# Simultaneous Estimation of Paracetamol, Ambroxol Hydrochloride, Levocetirizine Dihydrochloride, and Phenylephrine Hydrochloride in Combined Tablet Formulation by First-Order Derivative Spectrophotometry

Authors: K. Anandakumar; P. Veerasundari
Journal: ISRN Spectroscopy
 (2014)

---

## Abstract

Paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride are used in combination for the treatment of chronic sinusitis, rhinitis, fever, nasal discharge, sore throat, and wheezing. The present work deals with method development for simultaneous estimation of paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride in tablet formulation by first-order derivative spectrosphotometry. For determination of sampling wavelength, 10μg/mL of each of paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride was scanned in 200–400 nm ranges and sampling wavelengths were found to be 305.5 nm for paracetamol, 321 nm for ambroxol hydrochloride, 244 nm for levocetirizine dihydrochloride, and 280 nm for phenylephrine hydrochloride in first-order derivative spectrophotometry. In this method, linearity was observed in the ranges of 20–140 μg/mL for paracetamol and 10–70 μg/mL for ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride. The % recovery was within the range between 98 and 102%, and % relative standard deviation for precision and accuracy of the method was found to be less than 2%. The method is validated as per International Conference on Harmonization Guidelines. The method can be successfully applied for the simultaneous analysis of these drugs in pharmaceutical dosage forms.


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## Body

## 1. Introduction

Paracetamol (PARA), chemically known asN-(4-hydroxyphenyl)ethanamide (Figure 1(a)), inhibits the cyclooxygenase (COX) used as an analgesic, antipyretic, and nonnarcotic agent. Ambroxol hydrochloride (AMB), chemically known astrans-S-4-(2-Amino-3, 5-dibrombenzylamino)-cyclohexanol (Figure 1(b)), is an active N-desmethyl metabolite of the mucolytic bromhexine used as an oral mucolytic expectorant. Levocetirizine dihydrochloride (LEVO), chemically known as [2-[4-[(R)-(4-chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]-acetic acid dihydrochloride (Figure 1(c)), is L-enantiomer of cetirizine racemate. It works by blocking H1 histamine receptors; it is used for allergic rhinitis and conjunctivitis, hay fever, and pollinosis; it controls sneezing, runny but not blocked nose, and red, watering, and itchy eyes. Phenylephrine hydrochloride (PHEN), chemically known as (R)-3-hydroxy-alpha [(methylamino)methyl]benzenemethanol hydrochloride (Figure 1(d)), is a direct acting sympathomimetic agent. It is a selective α
1 adrenoceptor agonist and has negligible β action and is used as a nasal decongestant and for producing mydriasis when cycloplegia is not required. PARA, AMB, and PHEN are official in IP [1] and BP [2]. But LEVO is not official in any of the Pharmacopoeia.Chemical structure of (a) PARA, (b) AMB, (c) LEVO, and (d) PHEN.
(a)
(b)
(c)
(d)Literature survey revealed that there are several methods that have been reported for the estimation of these drugs in combination with other drugs by using UV spectrophotometry [3–12], RP-HPLC [13–36], HPTLC [36–39], and LC-MS [40, 41]. PARA, AMB, LEVO, and PHEN in combination are not official in any pharmacopoeia. As per literature, no analytical method could be traced for the analysis of PARA, AMB, LEVO, and PHEN in combined tablet dosage form. Therefore, simple, rapid, and reliable method for simultaneous estimation of these drugs in combination seemed to be necessary.Spectrophotometric methods of analysis are more economic and simpler, compared to methods such as chromatography and electrophoresis. Under computer-controlled instrumentation, derivative spectrophotometry is playing a very important role in the multicomponent analysis of mixtures by UV molecular absorption spectrophotometry. Quaternary mixture can be easily resolved by means of a spectrophotometric method, which is based on the simultaneous use of “zero crossing” method. The aim of this work was to investigate the utility of derivative spectrophotometry and to develop reliable spectrophotometric procedure for the simultaneous determination of PARA, AMB, LEVO, and PHEN in combined tablet dosage form without any prior separation of individual drugs. The presently developed method was validated as per International Conference on Harmonization guidelines (ICH) [42, 43].


## 2. Materials and Methods

### 2.1. Apparatus and Instrument

A double beam UV-Visible spectrophotometer (Shimadzu, model pharm spec 1700) having two matched quartz cells with 1 cm light path and electronic analytical balance (Shimadzu AUX-220) and ultrasonication (Soltec 2200) were used. Volumetric flasks and pipettes of borosilicate glasses were used in the study.


### 2.2. Chemicals and Reagents

Pure drug samples of PARA and LEVO were obtained as gift sample from Medopharm Pvt. Ltd., Chennai, Tamil Nadu, India; AMB was gifted by Madras Pharmaceuticals Pvt. Ltd., Chennai, Tamil Nadu, India; PHEN was gifted by APEX Pharmaceuticals, Chennai, Tamil Nadu, India. Methanol (HPLC grade) was purchased from Qualigens Pvt. Limited, Mumbai, India. Distilled water was obtained from the Double Distillation Unit in our laboratory.


### 2.3. Marketed Formulation

The marketed formulation studied was 1-AL total tablets manufactured by FDC Limited., Aurangabad, India. Each tablet contains 500 mg of PARA, 60 mg of AMB, 2.5 mg of LEVO, and 5 mg of PHEN.


### 2.4. Selection of Common Solvent

Methanol was selected as a common solvent to prepare the stock solution and further dilutions were made with distilled water for developing spectral characteristics of these drugs. The selection was made after assessing the solubility of these drugs in different solvents.


### 2.5. Preparation of Standard Solutions

50 mg of PARA and 25 mg of AMB, LEVO, and PHEN were weighed accurately, transferred into 25 mL volumetric flask individually, and dissolved with methanol and the volume was made up to 25 mL with methanol. The stock solution contains 2 mg/mL of PARA and 1 mg/mL of AMB, LEVO, and PHEN.


### 2.6. Spectrophotometric Conditions

(i)
Mode: spectrum
(ii)
Scan speed: medium
(iii)
Bandwidth: 1 nm
(iv)
Wavelength range: 400–200 nm
(v)
Absorbance scale: 0.00 A–2.15 A
(vi)
Initial baseline correction: distilled water.


## 2.1. Apparatus and Instrument

A double beam UV-Visible spectrophotometer (Shimadzu, model pharm spec 1700) having two matched quartz cells with 1 cm light path and electronic analytical balance (Shimadzu AUX-220) and ultrasonication (Soltec 2200) were used. Volumetric flasks and pipettes of borosilicate glasses were used in the study.


## 2.2. Chemicals and Reagents

Pure drug samples of PARA and LEVO were obtained as gift sample from Medopharm Pvt. Ltd., Chennai, Tamil Nadu, India; AMB was gifted by Madras Pharmaceuticals Pvt. Ltd., Chennai, Tamil Nadu, India; PHEN was gifted by APEX Pharmaceuticals, Chennai, Tamil Nadu, India. Methanol (HPLC grade) was purchased from Qualigens Pvt. Limited, Mumbai, India. Distilled water was obtained from the Double Distillation Unit in our laboratory.


## 2.3. Marketed Formulation

The marketed formulation studied was 1-AL total tablets manufactured by FDC Limited., Aurangabad, India. Each tablet contains 500 mg of PARA, 60 mg of AMB, 2.5 mg of LEVO, and 5 mg of PHEN.


## 2.4. Selection of Common Solvent

Methanol was selected as a common solvent to prepare the stock solution and further dilutions were made with distilled water for developing spectral characteristics of these drugs. The selection was made after assessing the solubility of these drugs in different solvents.


## 2.5. Preparation of Standard Solutions

50 mg of PARA and 25 mg of AMB, LEVO, and PHEN were weighed accurately, transferred into 25 mL volumetric flask individually, and dissolved with methanol and the volume was made up to 25 mL with methanol. The stock solution contains 2 mg/mL of PARA and 1 mg/mL of AMB, LEVO, and PHEN.


## 2.6. Spectrophotometric Conditions

(i)
Mode: spectrum
(ii)
Scan speed: medium
(iii)
Bandwidth: 1 nm
(iv)
Wavelength range: 400–200 nm
(v)
Absorbance scale: 0.00 A–2.15 A
(vi)
Initial baseline correction: distilled water.


## 3. First-Order Derivative Spectrophotometric Method

The standard stock solution of PARA, AMB, LEVO, and PHEN was appropriately diluted with distilled water to get the concentration 10μg/mL of all the four drugs. Spectra of these diluted solutions were scanned in the spectrum mode between 200 nm and 400 nm using distilled water as a blank. The zero-order spectra of PAR, AMB, LEVO, and PHEN were transformed to corresponding first derivative spectra in the range of 200–400 nm. The overlay spectra (zero- and first-order) of PARA, AMB, LEVO, and PHEN are shown in Figures 2 and 3.Figure 2
Overlain UV spectra of PARA, AMB, LEVO, and PHEN (10μg/mL concentration of each drug solution).Figure 3
Overlain first-order derivative UV spectra of PARA, AMB, LEVO, and PHEN (10μg/mL concentration of each drug solution).


### 3.1. Selection of Wavelengths

A signal at 305.5 nm of first-order derivative spectrum was selected for the quantification of PARA where no interference due to AMB, LEVO, and PHEN was observed. Similarly, 321 nm was selected for quantification of AMB where PARA, LEVO, and PHEN did not interfere with the estimation of AMB. At 244 nm, LEVO and PARA showed marked absorbance where as AMB and PHEN have zero crossing point. The absorbance of PARA was interfered in the analysis of LEVO. Hence, the absorbance of PARA was corrected for interference from the total absorbance value. With the help of corrected absorbance, the amount of LEVO was calculated at 244 nm. A signal at 280 nm PHEN, PARA, and LEVO showed marked absorbance and AMB has zero crossing point. The absorbance of PARA and LEVO was interfered in the analysis of PHEN. Hence, the absorbance of PARA and LEVO was corrected for interference from the total absorbance value. With the help of the corrected absorbance, the amount of PARA was calculated at 280 nm.


### 3.2. Calibration Curves for PARA, AMB, LEVO, and PHEN

The standard solutions of PARA (2 mg/mL), AMB (1 mg/mL), LEVO (1 mg/mL), and PHEN (1 mg/mL) were used to prepare the working standard solution of PARA (20–140μg/mL), AMB (10–70 μg/mL), LEVO (10–70 μg/mL), and PHEN (10–70 μg/mL), respectively. For, these aliquots of 1–7 mL of standard stock solutions of PARA, AMB, LEVO, and PHEN were transferred separately to a series of 100 mL volumetric flasks and diluted up to the mark with distilled water. The absorbance was measured at 305.5 nm for PARA, 321 nm for AMB, 244 nm for LEVO, and 280 nm for PHEN, respectively. The values of first derivative dA/dλ were plotted against corresponding concentrations to construct the calibration curves. The calibration curves are shown in Figure 4.Calibration curve for (a) PARA, (b) AMB, (c) LEVO, and (d) PHEN.
(a)
(b)
(c)
(d)


### 3.3. Analysis of Tablet Formulation

Marketed tablet formulation containing PARA (500 mg), AMB (60 mg), LEVO (2.5 mg), and PHEN (5 mg) was analyzed using this method. From the triturate of 10 tablets, the amount of tablet powder equivalent to 50 mg of PARA was accurately weighed and transferred into a series of 25 mL volumetric flasks. To that 4 mg/mL solution of AMB, 9.75 mg/mL solution of LEVO and 9.5 mg/mL solution of PHEN were added and dissolved with methanol and the solution was sonicated for 15 minutes. Then the final volume was made up to 25 mL with methanol. The solution was filtered through Whatmann filter paper Number 41. 3 mL of the stock solution was further diluted to 100 mL with distilled water to get the theoretical concentrations of 60μg/mL, 12 μg/mL, 12 μg/mL, and 12 μg/mL of PARA, AMB, LEVO, and PHEN, respectively. The absorbance values were measured at 305.5 nm, 321 nm, 244 nm, and 280 nm for PARA, AMB, LEVO, and PHEN, respectively. The concentration of each analyte was determined with the equations generated from calibration curve of respective drugs. The procedure was repeated for six times.


## 3.1. Selection of Wavelengths

A signal at 305.5 nm of first-order derivative spectrum was selected for the quantification of PARA where no interference due to AMB, LEVO, and PHEN was observed. Similarly, 321 nm was selected for quantification of AMB where PARA, LEVO, and PHEN did not interfere with the estimation of AMB. At 244 nm, LEVO and PARA showed marked absorbance where as AMB and PHEN have zero crossing point. The absorbance of PARA was interfered in the analysis of LEVO. Hence, the absorbance of PARA was corrected for interference from the total absorbance value. With the help of corrected absorbance, the amount of LEVO was calculated at 244 nm. A signal at 280 nm PHEN, PARA, and LEVO showed marked absorbance and AMB has zero crossing point. The absorbance of PARA and LEVO was interfered in the analysis of PHEN. Hence, the absorbance of PARA and LEVO was corrected for interference from the total absorbance value. With the help of the corrected absorbance, the amount of PARA was calculated at 280 nm.


## 3.2. Calibration Curves for PARA, AMB, LEVO, and PHEN

The standard solutions of PARA (2 mg/mL), AMB (1 mg/mL), LEVO (1 mg/mL), and PHEN (1 mg/mL) were used to prepare the working standard solution of PARA (20–140μg/mL), AMB (10–70 μg/mL), LEVO (10–70 μg/mL), and PHEN (10–70 μg/mL), respectively. For, these aliquots of 1–7 mL of standard stock solutions of PARA, AMB, LEVO, and PHEN were transferred separately to a series of 100 mL volumetric flasks and diluted up to the mark with distilled water. The absorbance was measured at 305.5 nm for PARA, 321 nm for AMB, 244 nm for LEVO, and 280 nm for PHEN, respectively. The values of first derivative dA/dλ were plotted against corresponding concentrations to construct the calibration curves. The calibration curves are shown in Figure 4.Calibration curve for (a) PARA, (b) AMB, (c) LEVO, and (d) PHEN.
(a)
(b)
(c)
(d)


## 3.3. Analysis of Tablet Formulation

Marketed tablet formulation containing PARA (500 mg), AMB (60 mg), LEVO (2.5 mg), and PHEN (5 mg) was analyzed using this method. From the triturate of 10 tablets, the amount of tablet powder equivalent to 50 mg of PARA was accurately weighed and transferred into a series of 25 mL volumetric flasks. To that 4 mg/mL solution of AMB, 9.75 mg/mL solution of LEVO and 9.5 mg/mL solution of PHEN were added and dissolved with methanol and the solution was sonicated for 15 minutes. Then the final volume was made up to 25 mL with methanol. The solution was filtered through Whatmann filter paper Number 41. 3 mL of the stock solution was further diluted to 100 mL with distilled water to get the theoretical concentrations of 60μg/mL, 12 μg/mL, 12 μg/mL, and 12 μg/mL of PARA, AMB, LEVO, and PHEN, respectively. The absorbance values were measured at 305.5 nm, 321 nm, 244 nm, and 280 nm for PARA, AMB, LEVO, and PHEN, respectively. The concentration of each analyte was determined with the equations generated from calibration curve of respective drugs. The procedure was repeated for six times.


## 4. Results and Discussion

### 4.1. Selectivity

The UV spectra of standard mixture (PARA—60μg/mL, AMB—12 μg/mL, LEVO—12 μg/mL, and PHEN—12 μg/mL) and sample solutions (tablet) were recorded between 200 and 400 nm and their dA/dλ value was measured. The selectivity of the method was assessed by comparing spectra obtained from formulation solution with that obtained from standard drug solution. The UV absorption spectra obtained from standard and sample solutions were found to be identical, confirming the selectivity of the method.


### 4.2. Linearity

Linear correlation was obtained between dA/dλ values versus concentrations of 20–140 μg/mL for PARA, 10–70 μg/mL for AMB, LEVO, and PHEN, respectively. Regression parameters are mentioned in Table 1.Table 1
Regression analysis data and summary of validation parameters for the proposed method.
Parameters
First derivative spectrophotometric method
PARA*
AMB*
LEVO*
PHEN*
Wavelength
305.5 nm
321 nm
244 nm
280 nm
Linearity range (μg/mL)
20–140
10–70
10–70
10–70
Slope (m)
0.00012
0.00026
0.00119
0.00051
Intercept (c)
−0.0002
−0.000064
0.000085
0.00031
Correlation coefficient (r
2)
0.9992
0.9990
0.9990
0.9995
LOD (μg/mL)
0.0352
0.0373
0.0645
0.0557
LOQ (μg/mL)
0.1070
0.1132
0.1954
0.1687
% Recovery
99.77–99.96
99.28–101.07
99.80–100.95
100.27–100.31
Precision ( RSD) %
Repeatability (n
=
6)
0.1661
1.9231
1.6141
1.1750
Interday (n
=
3)
0.0726
1.6244
0.0750
0.0763
Intraday (n
=
3)
0.4977
0.9453
0.0752
0.0765
*Mean of six observations.


### 4.3. Accuracy

Accuracy of the method was confirmed by recovery studies. Recovery studies were performed by standard addition method at three levels, namely, 80%, 100%, and 120%. Known amounts of pure PARA, AMB, LEVO, and PHEN were added to preanalyzed sample of marketed formulation, and they were subjected to analysis by the proposed method. The recovery was verified by estimation of drug in triplicate preparations at each specified concentration level and calculated %RSD. The recoveries were in the range of 99.77%–99.96%, 99.78%–101.07%, 99.80%–100.95%, and 100.27%–100.31% for PARA, AMB, LEVO, and PHEN, respectively. The low percentage RSD values indicated that there is no interference due to the excipients used in formulation. Hence the accuracy of the method was confirmed. The results of the recovery analysis are shown in Table2.Table 2
Recovery data for the proposed method.
Drug
Amount present (μg/mL)*
Amount added (μg/mL)*
Amount estimated (μg/mL)*
Amount recovered (μg/mL)*
Percentage recovered  (n
=
6)*
% RSD
PARA
59.6533
47.3221
107.8898
48.2365
101.93
0.2757
59.6533
60.0016
119.3033
59.6500
99.41
0.2891
59.6533
71.8727
131.4650
71.8117
99.92
0.1703
AMB
12.1440
9.6417
21.6966
9.5526
99.08
0.4154
12.1440
12.0892
24.2021
12.0581
99.74
0.3285
12.1440
14.3880
26.3979
14.2539
99.07
0.7261
LEVO
12.1176
9.5284
21.6460
9.5284
100.00
0.3518
12.1176
12.0916
23.9843
11.8667
98.14
0.3848
12.1176
14.3880
26.3683
14.2507
99.05
0.4868
PHEN
11.9592
9.5524
21.6182
9.6590
101.11
0.9930
11.9592
12.1304
24.2703
12.3111
101.49
0.6889
11.9592
14.3957
26.5306
14.5714
101.22
0.7734
*Mean of six observations. RSD: relative standard deviation.


### 4.4. Precision

#### 4.4.1. Repeatability

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solutions (n
=
6) of PARA (10 μg/mL), AMB (10 μg/mL), LEVO (10 μg/mL), and PHEN (10 μg/mL) without changing the parameters of the proposed method. The %RSD values for PARA, AMB, LEVO, and PHEN were found to be 0.6874, 1.8892, 0.6264, and 1.1750, respectively (Table 3). Low relative standard deviation (<2) indicates that the proposed method is repeatable.Table 3
Repeatability data for proposed method.
Serial number
Concentration (μg/mL)
Absorbance of PARA at 305.5 nm
Absorbance of AMB at 321 nm
Absorbance of LEVO at 244 nm
Absorbance of PHEN at 280 nm
1
10 ppm
0.0199
0.0027
0.0121
0.0054
2
10 ppm
0.0200
0.0028
0.0120
0.0053
3
10 ppm
0.0203
0.0027
0.0119
0.0052
4
10 ppm
0.0200
0.0027
0.0120
0.0052
5
10 ppm
0.0201
0.0028
0.0120
0.0051
6
10 ppm
0.0200
0.0027
0.0121
0.0053
Mean
0.0201
0.0027
0.0120
0.0052
SD
0.00014
0.000052
0.000075
0.0001
% RSD
0.6874
1.8892
0.6264
1.9977
SD: standard deviation; RSD: relative standard deviation.


#### 4.4.2. Intermediate Precision

Precision of both methods was determined in terms of intraday and interday variations (%RSD). Intraday precision (%RSD) was assessed by analyzing standard drug solutions within the calibration range, three times on the same day. Interday precision (%RSD) was assessed by analyzing drug solutions within the calibration range on three different days. The intraday and interday precisions were determined, results of which are given in Table4.Table 4
Intraday and interday precision data of PARA, AMB, LEVO and PHEN.
Drug
Amount   labeled (mg/tab)
Percentage obtained (n
=
6)
SD
% RSD
Intraday
Interday
Intraday
Interday
Intraday
Interday
PARA
500
99.47
99.60
500
98.61
99.48
0.4936
0.0723
0.4977
0.0726
500
99.46
99.61
Mean
99.18
99.56
AMB
60
98.56
97.85
60
100.44
100.83
0.9408
1.6197
0.9453
1.6244
60
99.57
100.44
Mean
99.52
99.72
LEVO
2.5
99.88
100.01
2.5
100.01
99.88
0.0750
0.0751
0.0752
0.0750
2.5
99.88
100.01
Mean
99.92
99.96
PHEN
5
98.21
98.34
5
98.34
98.22
0.0750
0.0752
0.0765
0.0763
5
98.21
98.34
Mean
98.25
98.29
SD: standard deviation; RSD: relative standard deviation; CI: confidence interval; SE: standard error.


### 4.5. LOD and LOQ

LOD and LOQ of the drug were calculated as per ICH guideline. LOD values for PARA, AMB, LEVO, and PHEN were found to be 0.0352μg/mL, 0.0373 μg/mL, 0.0645 μg/mL, and 0.0557 μg/mL, respectively. LOQ values for PARA, AMB, LEVO, and PHEN were found to be 0.1070 μg/mL, 0.1132 μg/mL, 0.1954 μg/mL and 0.1687 μg/mL, respectively (Table 1). These data show that the proposed method is sensitive for the determination of PARA, AMB, LEVO, and PHEN.


### 4.6. Analysis of PARA, AMB, LEVO, and PHEN in Marketed Formulation

Content of PARA, AMB, LEVO, and PHEN found in the marketed formulation from the proposed method is shown in Table5. The percentage purity was 99.62% ± 0.1655 for PARA, 101.02% ± 1.9427 for AMB, 100.98% ± 1.6300 for LEVO, and 99.66% ± 1.1711 for PHEN.Table 5
Assay results of marketed formulation.
Parameters
PARA
AMB
LEVO
PHEN
Actual concentration (μg/mL)
60
12
12
12
Percentage purity
99.62
101.02
100.98
99.66
% RSD
0.1661
1.9231
1.6141
1.1750


## 4.1. Selectivity

The UV spectra of standard mixture (PARA—60μg/mL, AMB—12 μg/mL, LEVO—12 μg/mL, and PHEN—12 μg/mL) and sample solutions (tablet) were recorded between 200 and 400 nm and their dA/dλ value was measured. The selectivity of the method was assessed by comparing spectra obtained from formulation solution with that obtained from standard drug solution. The UV absorption spectra obtained from standard and sample solutions were found to be identical, confirming the selectivity of the method.


## 4.2. Linearity

Linear correlation was obtained between dA/dλ values versus concentrations of 20–140 μg/mL for PARA, 10–70 μg/mL for AMB, LEVO, and PHEN, respectively. Regression parameters are mentioned in Table 1.Table 1
Regression analysis data and summary of validation parameters for the proposed method.
Parameters
First derivative spectrophotometric method
PARA*
AMB*
LEVO*
PHEN*
Wavelength
305.5 nm
321 nm
244 nm
280 nm
Linearity range (μg/mL)
20–140
10–70
10–70
10–70
Slope (m)
0.00012
0.00026
0.00119
0.00051
Intercept (c)
−0.0002
−0.000064
0.000085
0.00031
Correlation coefficient (r
2)
0.9992
0.9990
0.9990
0.9995
LOD (μg/mL)
0.0352
0.0373
0.0645
0.0557
LOQ (μg/mL)
0.1070
0.1132
0.1954
0.1687
% Recovery
99.77–99.96
99.28–101.07
99.80–100.95
100.27–100.31
Precision ( RSD) %
Repeatability (n
=
6)
0.1661
1.9231
1.6141
1.1750
Interday (n
=
3)
0.0726
1.6244
0.0750
0.0763
Intraday (n
=
3)
0.4977
0.9453
0.0752
0.0765
*Mean of six observations.


## 4.3. Accuracy

Accuracy of the method was confirmed by recovery studies. Recovery studies were performed by standard addition method at three levels, namely, 80%, 100%, and 120%. Known amounts of pure PARA, AMB, LEVO, and PHEN were added to preanalyzed sample of marketed formulation, and they were subjected to analysis by the proposed method. The recovery was verified by estimation of drug in triplicate preparations at each specified concentration level and calculated %RSD. The recoveries were in the range of 99.77%–99.96%, 99.78%–101.07%, 99.80%–100.95%, and 100.27%–100.31% for PARA, AMB, LEVO, and PHEN, respectively. The low percentage RSD values indicated that there is no interference due to the excipients used in formulation. Hence the accuracy of the method was confirmed. The results of the recovery analysis are shown in Table2.Table 2
Recovery data for the proposed method.
Drug
Amount present (μg/mL)*
Amount added (μg/mL)*
Amount estimated (μg/mL)*
Amount recovered (μg/mL)*
Percentage recovered  (n
=
6)*
% RSD
PARA
59.6533
47.3221
107.8898
48.2365
101.93
0.2757
59.6533
60.0016
119.3033
59.6500
99.41
0.2891
59.6533
71.8727
131.4650
71.8117
99.92
0.1703
AMB
12.1440
9.6417
21.6966
9.5526
99.08
0.4154
12.1440
12.0892
24.2021
12.0581
99.74
0.3285
12.1440
14.3880
26.3979
14.2539
99.07
0.7261
LEVO
12.1176
9.5284
21.6460
9.5284
100.00
0.3518
12.1176
12.0916
23.9843
11.8667
98.14
0.3848
12.1176
14.3880
26.3683
14.2507
99.05
0.4868
PHEN
11.9592
9.5524
21.6182
9.6590
101.11
0.9930
11.9592
12.1304
24.2703
12.3111
101.49
0.6889
11.9592
14.3957
26.5306
14.5714
101.22
0.7734
*Mean of six observations. RSD: relative standard deviation.


## 4.4. Precision

### 4.4.1. Repeatability

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solutions (n
=
6) of PARA (10 μg/mL), AMB (10 μg/mL), LEVO (10 μg/mL), and PHEN (10 μg/mL) without changing the parameters of the proposed method. The %RSD values for PARA, AMB, LEVO, and PHEN were found to be 0.6874, 1.8892, 0.6264, and 1.1750, respectively (Table 3). Low relative standard deviation (<2) indicates that the proposed method is repeatable.Table 3
Repeatability data for proposed method.
Serial number
Concentration (μg/mL)
Absorbance of PARA at 305.5 nm
Absorbance of AMB at 321 nm
Absorbance of LEVO at 244 nm
Absorbance of PHEN at 280 nm
1
10 ppm
0.0199
0.0027
0.0121
0.0054
2
10 ppm
0.0200
0.0028
0.0120
0.0053
3
10 ppm
0.0203
0.0027
0.0119
0.0052
4
10 ppm
0.0200
0.0027
0.0120
0.0052
5
10 ppm
0.0201
0.0028
0.0120
0.0051
6
10 ppm
0.0200
0.0027
0.0121
0.0053
Mean
0.0201
0.0027
0.0120
0.0052
SD
0.00014
0.000052
0.000075
0.0001
% RSD
0.6874
1.8892
0.6264
1.9977
SD: standard deviation; RSD: relative standard deviation.


### 4.4.2. Intermediate Precision

Precision of both methods was determined in terms of intraday and interday variations (%RSD). Intraday precision (%RSD) was assessed by analyzing standard drug solutions within the calibration range, three times on the same day. Interday precision (%RSD) was assessed by analyzing drug solutions within the calibration range on three different days. The intraday and interday precisions were determined, results of which are given in Table4.Table 4
Intraday and interday precision data of PARA, AMB, LEVO and PHEN.
Drug
Amount   labeled (mg/tab)
Percentage obtained (n
=
6)
SD
% RSD
Intraday
Interday
Intraday
Interday
Intraday
Interday
PARA
500
99.47
99.60
500
98.61
99.48
0.4936
0.0723
0.4977
0.0726
500
99.46
99.61
Mean
99.18
99.56
AMB
60
98.56
97.85
60
100.44
100.83
0.9408
1.6197
0.9453
1.6244
60
99.57
100.44
Mean
99.52
99.72
LEVO
2.5
99.88
100.01
2.5
100.01
99.88
0.0750
0.0751
0.0752
0.0750
2.5
99.88
100.01
Mean
99.92
99.96
PHEN
5
98.21
98.34
5
98.34
98.22
0.0750
0.0752
0.0765
0.0763
5
98.21
98.34
Mean
98.25
98.29
SD: standard deviation; RSD: relative standard deviation; CI: confidence interval; SE: standard error.


## 4.4.1. Repeatability

The precision of the instrument was checked by repeated scanning and measurement of the absorbance of solutions (n
=
6) of PARA (10 μg/mL), AMB (10 μg/mL), LEVO (10 μg/mL), and PHEN (10 μg/mL) without changing the parameters of the proposed method. The %RSD values for PARA, AMB, LEVO, and PHEN were found to be 0.6874, 1.8892, 0.6264, and 1.1750, respectively (Table 3). Low relative standard deviation (<2) indicates that the proposed method is repeatable.Table 3
Repeatability data for proposed method.
Serial number
Concentration (μg/mL)
Absorbance of PARA at 305.5 nm
Absorbance of AMB at 321 nm
Absorbance of LEVO at 244 nm
Absorbance of PHEN at 280 nm
1
10 ppm
0.0199
0.0027
0.0121
0.0054
2
10 ppm
0.0200
0.0028
0.0120
0.0053
3
10 ppm
0.0203
0.0027
0.0119
0.0052
4
10 ppm
0.0200
0.0027
0.0120
0.0052
5
10 ppm
0.0201
0.0028
0.0120
0.0051
6
10 ppm
0.0200
0.0027
0.0121
0.0053
Mean
0.0201
0.0027
0.0120
0.0052
SD
0.00014
0.000052
0.000075
0.0001
% RSD
0.6874
1.8892
0.6264
1.9977
SD: standard deviation; RSD: relative standard deviation.


## 4.4.2. Intermediate Precision

Precision of both methods was determined in terms of intraday and interday variations (%RSD). Intraday precision (%RSD) was assessed by analyzing standard drug solutions within the calibration range, three times on the same day. Interday precision (%RSD) was assessed by analyzing drug solutions within the calibration range on three different days. The intraday and interday precisions were determined, results of which are given in Table4.Table 4
Intraday and interday precision data of PARA, AMB, LEVO and PHEN.
Drug
Amount   labeled (mg/tab)
Percentage obtained (n
=
6)
SD
% RSD
Intraday
Interday
Intraday
Interday
Intraday
Interday
PARA
500
99.47
99.60
500
98.61
99.48
0.4936
0.0723
0.4977
0.0726
500
99.46
99.61
Mean
99.18
99.56
AMB
60
98.56
97.85
60
100.44
100.83
0.9408
1.6197
0.9453
1.6244
60
99.57
100.44
Mean
99.52
99.72
LEVO
2.5
99.88
100.01
2.5
100.01
99.88
0.0750
0.0751
0.0752
0.0750
2.5
99.88
100.01
Mean
99.92
99.96
PHEN
5
98.21
98.34
5
98.34
98.22
0.0750
0.0752
0.0765
0.0763
5
98.21
98.34
Mean
98.25
98.29
SD: standard deviation; RSD: relative standard deviation; CI: confidence interval; SE: standard error.


## 4.5. LOD and LOQ

LOD and LOQ of the drug were calculated as per ICH guideline. LOD values for PARA, AMB, LEVO, and PHEN were found to be 0.0352μg/mL, 0.0373 μg/mL, 0.0645 μg/mL, and 0.0557 μg/mL, respectively. LOQ values for PARA, AMB, LEVO, and PHEN were found to be 0.1070 μg/mL, 0.1132 μg/mL, 0.1954 μg/mL and 0.1687 μg/mL, respectively (Table 1). These data show that the proposed method is sensitive for the determination of PARA, AMB, LEVO, and PHEN.


## 4.6. Analysis of PARA, AMB, LEVO, and PHEN in Marketed Formulation

Content of PARA, AMB, LEVO, and PHEN found in the marketed formulation from the proposed method is shown in Table5. The percentage purity was 99.62% ± 0.1655 for PARA, 101.02% ± 1.9427 for AMB, 100.98% ± 1.6300 for LEVO, and 99.66% ± 1.1711 for PHEN.Table 5
Assay results of marketed formulation.
Parameters
PARA
AMB
LEVO
PHEN
Actual concentration (μg/mL)
60
12
12
12
Percentage purity
99.62
101.02
100.98
99.66
% RSD
0.1661
1.9231
1.6141
1.1750


## 5. Conclusion

In this proposed method, the linearity was observed in the concentration ranges of 20–140μg/mL, 10–70 μg/mL, 10–70 μg/mL, and 10–70 μg/mL while coefficients of correlation were r
2
=
0.9992, r
2
=
0.9990, r
2
=
0.9990, and r
2
=
0.9995 for PARA, AMB, LEVO, and PHEN at 305.5 nm, 321 nm, 244 nm, and 280 nm, respectively. The result of the analysis of combined mixture by the proposed method was found to be highly reproducible and reliable. The additive present in the combined mixture of the assayed samples did not interfere with determination of PARA, AMB, LEVO, and PHEN. So, the developed first derivative UV spectrophotometric method is simple, precise, accurate, and reproducible and can be effectively used for the simultaneous determination of PARA, AMB, LEVO, and PHEN in combined tablet dosage form.



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*Source: 248960-2014-03-30.xml*