part-a electrochemical studies of sodium levothyroxine...

Cfuipter-3

PART-A

ELECTROCHEMICAL STUDIES OF SODIUM LEVOTHYROXINE AT SURFACTANT MODIFIED

CARBON PASTE ELECTRODE

International Journal of Electrochemical Science 4 (2009) 223-237.

Sodium levothyroxine

Chapter 3 (part A)

3.1. Introduction

In the present chapter the electrochemical response of sodium levothyroxine (T4) at

carbon paste electrode in the presence of 0.1 M HCl as supporting electrolyte was

investigated by the cyclic voltammetry. The discussion involves the chemistry,

biological relevance of sodium levothyroxine and its oxidation behavior in 0.1 M

HCl solution at the bare and surfactant modified carbon paste electrode. It showed a

well-defined oxidation peak and two sensitive and indiscernible reduction peaks at

the bare carbon paste electrode. The effect of concentration and scan rate of sodium

levothyroxine was studied. The scan rate effect showed that the electrode process is

adsorption controlled. The effect of surfactants like sodium dodecyl sulphate (SDS),

cetyltrimethylammonium bromide (CTAB), and tritonX-100 (TX-lOO) were studied

by mobilization and immobilization methods. The concentration effect of all the

three surfactants was studied. Among these SDS showed excellent enhancement for

both oxidation peak and reduction peak currents. Thus this method offers a

promising, relatively uncomplicated protocol for the preparation of biosensors.

3.2. Chemistry of Sodium Levothyroxine

Sodium Levothyroxine (Scheme 3.1), also L-thyroxine, synthetic T4, or 3, 5,

3', 5'-tetraiodo-L-thyronine, is a synthetic form of thyroxine (thyroid hormone), used

as a hormone replacement for patients with thyroid problems. The natural hormone is

chemically in the chiral L-form, as is the pharmaceutical agent. T4, a form of thyroid

hormones, is the major hormone secreted by the follicular cells of the thyroid gland.

Thyroxine was first isolated in pure form in 1914 at the Mayo Clinic by Edward

Calvin Kendall from extracts of hog thyroid glands [1]. The hormone was

synthesized in 1927 by British chemists Charles Robert Harington and George

Barger. T4 is involved in controlling the rate of metabolic processes in the body and

influencing physical development. Administration of thyroxine has been shown to

significantly increase the concentration of nerve growth factor in the brains.

Thyroxine is a prohormone and a reservoir for the active thyroid hormone

triiodothyronine (T3), which is about four times more potent. T4 is converted in the

tissues by deiodinases, including thyroid hormone iodine peroxidase (TPO), to T3.

The "D" isomer is called "Dextrothyroxine" and is used as a lipid modifying agent

[2].

66

Chapter 3 (part A)

3.2.1. Biosynthesis of Sodium Levothyroxine

Thyroxine is synthesized via the iodination and covalent bonding of the

phenyl portions of tyrosine residues found in an initial peptide, thyroglobulin, which

is secreted into thyroid granules. These iodinated diphenyl compounds are cleaved

from their peptide backbone upon being stimulated by thyroid-stimulating hormone

(TSH). T4 is transported in blood, with 99.95% of the secreted T4 being protein-

bound, principally to thyroxine-binding globulin (TBG), and, to a lesser extent, to

transthyretin and serum albumin. The half-life of thyroxine once released into the

blood circulatory system is about 1 week. Thyroxine can be measured as free

thyroxine, which is an indicator of thyroxine activity in the body. It can also be

measured as total thyroxine, which also depends on the thyroxine that is bound to

TBG. A related parameter is the free thyroxine index, which is total thyroxine

multiplied by thyroid hormone uptake, which, in turn, is a measure of the unbound

thyroxine binding globulins. The normal human adult range of T4 in blood is 4 - 11

Mg/dL [3].

3.2.2. Biological Relevance of Sodium Levothyroxine

Thyroxine (T4) is an important biological component produced in the thyroid

glands. The practical significances of thyroxine measurements for the diagnosis of

hyperthyroidism and hypothyroidism have been known for many years.

Levothyroxine is typically used to treat hypothyroidism [4]. It may also be used to

treat goiter via its ability to lower thyroid-stimulating hormone (TSH), a hormone

that is considered goiter-inducing [5, 6]. Dosing must be carefully controlled to

achieve TSH levels within the normal reference range. Long-term suppression of

TSH values below normal values will frequently cause cardiac side-effects and

contribute to decreases in bone mineral density (high TSH levels are also well known

to contribute to osteoporosis) [7].

Patients prescribed too high a dose of levothyroxine may experience effects

that mimic hyperthyroidism. Overdose can result in heart palpitations, abdominal

pain, nausea, anxiousness, confiision, agitation, insomnia, weight loss, and increased

appetite. Allergic reactions to the drug are characterized by symptoms such as

difficulty breathing, shortness of breath, or swelling of the face and tongue. Acute

overdose may cause fever, hypoglycemia, heart failure, coma and unrecognized

adrenal insufficiency. Acute massive overdose may be life-threatening; treatment

67

Chapter 3 (part A)

should be symptomatic and supportive. Massive overdose may require beta-blockers

for increased sympathomimetic activity [8, 9]. Foods and other substances interfere

with absorption of thyroxine for example calcium and iron supplements and soy

products can reduce absorption of the drug. Grapefruit juice may delay the

absorption of levothyroxine, Other substances that reduce absorption are aluminium

and magnesium containing antacids, simethicone or sucralfate, cholestyramine,

colestipol, Kayexalate. A study suggests that coffee may interfere with the intestinal

absorption of levothyroxine, though at a level less than eating bran [10]. Different

substances cause other adverse effects that may be severe. Ketamine may cause

hypertension and tachycardia and tricyclic and tetracyclic antidepressants increase its

toxicity. On the other hand lithium can cause hyperthyroidism (but most often

hypothyroidism) by affecting iodine metabolism of the thyroid itself and thus inhibits

synthetic levothyroxine as well.

3.2.3 Review of Electrochemistry of Sodium Levothyroxine

Thyroxine (T4) (3, 5, 3', 5'-tetraiodothyronine) [Scheme.3.1] is a derivative

of the amino acid tyrosine & is unique in being the only iodine-containing compound

of importance [11-15]. Thyroxine is an important biological component produced in

the thyroid glands. The practical significances of thyroxine measurements for the

diagnosis of hyperthyroidism and hypothyroidism have been known for many years.

The usual methods for the determination of T4 were enzyme immunoassays

[16-18], time resolved fluroscence [19], radioimmunoassay (RIA) [20], capillary

electrophoresis with laser-induced fluroscence [21], HPLC [22], chemiluminescence

(CL) [23]. However these methods have some disadvantages such as expensive

instrumentation and time consuming complicated operations. Electrochemical

techniques have also been used for the detection of T4. Jacobsen & Fonahn [24]

reported a DPP method for the determination of T4 at HMDE. Cathodic reduction of

T4 on silver electrode was studied by Iwamoto & Co-workers [25] in comparison

with its multi-step reduction at HMDE.

Surfactants are a kind of amphiphilic molecules with a polar (hydrophilic)

head compatible with water on one side and a long hydrophobic tail compatible with

oil on the other side. The applications of surfactants in electroanalytical chemistry

have been widely reported. Surfactant hiodified carbon paste electrodes are used by

Hu's group for the determination of thyroxin in the presence of C T A 6 [26-28] and

68

Chapter 3 (part A)

also determination of thyroxine at the glassy carbon electrode modified with SWNTs

was reported by F. Wang et al [29]. The results showed that the electrochemical

response was greatly enhanced in the presence of trace surfactants.

3.3. Experimental

3.3.1. Apparatus

Electrochemical measurements were carried out with a model-201

electrochemical analyzer (EA-201 Chemilink system) in a conventional three-

electrode system. The working electrode was a carbon paste electrode, having a

cavity of 3mm diameter. A platinum wire and a saturated calomel electrode (SCE)

were used as the counter and the reference electrode respectively.

3.3.2. Chemicals and Reagents.

T4 (obtained from Sigma) was dissolved in methanol with 2% of dilute

orthophosphoric acid to prepare 0.5 mM standard stock solutions and stored at 4°C.

SDS, CTAB and TX-lOO were dissolved in water to form 1 \xM solutions. Other

chemicals used were of analytical grade except for spectroscopically pure graphite

powder. All solutions were prepared with double distilled water.

3.3.3. Preparation of bare carbon paste electrode.

The carbon paste electrode was prepared by hand mixing 70% graphite

powder and 30% silicon oil in an agate mortar for about 30 min to get homogeneous

carbon paste. This carbon paste was then packed into the cavity of a Teflon tube

electrode (3 mm in diameter). Before measurement the electrode was smoothened on

a piece of transparent paper to get a uniform, smooth and fresh surface.

3.3.4. Preparation of surfactant modified carbon paste electrode.

Bare carbon paste electrode was prepared as explained in the section 3.3.3,

and then the measured volumes of surfactant solutions were pipetted onto the surface

of bare carbon paste electrode and kept quiet for about 5min. Then the electrode

surface was carefully washed with distilled water to remove any unadsorbed particles

and air dried. Then a stable and uniform surfactant immobilized film was formed on

the surface of electrode and used for electrochemical analysis. To prepare surfactant

mobilized carbon paste electrode, measured volume of surfactant solutions were

69

Chapter 3 (part A)

pipetted into an electrochemical cell along with the T4 and the voltammograms were

recorded in the potential range 0 mV to 1000 mV.

3.4. Results and Discussion

3.4.1. The voltammetric behaviour of sodium levothyroxine at carbon paste

electrode

Fig.3.1a shows the cyclic voltammogram of T4 at carbon paste electrode,

which was investigated in 0.1 M HCl. When the potential initially sweeps from 0 to

l.OV a well-defined oxidation peak at 0.78V (Oi) in the positive scan and two

reduction peaks at 0.53 (Ri) and 0.32V (R2) on the reversal scan are observed in the

first cycle. In the second and following cycles the peak currents of Oi and Ri

decrease greatly and a new oxidation peak appears at about 0.48V (O2). The peak

currents of Oi and Ri decrease with the increasing of scan number while those of O2

and R2 remain stable. These results shows that the electrochemical behaviors of T4 at

carbon paste electrode are totally irreversible and that the products are strongly

adsorbed on the electrode surface, blocking the mass transfer of T4 from the solution

to the electrode surface.

According to Murphy [30], the appearance of Oi is due to the oxidation of

OH on the phenol moiety of T4. From the structure of T4 it is clear that Ri may be the

reduction peak of iodine atoms on T4. The O2 and R2 are the electrochemical

responses of the product of T4 produced from the oxidation of OH on T4. (Although

the proper electrochemical reactions involved in the oxidation/reduction of T4 have

been proposed, the hidden relationships between these responses are still unknown).

When electrode potential was scanned over the range of 0.5-l.OV, the OH signal (i.e.

Oi) is unchanged but no peaks were observed in the reverse scan. Such resuhs prove

that reduction of the iodine atoms on T4 is achieved only after the oxidation of OH

on T4 because the iodine atoms on the phenol group of T4 are activated after the

stable benzene ring is destroyed during the oxidation process.

3.4.2. Electrochemical response of sodium levothyroxine at carbon paste

electrode in presence of surfactants

It is well known that surfactants can be adsorbed on solid surfaces to form

surfactant film [31, 32] which may alter the over voltage of the electrode and

influence the rate of electron transfer. The electrochemical responses of T4 at carbon

70

Chapter 3 (part A)

paste electrode in the presence of trace amount of surfactants onto the surface

(immobilized form) as well as into the solution (mobilized form) were studied

(Fig.3.2a to Fig.3.2f) in O.IM HCl as supporting electrolyte with 100 mV/s scan rate.

The low signal (solid line) is the cyclic voltammogram of T4 in the absence of

surfactants. However the voltammetric response is apparently improved (dotted line)

in the presence of 10^L of SDS (Fig.3.2a and Fig.3.2b), CTAB (Fig.3.2c and

Fig.3.2d) and TX-lOO (Fig.3.2e and Fig.3.2f) both in mobilized and immobilized

forms respectively. Comparative Cyclic voltammograms of 10|xL TX-lOO, CTAB

and SDS are also shown in the Fig.3.3a (a-d) and Fig.3.3b (a-d) for both mobiUzed

and immobilized forms respectively When the cationic surfactant CTAB and non-

ionic surfactant TX-lOO were used, there was increase in both oxidation as well as

reduction peak currents both into the solution and onto the surface, shifting the

cathodic peak potential to the negative side and anodic peak potential to more

positive. But on the contrary when SDS is used large oxidation and reduction peak

currents obtained. These results show that anionic surfactants can more effectively

promote both oxidation as well as reduction of T4. This may be because T4 is an

amphipathic molecule and in strong basic solution carboxyl group in T4 is

completely ionized and negatively charged. When cationic surfactant CTAB is added

the adsorption of CTAB on the electrode surface may form a positively charged

hydrophilic film on the electrode, therefore the oxidation of T4 is facilitated by the

cationic surfactant CTAB [25, 27]. This explains that T4 exists in more positively

charged by ionization in acidic media i.e. in orthophosphoric acid-methanol T4 exists

in cationic form and interacts with negative-charged head groups SDS via

electrostatic interactions [33]. Therefore by preparing the Thyroxine in methanol

with 2% orthophosphoric acid (pH 4) makes possible the enhancement of both

oxidation as well as reduction peaks compared to CTAB and TX-lOO.

3.4.3. Effect of surfactant concentration on sodium levothyroxine

The effect of surfactant concentration on T4 oxidation/reduction peak currents

are shown in fig (Fig.3.4a and Fig.3.4b) for both mobilized and immobilized forms.

The peak current increases linearly with the concentration of surfactant for all the

three. The peak potential of oxidation peak (Oi) shifts towards positive side and the

peak potentials of reduction peaks (Ri) & (R2) tend to shift towards negative side in

all the three cases. The current response for all the three surfactants in both mobilized

71

Chapter 3 (part A)

and immobilized forms were similar and in both the cases the enhancement in the

current response for the SDS surfactant was more compared to CTAB and TX-100.

3.4.4. Effect of concentration of Hydrochloric acid

From the Fig.3.5 it is clear that as the concentration of HCl increased from

O.OIM to 0.25M the anodic peak potential was decreased from 0.79 V for O.OIM to

0.77 V for O.IM and again increased from 0.77 V for O.IM to 0.785 for 0.25M. The

oxidation was easier at O.IM HCl, therefore O.IM HCl was taken for further studies.

The cathodic peak potentials of Ri peak was negatively shifted and of R2 positively

shifted and the anodic peak potential of Oi is negatively shifted. Multiple

voltammograms indicate that the thyroxine got adsorbed on the surface of the

electrode during the redox process. This conclusion is supported by linear nature of

ipa vs V plots [32] (Fig .3.6a).

3.4.5. Effect of scan rate

The dependence of peak current (ipa) on the scan rate (v) was studied in the

range of 50-300mV/s, a linear relationship was observed suggesting the adsorption-

controlled process of the sodium levothyroxine (Fig.3.6a). The plot of ipa vs scan rate

indicate an increase in peak current with an increase in sweep rate (Fig.3.6b)

confirming that the electrode process at the electrode surface has some adsorption.

3.4.6. Effect of sodium levothyroxine concentration

The cyclic voltammetry showed successive enhancement of peak current on

increasing T4 concentration. The plot of peak current vs the respective concentration

of T4 was found to be linear in the range of 2x10"'' M to 1.2x10' M. The variation of

anodic peak current (ipa) with concentration shown in the fig (Fig .3.7a and Fig.3.7b).

72

Chapter 3 (part A)

3.5. Conclusion

• The present study has demonstrated the SDS, CTAB and TX-lOO surfactants

modified electrode by immobilization and mobilization methods for the

determination of thyroxine in 0.1 M HCl.

• The results shows that anionic surfactant, SDS showed more current

enhancement compared to cationic CTAB and non-ionic TX-100.

• This is because in acidic media T4 exists in cationic form and interacts with

negatively charged SDS through electrostatic interactions.

• Based on the different existing forms of T4 (cationic or anionic) depending on

the preparation media, suitable ionic surfactants can be used to improve the

sensitivity of determination of T4 using a carbon paste electrode.

• Due to good sensitivity, selectivity and reliability of the modified electrode,

there is a good possibility of extending the proposed method to the analysis of

thyroxine in real biological samples.

• With its low cost and ease of preparation the SDS surfactant modified CPE

seems to be of great utility for further sensor development.

73

Chapter 3 (part A)

OH

Scheine.3.1. Structure of sodium levothyroxine

11111111111111111111 i i i H m i i i i | i i i i i i H i i m i i | n i i i i n i i i i n i l

250 500 750 Potential (mV)

1000

Figure 3.1a. Cyclic voltammogram of 1x10'' M Thyroxine at CPE in

O.IM HCl; scan rate, lOOmV/s.

74

Chapter 3 (part A)

^ / ^

\

TlLZpA ^

^ ^ 1

0 250 "'l"""'| "1""

500 Potential (mV)

750 1000

Figure 3.2b. Cyclic voltammogram 1x10" M Thyroxine at the bare CPE (solid

line) and IxlO' M SDS surfactant immobilized CPE (dotted line).

M i l l i i i | i i i i i i i | i i i i i i i [ i i i i i i i i i i i i n I | i i i i n i | i i i i i i i | i I I I m

0 250 500 750 1000

Potential (mV)

Figure 3.2c. Cyclic voltammogram 1x10" M Thyroxine at the bare CPE (solid

line) and IxlO' M CTAB surfactant mobilized CPE(dotted line).

76

Chapter 3 (part A)

^ ^

I I I I I I 1 1 1 I I I I 1 1 1 [ 1 1 1 1 I I 1 1 1 1 1 I I 1 1 1 I I I I I I I I I I I I I I I 11111II111m 1111

0 250 500 750 1000

Potential (mV)

Figure 3.2d. Cyclic voltammogram 1x10" M Thyroxine at the bare CPE (solid

line) and lxlO"^M CTAB surfactant immobilized CPE (dotted line).

I I " I | i i i m i [ I | i i i i i i n i i i i i i i [

0 250 500 750 1000 Potential (mV)

Figure 3.2e. Cyclic voltammogram 1x10"'* M Thyroxine at the bare CPE (solid

line) and lxlO"^M TX-lOO surfactant mobilized CPE (dotted line).

77

Chapter 3 (part A)

I m i l i i | i i i i i i I I I I I i i i i | i i i i i i i | i i I i i i i | i i i i i i i | I I I I I I i | n i l I I I I

0 250 500 750 1000

Potential (mV)

Figure 3.2f. Cyclic voltammogram 1x10" M Thyroxine at the bare CPE (solid

line) and IxlO' M TX-lOO surfactant immobilized CPE (dotted line).

i i i i i i i i I I I I I I rXTTTJTTTn T rm rrpi

750

nmn ™1 1000 250 500

Potential (mV)

Figure 3.3a. Cyclic voltammograms for IxlO" M Thyroxine at (a-d). (a) is bare

CPE, (b) is 1x10 ^M TX-lOO, (c) is IxlO^M CTAB and (d) is IxlO^M SDS

surfactants mobilized CPE.

78

Chapter 3 (part A)

|lllllll|ll M M lllll II ll|lllllll|lllllll[lllllll|lllllll|llll Mil

0 250 500 750 1000 Potential (mV)

Figure 3.3b. Cyclic voltammograms for 1x10" M Thyroxine at (a-d). (a) bare

'M TX-100, (C) 1X10 ^M C T A B J

surfactants immobilized CPE.

CPE and (b) lxlO"^M TX-100, (c) 1x10 M CTAB and (d)lxlO M SDS

• a • b A c

I 1

140-

120-

100-

80

60-1

40

0 2 4 6 8

Surfactant(p,L) 10

Figure 3.4a. Effect of surfactant concentration variation on Thyroxine oxidation

peak current (a-c); (a) IxlO^ M TX-100 ,(b) IxlO^ M SDS and (c) IxlQ- M

CTAB at OfiM, 2fiM, 4nM, 6nM, SjiM, lOjiM mobilized CPE.

79

Chapter 3 (part A)

l O U -

160-• •

140- • •

^ 1 2 0 -

^ 1 0 0 -

80-

•

60-

40-1

• •

I • 1

X

' 1

X

' 1

1

1 r" ^ 1 t

0 2 4 6 8

Surfactant(jiL)

a b c

10

Figure 3.4b. Effect of surfactant concentration variation on Thyroxine oxidation

peak current (a-c); (a) IxlO^M TX-lOO ,(b) IxlQ- M SDS and (d) IxlO' M

CTAB at OnM, 2nM, 4\iM, 6^M, 8nM, lO^M immobilized CPE.

800-1

795

790

E 785

m 780

775

770 1 r 1 r 1 1 1 1 1 1 1 1 1

0.00 0,05 0.10 0.15 0.20 0.25 0.30

[HCI (M)]

Figure 3.5. Graph of the different concentration of Hydrochloric acid (a-f;

O.OIM, 0.05 M, O.IM, 0.15 M, 0.2M and 0.25M).

80

Chapter 3 (part A)

200- • 180- • 160-

- , 1 4 0 -

"3^120-

*100-•

•

80- • 60 J

40- • 1

' 1 1 1 ' 1 ' 1 '

50 100 150 200 250

v(mV) 3 0 0

Figure 3.6a. Graph of current vs scan rate.

| n i i i i H i i i i i i i | i i i i i i H i i i i i i i | i i i i i i H i i i i i i H i n i i i H i i i i n i l


1000

Figure 3.6b. Cyclic voltammograms of different scan rates (a-f; 50mV/s,

lOOmV/s, 150mV/s, 200mV/s, 250mV/s and 300mV/s).

81

Chapter 3 (part A)

1 1 1 1 I I I 1 1 1 1 I I I 1 1 1 1 1 1 1 1 1 1 | i 1 1 1 1 I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 H 1 1 1 1 1 1 1 1 1 1 1 1 I I I

0 250 500 750 1000

Potential (mV) Figure 3.7a. Cyclic voltammograms for the different concentration of T4 (a-g;

2x10'^M,3X10"M, 4X10''M,6X10"M,8X10""M,IxlO'^Mand 1.2X10-'M).

^

350-]

300

,250-

.200-

•'^150-1

100

50-

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Levothyroxine(mM)

v4 Figure 3.7b. Graph of different concentration of T4 (a-f; 2x10"" M, 4xl0'* M,

6X10"* M , 8X10-" M and 1.2 xlQ-^M).

82

Chapter 3 (part A)

3.6. References

[1] E.G. Kendall, J. Am. Med. Assoc, 64 (1915), 2042-2043

[2] Walker et al, 204 (1979) 427 - 429.

[3] Military Obstetrics & Gynecology > Thyroid Function Tests In turn citing:

Operational Medicine 2001, Health Care in Military Settings, NAVMED P-

5139, May 1, 2001, Bureau of Medicine and Surgery, Department of the

Navy, 2300 E Street NW, Washington, D.G., 20372-5300.

[4] B. Vaidya, S.H. Pearce, BMJ (Clinical research ed.) 337 (2008). aSOl.

[5] J. Svensson, U.B. Ericsson, P. Nilsson, The Journal of clinical endocrinology

and metabolism 91 (2006) 1729-34.

[6] M. Dietlein, K. Wegscheider, R. Vaupel, M. Schmidt, H. Schicha, Nuclear

medicine 46 (2007) 65-75.

[7] A. Frilling, G. Liu, F. Weber, Scandinavian journal of surgery, 93 (2004)

278-81.

[8] "Synthroid (Levothyroxine Sodium) Drug Information: Uses, Side Effects,

Drug Interactions and Warnings". RxList. http://www.rxlist.com/synthroid-

drug.htm. Retrieved 2010-07-18.

[9] Irizarry, Lisandro (2010-04-23). "Toxicity, Thyroid Hormone". WebMd.

http://www.emedicine.com/emerg/TOPIC800.HTM. Retrieved 2010-10-31.

[10] B. Salvatore, L. Bartolone, M.A. Pappalardo, A. Russo, D. Lapa, G.

Giorgianni, G. Saraceno, F. Trimarchi, Thyroid, 18 (2008) 293-301.

[11] F.H.frieden and N.W.Layman, J. Biol.chem. 229 (1957) 569.

[12] C.Long, Biochemists Handbook (Redwood Press, London) pp. 743-745.

[13] J. Oppenheimer and H. Samuels, eds.. Molecular Basis of Thyroid Hormone

Action (1983).

[14] ]R. Howard, ed., McGraw-Hill Encyclopedia of Sci. and Tech., Vol. 8,6* Ed.

(McGraw-Hill, New York, 1987).

[15] A.C.Guyton, Textbook of Medical Physiology, 6* Ed. (W.B. Saunders,

1981).

[16] M.R. Gates, W. Clarke, A. Zimlichll, D. S. Hage, Anal. Chim. Acta, 470

(2002) 37-50.

[17] H. Silvaieh, R. Wintersteiger, M.G.Schmid, O. Hofstetter, V. Schurig, G.

Gubitz, Anal Chim. Acta, 463 (2002) 5-14.

83

http://www.rxlist.com/synthroid-

http://www.emedicine.com/emerg/TOPIC800.HTM

Chapter 3 (part A)

[18] CD. Karapitta, A. Xenakis, A. Papadimitriou, T.G. Sotiroudis, Clin. Chim.

Acta, 308 (2001) 99-106.

[19] E. Taimela, V. Kairisto, P. Koskinen, A. Leino, K. Irjala, Eur. J. Clin. Chem.

Clin. Biochem. 35 (1997) 889.

[20] F. Tagliaro, M. Camilot, R. Valentini, F. Mengarda, F. Antoniazzi, L. Tato, /.

Chromatogr. B: Biomed. AppL, 716 (1998) 77.

[21] D. Schmalzing, L.B. Koutny, T.A. Taylor, W. Nashabeh, M. Fuchs, J.

Chromatogr.B: Biomed. AppL, 697 (1997) 175.

[22] M. Yang, S.A. Tomellini, Anal. Chim. Acta, 409 (2000) 45.

[23] E. Gok, S. Ates, Anal. Chim. Acta. 505 (2004) 125.

[24] E. Jacobsen, W.F. Fonahn, Anal. Chim. Acta, 119 (1980) 33.

[25] M. Iwamoto, A. Webber, R.A. Osteryoung, Anal. Chem., 56 (1984) 1202.

[26] C. Hu, X. Dang, S. Hu, J. Electroanal. Chem., 572 (2004) 161.

[27] Q. He, X. Dang, C. Hu, S. Hu, Colloid Surf. B: Biointer., 35 (2004) 93.

[28] C. Hu, Q. He, Q. Li, S. Hu, Analyt. Sci., 20 (2004) 1049.

[29] P. Wang, J. Fei, S. Hu, Colloid Surf. B: Biointer., 39 (2004) 95.

[30] M.A. Murphy, G.D.Wilcox, R.H.Dahm, and F.Marken. Electrochem.

Commun, 5 (2003) 51.

[31] S. Hu, Y. Yan, Z. Zhao, Anal.Chim.Acta 248 (1991) 103

[32] G.N. Kamau, T. Leipert, S. Shulkla, J.F. Rusling, J. Electroanal. Chem. 233

(1987) 173.

[33] S. Zhang, K. Wu, Bull. Korean Chem. Soc. 25 (2004) 1321.

84

http://Anal.Chim.Acta

Cfiapter-3

PART-B

ELECTROCATALYTIC OXIDATION OF SODIUM LEVOTHYROXINE WITH PHENYL HYDRAZINE

AS A MEDIATOR AT CARBON PASTE ELECTRODE: A CYCLIC VOLTAMMETRIC STUDY

Journal of Electroanalytical Chemistry 645 (2010) 10-15.

Sodium levothyroxine

Chapter 3 (part B)

3.7. Introduction

In this chapter the electrochemical oxidation of sodium levothyroxine (T4)

has been studied on carbon paste electrode (CPE) with phenyl hydrazine

homogenous as mediator. The discussion involves the chemistry, biological

relevance of sodium levothyroxine and its oxidation behavior in 0.1 M HCl solution

at the bare and modified carbon paste electrode. The effect of scan rate and

concentration of sodium levothyroxine in presence of trace phenyl hydrazine was

studied. The practical application of the phenyl hydrazine mediated CPE in the

determination of T4 in a commercial tablet sample demonstrated that it has good

selectivity and high sensitivity. Thus this method offers a promising, relatively

uncomplicated protocol for the preparation of biosensors.

3.8. Chemistry and Biological Relevance of Sodium Levothyroxine

The chemistry and biological relevance of sodium levothyroxine has been

explained in detail in chapter 3A section 3.2.

3.9. Review of Cyclic Voltammetry of Sodium Levothyroxine.

The design, fabrication and application of sensitive and selective

electrochemical sensors have been of considerable interests in recent years. The

electrochemical sensors based on chemically modified electrodes (CMEs) have

widely been used for the detection of biologically important organic compounds. The

modified electrodes using transition metal complexes [1], organic electron mediators

[2] and polymer-modified electrodes [3] have attracted the most attention in this

regard. One of the most important characteristics of the electron mediators, which are

used in modified electrodes, is sufficient sensitivity and selectivity [4].

Phenyl hydrazine was the first hydrazine derivative characterized, reported by

Emil-Fischer in 1875. Hydrazine's are nitrogen-containing compounds and constitute

an important class of xenobiotic agents occurring in natural organisms and the free

electron pairs of the nitrogen from the amino group, which are involved in charge

transfer phenomena, as a support for the electron conduction through biostructured

systems [5, 6].

As told in the previous chapter 3A section 3.2.3 the usual methods for the

determination of T4 were immunoassays, high performance liquid chromatography

(HPLC). However these methods have some disadvantages such as expensive

85

Chapter 3 (part B)

instrumentation and time consuming, complicated operations. Some electrochemical

techniques have also been used for the detection of T4 which minimize the sample

pre-treatment; reduce the cost and time of analysis [7-9].

3.10. Experimental

3.10.1. Apparatus

Electrochemical measurements were carried out with a model-201

electrochemical analyzer (EA-201 Chemilink system) in a conventional three-

electrode system. The working electrode was a carbon paste electrode, having cavity

of 3 mm diameter. The counter electrode was a bright platinum wire with a saturated

calomel electrode (SCE) completing the circuit.

3.10.2. Chemicals and Reagents.

T4 (obtained from Sigma, >99.0%) was dissolved in methanol with 2% of dilute

orthophosphoric acid to prepare 0.5 mM standard stock solutions and stored at 4 °C.

Phenyl hydrazine (>97%) was prepared by using double distilled water. Other

chemicals used were of analytical grade except for spectroscopically pure graphite

powder. All solutions were prepared with double distilled water.

3.10.3. Preparation of bare carbon paste electrode

The preparation of bare carbon paste electrode has been explained in detail in

chapter 3A section 3.3.3.

3.10.4. Preparation of phenyl hydrazine modified carbon paste electrode

Measured volume of 0.1 M HCl and phenyl hydrazine solution were pipetted

into an electrochemical cell. Then the standard solution of T4 was added to the cell.

The voltammograms were recorded for T4 in the potential range of 0.0 V - 1.0 V.

3.11. Results and Discussions

3.11.1. Electrocatalytic oxidation of sodium levothyroxine at carbon paste

electrode

Experimental results show that phenyl hydrazine acts as a suitable

intermediate for electron transfer in the oxidation of T4 at the surface of carbon paste

electrode. Fig.3.8a shows the cyclic voltammetric responses of 0.1 mM pheiiyi

86

Chapter 3 (part B)

hydrazine (curve a) and T4 in the absence (curve b) and in the presence (curve c) of

phenyl hydrazine in 0.1 M HCl as supporting electrolyte. T4 oxidation peak current

of (Oi) increases sharply in the presence of phenyl hydrazine.

In the absence of phenyl hydrazine, a well-defined oxidation peak appears at

780 mV (0|) in the positive scan when the potential initially sweeps from 0.0 mV to

1000 mV and two indiscernible reduction peaks ( Ri and R2) at 520 mV and 330 mV

are obtained on the reversal scan (Fig.3.8b). However the peak currents of Oi

decrease greatly and another oxidation peak (O2) at about 420 mV appears on the

second scan. During following successive cyclic scans, the peak Oi decrease all the

same with the increasing of scan number, resulting from the fact that electrode

surface is blocked by the strong adsorption of the reaction products. When the

electrode potential was scanned over the range of 500 - 1000 mV, the Oi signal is

unchanged but no peaks were observed in the reverse scan. All results show that

electrochemical oxidation of T4 is a totally irreversible process, which can be

explained by the strong adsorption of reduction products of T4 at the electrode

surface. The peaks O2 and R2 are ascribed to the electrochemical responses of the

product of T4 [10]. According to Iwamoto et al.'s report [11] R2and O2 are attributed

to the reduction and the oxidation of the iodine atoms on T4 respectively, and O2

always appears following the R2. As for the oxidation peak Oi is considered it may

be caused by the oxidation of the phenolic hydroxyl group on the T4 molecule and

the Ri is the reduction response of the products of T4 such as the hydroquinone-

benzoquinone redox system produced from the oxidation of phenolic hydroxyl group

onT4[12].

Fig.3.8a shows the electrochemical responses of T4 with phenyl hydrazine

blank (curve a), in the absence (curve b) and in the presence (curve c) of phenyl

hydrazine at CPE. It is clear from the fig.3.8a that, the anodic peak current of (Oi) of

T4 in the presence of phenyl hydrazine is much enhanced than at the bare CPE. Also

the oxidation peak potential of T4 in the presence of phenyl hydrazine shifts slightly

from 780 mV to 800 mV. The anodic peak current difference (Ipa) in the presence

and absence of phenyl hydrazine shows that phenyl hydrazine acts as a suitable

intermediate for electron transfer in the oxidation of T4.

The main difficulty in determining the exact mechanism is identification of

the intermediate in the oxidation process [13]. The hydrazines are easier to oxidize so

87

Chapter 3 (part B)

their oxidation behaviors were studied, and the result shows that the oxidation

process is based on the hydrazine moiety and not on their derivative groups [14-18].

The proposed sequence of reactions that occur between phenyl hydrazine and

T4 at carbon paste electrode is shown below.

Phenylhydrazine (aq) 1 ;;>> (Phenylhydrazine) ox (aq)

(Phenylhydrazine) ox (aq) + Sodium levothyroxine (aq) 1 ^ Phenylhydrazine

(aq) + (Sodium levothyroxine) ox (aq)

The above sequence of reactions between phenyl hydrazine and T4 can be

explained as follows. First phenyl hydrazine undergoes oxidation to diazenyl benzene

as shown in the step 1 of scheme.3.2 and attach to the electrode surface as shown in

step 1 of scheme.3.3 followed by two electron transfer to the electrode and the

oxidized phenyl hydrazine then helps the T4 to undergo oxidation probably either one

of the way (2a or 2b) as shown in scheme.3.3.

But when the system contains methanol (since the dilution media for sodium

levothyroxine is methanol) then there is a chance of formation of methoxy benzene,

hydrazine with dinitrogen as the leaving group in small amounts giving the overall

reaction as shown in scheme.3.2 [19-22].

3.11.2. Effect of phenyl hydrazine concentration.

The effect of phenyl hydrazine concentration on the anodic peak current was

studied for the range of 0.025— 0.2 mM phenyl hydrazine concentration, in the

solutions containing 0.1 mM T4 in 0.1 M HCl was shown in Fig.3.9a and Fig.3.9b.

The results showed that by increasing phenyl hydrazine concentration up to 0.1 mM

the anodic peak current increased, whereas higher concentration of phenyl hydrazine

caused a slight decrease in the peak current and almost keeps unchangeable. This

may be due to the fact that the adsorption of phenyl hydrazine at the carbon paste

electrode surface tends to saturation due to the formation of phenyl hydrazine

aggregations. Therefore 0.1 mM was selected as the optimal mediator concentration.

The peak potential for the system shifts slightly to a more positive potential.

3.11.3. Effect of sodium levothyroxine concentration.

The cyclic voltammogram showed successive enhancement of anodic peak

current with increase in concentration of T4. The variation of peak current (J a) with

T4 concentration (Fig. 3.10) in presence of 0.1 mM phenyl hydrazine was linear in

88

Chapter 3 (part B)

the range of 0.025— 0.1 mM with a correlation coefficient 0.997. It was also

observed that the anodic peak potential (Epa) was shifted towards positive potential

with increasing concentration showing adsorption of the oxidized product over the

electrode surface. The detection limit of T4 in the presence of 0.1 mM phenyl

hydrazine was found to be 2.5 |iM by cyclic voltammetric method.

3.11.4. Effect of scan rate

The effect of the potential scan rate on the electrocatalytic properties of

phenyl hydrazine in a 0.1 M HCl supporting electrolyte containing 0.1 mM T4 was

studied. The obtained results showed that the anodic peak current increased linearly

with the increase of scan rate in the range of 100-350 mV/s, it seems that the

electrode process is controlled by adsorption. This is consistent with the discussion

above, i.e. the decrease of the peak current of Oi with increase of scan numbers.

The dependence of the oxidation peak current (Ipa) as well as peak current

function (Ipa /v""^) and also peak potential on the scan rate (v) were studied in the

range 100-350 mV/s as shown in figs.3.11a to 3.11c. A linear relationship was

observed between log Ipa and log v with a correlation coefficient of 0.990 (Fig.3.1 la).

The plot of Ipa/v'*' vs. log v indicated an increase in peak current with an increase in

sweep rate (Fig.3.1 lb) confirming that the electrode process at the electrode surface

has some adsorption. Also, the plot of peak potential Epa vs. log v (Fig.3.1 Ic) was

linear with a correlation coefficient of 0.998. Fig.3.1 Ic shows the relationship

between the oxidation peak potential Epa and the log v and can be expressed by the

following equation:

Epa = 0.1329 log v +0.535 (R=0.998) (3.1)

It can be noted from Fig.3.1 Ic that, along with an increase in the scan rate,

the peak potential for the catalytic oxidation of T4 shifts to the more positive

potentials, suggesting a kinetic limitation to the reaction between the phenyl

hydrazine and T4.

The values of an<, (where ana is the charge transfer coefficient) were

calculated for the oxidation peak of T4 in 0.1 M HCl in the presence and absence of

phenyl hydrazine mediator at CPE, according to the following equation [23].

an„ = 0.0477 / (Epa-Epa/2), (3.2)

The values for ana were found to be 0.91 and 0.64 for the oxidation of T4 at CPE in

the presence and absence of phenyl hydrazine respectively. These results clearly

89

Chapter 3 (part B)

show that the rate of the electron-transfer process is greatly enhanced in presence of

mediator. This phenomenon is thus confirmed by large Ipa values recorded at the CPE

in presence of phenyl hydrazine.

3.11.5. Analytical application

In order to evaluate the applicability of proposed method, T4 was determined

in the commercially available Eltroxine IP tablets (declared content is 100 meg of T4

in one tablet). The average mass of 10 tablets were weighed accurately and finely

powdered and transferred to a 50 ml volumetric flask and dissolved in methanol. The

mixture was sonicated for 30 min and it was then filtered. After that a suitable

aliquot of the clear filtrate was quantitatively diluted with 0.1 M HCl solution and the

determination of sodium levothyroxine in tablets was carried out by applying a

calibration plot. A typical cyclic voltammogram for the determination of T4 in the

commercial Eltroxine tablets is as shown in the fig.3.12. T4 in commercial Eltroxine

IP tablets obtained from cyclic voltammetric determination are presented in

Table.3.1. The results were satisfactory, showing that the proposed method could be

efficiently used for the determination of T4 in pharmaceutical preparations.

90

Chapter 3 (part B)

3.12. Conclusion

• This is a new cyclic voltammetric method approach for the determination of

T4 using phenyl hydrazine as the mediator.

• The electrochemical oxidation of T4 at carbon paste electrode showed that the

oxidation peak current of T4 was improved in the presence of phenyl

hydrazine.

• In the presence of methanol with dilute orthophosphoric acid as the

preparation medium for T4, the oxidation peak was more selective for the

determination of T4.

• The electrochemical response is adsorption controlled and irreversible in

nature.

• The oxidation peak current of T4 was linear in range 0.025— 0.1 mM, with a

detection limit of 2.5 \iM. The proposed method has been practically and

successfully applied for the determination of T4 in commercial tablets.

• The proposed method could be efficiently used for the determination of T4 in

pharmaceutical preparations.

91

Chapter 3 (part B)

T21.25|lA

i i i i i i i i j i i i i i i i i i i i i i i i j i i i i m i i i i i i i i j i i i i i i i i i i i i i i H i i i i U N

250 500

E/mV

750 1000

Fig.3.8a. Cyclic voltammograms of 0.1 mM phenyl hydrazine (curve a) and

O.lmM Sodium Levothyroxine in the absence (b) and presence (c) of O.lmM

phenyl hydrazine at a carbon paste electrode in O.IM HCI with a scan rate of

100 mV/s, in the potential range O.OmV to lOOOmV.

TI6.25|JA "* ^ ^

| i i i i i i i | i i i i i i i i i i i i n i | i i i i i i i | i i i i i i i | i i i i i i i | m i i i i | i i i i n i l

0 250 500 750 1000

E/mV

Fig.3.8b. Appearance of new peak at around 480 mV (dashed line) after the first

scan in the electrochemical response of 0.1 mM Sodium Levothyroxine in O.IM

HCI; scan rate, lOOmV/s.

92

Chapter 3 (part B)

| l l l l l l l | l l l l l l l | l l l l l l l | l l l l l l l | l l l l l l l | l l l l l l l [ l l l l l l l | l l l l n i l

0 250 500 750 1000

E/mV

Fig.3.9a. Cyclic voltammograms for 0.1 mM Sodium levothyroxine at different

concentrations of phenyl liydrazine (a-e). (a) bare CPE, (b) 0.025,(c) 0.05, (d)

0.075 and (e) 0.1 mM phenyl hydrazine.

90- • 80-

• • 70-

^ s o • • • • ts

• 40-

30- • 20- — 1 —

0.00 0.05 0.10 0.15 0.20

Phenyl hydrazine /mM

Fig.3.9b. Plot of different concentrations of phenyl hydrazine v/s anodic peak

current in the presence of 0.1 mM sodium levothyroxine.

93

Chapter 3 (part B)

1

40-,

35-

30-

25-

20-

15'

10 " T — ' — I — > — I — I — I — ' — r 2 3 4 5 6

- I — I — I — ' — I — I — I — I — I

7 8 9 10 11

r.-5i Sodium levothyroxlne/10 M

Fig.3.10. Plot of concentration of sodium levothyroxine v/s anodic peak current

in the presence of 0.1 mM phenyl hydrazine.

< * c 4 •

• < n • a. c4' <i •

C M .

c4" Ui • O ^ .

c4"

p

• •

2.0 2.1 2.2 ' 1 ' 1 2.3 2.4

• 1 • 1 2.5 2.6

logv/mVs^ Fig.3.11a. Dependence of log Ipa on log v in the presence of 0.1 mM Sodium

levothyroxine and 0.1 mM phenyl hydrazine.

94

Chapter 3 (part B)

100i

T 90- m V)

> 80-s ^ 70. •

<^e<'- • 'I 50-> > 4 0 - • s. '^ 30- •

20- • 1 •

2.0 2.1 2.2 2.3 2.4 • - 1 — • 1

2.5 2.6

logv/mVs^ Fig.3.11b. Dependence of Ipa /v" ^ on log v in the presence of 0.1 mM Sodium


CO-i

o , • 0 0 .

• o •

> •

^ * C O . •

cq. d

•

o CO

d" • 2.0 : 2.1 2.2 2.3 2.4 2.5

1 2.6

logv/mVs^ Fig.3.11c. Dependence of Epa on log v in the presence of 0.1 mM Sodium


95

Chapter 3 (part B)

TsiiA

| i i i i i i i | i i i i i i i | i i i i i i i | i i i i i i i | i i i i i i i | i i i i i i i | i i i i i i i | i i i i n i l

0 250 500 750 1000

E/mV

Fig.l2. Typical cyclic voltammograms for tlie determination of sodium

levothyroxine in a commercial tablet sample in the absence (curve a) and in the

presence (curve b) of O.lmM phenyl hydrazine at a carbon paste electrode with

a scan rate of 100 mV/s.

—> ^ \—S=S + 2H*+ 2f"

\= / I H

Pheu]^ Hydraziiie Diazeiiyl Benzene

(1)

H

/ \ _ N = N + i O , + H* • / " ^ N S N + HjO

Diazenyl Benzene Benzenediazoniran

(2)

^ ^ — N S N + CHjOH-

Beuzenediazoiiimn

- • / \ - O C H 3 + N j + H*

MeUiojcy Benzene

(3)

H

OCH3 + H N = N H

Diazen]4 Benzene Methoxy Benzene Hydi-azine

(4)

Scheme.3.2. Proposed mechanism of phenyl hydrazine.

96

Chapter 3 (part B)

H H

h^'^O H'^'G' h^'^j' i J^^^JJ^^N; ^^^^^^^^^^^^^^^^^^^^^N^^^J^^^J^^^^^

(1)

CPE SiuTace

^ c

n ^ (2ii)

'J7 ^

a/t H - N - H

CPE Sui'fnce

^ ay t\/^^ '-

(2b)

\ N

I H—N-H

CPE Sinface

Scheine.3.3. Probable mechanism of phenyl hydrazine with Sodium

levothyroxine.

Sample No Specified

(meg/tab)

Detected (meg/tab) RSD% (n=3)

1 100 98 1.94

2 100 99 1.75

3 100 96 1.5

Table.3.1. Determination of sodium levothyroxine in the commercial Eltroxine

IP tablets

97

Chapter 3 (part B)

3.13. References

[I] P. Janda, J. Weber, L. Dunsch, A.B.P. Lever, Anal.Chem., 68 (1996) 960.

[2] S. Shahrokhian, M. Ghalkhani, Electrochim Acta, 51 (2006) 2599.

[3] R. Aguilar, M.M. Davila, M.P. Elizalde, J. Mattusch, R. Wennrich,

Electrochim. Acta, 49 (2004) 851.

[4] B. Ursula and P. E. T. Anthony, "Biosensors for Environmental Monitoring",

2000, Harwood Academic Publishers, Amsterdam, 428.

[5] R. A. Prough, S. Moloney, in: ' Bioactivation of foreign Compounds', M.W.

Andres (Ed.), Academic Press, New York, 1985, p. 433.

[6] G. Flores, E. Frieden, J. Pharmacol Exp.Ther., 174 (1970) 463.

[7] E. Jacobsen, W.F. Fonahn, Anal. Chim. Acta, 119 (1980) 33.

[8] M. Iwamoto, A. Webber, R.A. Osteryoung, Anal. Chem., 56 (1984) 1202.

[9] F. Wang, J. Fei, S. Hu, Colloid Surf. B: Biointer., 39 (2004) 95.

[10] M. A. Murphy, G. D. Wilcox, R. H. Dahm, F. Marken. Electrochem.

Commun., 5 (2003) 51.

[II] M. Iwamoto, A. Webber, R.A. Osteryoung, Anal. Chem., 56 (1984) 1202.

[12] S. Chitravathi, B. E. Kumara Swamy, E. Niranjana, Umesh Chandra, G. P.

Mamatha, B.S. Sherigara, Int. J. Electrochem. Sci., 4 (2009) 223.

[13] X. Cao, B. Wang, Q. Su, J. Electroanal. Chem., 361 (1993) 211.

[14] G. Cauquis, M. Genies, Tetrahedron Lett, 9 (1968) 3537.

[15] G. Cauquis, M. Genies, Tetrahedron Lett., 11 (1970) 2903.

[16] U. Eisner, Y. Zemer, J. Electroanal. Chem., 38 (1972) 381.

[17] U. Eisner, E. Gileadi, J. Electroanal. Chem., 28 (1970) 81.

[18] U. Eisner, N. Zommer, /. Electroanal. Chem., 30 (1971) 433.

[19] S. G. Cohen, J. Nicholson, 7. Am. Chem. Soc, 86 (1964) 3892.

[20] J. Nicholson, S. G. Cohen, /. Am. Chem. Soc, 88 (1966) 2247.

[21] A. M. Bond, A. F. Hollenkamp, S. B. Thomson, Anal. Chem., 60 (1988)

1023.

[22] A. A. Ensafi, E. Mirmomtaz. J. Electroanal. Chem., 583 (2005) 176.

[23] E. S. Takeuchi and R. W. Murray, / Electroanal. Chem., 189 (1985) 49.

98

part-a electrochemical studies of sodium levothyroxine...

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