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Spectrophotometric determination of chlorthalidone in pharmaceutical formulations using different order derivative methods
⁎Corresponding author. Tel.: +964 7504600445. rebwar_s75@uni-sci.org (Rebwar O. Hassan)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Simple, repaid and accurate zero-, first- and second-order derivative spectrophotometric methods have been developed for determination of chlorthalidone (CLT) in commercially available tablets. Normal spectrophotometric scan (zero order) shows maximum absorbance at 276 nm in methanol solution and a good linearity in the range of 10.0–75.0 μg/mL. Linear relations using first (D1) and second (D2) order derivative methods were obtained at 278 and 288 nm for D1 and 286 and 292 nm for D2.The calibration curves were constructed in the range of 1.0–25.0 μg/mL for D1 (R = 0.998) and D2 (R = 0.999). Different analytical validations were determined (accuracy, precision, specificity, recovery, stability and robustness) to demonstrate its suitability for routine quality control labs. All the developed methods were successfully applied to a tablet formulation and the results were compared statistically with each other and with those obtained by the HPLC reference method.
Keywords
Derivative spectroscopy
Chlorthalidone
Pharmaceutical formulation
1 Introduction
Chlorthalidone (first introduced in Switzerland in 1959) is a sulphamyl benzophenone derivative [2-chloro-5-(1-hydroxyl-3-oxo-2,3-dihydro-1H-isoindol-1-yl) benzene-1-sulfonamide] (Fig. 1) (Douglas et al., 1961). It is a diuretic agent used in the treatment of edema associated with congestive heart failure (Florey, 1985). Compared with other medications like thiazide class, chlorthalidone has a longer duration of action but a similar diuretic effect at maximal therapeutic doses (Akiful Haque et al., 2012).
Chemical structure of chlorthalidone.
Chlorthalidone analysis as anti-hypertensive drug is of great interest, since hypertension is very common disorder, particularly in the past middle age. Accordingly, the development and validation of new analytical methods for estimation of anti-hypertensive drug are required (Shaja et al., 2011). Literature survey reveals that there are various analytical methods for estimation of chlorthalidone individually or in combination with other drugs, that involves spectrophotometric (Barary et al., 1990; El-Maaboud and Salem; Florey, 1985; 2005; Luz Luis et al., 1999; Vetuschi and Ragno, 1990), fluorimetric (Ayad et al., 1996; Gonzaleza et al., 2009), chemiluminescence (Ciborowski and Catalá, 2004), high-performance liquid chromatography (HPLC) (Bauer et al., 1983; Elgawish and Mostafa, 2011; Khuroo et al., 2008; Mhaske et al., 2012; Mhaske et al., 2012; Sahoo et al., 2012), and gas chromatography (GC) (Magnar Ervik and Klas Gustavii, 1974). An effort was made to develop a rapid, economical, precise and accurate method for chlorthalidone determination in tablet formulations. For instance, derivative spectrophotometry is an analytical technique of great utility for extracting both qualitative and quantitative information from spectra composed of un-resolved bands, and eliminating the effect of baseline shifts and baseline tilts. This consists of calculating and plotting one of the mathematical derivatives of a spectral curve (Ojeda and Rojas, 2004).
The present method involves development of a zero-, first-, and second-order derivative spectrophotometric method for quantitative determination of chlorthalidone in pharmaceutical formulations without any necessity for sample pre-treatment and separation.
The developed methods were rapid, simple, accurate, reproducible, economic, and can be successfully employed in the routine analysis of this drug in different tablet dosage forms. The results obtained by those three present methods were statistically compared with those obtained by the HPLC method.
2 Materials and methods
2.1 Instrument
A CECIL UV–Visible digital spectrophotometer (model CE 3021), with a 1-cm matched quartz cell, was used for different derivative spectral measurements. However, the derivative mode was carried out with different instrumental parameters vs. spectral band width 1.8 nm, scan speed 3.0 nm/s and wavelength range 250–350 nm.
2.2 Chemicals and reagents
The CLT standard (98% purity) was obtained from Shang Hai DEMO Medical Tech Co., Ltd. Three types of commercial chlorthalidone tablets (50 mg) were obtained from different pharmacies in Erbil city (Table 1) and methanol was purchased from Schar Lab S.L.
Sample no.
1
2
3
Name
Hygroton 50.0 mg
Hygroton 50.0 mg
Hygroton 50.0 mg
Manufacture
mibe GmbH Arzneimittel
Mediterranean Pharmaceutical Industries (MPI) Under license from: NOVAETIS Pharma AG, Switzerland
Amdipharm Limited
Agents
Chlorthalidone 50 mg
Chlorthalidone 50 mg
Chlorthalidone 50 mg
Pharmaceutical form
Tablet
Tablet
Tablet
Products Country
Germany
Syria
Portugal
2.2.1 Standard solution
Standard CLT stock solution was prepared by dissolving 0.010 g drug in methanol and the volume was made up to 100 mL with methanol to get concentration of 100.0 μg/mL. Working solutions were freshly prepared by subsequent dilutions with the same solvent.
2.2.2 Sample preparation
Ten tablets were weighed and crushed into a fine powder using mortar and pestle. A portion of the powder equivalent to one tablet was weighed accurately, transferred into a 100 mL volumetric flask. About 50 mL of methanol was added, shaken and sonicated for 25 min for further dissolving. This solution was diluted up to volume with methanol. Sample solution was filtered through Whatman No. 42 to get a clear solution. The supernatant solution obtained was used as a stock sample solution. Dilution of the stock solution quantitatively with methanol brings about suitable working sample solutions for zero, first-, and second-order derivative measurements against the diluent methanol.
2.3 Procedure
2.3.1 Zero-order method
The zero absorbance spectrum of CLT shows two maximum absorbance peaks at 276 and 284 nm in methanol (Fig. 2A), when a series solutions of CLT scanned over the range of 260–290 nm.
Absorption spectra at different concentrations of standard CLT (A) Zero-order derivative (10.0, 25.0, 35.0 and 75.0 μg/mL. (B) First-order derivative ((1.0, 5.0, 10.0, 15.0, 20.0 and 25.0 μg/mL). (C) Second-order derivative (1.0, 5.0, 10.0, 15.0, 20.0 and 25.0 μg/mL).
2.3.2 First-order method
The two shouldered peaks obtained in normal spectrum are easily separated by derivative spectrophotometry. The first derivative spectrum (D1) appears in Fig. 2B, shows one maximum peak at 272 nm with two minimum peaks at 278 and 288 nm at wavelength range 250–350 nm.
2.3.3 Second-order method
The zero order spectrums were derivatized at wavelength range 250–350 nm to get second order derivative spectrum with two minimum peaks at 276 nm and 286 nm with one maximum peak at 292 nm (Fig. 2C).
2.3.4 HPLC method
Validation of the present methods was performed with the HPLC method reported by Singh et al. (2009). The method was carried out on reverse phase C18 column (250 × 4 mm, 5 μm) using a mixture of 50 mM disodium hydrogen phosphate:methanol:acetonitrile in the ratio of 70:30:05 (pH adjusted to 3.5 with orthophosphoric acid) as mobile phase. Flow rate was maintained at 1.0 mL/min. The UV detection was made at 220 nm and all analyses were done at column temperature (30 ± 2 °C) under isocratic conditions.
3 Results and discussion
3.1 Method development
Development of analytical methods and validation, play important roles in the discovery, development, and manufacture of pharmaceuticals to ensure a high efficacy and safety for the patients. Methods of derivative spectrophotometry offer alternative approaches to the enhancement of sensitivity and specificity in analysis. This technique has been frequently used to extract information from overlapping bands of the analytes and interferences. In the present work validation of three simple methods to quantify the drug in pharmaceutical formulations is considered. Different variables related to the derivative measurements are optimized. The derivative measurements are highly dependent on the smoothing factor (n), since broad peaks become sharper, signal to noise ratio increased and increase the sensitivity of the method by controlling the smoothing number. Accordingly, different n- values in the range 1–10 were tested in the D1 and D2 spectra of the drug. Optimum results were obtained with n = 10, scan speed = 1 nm/s, bandwidth = 1.8 nm in the wavelength range 250–350 nm.
3.2 Calibration curve
3.2.1 Zero-order method
The calibration curve for CLT standard in the concentration range 10.0–75.0 μg/mL at 276 nm was constructed by plotting the absorbance (A) versus concentration (Fig. 3I). Statistical data are shown in Table 2. D.L. = detection limit; r = Correlation coefficient.
Calibration curves for: (I) Zero order method at 276 nm. (II) D1 method at (a) 288 nm and (b) 278 nm. (III) D2 method at (a) 292 nm, (b) 286 nm.
Methods
Wavelength range (nm)
Concentration range (μg/mL)
λ (nm)
Regression equation
r
D.L. (μg/mL)
Zero-order method
260–290
10.0–75.0
276
y = 0.004x + 0.010
0.998
5.50
First-order derivative (D1)
250–350
1.0–25.0
278
y = 1.848x + 1.508
0.998
0.46
288
y = 4.111x + 0.190
0.998
0.35
Second-order derivative (D2)
250–350
1.0–25.0
286
y = 2.190x + 0.100
0.999
0.32
292
y = 2.378x + 0.454
0.999
0.15
3.2.2 Derivative methods
Calibration curves were constructed for the assay of CLT with the UV-spectrophotometric method, operated under D1 and D2 mode (Fig. 3II and III) in wavelength range 250–350 nm with different minimum and maximum peaks (Fig. 2B and C). The peak to zero method for calibration curve in the first- and second-order derivative spectrophotometric methods was applied. The curves were constructed at each minimum and maximum peak wavelength by plotting peak height versus concentration in the range of 1.0–25.0 μg/mL. Statistical calibration results for regression equation in the concentration range are evaluated by using the least-squares method (Table 2).
3.3 Validation methods
3.3.1 Accuracy and precision
The reliability of the present methods (precise %RSD and accuracy %E) under optimum experimental conditions was determined by performing the standard analysis at different time intervals in the same day (intraday) and in three different days (interday) of the analytical methods. The intraday precision and accuracy were determined by measuring five replicate analyses (n = 5) at two concentration levels (15, 20 μg/mL), respectively. Similarly, the interday precision and accuracy were determined by performing five replicate analyses at three different days at same mentioned concentration level (Table 3). X = Mean of five replicate (n = 5), SE = Standard error, SD = Standard deviation, %RSD = Relative standard deviation, %E = Relative error.
Method
λ (nm)
Added (μg/mL)
Intraday
Interday
Found (μg/mL) X ± SE, SD
%E
RSD%
Found (μg/mL) X ± SE, SD
%E
RSD%
Zero-order method
276
15
14.90 ± 0.148; 0.331
0.68
2.22
15.16 ± 0.143; 0.319
−1.04
2.10
20
20.28 ± 0.168; 0.377
−1.39
1.86
20.32 ± 0.178; 0.399
−1.61
1.96
First derivative (D1)
278
15
14.74 ± 0.142; 0.318
1.73
2.16
14.88 ± 0.091; 0.203
0.79
1.36
20
20.37 ± 0.225; 0.504
−1.85
2.47
20.25 ± 0.213; 0.476
−1.26
2.35
288
15
14.91 ± 0.073; 0.164
0.59
1.10
14.92 ± 0.102; 0.228
0.55
1.53
20
20.08 ± 0.185; 0.415
−0.39
2.06
20.22 ± 0.096; 0.214
−1.12
1.06
Second derivative (D2)
286
15
15.23 ± 0.151; 0.337
−1.53
2.21
15.10 ± 0.138; 0.309
−0.68
2.04
20
20.10 ± 0.023; 0.051
−0.49
0.25
20.10 ± 0.174; 0.389
−0.49
1.93
292
15
15.12 ± 0.085; 0.190
−0.81
1.26
15.25 ± 0.101; 0.226
−1.68
1.48
20
20.21 ± 0.080; 0.178
−1.05
0.88
20.3 ± 0.112; 0.251
−1.49
1.24
3.3.2 Selectivity/specificity
Selectivity/Specificity is the ability of a method to determine accurately and specifically the analyte of interest in the presence of other components in a sample matrix under the stated conditions of the present method. The importance of derivative methods arises from that it allows quantification of one or few analytes without initial expensive and fatigue separation or purification procedure. Excipients are only interferences in drug formulation. No difference in the maximum and minimum wavelength of all spectra was observed between the spectra of CLT standard (Fig. 2) and three tablet solutions (Fig. 4).
Spectra of three sample solutions: (A) (1, 2 and 3) are zero order containing 25 and 65 μg/mL of CLT; (B) (1, 2 and 3) and (C) (1,2 and 3) are first- and second-order derivative, respectively, containing 10 and 65 μg/mL of CLT where numbers 1, 2, and 3 are referred to the sample number as mentioned in Table 1.
3.3.3 Recovery
Recovery is best measured by adding different volumes of known concentrations of CLT to (a) the diluent solvent (methanol) and (b) the pre-analyzed sample (4.0 μg/mL), in a way that total concentration after addition and dilution equals to 12.0, 14.0, 16.0, 18.0 and 20.0 μg/mL. The former samples establish a reference calibration curve. The other samples are taken through the whole sample work-up procedure and yield a second calibration curve, the results obtained are tabulated in Table 4. No significance difference in the slope of the two curves shows good efficiency and selectivity of the present methods without any interferences effects (Meier and Zünd, 2000). X: mean of five replicate (n = 5); SE: standard error; SD: standard deviation; %RSD: relative standard deviation.
λ (nm)
Added (μg/mL)
Found (μg/mL) X ± SE, SD
Regression equation; slope
Recovery%
RSD%
Method I
Zero-order method
276
12
12.11 ± 0.187; 0.418
y = 0.004x + 0.010;
101.10
3.44
14
14.47 ± 0.245; 0.548
0.004
103.36
3.78
16
16.06 ± 0.133; 0.298
100.36
1.86
18
18.19 ± 0.173; 0.386
101.04
2.12
20
20.01 ± 0.167; 0.374
100.05
1.87
First-order derivative (D1)
288
12
12.20 ± 0.125; 0.279
y = 4.111x + 0.190;
101.63
2.29
14
13.81 ± 0.066; 0.147
4.111
98.66
1.07
16
16.00 ± 0.163; 0.0.366
99.98
2.29
18
18.04 ± 0.094; 0.209
100.22
1.16
20
20.37 ± 0.148; 0.331
101.83
1.62
Second-order derivative (D2)
292
12
11.68 ± 0.111; 0.247
y = 2.378x + 0.454;
97.37
2.12
14
13.81 ± 0.147; 0.329
2.378
98.61
2.38
16
16.06 ± 0.192; 0.430
100.36
2.68
18
18.39 ± 0.182; 0.407
102.17
2.21
20
20.38 ± 0.141; 0.316
101.88
1.55
Method II
Zero-order method
276
12
15.69 ± 0.218; 0.488
y = 0.004x + 0.008;
97.40
3.11
14
18.24 ± 0.244; 0.547
101.74
3.00
16
20.12 ± 0.137; 0.307
0.004
100.76
1.53
18
21.92 ± 0.141; 0.315
99.57
1.44
20
23.91 ± 0.156; 0.348
99.56
1.46
First-order derivative (D1)
288
12
16.06 ± 0.155; 0.346
y = 4.110x + 0.194;
100.53
2.15
14
18.14 ± 0.177; 0.397
101.03
2.19
16
20.15 ± 0.167; 0.597
4.110
100.94
2.96
18
21.87 ± 0.139; 0.310
99.26
1.42
20
24.19 ± 0.148; 0.331
100.97
1.37
Second-order derivative (D2)
292
12
16.10 ± 0.094; 0.210
y = 2.377x + 0.463;
100.82
1.30
14
17.99 ± 0.060; 0.133
99.91
0.74
16
20.06 ± 0.127; 0.248
2.377
100.40
1.41
18
21.99 ± 0.139; 0.312
99.93
1.42
20
23.95 ± 0.090; 0.202
99.73
0.84
3.3.4 Robustness
An analytical method is robust if results are not (very) sensitive to variations in the experimental conditions. Evaluation of the method robustness was performed by testing the stability of the drug solution at different temperatures and times. Two different volumes of known concentration of CLT were added to the pre-analyzed sample containing 10.0 μg/mL CLT, and then the recoveries of the added standard were measured against different temperatures and times. The results show that no significant change in the response of CLT was observed. Hence, the present methods are rugged and robust for determination of CLT (Table 5).
Method
Added (μg/mL)
At 35 °C (recovery% ± SE)
Room temperature (recovery% ± SE)
Refrigerator temperature (recovery% ± SE)
3 h
10 h
3 h
10 h
3 h
10 h
Zero-order (276 nm)
5
101.2 ± 1.23
102.36 ± 1.32
99.87 ± 0.89
100.31 ± 1.75
100.69 ± 1.89
99.14 ± 0.17
10
100.8 ± 0.92
101.55 ± 0.72
100.39 ± 0.19
101.23 ± 0.37
100.37 ± 0.39
100.59 ± 0.27
First-order (288 nm)
5
99.89 ± 0.36
100.41 ± 0.96
100.07 ± 0.26
101.75 ± 0.66
101.92 ± 0.87
101.39 ± 0.22
10
98.88 ± 1.62
101.03 ± 0.12
100.70 ± 0.79
100.11 ± 0.54
100.89 ± 1.26
101.34 ± 0.97
Second-order (292 nm)
5
99.76 ± 0.25
100.21 ± 1.79
99.38 ± 0.26
100.79 ± 1.03
100.9 ± 0.33
101.85 ± 0.21
10
99.49 ± 0.45
99.89 ± 0.13
99.88 ± 0.36
100.97 ± 0.98
99.12 ± 0.11
102.01 ± 0.89
3.3.5 Comparison of the present methods
The CLT in three different pharmaceutical products were successfully analyzed by the present methods, and the results are compared with the reference method (Table 6). The calculated Student’s t-test values and variance F-test values did not exceed the theoretical value, which indicates the absence of any difference between the methods compared (Harris, 2009). The present method gives good results and agreement with that obtained by the reference method. X: mean of five replicate (n = 5); SD: standard deviation; RSD: relative standard deviation; tabulate students t-test and variances F-test at 95% confidence limit (n = 5) were 2.78 and 6.39, respectively.
Sample no.
Methods
Found (mg/tablet) X ± SD
Recovery %
RSD %
F-test
t-test
1
Reference
50.30 ± 0.73
100.60
1.46
–
–
Zero-order (276 nm)
50.61 ± 1.67
101.21
3.31
5.19
0.41
D1
278 nm
50.32 ± 1.24
100.64
2.47
2.86
0.04
288 nm
51.30 ± 1.33
102.60
2.60
3.29
1.68
D2
286 nm
50.64 ± 1.19
101.28
2.36
2.64
0.64
292 nm
51.16 ± 1.30
102.32
2.54
3.13
1.48
2
Reference
51.02 ± 0.75
102.04
1.46
–
–
Zero-order (276 nm)
51.28 ± 1.54
102.55
3.01
4.28
0.37
D1
278 nm
49.79 ± 1.16
99.58
2.32
2.41
2.37
288 nm
50.48 ± 1.62
100.96
3.22
4.75
0.74
D2
286 nm
51.18 ± 1.60
102.36
3.12
4.60
0.22
292 nm
51.68 ± 0.84
103.35
1.63
1.28
1.74
3
Reference
51.90 ± 1.12
101.80
2.21
–
–
Zero-order (276 nm)
50.76 ± 1.99
101.52
3.91
3.12
0.16
D1
278 nm
50.58 ± 1.32
101.17
2.61
1.38
0.53
288 nm
50.29 ± 1.30
100.57
2.59
1.35
1.05
D2
286 nm
50.31 ± 1.20
100.62
2.39
1.15
1.09
292 nm
50.59 ± 1.33
101.18
2.63
1.40
0.51
4 Conclusions
Analytical methods based on measurements of UV or visible light absorption were considered as one of the most popular and most communes used in laboratory practice, and commercially available device are low-priced and easy for operation and control. The main disadvantage and limitation of the spectrophotometry is its low selectivity. Thus, this selectivity can simply be enhanced by derivatization of spectra. This operation allows removing spectral interferences and as a consequence leads to increased selectivity of assay. The derivatization of normal spectrum can lead to separation of overlapped signal and elimination of a background caused by presence of other compounds in a sample. Those properties can allow quantification of one or few analytes without initial separation or purification. The main objective of the present investigation was development of three methods for determination of CLT basing on the derivative method. There was no interference from the excipients present in the tablets, hence the present method can be directly applied to the pharmaceutical sample without any prior exhaustive treatment in the estimation of CLT in bulk and pharmaceutical dosage forms in a routine manner, when simplest and fastest method is desirable.
Acknowledgments
The authors would like to thank all staff in Chemistry Department- Science College- Salahaddin University -Erbil for their help during all research intervals involved in the work.
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