparticles modified glassy carbon paste
electrode was constructed and used for the determination of dopamine, levodopa, epinephrine and
norepinephrine. The electrochemical behaviour of these drugs was studied using cyclic voltammetry,
chronocoulometry and adsorptive stripping square-wave voltammetry techniques.
Table 3. Determination of drug by various electrochemical techniques
Technique

Drugs determined

Voltammetry

b-blocker drugs
Rosiglitazone
Leucovorin
Secnidazole
Dopamine
Atenolol
Nifedipine
Anti-cancer drug, Vitamin K3
Ciclopirox olamine
Diclofenac
Verapamil
N-acetyl-L-cysteine
Pentoxifylline

Polarography

Amperometry
Potentiometry

3. Conclusion
Pharmaceutical drugs play a vital role in our society and serves to mankind by making them free from
potential illness or preventing them from disease. For the medicine to serve its intended purpose they
should be free from impurity or other interference which might harm humans. The aim of this paper is
to highlight the role of various analytical techniques in the analysis of pharmaceuticals which includes
analysis of raw material, intermediates and final product and giving a thorough literature review of the
instrumentation involved in each pharmaceutical analysis method. The paper also highlights the
advancement of the analysis techniques beginning from the older titrimetric method and reaching the
advanced electrochemical methods.
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