Chapter-5 Hydrochlorothiazide INTRODUCTION Over the past...

Chapter-5 Hydrochlorothiazide

Department of Chemistry Dr. Hari Singh Gour Central University, Sagar (M.P.) 135

INTRODUCTION

Over the past decade several new drugs have been introduced to

treat hypertension, thus providing alternative options for treatment.

Many authorities and expert organizations worldwide, however, still

recommend thiazide diuretics and blockers as first line drugs for the

treatment of hypertension [1].

The selection of a new antihypertensive agent or a modification

of the commonly used stepped care approach to antihypertensive

treatment, however, presupposes that the new drug has been proved

to be superior, or at least equivalent to, the standard treatment. Thus

there is a need for long term studies of sufficient numbers of patients

to compare the antihypertensive efficacy, tolerability, and adverse

drug reactions of the new therapeutic regimen with the standard

treatment [2].

Hydrochlorothiazide (HCTZ) is widely prescribed diuretic, used

in congestive heart failure and hypertension [3]. In healthy fasting

volunteers the gastrointestinal uptake of the compound approximates

70 % and it is linear over the dose range 5-75 mg [4-6].

Structure

NH

NHSNH

2SO

2

Cl

OO

HCTZ, (6-chloro-3,4-dihydro-2H-1,2,4-benzo-thiadiazines- 7-

sulfonamide 1,1-dioxide) [7-8] is a widely prescribed diuretic. It is

indicated for the treatment of edema, control of essential hypertension

and management of diabetes insipidus [9-11].

Formula – C7H8ClN3O4S2

Solubility – Soluble in acetone, sparingly soluble in

ethanol (95 %), very slightly soluble in water.



It dissolves in dilute solution of alkali

hydroxides [12].

Mol. Wt. – 297.73 g/mol

Brand name – Hydrazide(H1), Aquazide(H2), Xenia(H3)

Identification – Identification of pure drug is performed by

FT-IR (Shimadzu 8400s) and compared with

standard one available in Indian

pharmacopeia [13].

Fig. 5.1: Reference IR Spectrum of HCTZ

Table 5.1: Characteristics absorption frequencies for identification

of pure HCTZ

S. No. Types of Vibrations Frequency (cm-1)

1. Ar. C – H Stretching 3066.92

2. Ar. C = C Stretching 1600.97

3. Ar. N – H Stretching 3263.66

4. N – H Stretching in NH2 3169.15

5. Asymmetric Stretching SO2 1330.93

6. Symmetric Stretching SO2 1180.47

7. S – N Stretching 908.5



Fig

. 5.2

: IR

spectr

um

of

pure

HC

TZ



Bioavailability - About 65-70%

Protein binding - 67.9%

Metabolism - HCTZ is not modified by organic biochemical processes.

Half life - 5.6 and 14.8 hrs.

Excretion - Elimination of HCTZ is mainly due to renal clearance that

occurs in about 320 mg/min. It is excreted unchanged in the urine.

HCTZ crosses the placental barrier and appears in breast milk.

History - Research in sulfonamide chemistry has brought a rich yield

of valuable therapeutics. One of the great successes was the discovery

of the benzothiadiazines as potent diuretics of low toxicity [14]. In

1958 De Stevens et al. [15] reported on the condensation product of 4-

amino-6-chloro-3, 5-disulfonamide and formaldehyde which was

found to be identical with the hydrogenation product of chlorothiazide

[16] and which soon became a widely used saluretic – Hydrochlorothiazide.

Adverse effects - The thiazide diuretic may cause a number of

metabolic disturbances. HCTZ may induce hyperglycemia and may

aggravate pre-existing diabetes mellitus. Thiazide diuretics increase

the concentrations of cholesterol and triglycerides in plasma by

unknown mechanism.

Uses - Hydrochlorothiazide is a diuretic which reduces the reabsorption

of electrolyte from the renal tubules.

Used to treat hypertension disease to manage the oedema due

to mild-to-moderate congestive heart failure. Oedema due to chronic

hepatic or renal disease may also respond favorably. It may also used

in patients with diabetes insipidus, due to a paradoxical effect.

Bio–Analytical methods -

Several analytical methods including LC-UV [17-18], spectro-

photometry and HPLC have already been reported for its determination,

either alone or in combination with other drugs [19-48].



Gotardo et al. have described diffuse reflectance spectroscopic

method for the determination of HCTZ in tablet formulations [49].

Kadam et al. have been reported quantitative analysis of

valsartan and HCTZ in tablets by high performance thin-layer

chromatography with ultraviolet absorption densitography [50].

Qutab and co-workers have described a simple and sensitive

LC-UV method for simultaneous analysis of HCTZ and candesrtan

cilexetil in pharmaceutical formulations [51].

Maillard et al. studied the fixed dose combination of telmisartan

and HCTZ to treat hypertension [52].

Satana et al. have determined simultaneously HCTZ and valsartan

in tablets by first-derivative ultraviolet spectrophotometry and LC

method [53].

Jordo et al. have been studied bioavailability and disposition of

metoprolol and HCTZ combined in one tablets and of separate doses

of HCTZ [54].



Pharmacodynamic concerns the relationship of formulations

and pharmacological effects, especially how solid dosage forms are

absorbed in vivo. Complicated factors are involved, among which, drug

disintegration and dissolution are very important ones. Due to its low solubility in water, the Federal Register of the

FDA categorizes it as questionable bioavailability tests [55].

Formulations of different brands have different types and/or amount

of adhesive, disintegrates, lubricants, or other excipients, as well as

different compression forces which affect the disintegration and

dissolution rate of a given formulation. Substantial related research

has been published such as Ibrahim H. G. who studied the influence

of compression forces on the dissolution profile of HCTZ and

phenylbutazone. It is proven that dissolution is positive proportional

to the logarithm of compression forces [56]. Tablet intensity is also

functionality relation with compression forces, so it is feasible to

establish in relationship of tablet intensity and dissolution rate. Desai

et al. documented the reduced dissolution stability of HCTZ

formulation containing sodium starch glycorate without which the

dissolution would be unacted [57].

Determination of λmax of pure HCTZ:

The pure form of HCTZ was accurately weighed 10 mg dissolved

in acetone and made up to the mark with distilled water in 100 mL of

volumetric flask. The stock solution was further diluted suitably with

water to give a concentration of 10 g/mL and scan on spectrophotometer

with data processing system. Maximum absorbance is obtained at 270

nm (Fig. 5.3).



Fig. 5.3: Determination of λmax of pure HCTZ

Verification of Beer’s Lambert law:

Aliquots of the standard solution of pure HCTZ was pipetted out

into 10 mL volumetric flask .The volume was made up to the mark

with water, to obtain a concentration of 0.2-25 µg /mL.

The absorbance of prepared solutions of HCTZ was measured at

270 nm using against appropriate blank. Averages of such eleven sets

of values were taken for standard calibration curve. Beer’s law obeyed

in concentration range of 0.4 – 25 g/mL.

Fig. 5.4: Verification of Beer’s Lambert law



Solubility measurements:

The solubility of HCTZ in different concentrations of surfactants

was determined by placing 75 mg of HCTZ in 25 mL of media and

gently agitated on a magnetic stirrer for 40 minutes at 37°C ± 0.5°C.

Supernatant was filtered through 0.45 μm syringe filter. 0.1 mL

sample was withdrew and diluted with distilled water up to the mark

in 25 mL volumetric flask and then analyzed at 270 nm by UV-visible

spectrophotometer. The mean results of triplicate measurements were

recorded.

Table 5.2: Solubility of HCTZ in different media

S. No.

Sample

Wt. of

drug (mg)

Overall volume

(mL)

Abs.

Solubility increase in fold

1. HCTZ + Distilled water 75 25 0.067 1.00

2. HCTZ + CTAB 75 25 0.075 1.12

3. HCTZ + PVP 44000 75 25 0.089 1.33

4. HCTZ + PEG 400/CTAB 75 25 0.095 1.41

5. HCTZ + SLS 75 25 0.080 1.20

6. HCTZ + PEG 4000/PVP 4000 75 25 0.081 1.21

6. HCTZ + PEG 4000 75 25 0.078 1.17

7. HCTZ + PEG 400 75 25 0.305 4.46

Fig. 5.5: Solubility determination of KTCZ in different fluids



In Vitro dissolution study:

1. Apparatus: Electrolab TDT – 08L USP apparatus.

2. Dissolution Media: 0.000125 M PEG 400.

3. Rotation speed: 100 rpm.

4. Preparation of HCTZ standard solution: 1.4 mg HCTZ USP

standard was weighed precisely, put in 100 mL volumetric flask

and made up to the mark with dissolution media.

5. Test preparation: Dissolution testing was performed on tablets

containing 12.5 mg HCTZ in 0.000125 M PEG 400 (37°C ± 0.5°C)

using paddle method. Sample of 5 mL were withdrawn at regular

time intervals, replaced by fresh medium and spectro-photometrically

analyzed at 270 nm after filtration through 0.45 m syringe filter.

All dissolution tests were performed in triplicate.

6. Time point: Dissolution amount was measured separately at 10,

20, 30, 40, 50, 60 and 70 minutes.

claim Lable

100

100

potency

dilution Test

dilution Std.

Std. of Absorbance

Sample of Absorbance

Table 5.3: Sample absorbance at different time intervals

S. No. Time (min) Absorbance

H1 H2 H3

1. 10 0.082 0.057 0.061

2. 20 0.099 0.079 0.074

3. 30 0.145 0.129 0.173

4. 40 0.234 0.221 0.216

5. 50 0.245 0.245 0.225

6. 60 0.256 0.267 0.235

7. 70 0.27 0.287 0.267

Standard Abs. 0.301



Table 5.4: % drug release of various formulations in polyethylene glycol 400 at different time

S. No. Time (min) % drug release

H1 H2 H3

1. 10 27.46 19.08 20.42

2. 20 33.48 26.45 24.78

3. 30 48.55 43.2 57.34

4. 40 78.36 74 72.33

5. 50 82.04 82.04 75.34

6. 60 85.73 89.41 78.61

7. 70 90.41 96.11 89.41

Table 5.5: log time, square root of time and log % of drug release

S. No.

Time (min)

log time

Square root of time

log % drug release

H1 H2 H3

1. 10 1.0 3.16 1.43 1.28 1.31

2. 20 1.30 4.47 1.52 1.42 1.39

3. 30 1.47 5.47 1.68 1.63 1.75

4. 40 1.60 6.32 1.89 1.86 1.85

5. 50 1.69 7.07 1.91 1.91 1.87

6. 60 1.77 7.74 1.93 1.95 1.89

7. 70 1.84 8.36 1.95 1.98 1.95



Fig. 5.6: Dissolution profile (n=3) of three commercial products of HCTZ in polymeric micellar media (Zero order plot)

Fig. 5.7: Regression plot for zero order



Fig. 5.8: First order plot

Fig.5.9: Regression plot for first order



Fig. 5.10: Korsmeyer Plot

Fig. 5.11: Regression plot Korsmeyer model



Fig. 5.12: Higuchi Plot

Fig. 5.13: Regression plot for Higuchi model



Table 5.6: Kinetic parameters for H1

S. No. Time

(min)

Rate Constant (k)

First order Korsmeyer Higuchi Zero order

1. 10 23.0 × 10-2 6.14 × 10-2 - -

2. 20 11.8 × 10-2 4.63 × 10-2 1.34 0.30

3. 30 8.5 × 10-2 5.06 × 10-2 3.85 0.70

4. 40 9.5 × 10-2 6.69× 10-2 8.04 1.27

5. 50 8.9 × 10-2 6.00 × 10-2 7.71 1.09

6. 60 10.3 × 10-2 5.53 × 10-2 7.52 0.97

7. 70 - 5.24 × 10-2 7.52 0.89

r2 0.8789 0.9338 0.9285 0.9277

Slope (n) 0.69


S. No. Time

(min)

Rate Constant (k)


1. 10 0.230 2.38 × 10-2 - -

2. 20 0.117 1.75 × 10-2 1.64 0.36

3. 30 0.084 1.97 × 10-2 4.40 0.80

4. 40 0.080 2.57 × 10-2 8.68 1.37

5. 50 0.075 2.32 × 10-2 8.90 1.25

6. 60 0.087 2.15 × 10-2 9.07 1.17

7. 70 - 2.00 × 10-2 9.20 1.10

r2 0.8984 0.9571 0.9537 0.9636

Slope (n) 0.92




S. No. Time

(min)

Rate Constant (k)


1. 10 0.230 3.34 × 10-2 - -

2. 20 0.1169 2.27 × 10-2 0.97 0.21

3. 30 0.0937 3.75 × 10-2 6.74 1.23

4. 40 0.0858 3.73 × 10-2 8.20 1.29

5. 50 0.0737 3.22 × 10-2 7.76 1.09

6. 60 0.0682 2.89 × 10-2 7.51 0.96

7. 70 - 2.89 × 10-2 8.24 0.98

r2 0.8196 0.9145 0.9307 0.9257

Slope (n) 0.83

RESULTS AND DISCUSSION

It is well known that the nature of the drug formulation can also

influence the dissolution process. To investigate this effect, solubility

of HCTZ at different concentration of surfactants was studied. This

was based on the assumption that polymer dissolution during the

time course of study changes the surface tension of the medium and

increase drug solubility. A significant increase in solubility was

observed in the concentration ranges studied (Table 5.2). This can be

attributed to the surface activity of the polymeric surfactant. The

surface tension of water (at 20°C) is 72 mN/m and that of PEG 400 at

the same temperature is 60.25 mN/m. This reduction in surface

tension can increase the wetting of the drug particles and as a result,

increase the solubility.

Dissolution of HCTZ from all the tablets was studied in PEG-

400. This dissolution media is selected on the basis of solubility

measurements. Different kinetic models were applied for study of

release rate.



Dissolution of HCTZ from all the tablets followed Korsmeyer

model. Plot of log percent release vs log time was found to be linear (r2

in the range 0.91-0.95). From the slopes of linear plots the dissolution

rates were calculated. The kinetic parameters of three brands of

tablets are summarized in table 5.6, 5.7, 5.8.

Depending on the dose size and solubility characteristics of low

solubility drugs, a meaningful and discriminatory power of

dissolution rate testing can be demonstrated.

Solubility of HCTZ increased directly with PEG 400

concentration.

For a 12.5 mg HCTZ tablet, PEG 400 at 0.000125 M level is

required for a discriminative dissolution test.



Preparation of stock solution of pure HCTZ drug:

Stock solution of HCTZ was prepared by dissolving 297 mg in

1:1 acetic acid and water (v/v) and made up to the mark in 100 mL

volumetric flask. The above stock solution was further diluted to get a

working standard solution of 1 to 50 mg/mL.

Preparation of calibration curve in presence of V(V):

Aliquot of reference standard solution of HCTZ from 0.1 – 8 mL

were pipetted in to series of 10 mL of volumetric flask. In each flask

0.125 mL of PEG 400, 0.1 mL V(V), 0.55 mL of H2SO4 were

successively added and diluted with distilled water up to the mark.

The contents of each flask were mixed well and allow standing for 10

min. The increase in absorbance at 365 nm was recorded as a

function of time against the reagent blank prepared similarly. The

amount of the drug was calculated from the calibration graph.

Fig. 5.14: Absorption maxima of V(V) and V(IV)



Table 5.9: Reaction mixture

Sample

Concentration (M)

Stock solution (M) Required (M)

[HCTZ] 0.01 1 × 10-4 – 8 × 10-3

[V(V)] 0.01 0.0001

[H+] 18 1

[PEG 400] 0.01 1.25 × 10-4

Overall volume 10 mL

Table 5.10: Absorbance of standard solutions of pure drug at

different concentrations in presence of V(V)

S. No. Concentration (M) Absorbance (at 365 nm)

1. 0.0001 0.124

2. 0.0004 0.126

3. 0.0008 0.128

4. 0.001 0.130

5. 0.002 0.133

6. 0.004 0.139

7. 0.006 0.148

8. 0.008 0.154



Fig. 5.15: Calibration curve for pure drug in presence of V(V)

Preparation of sample solutions:

Ten tablets of each commercial pharmaceutical brand to be

studied were weighted and finely powdered. A portion of this powder,

equivalent to 297 mg of HCTZ was accurately weighed. The sample

was shaken with 1:1 acetic acid and water in a magnetic stirrer for 10

minutes and filtered through 0.45 μm syringe filter paper. This

solution was then diluted with acetic acid and water (1:1) in calibrated

100 mL flask. The 0.4, 0.8 and 2.0 mL of solution was pipetted in 10

mL volumetric flask. The three solutions of HCTZ were obtained (4 ×

10-4 to 1 × 10-3 M) applying the same procedure drug solutions.



Table 5.11: Absorbance of sample solutions of different marketed brands at three concentrations

S.

No.

Concentration

(M)

Absorbance (at 365 nm)

Pure drug H1 H2 H3

1. 0.0004 0.126 0.127 0.125 0.124

2. 0.0008 0.128 0.130 0.125 0.129

3. 0.001 0.130 0.129 0.131 0.127

Table 5.12: Recovery Study

S.

No. Label claim in mg

Amount of drug found (%)

H1 H2 H3

1. 12.5 100.7 99.3 99.2

2. 12.5 101.6 100.9 101.0

3. 12.5 99.23 100.5 98.0

Average Recovery (%) 100.6 100.3 99.4

Standard Deviation 1.14 0.83 1.50

RESULTS AND DISCUSSION

Spectral studies:

The spectrum of reference pure drug of HCTZ in aqueous

solution shows one absorbance band at 270 nm. The addition of

acidic solution of V(V) to the drug solution causes change in the

absorption spectrum with new characteristic band appearing at 365

nm. HCTZ oxidizes by aqueous acidic vanadium (V). Simultaneously

V(V) was produced as reaction proceeds. The equilibrium is attained

in ~50 minute. The yellow color changes to light green and finally

becomes blue on the completion of reaction. This indicates formation



of an intermediate complex between V(V) and HCTZ. Therefore, a

kinetically based spectrophotometric method was developed for the

quantitative determination of HCTZ by measuring the increase in

absorbance at 365 nm as a function of time.

Linearity:

The linearity range was observed between from 0.029–3.18

mg/mL.

Application of the method to tablets:

The validity of the proposed method was presented by recovery

studies. For this purpose, a known amount of reference drug was

spiked to marketed tablets at three different concentration levels.

Each level was repeated five times. The results (Table 5.12) were

reproducible with low SD. No interference from the common excipients

was observed. The applicability of the proposed method for the

determination of HCTZ has been tested on commercially available

pharmaceutical formulations.

Limit of detection and quantification

The limit of detection (LOD) and quantification were evaluating

the minimum level at which the analyte could be readily detected and

quantified with accuracy, respectively. The LOD was 3.43 mg/mL and

LOQ was 11.45 mg/mL.



Table 5.13: Quality Control Parameters

S.No. Parameters HCTZ

1. max (nm) 365

2. Beer’s Range (mg/mL) 0.029 – 3.18

3. Molar Absorptivity (L mol-1 cm-1) 8.85 × 103

4. Sandell’s Sensitivity (g cm-2) 0.033

5. Regression equation 0.9898

6. Intercept 0.1253

7. Slope 0.1045

8. Limit of Detection (mg/mL) 3.28

9. Limit of Quantization (mg/mL) 10.95

10. Standard deviation of calibration line 0.114

PROPOSED MECHANISM

The equivalent concentration of reactants was taken in a

reaction vessel and the reaction was allowed to completion. The

reaction products formed were identified by the following procedure.

The formation of free radicals during the course of the reaction

was tested by polymerization of the acrylonitrile monomer. To a small

portion of the reaction mixture 1-2 mL of acrylonitrile monomer was

added and rest aside for 24 hours. Precipitation of polymerized

acrylonitrile was obtained. This confirms the possibility of the

formation of free radicals during the course of the reaction. The

confirmation of product was performed using the LC/MS analysis.



Fig. 5.16: LC/MS spectrum of standard HCTZ and HCTZ + V(V)

VO2 + 2H+ V (OH)2

3+

V (OH)2

+3+ HSO4

-V (OH)[

2 HSO4

]2+

R N V

H

H OH

OH

SO4H

Intermidiate

Intermidiate + H2OSlow

RNH + V(IV) + H3O+

RNH + HNRfast

CouplingRNH - NHR

Hydrazo derivative

RNH - NHR + 2 V(V) + H2Ofast

R-N=N-R + 2V(IV) + 4H +

Azoxy derivative

RNH2+[V(OH)2HSO4]2+

.

. .

+

2+

Where R =

[ ]

NH

NH

S

Cl

SO2

OO

(m/z 589)

HCTZ



The critical micellar concentration of PEG 400 was found to at

0.000125 M. The reaction shows that PEG and H+ both favors the

metabolic conversion of HCTZ.

HCTZ obeyed the Beer’s law from 0.029 – 3.18 mg/mL and from

liner equation slope, intercept, molar absorptivity, Sandell’s sensitivity

were also calculated (Table 5.13).

It has been reviewed by several researchers that HCTZ is

metabolized very low and except 4% of drug get excreted unchanged.

The reaction proceeded by free radical in micellar media. The

kinetic evidence shows that there is a complex formation between

substrate and oxidant. This is further confirmed by spectro-

photometrically.

As far as the oxidation of HCTZ is concerns, only few

mechanistic studies have been published. In this study oxidation of

HCTZ by V(V) in micellar medium have been proposed. During this

study it appeared that HCTZ dimers are formed (Fig. 5.16).

CONCLUSION

The proposed method was applied satisfactory to the

determination of HCTZ in pharmaceutical preparations.

The apparent molar absorptivity and Sandell’s sensitivity of the

resulting colored product were found to be 8.85 × 103 L mol-1

cm-1 – 0.033 g cm-1 respectively. The values of three parameters

can be considered satisfactory at least for the drug

concentration level examined.



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