CHAPTER 4

SPECTROPHOTOMETRIC DETERMINATION OF CARVEDILOL

IN PHARMACEUTICALS AND ITS SINGLE CRYSTAL XRD

STUDIES

4. 1 INTRODUCTION

4. 2 ANALYTICAL CHEMISTRY

4. 3 EXPERIMENTAL

4. 4 RESULTS AND DISCUSSION

4. 5 SINGLE CRYSTAL XRD OF CARVEDILOL

4. 6 CONCLUSIONS

4. 7 REFERENCES

4. 1 INTRODUCTION

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Carvedilol, 1-(9H-carbazol-4-yloxy) - 3-[2-(2-methoxyphenoxy) ethylamino]

propan-2-ol (CAR) is a non - selective β - adrenergic blocking agent with α1 -

blocking activity indicated for the treatment of mild to moderate congestive heart

failure (CHF). It is the first β-blocker labeled in United States especially for the

treatment of heart failure of ischemic or cardiomyopathic origin with significant

antioxidant acitivity1-3. It also displays α1-adrenergic antagonism in addition to

blocking both β1 and β2 - adrenergic receptors, which confers the added benefit of

reducing blood pressure through vasodilation. Relative to other β-blockers,

carvedilol (CAR) has minimal ‘inverse agonism’ indicating a reduced negative

chronotropic and inotropic effect, which decreases its potential to worsen symptoms

of heart failure4. At high dosages, it exerts calcium channel blocking activity5. The

benefits of using CAR in patients with CHF in both single-center and multicenter

trials have been reported in the literature6-8. The apparent mean terminal elimination

half-life of CAR ranges from seven to ten hours. CAR is primarily metabolized by

the liver, with less than 2 percent of a given dose excreted unchanged in urine.

Plasma concentrations of CAR are nevertheless increased in patients with renal

failure and are highly bound to plasma proteins. It inhibits the generation of oxygen

free radicals and prevents low-density lipoprotein (LDL) oxidation, which in turn,

reduces the uptake of LDL into the coronary vasculature. Based on paramagnetic

electron resonance studies, CAR directly and in a dose-dependent mode, removes

free radicals of oxygen and protects cardiac membranes from lipid peroxidation

induced by them, both in vivo and in vitro. It prevents vitamin E, gluthation and SH

protein depletion induced by oxidative stress, the main defense mechanisms against

tissue injury caused by free radicals9. In view of its antioxidant activity 10, 11, CAR is

efficient to suppress lipid peroxidation, protein oxidation or to inhibit the generation

of reactive oxygen species12, 13.

Over the past few years, β-blocker therapy has become one of the main

treatment modalities for heart failure. Initially, β-blocker therapy causes negative

inotropic and chronotropic effects while improvements in left ventricular function

develop over time. These unique properties of CAR, a third generation agent, which

causes multiple adrenergic (β1, β2 and α1) blockade, besides its antioxidative and

antiproliferative effects, may be important in preventing progressive deterioration of

left ventricular dysfunction and heart failure. Several analytical methods such as

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spectrophotometry14, 15, HPLC16-20, capillary electrophoresis21, 22, fluorometry23, 24,

synchronous fluorimetry25, HPLC-MS / MS26, differential pulse voltammetry27, GC-

MS28 have been widely used for the determination of CAR. In view of the

importance of CAR in the treatment of CHF, simple methods for the determination

of CAR have been described. The proposed methods are compared with the

published spectrophotometric methods and are summarized in Table 4. 1. The two

spectrophotometric methods reported recently14,15 are in the UV region and it is well

known that uv-spectrophotometry is not a selective method, and therefore excipients

can interfere with the method. Therefore the proposed method takes the advantage

over the uv-spectrophotometric method in terms of selectivity. The proposed

methods are simple, accurate and easy to apply in routine use.

4. 2 ANALYTICAL CHEMISTRY

Silva et al.,29 reported a novel flow injection (FI)-spectrofluorimetric

methodology for the determination of carvedilol in micro heterogeneous medium.

The method was applied to determine carvedilol in commercial pharmaceutical

formulations. The methodology developed showed high selectivity with respect to

the common excipients used in pharmaceuticals.

Rathod et al.,30 reported a simple, precise and sensitive HPLC procedure for

determination of carvedilol in human plasma. The developed method was specific

and had a linearity range of 1-128 ng mL-1 with intra- and inter-day precision less

than 15 %.

Zarghi et al.,31 reported a simple, rapid and sensitive isocratic RP-HPLC

method with fluorescence detection. The calibration curve was linear over the

concentration range of 1-80 ng mL-1. The coefficients of variation for inter-day and

intra-day assay were found to be less than 8.0 %.

Jeong et al.,32 reported a rapid, sensitive and selective method for the

determination of carvedilol in human plasma using hydrophilic interaction liquid

chromatography with tandem mass spectrometry (HILIC-MS/MS). The lower limit

of quantification for carvedilol was 0.1 ng mL-1 using 50 μL plasma sample. The

coefficient of variation and relative error for intra- and inter-assay at four QC levels

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were 1.6- 4.5 % and -6.4 - 4.8 %, respectively. The method was successfully applied

to the bioequivalence study of carvedilol in humans.

A sensitive and rapid extractive spectrophotometer method has been

developed by Verma and Syed33 for the assay of carvedilol in pharmaceutical

formulations. The method was based on the formation of a chloroform soluble ion-

pair complex between carvedilol and bromocresol green in acidic medium. Beer's

law obeyed in the concentration range of 5-25 µg mL-1. The results obtained by the

proposed method were validated statistically and by recovery studies.

Gannu et al.,34 developed a simple and sensitive analytical method for

carvedilol in human serum by using high performance liquid chromatography

(HPLC). The method employs a liquid-liquid extraction for isolation and sample

concentration followed by reversed phase liquid chromatography (RPLC) analysis

using ultraviolet (UV) detection at 238 nm. The calibration curve was linear over a

concentration range of 5-500 ng mL-1. The extraction recovery of carvedilol is more

than 75 %. The validated method was applied to a pharmacokinetic study of

carvedilol in human serum following the administration of a single carvedilol tablet

(6.25 mg).

Cardoso et al.,35 reported a simple extraction-free spectrophotometric

methods for the determination of carvedilol (CAR). The methods were based either

on charge-transfer reaction of the drug with the σ-acceptor iodine, in acetonitrile, or

on ion-pair formation with the acidic sulphophthalein dyes bromothymol blue (BTB)

and bromocresol green (BCG), in chloroform. The proposed methods were applied

for the determination of CAR in tablets and compounded capsules.

Two simple, specific, accurate and precise methods, namely, reverse phase

high performance liquid chromatography and high performance thin layer

chromatography were developed by Patel et al.,36 for estimation of carvedilol in bulk

drug and pharmaceutical formulations. The methods were validated in terms of

linearity, accuracy and precision. The linearity curves were found to be linear over

1-35 μg mL-1 for high performance liquid chromatography and 50-300 ng spot-1 for

high performance thin layer chromatography. The limit of detection and limit of

quantification for high performance liquid chromatography were found to be 0.2 and

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0.85 µg mL-1, respectively, and for high performance thin layer chromatography, 10

and 35 ng spot-1 respectively. The proposed methods were used to determine the

drug content of marketed formulations.

Pires et al.,37 reported a simple, fast, sensitive and selective flow based

procedure for the chemiluminometric determination of carvedilol. The methodology

took the advantage of the antioxidant capacity of carvedilol to inhibit the

chemiluminescence response resulting from the oxidation of luminal by

hypochlorite, by acting as a hypochlorite scavenger.

Iegglli et al.,38 reported ultraviolet spectrophotometric and nonaqueous

volumetric methods for the determination of carvedilol in pharmaceutical

formulations. The methods were evaluated for linearity, accuracy and precision. The

methods were applied to tablets and compounded capsules.

A sensitive and efficient method was developed by Myung and Jo39 for the

determination of carvedilol and its metabolites in human urine by gas

chromatography-mass spectrometry (GC-MS) The method was effective for the

determination of carvedilol and its three metabolites in human urine.

Hokama et al.,40 reported the use of high-performance liquid chromatography

(HPLC) with spectrofluorometric detection, using a solid-phase extraction for a

simple, rapid and sensitive determination of plasma carvedilol levels in rats. The

detection limit was 3.6 ng mL-1 in the plasma.

Ptacek et al.,41 developed a high-performance liquid chromatographic

method for the quantitation of carvedilol in human plasma. The method involved

protein precipitation with methanol, concentration of the supernatant by evaporation

and reversed-phase chromatography with fluorimetric detection. The assay was used

for pharmacokinetic studies.

4. 3 EXPERIMENTAL

4. 3. 1 Apparatus

A Shimadzu UV-2550 UV-VIS Spectrophotometer with 1cm matched quartz

cells was used for absorbance measurements. A Stoe IPDS-II two-circle

diffractometer was used for X-ray data collection. For data deduction X-AREA was

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used. SHELXS97 and SHELXL97 programs were used to solve structure and to

refine the structure and for molecular graphics PLATON and XP in SHELXTL-Plus

was used42-45.

4. 3. 2 Reagents and Solutions

All reagents used were of analytical reagent grade and distilled water is used

for the preparation of all solutions. A 1000 µg mL-1 standard solution of CAR

(Cadila Pharmaceuticals, Gujarat, India) was prepared in 50 % ethanol and diluting

to the mark in a 100 mL calibrated flask. The stock solution was diluted

appropriately to get the working concentration. Ninhydrin (0.3 %), NaOH (1 %),

Na2CO3 (2 %), sodium nitroprusside (1 %) and CH3CHO (10 %) were used.

4. 3. 3 Procedures

4. 3. 3. 1 Determination of carvedilol using ninhydrin as reagent

Aliquots containing (0.2 – 1.2 mL) 10 µg mL-1 of CAR were transferred into

a series of 10 mL calibrated flasks by means of a micro burette. To this 2 mL of 0.3

% ninhydrin was added followed by 0.5 mL of 1% NaOH solution. The contents

were shaken well and were set aside for 5 minutes and diluted up to the mark with

distilled water and mixed well. The absorbance of each solution was measured at

402 nm against the corresponding reagent blank.

4. 3. 3. 2 Determination of carvedilol using acetaldehyde as reagent

Aliquots containing (0.6 – 2.0 mL) 10 µg mL-1 of CAR were transferred into

a series of 10 mL calibrated flasks by means of a micro burette. To this 1 mL of

freshly prepared acetaldehyde solution (10 %) and 1 mL of sodium nitroprusside (1

%) were added followed by 1 mL of Na2CO3 (2 %) solution. The contents were

shaken well and were set aside for 15 minutes and was heated on a water bath for

about 5 minutes and diluted up to the mark with distilled water and mixed well. The

absorbance of each solution was measured at 558 nm against the corresponding

reagent blank.

4. 3. 3. 3 Analysis of dosage forms

Two different CAR dietary supplement products were purchased and a

sample stock solution of each was prepared by grinding an amount of powdered

tablets equivalent to 0.1 g of CAR to a fine powder using a mortar and pestle and

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transferring into a 100 mL volumetric flask by washing with methanol and making

up to the mark with methanol. A convenient aliquot was then subjected to analysis

by the proposed method.

4. 3. 3. 4 Preparation of carvedilol crystals

Carvedilol was obtained as a gift sample from Cadila Pharmaceuticals,

Gujarat, India. X-ray quality crystals were obtained from toluene by slow

evaporation, M.P = 385-387 K.

4. 4 RESULTS AND DISCUSSION

The determination of CAR is based on the two simple reagents. Method I

involve the reaction of CAR with ninhydrin in presence of a base to form yellowish-

orange colored iminium salt, which has an absorption maximum at 402 nm (Figure

IV. 1) and is given in Scheme 4. 1. Method II is based on the reaction of the former

with acetaldehyde in presence of sodium nitroprusside in basic medium to form an

adduct, which has an absorption maximum at 558 nm (Figure IV. 2) and is given in

Scheme 4. 2.

4. 4. 1 Optimization of Experimental Conditions

Preliminary experiments are performed to fix the concentration of the

reagents that could be measured spectrophotometrically and are found to be 0.3 % (2

mL), 10 % (1 mL), and 1 % (1 mL) for ninhydrin, acetaldehyde and sodium

nitroprusside respectively. For method I, NaOH medium is found to be ideal and

0.5 mL of 10 % NaOH in a total volume of 10 mL is adequate for the reaction to

take place and for method II, Na2CO3 is found ideal and 1mL of 2 % Na2CO3 is

found to be optimum. A 5 minute standing time is found necessary for the complete

reaction to take place. Under these conditions, the system is stable for a period of

over 8 hrs.

4. 4. 2 Analytical Data

A linear correlation is found between absorbance and concentration of the

drug (Figure IV. 3 & IV. 4). The optical parameters such as molar absorptivity,

Beer’s law limits and Sandell’s sensitivity values are calculated and are given in

Table 4. 2. Correlation coefficients, intercepts and slopes for the calibration graphs

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are also compiled in Table 4. 2. The limit of detection and quantification calculated

according to ICH guidelines are also incorporated in Table 4. 2.

4. 4. 3 Method Validation

To evaluate the accuracy and precision of the method, pure drug within the

working limits is analyzed, each determination being repeated five times. The RE

(%) and RSD (%) values are less than 2 and indicate the high accuracy and precision

for the methods and are presented in Table 4. 3. For a better picture of

reproducibility on a day-to-day basis, a series is run in which standard drug solution

at five levels are determined each day for a week, preparing all solutions fresh. The

relative standard deviation values are less than 2 % and represent the best appraisal

of the procedures in daily use. The proposed method is applied to the assay of CAR

in two commercial dietary supplements. An aliquot containing 0.8 mL (10 µg mL-1)

drug solution is taken and assayed according to the proposed methods. The content

of the tablet formulation is calculated by applying suitable dilution factor. The

accuracy of the proposed method is checked by a thorough analysis of each spiked

sample and is given Table 4. 4. The results are compared statistically with those of

reference method at 95 % confidence level. The calculated student’s t-test and F-test

values did not exceed the tabulated value, indicating that there is no significant

difference between the proposed method and the reference method in respect to

accuracy and precision.

4. 4. 4 Interference Study

In the pharmaceutical analysis, it is important to test the selectivity towards

the excipients and fillers added to the pharmaceutical preparations. Several species

that can occur in the real samples together with drug were investigated. The level of

interference is considered tolerable (Table 4. 5). From this study it is apparent that

the usual co-formulated substances seldom interfere in the proposed methods.

4. 5 SINGLE CRYSTAL XRD OF CARVEDILOL

Many pharmaceutical materials exhibit polymorphism. Polymorphism is

characterized as the ability of a drug substance to exist as two or more crystalline

phases that have different arrangements and / or conformations of the molecule in

the crystal lattice46-49. Depending upon the condition used to generate the crystalline

forms, the drug may exhibit one or more unstable, polymorphic crystalline states.

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The existence of these polymorphic crystalline states is important for many

pharmaceutical materials, as they can have a major effect upon the uptake of the

active drug into the blood-stream once ingested and the shelf life of the drug.

Polymorphism in drugs can also have direct medical implications. Medicine is often

administered orally as a crystalline solid and dissolution rates depend on the exact

crystal form of polymorphs. Understanding the physical and chemical factors that

underlie the formation of different polymorphs of a given molecule is fundamentally

important as, in principle, any solid state property may differ between different

polymorphic forms of the same molecule50-52.

Chen et al., reported the crystal structure of carvedilol53. It crystallizes in P21/

c space group. The second polymorph of carvedilol in the same space group as the

first differs in two key torsional angles (Figure IV. 5). The bond lengths and angles

can be regarded as normal. The structure of second polymorph (I) shows several

significant differences from the already known polymorph (II). Although both

structures are monoclinic and crystallizes in same space group, the cell parameters

are completely different [for II, a = 9.094(1) Å, b= 12.754 (1) Å, c= 18.330 (2) Å

and β = 97.36 (1)º]. The molecular conformations of I and II are totally different,

but a closer outlook reveals that only two torsion angles are infact responsible for

this difference (Table 4. 6 ). The conformation about the C2-C3 bond is

antiperiplanar in I and it is synclinal in II, and the conformation about O8-O81 is

anticlinal in I and synclinal in II. A least-square fit (Figure IV. 6), of matching

torsion angles shows similarities and differences of I and II.

Nevertheless, the two classical hydrogen bonds in I are also present in II and

are given in Table 4. 7 and 4. 8. The carbazole N atom forms an intermolecular

hydrogen bond to the methoxy O atom (O87) and the hydroxyl group forms an

intermolecular hydrogen bond to amino N atom. However, whereas it is a 21 screw

axis that generates the second molecule for the Nc-H—O (c = carbazole) hydrogen

bond in I and II, the symmetry operation for generating the second molecule for the

O-H---N hydrogen bond is a c-glide plane in I, but an inversion centre in II. The

amino H atom has close contacts to the hydroxyl O atom and the ether O atom,

(Tables 4. 7 and 4. 8) but the H---O distances are rather long and the N-H---O

angles are rather small.

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The H atoms are found in a difference map. Those bonded to C are relocated

in idealized locations (C-H = 0.95 – 1.00 Å ) and refined as riding with Uiso(H)= 1.2

Ueq (C) or 1.5 Ueq(methyl C). The positions and Uiso values for the H atoms bonded

to N and O are freely refined.

FIGURE IV. 5 - THE MOLECULAR STRUCTURE OF I SHOWING 50%

PROBABILITY DISPLACEMENT ELLIPSOIDS

FIGURE IV. 6 - LEAST-SQUARES OVERLAP OF THE MAIN BACKBONES

OF I (FULL BONDS) AND II (OPEN BONDS)

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The crystal data and other relevant parameters regarding data collection, data

reduction, structure solution and refinement are given in Table 4. 9. The atomic

coordinates with their equivalent displacement parameters are presented in Table 4.

10. The anisotropic displacement parameters are listed in Table 4. 11. The bond

lengths, hydrogen bonding geometry with isotropic displacement and torsion angles

are compiled in Table 4. 12, 4. 13 and 4. 14 respectively.

4. 6 CONCLUSIONS

Simple and rapid method for the spectrophotometric determination of CAR

have been developed using two reagents.

The reagents are cheap and easily available.

The ingredients usually present in the pharmaceutical formulations of these

drugs seldom interfere in the proposed method.

The proposed method has been successfully applied for the determination of

CAR in two dietary supplements.

The single crystal x-ray studies of carvedilol were performed and analysis

led to the conclusion of the existence of second polymorphic form.

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TABLE 4. 1 – COMPARISON TABLE FOR PROPOSED METHOD AND

REPORTED METHOD

ReagentRange

µg mL-1λ max (nm)

Molar

absorptivity

(L mol-1 cm-1)

Reaction

conditionsRef

- 4.0 – 36.0 285 1.539 × 104 Methanol

(as solvent)

[14]

- 2.0 – 20.0 242 2.193 × 104 Methanol

(as solvent)

[15]

- 1.0-10.0 245 4.453 × 104 PEG- 400 +

Water

(as solvent)

[15]

Proposed MethodNinhydrin 0.2 – 1.2 402 2.571 × 104 NaOH medium -Acetaldehyde

+

SNP

0.6 – 2.0 558 1.617 × 104 Na2CO3

medium

-

PEG - Polyethylene glycol

SNP – Sodium nitroprusside

TABLE 4. 2- ANALYTICAL PARAMETERS

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Parameters CAR-NIN CAR-AA

λmax (nm) 402 558

Beer’s law limits (μg mL-1) 0.2 – 1.2 0.6 – 2.0

Molar absorptivity (L mol-1 cm-1) 2.571 × 104 1.617 × 104

Sandell’s sensitivity (μg cm-2) 1.58 × 10-2 2.51 × 10-2

Limit of detection** (μg mL-1) 0.139 0.157

Limit of quantification** (μg mL-1) 0.418 0.222

Regression equation* Y= bX + a Y= bX + a

Slope (b) 0.024 0.021

Intercept (a) 0.047 0.045

Correlation coefficient (R) 0.9996 0.9990

* Y is the absorbance and X is the concentration in µg mL-1

** Calculated using ICH - Guidelines

TABLE 4. 3 -EVALUATION OF ACCURACY AND PRECISION

Using Ninhydrin

Amount taken(µg mL-1)

Amount found*(µg mL-1)

SD(µg mL-1)

RE(%)

RSD(%)

0.4 0.401 0.002 0.150 0.4540.6 0.601 0.003 0.067 0.4790.8 0.801 0.002 0.125 0.234

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1.0 1.006 0.011 0.560 1.1311.2 1.205 0.009 0.383 0.718

Using Acetaldehyde

Amount taken(µg mL-1)

Amount found*(µg mL-1)

SD(µg mL-1)

RE (%)

RSD (%)

0.6 0.603 0.001 0.433 0.1890.8 0.801 0.002 0.100 0.1851.0 1.008 0.008 0.800 0.8301.2 1.206 0.009 0.500 0.7421.4 1.405 0.011 0.383 0.796

* Mean value of five determinations

RE – Relative Error; SD – Standard Deviation; RSD – Relative Standard

Deviation

TABLE 4. 4 - RESULT OF ASSAY OF FORMULATIONS BY THE

PROPOSED METHODS

Brand Name Labeledamount (mg)

Found* ± SDUsing NIN

Found* ± SDUsing AA

ReferenceMethod

Carloc 25 25.024.88 ± 0.08at = 2.22 bF = 3.14

24.98 ± 0.17a t = 2.63 bF = 1.47

24.72 ± 0.14

Carvidas 25 25.024.89 ± 0.08at = 2.25 bF = 1.44

24.92 ± 0.09a t = 2.63 bF = 1.77

24.78 ± 0.07

* Mean of five determinations

aTabulated t-value at 95% confidence level is 2.78

bTabulated F-value at 95% confidence level is 6.39

TABLE 4. 5 – AMOUNT OF TOLERANCE OF EXCIPIENTS

Excipients Amount (mg)Lactose 65Starch 60Talc 30Sucrose 40

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Stearic acid 30Fructose 55Glucose 60

TABLE 4. 6- SELECTED TORSION ANGLES (º) FOR (I) AND (II)

I II

C12-C11-O1-C2 164.14(11) -175.2

C11-O1-C2-C3 -173.50(11) 177.2

O1-C2-C3-C4 170.44(11) 59.2

C2-C3-C4-N5 178.21(12) 175.0

C4-N5-C6-C7 169.95(12) 167.3

C6-C7-O8-C81 161.69 (13) 159.8

C7-O8-C81-C82 129.41(15) -150.7

TABLE 4. 7- HYDROGEN BOND GEOMETRY (Å,º)

D-H---A D-H H---A D----A D-H---A

O31-H31---N5i 0.92 (2) 2.09 (2) 2.9633 (16) 158 (2)

N1-H1----O87ii 0.94 (2) 2.11 (2) 3.0336 (19) 167.4 (18)

N5-H5----O8 0.910(19) 2.456 (18) 2.8619 (17) 107.3 (13)

N5-H5----O31 0.910 (19) 2.443 (17) 2.8198 (17) 105.0 (13)

Symmetry codes: (i) x, -y+ ½ , z + ½, ; (ii) –x + 1, y + ½, -z + 3/2

TABLE 4. 8 - HYDROGEN BOND PARAMETERS FOR (II)

D-H---A D-H H---A D----A D-H---AO-H---Niii 1.14 1.73 2.837 173

Ncarbazole-H--Oiv 0.90 2.35 3.193 156Namino-H---Oether 0.90 2.48 2.828 103

Namino-H5---Ohydroxyl 0.90 2.70 2.872 92

The geometrical values for the O-H---N bond were taken from the original

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publication. The other values were determined by us with H atoms placed in

calculated positions. Symmetry codes: (iii) 1-x, -y, -z ; (iv) 1–x, ½ + y, ½ -z .

TABLE 4. 9 - CRYSTAL DATA AND STRUCTURE REFINEMENT

Temperature 173(2) KWavelength 0.71073 ÅCrystal system MonoclinicSpace group P 21/cUnit cell dimensions a = 15.4817(9) Å α= 90°.

b = 15.1670(10) Å β= 100.841(4)°.c = 9.0986(5) Å γ = 90°.

Volume 2098.3(2) Å3

Z 4

Density (calculated) 1.287 Mg/m3

Absorption coefficient 0.088 mm-1

F(000) 864

Crystal size 0.38 x 0.19 x 0.19 mm3

Theta range for data collection 2.65 to 27.13°.Index ranges -19<=h<=19, -19<=k<=19, 0<=l<=11Reflections collected 28974Independent reflections 4520 [R(int) = 0.0871]Completeness to theta = 25.00° 99.9 % Absorption correction NoneMax. and min. transmission 0.9835 and 0.9673

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 4520 / 0 / 286

Goodness-of-fit on F2 1.060Final R indices [I>2sigma(I)] R1 = 0.0655, wR2 = 0.1482R indices (all data) R1 = 0.0755, wR2 = 0.1582Extinction coefficient 0.018(3)

Largest diff. peak and hole 0.276 and -0.182 e.Å-3

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TABLE 4. 10 - ATOMIC COORDINATES ( X 104) AND EQUIVALENT ISOTROPIC

DISPLACEMENT PARAMETERS (Å2X 103). U(EQ) IS DEFINED AS ONE THIRD OF THE

TRACE OF THE ORTHOGONALIZED UIJ TENSOR.

x y z U(eq)

O(1) 5774(1) 7189(1) 7456(2) 31(1)O(2) 3966(1) 7495(1) 5884(2) 35(1)O(3) 2020(1) 9145(1) 2381(2) 40(1)O(4) 494(1) 9791(1) 2659(2) 49(1)N(1) 7980(1) 5441(1) 10019(2) 40(1)N(2) 3806(1) 8658(1) 3440(2) 32(1)C(1) 5458(1) 8030(1) 6886(2) 32(1)C(2) 4745(1) 7871(1) 5522(2) 30(1)C(3) 4514(1) 8749(1) 4747(2) 32(1)C(4) 3537(1) 9524(1) 2799(2) 35(1)C(5) 2708(1) 9445(2) 1632(3) 42(1)

C(11) 6486(1) 7182(1) 8606(2) 29(1)C(12) 6913(1) 6368(1) 8873(2) 29(1)C(13) 7647(1) 6290(1) 10040(2) 34(1)C(14) 7953(1) 6998(2) 10974(2) 40(1)C(15) 7520(1) 7792(2) 10676(2) 39(1)C(16) 6798(1) 7897(1) 9499(2) 34(1)C(21) 6194(1) 5199(1) 6887(2) 35(1)C(22) 6793(1) 5524(1) 8120(2) 31(1)C(23) 7467(1) 4972(1) 8874(2) 37(1)C(24) 7559(2) 4105(1) 8420(3) 47(1)C(25) 6964(2) 3800(1) 7203(3) 49(1)C(26) 6287(2) 4332(1) 6442(3) 44(1)C(31) 1293(1) 8791(1) 1468(2) 38(1)C(32) 474(1) 9120(2) 1646(3) 41(1)C(33) -280(2) 8743(2) 816(3) 60(1)C(34) -214(2) 8054(2) -168(3) 73(1)C(35) 588(3) 7738(2) -338(4) 72(1)C(36) 1350(2) 8108(2) 477(3) 55(1)C(37) -326(2) 10183(2) 2799(4) 72(1)

TABLE 4. 11- ANISOTROPIC DISPLACEMENT PARAMETERS (Å2X 103). THE

ANISOTROPIC DISPLACEMENT FACTOR EXPONENT TAKES THE FORM:

-2P2[ H2A*2U11 + ... + 2 H K A* B* U12 ]

U11 U22 U33 U23 U13 U12

O(1) 27(1) 29(1) 35(1) 1(1) 1(1) 1(1)O(2) 28(1) 38(1) 40(1) 5(1) 7(1) -2(1)O(3) 27(1) 51(1) 41(1) -7(1) 6(1) -1(1)O(4) 29(1) 66(1) 52(1) -7(1) 9(1) 8(1)N(1) 31(1) 45(1) 44(1) 13(1) 5(1) 5(1)

135

N(2) 28(1) 32(1) 36(1) 1(1) 5(1) 2(1)C(1) 31(1) 28(1) 36(1) 1(1) 5(1) 2(1)C(2) 27(1) 30(1) 34(1) -2(1) 7(1) 2(1)C(3) 28(1) 32(1) 35(1) -1(1) 5(1) 1(1)C(4) 30(1) 34(1) 41(1) 4(1) 8(1) 2(1)C(5) 32(1) 49(1) 44(1) 11(1) 6(1) 2(1)

C(11) 24(1) 34(1) 29(1) 0(1) 6(1) -1(1)C(12) 25(1) 35(1) 29(1) 2(1) 7(1) -1(1)C(13) 26(1) 44(1) 32(1) 6(1) 7(1) 0(1)C(14) 29(1) 59(1) 31(1) -1(1) 2(1) -5(1)C(15) 33(1) 51(1) 36(1) -12(1) 9(1) -11(1)C(16) 29(1) 36(1) 38(1) -5(1) 10(1) -3(1)C(21) 38(1) 34(1) 35(1) 0(1) 10(1) -5(1)C(22) 31(1) 31(1) 34(1) 4(1) 11(1) -1(1)C(23) 33(1) 37(1) 43(1) 10(1) 14(1) 1(1)C(24) 46(1) 34(1) 65(2) 14(1) 24(1) 8(1)C(25) 62(2) 31(1) 62(2) 0(1) 33(1) -2(1)C(26) 53(1) 36(1) 46(1) -4(1) 21(1) -10(1)C(31) 38(1) 37(1) 37(1) 0(1) 2(1) -4(1)C(32) 33(1) 50(1) 37(1) 5(1) 3(1) -7(1)C(33) 37(1) 93(2) 47(1) 9(1) -2(1) -21(1)C(34) 73(2) 93(2) 47(2) 0(2) -7(1) -46(2)C(35) 97(2) 61(2) 56(2) -14(1) 10(2) -33(2)C(36) 66(2) 42(1) 56(2) -8(1) 12(1) -5(1)C(37) 40(1) 97(2) 82(2) 6(2) 23(1) 24(1)

TABLE 4. 12 - BOND LENGTHS [Å] AND ANGLES [°]

O(1)-C(11) 1.370(2) O(1)-C(1) 1.428(2)

O(2)-C(2) 1.428(2) O(2)-H(2) 0.88(3)

O(3)-C(31) 1.376(2) O(3)-C(5) 1.443(3)

O(4)-C(32) 1.370(3) O(4)-C(37) 1.430(3)

N(1)-C(23) 1.383(3) N(1)-C(13) 1.388(3)

N(1)-H(1) 0.97(3) N(2)-C(3) 1.465(3)

N(2)-C(4) 1.466(2) N(2)-H(2N) 0.93(3)

C(1)-C(2) 1.518(3) C(1)-H(1A) 0.9900

C(1)-H(1B) 0.9900 C(2)-C(3) 1.517(3)

C(2)-H(2A) 1.0000 C(3)-H(3A) 0.9900

C(3)-H(3B) 0.9900 C(4)-C(5) 1.508(3)

C(4)-H(4A) 0.9900 C(4)-H(4B) 0.9900

C(5)-H(5A) 0.9900 C(5)-H(5B) 0.9900

C(11)-C(16) 1.387(3) C(11)-C(12) 1.400(3)

C(12)-C(13) 1.407(3) C(12)-C(22) 1.447(3)

C(13)-C(14) 1.397(3) C(14)-C(15) 1.380(3)

C(14)-H(14) 0.9500 C(15)-C(16) 1.403(3)

C(15)-H(15) 0.9500 C(16)-H(16) 0.9500

C(21)-C(26) 1.391(3) C(21)-C(22) 1.404(3)

136

C(21)-H(21) 0.9500 C(22)-C(23) 1.411(3)

C(23)-C(24) 1.394(3) C(24)-C(25) 1.380(4)

C(24)-H(24) 0.9500 C(25)-C(26) 1.398(4)

C(25)-H(25) 0.9500 C(26)-H(26) 0.9500

C(31)-C(36) 1.387(3) C(31)-C(32) 1.401(3)

C(32)-C(33) 1.389(3) C(33)-C(34) 1.392(5)

C(33)-H(33) 0.9500 C(34)-C(35) 1.367(5)

C(34)-H(34) 0.9500 C(35)-C(36) 1.387(4)

C(35)-H(35) 0.9500 C(36)-H(36) 0.9500

C(37)-H(37A) 0.9800 C(37)-H(37B) 0.9800

C(37)-H(37C) 0.9800 C(11)-O(1)-C(1) 117.09(14)

C(2)-O(2)-H(2) 112(2) C(31)-O(3)-C(5) 115.56(17)

C(32)-O(4)-C(37) 117.4(2) C(23)-N(1)-C(13) 108.91(17)

C(23)-N(1)-H(1) 125.7(18) C(13)-N(1)-H(1) 123.7(19)

C(3)-N(2)-C(4) 110.60(15) C(3)-N(2)-H(2N) 106.0(17)

C(4)-N(2)-H(2N) 110.6(17) O(1)-C(1)-C(2) 107.59(15)

O(1)-C(1)-H(1A) 110.2 C(2)-C(1)-H(1A) 110.2

O(1)-C(1)-H(1B) 110.2 C(2)-C(1)-H(1B) 110.2

H(1A)-C(1)-H(1B) 108.5 O(2)-C(2)-C(3) 108.84(15)

O(2)-C(2)-C(1) 113.03(16) C(3)-C(2)-C(1) 108.21(15)

O(2)-C(2)-H(2A) 108.9 C(3)-C(2)-H(2A) 108.9

C(1)-C(2)-H(2A) 108.9 N(2)-C(3)-C(2) 111.67(15)

N(2)-C(3)-H(3A) 109.3 C(2)-C(3)-H(3A) 109.3

N(2)-C(3)-H(3B) 109.3 C(2)-C(3)-H(3B) 109.3

H(3A)-C(3)-H(3B) 107.9 N(2)-C(4)-C(5) 110.46(17)

N(2)-C(4)-H(4A) 109.6 C(5)-C(4)-H(4A) 109.6

N(2)-C(4)-H(4B) 109.6 C(5)-C(4)-H(4B) 109.6

H(4A)-C(4)-H(4B) 108.1 O(3)-C(5)-C(4) 107.41(18)

O(3)-C(5)-H(5A) 110.2 C(4)-C(5)-H(5A) 110.2

O(3)-C(5)-H(5B) 110.2 C(4)-C(5)-H(5B) 110.2

H(5A)-C(5)-H(5B) 108.5 O(1)-C(11)-C(16) 125.69(17)

O(1)-C(11)-C(12) 115.18(16) C(16)-C(11)-C(12) 119.13(18)

C(11)-C(12)-C(13) 119.56(18) C(11)-C(12)-C(22) 133.09(18)

C(13)-C(12)-C(22) 107.26(17) N(1)-C(13)-C(14) 129.57(19)

N(1)-C(13)-C(12) 108.52(18) C(14)-C(13)-C(12) 121.88(19)

C(15)-C(14)-C(13) 117.05(19) C(15)-C(14)-H(14) 121.5

C(13)-C(14)-H(14) 121.5 C(14)-C(15)-C(16) 122.46(19)

C(14)-C(15)-H(15) 118.8 C(16)-C(15)-H(15) 118.8

C(11)-C(16)-C(15) 119.88(19) C(11)-C(16)-H(16) 120.1

C(15)-C(16)-H(16) 120.1 C(26)-C(21)-C(22) 118.3(2)

C(26)-C(21)-H(21) 120.8 C(22)-C(21)-H(21) 120.8

C(21)-C(22)-C(23) 119.81(19) C(21)-C(22)-C(12) 134.09(18)

C(23)-C(22)-C(12) 106.09(18) N(1)-C(23)-C(24) 129.2(2)

N(1)-C(23)-C(22) 109.21(18) C(24)-C(23)-C(22) 121.6(2)

137

C(25)-C(24)-C(23) 117.6(2) C(25)-C(24)-H(24) 121.2

C(23)-C(24)-H(24) 121.2 C(24)-C(25)-C(26) 122.0(2)

C(24)-C(25)-H(25) 119.0 C(26)-C(25)-H(25) 119.0

C(21)-C(26)-C(25) 120.7(2) C(21)-C(26)-H(26) 119.6

C(25)-C(26)-H(26) 119.6 O(3)-C(31)-C(36) 122.7(2)

O(3)-C(31)-C(32) 116.48(19) C(36)-C(31)-C(32) 120.7(2)

O(4)-C(32)-C(33) 125.6(2) O(4)-C(32)-C(31) 115.89(18)

C(33)-C(32)-C(31) 118.5(2) C(32)-C(33)-C(34) 120.2(3)

C(32)-C(33)-H(33) 119.9 C(34)-C(33)-H(33) 119.9

C(35)-C(34)-C(33) 121.0(3) C(35)-C(34)-H(34) 119.5

C(33)-C(34)-H(34) 119.5 C(34)-C(35)-C(36) 119.8(3)

C(34)-C(35)-H(35) 120.1 C(36)-C(35)-H(35) 120.1

C(31)-C(36)-C(35) 119.9(3) C(35)-C(36)-H(36) 120.1

O(4)-C(37)-H(37B) 109.5 H(37A)-C(37)-H(37B) 109.5

O(4)-C(37)-H(37C) 109.5 H(37A)-C(37)-H(37C) 109.5

H(37B)-C(37)-H(37C) 109.5

120.1 O(4)-C(37)-H(37A) 109.5

138

TABLE 4. 13 - HYDROGEN COORDINATES ( X 104) AND ISOTROPIC DISPLACEMENT

PARAMETERS (Å2X 103)

x y z U(eq)H(2) 4080(20) 7160(20) 6680(40) 68(10)H(1) 8400(20) 5190(20) 10830(40) 67(9)

H(2N) 3344(18) 8387(18) 3780(30) 50(7)H(1A) 5944 8377 6606 38H(1B) 5217 8361 7655 38H(2A) 4978 7467 4821 36H(3A) 4330 9169 5463 38H(3B) 5042 8993 4427 38H(4A) 4013 9775 2337 42H(4B) 3435 9929 3602 42H(5A) 2551 10024 1153 50H(5B) 2795 9019 849 50H(14) 8438 6936 11779 48H(15) 7717 8286 11291 47H(16) 6525 8457 9315 41H(21) 5735 5562 6369 42H(24) 8015 3736 8931 56H(25) 7015 3212 6872 59H(26) 5887 4099 5612 52H(33) -843 8955 921 72H(34) -733 7800 -730 88H(35) 624 7266 -1011 86H(36) 1909 7894 355 65

H(37A) -615 10418 1827 108H(37B) -222 10662 3533 108H(37C) -705 9736 3132 108

TABLE 4. 14 - TORSION ANGLES [°]

C(11)-O(1)-C(1)-C(2) 173.59(15)

O(1)-C(1)-C(2)-O(2) 69.08(19)

O(1)-C(1)-C(2)-C(3) -170.30(15)

C(4)-N(2)-C(3)-C(2) 174.51(16)

O(2)-C(2)-C(3)-N(2) -54.9(2)

C(1)-C(2)-C(3)-N(2) -178.10(16)

139

C(3)-N(2)-C(4)-C(5) -170.03(17)

C(31)-O(3)-C(5)-C(4) -161.71(17)

N(2)-C(4)-C(5)-O(3) 63.7(2)

C(1)-O(1)-C(11)-C(16) 16.4(3)

C(1)-O(1)-C(11)-C(12) -164.18(17)

O(1)-C(11)-C(12)-C(13) -179.37(17)

C(16)-C(11)-C(12)-C(13) 0.1(3)

O(1)-C(11)-C(12)-C(22) 4.6(3)

C(16)-C(11)-C(12)-C(22) -175.9(2)

C(23)-N(1)-C(13)-C(14) -178.8(2)

C(23)-N(1)-C(13)-C(12) -0.7(2)

C(11)-C(12)-C(13)-N(1) -176.49(17)

C(22)-C(12)-C(13)-N(1) 0.5(2)

C(11)-C(12)-C(13)-C(14) 1.7(3)

C(22)-C(12)-C(13)-C(14) 178.69(18)

N(1)-C(13)-C(14)-C(15) 175.9(2)

C(12)-C(13)-C(14)-C(15) -1.9(3)

C(13)-C(14)-C(15)-C(16) 0.4(3)

O(1)-C(11)-C(16)-C(15) 177.82(18)

C(12)-C(11)-C(16)-C(15) -1.6(3)

C(14)-C(15)-C(16)-C(11) 1.4(3)

C(26)-C(21)-C(22)-C(23) -0.1(3)

C(26)-C(21)-C(22)-C(12) 178.8(2)

C(11)-C(12)-C(22)-C(21) -2.7(4)

C(13)-C(12)-C(22)-C(21) -179.0(2)

C(11)-C(12)-C(22)-C(23) 176.3(2)

C(13)-C(12)-C(22)-C(23) 0.0(2)

C(13)-N(1)-C(23)-C(24) 179.0(2)

C(13)-N(1)-C(23)-C(22) 0.7(2)

C(21)-C(22)-C(23)-N(1) 178.76(18)

C(12)-C(22)-C(23)-N(1) -0.4(2)

C(21)-C(22)-C(23)-C(24) 0.3(3)

C(12)-C(22)-C(23)-C(24) -178.85(19)

N(1)-C(23)-C(24)-C(25) -178.2(2)

C(22)-C(23)-C(24)-C(25) -0.1(3)

C(23)-C(24)-C(25)-C(26) -0.2(3)

C(22)-C(21)-C(26)-C(25) -0.2(3)

C(24)-C(25)-C(26)-C(21) 0.4(3)

C(5)-O(3)-C(31)-C(36) 53.9(3)

140

C(5)-O(3)-C(31)-C(32) -129.6(2)

C(37)-O(4)-C(32)-C(33) -4.9(4)

C(37)-O(4)-C(32)-C(31) 176.0(2)

O(3)-C(31)-C(32)-O(4) 3.2(3)

C(36)-C(31)-C(32)-O(4) 179.7(2)

O(3)-C(31)-C(32)-C(33) -176.0(2)

C(36)-C(31)-C(32)-C(33) 0.5(4)

O(4)-C(32)-C(33)-C(34) -179.2(2)

C(31)-C(32)-C(33)-C(34) -0.2(4)

C(32)-C(33)-C(34)-C(35) 0.0(5)

C(33)-C(34)-C(35)-C(36) -0.2(5)

O(3)-C(31)-C(36)-C(35) 175.5(2)

C(32)-C(31)-C(36)-C(35) -0.8(4)

C(34)-C(35)-C(36)-C(31) 0.6(5)

141

Wavelength (nm)

340 360 380 400 420 440 460 480

Abs

orba

nce

0.04

0.06

0.08

0.10

0.12

0.14

0.16

FIGURE IV. 1- ABSORPTION SPECTRUM OF COLORED CAR-NIN ADDUCT

Wavelength / nm

200 300 400 500 600 700 800

Ab

sorb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

FIGURE IV. 2- ABSORPTION SPECTRUM OF COLORED CAR-AA ADDUCT

142

Concen (g mL -1)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

Abso

rbance

0.050

0.055

0.060

0.065

0.070

0.075

0.080

FIGURE IV. 3 – ADHERENCE OF BEER’S LAW FOR THE DETRERMINATION

OF CAR USING NINHYDRIN AS A REAGENT

Concentration (g mL-1)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Abso

rbanc

e

0.04

0.05

0.06

0.07

0.08

FIGURE IV. 4 – ADHERENCE OF BEER’S LAW FOR THE DETRERMINATION

OF CAR USING ACETALDEHYDE AS A REAGENT

143

N

O

H

N

O

OH

H

H3CO

+O

O

HO

HO

NaOH

N

O

H

N

O

OH

H3CO

O

O

+HO-

SCHEME 4. 1- REACTION OF CAR WITH NINHYDRIN IN PRESENCE OF

BASE

144

N

O

H

N

O

OH

H

H3CO

+ CH3

H

O

Na2CO3

N OH

N

O

OH

H3CO

CH2

Na2[Fe(CN)5NO].2H2O

(CAR-A)

+ NO + 2H2ONa2[Fe(CN)5(CAR-A)]

SCHEME 4. 2 - REACTION OF CAR WITH ACETALDEHYDE AND SODIUM

NITROPRUSSIDE IN PRESENCE OF BASE

145

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CHAPTER 5

SPECTROPHOTOMETRIC DETERMINATION OF NEVIRAPINE

USING TETRATHIOCYANATOCOBALT(II) ION AS A REAGENT

AND SINGLE CRYSTAL XRD STUDIES OF ITS DERIVATIVE

5. 1 INTRODUCTION

5. 2 ANALYTICAL CHEMISTRY

5. 3 EXPERIMENTAL

5. 4 RESULTS AND DISCUSSION

5. 5 SINGLE CRYSTAL XRD OF NEVIRAPINE DERIVATIVE

5. 6 APPLICATIONS

5. 7 CONCLUSIONS

5. 8 REFERENCES

149