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Sequential spectrophotometric method for the simultaneous determination of amlodipine, valsartan, and Hydrochlorothiazide in coformulated tablets.

1. Introduction

Many analytical methods have been introduced for the analysis of mixtures among which the molecular absorption spectroscopy was the most simple, fast, and applicable in laboratories. Molecular absorption spectroscopy has been extensively used for the determination of drugs in pharmaceutical preparations with a view to the development of analytical methods. The use of this technique for pharmaceutical analyses has the inherent constraint that most active drugs absorb in the UV region and exhibit strongly overlapped spectra that impede their simultaneous determination.

Direct spectrophotometry cannot resolve the drugs in their mixtures with impurities or other drugs, so several manipulations were performed to enable resolution of binary and ternary mixtures. Binary mixtures can be determined using different order derivatives [1], methods manipulating ratio spectra [2, 3] or dual wavelength [4-6], and isoabsorptive method [7]. For ternary mixtures, few spectrophotometric methods could resolve the overlap in their spectra, namely, Derivative Ratio Zero Crossing [8] and Double Divisor Ratio Spectra-Derivative Spectrophotometry [9] methods.

This paper describes the development and subsequent validation of a novel, simple, and rapid spectrophotometric method "Sequential Spectrophotometry" for simultaneous quantitation of ternary mixtures. The method was applied on a ternary mixture of AML, VAL, and HCT in bulk powder and pharmaceutical dosage forms. The linearity of response, accuracy, intermediate precision, and robustness of the described method for assay of AML, VAL, and HCT have been checked.

Amlodipine (AML), 2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine carboxylic acid 3-ethyl 5-methyl ester [10], (Figure 1), is a dihydropyridine derivative with calcium antagonist activity. It is used in the management of hypertension, chronic stable angina pectoris, and prinzmetal's variant angina [11].

Valsartan (VAL) (chemically described as N-[p-(o-1Htetrazol-5-ylphenyl)benzyl]-N-valeryl-L-valine [10] Figure 1), is a potent and specific competitive antagonist of the angiotensin-II [AT.sub.3]-receptor. It is used for treatment of hypertension, heart failure, and post-myocardial infarction [12].

Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide [10] Figure 1, is a diuretic of the class of benzothiadiazines widely used in antihypertensive pharmaceutical formulations, alone or in combination with other drugs [13].

Literature survey revealed that Amlodipine besylate and Hydrochlorothiazide are official in the British Pharmacopoeia [14], Valsartan, Hydrochlorothiazide, and their mixture are official in the United States Pharmacopoeia [15]. There are reported methods for the determination of AML, VAL, or HCT in different dosage forms [2, 4, 16-19] and in their binary mixtures [20-22]. Few methods were reported for the simultaneous estimation of AML, VAL, and HCT in their ternary mixture [23-26] and only one spectrophotometric method was developed for the determination of this mixture [27]. In previous work, the authors developed derivative and chemometric methods for the analysis of the same mixture [28].

2. Experimental

2.1. Samples

2.1.1. Pure Samples

(a) Pure Amlodipine. It was kindly supplied by Al-Hekma pharmaceutical Company, Cairo, Egypt; its purity was certified to be 99.89 [+ or -] 0.691.

(b) Pure Valsartan. It was kindly supplied by Novartis pharmaceutical Company, Cairo, Egypt; its purity was certified to be 99.69 [+ or -] 0.231.

(c) Pure Hydrochlorothiazide. It was kindly supplied by Al-Hekma pharmaceutical Company, Cairo, Egypt; its purity was certified to be 99.78 [+ or -] 0.364.

2.1.2. Market Samples. Three EXFORGE HCT tablet dosage forms, labeled to contain 5(AML)/160(VAL)/12.5(HCT) mg batch number 5002125, 5/160/25 mg batch number 5002141, and 10/320/25 mg batch number 5002159, manufactured by Novartis Pharmaceuticals Corporation, USA. They were procured from the USA. market.

2.2. Reagents

Methanol. It was purchased from El-NASR, Egypt.

2.3. Instruments. SHIMADZU dual beam UV-visible spectrophotometer (Kyoto/Japan), model UV-1650 PC connected to IBM compatible and an HP1020 laserjet printer were used. The bundled software, UV-Probe personal spectroscopy software version 2.21 (SHIMADZU) was used. The spectral band was 2nm and scanning speed is 2800nm/min with 0.1 nm interval.

2.4. Procedures

2.4.1. Standard Solutions

(a) Standard stock solutions of AML, VAL, and HCT 1 mg/mL in methanol were used.

(b) Standard working solutions for AML and VAL 80 [micro]g/mL and for HCT 62.5 [micro]g/mL were prepared from stock solutions by appropriate dilutions with methanol.

2.4.2. Spectral Characteristics of AML, VAL, and HCT. The zero order ([D.sub.0]) absorption spectrum of AML, VAL, and HCT (20 [micro]g/mL for each) solutions was recorded against methanol as a blank over the range of 200-400 nm.

The zero order ([D.sub.0]) absorption spectrum of 16 [micro]g/mL VAL, 16 [micro]g/mL HCT, and a binary mixture of VAL and HCT (8 [micro]g/mL for each) were recorded against methanol as a blank over the range of 200 to 400 nm.

2.4.3. Construction of Calibration Curves. Aliquots equivalent to 40-320 [micro]g AML, 40-440 [micro]g VAL and 20-200 [micro]g HCT were accurately transferred from their standard working solutions into three separate series of 10-mL volumetric flasks then completed to volume with methanol. The spectra of the prepared standard solutions were scanned from 200-400 nm and stored in the computer.

For the determination of AML in presence of VAL and HCT, a calibration curve was constructed relating the absorbance of zero order spectra of AML at 359.4 nm to the corresponding concentration of AML and the regression equation was computed.

For the determination of HCT in presence of AML and VAL, a calibration curve was constructed relating the absorbance of zero order spectra of HCT at 316.4 nm to the corresponding concentration and the regression equation was computed.

For the determination of VAL in presence of AML and HCT, the isoabsorptive method was applied by measuring the absorbance of VAL and HCT at 256.8 nm ([A.sub.iso]). Two calibration curves relating the absorbance to the corresponding drug concentrations were constructed, and the corresponding regression equations were computed.

2.4.4. Analysis of AML, VAL, and HCT in Laboratory Prepared Mixtures (Specificity). Aliquots of AML, VAL, and HCT were transferred from their standard working solutions into a series of 10 mL measuring flasks, completed to volume with methanol to prepare mixtures containing different ratios of AML, VAL, and HCT. The spectra of these mixtures were scanned from 200 to 400 nm and stored in the computer.

For the determination of AML in presence of VAL and HCT, the same procedure under linearitywas applied and the concentration of AML was calculated from the corresponding regression equation.

For the determination of HCT in the presence of AML and VAL, the stored mixture spectra were divided by the spectrum of normalized AML, and the constant was subtracted from the ratio spectra followed by multiplication of the obtained spectra by the divisor. The result of these steps is the spectra ofVAL and HCT binary mixture. The absorbance values at 316.4 nm (where VAL shows no interference) were recorded and the concentration of HCT was calculated from the corresponding regression equation.

For the determination of VAL in presence of HCT, the absorbance at ([A.sub.iso]) in the binary mixture ofVAL and HCT (after removal of AML) was measured, and then the total content ofVAL and HCT was calculated from the corresponding regression equation. Subtraction of HCT concentration from the total concentration yields the actual concentration ofVAL in the mixture.

2.4.5. Analysis of AML, VAL, and HCT in Exforge HCT Tablets. Five tablets of each Exforge HCT formulation were accurately weighed and finely powdered. An amount of the powder equivalent to 8 mg VAL was weighed and dissolved in methanol by shaking in ultrasonic bath for about 30 minutes. The solutions were filtered into separate 100 mL measuring flasks, and the volume was completed with methanol. Five mL was transferred into 10 mL measuring flasks, suitable aliquots of AML and HCT were transferred from their standard working solutions for spiking the solution to reach concentrations of linearity range, and then volume was completed with methanol. The spectra of these solutions were scanned from 200 to 400 nm and stored in the computer. The same procedure under laboratory prepared mixtures was applied and the concentrations of AML, VAL, and HCT were calculated from the corresponding regression equations.

3. Results and Discussion

Sequential spectrophotometry depends on the presence of extended spectrum of one of the three drugs, so ratio subtraction technique can be used to remove this extended spectrum producing spectrum of the other two drugs. The second step depends on determination of the other two drugs by any spectrophotometric method that can resolve binary mixtures [6, 7, 29-33].

In this work, after direct determination of AML, we applied ratio subtraction to remove its extended spectrum producing a spectrum of HCT and VAL, where HCT was determined directly. Then the total concentration of HCT and VAL was calculated at their isoabsorptive point and VAL concentration was calculated by subtraction.

3.1. Determination of AML. The absorption spectra of the three compounds, AML, VAL, and HCT, show highly overlapped spectra in the region 200-300 nm (Figure 2). AML can be determined at 359.4 nm directly where VAL and HCT show no absorbance.

A linear correlation was obtained between the absorbance and the corresponding concentration of AML at 359.4 nm. The regression equation was calculated as follows:

[P.sub.AML] = 0.0162C +0.0010, r = 0.9998, (1)

where C is the concentration of AML in [micro]g/mL, P is the peak amplitude of the zero order spectrum of AML at 359.4 nm, and r is the correlation coefficient.

3.2. Determination of HCT. For the determination of both HCT and VAL, ratio subtraction technique was applied for removing AML spectrum.

The method depends on that when a mixture of VAL + HCT (X) and AML (Y) where the spectrum of (Y) is more extended (Figure 2), the determination of (X) could be done by scanning the zero order absorption spectra of the laboratory prepared mixtures (AML and VAL + HCT), dividing them by carefully chosen concentration of standard AML (Y' = divisor) producing a new ratio spectra that represent (X/Y') + constant as shown in Figure 3, then subtraction of the absorbance values of these constants (Y/Y') in plateau as shown in Figure 4, followed by multiplication of the obtained spectra by the divisor (Y'). Finally, the original spectra of (X) could be obtained Figure 5), which were used for direct determination of HCT at 316.4 nm, where VAL shows no absorbance (Figure 2), and calculation of the concentration from the corresponding regression equation was done.

The constant can be determined directly from the curve (X + Y)/Y' by the straight line which is parallel to the wavelength axis in the region where (Y) is extended.

The linearity was checked between absorbance at the selected wavelength 316.4 nm and the corresponding concentration of HCT. A linear correlation was obtained and the regression equation was found to be

[P.sub.HCT] = 0.0108C + 0.0030, r = 0.9998, (2)

where C is the concentration of HCT in [micro]g/mL, P is the peak amplitude of the zero order spectrum of HCT at 316.4 nm, and r is the correlation coefficient.

3.3. Determination of VAL. The absorbance spectra of 16 [micro]g/mL VAL, 16 [micro]g/mL of HCT, and a mixture containing equal concentrations of VAL and HCT (8 [micro]g/mL of each) showed isoabsorptive point at 256.8 nm (Figure 6).

By measuring the absorbance value at the chosen isoabsorptive point in the zero order absorption spectrum obtained from ratio subtraction method, the total content of VAL and HCT in the mixture can be calculated, while the content of HCT alone can be calculated using the zero order absorption spectrum obtained from ratio subtraction method without any interference from VAL. Thus the content of VAL can be calculated by subtraction.

By applying the isoabsorptive point, linear correlation was obtained between the absorbance values at 256.8 nm ([A.sub.iso]) and corresponding concentrations of VAL and HCT. The regression equations were computed as follows:

[P.sub.VAL] = 0.0258C + 0.0051, r = 0.9998, (3)

[P.sub.HCT] = 0.0254C-0.0031, r = 0.9993,

where C is the concentration of VAL and HCT in [micro]g/mL, P is the peak amplitude of the zero order spectrum of VAL and HCT at 256.8 nm, and r is the correlation coefficient.

The proposed method was found to be valid in the range of 4-32 [micro]g/mL, 4-44 [micro]g/mL, and 6-20 [micro]g/mL for AML, VAL and HCT, respectively, as shown by the small intercept and correlation coefficient approaching unity in the regression equations.

The specificity of the proposed method was assessed by the analysis of laboratory prepared mixtures containing different ratios of the drugs, where satisfactory results were obtained over the calibration range as shown in Table 1.

The proposed method was also applied for the determination of AML, VAL, and HCT in Exforge HCT tablets and the validity of the proposed method was further assessed by applying the standard addition technique as presented in Table 2.

Results obtained by the proposed method for the determination of the drugs in Exforge HCT tablets were statistically compared [34] to those obtained by the reported HPLC method [23]; no significant differences between the results were obtained as presented in Table 3. A validation sheet according to ICH guidelines was also presented in Table 4.

The method shows simplicity over our previous work on the same mixture [28], where the determination of the three drugs needs more steps of division and derivatization or difficult calculations as in chemometrics. Also, the method is preferred over chromatographic methods as no expensive solvents or sophisticated instruments are required.

4. Conclusion

From the previous discussion, it could be concluded that the new sequential spectrophotometry is simple, accurate, and specific and can be used for quantitation of ternary mixtures. The method was successfully applied for determination of a ternary mixture of AML, VAL, and HCT. The method is more rapid than chromatographic methods and does not need sample preparation or sophisticated techniques and instruments. It is also sensitive and selective and can be used for routine analysis of AML, VAL, and HCT in their pure powder and dosage forms.

Disclosure

The authors of the paper are academic staff in Cairo University and do not have a direct financial relationship with the commercial identities mentioned in the paper.

http://dx.doi.org/10.1155/2013/273102

References

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Correspondence should be addressed to Said A. Hassan; saidmonem84@yahoo.com

Received 27 February 2013; Accepted 21 August 2013

Academic Editor: Karol Jackowski

Hany W. Darwish, (1,2) Said A. Hassan, (1) Maissa Y. Salem, (1) and Badr A. El-Zeany (1)

(1) Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, KasrEl-Aini Street, Cairo 11562, Egypt

(2) Pharmaceutical Chemistry Department, College of Pharmacy, KingSaud University, PO. Box 2457, Riyadh 11451, Saudi Arabia

TABLE 1: Determination of AML, VAL, and HCT in laboratory
prepared mixtures by the proposed method.

Concentration              Proposed method
([micro]g/mL)               recovery % (a)

 AML    VAL    HCT     AML      VAL      HCT

4 (b)    40    6.25   101.01   101.38   97.78
 12      40    6.25   100.91   98.20    100.74
 20      40    6.25   99.53    100.04   100.74
 12      20    6.25   101.37   100.16   99.26
 12      4     6.25   101.55   97.34    100.74
 12      20    12.5   101.22   100.12   101.48
  4      4     12.5   98.26    99.82    100.74
        Mean          100.55   99.58    100.21
        SD            1.208    1.357    1.262
        RSD%          1.201    1.363    1.260

(a) Average of three determinations.

(b) The ratio of dosage form.

TABLE 2: Determination of AML, VAL, and HCT in Exforge HCT
tablets by the proposed method and the reported HPLC method [23]
and application of standard addition technique.

Product          Drug      Proposed method      Reported method (a)

Exforge          AML    99.91 [+ or -] 1.069    100.40 [+ or -] 0.636
HCT 5/160/12.5

                 VAL    99.50 [+ or -] 0.951    100.13 [+ or -] 0.884

                 HCT    99.83 [+ or -] 1.282    100.13 [+ or -] 0.729

Exforge          AML    101.01 [+ or -] 1.650   99.85 [+ or -] 0.970
HCT 5/160/25

                 VAL    100.27 [+ or -] 1.403   100.00 [+ or -] 0.859

                 HCT    99.26 [+ or -] 1.493    99.71 [+ or -] 0.693

Exforge          AML    100.20 [+ or -] 1.173   100.37 [+ or -] 0.712
HCT 10/320/25

                 VAL    99.93 [+ or -] 0.897    100.55 [+ or -] 0.595

                 HCT    99.75 [+ or -] 1.045    100.23 [+ or -] 0.825

Product          Drug          Standard addition

                            Taken           Added
                        ([micro]g/mL)   ([micro]g/mL)

Exforge          AML          6               4
HCT 5/160/12.5                                6
                                              8

                 VAL         16              12
                                             16
                                             20

                 HCT        9.375           6.25
                                            9.375
                                            12.5

Exforge          AML          6               4
HCT 5/160/25                                  6
                                              8

                 VAL         16              12
                                             16
                                             20

                 HCT        9.375           6.25

                                            9.375
                                            12.5

Exforge          AML          6               4
HCT 10/320/25                                 6
                                              8

                 VAL         16              12
                                             16
                                             20

                 HCT        9.375           6.25
                                            9.375
                                            12.5

Product          Drug               Standard addition

                              Found            Recovery % (b)
                          ([micro]g/mL)

Exforge          AML          3.994                 99.85
HCT 5/160/12.5                6.074                101.23
                              7.884                 98.55
                        Mean [+ or -] RSD   99.88 [+ or -] 1.344

                 VAL         12.259                102.16
                              16.14                100.88
                             20.175                100.88
                        Mean [+ or -] RSD   101.30 [+ or -] 0.730

                 HCT          6.329                101.27
                              9.319                 99.4
                             12.645                101.16
                        Mean [+ or -] RSD   100.61 [+ or -] 1.041

Exforge          AML          3.985                 99.61
HCT 5/160/25                  6.121                102.02
                              7.987                 99.84
                        Mean [+ or -] RSD   100.49 [+ or -] 1.326

                 VAL         11.935                 99.46
                             16.379                102.37
                             20.171                100.86
                        Mean [+ or -] RSD   100.89 [+ or -] 1.440

                 HCT          6.334                101.34
                              9.533                101.68
                             12.524                100.19
                        Mean [+ or -] RSD   101.07 [+ or -] 0.774

Exforge          AML          4.01                 100.24
HCT 10/320/25                 5.934                 98.91
                              8.038                100.47
                        Mean [+ or -] RSD   99.87 [+ or -] 0.846

                 VAL         12.101                100.84
                             15.949                 99.68
                             19.797                 98.99
                        Mean [+ or -] RSD   99.84 [+ or -] 0.939

                 HCT          6.344                 101.5
                              9.27                  98.88
                             12.761                102.09
                        Mean [+ or -] RSD   100.82 [+ or -] 1.695

(a) HPLC method using Luna [C.sub.18] column, a mobile phase
consisting of methanol/phosphate buffer (30 mM, pH 5.5) (62:38 by
volume) at a flow rate of 1 mL/min, and UV detection at 234 nm.

(b) Average of three determinations.

TABLE 3: Statistical comparison for the results obtained by the
proposed spectrophotometric method and the reported method [23]
for the analysis of AML, VAL, and HCT in Exforge HCT tablets.

Value                          Proposed method

                    AML             VAL             HCT

Mean              100.37           99.90           99.61
SD                 1.252           1.015           1.141
RSD%               1.247           1.016           1.145
n                    9               9               9
Variance           1.567           1.030           1.302
Student's      0.343 (2.16)    0.788 (2.131)   0.927 (2.16)
  t test (b)
F value (b)    2.921 (3.438)   1.933 (3.438)   2.710 (3.438)

Value             Reported method (a)

                AML      VAL      HCT

Mean           100.21   100.23   100.03
SD             0.733    0.730    0.693
RSD%           0.731    0.728    0.693
n                9        9        9
Variance       0.537    0.533    0.480
Student's        --       --       --
  t test (b)
F value (b)      --       --       --

(a) HPLC method using Luna [C.sub.18] column, a mobile phase
consisting of methanol-phosphate buffer (30 mM, pH 5.5) (62:38 by
volume) at a flow rate of 1 mL/min, and UV detection at 234 nm.

(b) The values in the parenthesis are the corresponding
theoretical values of t and F at P = 0.05.

TABLE 4: Assay validation sheet of the proposed spectrophotometric
method for the simultaneous determination of AML, VAL, and HCT.

Parameter                            Proposed method

                              AML                    VAL

Accuracy (mean        99.93 [+ or -] 1.188   99.66 [+ or -] 0.956
  [+ or -] RSD)
Precision
  Repeatability (a)          0.535                  0.651
  Intermediate               1.061                  1.225
    precision (b)
Robustness (c)               0.704                  0.633
Linearity
  Slope                      0.0162                 0.0258
  Intercept                  0.0010                 0.0051
  Correlation                0.9998                 0.9998
    coefficient (r)
  Range                       4-32                   4-44
    ([micro]g/mL)

Parameter                Proposed method

                               HCT

Accuracy (mean        100.24 [+ or -] 0.679
  [+ or -] RSD)
Precision
  Repeatability (a)           0.867
  Intermediate                1.002
    precision (b)
Robustness (c)                0.807
Linearity
  Slope                      0.0108
  Intercept                  0.0030
  Correlation                0.9998
    coefficient (r)
  Range                       6-20
    ([micro]g/mL)

(a) The intraday (n = 3) average of three concentrations (12, 16,
20 [micro]g/mL) for AML, VAL, and HCT repeated three times within
the day.

(b) The interday (n = 3) average of three concentrations (12, 16,
20 [micro]g/mL) for AML, VAL, and HCT repeated three times in
three days.

(c) Robustness (n = 3) average of three concentrations (12, 16,
20 [micro]g/mL) for AML, VAL, and HCT analyzed using 75 and 70%
methanol.
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Title Annotation:Research Article
Author:Darwish, Hany W.; Hassan, Said A.; Salem, Maissa Y.; Zeany, Badr A. El-
Publication:International Journal of Spectroscopy
Article Type:Report
Geographic Code:7EGYP
Date:Jan 1, 2014
Words:4639
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