Indian Journal of Chemistry Vol. 42A, December 2003. pp. 3036-3040

Microdetermination of anti-hypertensive drug Captopril using 2,6-dichlorophenol indophenol

Ashutosh Srivastava*, Bindu Khare, RupaJi Argal & Sandeep Patel

Department of Che mi stry, Rani Durgavati Uni versity, Jabalpur, Madhya Pradesh 482 00 I, India

Received 15 l a l1//(//)' 2003

A method has been developed for quantitative microdetermination of antihypertensive drug Captopril in l'itro as well as in vivo samples, spectrophotometrically using 2,6-dichloropheno l indopheno l [2,6-DCPIP) . The sulphydryl moiety , of the Captopril is quantitati vely oxidized to its correspo nding [I, I' -dithiobi s'(2-methyl-I-oxopropane-3, 1 diyl)bi s-L­pro lineldi sulphide by 2,6-DCPIP ex hibiting stoichio metry of 2: I. The reaction product has been characterized by FrIR and NMR spectral studies. The method is rapid, accurate with high precision and can be applied for determining drugs in pharmaceutical preparations as well as in-vivo samples.

Angiotensin Convelting Enzymes (ACE) inhibitors' are one of the most important class of drugs for treating hypertension and chronic heart failure. Captopril , 1-(3-mercapto-2-D-methyl)-1-oxo-propyl is an anti­hypertensive drug containing sulphydryl functional group. It is a potent specific and orally active inhibitor of angiotensin converting enzyme 203 . Clinically it is highly efficacious in the treatment of congestive heart failure4,s.

Quantitatively captopril is determined by its derivatisation with pentafluorobenzylbromide6 in acetonitrile using potass ium carbonate as a base catalyst. The resulting derivative is then separated and monitored with flame ionisation detector. Spectrophotometrically captopril can be determined either using 2,6-dichloroquinone-4-chlorimide7 atpH 7, or 2,2-diphenyl­I -picrylhydrazyls. Determination of captopril fluorometrically (HPLC) using N-[4-(5,6-dimethoxy-2-phthalimidyl)phenyl]maleirnide7 or colorimetrically either using n-bromophthalimide9 in H3P04 (50%) solution or using molybdophosphoric acid(MPA) has already been developed. Quantitatively Captopril is determined by titrimetric'o amperometric" , gas liquid h h 12 d· · ' 3 fl . 14

C romatograp y , ra 100mmunoassay , uorometnc , colourimetric's, HPLC'6, chromatography and spectro­photometric' 7 methods. Toshio Nambara et at. developed a method for quantitation of captopril in human blood by derivatising with N-(4-dimethyl amino phenyl)maleimide(DAPM) into the electrochemically active adduct. The derivatives are separated and then determined by HPLC with an electrochemical detector on a reversed phase column' S. All the above-proposed methods for quantitative determination of captopril

require sophisticated instrumental techniques and also most of them are linear over a limited range of concentration only.

2 ,6-Dichlorophenol indophenol [2,6-dichloro-N-(p­hydroxy phenyl)p-benzo quinone amine] is a well known titrant of Vitamin C in biochemical studies l9

.

This titrant has been used in a variety of reactions of pharmaceutical and analytical importance20.21. Basford and Huennekens22 first reported its reaction with sulphydryl functional group bearing compounds, thiol which were later reported by Hadler et at. 23 . These findings were exploited in biochemical researches to determine the membrane sulphydryl g roup24. Due to its resemblance with quinonoid moiety it is al so reported to be used in cancer cobalt thcrapy2s .

The proposed method circumvents, the shortcomings of earlier reported methods for the determination of I (3-mercapto,2-D-methyl-l-oxo­propyl)L-proline. The proposed method involves spectrophotometric assay of Captopril by oxidation of its sulphydryl group to the corresponding disulphide i.e. (S ,S) 1,] ' dithio bis(2-methyl-l -oxo propane-3 ,1 diyl) bi s-L-proline with 2,6-dichlorophenolindo­phenol, having stoichiometry of 2 : 1 following the reaction given in Scheme 1.

The fading of intense blue colour at 610 nm is made the basis of determination over the entire range of Beer' s Law. The method evolved was applied to the in vivo samples of whole blood and serum of two confirmed hypertensive patients aged 6 1 and 42 years. The method is si mple, rapid, preci se nd accurate and may be adopted in the pathological laboratories. No

SRIV AST A V A et al.: MICRODETERMINATION OF CAPTOPRIL 3037

OH

Ht::-C-CH2-S-S-CH 2- C-CH I ~ ~ C'9C' I I 3 C=O C=O ~ I I + ~COOH ~COOH N-H

¢ OH CAPTOPRIL DISULPHIDE

Scheme 1

interference has been found to hamper the determination by the binders present in the pharmaceutical samples, which makes the method more adaptable.

Materials and Methods

Preparation of Captopril solution Gift samples obtained from Bristol Meyer Squibb

Company, Princeton, New Jersey U.S.A. and tablets of Lupin Laboratories, Bhopal (M.P.) were considered. Accurately weighed, finally powdered 20 tablets equivalent to ca. 25 mg of captopril were thoroughly mixed with 15 ml of distilled water. The resulting mixture was filtered through Whatman filter paper No. 40 and diluted to 100 ml with distilled water.

Preparation of leuco 2,6-dichlorophenol indophenol (2,6 DCPIP) solution

To 20.0 ml of a 0.05% solution of the sodium salt of the oxidising salt, 5ml of phosphate buffer was added and the blue colour of the solution was bleached by adding 0.05% of ascorbic acid solution dropwise. The reduced indicator was oxidised when exposed to air and it is essential to decolouri se the solution with ascorbic acid before use.

Phosphate buffer pH=7 was prepared by dissolving 117.7 gm of K2HP04 and 44.1gm of KH2P04 in one litre of doubly disti.lled water.

In vivo sampling of Captopril The subjects were two male patients suffering with

hypertension aged 61 and 42 years. They weighed between 70 to 82 kgs (x=76.0 kgs). The patients received no medication for at least two weeks before the study . Each subject was orally given a single tablet of Captopril 25 mg with water. The clinical studies were carried out on patients kept on fast for 3 h.

Two blood samples were collected from patient suffering from hypertension. First blood sample, 10.0 ml (without drug), was collected before giving the drug. Second blood sample, 10 ml (with drug), was collected after 30 min of administration of captopril, a 25 mg tablet. All samples were frozen in dry ice- bath until analyzed.

Serum sample can be separated by treating pure blood and drug containing blood separately with 1.0 ml of 2% trichloroacetic acid. The sample was then centrifuged for 45 mins at 360 rpm. The supernatant clean liquid, is the serum and it was taken in varying aliquots.

Procedure Analysis of Captopril in pharmaceutical samples­

To different aliquots from 0.5-5.0 ml containing 0.125-l.25 ppm of Captopril, 10ml of 2,6 DCPIP having molarity 2.46xlO·s, £ = 8xl03 cm2/mole was added. The volume was made upto 15 ml with doubly distilled water. The absorbance of different sets was recorded at Amax of 2,6 DCPIP i.e. 610 nm. A Beer Lambert graph was plotted by taking known concentrations of the drug, Captopril with 2,6 DCPIP. The concentration of drug samples of the unknown solutions were determined from the plot.

Analysis of Captopril in drug injected whole blood and serum samples-For in vivo studies different sets of aliquots containing 0.2-8.0 ml of whole blood sample containing 0.6-20.7 ppm of drug were taken in 150 ml Erlenmeyer flasks. To each aliquot 10 ml of 2.46xlO-s M 2,6-DCPJP, £=8x103 cm2/mole was added. It was then diluted to 18 ml by adding doubly distilled water. The absorbance values of all such aliquots were recorded at 610 nm and unknown concentrations were calculated as mentioned in the above procedure.

3038 INDIAN J CHEM, SEC A, DECEMB ER 2003

The same procedure is followed fo r the serum with drug inj ected samples.

Results and Discussion Spectrophotometri c determination of Captopril can

be successfull y perfo rmed as direct method involving two elec tron change. Captopril undergoes ox idation and the thiol functi onal group is converted into corresponding disulphide confirming the 2: I stoichiometry . Beer-Lambert plot for the spectrophotometric determination of Captopril usmg 2,6-DCPIP was fo und to be linear over the range 0.37- 1.25 ppm.

In case of the ill vivo samples of whole blood, linear plots are observed in the range 0.67-20.78 ppm and in serum sample fro m 0 .4-4.0 ppm. The linearity of plots fo r pure whole blood and serum samples may be due to the presence of mixed di sulphide or glutathi one, cysteine present in human sys tem. The amount of Captopril thus assimil ated can be determined by the comparati ve studi es . of pure and

Table I- Analys is of drug inj ected whole blood using 2,6-dichlorophenol indophenol

Blood sample Drug (ppm) (ppm)

Taken Found

0.6-20.0 0.6 0.6 1.7-20.0 1.7 1.7 3.4-20.0 3.4 3.4 6.9-20.0 6.9 6.9 10.3-20.0 10. 3 10.34 13.8-20.0 13.8 13.8 1 17.2-20.0 17.2 17.22

20.0 20.7 20.78 *Data are an average of ten determinations.

Error (0/0 )

0.00 0.000 0.00 0.00 0.40

0.072 0. 116 0.40

Tab le 2- Analys is of drug injected serum wi th 2.6-dichlorophenol indophenol

Serum sample Drug (ppm) (ppm)

Taken Found

0.4-4.0 0.40 0.40 0.8-4.0 0.80 0.80 1.2-4.0 1.2 1.2 1.6-4.0 1.6 1.6 2.0-4 .0 2.0 2.004 2.5-4.0 2.5 2.52 2.9-4.0 2.9 2.9 3.3-4.0 3.3 3.3 3.7-4.0 3.7 3.7 1

4.0 4.0 4.0 "Data are an average o f eight determinations.

Error (0/0)

0.00 0.00 0.00 0.00 0.20 0.80 0.00 0.00 0.27 0.00

drug injected blood and serum sample plots. The proposed method showed the error of 0.15% for whol e blood and 0.16% for serum samples. (Tables 1 and 2)

Bas ically, the metabolite of Captopril formed in the body needs to be removed. This prompted us to develop a method fo r finding out the blood and serum level of drug ass imilation and also the level of unass imilated drug. For this purpose, solid products of the in vivo samples were analyzed by FTIR and high resolution proton nuclear magn~ti c resonance spectrophotometric studies.

FTIR spectrum of pure Captopril in chloroform as KBr pellets indicates characteri stic frequencies at 945cm-' due to -CN bending vibration; at 1550 cm-' due to ring nitrogen; at 1650 cm-' due to C=O and at 1760 cm-' indicating carboxylate ion COO- (of COOH group). Characteri stic peak at 2550 cm-' indicates the presence of - SH group of captopril.

In FTIR spectrum of drug injected whole blood sample a new peak appears at 654 cm ' confirming the ox idation of - SH group to S-S (di sulphide) . Peak at 930 cm-' due to metabolite of whole blood, at 1547 cm-' due to -N H bending and at 1656 cm-' due to C=N vibrati on, are with shi fted frequencies in human blood system. In drug injected serum sample, peak at 2550 cm-' indicates conversion of - SH group of captopril into -C=S ; a new peak at 1167 cm-' indicates - C=S stretch another peak at 669 cm-' indicates presence of C-l bond . The peaks at 1652 and 1558 cm-' indicate-NH bending and C=N vibration and a characteri stic peak at 443 cm-' indicates fo rmation of - S-S bond in drug injected serum sample.

FTrR spectrum of 2,6-DCPIP shows a broad band at 3407 cm-' indi cating - OH stretch of phenol group, a band at 2940 cm-' show ing -CH2 vibration, at 1505.7 cm-' showing C=N vibration and at 84 1.0 cm-' showing -C-CI group stretching. In the drug injected whole blood sample of patient and on oxidati on of the sample wi th 2,6-DCPIP, a characteri sti c peak at 34 14.3 cm-' ind icating - OH group gets shi fted by 7.4 cm-'. Peak at 2920.7 cm-' shows-CH2 stretch of benzene , at 286 1 cm-' show CH2-COO- stretch of hemoglobin , at 2 130 cm-' indicate C=C stretch of hemoglobi n, at 159 1 cm-' shows linkage of - SH bond to -N-C of hemog lobin as - N-C=S and band at 1130 em-' shows C6HS-OH stretching frequency . Band at 669.8 cm-I show - S-S bond and at 939.7 cm-' indicates C-CI bond.

SRI V ASTA V A et al.: MICRODETERMINATION OF CAPTOPRIL 3039

Table }-Characteri sti c 'H-NMR chemical shift (299.9 MHz) of pure serum:2,6-dcpip and drug serum:2,6-dcpip

S No NMR of pure serum : 2,6-DCPIP

NMR of drug serum: 2,6-DCPIP

Assignment of signals

0.8-1.0 ppm 0. 9- 1. 1 ppm R-CH) protons of serum overlapping with CH3 of drug

2 1.15- 1.25 ppm Serum protons

3 1.3 ppm -CHz-CI protons o f 2,6DC PIP and CH-CI of serum

4 1.5, I. 9-2.2 ppm Serum protons

5 1.45 ppm R -CH2 serum protons

6 2.4 ppm C6HS-CH proton of 2,6DCPIP

7 2.7-3.0 ppm Weak serum proton signal

8 2.6, 2.65,2.7 and 2.75 ppm Overlapping serum proton signal with non-equi valent protons of indo le group of 2,6DCPIP

9 2.8 ppm

10 2.8-2 .85 ppm

II 3.5-3.8 ppm 3.0 ppm

12 3.85-3.9 ppm(m)

13 3.6-3.7 ppm(m)

14 4.8 ppm 4 .8 ppm

15 7.4 ppm(m)

In FfIR spectrum of drug inj ected serum with 2,6-DCPIP, bands at 3401.2 cm·1 and 3289.2 cm·1 indicate free-OH group of 2,6DCPIP and of captopril. Sharp peak at 525 cm' l indicates formation of H2C-S-CH2 linkage (thioacetal) and a peak at 709.3 cm,l show formation of -S-S bond of captopril di sulphide.

Proton nuclear magnetic resonance spectroscopic technique was adopted for further quantitati ve measurements of pharmaceutical and in vivo preparations. The 300 MHz 'H-NMR spectra of the product were obtained in D20 solvent from RSIC, Bombay. Results of spectral studies are summari sed in Table 3

A perusal of seven NMR spectra of pure drug, reagent, blood sample and drug injected whole blood and serum sampl es, confirm the derivatisation of -SH group by disappearance of its signal at 1.9 ppm and also by appearance of signals at 3.65 ppm and 7.4ppm

O=C(R)-CH3 o f serum prote in

-NH proton of drug interaction with -CH proton o f aromatic benzene of 2,6DCPIP

RCH 21 proton interaction with indo le of 2,6DCPIP

Non-equivalent protons of indole and aromat ic ring

-(S-CH2)2 di sulphide proton signal s

S ignals of conjugated carbon proton of serum

Signal of Captopril hemithi oacetal , i.e., CHz-S-CH20H protons

confirming C-S bond and thiohemiacetal protons. Various oxidising agents like NBCL, ICL, N­

chlorosuccinamide and 2,6-DCPIP have been considered for determining the drug by titrimetry also. Stoichiometry of the products by various halogenating reagents like NBCL,NCS showed 1: 1 combination while ICI and 2,6-DCPIP indicated 1:2 stoichiometry with captopril.

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