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METHODS FOR THE ESTIMATION AND VALIDATION OF MULTICOMPONENT FORMULATION

 

Clinical courses

About Authors:
B. Madhavi*, Dr. P.Venkateswara Rao, P.Rama Bharathi, T.Swathi, BH. Ramya Reddy
A.M Reddy Memorial College of Pharmacy, Narasaraopet
ANU University, Guntur.
*madhavi.pharm@gmail.com

Abstract:
The aim of the present work was focused on development of analytical methods for the estimation of drugs in multi component dosage form. There is a plethora of analysis of such formulations without prior separation. For the estimation of multi component formulation, the instrumental techniques, which are commonly employed, are spectrophotometry, Gas liquid chromatography (GLC), high performance thin layer chromatography (HPTLC), high performance liquid chromatography (HPLC) etc. These methods are based upon the measurement of specific and non specific physical properties of the substances. Chromatographic separation techniques are one of the most widely used technique for analysis of a multi component formulation. HPLC techniques allows for the separation as well as analysis of different drugs that are present in a combined formulation. Validation studies were performed in order to assess the validation parameters for the analytical method developed in accordance to ICH Guidelines.

Reference ID: PHARMATUTOR-ART-1955

Introduction:
Analytical chemistry is primarily concerned about determining the qualitative and quantitative composition of material understudy. Both these aspects are necessary to understand the sample material. Analytical chemistry is divided into two branches quantitative and qualitative. A qualitative analysis gives us the information about the nature of sample by knowing about the presence or absence of certain components. A quantitative analysis provides numerical information as to the relative amount of one or more of this component. For analyzing the drug samples in bulk, pharmaceutical formulations and biological fluids, different analytical methods are routinely being used.


In non-instrumental, the conventional and physicochemical property are use to analyse the sample. The instrumental methods of analysis are based upon the measurements of some physical property of substance using instrument to determine its chemical composition. The instrumental methods are simple, precise, and reproducible as compared to classical methods. Therefore, analytical methods developed using sophisticated instruments such as spectrophotometer, HPLC, GC and HPTLC have wide applications in assuring the quality and quantity of raw materials and finished products.

1. Spectrophotometric Method:
Absorption spectroscopy is one of the most useful and widely used tools available to the analyte for quantitative analysis. The relation between the concentration of analyte and the amount of light absorbed is the basis of most analytical application of molecular spectroscopy. This method of analysis i gaining importance due to simple, rapid, precise, highly accurate and less time consuming. Spectrophotometric multi-component analysis can be applied where the spectra of drugs overlaps. In such cases overlapping spectra, simultaneous equation can be framed to obtain the concentration of individual component; otherwise multi-component analysis can be applied on any degree of spectral overlap provided that two or more spectra are not similar exactly. Some of the examples are given in table-1.


1.1 The various spectroscopic techniques used for multi-component analysis are as follows

Simultaneous equation method (Vierodt’s method)
Concentration of several components present in the same mixture can be determined by solving a set of simultaneous equation even if their spectra overlap. If Beer’s law is followed, these equations are linier.

Two wavelength method
The method can be used to calculate the concentration of component of interest found in a mixture containing it along some unwanted interfering component. The absorption different between two points on the mixture spectra is directly proportional to the concentration of the component to be determined irrespective of the interfering component.

The absorption ratio method
The absorbance ratio method is a modification of the simultaneous equation procedure. It depends on the property that for a substance, which obeys Beer’s law at all wavelength, the ratio of absorbance at any two wavelengths is constant value independent of concentration or path length. e.g. Two dilutions of the same substance give the same absorbance ratio A1 / A2. In the USP, this ratio is referred to as Q value. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbances are measured at two wavelengths. One being the λ max of one of the components (λ2) and the other being a wavelength of equal absorptivity of the two components (λ1), i.e., an iso-absorptive point.

Geometric correction method
A number of the mathematical correction procedures have been developed which reduce or eliminate the background irrelevant absorption that may be present in the samples of biological origin. The simplest of this procedure is the three-point geometric procedure, which may be applied if the irrelevant absorption is linier at the three wavelengths selected. This procedure is simply algebraic calculations of what the baseline technique in infrared spectrophotometry dose graphically.

Absorption factor method (Absorption correction method)
It is further modification of simultaneous equation method. Quantitative determination of one drug is carried out by E (1%, 1 cm) value and quantitation of another drug is carried out by subtraction absorption due to interfering drug using absorption factors.

Orthogonal polynomial method
The technique of orthogonal polynomials is another mathematical correction procedure, which involves complex calculation than the three-point correction procedure. The basis of the method is that an absorption spectrum may be represented in terms of orthogonal functions.

Difference spectrophotometry
Difference spectrophotometry provides a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in mixture. The essential feature of difference spectrophotometric assay is that the measured value is the difference absorbance (?A) between two equimolar solutions of the analyte in different chemical forms, which exhibits different spectral characteristics.

Derivative spectrophotometry
Derivative spectrophotometry is useful means of resolving two overlapping spectra and eliminating matrix interference due to an indistinct shoulder on side of an absorption bands. It involves conversion of normal spectrum [A= f (λ)]to its first [dA/ dλ = f (λ)], second [d2A/ dλ2 = f (λ)]and higher derivatives spectra where the amplitude in the derivative spectrum is proportional to the concentration of the analyte provided that Beer’s law is obeyed by the fundamental spectrum.

Area under curve method
In this method, the absorptivity values (ε1 andε2) of each of the two drugs were determined at the selected wavelength range. Total area under curve of a mixture at wavelength range is equal to the sum of area under the individual component at that wavelength range. This method is applicable when the λ max of the two components are reasonably dissimilar, the two components do not interact chemically and both the component must be soluble in same solvent.

Table- 1

List of multi-component formulation estimated by UV-visible spectrophotometer with respective reported references.

Sr. NO.

Combination of drugs

Therapeutic Use

Ref. No.

1

Acetyl salicylic acid, caffeine and codeine phosphate

NSAID

7

2

Acrivastine and pseudo ephedrine HCl

Respiratory System

8

3

Ambroxol HCl and cetrizine

Respiratory System

9

4

Amiloride, hydrochlorothiazide and atenolol

CVS

10

5

Amlodipine besylate and enalapril maleate

CVS

11

6

Amlodipine besylate and lisinopril

CVS

12

7

Amlodipine besytale and lisinopril

CVS

13

8

Amoxycillin and probencid

Antiinfective

14

9

Amoxycillin, ampicillin and cloxacillin

Antiinfective

15

10

Ampicillin and cloxacillin

Antiinfective

16

11

Aspirin compound tablet

NSAID

17

12

Aspirin, acetaminophen and ascorbic acid

NSAID

18

13

Atenolol and nefedipine

CVS

19

14

Benazepril and amlodipine besylate

CVS

20

15

Benazepril and hydrochlorthiazide

CVS

21

2.  Chromatographic methods:
Chromatography is a technique employed for separation of the components of mixture by continues distribution of the component between two phases. One phase moves (mobile phase) over the other phase (stationary phase) in a continuous manner. When the stationary phase is a solid support of adsorptive nature and mobile phase is liquid or gaseous phase it is called Adsorption chromatography. When the stationary phase is liquid in nature and the mobile phase is liquid or gaseous it is called Partition Chromatography.

2.1 Theory of Chromatography:
Two theoretical approaches have been developed to describe the processes involved in the passage of solutes through a chromatographic system.

1. The plate theory:
According to martin and synge, a chromatographic system consists of discrete layers of theoretical plates. At each of these, equilibration of the solute between the mobile and stationary phases occurs. The movement of solute is considered as a series of stepwise transfers from plate to plate.

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2. The rate theory:
This theory considers the dynamics of the solute particles as it passes through the void space between the stationary phase particles in the system as a well its kinetic as it is transferres to and from the stationary phase.

2.2 Phases of Chromatography:

1. Normal Phase Mode:
In normal phase mode the stationary phase is polar and the mobile phase is non polar in nature. In this technique, non polar compounds travel faster and are eluted first. This is because of the lower affinity between the non polar compounds and the stationary phase. Polar compounds are retained for longer times because of their higher affinity with the stationary phase. These compounds, therefore take more time to elute. Normal phase mode of separation is therefore, not generally used for pharmaceutical applications because most of the drug molecules are polar in nature and hence take longer time to elute.

2. Reversed Phase Mode:
It is the most popular mode for analytical and preparative separations of compound of interest in chemical, biological, pharmaceutical, food and biomedical sciences. In this mode, the stationary phase is non polar hydrophobic packing with octyl or octa decyl functional group bonded to silica gel and the mobile phase is polar solvent. The polar compound gets eluted first in this mode and non polar compounds are retained for longer time. As most of the drugs and pharmaceuticals are polar in nature, they are retained for longer times and hence elute faster. The different columns used are Octa Decyl Silane (ODS) or C18, C8, C4 ( in the order of ncreasing polarity of the stationary phase). An aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as ionization control, ion pairing and complexation) to control retention and selectivity.

3. Ion exchange chromatography:
The stationary phase contains ionic groups like NR3+ or SO3-, which interact with the ionic groups of the sample molecules. This is suitable for the separation of charged molecules only. Changing pH and salt concentration can modulate the retention. By definition, ion exchange is used mainly for selective removal (in purification) or fractionation (in separation) of ionic species.

4. Ion Pair Chromatography:
This technique is also referred to as Reversed Phase Ion Pair Chromatography or Soap Chromatography. It may be used for the separation of ionic compounds and this method can also substitute for Ion Exchange Chromatography. Strong acidic and basic compounds can be separated by reversed phase mode by forming ion pairs with suitable counter ions.

5. Affinity Chromatography:
This technique uses highly specific biochemical interactions for separation. The stationary phase contains specific groups of molecules which can adsorb the sample if certain steric and charge related conditions are satisfied. This technique can be also used to isolate proteins, enzymes as well as antibodies from complex mixtures.

6. Size Exclusive Chromatography:
It separates molecules according to their molecular mass. Largest molecules are eluted first and the smallest molecules last. This method is generally used when a mixture contains compounds with a molecular mass difference of at least 10%. This mode can be further subdivided into gel permeation chromatography (with organic solvents) and gel filtration chromatography (with aqueous solvents).

2.3 Separation techniques:
1.
Isocratic elution:
When the mobile –phase composition does not change throughout the course of the run, it is said to be isocratic. A mixed mobile phase can be delivered at a constant ratio by the pumps themselves or the solvent mixture can be prepared prior to analysis and pumped through a single reservoir. This is the simplest technique and should be the method of first choice when developing a separation.

2. Gradient Elution:
HPLC can be performed with changes in composition of mobile phase over time (gradient elution). The elution strength of the eluent is increased during the gradient run by changing polarity, pH, or ionic strength. Gradient elution can be a powerful tool to separate mixtures of compounds with widely different retention.

2.4 High performance thin layer chromatography (HPTLC):
The principle is based on plane chromatography. The mobile phase normally is driven by capillary action. The prominent advantages of this technique includes possibilities of separating of up to 70 samples and standard simultaneously on a single plate leading to high throughout, low cost analogs and the ability to construct calibration curves from standard chromatography under the same condition as the sample. Analyzing a sample by use of multiple separation steps and static post chromatographic detection procedures with various universal and specific visualization regents that are possible because all the sample components are stored on the layer without the chance of loss. Some of the examples are given in table-2.

Table-2

List of multi-component formulation estimated by reversed phase high performance thin layer chromatography (HPTLC) with respective reported references.

Sr. NO.

Combination of drugs

Therapeutic Use

Ref. No.

1

Cephalexin acid cefadoxil

Antiinfective

59

2

Cinnarzine and domperidon maleate

GIT

60

3

Gliclazide and metformine HCl

Antidibetics

61

4

 Gidazide and rosiglitazone

Antidibetics

62

5

Lignocaine and phenylephrine HCl

Respiratory System

63

6

 L-lysine HCl and DL- methionone

Endocrine System

64

7

Methocarbamol and nimesulide

NSAID

65

8

Paracetamol and mefanamic acid

NSAID

66

9

Pseudoephedrine sulphate and laratadine

Respiratory System

67

10

Rifluoperazine HCl, trihexylphenidyl HCl and chlorpromazine HCl

Respiratory System

68

2.5 Gas chromatography (GC):
GC is one of the most extensively used separation technique in which separation is accomplished by partitioning solute between a mobile gas phase and stationary phase, either liquid or solid. The chief requirement is same degrees of stability at the temperature necessary to maintain the substance in gas state. Some of the examples are given in the table-3.

Table-3

List of multi-component formulation estimated by gas chromatography (GC) with respective reported references.

Sr. NO.

Combination of drugs

Therapeutic Use

Ref. No.

1

Acetaminophen, salicylamide, phenyltoloxamine

NSAID

70

2

Benazepril HCl and hydrochlorthaizide

CVS

71

3

Codeine and ethyl morphine HCl

Respiratory System

72

4

Diclofenic sodium and chlorzoxazone

NSAID

73

5

Fluoxetine, fluvoxamine and clomipramine

Antipsycotic

74

2.6High performance liquid chromatography (HPLC):

This technique is based on the same method of separation as classical column chromatography. i.e. adsorption, partition, ion exchange and gel permeation but it differ from column chromatography, in that mobile phase is pumped through the packed column under high pressure. The technique is most widely used for all the analytical separation technique due to its sensitivity, its ready adaptability to accumulate quantitative determinations, its suitability for separating nonvolatile species or thermally fragile ones. In normal HPLC, polar solids such as silica gel; alumina (Al2O3) or porous glass beads and non-polar mobile phase such as heptane, octane or chloroform are used but if the opposite case holds, it is called as reversed phase HPLC. Some of the examples are given in the table 4 & 5.

Table-4

List of multi-component formulation estimated by high performance liquid chromatography (HPLC) with respective reported references.

8

Bromohexine HCl and Cephalexine

Respiratory System

22

9

Bromohexine HCl and methyl and propyl hydroxybenzote and determianation dextromethorphen hydrobromide

Respiratory System

23

10

Bromohexine HCl, phnyl praopalamine HCL and Chlorphenaramine meleate

Respiratory System

24

11

Caffeine and paracetamol

NSAID

25

12

Ceprofloxacin and tinidazole

Antiinfective

26

13

Ciprofloxacin HCl and tinadazole

Antiinfective

27

14

Ciprofloxacin HCl and tinidazole

Antiinfective

28

15

Codeine phosphate, ephedrine HCl and chlorphenaramine maleate

Respiratory System

29

16

 Dextromethorphen and pseudo ephedrine

Respiratory System

30

17

Diclofenic sodium, paracetamol and chlormerzonone

NSAID

31

18

Diloxanide furoate and tinidazole

Antiinfective

32

19

Ethniyl estradiol and levonorgestral

Endocrine System

33

20

Frusimide and spironolactone

CVS

34

21

Guaifenesin and codeine phosphate

Respiratory System

35

22

Guaifenesine, pseudoephedrine HCl and dextromethorphen hydrobromide

Respiratory System

36

23

 Hydrochlorthaizide and losartan potassium

CVS

37

24

Ibuprofen and dextromethorphen HCl

NSAID

38

25

Lavnivudine and zidovudine

Antiinfective

39

26

Metranidazole, clotimazole and chlorhexidne acetate

Antiinfective

40

27

Nimuselide and camplofine

NSAID

41

28

Norfloxacin and metranidazole

Antiinfective

42

29

Paracetamol, caffeine and prophyphenazone

NSAID

43

30

Paracetamol, chlorphenaramine maleate, phenylepherine and caffeine

NSAID

44

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Table-5

List of multi-component formulation estimated by reversed phase high performance liquid chromatography (RP-HPLC) with respective reported references.

Sr. NO.

Combination of drugs

Therapeutic Use

Ref. No

1

Amlodipine and benazepril

CVS

45

2

Amlodipine and losarton potassium

CVS

46

3

Amoxicillin and clavulanate

Antiinfective

47

4

Amoxycillin and clavulanate potessium

Antiinfective

48

5

Amoxycillin, probencid and tinidazole

Antiinfective

49

6

Ampicilline and probencid

Antiinfective

50

7

Cefazoline and cefotoxime

Antiinfective

51

8

Cefelexine and trimethoprim

Antiinfective

52

9

Ceprofloxacine and arnidazole

Antiinfective

53

10

Cetrizine and pseudo ephedrine HCl

Respiratory System

54

11

Chlophenaramine meleate, phenylepherine HCl and caffeine and acetaminophen

Respiratory System

55

12

Cinnarizine and domperidone

GIT

56

13

Ciprofloxacin and tinadazole

Antiinfective

57