INTRODUCTION:

Quantitative determination of drugs has been a subject of considerable importance both in bulk drug and pharmaceutical industries. Different physical methods viz., HPLC, TLC, GC-MS, LC- MS, spectroscopic techniques such as IR NMR UV-Vis spectrophotometry have been widely used for quantification studies. Out of these, UV- Vis spectrophotometry is simple, sensitive and accurate as well as cost effective due to common availability of spectrophotometers. The study involves measurement of absorbance and its variation with concentration of drug at 1) l_max of the drug, 2) at the l_max of chromophore generated by the interaction of drug with a chromogen ^1,2. The latter method involving p acceptors as chromogens is simple and non tedious and hence attracted the attention of researchers in the field ^3-6.

Thorough survey of literature on the following drugs revealed that quantification using p-CA as analytical reagent has not been reported yet although the reagent is common, known to offer simple, sensitive method of quantification for drugs. This prompted the author to develop quantification methods for the above mentioned drugs.. Thus the author has chosen p-chloranilic acid (p-CA ) as a chromogen and tested them for the formation of charge transfer complexes which is expected to form a basis for the quantification of the drugs (Scheme 1). The physiological activity of the drugs and methods used for their quantification are:

Metoprolol tartrate, is chemically a bis[(2 RS)-1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl) amino] propane-2-ol] (2R,3R)-2,3-dihydroxybutanedioate. It is a prototype β1-antiadrenergic drug which has the potency to increase the heart rate and decrease renin release from kidney. It is also quantified recently by direct UV method ⁷ and earlier by many physical methods cited therein such as Flourimetric,. HPLC. LC/MS.GC/MS.

Torsemide is recently quantified by a method ⁸based on the formation of blue colored chromogen when the drug reacts with Folin-Ciocalteu (F-C) reagent in alkaline medium. The earlier methods for quantification are quoted

Diclofenac sodium 2-[(2, 6-dichlorophenyl) amino] phenyl acetate, is a broadly used non-steroidal anti-inflammatory drug for the treatment of inflammatory conditions such as rheumatoid arthritis, osteoarthritis and ankylosing spondilytis . Recent determination by UV method ⁹ is preceded by many methods cited therein

Chlorpheniramine is an alkylamine derivative with the actions and uses of the antihistamines.

Derivative spectrophotometric method ¹⁰ is reported recently for the determination of chlorpheniramine maleate after citing many earlier methods of quantification.

Loratadine chemically(Ethyl 4-(8-chloro-5, 6-dihydro-11H-benzo^5,6 cyclohepta [1, 2-b] pyridine-11-ylidine)-1-piperidinecarboxylate) is a last generation of H1-antihistamine drug used to treat allergies, and marketed for its non-sedating properties. It is quantified recently by UV method ¹¹and earlier by many physical methods as cited therein

Irbesartan is an angiotensin receptor antagonist1, and it is chemically 2-butyl -3[(29-(1H-tetrazol-5yl) [1, 19-biphenyl]-4-yl] methyl] 1, 3-diazaspirol [4, 4] non-1-en-4-one. Recent determination by UV method ¹² is preceded by many methods cited therein

Structures of drugs Schem-1

1 Metoprolol (MT)

2 Torsemide (TS)

3 Diclofenac (DF) 4 Chlorpheniramine (CP)

5 Loratadine (LR) 6 Irbesartan(IB)

EXPERIMENTAL:

p-Chloranilic acid was obtained from Sd Fine Chem India Ltd.(mp 283-284⁰C). It was recrystallised twice from dry benzene. A stock solution of 200mg/100ml w/v (9.569 x 10^-3M) in acetoniltrile was freshly prepared. . The drugs used in study are procured from Hetero drugs pvt.ltd Hyderabad Most of the drugs procured are in the form of their acid salts. They have been neutralized by adding calculated amount of NaOH/NH₄OH as required followed by extraction with ether or CHCl₃. They were recrystallized from suitable solvent till TLC pure. Dichlofenac sodium is recrystallised from methanol before use. Stock solutions of drugs are prepared first (1mg/ml) and are further diluted according to the requirement for their analysis. The spectra (Fig 1) of each of the solution was recorded on scan mode against blank for 2 or 3 different concentrated samples as typical and absorbance was noted at 520nm for the remaining samples on fixed mode of instrument

Stability constants of ion – pair charge transfer complexes:

In literature the author noticed that Benesi - Hildebrand method (BH) [ ] is widely used for determination of stability constant K and molar absorption coefficient, e.

Ao/ D = 1/ K (Do) e +1/ e

Above equation is known as BH equation and a plot of Ao/ d Vs 1/ Do is a straight line from whose slope and intercept the K and e are determined. The BH method however demands the concentration of donor Do > > Ao ( Do should be 20 to 100 times the acceptor concentration) and many times the correct seperation of K and eis also doubtful.

Many workers used the Benesi - Hildebrand method without fulfilling the condition Do > > Ao and the values of e obtained varied widely. The e reported for p-CA^- : are 9.3 x 10⁴ to 1.8 x 10³(6). .

It is surprising that the molar absorption coefficient of an ion which is expected to be constant and charecteristic of that ion is widely varied. Therefore it is thought worth to determine the molar absorption coefficients of acceptor anions and then use the values to determine the stability constant K. To accomplish this, different volumes of dilute solutions of p-CAwere transfered to 25ml standard volumetric flask and excess drug was added and optical density was noted. The addition of drug continued until there is no appreciable increase in the optical density. A plot of d Vs concentration of acceptor gave a straight line from whose slope the molar absorption coefficient of anion of p-CAwas determined. This experiment was repeated at least with three drugs and each experiment was repeated three to four times until constant value of molar absorption coefficient (2000 L mol^-1cm^-1) was observed. The stability constant K

K = (d / e) / [(Ao - (d / e)] [Do - (d / e)]

is calculated using the molar extiction cofficient obtained from above experiment.

Extraction of drugs from pharmaceuticals:

1. Metroprolol tartrate (MT): Commercial formulation, Cap-I and Cap-II was purchased from a local pharmacy. Twenty capsules of each brand containing 25 mg of MT was weighed and finely powdered in a mortar. A quantity of powder equivalent to 100 mg of MT was weighed accurately and dissolved in 100 ml of methanol The solution was then filtered through Whatmann filter paper to get a clear solution. Methanol was .evaporated to dryness and the residue was dissolved in acetonitrile for analysis.

2 Torsemide (TS):

Twenty tablets (Dytor-10mg) were powdered, weighed and average weight of the tablets were determined. An amount of powder equivalent to 50mg of torsemide was taken into a 100ml volumetric flask. 100ml of methanol was added and shaken thoroughly for about 10 minutes and filtered using a quantitative filter paper in a beaker. The residue was washed with methanol for complete recovery of the drug. Methanol was evaporated by heating on water bath and acetonitrile is added and heated on water bath for the complete dissolution of the drug. The solution is diluted for the analysis.

3 Diclofenac sodium (DF):

Four brands of tablets containing DS from different manufacturers were purchased from local market and analyzed by using the current method. Ten tablets from each brand were finely powdered and mixed. An amount equal to the average weight of one tablet was collected randomly, transferred to a 100 ml volumetric flask, dissolved and made up with methanol. The solvent was evaporated to dryness and the residue was dissolved in acetonitrile for analysis

4 Chloropheniramine maleate (CP):

The content of twenty tablets (Dexodil-2.5mg) were crushed, powdered , weighed and average weight of the tablets were determined. The content of chloropheniramine maleate was stirred well with water and filtered with Whatmann filter paper. Residue was washed thrice for complete dissolution of the drug. The filtrate is taken in a separating funnel containing chloroform and 0.1 N NaOH is added for neutralisation. The content is shaken for 5 minutes. Chloroform layer is separated and extraction continued in two portions with chloroform. The chloroform was evaporated and the residue was dissolved in acetonitrile to prepare stock which was further diluted accordingly

5 Loratadine (LR):

Twenty tablests of each containing 5mg of loratadine were weighed accurately and finely powdered. and transferred to 50ml volumetric flask containing 25ml methanol shaken for 10min, then volume was made up to50ml with methanol and filtered through whatman filter paper No41 The residue was washed with methanol for complete recovery of the drug and the solvent was evaporated to dryness and the residue was dissolved in acetonitrile for analysis

6 Irbesartan ( IB):

The powdered contents of two tablets (Irbest-150mg) of strength were weighed and grounded. The powder equivalent to 50mg irbesartan was stirred well with acetonitrile. The solution was filtered through Whatmann filter paper in a 100ml volumetric standard flask and the residue was washed well with acetonitrile for complete recovery of the drug. The content of the standard flask was then diluted with acetonitrile to get required concentration for the analysis of the drug.

Instrument:

The spectra of individual components and charge transfer complexes were recorded on Shimadzu 140 double beam spectrophotometer as well as on Thermo Nicolet 100 and Elico 159 UV- Visible single beam spectrophotometers using matched pair of Quartz cells of 10mm path length.

Spectra:

The spectra (Fig 1) of ion – pair Charge transfer complexes were recorded in CH₃CN for quantification studies as well as to evaluate other parameters like stability constants and stoichiometry of the complexes from absorption studies on characteristic absorption band of anions of the acceptor.

Fig 1Absorption spectrum of P-CA with diclofenc

Procedure:

Different aliquots of solution of drugs were transferred to 10ml calibrated standard flask containing a constant volume of reagent solution and volume was made to 10ml by the solvent. The concentration of drug was varied so as to produce charge transfer complexes with absorbance between 0.06 to 1.5 absorbance units.

The stoichiometry of each of the complex was determined from Job’s continuous variation method (Fig 2) by using equimolar solutions of drug and acceptors.

RESULTS:

p- Chloranilic acid has absorption maximum at 430nm . When solution of any donor, in CH₃CN is added to it the l_max shifts to 520nm where neither the p-Chloranilic acid nor donor have any absorbance The intensity of the band increases with the increase in the concentration of donor and formed a basis for quantification study of donor. The absorbance at 520nm is attributed to the anion of p-chloranilic acid formed by the abstraction of electron from donor^5,6.

In the present study p-chloranilic acid was tested to form ion-pair charge transfer complexes with six drugs viz., Metroprolol tartrate (MT), Torsemide (TS), Diclofenac sodium (DF, Chloropheniramine maleate (CP), 5 Loratadine (LR) Irbesartan ( IB) showed color changes when mixed with p-chloranilic acid in CH₃CN. The purple color of solution is characteristic of anion of p-chloranilic acid and exhibited a band at 520nm as observed by other workers

Calibration curve:

Different aliquots of drugs have been transferred to 10ml calibrated standard flask containing 2ml of p-Chloranilic acid (9.569 x10^-3M) and the remaining volume was made up by solvent. The range concentrations of various drugs used have been mentioned in Table –1.

Table 1 The concentration range of used for the study

S. No.

Name of the drug

Concentration (g ml^-1)

50 to 300

25.to 230

25 to 175

30 to 250

50 to 350

Each drug sample is analyzed at least for five times. Calibration curves (Fig. 2) were linear for all the drugs whose limits are mentioned in Tables.2. The slope , intercept, correlation coefficient of the Calibration curves (d Vs mg/ml as well as d Vs moles/lit) are calculated and tabulated. Sandell’s sensitivity “Milligrams of drug per liter required to produce a change in the absorbance by 0.001 absorbance units” have been calculated for all the drugs. Limit of Detection “The lowest amount of analyte in a sample that can be detected, but not necessarily quantitated as an exact value” and Limit of Quantification “ The lowest amount of analyte in a sample that can be quantified using Calibration curves” have been calculated by using equations available in the literature.

LOD = 3.3s/S

LOQ = 10s/S.

Where s= standard deviation of the intercept ( n = 5)

S = slope of Calibration plot

The stoichiometry of each of the complex has been determined from Job’s continuous variation method and found to be 1:1 in each case. A typical Job’s plot of p-CA with Torsemide is presented in (Fig.3).

Formation constants¹³ (K) of the complexes have been determined for all the drugs by taking different volumes of p-CA of a stock (1.388x10^-3 M) in acetonitrile and transferred to 25ml standard flask and excess of drug was added so that entire p-CA is converted into p-CA anion and spectra was recorded. The concentration of p-CA Vs d gave a straight line from whose slope ε of anion of p-CA is determined. The ε of p-CA anion found is 2000 lit mole^-1cm^-1.

Optimisation of the parameters of quantification:

Effect of concentration of reagent:

When various concentrations of 2 % of p – Chloranilic acid ( 0.2, 0.4, 0.6, 0.8, 1.0,1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4 ml ) was added to fixed concentration of various drugs viz., 300 μg/ml of MT, 230 μg/ml of TS, 175 μg/ml of DF, 270 μg/ml of CP, 350 ug/ml of LR and 350 μg/ml of IB. A plot of volume of reagent and the absorbanceb showed that 1.8ml of reagent solution is enough ( 9.569 x 10^-3M) to develop the purple colour to its maximum intensity after that a plateau was observed. Therefore an excess of reagent i.e. 2ml of reagent in a total volume of 10 ml was used throughout the work (Fig.4). Effect of concentration of Drug

Table2. Analytical parameters for the charge transfer complex Of p- CA with Drugs in Acetonitrile

Parameter	MT	TS	DF	CP	LR	IB
λ_max (nm)	520	520	520	520	520	520
Beer’s law limits (μgml^-1)	50-300	25-230	25-175	30-250	50-350	50-350
Molar absorptivity (L mol^-1 cm^-1)	1176	1300	1075	852	880	901
Formation constant, K, M^-1	410	350	340	300	280	270
Sandell sensitivity (μg cm^-2)	0.227	0.27	0.294	0.322	0.434	0.476
Slope b	0.0044	0.0037	0.0034	0.0031	0.0023	0.0021
Intercept (a)	0.1066	0.00112	-0.00116	0.00118	0.010	0.005
Correlation coefficient	0.999	0.999	0.998	0.999	0.998	0.999
Standard deviation of intercepts (% n=5)	0.00364	0.001603	0.001442	0.00155	0.001952	0.001718
Limit of detection, μgml^-1	2.73	1.43	1.40	1.65	2.8	2.7
Limit of quantification μgml^-1	8.2	4.3	4.21	4.97	8.4	8.1
Reegression equation Y= bx+a	0.0044c+0.1066	0.0037c +0.00112	0.0034c -0.00116	0.0031c +0.00118	0.0023c +0.010	0.0021c +0.05

Different volumes of drug of random concentration was added to a fixed volumes of acceptor. Solutions developed coloration. Absorbance of solutions was measured at 520nm. Beer’s law was obeyed upto certain extent of concentration above which linearity was not observed. This concentration was taken as optimum concentration and stock was prepared. The stock was further diluted to get a minimum of 8 to 10 points in the range of Beer’s law plot. Similarly when the concentration is below certain limit points scattered. This was taken roughly a measure of limit of detection which is further checked by following the procedure for the determination of LOD and LOQ.

Effect of time:

The interaction of p-CA with drugs resulted in the formation of colored product which stabilized within 2 mints of mixing. The developed color remained stable at room temperature for about an hour. After two hours many solutions turned purple. After a day all solutions turned black hence the measurements were made immediately after mixing the solutions.

Effect of organic solvent:

Various solvents such as carbon tetrachloride, chloroform, 1,2 dichloroethane, methanol and acetonitrile have been tried to select suitable solvent for the analysis of the drug. Acetonitrile is found to be the suitable solvent as it produces maximum optical density with a fixed concentration of drug while other solvents mentioned above are found to be unsuitable as they produced lower absorbances due to incomplete dissociation of complex. Hence acetonitrile is used throughout the work (Table 3).

Table 3 The effect of solvent on the Optical density of charge transfer band of p-CA with ddddiclofenac (175g/ml)

S. No

Solvent

Optical density

Acetonitrile

Methanol

1,2- dichloroethane

Chloroform

Carbon tetrachloride

0.63

0.58

0.30

0.12

0.07

Validation of the proposed methods:

The methods developed have been validated in terms of guidelines of international conference of harminisation (ICH) ¹⁵viz., selectivity, .precision , accuracy linearity, LOD, LOQand robustness. The methods are selective and can differentiate the analyte from the excipients.. The precision is tested by repeating each experiment at least 6 times while the accuracy has been tested by taking known weight of sample and performing recovery experiments. The values %RSD and t- and F tests are in the permissible range of experimental errors. (Table 4)

Limits of calibration, LOD, LOQ, have been determined as mentioned earlier.. The robustness of the methods are examined by performing the experiments on three different spectrophotometers with excellent tally of absorbance values. The methods developed have also been applied for the analysis of pharmaceuticals. The recovery experiments performed show high accuracy and precision and the results are compared to the available validated reported methods on each drug. The values %RSD and t- and F tests are in the permissible range of experimental errors. (Table5) and show that the methods can be used in both pharmaceutical and drug industries

DISCUSSION:

p- Chloranilic acid (p-CA) is a π – electron acceptor and has been used for the determination of amino acids, aliphatic, aromatic and tertiary amines ¹⁴. Hydrochloride salts of amines did not react with p- chloranilic acid because they do not posses a lone pair of electrons. Such drugs have been first neutralised and then p- chloranilic acid was added. Therefore, the addition of p- chloranilic acid to the drugs possessing a lone pair of electrons results in the formation of a charge transfer complex of the n – π type. The complex is formed by the lone pair of electrons donated by an n - donor to p- Chloranilic acid, a charge transfer reagent as an electron acceptor, through which a partial ionic bond ( D⁺A^-) is assumed to be formed.

Table 4 Application of proposed method for the analysis of the studied drugs in pure form

Drug

Taken

(mg/ml)

Found

(mg/ml)

Recovery

(%)

RSD

(%)

Proposed method

Mean± SD

Reference method

Mean± SD

t-test

F-test

100

150

200

49.60

100.2

149.5.0

200.6

99.2

100.2

99.5

100.3

1.47

1.33

1.02

0.52

99.8±0.53

98.74 ± 0.81

2.60

0.42

100

24.6

50.12

75.25

100.5

98.4

100.24

100.33

100.5

1.54

1.30

1.60

1.03

99.86±0.98

100.13±0.65

0.52

2.27

100

24.5

50.5

75.4

100.4

98.0

101.0

100.5

100.4

1.15

0.76

0.95

0.35

99.97±1.34

101.21 ± 1.1

1.65

1.48

120

29.8

60.5

90.5

120.6

99.3

100.8

100.5

1.14

0.51

0.35

0.12

100.27±0.66

102.0 ± 2.2

1.82

0.09

100

150

200

50.4

100.6

148.8

198.0

100.8

100.6

99.2

99.0

1.15

1.79

1.06

1.05

99.9±0.93

99.5±0.56

0.84

2.75

100

150

200

49.6

98.9

151.2

198.5

99.2

98.9

100.8

99.25

0.65

0.84

0.35

0.19

99.53±0.85

99.88±0.71

0.69

1.43

Table 5 Application of proposed method for the analysis of the studied drugs in pharmaceutical formulations

Drug

Taken

(g/ml)

Found

(g/ml)

Recovery

(%)

RSD

(%)

Proposed method

Mean± SD

Reference method

Mean± SD

t-test

F-test

100

150

200

49.8

100.2

149.6

200.6

99.6

100.2

99.73

100.3

0.62

0.82

0.30

0.17

99.95±0.5 4

98.74 ± 0.81

3.2

0.44

100

25.2

49.7

75.3

99.7

100.8

99.0

100.4

99.7

1.14

1.89

1.06

1.05

99.97±0.79

100.13±0.65

0.36

1.47

100

25.2

50.4

74.4

99.1

100.8

99.2

99.1

1.15

0.76

0.96

0.36

99.97±0.95

101.21 ± 1.1

2.00

0.74

120

30.4

59.8

90.5

119.4

101.3

99.6

100.5

99.5

1.03

0.53

0.35

0.10

100.22±0.84

102.0 ± 2.2

2.28

0.14

100

150

200

50.3

99.8

149.4

200.6

100.6

99.8

99.6

100.3

1.54

1.30

1.60

1.03

100.07±0.45

99.5±0.56

2.03

0.64

100

150

200

50.3

100.8

148.8

198.6

100.6

100.8

99.2

99.3

1.47

1.33

1.02

0.52

99.98±0.84

99.88±0.71

0.21

1.39

Reaction Mechanism:

Foster ¹⁴ suggested the reaction of p- chloranilic acid as electron acceptor and base as electron donor. The mechanism of interaction between p- chloranilic acid and donor may be as represented in the Scheme 1.

Drug Acceptor Donor-acceptor complex anion cation

(Donor) (p-Chloranilic or

acid) Charge transfer complex

Scheme 2

From the slopes of Calibration curve and from stability constants and it is clear that the donor ability of the drug is in the order: MT>TS>DF>CP>LR>IB

From the structures of the drugs it is clear that MT,TS and DF are secondary amines while, CP, IB, LR are tertiary amines. It is expected that the former set is more basic than latter set. In the former set too, MT is expected to show highest basicity. The secondary amine group of TS is adjacent to an electron withdrawing CO group, is expected to show lesser basicity than MT. The DF is also secondary amine but the electrons of N are delocalized into benzene rings which reduces the basicity of N. Among the tertiary amines CP is expected to show more basicity than LR and IB as teriary N in both off them is adjacent to electron withdrawing CO group.

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Received on 02.06.2011 Modified on 23.06.2011

Asian J. Research Chem. 4(8): August, 2011; Page 1311-1317