Thiouracil

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Thiouracil Comprehensive profile Nasr Y. Khalil, Haitham K. AlRabiah, Mohammed S. Almousa, Ahmed Bari, Hamad M. Alkahtani Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud, University, Riyadh, Saudi Arabia

Contents 1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Elemental analysis 1.4 Appearance 1.5 Uses and applications 2. Methods of preparation 2.1 Procedure for the preparation of 2-thiouracil 2.2 Procedure for the manufacture of 6-alkyl substituted thiouracils 3. Physical characteristics 3.1 Ionization constant 3.2 Solubility characteristics 3.3 Thermal methods of analysis 3.4 X-ray powder diffraction pattern 3.5 Spectroscopy 4. Methods of analysis 4.1 British pharmacopeia compendial methods 4.2 United States pharmacopeia compendial methods for propylthiouracil 4.3 Electrochemical methods of analysis 4.4 Spectroscopic methods of analysis 4.5 Chromatographic methods of analysis 5. Pharmacokinetics, metabolism, and excretion 5.1 Pharmacokinetics 5.2 Metabolism 5.3 Excretion 6. Pharmacology References

Profiles of Drug Substances, Excipients, and Related Methodology ISSN 1871-5125 https://doi.org/10.1016/bs.podrm.2018.11.006

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2019 Elsevier Inc. All rights reserved.

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1. Description 1.1 Nomenclature 1.1.1 Systematic chemical name [1] 2-Sulfanylidene-1H-pyrimidin-4-one. 1.1.2 Nonproprietary names Thiouracil. 1.1.3 Proprietary names [2] Antiroid (South Korea), Bingsaiyou (China), Lothisil (Viet Nam), Peteyu (Thailand), Procarbizol (Pakistan), Procil (Taiwan), Propycil (Bulgaria, Switzerland, Czech Republic, Germany, Honduras, Hungary, Portugal, Slovakia, Turkey), Propylthiocil (Israel), Propylthiouracile (Belgium), Propylthiouracil (Medic) (Denmark), Proracyl (France), Prothiucil (Austria), Prothuril (Greece), PTU (Australia, India, Uruguay), Thyrosan (Poland), Tiotil (Estonia, Sweden), Tirostat (Colombia, Ecuador).

1.2 Formulae 1.2.1 Empirical formula, molecular weight, and CAS number [1] C4H4N2OS ¼ 128.149 [141-90-2]. 1.2.2 Structural formula NH

O

N S H Structure of thiouracil

1.3 Elemental analysis [3] Carbon 37.49% Hydrogen 3.15% Nitrogen 21.86% Oxygen 12.48% Sulfur 25.02%

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1.4 Appearance [1] Minute crystal, Prisms, or White to pale cream-colored powder.

1.5 Uses and applications Thiouracil was introduced in 1943 as the first thionamide anti-thyroid drug. The usual dose was 1–2 g/day in divided doses. Owing to a high frequency of adverse reactions, especially agranulocytosis, its use was abandoned in favor of other, less toxic drugs, such as propylthiouracil and methimazole. Thiouracil is not currently used as a thyrostatic drug in humans [4]. Propylthiouracil is a thiourea antithyroid drug that acts by blocking the production of thyroid hormones; it also inhibits the peripheral deiodination of thyroxine to tri-iodothyronine. It is used in the management of hyperthyroidism, including the treatment of Graves’ disease, preparation of hyperthyroid patients for thyroidectomy, as an adjunct to radioiodine therapy and the treatment of thyroid storm. Propylthiouracil is usually given orally. Initial doses range from 150 to 450 mg daily (the BNF recommends 200–400 mg daily), although in severe cases initial doses of 600–1200 mg daily have been used. It has often been given in divided daily doses but once daily dosage is also possible. Improvement is usually seen in 1–3 weeks and control of symptoms is achieved in 1–2 months. When the patient is euthyroid the dose is gradually reduced to a maintenance dose, usually 50–150 mg daily. Treatment is usually continued for 1–2 years. In the United Kingdom, the BNFC recommends the following initial doses by mouth for children: • In neonates: 2.5–5 mg/kg twice daily • In those aged 1 month to 1 year: 2.5 mg/kg three times daily • In those aged 1–5 years: 25 mg three times daily • In those aged 5–12 years: 50 mg three times daily • In those aged 12–18 years: 100 mg three times daily These doses are given until the patient is euthyroid and then adjusted as needed; higher doses may be required, especially in thyrotoxic crises. Doses should be reduced in renal impairment (below). Doses may also need to be reduced in hepatic impairment. Administration in renal impairment: The dosage of propylthiouracil should be reduced in patients with renal impairment according to creatinine clearance (CC) as follows: • CC 10–50 mL/min, doses should be reduced by 25% • CC less than 10 mL/min, reduce doses by 50%.

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Alcoholic liver disease: Propylthiouracil has been said to reduce hyperoxic liver injury in hypermetabolic animals and despite reports of hepatotoxicity, including some fatalities, associated with propylthiouracil, it has been investigated in the treatment of patients with alcoholic liver disease. However, it was concluded that there is no evidence to substantiate this use. Propylthiouracil was associated with adverse effects and it could not be shown to have any significant effects on mortality, liver related mortality, liver complications, and liver histology. Several reports have described benefit in patients with psoriasis given propylthiouracil. An oral dose of 300 mg daily for 8–12 weeks has been used and is said not to produce clinical hypothyroidism [5].

2. Methods of preparation 2.1 Procedure for the preparation of 2-thiouracil This first involves preparation of ureide (2) and thioureide (3); A solution of Meldrum’s acid (1, 20.8 mmol) in trimethyl orthoformate (12 mL) was heated to reflux for 2 h and urea (22.9 mmol) was added directly. The reaction mixture was refluxed with stirring for two more hours and cooled. The corresponding ureide was filtered and isolated then washed with ethyl ether and dried to give 3.66 g of (2) (1.71 mmol, 82%). In a same manner, 3.45 g (15.0 mmol, 72%) of (3) were obtained from thiourea (22.9 mmol). Single recrystallization of crude products from ethanol/dimethylformamide gave analytically pure compounds. Ureide (2): Silky white needles, m.p. 209–210 degrees (d). IR (KBr) v 3600–3000 br, 1720, 1700, 1670, 1580, 1390 and 1270 cm1. 1H NMR (DMSO-d6)δ: 10.88 (br d, 1H, J  14 Hz, exchangeable with deuterium oxide), 8.66 (d, 1H, J  14 Hz), 7.9–7.5 (br, 2H, exchangeable with deuterium oxide) and 1.68 (s, 6H)ppm. Analytically calculated for C8H10O5N2: C, 44.86; H, 4.71; N, 13.08. Found: C, 44.42; H, 4.70; N, 12.90. Thioureide (3): White needles, m.p. 223–226 degrees (d). IR (KBr) v: 3600–3000 br, 1740, 1670, 1620, 1610, 1510, 1400, 1380, 1360, 1260 and 1220 cm1. 1H NMR(DMSO-d6) δ: 11.49 (br d, 1H, J  13 Hz, exchangeable with deuterium oxide), 9.52 (br s, 1H, exchangeable with deuterium oxide), 9.27 (d, 1H, J  13 Hz) and 1.71 (s, 6H) ppm. Analytically Calculated for C8H10O4N2S: C, 41.73; H, 4.38; N, 12.17; S, 13.93. Found: C, 41.62; H, 4.43; N, 12.31; S, 13.97.

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Thiouracil

O

O X

O 1. HC(OMe)3

O

H2N-C-HN-HC

2. (NH2) C=X O

O

O

O 1

Ph2O heat H N

2. X=O 3. X=S

X

NH

O 4. X=O 5. X=S

Scheme 1 Preparation of uracil and thiouracil from a mixture of ureide (or thioureide) and diphenyl ether.

The method for preparation of uracil and 2-thiouracil involves heating and refluxing a mixture of the ureide or thioureide (10 mmol) and diphenyl ether (45 g) for 5 min. The cooled mixture was poured on low-boiling petroleum ether and the precipitate was filtered, washed with ethyl ether and dried in vacuo (Scheme 1). The crude uracil (4) or 2-thiouracil (5) which did not require further purification was obtained in 68% and 70% yield, respectively. These synthetic compounds were characterized by the physical and spectral properties previously described [6].

2.2 Procedure for the manufacture of 6-alkyl substituted thiouracils Sodium metal (2.3 g, 0.10 g atom) was dissolved in 50 mL of anhydrous ethanol and 5.33 g (0.07 mol) of thiourea and 0.05 mol of the appropriate β-oxo ester were added to the clear solution. Then the mixture was heated on a steam-bath until a clear solution is obtained in about 10 min. Afterward a precipitation will begin to form by time. The total time of heating is 6–7 h after which the mixture is allowed to stand overnight. Then it is

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O

S

N

co2et + npr

npr

H2N

ethyl 3-oxohcxanoate

SH

S

H2N

N

NH

NH2

thiourea

OH O

O

Propylthiouracil

Scheme 2 Synthesis of propylthiouracil from ethyl 3-oxohexanoate and thiourea.

distilled in vacuo at 40–50 °C until near dryness and the residue is dissolved in 50 mL of water. The product is precipitated by adding 7 mL of concentrated hydrochloric acid and subsequent acidification by glacial acetic acid to pH 4. Then the crude substituted thiouracil is filtered off, washed and dried at 80 °C. Scheme 2 shows the preparation of propylthiouracil. Most of the crude thiouracils are purified by recrystallization from boiling water [7].

3. Physical characteristics 3.1 Ionization constant pKa of thiouracil is 7.46 at 25.0 °C pKa of propylthiouracil is 7.76 at 25.0 °C [8]

3.2 Solubility characteristics Thiouracil is very slightly soluble in water, sparingly soluble in alcohol. It dissolves in solutions of alkali hydroxides [8]. Propylthiouracil is slightly soluble in water, sparingly soluble in ethanol and acetone.

3.3 Thermal methods of analysis 3.3.1 Melting behavior The melting point of thiouracil is about 300–340 °C (with decomposition). The melting point of propylthiouracil is 217–221 °C [9]. 3.3.2 Differential scanning calorimetry Differential scanning calorimetric (DSC) analysis of thiouracil (Fig. 1) was performed using a Perkin Elmer differential scanning calorimeter (DSC8000). About 5 mg sample was added in a 40 μL aluminum pan which was sealed and heated over the range of 40–400 °C, at a heating rate of

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Fig. 1 DSC thermogram of thiouracil.

10 °C/min. An empty aluminum pan was used as the reference standard. Analysis was carried out under nitrogen purge. The thermogram showed a sharp endothermic peak at 328.23 °C corresponding to the reported melting point of thiouracil (300–340 °C).

3.4 X-ray powder diffraction pattern The X-ray powder diffraction pattern of thiouracil has been obtained on an Ultima IV X-ray diffraction (Rigaku, Japan) diffractometer equipped with ˚ ). The scintillation detector using copper radiation (wavelength ¼ 1.5406 A sample was scanned over a range of 3–60 degrees 2θ, at scanning speed of 0.5 degree/min (Fig. 2). A full summary of the crystallographic data is compiled in Table 1.

3.5 Spectroscopy 3.5.1 Ultraviolet spectroscopy The UV spectrum of thiouracil (at a concentration of 2 μg/mL in methanol) was obtained using a Shimadzu spectrophotometer (model UV-1800). The

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Fig. 2 X-ray powder diffraction pattern of thiouracil.

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Thiouracil

Table 1 X-ray crystallographic data of thiouracil. Peak no. 2θ Flex width d-Value Intensity

I/Io

1

15.200

0.235

5.8241

1354

93

2

17.400

0.235

5.0924

212

15

3

19.600

0.235

4.5255

1213

84

4

21.700

0.235

4.0290

254

18

5

25.300

0.118

3.5173

173

12

6

25.700

0.118

3.4635

490

34

7

26.400

0.235

3.3732

757

52

8

27.800

0.235

3.2065

1460

100

9

29.500

0.235

3.0254

71

5

10

29.900

0.235

2.9859

215

15

11

30.800

0.353

2.9006

551

38

12

32.100

0.235

2.7861

236

17

13

34.400

0.353

2.6049

366

26

14

35.300

0.235

2.5405

266

19

15

37.800

0.235

2.3780

167

12

16

46.700

0.235

1.9435

158

11

17

50.100

0.353

1.8058

103

8

18

50.500

0.588

1.8058

127

9

19

52.200

0.471

1.7509

73

5

solution was scanned over the wavelength range of 200–400 nm, and the resulting spectrum is shown in Fig. 3. The principle peak was found to be around 272 nm. 3.5.2 Vibrational spectroscopy The infrared (IR) spectrum of thiouracil in a KBr pellet was obtained using a Perkin Elmer FTIR BX apparatus. Fig. 4 shows the IR spectrum, which contains peaks at 1225 cm1 (C]S mode), 1710 cm1 (C]O mode), and 3448 m1 (NH mode).

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Fig. 3 UV spectrum of thiouracil (2 μg/mL) in methanol.

3.5.3 Nuclear magnetic resonance (NMR) spectrometry 3.5.3.1 1H NMR spectrometry

The 1H NMR spectrum of thiouracil (dissolved in DMSO-d6) was scanned on a Bruker NMR spectrometer operating at 700 MHz. In Table 2 and Fig. 5, the chemical shifts are expressed in δ-values (ppm), and the coupling constants (J) are expressed in Hz. 3.5.3.2

13

C NMR spectrometry

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The C NMR spectrum of thiouracil (dissolved in DMSO-d6) was scanned on a Bruker NMR spectrometer operating at 176.05 MHz. In Table 3 and Fig. 6, the chemical shifts are expressed in δ-values (ppm). 3.5.4 Thermo gravimetric analysis (TGA) The TGA thermogram of thiouracil was obtained using a Perkin Elmer Pyris 1 TGA instrument. As shown in Fig. 7, thiouracil begins to lose weight when heated beyond 225 °C and was seen to lose all mass through decomposition when heated beyond 325 °C. This behavior is consistent with that of a non-solvated compound. 3.5.5 Mass spectrometry The mass spectrum of thiouracil was obtained using a Perkin Elmer Clarus 600 gas chromatography equipped with an EI (electro ionization) source. Fig. 8 shows the resulting mass spectrum of thiouracil, which contained the highest peaks at m/z ¼ 128; [M]+ and 129 [M + 1]+. The second highest peak was seen at m/z ¼ at 69.09, and the third highest peak at m/z ¼ 42.06.

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Fig. 4 IR spectrum of thiouracil.

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Table 2 1H NMR of thiouracil (DMSO-d6). Signal Location (δ) Shape Integration

Correspondences

1

5.83

d, J ¼ 7.7 Hz

1H

–CH2

2

7.40

d, J ¼ 7.7 Hz

1H

–CH3

3

12.30

Br-d

1H

2xNH

4. Methods of analysis 4.1 British pharmacopeia compendial methods The official substance in the British Pharmacopeia is propylthiouracil; hence the tests below are performed on propylthiouracil. 4.1.1 Identification 1. Infrared absorption spectrophotometry. Complies with the requirements for Monographs of the British Pharmacopeia for the infrared absorption spectrophotometry [9]. 4.1.2 Impurity A (thiourea) and related substances Carry out the method for thin-layer chromatography, using the following procedure: Test solution (a): Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 10 mL with the same solvent. Test solution (b): Dilute 1 mL of test solution (a) to 10 mL with methanol R. Reference solution (a): Dissolve 10 mg of propylthiouracil CRS in methanol R and dilute to 10 mL with the same solvent. Reference solution (b): Dissolve 50 mg of thiourea R in methanol R and dilute to 100 mL with the same solvent. Dilute 1 mL of this solution to 100 mL with methanol R. Reference solution (c): Dilute 1 mL of test solution (a) to 100 mL with methanol R. Apply separately to the plate 10 μL of each solution. Develop over a path of 15 cm using a mixture of 0.1 volumes of glacial acetic acid R, 6 volumes of 2-propanol R and 50 volumes of chloroform R. Allow the plate to dry in air. Examine under ultraviolet light at 254 nm. Expose the plate to iodine vapor for 10 min. In the chromatogram obtained with test solution (a), any spot corresponding to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.05%) and any spot apart from the principal spot and any spot corresponding to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (c) (1.0%) [9].

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Fig. 5 1H NMR spectrum of thiouracil.

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Table 3 Signal

13

C NMR of thiouracil (DMSO-d6). Location (δ) Correspondences

1

105.75

–CH2

2

142.57

–CH3

3

161.48

C]O

4

176.50

C]S

4.1.3 Assay To 0.300 g of the test sample add 30 mL of water R and 30.0 mL of 0.1 M sodium hydroxide. Boil and shake until dissolution is complete. Add 50 mL of 0.1 M silver nitrate while stirring, boil gently for 5 min and cool. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). The volume of 0.1 M sodium hydroxide used is equal to the sum of the volume added initially and the volume used in the final titration. 1 mL of 0.1 M sodium hydroxide is equivalent to 8.511 mg of propylthiouracil, C7H10N2OS [9].

4.2 United States pharmacopeia compendial methods for propylthiouracil 4.2.1 Identification Infrared absorption spectrophotometry. Complies with the requirements for Monographs of the United States Pharmacopeia for the Infrared absorption spectrophotometry [10]. 4.2.2 Related substances Ordinary Impurities: Sample solution: Methanol. Application volume: 10 μL. Eluent: Toluene, ethyl acetate, and formic acid (50:45:5), in a nonequilibrated chamber. Test solution: Propylthiouracil 10 mg/mL in methanol. Standard solutions: Propylthiouracil concentrations of 0.01 mg mL1, 0.05 mg mL1, 0.1 mg mL1, and 0.2 mg mL1. Visualization: Use UV light at 254 nm and at about 366 nm. Procedure: Use a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture, and the eluent specified in the monograph. Apply equal volumes (10 μL for each) of the test

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Fig. 6

13

C NMR spectrum of thiouracil.

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Fig. 7 TGA thermogram of thiouracil.

solution and standard solutions to the plate, using a stream of nitrogen to dry the spots. Allow the chromatogram to develop in a pre-equilibrated chamber until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber and air-dry. View the plate using the visualization technique(s): (Use UV light at 254 nm and at about 366 nm). Locate any spots other than the principal spot in the chromatogram of the test solution and determine their relative intensities by comparison with the chromatograms of the appropriate standard solutions [10]. 4.2.3 Assay Procedure: Sample: 300 mg of propylthiouracil Analysis: Transfer the Sample into a 500-mL conical flask and add 30 mL of water. Add from a burette 30 mL of 0.1 N sodium hydroxide VS, heat to boiling, and agitate until solution is complete. Wash down any particles on the wall of the flask with a few mL of water, and then add

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Fig. 8 Mass spectrum of thiouracil.

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50 mL of 0.1 N silver nitrate while mixing, and boil gently for 7 min. Cool to room temperature. Titrate with 0.1 N sodium hydroxide VS, using a glass-calomel electrode system. Each mL of 0.1 N sodium hydroxide is equivalent to 8.512 propylthiouracil, C7H10N2OS. Acceptance criteria: 98.0–100.5% [10].

4.3 Electrochemical methods of analysis 4.3.1 Voltammetry The voltammetric behavior of two mercaptopyrimidine derivatives (2-thiouracil and 2-thiobarbituric acid) has been studied by cyclic voltammetry at a cobalt phthalocyanine (CoPc)-modified carbon-paste electrode. The results of voltammetric determinations showed that the CoPc in the matrix of modified electrode acts as catalyst for electro-oxidation of these thiols (RSH), lowering the overpotential of the reaction and significantly increasing the sensitivity for detection of thiols in neutral conditions. The results of voltammetric and polarization measurements in solutions at various pH values were used for prediction of the mechanism of electrocatalytic oxidation at the surface of modified electrode. These results showed that at the modified electrode, electrochemical oxidation of thiolate anion (RS) is the rate-determining step. It was found that the modified electrode exhibits good selectivity for catalytic oxidation of mercaptopyrimidines over other biologically important mercaptans, such as cysteine, glutathione and thioglycolic acid. The results demonstrate that the peak current for thiol oxidation has a linear variation with the concentration in the range of 1  102–1  105 M. This system can be used for sensitive and selective voltammetric detection of mercaptopyrimidine derivatives [11]. Another simple and rapid method was developed using cyclic and square wave voltammetric techniques for the determination of the trace-level sulfur containing compound (2-thiouracil) at a glassy carbon electrode. 2-Thiouracil produced two anodic peaks at 0.334 and 1.421 V and a cathodic peak at 0.534 V. Square wave voltammetry of 2-thiouracil gave a good linear response over the range of 1–20 μM, with a detection limit of 0.16 μM and quantification limit of 0.53 μM (0.0679 μg/g), which is in good agreement as per IUPAC definition of trace component analysis (100 μg1). The obtained recoveries range from 98.10% to 102.1%. The proposed method was successfully used for its quantitative determination in pharmaceutical formulations and urine as real samples [12]. In another study, the reactions of 2-thiouracil with a silver electrode during anodic deposition and cathodic stripping were investigated using a

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rotating silver disk electrode and linear potential scan voltammetry. The formation of two different insoluble silver salts with 2-thiouracil and its dimerization product (2-thiouracil disulfide), the dependence of the stripping peak current on the concentration of 2-thiouracil, and the potential range were discussed. It has been found that these sparingly soluble silver compounds of 2-thiouracil formed during anodic deposition can be stripped by scanning the potential in a negative direction. The influence of parameters on the shape and the height of the stripping peaks has been determined. For concentrations of 2-thiouracil less than 6  106 mol dm3, one stripping peak was observed. Two stripping peaks were observed in the intermediate concentration range 1  105–5  105 mol dm3. A random stripping process with repulsive lateral interactions in the film was observed at concentrations of 2-thiouracil 107 mol dm3. Experimental data were found to fit a Frumkin isotherm with a Temkin coefficient, g, of 5 [13]. The electrochemical behavior of catechol in the presence of propylthiouracil (PTU) and methylthiouracil (MTU) at a glassy carbon electrode was thoroughly investigated. The cyclic voltammetric studies of the systems were performed at different pH values and sweep rates. The results of the voltammetric foundations showed an electrocatalytic oxidation of catechol in the presence of PTU and MTU, leading to a remarkable increase in the anodic peak current of catechol together with the contemporaneous disappearance of the corresponding cathodic wave. The presented mechanism showed the nucleophilic addition/reduction of the electro-generated o-quinone by the PTU or MTU, which produced chemically reduced adducts whose subsequent re-oxidation at the electrode surface led to a considerable amplification of the anodic peak current. The increasing of the anodic peak current showed a linear correlation with the concentration of PTU and MTU. The spectrophotometric investigations were used to confirm the interaction of the thiols with the electrochemically produced o-quinone during the controlled-potential coulometry. The effect of various biological thiols, such as cysteine penicillamine, N-acetyl cysteine, glutathione and captopril on the electrochemical behavior of catechol, was investigated at the same conditions. The results represented the unique electrocatalytic effect of PTU and MTU for improving the anodic peak current of catechol and exhibited a very sensitive and relatively selective method for the voltammetric detection of the pointed thiols. A linear range from 0.1 to 10.0 μM with a detection limit of 0.05 μM is obtained for the determination of PTU. The linear range for MTU, using differential pulse

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voltammetry, was from 0.1 to 5.0 μM with a detection limit of 0.07 μM. The proposed method showed to be useful for the sensitive detection of PTU and MTU among pharmaceutical and biological thiols [14]. 4.3.2 Polarography The mechanism of the polarographic reduction of 2-thiouracil and its S-methyl and ethoxy derivatives with stabilized 2-thiol-4-keto or 2-thione4-enol structure has been studied. 2-Thiouracil is not electro-reducible in aqueous solutions. However, it exhibits a strong tendency to adsorption and association on the mercury electrode. 2-Thio-4-ethoxypyrimidine undergoes a 4e, 4H+ reduction which involves 2e reduction of the 3,4 N]C bond, elimination of the ethoxy substituent to form 2-thiopyrimidine, and a 2e, 2H+ reduction of the latter to 3,4dihydroderivative. For 2-thiomethyluracil, a model compound of the 2-thiol-4-keto form, the electroactive center is shifted to C-2 and in a 2e, 2H+ reduction process. The sulfur substituent undergoes elimination, to give pyrimidone-4; the latter can further be reduced at more negative potentials to tetrahydroderivatives (a 4e-process) [15]. Although potentiometric studies of cupric ion-thiouracil mixtures were unable to permit the estimation of stability constants of the resultant complexes due to their instantaneous precipitation, the stoichiometric titer provided evidence for the formation and precipitation of either the mixed ligand complex of cupric ion with thiouracil and hydroxide ions (CuUOH) or the dimer, containing two cupric ions and two thiouracil anions (Cu2(U)2), and the complex of one cupric and two thiouracil ions (Cu(U)2). Synthesis, isolation, and characterization of precipitated complexes gave evidence of the formed mixed ligand complex (CuUOH) that lost a molecule of water on drying to give bis(2-thiouracil)-μ-oxodicopperII (Cu(U)2O). Polarographic studies showed a one-electron change attributable to the reduction of the singly charged complex of a cupric ion and a thiouracil anion (Cu(U)+) to an uncharged complex of a cuprous ion and a thiouracil anion at the dropping mercury electrode. The one-electron change could not be attributed to the reduction of the latter complex to copper metal since thiouracil reduction of cupric ion does not occur in acidic solution. Analyses of the ΔE1/2, data for the difference between complex half-wave potential and the half-wave potential of cupric-ion reduction to the cuprous state provided estimates of approximately 0.1 for the ratio of the dissociation constant of the singly charged complex of a cupric ion and a thiouracil anion (Cu(U)+) to the

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dissociation constant of the uncharged complex of a cuprous ion and a thiouracil anion. It was not possible to determine the absolute values of the cupricion complexes’ stability constants from polarography. The relative tendency of metal ions to complex with thiouracils, Cu2+ > Pb+2 > Cd+2 > Ni+2  Zn+2, is consistent with the “natural order” of such transition metals, their relative ionic radii, their electronic structure, and the concept that “soft acids” prefer to complex with “soft bases.” The presence of a free sulfhydryl group at the 2-position of pyrimidines appears necessary both for metal complexation and for antithyroid activity. Substituents on the ring nitrogens of thiouracil or alkyl substitution on the sulfur, which prevented the possibility of hydrogen tautomerism to the sulfur atom, destroyed complexation ability and biological activity. Substituents at the 5-position with high electronegativity decreased the pK0 a of 2-thiouracils, reduced the stability constants of the metal complexes, and minimized the antithyroid activities [16].

4.4 Spectroscopic methods of analysis 4.4.1 Spectrophotometry The ultraviolet absorption spectra of several 2-thiouracil derivatives were measured at various pH values and apparent dissociation constants determined from these spectra [17]. General comparisons were made with corresponding uracil analogs. The structure of 2-thiouracil was shown to be in the di-keto form in acid and near-neutral aqueous solutions. With further increase in pH, two equilibria were demonstrated spectrophotometrically, referring to tautomerism and concomitant ionization at the 2 and 4 positions, respectively. The spectra and structure of other 2-thiouracil derivatives were discussed. A comparison of the degrees of dissociation of 2-thiouracil and its 6-methyl analog at blood pH values as determined from their spectra places in question the necessity of assuming appreciable dissociation of these compounds for iodine absorption in anti-thyroid activity. A spectrophotometric method for determination of sodium 2-sulfanylethanesulfonate, MeSNa (MA), 6-mercaptopurine (MP), 6-thioguanine (TG), and 6-propyl-2-thiouracil (PTU) has been developed [18]. The method was based on the reaction of thiouracils with N,Ndimethyl-p-phenylenediamine (DMPD) in acidic solution in the presence of the Fe3+ ion, an oxidizing agent. Absorbance of the obtained colored products was measured at the corresponding optimum wavelengths: 490 nm for MA (ε ¼ 1.9  103 L mol1 cm1), 455 nm for MP (ε ¼ 1.9  103 L mol1 cm1), 1460 nm for TG (ε ¼ 1.6  103 L mol1 cm1), and 465 nm for PTU

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(ε ¼ 2.1  103 L mol1 cm1). Beer’s law was obeyed in the following concentration ranges: 8.2  103–9.0  102 g L1 for MA, 6.0  103 –3.8  102 g L1 for MP, 4.2  103–4.2  102 g L1 for TG and 4.2  103–3.9  102 g L1 for PTU. The method has been applied for determination of thiols in pharmaceuticals. In another study, two multivariate calibration methods, partial least squares (PLS) and principal component regression (PCR), were applied to the spectrophotometric simultaneous determination of 2-mercaptobenzimidazole (MB) and 2-thiouracil (TU) [19]. A genetic algorithm (GA) using partial least squares was successfully utilized as a variable selection method. The concentration model was based on the absorption spectra in the range of 200–350 nm for 25 different mixtures of MB and TU. The calibration curve was linear across the concentration range of 1–10 μg/mL and 1.5–15 μg/mL for MB and TU, respectively. The values of the root mean squares error of prediction (RMSEP) were 0.3984, 0.1066, and 0.0713 for MB and 0.2010, 0.1667, and 0.1115 for TU, which were obtained using PCR, PLS, and GA-PLS, respectively. Finally, the practical applicability of the GA-PLS method was effectively evaluated by the concurrent detection of both analytes in animal tissues. It should also be mentioned that the proposed method is a simple and rapid way that requires no preliminary separation steps and can be used easily for the analysis of these compounds, especially in quality control laboratories.

4.4.2 Colorimetry A direct colorimetric method was described for the determination of propylthiouracil in serum, which was based on reaction of the drug at pH 8.0 with 2,6-dichloroquinone-chloroimide to produce a colored chloroformsoluble compound [20]. After isolation from the aqueous layer with phase-separating filter paper, the chloroform solution was measured against a chloroform blank at 435 nm. Propylthiouracil added to drug-free serum was used as a control sample. The method obeyed Beer’s law up to a concentration of at least 10 mg/L. In another study, propylthiouracil (2-thio-4-oxo-6-propy1-1,3pyrimidine) was reacted with sodium nitroprusside in presence of sodium hydroxide, and on treatment with acetic acid, it gave a greenish blue color measurable at 600 nm. The effects of pH, time, concentrations of sodium nitroprusside, sodium hydroxide and acetic acid have been studied. The developed color was used as a basis of a colorimetric method for the

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determination of pure propylthiouracil and in pharmaceutical formulations. The method can be adopted for concentrations ranging from 8 to 32 μg/mL, with 100  2.0% accuracy [21]. 4.4.3 Flow-injection method A flow injection analysis procedure with chemiluminescence detection for the determination of both thiol-containing drugs and the amino acid cysteine was described. Procedures were based on the inhibitory effect of the drugs on the chemiluminescence generated in the copper-catalyzed oxidation of luminol by hydrogen peroxide. The proposed methods were applied to the determination of cysteine, N-acetylcysteine, penicillamine, 2-mercaptopropionylglycine and thiouracil in pharmaceuticals [22].

4.5 Chromatographic methods of analysis 4.5.1 Electrophoresis A method was developed based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of thiouracil (TU) and phenylthiouracil (PhTU) with high sensitivity (nanomolar range, i.e., attomoles detected). After derivatization with 5-iodoacetamidofluorescein, the analytes were separated by capillary zone electrophoresis using 20 mM phosphate buffer (pH 10.0) and quantified by fluorescence detection. The linearity range, precision, recovery, and detection limits were determined, and the method was shown to be applicable for the determination of TU and PhTU in spiked feed samples and urine [23]. Another capillary zone electrophoresis (CZE) was optimized for the separation of thiouracil, methylthiouracil and propylthiouracil. Methylthiouracil could be determined within 15 min in various types of urine (human, bovine, horse), either without any pretreatment or in ethyl acetate extracts. For identification, the simultaneous detection at three UV wavelengths (216, 245 and 278 nm) was advantageously used, while for quantification the wavelength of the absorbance maximum at 278 nm was preferred. Under optimized conditions a linear response of the detector in the concentration range 0.1–100 ppm was obtained. On analysis of untreated urine, a detection limit of 0.5 ppm was found; for urine extracts the detection limit was 0.1 ppm. Univocal peak identification, based on absorption at three wavelength, was only possible above 2 ppm. Relative standard deviation for standard solutions of methylthiouracil, diluted in the background electrolyte, was 1%; for methylthiouracil in extracts dissolved in the background electrolyte it was 4.5%, and for methylthiouracil in untreated urine, it was 12.7% [24].

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In another study, a new capillary zone electrophoresis (CZE) method has been developed for the analysis of thyreostatic drugs (TD) such as methylthiouracil (MTU), propylthiouracil (PTU), and thiouracil (TU) in urine specimens. An untreated fused-silica capillary tube (75 μm i.d., 57 cm total length, 49.5 cm length to the detector) was used in all of the experiments. Optimal conditions were found using 50 mM borate buffer, pH 7.6, applied voltage 15 kV, 25 °C, and 30 s of hydrodynamic injection. UV detection at 276 nm was employed. Ethyl acetate extraction of the compounds was made before the analysis of each urine sample. An electrostacking procedure to increase the sensitivity was applied. The method developed was used for the analysis of several thousands of samples as part of a veterinary control procedure (Institute for State Control of Veterinary Biological and Medicaments, Brno, Czech Republic). The following detection limits were reached: 0.3 ppm (PTU and MTU) and 0.5 ppm (TU). The relative standard deviations for PTU, MTU, and TU determination were 1.12%, 0.98%, and 3.8%, respectively [25]. CE coupled to electrochemiluminescence detection has been established, based on the electrochemiluminescence enhancement of tris(2,2-bipyridyl)ruthenium(II) with four of the most used thyreostatic drugs (methimazole, 2-thiouracil, 6-methyl-2-thiouracil, and 6-propyl-2-thiouracil). The method was rapid, simple, and practical. Parameters that affect separation and detection were optimized. Under the optimum experimental conditions, the four analytes could be well separated within 11 min at the separation voltage of 16 kV in a running solution containing 20 mM phosphate buffer (pH 9.0) and 1.0  104 M Ru(bpy)32 +, with a solution of 20 mM phosphate buffer (pH 12.0) containing 1.0  104 M Ru(bpy) 32 + in the electrochemiluminescence detection cell. The detection limits for methimazole, 6-methyl-2-thiouracil, 6-propyl-2-thiouracil, and 2-thiouracil were 0.1, 0.05, 0.05, and 0.01 μM, respectively. The proposed method was applied to analyze these drugs in spiked animal feed samples. The recoveries were 88.2–99.0% and 86.4–98.7% for the intraday and interday analyses, respectively. The RSDs were 2.7–4.8% and 1.8–5.0% for the intraday and interday analyses, respectively. The results demonstrate that the proposed method has promising applications in the detection of thyreostatic drugs in animal feeds [26]. 4.5.2 High performance liquid chromatography Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with

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acidic potassium permanganate, and the combination technique of highperformance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1–20 μg/mL for MTU and 0.1–10 μg/mL for PTU. The detection limits were 0.03 μg/mL for PTU and 0.03 μg/mL for MTU. Finally, the quantification limits were 0.1 μg/mL for PTU and 0.1 μg/mL for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples [27]. Another method was described for determination of biological thiols at the picomole level based upon conversion of thiols to fluorescent derivatives by reaction with monobromobimane and separation of the derivatives by reverse-phase high-performance liquid chromatography. Thiols separated by the procedure were N-acetylcysteine, coenzyme A, coenzyme M, cysteamine, cysteine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, γ-glutamylcysteine, homocysteine, hydrogen sulfide, 2-mercaptoethanol, mercaptopyrimidine, methanethiol, pantetheine, 40 -phosphopantetheine, thiosulfate, and 2-thiouracil. Since monobromobimane has little fluorescence and reacts very selectively with thiols to produce fluorescent derivatives, crude extracts can be derivatized and analyzed without pre-purification of the thiols, the entire process requiring only 1–2 h. The technique was illustrated by determination of the thiol levels in red blood cells [28]. In addition, other high-performance liquid chromatography (HPLC) methodologies were evaluated for the detection and quantification of thyreostatic drug residues in cattle serum and thyroid tissue. The paper details a protocol, using a simple ethyl acetate extraction for the determination of thiouracil, tapazole, methyl thiouracil, propylthiouracil and phenyl thiouracil in thyroid tissue. Using two sequential HPLC injections, and quantitative analysis, in two steps, all five thyreostats were detectable at concentrations greater than 2.45–4.52 ng/g. Modifications to a published method for detection of thyreostatic residues in serum involving the addition of mercaptoethanol and a freezing step were described. The modifications improved sensitivity and allowed detection of the five thyreostats at levels greater than 16.98–35.25 ng/mL. Young bulls were treated with thyreostats to demonstrate the validity of the methodologies described. Administered thyreostats were not absorbed equally by the test animals and the compounds were not all detected in the serum samples removed at 7 days following drug withdrawal. These experiments indicate the necessity to be able to detect thyreostat residues in a variety of matrices [29].

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In another study, a procedure was described for the concurrent assay of thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil and methimazole in bovine plasma. In this procedure, reversed-phase high-performance liquid chromatography was applied after liquid-liquid extraction of plasma with ethyl acetate. Thiouracil, methylthiouracil, propylthiouracil and phenylthiouracil were quantified by ultraviolet detection at 276 nm, while methimazole was assayed at 258 nm. The linearity range, precision, recovery and detection limits were determined, and the method was shown to be applicable to samples of plasma from young bulls experimentally treated with methylthiouracil [30]. A method which was asserted to be simple and rapid for the determination of nanomole levels of biological thiols was described. The analysis was based on the combination of reverse-phase high-performance liquid chromatography with a post-column reaction with 6,60 -dithiodinicotinic acid. Thiols, including cysteine, cysteamine, thiolhistidine, homocysteine, glutathione, penicillamine, ergothioneine, and thiouracil, were separated by eluting with 33 mM KH2PO4 at pH 2.2. Glutathione, cysteine, cysteamine, homocysteine, and penicillamine were quantitatively determined with detection limits of 0.1 nmol, while the quantitative detection of thiolhistidine, ergothioneine, and thiouracil was not successful. The method was applied to the assay of glutathione in human erythrocytes and Escherichia coli [31]. A reversed-phase high-performance liquid chromatographic analysis was developed for propylthiouracil in plasma (1 mL sample size). After protein precipitation with acetonitrile, the solution was diluted with water and injected into a liquid chromatograph equipped with sequential C18 and C8 columns. The peak area was linear over the 0.25–10 mg/L range, and the recovery was 101  4.5%. This assay has the advantages of specificity, simplicity, speed over previously published methods, and requires smaller sample volumes. None of 19 drugs tested interfered with the assay [32]. In an attempt to assay propylthiouracil in human breast milk, a highperformance liquid chromatographic (HPLC) method was developed. After filtration with a membrane filter (Molcut II), the eluent was injected into a liquid chromatograph equipped with C18 precolumn and analytical column in series using a column switching technique. This method is sufficiently sensitive for most pharmacokinetic studies in human breast milk. The concentration of propylthiouracil was linear over the range of 50–5000 ng/mL. The recovery and the coefficient of variation were 92.0–100.6% and 1.6–2.9%, respectively. This assay has the advantages

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of specificity, simplicity and reproducibility for the measurement of propylthiouracil in human breast milk [33]. A sensitive, selective and simple post-column detection method for the determination of propylthiouracil (PTU) was developed. The method was based sensitization induction of the iodine–azide reaction combined with high-performance liquid chromatography. The analysis was conducted in the optimum conditions for iodine–azide detection system and HPLC separation. The linear range, the lower limit of detection and quantification for PTU in urine were established at the levels of 0.4–1.0 nmol/mL urine, 0.3 nmol/mL urine, and 0.4 nmol/mL urine, respectively [34]. Carrier-mediated three-phase hollow fiber microextraction combined with high-performance liquid chromatography-ultra violet detection (HPLC-UV) was applied for the extraction and determination of propylthiouracil in biological samples. Propylthiouracil (PTU) was extracted from 7.5 mL of the basic solution (the source phase) with pH 12 into an organic phase (n-octanol containing 6% (w/v) of Aliquat 336 as the carrier) impregnated in the pores of a hollow fiber, and finally was back extracted into 24 μL of the acidic solution located inside the lumen of the hollow fiber (the receiving phase). The extraction was performed through the gradient of counter ion from the source to the receiving phase. The effects of different variables on the extraction efficiency were studied simultaneously using an experimental design. A half-fractional factorial design was employed for screening to determine the variables significantly affecting the extraction efficiency. Then, the factors with significant effect were optimized using a central composite design (CCD) and the response surface equations were developed. The optimal experimental conditions obtained from this statistical evaluation included: source phase, pH 12; temperature, 25 °C; extraction time, 40 min; counter ion concentration, 2 mol/L of NaClO4; organic solvent 6% of octanol, and without salt addition in the source phase. Under the optimized conditions, the preconcentration factors were between 125 and 198 and also the limit of detections (LODs) ranged from 0.1 to 0.4 μg/L in different biological samples. The calibration curve was linear (r2 ¼ 0.998) over the concentration range of 0.5–1000 μg/L. Finally, the feasibility of the proposed method was successfully confirmed by extraction and determination of PTU in human plasma and urine as well as the bovine milk and meat samples in microgram per liter, and suitable results were obtained (RSD values less than 6.3%) [35]. A high-performance liquid chromatography method for the analysis of propylthiouracil in plasma was validated, and which used methylthiouracil as the internal standard. A one step extraction procedure and an isocratic

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HPLC method with UV detection were used. No plasma components were found to interfere in the assay. Linear calibration (r2 > 0.99) curves using water and plasma as matrices in the range of 0.05–15 μg/mL were obtained. Good recoveries for propylthiouracil (>85%) and methylthiouracil (>65%) were seen both in water and in plasma. The coefficient of variation for repeatability was less than 7%, less than 8% for reproducibility, and less than 6% for accuracy. The limit of detection was 2 ng/mL in water and 5 ng/mL in plasma. Poor stability of propylthiouracil was observed even at 20 °C, and thus it is recommended to perform the analysis shortly after sampling [36]. Urine analysis for the content of endogenous thiols and thiol drugs by HPLC with ultraviolet detection was described in a study. Other methodologies for detection and determination of thiols in urine were only mentioned. Outline of metabolism, role of main biological thiols in physiological and pathological processes and their reference concentrations in urine were presented. In particular, urine sample preparation procedures, including reduction of thiol disulfides, chemical derivatization and reversed-phase HPLC separation steps were discussed. Some experimental details of analytical procedures for determination of endogenous thiols cysteine, cysteinylglycine, homocysteine, N-acetylcysteine, thioglycolic acid; and thiol drugs cysteamine, tiopronin, D-penicillamine, captopril, mesna, methimazole, propylthiouracil and thioguanine were reviewed [37]. A simple and sensitive HPLC method for the determination of PTU in blood was described, along with a basic pharmacokinetic analysis after a single intraperitoneal administration to rats. The blood samples were deproteinized with phosphotungstic acid (PTA) reagent, and the clear supernatant obtained after centrifugation was directly analyzed in the HPLC system [38]. A high-performance liquid chromatographic method with a postcolumn iodine-azide reaction has been chosen and tested for validity in quantitative determination of propylthiouracil in tablets. A mobile phase with a flow rate of 1.4 mL/min was conducted in the form of isocratic chromatography on a C18 column with acetonitrile-water-sodium azide solution (2.5%; pH 5.5), 24:26:50 (v/v/v). Unreacted iodine from the post-column iodine-azide induced by-reaction was monitored with detection at λ ¼ 350 nm. The method proved to be linear within the range of 8–100 nM (r2 > 0.9988) with satisfactory results of inter-day precision (RSD < 4.2%) and accuracy (recovery exceeding 91%). The limits of detection (LOD) and quantification (LOQ) were 5 and 8 nM, respectively.

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The validation of the method comprised also its specificity. The results obtained proved the suitability and appropriateness of the suggested method for intended use [39]. A stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for the assay of propylthiouracil (PTU). The method was used to quantify PTU in topical formulations and in tablets. Excellent linearity was observed between PTU concentration and the peak area (r2 ¼ 0.999). The limit of detection was 1 ng, and the limit of quantitation was 1.2 ng. The method proved to be selective. Selectivity was validated by subjecting a stock solution of PTU to acidic, basic, and oxidative degradations. The peaks of the degradation products did not interfere with the peak of PTU. Excipients present in the dosage forms did not interfere with the analysis, and the recovery of PTU from each dosage form was quantitative [40]. A rapid and sensitive high-performance liquid chromatographic method was developed for the determination of propylthiouracil. The chromatographic system utilized a C8 column with the detector wavelength set at 276 nm. The mobile phase was composed of water and methanol in a 70:30 (v/v) ratio and was delivered at a flow rate of 0.5 mL/min. The analytical run time was less than 7 min. The standard curves were linear over a range of 1–100 μg/mL, with a correlation coefficient(r) of 0.9958. The limit of quantification was assessed to be 15 ng and the limit of detection 4.5 ng. The RSD values for the intra- and inter-day precision studies were 0.11–1.28% and 1.65%, respectively. The accuracy was 5% over the concentration range 1–100 μg/mL. The stability-indicating nature of the assay method was determined by subjecting a stock solution of propylthiouracil to extremes of acidic and basic degradation. It was found that the method was specific for propylthiouracil. Hence, a simple, rapid, sensitive, stabilityindicating, and reproducible assay method has been developed for the assay of propylthiouracil in a pharmaceutical dosage form [41]. 4.5.3 High performance thin layer chromatography (HPTLC) In this study, the application of a post-chromatographic iodine-azide reaction, followed by high-performance thin-layer chromatography (HPTLC), is presented. The method was applied for the determination of three thiouracils (6-benzyl-, 6-methyl-, and 6-propyl-2-thiouracil). The HPTLC plates developed by methanol were sprayed with a freshly prepared mixture of sodium azide, potassium iodide, and starch solution adjusted to pH 5.5, and exposed to iodine vapor for 5 s. Because of the induction properties of

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the C–S and C]S bonds of thiouracil molecule in the iodine-azide reaction, the spots became visible as white spots on a violet-brown background. Scanning of the HPTLC plates was performed on a PC scanner and analyzed by a special software. Determination range was 7–16 pmol per spot, 80–160 nmol/mL of urine, or 133–266 nmol/mL of serum [42]. In another study, application of iodine–azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapor for 5 s. The detection limits were established at the pmol level. The factors depending on the detection limits were described. A comparison of iodine–azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated [43].

4.5.4 Liquid chromatography–mass spectrometry A method for extraction of tapazol, thiouracil, methylthiouracil, propylthiouracil, and mercaptobenzimidazol (MBI) from thyroid tissue was described. The solid-phase extraction procedure was optimized to obtain the maximum results for the main thyreostats, including MBI. Different combinations of sample application, column conditioning and wash steps were tested. The analytes were extracted from the matrix with methanol. After solid-phase extraction they were derivatized with 7-chloro-4-nitrobenzo-2furazan. Determination was carried out using liquid chromatography– electrospray mass spectrometry. The identification of the analytes was performed according to the final revision of the European Union (EU) criteria (93/256/EC decision). The detection capability was 20 μg/kg for all mentioned thyreostats [44]. Another method using liquid chromatography–atmospheric pressure ionization mass spectrometry was described for the determination of five thyreostats in urine and thyroid tissue. Samples were extracted with ethyl acetate and the extracts were cleaned up on silica solid-phase cartridges. They were then concentrated and injected into the LC-MS system, monitoring the [M + H] + ions for each compound. Additional fragment ions for confirmation purposes could be obtained by increasing the cone voltage on the LC-MS system. Detection limits were in the region of 25 ng/g and recoveries ranged from 41% for the more hydrophilic compounds such

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as thiouracil to above 100% for the more hydrophobic compounds such as propyl- or phenylthiouracil [45]. Also, a method based on ultra-performance liquid chromatography– electrospray ionization-tandem mass spectrometry (UPLC-MS/MS) for the determination of six thyreostatic drugs in thyroid tissue has been optimized and validated in accordance with Decision 2002/657/EC. Sample extraction was evaluated in methanol and in ethyl acetate, the latter gave better results. Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation chromatography (GPC). Recoveries ranged from 40% to 79% for the SPE approach and from 80% to 109% for GPC. Quantification was performed with blank tissue samples spiked with the analytes in the range 50–500 μg/kg. 5,6-Dimethyl-2thiouracil and 2-mercaptobenzimidazole-d4 were used as internal standards. Decision limit (CCα) and detection capability (CCβ) ranged from 1 to 15 μg/kg and from 6 to 25 μg/kg, respectively. The accuracy of the method was calculated as percent error, which was less than 10%. The relative standard deviation in reproducibility conditions ranged between 2% and 14% [46]. In an another study, a sensitive and selective analytical method for the determination of four thyreostats (tapazol, thiouracil, methylthiouracil and propylthiouracil) in cow’s milk, lamb’s milk, and goat’s milk was developed and validated according to 2002/657/EC criteria. Proteins in milk samples were precipitated by acetonitrile and analytes were derivatized with 3-iodobenzylbromide. Afterward, derivatives were separated from the matrix by liquid-liquid extraction with ethyl acetate as an organic solvent and analysis was carried out using LC-MS/MS in a positive electrospray mode. The method provides, for all determined analytes, decision limits CCα below 1 ng/mL and a detection capability CCβ value below 1.5 ng/mL. The stability of analytes in sample extracts stored at various conditions was also tested and evaluated [47]. It is widely known that thyreostatic compounds could be illegally administered to animals in order to obtain a weight gain due to a higher retention of water in the edible tissue and the gastrointestinal tract. In the European Union, their use for animal production has been banned since 1981. Recently a highly sensitive method exploiting the determination of thyreostats with 3-iodobenzylbromide prior to purification to determine thyreostats in urine and other matrices was reported. For the first time, UPLC instrumentation was used to separate the 3-iodobenzyl derivatives of various thyreostats. The deuterated internal standards tapazole-d3 and propylthiouracil-d5 were

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used for the quantification of tapazole, thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil and mercaptobenzimidazole. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be for all compounds below the recommended value of 10 μg/kg [48].

5. Pharmacokinetics, metabolism, and excretion 5.1 Pharmacokinetics The half-life of propylthiouracil was determined in seven normal subjects after intravenous injection of 400 mg of the drug. An average value of 77  17 min (mean  S.D.) was found. As the concentration of the drug in the blood declined by a rapid phase followed by a more slow phase a two-compartment model was used for further calculations, and the rate constants between the two compartments and the overall elimination constant have been calculated. The same dose of propylthiouracil was later administered per os to the same seven persons and the plasma concentration was followed to non-measurable values. An average maximum concentration of 9.1  2.5 μg/mL was obtained after a period of 57  22 min. The area under the “peroral” curve has been calculated in per cent of the area under the “intravenous” curve. An average value of 77  13% was found expressing the bioavailability of the drug after oral administration [49]. The simultaneous occurrence of thyrotoxicosis and renal failure has rarely been reported in the literature, and data concerning appropriate anti-thyroid drug management in this circumstance are limited. A pharmacokinetic study on propylthiouracil was conducted in one such patient while the patient was receiving hemodialysis. On a day when the patient was not receiving hemodialysis, propylthiouracil serum levels were high, but serum propylthiouracil half-life was not prolonged. During hemodialysis, serum propylthiouracil levels were normal, and the time to peak serum levels was delayed; the disappearance of the drug from the serum was normal after hemodialysis was completed. The amount of propylthiouracil that appeared in the dialysate was approximately 5% of the administered dose. These data suggested that propylthiouracil can be administered in standard dosages to patients with thyrotoxicosis and renal failure [50]. The kinetics of propylthiouracil have been estimated in 10 hyperthyroid subjects according to a two-compartment model by giving the drug initially

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by the intravenous and subsequently by the oral route. The apparent half-life was 77  34 min (mean  S.D.). This value and other pharmacokinetic parameters were compared with the corresponding values obtained in a previous study in normal subjects. No significant differences were found. Study results correlating drug metabolism and thyroid status in humans were presented [51]. The pharmacokinetics of propylthiouracil was evaluated in nine elderly patients and compared to previous results from six younger subjects. By giving the drug both by the intravenous and oral route of administration, it was possible to estimate the rate and extent of bioavailability. The various kinetic parameters were calculated according to a two-compartment model by use of two different methods: a graphical hand drawn one and by a special developed computer program based on a least squares mineralization. While no significant differences could be demonstrated between the two age groups concerning volumes of distribution, clearance and extent of absorption, a large difference was found with regard to the absorption rate constant (ka), which was about three times higher in the younger than in the elderly subjects, presumably due a reduced gastric emptying time. Considering the comparison between the two methods of calculations all the kinetics parameters were similar except ka, and the slow disposition rate constant (β) which were underestimated by the graphical method. It was concluded that no age-dependent changes existed concerning the kinetics of propylthiouracil except for a decreased rate of absorption. Graphical methods in pharmacokinetics are useful in obtaining distribution and elimination data but seem often biased for the evaluation of absorption rate constants [52].

5.2 Metabolism Propylthiouracil (PTU) is associated with idiosyncratic agranulocytosis that may be due to reactive metabolites generated from oxidative metabolism by neutrophils. Therefore, the metabolism of PTU was investigated in activated neutrophils. Three oxidized metabolites were observed on HPLC: PTU-disulfide, propyluracil-2-sulfinate, and propyluracil-2-sulfonate (PTUSO3). No metabolism was detected in cells that had not been activated. Metabolism was inhibited by sodium azide and by catalase. The same products were produced by myeloperoxidase (MPO) in an MPO/H2O2/Cl system. PTU inhibited its own metabolism; however, complete conversion to PTUSO3 could be achieved with optimal PTU concentrations. MPO/H2O2 without Cl produced only slight metabolism. The PTU-sulfonyl chloride

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is a postulated intermediate. In the absence of chloride, oxidation might proceed through propyluracil-2-sulfonic acid. The sulfonyl chloride and PTUSO 3 are both chemically reactive with sulfhydryl compounds such as N-acetylcysteine. Such reactive metabolites, generated by activated neutrophils, may be involved in hypersensitivity reactions associated with PTU, such as agranulocytosis [53].

5.3 Excretion Propylthiouracil (PTU) concentrations were measured in blood and milk from nine lactating women after oral administration of 400 mg of PTU. 90 min after PTU ingestion, the mean serum-PTU level reached 7.7 μg/mL, while the mean concentration of PTU in milk reached only 0.7 μg/mL. The mean total amount of PTU excreted during 4 h was 99 μg (i.e., 0.025% of the administered dose). One of the suckling babies was studied for 5 months, during which the mother received 200–300 mg of PTU daily. It was noted that there were no changes in any of the thyroid parameters. PTU is not concentrated in human breast milk and recommended dosages to the mother result in minimal and presumably clinically insignificant doses to the suckling infant [54]. 6-n-Propyl-2-thiouracil-6-14C was administered to Sprague-Dawley rats of either sex (i.v., i.p. or p.o.) at doses of 20 mg/kg. Sustained plasma levels of radioactivity resulted after i.p. and p.o. doses. Equilibrium dialysis indicated that 57% of the drug in the plasma was protein bound. The drug had no particular affinity for any tissue. Propylthiouracil was found to have low aqueous solubility and chloroform/water partition coefficients. Between 75% and 90% of the administered radioactivity was excreted in the urine and approximately 15% appeared in the bile. Since negligible radioactivity appeared in the feces, the drug was determined to be completely absorbed, and enterohepatic circulation was present. The half-life of urinary excretion of radioactivity was 4–6 h regardless of the route of administration. Only 9–15% of the administered dose was excreted as unchanged drug in the 24-h urine. The major urinary metabolite was propylthiouracil glucuronide, which accounted for 40–48% of the administered dose in 24-h urine samples. The other urinary metabolite, which accounted for 10–16% of the administered dose in 24 h, has not yet been completely characterized. The major biliary metabolite was determined to be a glucuronide conjugate of propylthiouracil but different from the major urinary metabolite [55].

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6. Pharmacology The mechanism of inhibition of human thyroid iodide peroxidase (TPO) by 6-propyl-2-thiouracil (PTU) and l-methyl-2-mercaptoimidazole (MMI) used in the therapy of hyperthyroid patients was studied in vitro. The inhibition of TPO by MMI was not restored either by dialysis or by dilution, but the inhibition by PTU was restored by both treatments. PTU interacted directly with the product of TPO action (oxidized iodide) in the reaction mixture without significantly affecting TPO activity. MMI interacted directly with TPO and inhibited enzyme activity, rather than interacting with the product (oxidized iodide). The inhibition was irreversible with MMI, but reversible with PTU. The concentrations of PTU and MMI producing 50% inhibition of TPO were 2  106 and 8  107 M, respectively. 2-Mercaptoimidazole inhibited TPO reversibly but 1-methylimidazole and imidazole did not. Both the methyl and mercapto residues in MMI moiety are thought to be essential to its irreversible inhibition of TPO. The in vivo effect of MMI and PTU on TPO activity was also studied. TPO activities in the thyroid homogenate of rats, to which MMI (2 mg per rat) or PTU (10 mg per rat) had been administered intra peritoneally, were determined before and after dialysis against buffer. TPO activity in the PTU treated thyroid homogenate was significantly lower than that in the control before dialysis, but the activity was restored to the control value after dialysis. On the contrary, TPO activity in the MMI treated thyroid homogenate was significantly lower than that in the control and was not affected by dialysis. These data may explain why MMI is a more potent inhibitor of iodination than PTU and may fit the clinical results observed when hyperthyroid patients are treated with these agents [56]. In a previous study it was shown that the thioureylene antithyroid drugs 6-propyl-2-thiouracil (PTU) and l-methyl-2-mercaptoimidazole (MMI) can inactivate thyroid peroxidase (TPO) in a model iodination system containing relatively high concentrations of iodide. The same authors further continued their work in the current study to determine whether these drugs may also inactivate TPO in vivo in rats. Assays for total TPO activity after injection of PTU or MMI did not prove to be a valid approach. As TPO inactivation might be expected to result in a relatively prolonged inhibition of enzyme activity, most of the experiments involved measurement of the duration of the inhibitory effect of a single injection of the drug. Young rats were injected with low doses of PTU or MMI, and the effect on thyroidal

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organic iodine formation was determined at intervals after injection, either by 1-h pulse labeling with 131I in vivo or by incubation of excised thyroid lobes in a medium containing 131I. Results of both types of the experiments demonstrated that the inhibitory effect of a small dose of PTU (1 μmol/100 g body weight) was still very marked 17–18 h after injection. Moreover, an inhibitory effect of this small dose of PTU on the metabolism of [35S] MMI could also be demonstrated. Administration of MMI to rats, on the other hand, did not show the prolonged inhibitory effect observed with PTU. This is most likely attributable to the much lower thyroidal uptake of MMI than of PTU in rats. Intrathyroidal metabolism of [35S]PTU and [35S]MMI was also investigated. In contrast to the rapid disappearance of 35 S from plasma, both drugs showed accumulation and retention of 35S in the thyroid. However, they obtained no evidence that thyroidal accumulation of PTU or one of its metabolites could explain the prolonged inhibitory effect of this drug. It seemed more likely that this was attributable to TPO inactivation. The clinical implications of their findings were discussed with relation to the dosage schedule commonly employed in the treatment of Graves’ disease with antithyroid drugs [57].

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