SPECTROPHOTOMETRIC DETERMINATION OF NIACIN THIAMINE GLIBENCLAMIDE ERYTHROMYCIN AND PARA AMINO BENZO IC ACID USING 2 3 – DICHLORO – 5 6 – DICYANO – 1 4 – BENZOQUINONE

ABSTRACT

A simple and sensitive spectrophotometric method is described for the assay of the drugs; niacin, glibenclamide, erythromycin, thiamine and 4-aminobenzoic acid.  The method is based on charge transfer complexation (CT) reaction of niacin, glibenclamide, erythromycin,  thiamine  and  4-aminobenzoic  acid  as  n-electron  donors  with  2,3- dichloro-5,6-dicyno-1,4-benzoquinone(DDQ) as л-electron acceptor in methanol. Intensely coloured charge transfer complexes with niacin (reddish brown, max ;464 nm;

εmax, 1.02×103 dm3mol-1cm-1) thiamine (reddish brown ,max  ;474 nm; εmax,  1.08×103

dm3mol-1cm-1), glibenclamide (reddish brown , max  ;474 nm; εmax,0.99×103   dm3mol-

1cm-1) erythromycin(reddish brown , max  ;464 nm; εmax, 1.27×103  dm3mol-1cm-1)  4- aminobenzoic acid(reddish brown, max  ;474nm; εmax, 1.06×103 dm3mol-1cm-1) all in  a

1:1 stoichiometric ratio. Condition for complete reactions and optimum stability of complexes were niacin (70 min, 60 OC) thiamine (25 min, 40 OC), glibenclamide (35 min, 40 OC), erythromycin (15 min, 60 OC) and 4-aminobenzoic acid (15 min, 60 OC) as absorbances of the complexes remained invariant within these conditions. Formation

and stability of the complexes of niacin, thiamine, 4-aminobenzoic acid and erythromycin were optimum at  pH 8. For glibenclamide pH 2.0 favoured optimum stability and formation. The bands distinguished for the donors to donor-acceptor CT complexes displayed small changes in band intensities and frequency values in the IR spectra ,The –NH2  group vibration occurring at 3609 cm-1  shifted   to 3610 cm-1  in

thiamine, PABA (3222 cm-1  to 3183 cm-1), ѵ (N-H) occurring at 3331cm-1  shifted to

3371 cm-1  in glibenclamide, ѵ(C= N) occurring at 2936 cm-1 shifted to 2944 cm-1  in niacin, ѵ (CH3-N) occurring at 2948 cm-1  shifted to 2939 cm-1  in erythromycin. The

vibration ѵ (C=  O) of DDQ observed at 1665 cm-1  shifted to 1669 cm-1  in the CT

complex for thiamine, PABA(1665 cm-1  to 1670 cm-1), glibenclamide(1675 cm-1  to

1676 cm-1), erythromycin(1665 cm-1  to 1674 cm-1), niacin(1665 cm-1  to 1655 cm-1)

respectively. Adherence to Beer’s Law was within the concentration range for niacin (5-

130 μg/cm3), thiamine (5-80 μg/cm3), glibenclamide (9-100 µg/cm3), erythromycin

(5-150   µg/cm3),   4-aminobenzoic   acid(5-90   µg/cm3).   Limit   of   detection   and quantification of the drugs based on this method is niacin (1.78 and 5.4), thiamine (1.23 and 3.37), glibenclamide (3.47 and 10.5), erythromycin (2.11 and 6.40), 4-aminobenzoic acid (0.55 and 1.67) respectively. Evaluation of the degree of interference by excipients used in the drugs manufactured indicates tolerance to certain concentrations. A detailed study on the interference of different excipients was made. No significant interference was observed in magnesium stearate (30 µg/cm3), Talc (15-25µg/cm3, 35-40 µg/cm3) with thiamine-DDQ complex. There were no significant interference in stearic acid (35 µg/cm3) but tolerable interference was seen in magnesium stearate (20 µg/cm3) and calcium phosphate (15 µg/cm3) with niacin-DDQ complex. For glibenclamide – DDQ complex, no significant interference was seen with calcium phosphate (30 µg/cm3) but there were tolerable interference present in stearic acid (40 µg/cm3). In 4-aminobenzoic acid, no significant interference was observed with magnesium stearate (30 µg/cm3) and talc (35 -40µg/cm3) but tolerable interference was observed in corn starch (15 µg/cm3). Also no significant interference was seen in corn starch (35 µg/cm3) with erythromycin- DDQ complex but there was tolerable interference in talc (10 µg/cm3).  The Pearson correlation coefficient for the compliance of the method as regards the pure and commercial forms of niacin, thiamine, glibenclamide, erythromycin and 4-aminobenzoi

acids are 0.993, 0.977, 0.987, 0.998 and 0.993 respectively which shows significance with p < 0.01.   The analysis of variance test revealed the non-significance of niacin, thiamine, glibenclamide, erythromycin and 4-aminobenzoic acid with p > 0.01.   The mean percentage recoveries were 98.94 ±  0.016, 96.2  ±  0.016, 98.24  ±  0.011, 107.4 ±  0.023 and 102.35 ±  0.014 for niacin, thiamine, glibenclamide, erythromycin and 4- aminobenzoic acid respectively. Kinetics of the reactions infer that the rate of formation of the  CT  complexes did  not  vary significantly with  increase  in  concentration of glibenclamide,  erythromycin,  thiamine,  niacin  and  4-aminobenzoic  acid  indicating likely zeroth order dependence of the rate with respect to concentration of the drugs. However, the linearity of the pseudo-first order plot points to first order dependence of rate on [DDQ].The overall rate equation for the reactions can be given as Based on the limit of detection and quantification, adherence to Beer-Lambert’s law and low degree of interference, the method is recommended for the analysis of these drugs.

CHAPTER ONE

1.0        Introduction

1.1       Charge Transfer Complexation

Acceptors are aromatic systems containing  electron withdrawing  substituents such as nitro, cyano and halogen groups (Foster, 1967). Electron donors are systems that are electron rich (Ajali and Chukwurah, 2001). The  interaction between electron donor and electron acceptor results in formation of charge transfer complex (Ajali et al,

2008). The term charge transfer denotes a certain type of complex which results from interaction of an electron acceptor and an electron donor with the formation of weak bonds (Hassib and Issa, 1996).    However  the nature of the interaction  in a charge transfer  complex  is not  a stable  chemical bond  and is much weaker  than covalent forces. It is better characterized as a weak electron resonance. As a result, the excitation energy  of  this  resonance   occurs  very  frequently   in  the   visible  region  of  the electromagnetic  spectrum. This produces the usually intense colour characteristic  for these complexes. These optical absorption bands are often referred to as charge transfer bands.  Molecular  interactions  between  electron  donors  and acceptors  are generally associated with the formation of intensely coloured charge transfer complexes which absorb radiation in the visible region.Charge transfer (CT) complexes have been widely studied (Ezeanokete et al, 2013; Hala et al, 2013; Frag et al, 2011; Ramzin et al, 2012; Farha, 2013). Charge  transfer complexes  are known to take part in many chemical reactions like addition, substitution and condensation reactions (Van et al, 2006).

Donor  acceptor  properties  are  prerequisites  for  the  formation  of  charge  transfer complexes. Most drugs have –NH or –NH2  groups which behave as  bases (electron donors) and could form complexes with acids (electron acceptor).Various cases have been reported.  The charge-transfer  complexes  formed  between the ephedrine  (Eph)

drug  as a donor  with picric  acid  (Pi) and quinol (QL)  as      –acceptors  have  been synthesized in methanol as a solvent at room temperature and spectroscopically studied

as shown in scheme 1:

Quinol                           Ephedrine

[(EPh) (QL)] Complex

Scheme 1:       Interaction  of  Ephedrine  with  Quinol  to  form  the  charge  transfer complex

Spectrophotometry is widely used to monitor the progress of reactions and the position of equilibrium. Its measurement is often straight forward to make and the technique is sensitive and precise provided that relevant limitations (such as the regions over which Beer’s law is valid) are recognized. Spectrophotometric technique continues to be the most  preferred  methods  for  routine  analytical  work  due  to  their  simplicity  and reasonable sensitivity with significant economical advantages (Raza, 2006).

1.1.2:   Analysis of Drugs

A  spectrophotometric  method  has  been  employed  for  the  determination  of allopuriol using DDQ through charge transfer  formation.  The absorption  spectra  of allopuriol-DDQ complex in acetonitrile solvent showed three maxima  at  (ʎmax  = 450

nm; ε1  = 1.95 x103  Lmol-1cm-1), 540 nm (ε2  = 0.80 x 103  Lmol-1cm-1) and 580  nm

(ε3  = 0.69 x 103  Lmol-1cm-1) with a 1:1 stoichiometric  ratio between allopuriol and

Allopurinol -DDQ Charge Transfer Complex

Scheme 2: Interaction of Allopurinol with DDQ to form the charge transfer complex

DDQ (2,3 – dichloro – 5, 6 – dicyano -p- benzoquinone)  acts as an  oxidizing (Braude et al, 1956) as well as dehydrating agent in synthetic organic chemistry. It is known for its interaction with drugs having donor sites in their structures and form Ion- Pair  charge  transfer  complexes  which  offers  a  basis  for  quantification  of  drugs (Ghabsha et al, 2007; Vmsi and Gowri, 2008; Rehman et al, 2008; Rahman and Kashif,

2005;  Khaled,  2008;  Walash,  2004;  El-Ragehy  et  al,  1997).    DDQ  as  π-electron acceptors   often   forms   highly  coloured   electron-donor,   electron-acceptor   or  CT complexes with various donors which provide the possibility of determination of drugs by spectrophotometric methods.

Vitamin B1 (Thiamine) has its chemical name as 2-[3-[(4-Amino-2-methyl- pyrimidin-

5-yl) methyl]  -4-methyl – thiazol – 5 – yl] ethanol.  Vitamin  B1  is a water  soluble vitamin.  It  plays  an  important  biological  role  in  the  metabolic   process  of  the carbohydrate  in  the  human  body  (Khaled,  2008).  Previous  studies  have  utilized different  techniques  for  the  estimation  of  thiamine  hydrochloride  which  includes:

normal flow injection (Mouayed, 2012), electrochemical  analysis method  (Akyilmaz and  Dinckaya,  2006)  high  performance  liquid  chromato  graphy  (Ghasemi,  2005) spectrofluorimetry (Hassan, 2001) polarimetry. Also direct spectrophotometric method has been described for the determination of thiamine hydrochloride in the presence of its degradation products (Wahbi et al, 1981).

Vitamin B3  (Niacin) chemically designated as [pyridine -3- carboxylic acid] is one of the water soluble vitamins of the B-complex. It is an essential vitamin that is widely available  in  drug  and  health  food  stores.  Niacin  is  sometimes  prescribed  in  high dosages to lower cholesterol. People also take niacin supplements because they think niacin helps ease gastrointestinal  disturbances.  It  is widely distributed  among plants and animals. Some analytical methods have been developed for determination of niacin which includes HPLC, flow injection TLC (Sarangi et al, 1985) HPTLC (Tiwari, 2010; Zarzycki et al, 1995; Hsieh, 2005).

Furthermore,   Spectrophotometric    methods   have   been   reported   for   the simultaneous estimation of Atorvastation and niacin based on simultaneous  equation and absorbance ratio method (Sawart et al, 2012).

PABA [4-aminobenzoic acid] was used as a component of some medicines e.g analgesic or anesthetic preparations,  sunscreen agents and bentiromide (Imondi et al,

1972; Cyr et al, 1976; Charles et al, 1977).

It  is  an  essential  factor  for  the  growth  of  bacteria.  It  is  possessed  of  an  anti- sulfanilamide activity (Zhang et al, 2005). Various methods used for the  analysis of PABA include HPLC (Zhang et al, 2005) GC (Zhou and Zhang, 1998; Schmidt et al,

1997;  Lambropoulon,  2002).    Spectrophotometric  methods  have  been used  for  the determination of PABA; most of the methods are based on diazotization of PABA and coupling  the  corresponding  agent  such  as  Braton  Marshall  reagent  (Othaman  and

Mansor, 2005), 4–dimethylaminobenzaldehyde  (Yamato  and Kinoshita,  1979),  N-(I- napthyl) ethylediamine dihydrochloride (Fister and Drazin, 1973) and phyloroglucinol (Othaman and Mansor, 2005).Indirect spectrophotometric method for the determination of    PABA    has    been    reported    (Salvandor    et    al,    2003).A    flow    injection spectrophotometric  determination  of propoxur  with  diazotized-  4-aminobenzoic  acid oxidation (Mirick, 1943) methods has been reported.

Erythromycin (3R, 4S, 5S, 6R, 7R, 9R, 11R, 12R, 13S, 14R) – 4-[(2,6-dideoxy -3- C- methyl-3-o-methyl-a-L-ribo-hexopy-ransoyl)  oxy]  –  14  –  ethyl  –  7  ,  12  ,  13  – trihydroxy -3,5,7,9,11,13-hexamethyl- 6 – [ ( 3 , 4 , 6 – trideoxy – 3 – dimethylamino–β- D-xylo-hexopyranosyl)-oxy]oxa   cyclotetradecane   -2,  10-   dione     is  a  macrolide antibiotic that has an antimicrobial spectrum similar to or slightly wider than that of penicillin.  It  has  better  coverage  of a  typical  organism  and  occasional  used  as  a prokinetic  agent.  It  inhibits  bacterial  reproduction  but  does  not  kill  bacterial  cells. Literature revealed different techniques for the analysis of the studied macrolides.

The British Pharmacopeia stated the liquid chromatography method for the assay  of erythromycin.  Other  method of analysis  includes  spectrofluormetry  (Pakinaz,  2002) and  (Nawal  et  al,  2006)  capillary  electrophoresis    HPLC  (Maria  and  Britt,  1995; Dubois et al, 2001; Ramakrishna et al, 2005) voltametry (Faryhaly and Mohammed,

2004) microbiological method (Bernabaeu et al, 1999), spectrophotometry (Tasmin et al, 2008; Carlos et al, 2010; Safwan Roula, 2012; Magar et al, 2012).

Glibenclamide   chemically   known   as   5-chloro-n-[2-[4[(cyclohexylamino) carbonyl]  -amino]  sulphonyl]  phenyl]  –ethyl]  -2-methoxy  benzamide  is  a second generation sulphonyl ureas drug widely used in treatment of type 2 diabetic patient (Parmeswararo  et al,  2012).  The  literature  survey  shows  that  spectrophotometric methods  have  been  employed  for  the  determination  of  glibenclamide  based  on

derivatization technique or coupling with another reagent (Nalwaya, 2008), (Bediar et al, 1990; Lopez et al, 2005; Goweri et al, 2005; Martins, et al,  2007; Gianotto et al,2007) High pressure liquid chromatography methods are the most commonly used for  the  determination  of  glibenclamide  and  different  methods  coupled  with  UV detection. Fluorescence (Khtri et al, 2001) detection or mass spectrometry (Smgh and Taylor, 1996) .Thin layer chromatography  has  been employed  for the detection of glibenclamide    (Kumasak    et   al,   2005),    voltametric    method   (Radi,   2004). Spectrofluorimetric method  have all been reported. Erythromycin, thiamine, niacin, p-Aminobenzoic  acid, and  glibenclamide  are all bases with –NH2  or –NH groups

which have donor sites and can form charge transfer complexes.

1.1.3               Justification of the Study

In order to solve the problem of fake drugs which is rampart in Nigeria, there is need for a method of drug analysis which is simple, fast and cost effective. However,

this new method of analysis will bring about easier analysis of drugs that is simple, fast and  of  low  cost  which  will  invariably  reduce  importation  and   manufacture  of substandard drugs in Nigeria.

Secondly,  the new method will solve the problem of interferences caused  by drug excipients.

1.1.4:               Problem of the Study

These drugs are easily adulterated  due to their nature and their  high demand.  This requires that their degree of purity be certified before usage. Also the methods used in determining  these  drugs  like  the  flow  injection  spectrophotometric  method  ,  High performance liquid chromatography, voltammetry, polarimetry and spectrofluorimetry all require costly equipment, laborious, involve rigid pH control and use large amounts

of  organic  solvents  which  are  expensive,  hazardous  to  health  and  harmful  to  the environment. Methods like spectrophotometry based on charge transfer complexation, which are fast, less laborious and economical, are required for the assay of these drugs.

1.1.5      Aims and Objectives

The aim of this research was to determine a method based on formation of CT complex between the drugs and DDQ that is simple, fast, economical and less laborious. The objectives of this research are to:

(i)         establish the degree of CT complex formation between the drugs and DDQ

(ii)        determine the stability of the CT complexes with respect to time, temperature and pH.

(iii)       apply the CT complexes in spectrophotometric determinations of the drug

(iv)       determine the average recoveries of the drugs in pure and commercial forms

(v)         validate    the    proposed    method    using    International    Conference    on

Harmonization Guideline.

(vi)       determination  of  the  kinetic  model  for  the  charge  transfer  complexation reactions.

(vii)     characterization   of  the  CT  complex  using  Fourier  transformer   infra   red spectrometer.

1.1.6:   Scope of Study

•    U.V  Absorption spectra

•     I.R  Absorption spectra

•    Establishment  of    -max

•     Stoichiometric  relationships

•     Optimum conditions(time , temperature ,  pH)

•     Establishment  of standard curves

•      Applying the charge transfer complex in the spectrophotometric determination of the drugs

•     Validation   of   the   proposed   method   using   international   conference   on harmonization guidelines

•      Statistical  analysis  (One-way  analysis  of  variance  and  Pearson  correlation coefficient)

•    Determination of order of reactions (zero and 1st   order)

•     Characterization of charge transfer complexes using Fourier transformer  infra red spectrometer.

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