**4. Analytical application of derivative spectrophotometry**

Derivative spectrophotometry (DS) has found a wide application in quantitative chemical analysis. As the latest applications have been gathered and described in reviews published previously [8,9], this part is focused on the recent use of DS. Based on scientific literature the following fields of application of derivative spectrophotometry can be distinguished:

Basic Principles and Analytical Application of Derivative Spectrophotometry 259

**C**

190 240 290 340 390

**Wavelength, nm**

Fig. 2. Zero order (A) and fifth derivative order spectra of ethanolic solution of retinol acetate (10 ppm). Spectrum B has been obtained by gradual derivatisation, spectrum C by direct generation of fifth derivative from zero-order spectrum. Apparatus working conditions: Hewlett-Packard HP-8452A diode array spectrophotometer with following working parameters: integration time 1 s, spectral bandwidth 2nm, spectrum scan 0.1 s. Derivative spectra were generated using Savitzky Golay algorithm by PC computer equipped with Excel for Microsoft Windows (∆λ=14 nm, fourth polynomial degree).

Derivative spectrophotometry (DS) has been mainly used in pharmaceutical analysis for assaying of a main ingredient in a presence of others components or its degradation product. Pharmaceutical samples are characterised by high level of constituents and presence of a relatively simple and stable matrix. The spectral influences of disturbing compounds are easy to remove by derivatisation of spectra. The most numerous procedures based on derivative spectra have been devoted for determination of one components without sample purification. Another field of DS application is the use of it for simultaneous determination of two or more components. As a form of derivative spectrum is more complicated in comparison to its initial zero order, usually derivatives of low orders are employed for analytical purposes. Procedures used DS for pharmaceutical analysis are

Derivative spectrophotometry was applied in different than pharmaceutical analysis areas of analysis. This method was utilised for the determination of amphothericin in various biological samples like plasma, serum, urine and brain tissue [40]. The combination of ratio spectra with their derivatisation allowed to remove spectral interferences caused by a

a. Multicomponent analysis




0

0.1

**Fifth derivative**

0.2

0.3

0.4

assembled in Tables 1 and 2.

presence of bilirubin in plasma [40].

b. Others applications


Fig. 2. Zero order (A) and fifth derivative order spectra of ethanolic solution of retinol acetate (10 ppm). Spectrum B has been obtained by gradual derivatisation, spectrum C by direct generation of fifth derivative from zero-order spectrum. Apparatus working conditions: Hewlett-Packard HP-8452A diode array spectrophotometer with following working parameters: integration time 1 s, spectral bandwidth 2nm, spectrum scan 0.1 s. Derivative spectra were generated using Savitzky Golay algorithm by PC computer equipped with Excel for Microsoft Windows (∆λ=14 nm, fourth polynomial degree).

#### a. Multicomponent analysis

258 Macro to Nano Spectroscopy

a. Multicomponent analysis. This group is the most numerous. The goal of proposed methods is application of DS for determination of one analyte in presence of matrix or

b. Calculation of some physico-chemical constants, e.g. reaction, complexation or binding

**A**

190 240 290 340 390

**B**

190 240 290 340 390

**Wavelength, nm**

**Wavelength, nm**

for simultaneous assaying of few analytes.

c. Application for investigation of some processes kinetics[11, 12].

constants [10].

0.00E+00




**Fifth derivative**

0.005

0.01

0

2.00E-01

4.00E-01

6.00E-01

**Absorbance**

8.00E-01

1.00E+00

1.20E+00

1.40E+00

Derivative spectrophotometry (DS) has been mainly used in pharmaceutical analysis for assaying of a main ingredient in a presence of others components or its degradation product. Pharmaceutical samples are characterised by high level of constituents and presence of a relatively simple and stable matrix. The spectral influences of disturbing compounds are easy to remove by derivatisation of spectra. The most numerous procedures based on derivative spectra have been devoted for determination of one components without sample purification. Another field of DS application is the use of it for simultaneous determination of two or more components. As a form of derivative spectrum is more complicated in comparison to its initial zero order, usually derivatives of low orders are employed for analytical purposes. Procedures used DS for pharmaceutical analysis are assembled in Tables 1 and 2.

b. Others applications

Derivative spectrophotometry was applied in different than pharmaceutical analysis areas of analysis. This method was utilised for the determination of amphothericin in various biological samples like plasma, serum, urine and brain tissue [40]. The combination of ratio spectra with their derivatisation allowed to remove spectral interferences caused by a presence of bilirubin in plasma [40].

Basic Principles and Analytical Application of Derivative Spectrophotometry 261

Compound Characteristic of the method Reference agreement with those obtained by HPLC and TLC methods.

> Derivative spectrophotometry was used for quantification of fluphenazine, pernazine and haloperidol in their preparations. First and second derivative were applied for determination of active ingredients in pharmaceutical

methods were proposed and utilised for determination of

First derivative of ratio spectra was used for determination

of analyte in presence of its degradation product.

were applied for determination of ertapenem in the presence of its degradation product. The analyte was assayed by the first method at 316 nm in the range 4-60 μg mL-1. The second method allowed ertapenem determination at 298 nm and 316 nm in the same concentration range using spectrum of degradant at 28 μg mL-1as a divisor.

Compound Characteristic of the method Reference

The quantification was achieved using first-order derivative method. Rupatadine was determined at 273.46 nm, while montelukast at 297.27 nm. The method was applied for determination of both compounds in their combined dosage

First derivative of ratio spectra method was applied for

The first-derivative method was proposed for simultaneous determination of both compounds. The measurements of amplitude was done at 230.5 and 280 nm for tramadol (Trama) and ibuprofen (Ibu), respectively. The linearity was obeyed in the rage 5-50 μg mL-1 for Trama and 5-100 μg mL-

First derivative of ratio spectra method was applied for simultaneous determination of both analyte. The amplitudes at 231 nm and 260 nm were used for

simultaneous determination of both analytes in pharmaceutical formulations and in laboratory-made

First derivative spectra were used for simultaneous determination of drugs in synthetic mixtures. The linearity in the ranges 10-40 μg mL-1 and 10-50 μg mL-1 were obeyed for democlocycline and minocycline, respectively. The method was applied for the analysis of these drugs in

clinical samples, urine and honey.

Ertapenem First derivative and first derivative of ratio spectra methods

22

23

24

25

26

27

28

29

30

Fluphenazine Pernazine Haloperidol Promazine

Oxybutynin hydrochloride

Democlocycline and minocycline

Rupatadine and montelukast

Ambroxol and doxycycline

Tramadol and ibuprofen

Sodium

itopride

rabeprazole and

preparations.

Table 1. Determination of one component in sample

form.

mixtures.

1 for Ibu.

Ezetimibe First, second and third derivative spectrophotometric

ezetimibe in pharmaceuticals.


chloranilic acid. First derivative spectrophotometry has been evaluated by measuring the derivative signal at 475.72 nm – 588.40 nm (peak to peak amplitude). Calibration graph was

at wavelengths of 263.8 and 255.4 nm for third- and fourthderivative, respectively. The method was found to be linear (r2> 0.999) in the range of 10-100 μg mL-1 for tropicamide in the presence of excipients. The method was applied for

Derivative spectrophotometry used for determination of

second derivative values measured at 230 nm (n=6) were used for the quantitative determination of the drug . Calibration graphs were linear in the concentration range of

proposed for determination of galanthamine. Absorbance was measured at 277.4 nm. It obeyed Lambert-Beer's law in

determination of doripenem in pharmaceuticals in the presence of its degradation products. The Beer low was

quantification was done by measurement of first-derivative amplitude at 271.9 nm. The obtained results were in a good

obeyed in the range (0.42-11.30)x10-2 mg L-1.

Tropisetron The first derivative spectra were applied for determination of analyte in the presence of its degradates. The

olanzapine using 2-10 μg mL-1 for first and second

The proposed methods were based on the reaction of gemifloxacin with chloranilic acid and parachloranil to give highly coloured complexes. The coloured products were quantified spectrophotometrically at 530 nm and 540 nm at zero order, 590 and 610 nm for the first derivative and 630 and 650 nm for second order derivative. Beer's law was obeyed in the concentrations range of 10 to 60 μg mL-1, 5 to25 μg mL-1 at zero order, 5 to 25 μg mL-1, 5 to 40 μg mL-1 at first order and 2 to 20 μg mL-1 and 2 to 14 μg mL-1 at second

13

14

15

16

17

18

19

20

21

Compound Characteristic of the method Reference

Sertraline The proposed method is based on reaction of sertraline with

established for 5-100 μg mL-1 of sertraline

analyte determination in eye drops.

nebivolol in bulk and in preparates.

Olanzapine The first derivative values measured at 222 nm and the

derivative spectrophotometric method.

Galanthamine The 1st derivative zero crossing spectrophotometry was

Doripenon The first derivative spectrophotometry was used for

the range of 30-80 μg mL-1.

0.40 mg mL-1.

order.

Tropicamide The measurements were carried out

The first-order derivative spectra were used for determination of estradiol in tablet. Measurements of derivative were made at 270 nm. The method showed specificity and linearity in the concentration range of 0.20 to

Estradiol valerate

Nebivolol hydrochloride

Gemifloxacin mesylate


Table 1. Determination of one component in sample


Basic Principles and Analytical Application of Derivative Spectrophotometry 263

Compound Characteristic of the method Reference

of Tween 80 was applied. Next the first and second derivative spectra of complexes were applied for

The reaction of studied ions with pyrogallol red at presence

37

38

39

quantification of calcium and magnesium in multivitamin preparations, samples of human serum and in drinking

The proposed method utilise the reaction of studied ions with morpholinedithiocarbamate (MDTC). Derivative spectra of generated complexes allowed simultaneous determination of Cu and Pd in pharmaceutical samples, synthetic mixtures, alloys and biological samples.

First to fourth derivative spectra of components were subjected to chemometric analysis (principal component regression, PCR; partial least squares with one dependent variable, PLS-1; three dependent variables, PLS2) and adopted for multicomponent analysis. The third derivative spectra of all ingredients became a basis of quantification

The fourth-derivative spectra of molybdenum complexes of tetramethyldithiocarbamate (tiram) fungicide were used for its quantification in commercial samples and in wheat grains [41]. Atrazine and cyanazine were assayed in food samples by first- derivative spectrophotometry [42]. In order to improve results of assay, the first-derivative spectra of the binary mixture were subjected to chemometric treatment (classical least squares, CLS; principal component regression, PCR and partial least squares, PLS). A combination of firstderivative with PCR and PLS models were applied for determination of both herbicides in biological samples [42]. A first-derivative spectrophotometry was used as a reference method for simultaneous determination Brillant Blue, Sunset Yellow and Tartrazine in food [43].

First derivative of ratio spectra was applied for determination of strontium, magnesium and calcium in Portland cement [44]. The proposed procedure was based on complexation of

As it is mentioned above, derivative spectrophotometry seems to be a very useful tool for physico-chemical studies. It can be applied for investigation of reaction kinetics [11,12], or

First derivative spectra of levomepromazine (LV) and its sulphoxide were employed for investigation of LV photodegradation [11]. The degradation process of biapenem was monitored by measurement of first-derivative amplitude at 312 nm [12]. The determined rate constants for studied process were in good agreement with those obtained by HPLC method [12]. The second-order derivative spectrophotometric method was used for investigation of solvolytic reaction 2-phenoxypropionate ester of fluocinolone acetonide [45]. The run of process was observed by measurement the second-order amplitude at 274.96 nm corresponded to fluocinolone acetonide. The solvolysis rate constant was calculated using

derivative method and compare with those obtained by HPLC methods [45].

Calcium and magnesium ions

Copper and palladium

Paracetamol, propiphenazone and caffeine

water.

method.

studied ions with Alizarin Complexone.

for determination of chemical reaction constants.

Table 2. Application of DS for multicomponent analysis


Compound Characteristic of the method Reference

simultaneous estimation of alprazolam (ALP) and fluoxetine hydrochloride (FXT) in pure powder and formulation. Quantitative determination of the drugs was performed at 232.14 nm and at 225.25 nm for ALP and FXT,

respectively. Quantification was achieved over the

The second-order derivative spectra were used for simultaneous determination of drotaverine (DRO) and mefenamic acid (MEF). Calibration graphs were constructed over the concentration range of 4-24 μg/mL-1 for DRO and MEF. Detection and quantitation limit were 0.4348 and 1.3176 μg/mL-1 for DRO and 0.6141and 1.8611 μg/mL-1 for MEF. The method was applied for determination of both

Second derivative spectrophotometric method was proposed for simultaneous determination of

PSE and TRI were measured at 271 and 321 nm,

pseudoephedrine hydrochloride (PSE) and triprolidine hydrochloride (TRI). The second derivative amplitudes of

respectively. The calibration curves were linear in the range of 200 to 1,000 μg mL-1 for PSE and 10 to 50 μg mL-1for TRI.

The method was based on the second-derivative spectra of both ingredients. The amplitude at 254.0 nm was used for clopidogrel bisulphate, while at 216.0 nm for aspirin. The linearity was obeyed in the range 5.0- 30.0 μg mL-1 for both

The first-order derivative spectrophotometric method was proposed for simultaneous determination of analytes in their mixtures. The measurements were carried out at 219 and 265 nm for simvastatin and ezetimibe respectively. The validation of method was done. The range of application was estimated to be 2-40 μg mL-1 for simvastatin in the presence of 10 μg mL-1 ezetimibe and 1-20 μg mL-1 of ezetimibe in the presence of 20 μg mL-1 of simvastatin.

The proposed method was based on the first derivative spectra of Al3+ and Fe3+complexes with chrome azurol S. The proposed procedure was successfully applied for simultaneous determination of studied ions in standard mixtures, pharmaceuticals and in post-haemodialysis

ingredients in combined dosage forms.

Second derivative spectrophotometry (D2) was applied for

31

32

33

34

35

36

concentration range 4-14 μg mL-1 for both drugs with mean recovery of 99.36 ± 0.84 and 99.60 ± 0.93 % for ALP and FXT,

hydrochloride quantification of rabeprazole and itopride, respectively.

respectively.

compounds.

samples.

Alprazolam and fluoxetine hydrochloride

Drotaverine hydrochloride and mefenamic

Triprolidine hydrochloride

Clopidogrel bisulphate and aspirin

Simvastatin and ezetimibe

Fe(III) and Al(III)

ions

pseudoephedrine hydrochloride

acid

and


Table 2. Application of DS for multicomponent analysis

The fourth-derivative spectra of molybdenum complexes of tetramethyldithiocarbamate (tiram) fungicide were used for its quantification in commercial samples and in wheat grains [41]. Atrazine and cyanazine were assayed in food samples by first- derivative spectrophotometry [42]. In order to improve results of assay, the first-derivative spectra of the binary mixture were subjected to chemometric treatment (classical least squares, CLS; principal component regression, PCR and partial least squares, PLS). A combination of firstderivative with PCR and PLS models were applied for determination of both herbicides in biological samples [42]. A first-derivative spectrophotometry was used as a reference method for simultaneous determination Brillant Blue, Sunset Yellow and Tartrazine in food [43].

First derivative of ratio spectra was applied for determination of strontium, magnesium and calcium in Portland cement [44]. The proposed procedure was based on complexation of studied ions with Alizarin Complexone.

As it is mentioned above, derivative spectrophotometry seems to be a very useful tool for physico-chemical studies. It can be applied for investigation of reaction kinetics [11,12], or for determination of chemical reaction constants.

First derivative spectra of levomepromazine (LV) and its sulphoxide were employed for investigation of LV photodegradation [11]. The degradation process of biapenem was monitored by measurement of first-derivative amplitude at 312 nm [12]. The determined rate constants for studied process were in good agreement with those obtained by HPLC method [12]. The second-order derivative spectrophotometric method was used for investigation of solvolytic reaction 2-phenoxypropionate ester of fluocinolone acetonide [45]. The run of process was observed by measurement the second-order amplitude at 274.96 nm corresponded to fluocinolone acetonide. The solvolysis rate constant was calculated using derivative method and compare with those obtained by HPLC methods [45].

Basic Principles and Analytical Application of Derivative Spectrophotometry 265

spectrum are strongly magnified in derivative spectrum. Application of derivative spectrophotometry requires from analyst knowledge about its specific properties. The main disadvantage of derivative spectrophotometry is its poor reproducibility. It is result of strong dependence of derivative spectrum on recording parameters of used spectrophotometer like scan rate, spectral width of beam, integration time and interpoint distance[1, 5, 7]. Zero-order spectra of the same substance obtained on different spectrophotometers can be identical, but derivatisation of them gives different results. The generated derivative spectra can derived in intensity, shape and positions of maxima and minima. So restoration of given literature method requires to use the same type of apparatus with the same working parameters described in an article or reoptimisation parameters of

Optimisation of used working spectrophotometer parameters should be done when a new derivative-spectrophotometric method is elaborated. A construction of some spectrophotometers does not allow to check influence of whole factors, but if more

As a result of derivatisation is closely connected with geometrical features of a zero-order spectrum, it is obvious that a method of spectrum registration is a key-point. The use of broad beams gives the averaged smoothed zero order spectra. Application of narrow beams results in intensification and narrowing of absorption bands. But from the other hands, the narrowing of monochromator' slit increases an effects connected with beams bending on edges of the slit. The edge phenomenon causes additional noises which are recorded with absorbance. So the absorption spectrum recorded with too narrow monochromator' slit can

Interpoint distance of registered spectrum is very important parameter. Absorption spectrum obtained by spectrophotometer possess a digital structure which is the result of construction of a monochromator and a manner of registration. Spectra registered with large

A level of noise enclosed in zero order spectrum directly influences a quality of generated derivative spectrum. It was proved that spectra registered with low scan rates and long integration times are less biased by noise. This is advantageous if high order derivative are

Taking into account above information, it is obvious that reproducibility of method based on derivative spectrophotometry depends on reproducibility of parameters of registration of zero-order spectra. So, adaptation of elaborated in another laboratory derivative spectrophotometric method, requires application the same working parameters as used by authors. But this problem is completely ignored by scientists. Based on analysis of articles concerned on application of derivative spectrophotometry it could be stated that working parameters of spectra registration are very rarely given [8]. There is noticeable lack of standardisation in description of procedures based on derivative spectra. Very often, authors of scientific articles give only information what model of apparatus they used without any details of its working parameters as well as algorithm for derivatisation of spectra. In this case the published procedure can be used only if our laboratory is equipped with the same model of spectrophotometer supplied with the same software. Otherwise verification of literature' method requires reoptimisation, adaptation to our conditions

interpoint distance are averaged, flat without many spectral details.

method on an own spectrophotometer.

be distorted by high level of noise.

generated [7].

advanced equipment is available it is worth to do.

An interesting application of derivative spectrophotometry was described by Wu and Zivanovic [46]. They proposed the use of the first derivative spectra for determination of the degree of acetylation of chitin and chitosan. They employed the evaluated procedure for commercial samples.
