ejpmr, 2016,3(2), 348-360 SJIF Impact Factor 2.

026
Review Article
Kamal et al.
EUROPEAN JOURNAL OF PHARMACEUTICAL
European Journal of Pharmaceutical and Medical Research
AND MEDICAL RESEARCH ISSN 3294-3211
www.ejpmr.com EJPMR

A REVIEW ON UV SPECTROPHOTOMETRIC METHODS FOR SIMULTANEOUS

MULTICOMPONENT ANALYSIS

Amira H. Kamal*, Samah F. El-Malla and Sherin F. Hammad

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Egypt

*Author for Correspondence: Dr. Amira H. Kamal
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Egypt

Article Received on 15/12/2015 Article Revised on 06/01/2016 Article Accepted on 27/01/2016

ABSTRACT
In present era, market is floated with various combinations dosage forms and the number is increased day by day.
These multicomponent formulations are gaining interest due to greater patient acceptability, increased potency,
multiple action, fewer side effects, and quicker relief. Therefore, it is desired that these formulations meet the entire
standards related to their quality, safety, and efficacy. This can only be possible if different analytical techniques are
available for their determination. Different UV spectrophotometric methods are used in simultaneous
multicomponent analysis. Such methods are based on recording and mathematically processing absorption spectra.
This review is mainly focused on simultaneous equation method, difference spectrophotometry, derivative
spectrophotometry, absorbance ratio spectra, derivative ratio spectra, double divisor ratio spectra derivative method,
successive ratio - derivative spectra, Q-absorbance ratio method, isosbestic point method, absorpitivity factor
method, dual wavelength method, ratio subtraction method, mean centering of the ratio spectra, absorption factor
method and multivariate methods. An overview of theories and some applications of these methods are presented.

KEYWORDS: spectrophotometric methods, multicomponent analysis, double divisor, successive ratio-derivative,

dual wavelength, ratio subtraction, multivariate methods.

INTRODUCTION incomplete separation. The procedure may be expensive

Combination drug products occupy a time-honored and and time consuming.[2]
important role in therapeutics. When rationally
formulated, fixed-combination drugs may produce UV spectrophotometric techniques are mainly used for
greater convenience, lower cost, and sometimes greater multicomponent analysis thus minimizing the
efficacy and safety.[1] cumbersome task of separating interferents and allowing
the determination of an increasing number of analytes,
Analysis of samples with numerous components presents consequently reducing analysis time and cost.[5]
a major challenge in modern analysis.[2] Multi-
component analysis has become one of the most Multicomponent UV spectrophotometric methods are
appealing topics for analytical chemists in the last few based on recording and mathematically processing
years, in fields as clinical chemistry, drug analysis, absorption spectra. They offer the following advantages:
pollution control,…. etc.[3] [6]
avoiding prior separation techniques e.g. extraction,
concentration of constituents, and cleanup steps that
Different analytical techniques can be applied for might be required; spectral data are readily acquired with
multicomponent analysis including; spectrophotometry, ease; the process is fast, accurate, and simple; wide
chromatography, and electrophoresis. UV applicability to both organic and inorganic systems;
spectrophotometric methods for simultaneous typical detection limits of 10-4 to 10-5 M and moderate to
determination of drugs are highlighted in this review. high selectivity.

Because most analytes of interest are accompanied in Different UV spectrophotometric multicomponent

their dosage forms by other compounds absorbing in the analysis methods include
same spectral region, classical UV spectral 1- Simultaneous equation method
measurements could not be used for their If a sample contains two absorbing drugs (x and y) each
determination.[4] The use of traditional methods like of which absorbs at the λ max of the other, it may be
extraction is quite difficult because extraction techniques possible to determine both drugs by the technique of
require large solvent consumption, with accompanying simultaneous equation (Vierordt’s method) provided that
risks of analyte loss or contamination, and possibility of certain criteria apply.

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The information required is 1- Reproducible changes may be introduced in the

 The absorptivities of x at λ1 and λ2, ax1 and ax2 spectrum of the analyte by the addition of one or more
respectively reagents.
 The absorptivities of y at λ1 andλ2, ay1 and ay2 2- The absorbance of the interfering substances is not
respectively altered by that reagent.
 The absorbance of the diluted samples at λ1 and λ2,
A1 and A2 respectively. The simplest and the most commonly employed
techniques for altering the spectral properties of the
Let Cx and Cy be the concentration of x and y analyte is the adjustment of the pH by means of aqueous
respectively in the diluted samples. Two equations are solutions of acids, alkalies or buffers.[7]
constructed based upon the fact that at λ1, the absorbance
of the mixture is the sum of the individual absorbance of 3- Derivative spectrophotometry (DS)
x and y. DS involves the conversion of a normal spectrum
……………. (1) (fundamental, zero-order spectrum) to its first, second or
higher derivative spectra by differentiating absorbance of
……………. (2) the sample with respect to wavelength(.[7] The
For measurements in 1 cm cells, b =1cm. Rearrange differentiation of zero-order spectrum can lead to
Eq.(2) separation of overlapped signals, elimination of
background caused by presence of other compounds in a
sample, [10] improvement of resolution of mixtures as it
……………. (3) enhances the detectability of minor spectral features, and
enhancement of sensitivity and specificity. [3]
Substituting for Cy in eq. (1) and rearranging gives
Derivative spectra yield a more characteristic profile in
……………. (4) comparison to the parent one; new maxima and minima
appeared and points where derivative spectra cross the X-
……………. (5) axis. [10]

DS keeps all laws of classical spectrophotometry, e.g.

Criteria for obtaining maximum precision, based upon dependence of derivative value on analyte concentration
absorbance ratios, have been suggested that place limits and additivity law. These features allow the
on the relative concentrations of the components of the determination of several components in a mixture by
measuring the amplitude of derivative spectrum of
mixture. The criteria are that the ratios and mixture at several wavelengths. If the measured height of
derivative peak of analyte is performed at those
should lie outside the range 0.1-2.0 for the wavelengths at which spectra of other components
undergo zeroing, the measured amplitude is proportional
precise determination of y and x respectively. These
only to concentration of this analyte. This approach of
criteria are satisfied only when the λ max of the two
quantitative determination is called “zero-crossing
components reasonably dissimilar and if the two
technique”.[10] DS has been used for simultaneous
components do not interact chemically, thereby negating
determination of different mixtures in pharmaceutical
the initial assumption that the total absorbance is the sum
formulation, e.g. loratadine and pseudoephedrine
of the individual absorbances. [7] Simultaneous equation
sulfate,[11] aceclofenac and tramadol with paracetamol,[12]
method was developed for simultaneous determination of
tramadol and ibuprofen[13] or dexketoprofen, [14]
several mixtures, e.g. atenolol and indapamide, [8] and
paracetamol and tapentadol, [15] naproxen and
dexibuprofen and paracetamol.[9]
acetaminophen [16] or diphenhydramine,[17] phenylephrine
and ketorolac, [18] and amiloride and hydrochlorothiazide
2- Difference spectrophotometry
with timolol.[19]
The selectivity and accuracy of spectrophotometric
analysis of samples containing absorbing interferents
4- Absorbace ratio spectra method
may be markedly improved by the technique of
Consider a mixture of two compounds x and y. The
difference spectrophotometry. The essential feature of
absorption spectrum of the mixture "measured in 1 cm
this method is that the measured value is the absorbance
cell" is defined by the equation[20]
difference (A) between two equimolar solutions of the
analyte in different chemical forms which exhibit ………………….(6)
different spectral characteristics.
Where; AM is the absorbance of the mixture,
The criteria for applying difference spectrophotometry to are the molar absorptivities, Cx and Cy are
the assay of the substance in the presence of other
absorbing substances are that the concentrations of x and y, respectively. If the
absorbance of the mixture is divided by the absorbance

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of a standard solution of x (its absorbance 5- Derivative ratio spectra method

), the following equation results This simple spectrophotometric method, developed by
Salinas et al.,[20] is based on the derivation of the ratio
………………………..(7) spectra for resolving binary mixtures. It permits the use
of the wavelength of highest value of analytical signals
with several maxima and minima, which give an
The ratio is a constant value (Fig. 1) which can be opportunity for the determination of active compounds in
the presence of other compounds and excipients which
eliminated by taking the difference in absorbance ratio could possibly interfere in the assay.[21, 24]
amplitudes between two wavelengths λ 1 and λ 2 (peak to
peak measurement) Calculation of the first derivative will remove the
constant value due to in Eq. 9, so concentration of y
…………….(8) can be easily determined without any interferences from
the drug x.

Eq. (8) illustrates that the amplitude difference in the ……………….. (9)
mixture absorbance ratio between two wavelengths 1
and 2 (termed; peak to peak, peak to trough, maximum The difference between the two spectra and
to minimum measurement, or ratio difference
spectrophotometric method) is equal to the same (Fig. 1) is due to the constant interference value due to
amplitude difference for compound y after canceling the compound x ( ). Elimination of such interference can
constant interference due to compound x. The
concentration of compound y (Cy) is proportional to the be done by measurement of ratio spectra difference
peak to peak amplitudes of its absorbance spectra. A between two wavelengths or calculating derivative of the
calibration graph is obtained by recording and storing the ratio spectra.[21] Second derivative of the ratio spectra
spectra of solutions of different concentrations of pure y, may be also used to improve linearity, mean %
and the spectrum of a solution of pure x (the divisor x0). recoveries and decrease relative standard deviation.[25]
The stored spectra of the solutions of pure y are divided Derivative ratio spectra was modified for the
by the standard spectrum of the divisor (x0). In the determination of ternary mixtures using the derivative
generated ratio spectra, the peak to peak amplitudes ratio spectra zero-crossing method. This method is
between the selected wavelengths are measured and realized by measurement of amplitudes at the zero-
plotted against Cy to obtain the calibration graph. By crossing points in the derivative ratio spectra.[19,26-29]
using the calibration graph, the concentration of
compound y in the mixture is determined after similar 6- Double divisor ratio spectra derivative method
treatment for the mixture solution. The concentration of This method is based on the use of the derivative of the
x in the mixture is determined by an analogous ratio spectrum obtained by dividing the absorption
procedure. Ratio spectra method was developed for the spectrum of the ternary mixture by a standard spectrum
simultaneous determination of several binary mixtures of a mixture of two of the three compounds in the
e.g., emtricitabine and tenofovir, [22] diclofenac and mixture, and the measuring at either the maximum or
pantoprazole [21] and ternary mixtures, e.g., omeprazole, minimum wavelengths. It can only be used for the
tinidazole and clarithromycin. [23] mixtures that the ratio of the concentrations of two
interfering compounds (used as double divisor) is
known. In other words, the ratio of the concentrations of
two interfering compounds should be the same in
calibration, prediction and unknown samples. It is
obvious that the ratio of the concentration of the analytes
in real samples is always unknown.

Theory
If a mixture of three compounds (x, y and z) is
considered, if Beer's law is obeyed for all compounds
over the whole wavelength range used and if the path
length is 1 cm, the absorption spectrum of the ternary
mixture at wavelength λcan be written in form of the
equation
Fig. 1: Ratio spectra of a standard solution of y and a
mixture solution (x and y) containing the same …………………. (10)
concentration of y, using x0 as a divisor.[21]

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where Am is the absorbance of the mixture, independent of the concentration values CX and Cy in the
are the absorptivities of x, y and z, ternary mixture. The concentrations of x and y are
determined by analogous procedures. [27]
and are the concentrations of x, y
and z, respectively. 7- Successive ratio - derivative spectra method
This method is used for simultaneous determination of
A similar equation for two compounds in the same the three compounds in ternary mixtures without need to
ternary mixture as in a standard binary mixture can be know the ratio of concentration of species. It is based on
written as the successive derivative of ratio spectra in two
………………………. (11) successive steps.

Theory: consider a mixture of three compounds x, y and

If Eq. (10) is divided by Eq. (11) corresponding to the
z. If Beer’s law is obeyed in the whole wavelength range
spectrum of a standard solution of two of the components
used and by considering the path length as 1 cm, the
in the ternary mixture, the ratio spectrum is obtained in
absorbance of the ternary mixture at each wavelength
the form of Eq. (12) can be written as
………. (12)
…………… (17)

The ratio of the sum of ( to the sum

Where: Amis the vector of the absorbance of the mixture,
of ( is equal to a constant (k) with are the absorptivity vectors of x, y
respect to ,. If the above constant is replaced in Eq. and z, and are the concentrations of
(12), we obtain Eq. (13) x, y and z, respectively. If Eq. (17) is divided by
corresponding to the spectrum of a standard solution of z
in ternary mixture, the first ratio spectrum is obtained in
the form of Eq. (18) (for possibility of dividing
…………………. (13)
operation, the zero values of should not be used in the
However, if the standard concentrations of and in
divisor)
Eq. (11) are equal or very close to each other, =
or ≈ , we could write ……………….. (18)
……….. (14)
If the first derivative of Eq. (18) is taken, since the
derivative of a constant (CZ) is zero, Eq. (19) would be
When Eq. (14) is substituted into Eq. (13), Eq. (15) is obtained
obtained
……(19)
…………. (15)
By dividing Eq. (19) by (d/dλ)(ay/az), corresponding to
If the first derivative of Eq. (15) is taken, since the the derivative of the ratio of the spectra of the standard
derivative of a constant is zero, Eq. (16) would be solutions of y and z, the second ratio spectrum is
obtained obtained as Eq. (20) (for possibility of dividing
operation, the zero values of (d/dλ)(ay/az) should not be
…………. (16) used in the divisor)
...(20)
Eq. (16) is the mathematical foundation of
multicomponent analysis which permits the
determination of the concentration of each of the active If the first derivative of Eq. (20) is taken since the
compounds in solution without interference from the derivative of a constant (Cy) is zero, Eq. (21) would be
other components of the ternary system. In practice, Eq. obtained
(16) corresponding to the first derivative ratio spectrum …………………. (21)
of z is obtained by dividing the absorption spectrum of
the ternary mixture of x, y and z by the standard
spectrum of two of the compounds in the ternary Eq. (21) is the mathematical foundation of
mixture. Also, in Eq. (16), the derivative signal of the multicomponent analysis that permits the determination
ratio spectrum of the ternary mixture is dependent only of concentration of each of the active compounds in the
on the concentration values CZ and , but is solution (x in this equation) without interference from

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the other compounds of the ternary system (y and z in measured using other spectroscopic method (DS), the
these equations). As Eq. (21) shows there is a linear concentration of the other could be calculated by
relation between the amount of dD/dλ and the subtraction. A linear correlation was obtained between
concentration of x in the solution. A calibration curve the absorbance values and the corresponding drug
could be constructed by plotting dD/dλ against concentrations. Consider you have a mixture of two
concentration of x in the standard solutions of x or in the drugs x and y. The absorbance of each drug can be
standard ternary mixtures. For more sensitivity the calculated at any wavelength (λ) from the equation
amount of dD/dλ corresponding to maximum or
…………. (27)
minimum wavelength should be measured. Calibration
graphs for y and z could be also constructed as described
for x. [30] Therefore, for drug x: ……(28)

8- Q-absorbance ratio method and for drug y: …………. (29)

This method "also termed absorption ratio method" is a Where Ax and Ay are the absorbance of x and y,
modification of the simultaneous equations method. respectively; Cx and Cy are the concentrations of x and y,
According to this method, the ratio of absorbance at any
two wavelengths for a substance, which obeys Beer's respectively; and are the
law, is a constant value independent of the concentration absorbtivities when the path length (b) is 1 cm and
and path length. This constant is termed as "Hufner's concentration is 1 g/100 mL for x and y, respectively. If
Quotient" or Q-value. The method involves the Cx = Cy, and Ax = Ay, this λ is called the isosbestic point,
measurement of absorbance at two wavelengths, one and at this λ
being the λ max of one of the components (λ 2) and the ……………. (30)
other being a wavelength of equal absorptivity of the two
components (λ 1), called the iso-absorptive point.[7, 9]
The concentration of each component can be calculated
by mathematical equations
Cx = (Qm-Qy/Qx-Qy) * A/a1 ………………… (22)
Cy = (Qm-Qx/Qy-Qx) * A/a2 ………………… (23)
where; Cx and Cy are the concentrations of x and y
respectively, A is absorbance of sample at isoabsorpitive
wavelength and a1 and a2 are the absorptivity of x and y
respectively at isoabsorpitive wavelength.

……(24)

Fig. 2: Zero order absorption spectra of 20 μg. ml-1 of

…………………… (25) metronidazole (___), 20 μg. ml-1 diloxanide furoate (- -
- - -) and (1:1) mixture containing 10 μg. ml-1 of each
(…….) using methanol as a blank. [32]
…………………... (26)
For a mixture of both drugs, the absorbance at this λ can
9- Isosbestic " isoabsorptive" point method be calculated from the equation
Erram and Tipnis [31] developed the isosbestic point ……(31)
method. This technique can be used only if the spectra of ..(32)
the same concentration of the two studied drugs cross at
a point called isosbestic or isoabsorptivity point. At the
Where AM is the absorbance of their mixture at isosbestic
isosbestic point both drugs have equal absorptivities and
their mixture acts as a single component and gives the point and and are the concentrations of drugs x
same absorbance as pure drug. and y in the mixture, respectively, and CTM is the
concentration of their mixture.
This theory can be confirmed experimentally by
recording the absorbance spectra of a certain Therefore we can conclude that
concentration of the two drugs and the absorbance ……………. (33)
spectra of a binary mixture containing the same
Thus, having the total concentration of both drugs, if the
concentration. The absorbance value at the isosbestic
concentration of one of them can be determined
points (Aiso) was determined, and the total concentration
separately by any other method, the concentration of the
of both drugs was calculated (Fig. 2). Since the
second drug can be calculated by subtraction.[31]This
concentration of one of them in this mixture can be

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Kamal et al. European Journal of Pharmaceutical and Medical Research

method has was successfully applied for the where, F is the absorptivity factor, ax and ay are the
simultaneous determination of several binary mixture, absorptivities of x and y respectively.
e.g., metronidazole and diloxanide furoate,[32] ezetimibe
and atorvastatin,[33] and sitagliptin and metformin.[34] For mixture of x and y, the total absorbance of x and y at
absorptivity factor point kF can be expressed as follows
10-Absorpitivity factor method …………………… (39)
The absorptivity factor (modification of the classical
isoabsorpitive method) is applied for the analysis of …… (40)
binary mixture if only there is a large difference in the …………………… (41)
absorptivity between both drugs, so there is no
occurrence of an isoabsorptive point.
Where Ax, Ay and Am are the absorbance of x, y and their
mixture at kF respectively, Cx and Cy are the
In isoabsorptive technique (Fig. 3) the spectra of the
concentrations of x and y respectively, ax and ay are the
same concentration of the two studied drugs should cross
absorptivities of x and y at kF respectively. When ax is
at a point called isoabsorptivity point at which they have
substituted by Fay
equal absorptivities while in absorpitivity factor method
the crossing point did not occur at equal concentration.
Crossing point is obtained only between different …………………… (42)
concentrations of the two drugs at which the …………………….... (43)
absorptivities of the two drugs are not equal but they are
equal to the inverse of the ratio of the used
So, the total concentration of the mixture (FCx +Cy) can
concentrations.[35]
be calculated by using a regression equation representing
the linear relationship between the absorbance of y and
its corresponding concentration at the absorptivity factor
point. The concentration of x can be determined after
subtraction of concentration of y and multiplication by
the inverse of F
……………. (44)

Absorpitivity factor method was successfully applied for

determination of salmeterol and fluticasone,[35] and
sodium cromoglicate and fluorometholone [36] in their
dosage forms.

11-Dual wavelength method

Fig. 3 : Zero order spectra of 8 μg. mL-1 salmeterol Dual wavelength method "also known as two
and 16 μg. mL-1 fluticasone showing the absorptivity wavelengths method" facilitates analyzing a component
factor points.[35] in presence of an interfering component by measuring
the absorbance difference (A) between two points in
Theory: For two drugs x and y, in the mixture, where the mixture spectrum. In this method (Fig. 4); one of the
concentration of y can be determined by using any of the drugs is considered as a component of interest and the
well established spectrophotometric methods; drug x can other drug is considered as an interfering component and
be determined by the absorpitivity factor method. This vice-versa. The basis for such method is the selection of
method depends on the calculation of the absorptivity two wavelengths where the interfering component shows
factor which is the ratio between the two absorptivities the same absorbance (A equals zero) whereas the
(ax, ay) at intersection point with the same absorbance component of interest shows significant difference in
value. This point is called the absorbtivity factor point absorbance with concentration. A between two points
(kF). This is summarized as follows[35] on the mixture spectra is directly proportional to the
concentration of the component of interest independent
………………………... (34) of interfering component. [37] This method was used for
simultaneous determination of different drugs, e.g.,
……………. (35) atenolol and indapamide, [8] drotaverine and aceclofenac,
[37]
Where atorvastatin and ezetimibe,[38] chlorphenir-amine and
phenylpropanolamine,[39] dexketoprofen and tramadol.[14]
………….………. (36)

………….…..………. (37)

…………………………. (38)

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(the divisor), therefore we can obtain the zero order

absorption spectrum (D0) of y (original spectrum of y).
…….. (48)

Concentration of y is calculated[42] by using the

regression equation representing the linear relationship
between the absorbance at its λmax versus the
corresponding concentration of y.

13-Mean centering of the ratio spectra

This method is applied for further improvement of the
selectivity to resolve the overlap present between drugs
Fig. 4: Selection of wavelengths for dual wavelength in binary and ternary mixtures. This eliminates the
method. [14] derivative step and therefore the signal-to-noise ratio is
enhanced. To explain the mean centering expression, let
12- Ratio subtraction method (RSM) us consider a three-dimensional vector
If you have a mixture of 2 drugs, x and y with
overlapping spectra, and the spectrum of y is extended ……………….. (49)
more than x, the determination of x can be done by
dividing the spectrum of the mixture by a certain We center or mean center (MC) this column by
concentration of y as a divisor (y0). subtracting the mean of three numbers calling
The division will give a new curve that
……………….. (50)
represents . If we subtract this
constant, then multiply the new curve obtained after
subtraction by y0, we will obtain the original zero-order ……….. (51)
D0 spectrum of x. This can be summarized in the
following equations
It could be proved that if the vector y is multiplied by n
Step1: ….. (45) (a constant number), the mean centered vector is also
multiplied by n and also if a constant number is added to
Step2: ….. (46) the vector y, the mean center of this vector is not
changed.[43] If there is no interaction among two
Step 3: ……………….. (47) components of a mixture, that is, x and y, and if Beer’s
law is obeyed for each compound, it can be expressed as
follows
The constant can be determined directly from the curve
……………….. (52)
by the straight line that is parallel to the wavelength
axis in the region where y is extended. [40] RSM was where AM is the absorbance of the mixture, a x and a y are
successfully applied for determination of the molar absorpitivies of x and y; and and are
multicomponent pharmaceutical products containing e.g.,
the concentrations of x and y, respectively. If Eq.
metronidazole and diloxanide,[32] amlodipine and
atorvastatin.[41] (52) is divided by , the ratio spectrum is obtained in
the form of the following equation
To determine the second component (y), an extension of
the already developed method has been established as a ……………….. (53)
new approach, and known as extended ratio subtraction
method ERSM, in which y could be determined by
dividing the obtained D0 spectrum of x by a known Since the mean centering of a constant is zero, mean
concentration of x as a divisor (x0) to get the value of the centering (MC) of eq. (53) would be obtained as
constant . Dividing the spectrum of the mixture (x + ……………….. (54)
y) by the same divisor ( ). The division will give a new Eq. (54) illustrates the mathematical explanation for
curve that represents , where is the analysis of binary components that permits the
determination of concentration of one compound without
previously obtained constant. If we subtract this constant, interference from the other compound of the binary
then multiply the obtained curve after subtraction by system, and vice versa.[21,38,41,43,44]

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recognizes that it is often better to measure many

14-Absorption factor method (AFM) nonselective signals and then combine them in
This method describes the analysis of a binary mixture multivariate model (multivariate analysis), whereby
where the two components x and y have overlapped multiple variables are considered simultaneously. A
spectra. Y shows interference at λ max of x, while x shows multivariate measurement is defined as one in which
no interference with y at another wavelength (λ 2). multiple measurements are made on a sample of interest.
So, more than one variable or response are measured for
each sample.[51,52] Multivariate methods include multiple
linear regression (MLR) methods and factor-based
methods.

The nature of the work makes it extremely convenient to

organize the data into matrices. In particular, it is useful
to organize the dependent and independent variables into
separate matrices. In the case of spectroscopy, if the
absorbance spectra of a number of samples of known
composition are measured, all these spectra are
assembled into one matrix called the absorbance
matrix. All of the concentration values for the
components of the sample are also assembled into a
Fig. 5: Zero order spectra of x and y and their separate matrix called the concentration matrix.
mixture.[36] Generally, MLR and PCR techniques employ data
organized as matrices of column vectors, while PLS
As shown in Fig. 5, the absorption spectra of x and y technique employs data organized as matrices of row
show severe overlap in the wavelength region of 200– vectors. [47] The data of matrices are organized into pairs;
300 nm. So, the absorption spectra of the standard each absorbance matrix is paired with its corresponding
solutions of the y with different concentrations were concentration matrix. The pair of matrices comprises a
recorded in the wavelength range of 200–400 nm, and data set. Data sets have different names depending on
their origin and purpose. [47] Training set is a data set
the average value of absorption factor ( ) was containing measurements on a set of known samples. It
is used to develop the calibration which is applied to
calculated. Since the absorbance of the mixture (x + y) at
predict the concentrations of unknown samples.
λ 2 nm is equal to that of pure y due to lack of
Training set should contain all expected components,
contribution of x at this wavelength, the absorption of x
span the concentration ranges of interest and contain
at could be calculated using the following equation: mutually independent samples. [47,51] Validation set is an
additional data set containing independent measurements
……… (55)
on samples that are independent from the samples used
to create the training set. Validation set is used to test
the validity of the calibration developed with the training
Where; and are the
set. The developed calibration is used to predict the
absorbance values of mixture at and concentrations of the components in the validation
samples. Then these predicted concentrations are
respectively, and is the absorption factor of pure y. compared to the actual concentrations. [47,51] The
absorbance matrix containing the unknown(s) spectra
The concentrations of x and y were calculated from the together with the corresponding result matrix containing
corresponding regression equation obtained by plotting the predicted concentrations comprise an unknown set.
the absorption values of the zero order spectra, at
and , against the corresponding concentrations, (I) Multiple Linear Regression (MLR) [53]
(a) Classical Least Squares (CLS) or (K-matrix)
respectively. AFM method has been developed for the
Classical least squares (CLS), sometimes known as K-
simultaneous determination of severalmixtures, e.g.,
matrix calibration, is so called because, originally, it
ramipril and olmesartan,[45] perindopril and
[46] involved the application of multiple linear regression
amlodipine, sodium cromoglicate and
(MLR) to the classical expression of the Beer-Lambert
fluorometholone.[36]
law of spectroscopy: [47]
A = KC …………………………….(56)
15- Multivariate chemometric methods
Chemometrics, in the most general sense, is the art of
Computing the calibration
processing data with various numerical techniques in
To produce a calibration using classical least squares, a
order to extract useful information.[47,48] Drug separation,
training set consisting of a concentration matrix, C, and
identification, determination and validation have been
an absorbance matrix, A, for known calibration samples
studied using chemometrics.[49,50] Chemometrics

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is required. We then solve for the matrix, K. Each C AT [AAT]-1 = P ……………………(71)

column of K will hold the spectrum of one of the pure
components. Since the data in C and A contain noise, Predicting unknowns [47]
there will, in general, be no exact solution for Eq. (56). Cunk = P Aunk ………………(72)
So, the best least squares solution for Eq. (56) has to be
found. In other words, K is found such that the sum of (II) Factor-based methods
the squares of the errors is minimized. The errors as in A factor space is nothing more than a particular
Eq. (57) are the difference between the measured spectra, coordinate system that offers certain advantages. When
A, and the spectra calculated by multiplying K and C. we operate in a factor space, instead of the native data
errors = KC – A……………….....(57) space, we are simply mapping our data into a new
To solve for K, each side of Eq. (56) is post-multiplied coordinate system. We are not actually changing the
by CT, the transpose of the concentration matrix. data itself. [47] Principal component regression (PCR) is
sometimes described as performing a least squares
A CT = KC CT…………………….(58) regression of the projections of the data onto the basis
A CT [CCT]-1 = K [CCT] [CCT]-1……..…………….. (59) vectors of a factor space using inverse least
A CT [CCT]-1 = K ……..……………….(60) squares.[47,20,55,56] Partial least squares (PLS) method is a
multivariate calibration method based on factor analysis.
Predicting unknowns The basic concept of PLS regression was originally
Aunk = K Cunk …………………….(61) developed by Wold. [57] A detailed description of the
KT Aunk = KT K Cunk …………..….(62) mathematical principles of the PLS algorithm was
[KTK]-1 KT Aunk = [KTK]-1 [KTK] Cunk ………(63) reported by Martens and Naes.[58] PLS method involves
[KTK]-1 KT Aunk = Cunk ………………………. (64) simultaneously the independent and the dependent
Kcal = [KTK]-1 KT ………………(65) variables in the data compression and decomposition
Cunk = Kcal Aunk ..…………………… (66) operations.[47,48] There are several reasons to use the
coordinate system of a factor space rather than the native
Kcal is called the calibration matrix or the regression space comprised of physically meaningful coordinates.
matrix. It contains the calibration, or regression, These reasons are numerical conditioning, reduced
coefficients which are used to predict the concentrations assumptions, noise rejection, new insights into the data
of an unknown from its spectrum. CLS can work quite and data compression.[47]
well under the right conditions. In particular, it is
important that the concentration values for all of the Any pair of axes lying in the plane which holds the
components present in the training samples are provided. spectra comprises a factor space for that data. Each axis
[47,51,54]
is a factor of the data space. These are usually called
abstract factors because they usually do not have an
(b) Inverse Least Squares (ILS) or (P-matrix) easily interpretable physical meaning. Instead of
Inverse least squares (ILS), sometimes known as P- specifying each spectrum in terms of all its wavelengths,
matrix calibration, is so called because, originally, it it will be specified in terms of its projections onto the
involved the application of multiple linear regression factors. These projections are often called the scores.
(MLR) to the inverse expression of the Beer-Lambert The projection of a spectrum onto the factors is nothing
law of spectroscopy: [47] more than the distance of that spectrum along the
C = PA ………………….(67) direction of each factor. We could find as many factors
as there are wavelengths in the spectra. Each factor will
Computing the calibration capture the maximum variance of the data that was not
P matrix will contain one row of coefficients for each yet spanned by the earlier factors and it must be mutual
component being predicted. Each row will have one orthogonal to all the factors that precede it.[47] The first
coefficient for each spectral wavelength. Thus, P will eigenvector spans the maximum variance of the data that
have as many columns as there are spectral wavelengths. can be spanned with a single vector. That is why this
Since the data in C and A contain noise, there will, in eigenvector is also called the first principal component of
general, be no exact solution for Eq. (67), so, the best the data set. The second eigenvector of the data will
least squares solution for Eq. (67) has to be found. In span the maximum possible amount of the remaining
other words, P is found such that the sum of the squares variance that was not spanned by the first factor. Each
of the errors is minimized. The errors are the difference eigenvector has an eigenvalue associated with it. The
between the measured concentrations, C, and the eigenvalue of an eigenvector is equal to the sum of the
concentrations calculated by multiplying P and A: squares of the projections of the data onto the
errors = PA – C ……………..(68) eigenvector [47] as shown in Fig. 6. These calibrations
involve multiple steps. First, a set of mutually
To solve for P, each side of Eq. (67) is post-multiplied by orthogonal vectors (factors) is found that spans the data
AT, the transpose of the absorbance matrix. space. Next, it is decided how many factors should be
C AT = P AAT ……..………………(69) kept to use in the calibration. Finally, calibration
C AT [AAT]-1 = P [AAT] [AAT]-1 ……………….(70)

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Kamal et al. European Journal of Pharmaceutical and Medical Research

matrices and/or coefficients are generated using the

selected number of factors.[53]

EV=30
Y
PC1

EV=00
X
PC3

PC2 EV=1.7 Fig. 7: PRESS versus number of factors for a PCR

model.[47]
Fig. 6: Mapping of the data of a binary mixture into a
new coordinate system then, plotting of the first Computing the calibration [47]
principal component (PC1) of highest eigenvalue, the Aproj = VcTA …………………(73)
second principal component (PC2) perpendicular to
the first one (PC1) and the third principal component where: Aproj is the matrix containing the new
(PC3) perpendicular to both (PC1) and (PC2) for data coordinates (the projections), A is the original training
of a binary mixture. [47] set absorbance matrix, Vc is the matrix containing the
basis vectors, one column for each factor retained.
(a) Principal Component Regression (PCR) Substitute Aproj into Eq. (67) in place of A; and P matrix
Principal component regression is sometimes described here in PCR is called F matrix
as performing a least squares regression of the C = F Aproj …………………..(74)
projections of the data onto the basis vectors of a factor C ATproj = F Aproj ATproj ……………….. (75)
space using ILS. [47] CATproj[Aproj ATproj ]-1= F AprojATproj[Aproj ATproj]-1
…………..(76)
Generating the abstract factors (eigenvectors) C ATproj [Aproj ATproj]-1 = F........................ (77)
All of the factors for the data matrix can be calculated
using a number of different algorithms. Strictly Predicting unknowns [47]
speaking, it is not generally needed to calculate all of the Cunk = F VcT Aunk ……………….. (78)
factors. The first N factors need only to be calculated Cunk = Fcal Aunk ……………….. (79)
where N is large enough to determine how many factors Where: Fcal (PCR calibration matrix) = the quantity VcT
should be included in the basis space. [47] F pre-calculated at calibration time.
(b) Partial Least Squares (PLS)
Keeping the significant factors: if the number of factors
retained is more than the required, more noise will be Like PCR, PLS involves the generation of abstract
added to the calibration and errors may occur. On the factors. However, instead of generating factors for the
other hand, if the number retained is too small, spectral information only, it also generates factors for the
meaningful data that could be necessary for the concentration information. Then, it rotates the spectral
calibration might be discarded.[47,52,53] Several indicators and concentration factors towards each other in order to
or functions offered by Chemometrics TOOLBOX for optimize the regression between the spectral data and the
use with MATLAB [53] could be used for determining the concentration data. [47,53] Since the spectral noise is
optimum number of factors (rank). independent from the concentration noise, a perfectly
For example, PCAPRESS indicator which generates a linear relationship is no longer possible. So, the best
calibration for every possible rank (number of factors which can be done is to restore optimum congruence in
retained). Then, use each calibration to predict the the least squares sense. The main advantage of the PLS
concentrations for a set of independently measured, method is based on the interrelated decomposition of the
independent validation samples. The predicted residual concentration matrix C and the absorbance matrix A, so
error sum-of-squares, or PRESS, for each calibration, is that with this algorithm the most robust calibration at
calculated and the calibration that provides the best present can be obtained. PLS calibration can be used for
results is selected. The number of factors used in that very complex mixtures since only knowledge of
calibration is the optimal rank for that system [47,51,59] as constituents of interest is required. [47,48,51]
shown in Fig. 7. Computing the calibration: [20, 47,48] absorbance values
obtained over a preset wavelength range are used to
construct an absorbance matrix A of n x m dimensions
(where n is the number of mixtures and m that of

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Kamal et al. European Journal of Pharmaceutical and Medical Research

wavelengths). Analyte concentrations are also arranged 6. Skoog DA,Holler FJ,Crouch SR. Principles of
in a matrix C of n x p dimensions (where p is the number Instrumental Analysis. 6th. ed., Canada; Thomson
of mixture components). Both A and C are resolved into Corporation: 2007.
two smaller matrices S and L (the scores and loadings 7. Beckett AH,Stenlake JB. Practical Pharmaceutical
matrices, respectively) plus an error matrix (E) Chemistry. part II, 4th. ed., London; Bloomsbury
A = SALA + EA………………. (80) Publishing: 2001.
C = SCLC + EC ........................ (81) 8. Fernandes N,Nimdeo MS,Choudhari VP,Kulkarni
RR,Pande VV,Nikalje AG. (Dual wavelength and
where SA is the absorbance score matrix, LA is the simultaneous equation spectrophotometric methods
absorbance loading matrix, SC is the concentration score for estimation of atenolol and indapamide in their
matrix, LC is the concentration loading matrix; E A and EC combined dosage form ). Int J Chem Sci, 2008; 6(1):
are the error matrices, the dimensions of which coincide 29-35.
with those of the original absorbance matrix (n x m) and 9. Chitlange SS,Soni R,Wankhede SB,Kulkarni AA.
concentration matrix (n x p), respectively. Matrices L A (Spectrophotometric methods for simultaneous
and LC can be related via a diagonal matrix estimation of dexibuprofen and paracetamol). Asian
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Co = Ao (SCA)-1 VLC …......................(83) AA. (Simultaneous determination of loratadine and
Where SC, LC and A can be obtained from the calibration pseudoephedrine sulfate in pharmaceutical
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