# Selective Spectrophotometric and Spectrofluorometric Methods for the Determination of Amantadine Hydrochloride in Capsules and Plasma  via Derivatization with 1,2-Naphthoquinone-4-sulphonate

Authors: Ashraf M. Mahmoud; Nasr Y. Khalil; Ibrahim A. Darwish; Tarek Aboul-Fadl
Journal: International Journal of Analytical Chemistry
 (2009)

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## Abstract

New selective and sensitive spectrophotometric and spectrofluorometric methods have been developed and validated for the determination of amantadine hydrochloride (AMD) in capsules and plasma. The methods were based on the condensation of AMD with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product. The spectrophotometric method involved the measurement of the colored product at 460  nm. The spectrofluorometric method involved the reduction of the product with potassium borohydride, and the subsequent measurement of the formed fluorescent reduced AMD-NQS product at 382  nm after excitation at 293  nm. The variables that affected the reaction were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9972–0.9974) and low LOD (1.39 and 0.013μg mL−1) were obtained in the ranges of 5–80 and 0.05–10
μg mL−1 for the spectrophotometric and spectrofluorometric methods, respectively. The precisions of the methods were satisfactory; RSD
≤2.04%. Both methods were successfully applied to the determination of AMD in capsules. As its higher sensitivity, the spectrofluorometric method was applied to the determination of AMD in plasma; the recovery was  96.3–101.2±0.57–4.2%. The results obtained by the proposed methods were comparable with those obtained by the official method


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## Body

## 1. Introduction

Amantadine hydrochloride (AMD), Scheme1, is an antiviral
agent used against infection with influenza type A virus and to ameliorate
symptoms when administered during the early stages of infection as well as in
the management of herpes zoster [1]. It has mild anti-Parkinsonism activity and thus it has been
used in the management of Parkinsonism, mainly in the early disease stage and
when the symptoms are mild. AMD is usually given by mouth as the hydrochloride
salt [2].Scheme 1Spectrophotometry and spectrofluorometry are
considered as the most convenient analytical techniques in  pharmaceutical
analysis because of their inherent simplicity and availability in most quality
control and clinical laboratories [3–9]. However, AMD
does not possess any chromophore or fluorophore in its molecule, which are the
essential requirements for the direct analysis by either spectrophotometric or spectrofluorometric
techniques. Therefore, derivatization of AMD was necessary for its
determination by either of the two techniques. For spectrophotometric determination
of AMD, it has been derivatized with different reagents. The involved derivatization
reactions that have been published prior to 1983 have been reviewed by
Kirschbaum [10]. The derivatizing reagents used thereafter included iodine [11], acetaldehyde/chloranil [11], α,α-diphenyl-β-picrylhydrazyl [12], bromocresol green [13], tetracyanoethylene
[14], iron(III) [15], and cyclodextrin [16]. Few spectrofluorometric methods have
been reported for the analysis of AMD. These methods were based on its oxidation
with cerium(IV) [7] or its derivatization with 2,3-diphenylquinolizinium
bromide [17], fluorescamine [18], and 9-isothiocynatoacridine [19]. As well, many derivatization techniques coupled with
chromatography have been established for the determination of AMD in the dosage
forms and biological matrices: TLC [20], HPLC [21–23], GC [24], and
capillary electrophoresis [25]. 1,2-Naphthoquinone-4-sulphonate (NQS) has been used for the determination
of many compounds [26–28]. The reaction
between NQS and AMD has not been investigated yet. Therefore, the present study was
devoted to explore NQS as a derivatizing reagent in the development of
selective and sensitive spectrophotometric and spectrofluorometric methods for
the determination of AMD in capsules and plasma.


## 2. Experimental

### 2.1. Apparatus

UV-1601 PC
(Shimadzu, Kyoto, Japan) ultraviolet-visible
spectrophotometer with matched 1 cm quartz cells  was used for all spectrophotometric
measurements. Spectrofluorimeter, Kontron SFM 25 equipped with a 150 W xenon
high-pressure lamp was used for measuring the fluorescence intensity. MLW type
thermostatically controlled water bath (Memmert GmbH, Co. Schwa bach, Germany).
Biofuge Pico centrifuge (Heraeus Instruments, Germany).


### 2.2. Chemicals and Materials

Amantadine
hydrochloride (AMD; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was obtained and used as received, its purity was100.02±1.25%. 1,2-naphthoquinone-4-sulphonate; (NQS; El-Nasr Pharmaceutical
Chemical Co., Abo-Zaabal, Egypt). Potassium borohydride (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany).
Adamine capsules (Rameda Co. for Pharmaceutical Industries & Diagnostic
Reagents, Cairo, Egypt) are labeled to contain 100 mg of AMD per capsule. Human plasma samples were collected from normal
healthy volunteer at King Khaled University Hospital (Riyadh,
Kingdom of Saudi Arabia), and they were stored at −20°C until
analysis. All solvents and materials used throughout
this study were of analytical grade. Double
distilled water was obtained through WSC-85 water purification system (Hamilton
Laboratory Glass Ltd., Ky, USA),
and used throughout the work.


### 2.3. Preparation of Standard and Sample Solutions

#### 2.3.1. Amantadine Hydrochloride (AMD) Standard Solutions

An accurately weighed amount (100 mg) of AMD
was quantitatively transferred into a 50 mL calibrated
flask, dissolved in 30 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mgmL−1. This stock solution was
further diluted with water to obtain working
solutions in the ranges of 50–800 and 0.5–100 μgmL−1 for the spectrophotometric and spectrofluorometric
methods, respectively.


#### 2.3.2. 1,2-Naphthoquinone-4-Sulphonate (NQS) Derivatizing Reagent

Accurately weighed amount of NQS (150 mg) was transferred into a 25 mL
calibrated flask, dissolved in 5 mL distilled water, completed to volume with water
to obtain a solution of 0.6% (w/v). The solution was freshly prepared and
protected from light during use.


#### 2.3.3. Capsules Sample Solution

Twenty capsules were carefully evacuated; their
contents were weighed and finely powdered. An accurately weighed quantity of
the capsule contents equivalent to 100 mg of AMD was transferred into a 100 mL
calibrated flask, and dissolved in about 40 mL of distilled water. The contents
of the flask were swirled, sonicated for 5 minutes, and then completed to
volume with water. The contents were mixed well and filtered rejecting the
first portion of the filtrate. The prepared solution was diluted quantitatively
with distilled water to obtain a suitable concentration for the analysis.


#### 2.3.4. Spiked Plasma Samples

Aliquots of 1.0 mL of plasma were spiked with different
concentration levels of AMD. The spiked plasma samples were treated with 0.1 mL
of 70% perchloric acid and vortexed for 1 minute. The samples were centrifuged
for 20 minutes at 13000 rpm. The supernatants were transferred into test tubes
and neutralized with 1 M NaOH solution.


### 2.4. General Recommended Procedure

#### 2.4.1. Spectrophotometric Method

One milliliter of the standard or sample
solution (50–800μg mL−1) was transferred into a test tube. One
milliliter of 0.01 M NaOH and 1 ml of NQS reagent (0.6%, w/v) were added. The
contents of the tubes were heated in a water bath at 80±5°C for 45 minutes
and then cooled in ice water for 2 minutes. The contents of the test tubes were
transferred quantitatively into a separating funnel containing anhydrous sodium
sulphate and extracted with two portions (5 mL) of chloroform. The combined
chloroformic extracts were transferred into 10 mL calibrated flasks and the
solutions were completed to volume with chloroform if necessary. The
absorbances of the resulting solutions were measured at 460 nm against reagent
blanks treated similarly.


#### 2.4.2. Spectrofluorometric Method

One milliliter of the standard or sample solution
(0.5–100μg mL−1) was transferred into a test tube. One milliliter
of 0.01 M NaOH and 1 mL of NQS reagent (0.6%, w/v) were added. The contents of the test tubes
were heated in a water bath at 80±5°C for 45 minutes and then cooled in ice
water for 2 minutes. The contents of the test tubes were transferred
quantitatively into a separating funnel and extracted with two portions (5 mL) of
chloroform. The combined chloroformic extracts were evaporated under stream of
air and the residues were reconstituted in 2 mL of methanol and quantitatively transferred
into 10 mL calibrated flask. A one milliliter of KBH4 solution (0.03%, w/v in methanol) was added and the reaction was allowed to
proceed for 5 minutes at room temperature (25±5°C). The solution was diluted
to volume with 0.025 M ethanolic HCl and the fluorescence intensity of the
resulting solution was measured at 382 nm after excitation at 293 nm against
reagent blanks treated similarly.


### 2.5. Determination of the Molar Ratio of the Reaction

The Job’s method of continuous variation [29] was employed. Master
equimolar (2.5×10−2 M) aqueous solutions of AMD and
NQS were prepared. Series of 5 mL portions of
the master solutions of AMD and NQS were made up comprising different
complementary proportions (0:10, 1:9, …, 9:1, 10:0, inclusive) in test tubes. One
milliliter of 0.01 M NaOH was added to each tube, and the tubes were further
manipulated as described under the general recommended procedure for the
spectrophotometric method.


## 2.1. Apparatus

UV-1601 PC
(Shimadzu, Kyoto, Japan) ultraviolet-visible
spectrophotometer with matched 1 cm quartz cells  was used for all spectrophotometric
measurements. Spectrofluorimeter, Kontron SFM 25 equipped with a 150 W xenon
high-pressure lamp was used for measuring the fluorescence intensity. MLW type
thermostatically controlled water bath (Memmert GmbH, Co. Schwa bach, Germany).
Biofuge Pico centrifuge (Heraeus Instruments, Germany).


## 2.2. Chemicals and Materials

Amantadine
hydrochloride (AMD; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was obtained and used as received, its purity was100.02±1.25%. 1,2-naphthoquinone-4-sulphonate; (NQS; El-Nasr Pharmaceutical
Chemical Co., Abo-Zaabal, Egypt). Potassium borohydride (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany).
Adamine capsules (Rameda Co. for Pharmaceutical Industries & Diagnostic
Reagents, Cairo, Egypt) are labeled to contain 100 mg of AMD per capsule. Human plasma samples were collected from normal
healthy volunteer at King Khaled University Hospital (Riyadh,
Kingdom of Saudi Arabia), and they were stored at −20°C until
analysis. All solvents and materials used throughout
this study were of analytical grade. Double
distilled water was obtained through WSC-85 water purification system (Hamilton
Laboratory Glass Ltd., Ky, USA),
and used throughout the work.


## 2.3. Preparation of Standard and Sample Solutions

### 2.3.1. Amantadine Hydrochloride (AMD) Standard Solutions

An accurately weighed amount (100 mg) of AMD
was quantitatively transferred into a 50 mL calibrated
flask, dissolved in 30 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mgmL−1. This stock solution was
further diluted with water to obtain working
solutions in the ranges of 50–800 and 0.5–100 μgmL−1 for the spectrophotometric and spectrofluorometric
methods, respectively.


### 2.3.2. 1,2-Naphthoquinone-4-Sulphonate (NQS) Derivatizing Reagent

Accurately weighed amount of NQS (150 mg) was transferred into a 25 mL
calibrated flask, dissolved in 5 mL distilled water, completed to volume with water
to obtain a solution of 0.6% (w/v). The solution was freshly prepared and
protected from light during use.


### 2.3.3. Capsules Sample Solution

Twenty capsules were carefully evacuated; their
contents were weighed and finely powdered. An accurately weighed quantity of
the capsule contents equivalent to 100 mg of AMD was transferred into a 100 mL
calibrated flask, and dissolved in about 40 mL of distilled water. The contents
of the flask were swirled, sonicated for 5 minutes, and then completed to
volume with water. The contents were mixed well and filtered rejecting the
first portion of the filtrate. The prepared solution was diluted quantitatively
with distilled water to obtain a suitable concentration for the analysis.


### 2.3.4. Spiked Plasma Samples

Aliquots of 1.0 mL of plasma were spiked with different
concentration levels of AMD. The spiked plasma samples were treated with 0.1 mL
of 70% perchloric acid and vortexed for 1 minute. The samples were centrifuged
for 20 minutes at 13000 rpm. The supernatants were transferred into test tubes
and neutralized with 1 M NaOH solution.


## 2.3.1. Amantadine Hydrochloride (AMD) Standard Solutions

An accurately weighed amount (100 mg) of AMD
was quantitatively transferred into a 50 mL calibrated
flask, dissolved in 30 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mgmL−1. This stock solution was
further diluted with water to obtain working
solutions in the ranges of 50–800 and 0.5–100 μgmL−1 for the spectrophotometric and spectrofluorometric
methods, respectively.


## 2.3.2. 1,2-Naphthoquinone-4-Sulphonate (NQS) Derivatizing Reagent

Accurately weighed amount of NQS (150 mg) was transferred into a 25 mL
calibrated flask, dissolved in 5 mL distilled water, completed to volume with water
to obtain a solution of 0.6% (w/v). The solution was freshly prepared and
protected from light during use.


## 2.3.3. Capsules Sample Solution

Twenty capsules were carefully evacuated; their
contents were weighed and finely powdered. An accurately weighed quantity of
the capsule contents equivalent to 100 mg of AMD was transferred into a 100 mL
calibrated flask, and dissolved in about 40 mL of distilled water. The contents
of the flask were swirled, sonicated for 5 minutes, and then completed to
volume with water. The contents were mixed well and filtered rejecting the
first portion of the filtrate. The prepared solution was diluted quantitatively
with distilled water to obtain a suitable concentration for the analysis.


## 2.3.4. Spiked Plasma Samples

Aliquots of 1.0 mL of plasma were spiked with different
concentration levels of AMD. The spiked plasma samples were treated with 0.1 mL
of 70% perchloric acid and vortexed for 1 minute. The samples were centrifuged
for 20 minutes at 13000 rpm. The supernatants were transferred into test tubes
and neutralized with 1 M NaOH solution.


## 2.4. General Recommended Procedure

### 2.4.1. Spectrophotometric Method

One milliliter of the standard or sample
solution (50–800μg mL−1) was transferred into a test tube. One
milliliter of 0.01 M NaOH and 1 ml of NQS reagent (0.6%, w/v) were added. The
contents of the tubes were heated in a water bath at 80±5°C for 45 minutes
and then cooled in ice water for 2 minutes. The contents of the test tubes were
transferred quantitatively into a separating funnel containing anhydrous sodium
sulphate and extracted with two portions (5 mL) of chloroform. The combined
chloroformic extracts were transferred into 10 mL calibrated flasks and the
solutions were completed to volume with chloroform if necessary. The
absorbances of the resulting solutions were measured at 460 nm against reagent
blanks treated similarly.


### 2.4.2. Spectrofluorometric Method

One milliliter of the standard or sample solution
(0.5–100μg mL−1) was transferred into a test tube. One milliliter
of 0.01 M NaOH and 1 mL of NQS reagent (0.6%, w/v) were added. The contents of the test tubes
were heated in a water bath at 80±5°C for 45 minutes and then cooled in ice
water for 2 minutes. The contents of the test tubes were transferred
quantitatively into a separating funnel and extracted with two portions (5 mL) of
chloroform. The combined chloroformic extracts were evaporated under stream of
air and the residues were reconstituted in 2 mL of methanol and quantitatively transferred
into 10 mL calibrated flask. A one milliliter of KBH4 solution (0.03%, w/v in methanol) was added and the reaction was allowed to
proceed for 5 minutes at room temperature (25±5°C). The solution was diluted
to volume with 0.025 M ethanolic HCl and the fluorescence intensity of the
resulting solution was measured at 382 nm after excitation at 293 nm against
reagent blanks treated similarly.


## 2.4.1. Spectrophotometric Method

One milliliter of the standard or sample
solution (50–800μg mL−1) was transferred into a test tube. One
milliliter of 0.01 M NaOH and 1 ml of NQS reagent (0.6%, w/v) were added. The
contents of the tubes were heated in a water bath at 80±5°C for 45 minutes
and then cooled in ice water for 2 minutes. The contents of the test tubes were
transferred quantitatively into a separating funnel containing anhydrous sodium
sulphate and extracted with two portions (5 mL) of chloroform. The combined
chloroformic extracts were transferred into 10 mL calibrated flasks and the
solutions were completed to volume with chloroform if necessary. The
absorbances of the resulting solutions were measured at 460 nm against reagent
blanks treated similarly.


## 2.4.2. Spectrofluorometric Method

One milliliter of the standard or sample solution
(0.5–100μg mL−1) was transferred into a test tube. One milliliter
of 0.01 M NaOH and 1 mL of NQS reagent (0.6%, w/v) were added. The contents of the test tubes
were heated in a water bath at 80±5°C for 45 minutes and then cooled in ice
water for 2 minutes. The contents of the test tubes were transferred
quantitatively into a separating funnel and extracted with two portions (5 mL) of
chloroform. The combined chloroformic extracts were evaporated under stream of
air and the residues were reconstituted in 2 mL of methanol and quantitatively transferred
into 10 mL calibrated flask. A one milliliter of KBH4 solution (0.03%, w/v in methanol) was added and the reaction was allowed to
proceed for 5 minutes at room temperature (25±5°C). The solution was diluted
to volume with 0.025 M ethanolic HCl and the fluorescence intensity of the
resulting solution was measured at 382 nm after excitation at 293 nm against
reagent blanks treated similarly.


## 2.5. Determination of the Molar Ratio of the Reaction

The Job’s method of continuous variation [29] was employed. Master
equimolar (2.5×10−2 M) aqueous solutions of AMD and
NQS were prepared. Series of 5 mL portions of
the master solutions of AMD and NQS were made up comprising different
complementary proportions (0:10, 1:9, …, 9:1, 10:0, inclusive) in test tubes. One
milliliter of 0.01 M NaOH was added to each tube, and the tubes were further
manipulated as described under the general recommended procedure for the
spectrophotometric method.


## 3. Results and Discussion

### 3.1. Strategy for Assays Development, Involved Reaction, and Spectral Characteristics

Because of the absence of any
chromophoric group in the AMD molecule, it has no absorption in the
ultraviolet-visible region above 200 nm, and it has no native fluorescence as
well. Therefore, direct spectrophotometric and fluorimetric determination of
AMD were not possible. Therefore, derivatization of AMD was attempted in the
present study for the development of both spectrophotometric and spectrofluorometric
methods for its determination. NQS has been used as chromogenic
and fluorogenic reagent for primary
and secondary amines [26–28, 30], however,
its reaction with AMD has not been investigated yet. Therefore, the present
study was devoted to explore NQS as a derivatizing reagent in the development
of spectrophotometric and spectrofluorometric methods for the determination of
AMD in capsules and plasma. Our preliminary
experiments in investigating the reaction between AMD and NQS revealed that
NQS-AMD product is orange colored exhibiting a maximum absorption at 460 nm and
it is insoluble in water, but soluble in organic solvents. Since the present
work was directed to involve plasma samples, the interference of endogenous
amines was a major concern. It is well known that NQS reacts with the
endogenous amines (e.g., amino acids) and yields water-soluble products [30]. For
this reason, an extraction step was necessary for the development of selective methods for the determination
of AMD in the presence of the endogenous amines. As well, the reduced AMD-NQS derivative
was found to be fluorescent and exhibited one emission maximum at 382 nm and
three excitation maxima at 293, 325, and 344 nm. The highest fluorescence
intensity was obtained after excitation  293 nm, thus the excitation in the present study was
performed at this wavelength. Scheme 2 shows the reaction pathway between AMD and
NQS, and Figure 1 shows the absorption, excitation, and emission spectra of the
reaction product. The following sections describe the optimization of the assay
variables and validation for the performance of both spectrophotometric
and spectrofluorometric methods.Scheme 2
Scheme for
the reaction pathway of amantadine hydrochloride (AMD) with
1,2-naphthoquinone-4-sulphonate (NQS).Absorption,
excitation, and emission spectra on the
reaction products of AMD with NQS (0.06%, w/v). (a) Absorption spectrum of the reaction
product of AMD (65μg mL−1) with NQS after extraction with
chloroform. (b) (1) Excitation and (2) emission
spectra of the reduced reaction product. RFI is the
relative fluorescence intensity.
(a)(b)


### 3.2. Method Development

#### 3.2.1. Optimization of Derivatization Reaction and Spectrophotometric Procedure

The factors affecting the derivatization reaction (the concentrations of
NQS and NaOH, reaction time, temperature, diluting solvent, and the extracting
solvent) were investigated by altering each variable in a turn while keeping
the others constant. The studying of NQS concentrations revealed that the reaction
was dependent on NQS reagent (Figure2). The highest absorption intensity was
attained when the concentration of NQS was 0.06–0.075% (w/v) in
the final solution; further experiments were carried out at NQS concentration
of 0.06% (w/v). The results of investigating the effect of NaOH concentration
on the reaction revealed that the optimum NaOH concentration was 0.01 M (Figure
2). The effect of temperature on the derivatization reaction was studied by carrying
out the reaction at different temperatures (25–100°C) and the
maximum readings were obtained at 70–100°C (Figure 3).
For more precise readings, further experiments were carried out at 80±5°C. The
effect of heating time on the formation of the reaction product was
investigated by carrying out the reaction at different times. The maximum absorbance
intensity was attained after 40 minutes, and longer reaction time did not
affect the absorbance intensity (Figure 3).
For more precise results, further experiments were carried out at 45 minutes.Figure 2
Effect of NaOH (∘) and NQS(•) concentrations on the reaction of
AMD (45 μg mL−1) with NQS.Figure 3
Effect of heating temperature (∘) and time (•) on
the reaction of AMD (45 μg mL−1)
with NQS.It was found that the colored AMD-NQS product is insoluble in the aqueous
reaction medium. For measurements, the reaction product might be either dissolved
in a miscible organic solvent of lower polarity than water or extracted with an
immiscible extractive solvent. Different solvents were tested for dilution;
methanol, ethanol, acetonitrile, dimethylsulphoxide, isopropanol, 1,4-dioxane,
and acetone. The highest readings were obtained when dioxane was used for
dilution (data not shown). As well, different nonmiscible solvents were tested
for the extraction of the AMD-NQS product: carbon tetrachloride, chloroform, dichloromethane,
ethyl acetate,
toluene, and benzene. The highest readings were obtained when chloroform was
used for extraction (Table1). The results revealed that the extractive
procedure is more sensitive (1.5 fold) than the nonextractive procedure. This
was attributed to the effective decrease in the blank readings and consequently
enhanced the sensitivity of the assay.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of AMD with NQS
(0.06%, w/v). Values for all
solvents are mean of three determinations; the RSDs for the readings were<3.
Spectrophotometric
methodSpectrofluorometric
methodExtracting
solventAbsorbanceDiluting solventFluorescence
intensityCarbon tetrachloride0.487Methanol65.66Chloroform0.718Ethanol78.38Dichloromethane0.616Isopropanol63.02Ethyl-acetate0.362Acetone2.00Toluene0.267Acetonitrile16.18Benzene0.239Dimethylformamide3.941,4-Dioxane76.11


#### 3.2.2. Optimization of Spectrofluorometric Procedure

For developing the spectrofluorometric procedure, a reduction step for AMD-NQS
product was necessary. However, the reduced NQS reagent itself is also
fluorescent and it had the same excitation and emission maxima of the AMD-NQS product,
therefore, a selective extraction step for the AMD-NQS product from the
remaining NQS reagent was necessary before carrying out the reduction step. Furthermore,
the extraction step is essential to provide the required selectivity for
analyzing the plasma samples as the NQS products with endogenous amines were
water soluble. Based on the reported efficiency [30], potassium borohydride as
a reducing reagent was selected for NQS-derivatives. In order to investigate
the effect of potassium borohydride concentration on the reduction, the reaction
was performed using varying concentrations (0.0005–0.01%, w/v). The highest fluorescence
intensity was obtained when the concentration was 0.003% in the final solution
(1 mL of 0.03%, w/v). Concentrations more than 0.003% did not affect the
fluorescence intensity (Figure 4). The effect of pH on the fluorescence
intensity was also studied and the results showed that the highest fluorescence
intensity was obtained at pH 2.0 (Figure 5). This pH could be attained by
diluting the reaction mixture with 0.025 M ethanolic HCl solution.Figure 4
Effect ofKBH4 concentration on the fluorescence intensity of the reaction product
of AMD (1 μg mL−1) with NQS (0.06%, w/v).Figure 5
Effect of pH on the fluorescence intensity of the reduced reaction product of AMD-NQS. RFI
is the relative fluorescence intensity.


### 3.3. Stoichiometry of Derivatization Reaction

Under
the optimum conditions, the stoichiometry of the reaction between AMD and NQS was investigated by Job’s method [29] and was found
to be 1:1 because AMD molecule contains only one center (primary amino group)
available for this condensation reaction. Based on this ratio, the reaction
pathway was postulated to be proceeded as shown in Scheme 2.


### 3.4. Method Validation

#### 3.4.1. Linearity, Limits of Detection and Quantitation

In the proposed methods, linear plots (n=6) with good correlation coefficients (0.9974 and 0.9972) were obtained in the
concentration ranges of 5–80 μg mL−1 for and 0.05–10 μg mL−1 for the
spectrophotometric and the spectrofluorometric methods, respectively (Table 2).
The limits of detection (LOD) and quantitation (LOQ) were determined [31] using
the formula LOD or LOQ = κSDa/b, where κ= 3.3
for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b
is the slope. The LOD values were 1.39 and 0.013 μg mL−1 for the
spectrophotometric and spectrofluorometric methods, respectively (Table 2).Table 2
Quantitative parameters and statistical data
for determination of amantadine hydrochloride by the proposed
spectrophotometric and spectrofluorometric methods.
ParameterSpectrophotometric methodSpectrofluorometric methodRange (μg mL−1)5.00–80.00.05–10.0Intercept (a)0.0759±0.0041−2.457±0.332Slope (b)0.00974±0.0003581.969±2.154Correlation coefficient (r)0.99740.9972ε×103(L mol−1cm−1)2.058—LOD (μg mL−1)1.390.013LOQ (μg mL−1)4.210.041


#### 3.4.2. Precision and Accuracy

The precision of the proposed methods was determined by replicate
analysis of five separate sample solutions at three concentration levels of AMD.
The relative standard deviations (RSDs) were 0.83–0.96 and 0.46–1.01% for the spectrophotometric
and spectrofluorometric methods, respectively (Table3), indicating the good
reproducibility of the proposed methods. Furthermore, the inter- and intra-assay precisions
of the proposed spectrofluorometric method were determined from the recovery
studies of spiked human plasma samples. The RSD values of the recovery were
0.57–2.04 and 0.72–4.20% for the intra-
and inter-assay determinations, respectively (Table 4). The accuracy of the proposed methods
was evaluated by standard addition method. The obtained recovery values were 98.8–100.2±1.04–1.54% indicating the
high accuracy of the proposed methods. Moreover, the accuracy of the proposed spectrofluorometric
method was evaluated by the recovery studies of spiked human plasma samples.
The obtained recovery values were 96.3–101.2±0.57–4.2% (Table 4).
These recovery results of the spiked human plasma indicate the suitability of
the proposed spectrofluorometric method for the analysis of AMD in human
plasma.Table 3
The precision of the proposed methods at three
concentration levels of AMD.
MethodNominal concentration (μg mL−1)RSD%(a)Spectrophotometric8.00.9640.00.8360.00.87Spectrofluorometric0.11.014.00.588.00.46Values(a) are mean of five determinations.Table 4
Recovery studies for the proposed spectrofluorometric method for the determination
of AMD in spiked human plasma.
Spiked concentration (μg mL−1)Recovery(%±RSD)(a)Intra-assayInter-assay0.0598.2±2.0496.3±4.200.1099.3±1.01101.1±2.00.20100.5±0.9999.1±1.500.40100.9±0.7498.5±1.520.80100.1±0.75101.2±1.441.6098.6±0.82100.3±1.123.2098.9±0.5799.7±0.72Values(a) are mean of three and five determinations for intra-
and inter-assay, respectively.


#### 3.4.3. Interference Studies

The results of the interferences study showed that no interferences were
found from any of the excipients studied; lactose, sucrose, starch, talc, gum
acacia, glucose, and magnesium stearate; the recovery of AMD was 98.15–100.72%. This indicated the absence of interferences
from these excipients. Moreover, the interferences from the amino acids with
the assay procedures were also studied using glycine as an example for the
amino acids. The results of this study revealed that the
amino acids could interfere with the spectrophotometric procedures. However, there
is no any interference coming from the amino acids after an extraction step for
the derivatized AMD product because the derivatized amino acids products are
water soluble. Therefore, the extraction step increased both the sensitivity
and selectivity by removing the interferences caused by both amino acids and
proteins in the plasma samples.


#### 3.4.4. Robustness and Ruggedness

Robustness was examined
by evaluating the influence of small variation of method variables, including
concentration of analytical reagents and reaction time on the performance of
the proposed methods. In these experiments, one parameter was changed whereas
the others were kept unchanged, and the recovery percentage was calculated each
time. It was found that small variation of method variables did not significantly
affect the procedures. This provided an indication for the reliability of the
proposed method during its routine application for the analysis of AMD.
Ruggedness was also tested by applying the proposed methods to the assay of AMD
using the same operational conditions but using two different instruments at
two different laboratories and different elapsed time. Results obtained from
lab-to-lab and day-to-day variations were found to be reproducible, the full
range of recovery values was 98.4–101.3% and the
RSD was 0.73 and 1.06% for the spectrophotometric and spectrofluorometric methods,
respectively.


### 3.5. Application of the Proposed Method to Analysis of AMD in Capsules

It is evident from the above-mentioned results that the proposed methods
gave satisfactory results with AMD in bulk. Thus, its capsules were subjected
to the analysis of their contents from the active ingredient by the proposed
methods and the official (nonaqueous titration) method [32]. The capsule contents, as percentage, were
98.70±1.79
and 98.91±1.93%
for the spectrophotometric and spectrofluorometric methods, respectively (Table 5). These results were compared with those obtained from the official method by
statistical analysis with respect to the accuracy (t-test) and precision
(F-test). No significant differences were found between the calculated and
theoretical values of t- and F-tests at 95% confidence
limit proving similar accuracy and precision in the analysis of AMD in its
dosage form.Table 5
Analysis of AMD in capsules by the proposed
and official methods.
MethodRecovery(%±SD)(a)t-value(b)F-value(b)Spectrophotometric98.70±1.790.421.64Spectrofluorometric98.91±1.910.191.85OfficialHPLC(c)99.13±1.41——Values(a) are mean of five determinations.
Theoretical(b) values for t and F at 95% confidence limit and n=5 were 2.31 and 6.39, respectively.
Reference(c) [32].


## 3.1. Strategy for Assays Development, Involved Reaction, and Spectral Characteristics

Because of the absence of any
chromophoric group in the AMD molecule, it has no absorption in the
ultraviolet-visible region above 200 nm, and it has no native fluorescence as
well. Therefore, direct spectrophotometric and fluorimetric determination of
AMD were not possible. Therefore, derivatization of AMD was attempted in the
present study for the development of both spectrophotometric and spectrofluorometric
methods for its determination. NQS has been used as chromogenic
and fluorogenic reagent for primary
and secondary amines [26–28, 30], however,
its reaction with AMD has not been investigated yet. Therefore, the present
study was devoted to explore NQS as a derivatizing reagent in the development
of spectrophotometric and spectrofluorometric methods for the determination of
AMD in capsules and plasma. Our preliminary
experiments in investigating the reaction between AMD and NQS revealed that
NQS-AMD product is orange colored exhibiting a maximum absorption at 460 nm and
it is insoluble in water, but soluble in organic solvents. Since the present
work was directed to involve plasma samples, the interference of endogenous
amines was a major concern. It is well known that NQS reacts with the
endogenous amines (e.g., amino acids) and yields water-soluble products [30]. For
this reason, an extraction step was necessary for the development of selective methods for the determination
of AMD in the presence of the endogenous amines. As well, the reduced AMD-NQS derivative
was found to be fluorescent and exhibited one emission maximum at 382 nm and
three excitation maxima at 293, 325, and 344 nm. The highest fluorescence
intensity was obtained after excitation  293 nm, thus the excitation in the present study was
performed at this wavelength. Scheme 2 shows the reaction pathway between AMD and
NQS, and Figure 1 shows the absorption, excitation, and emission spectra of the
reaction product. The following sections describe the optimization of the assay
variables and validation for the performance of both spectrophotometric
and spectrofluorometric methods.Scheme 2
Scheme for
the reaction pathway of amantadine hydrochloride (AMD) with
1,2-naphthoquinone-4-sulphonate (NQS).Absorption,
excitation, and emission spectra on the
reaction products of AMD with NQS (0.06%, w/v). (a) Absorption spectrum of the reaction
product of AMD (65μg mL−1) with NQS after extraction with
chloroform. (b) (1) Excitation and (2) emission
spectra of the reduced reaction product. RFI is the
relative fluorescence intensity.
(a)(b)


## 3.2. Method Development

### 3.2.1. Optimization of Derivatization Reaction and Spectrophotometric Procedure

The factors affecting the derivatization reaction (the concentrations of
NQS and NaOH, reaction time, temperature, diluting solvent, and the extracting
solvent) were investigated by altering each variable in a turn while keeping
the others constant. The studying of NQS concentrations revealed that the reaction
was dependent on NQS reagent (Figure2). The highest absorption intensity was
attained when the concentration of NQS was 0.06–0.075% (w/v) in
the final solution; further experiments were carried out at NQS concentration
of 0.06% (w/v). The results of investigating the effect of NaOH concentration
on the reaction revealed that the optimum NaOH concentration was 0.01 M (Figure
2). The effect of temperature on the derivatization reaction was studied by carrying
out the reaction at different temperatures (25–100°C) and the
maximum readings were obtained at 70–100°C (Figure 3).
For more precise readings, further experiments were carried out at 80±5°C. The
effect of heating time on the formation of the reaction product was
investigated by carrying out the reaction at different times. The maximum absorbance
intensity was attained after 40 minutes, and longer reaction time did not
affect the absorbance intensity (Figure 3).
For more precise results, further experiments were carried out at 45 minutes.Figure 2
Effect of NaOH (∘) and NQS(•) concentrations on the reaction of
AMD (45 μg mL−1) with NQS.Figure 3
Effect of heating temperature (∘) and time (•) on
the reaction of AMD (45 μg mL−1)
with NQS.It was found that the colored AMD-NQS product is insoluble in the aqueous
reaction medium. For measurements, the reaction product might be either dissolved
in a miscible organic solvent of lower polarity than water or extracted with an
immiscible extractive solvent. Different solvents were tested for dilution;
methanol, ethanol, acetonitrile, dimethylsulphoxide, isopropanol, 1,4-dioxane,
and acetone. The highest readings were obtained when dioxane was used for
dilution (data not shown). As well, different nonmiscible solvents were tested
for the extraction of the AMD-NQS product: carbon tetrachloride, chloroform, dichloromethane,
ethyl acetate,
toluene, and benzene. The highest readings were obtained when chloroform was
used for extraction (Table1). The results revealed that the extractive
procedure is more sensitive (1.5 fold) than the nonextractive procedure. This
was attributed to the effective decrease in the blank readings and consequently
enhanced the sensitivity of the assay.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of AMD with NQS
(0.06%, w/v). Values for all
solvents are mean of three determinations; the RSDs for the readings were<3.
Spectrophotometric
methodSpectrofluorometric
methodExtracting
solventAbsorbanceDiluting solventFluorescence
intensityCarbon tetrachloride0.487Methanol65.66Chloroform0.718Ethanol78.38Dichloromethane0.616Isopropanol63.02Ethyl-acetate0.362Acetone2.00Toluene0.267Acetonitrile16.18Benzene0.239Dimethylformamide3.941,4-Dioxane76.11


### 3.2.2. Optimization of Spectrofluorometric Procedure

For developing the spectrofluorometric procedure, a reduction step for AMD-NQS
product was necessary. However, the reduced NQS reagent itself is also
fluorescent and it had the same excitation and emission maxima of the AMD-NQS product,
therefore, a selective extraction step for the AMD-NQS product from the
remaining NQS reagent was necessary before carrying out the reduction step. Furthermore,
the extraction step is essential to provide the required selectivity for
analyzing the plasma samples as the NQS products with endogenous amines were
water soluble. Based on the reported efficiency [30], potassium borohydride as
a reducing reagent was selected for NQS-derivatives. In order to investigate
the effect of potassium borohydride concentration on the reduction, the reaction
was performed using varying concentrations (0.0005–0.01%, w/v). The highest fluorescence
intensity was obtained when the concentration was 0.003% in the final solution
(1 mL of 0.03%, w/v). Concentrations more than 0.003% did not affect the
fluorescence intensity (Figure 4). The effect of pH on the fluorescence
intensity was also studied and the results showed that the highest fluorescence
intensity was obtained at pH 2.0 (Figure 5). This pH could be attained by
diluting the reaction mixture with 0.025 M ethanolic HCl solution.Figure 4
Effect ofKBH4 concentration on the fluorescence intensity of the reaction product
of AMD (1 μg mL−1) with NQS (0.06%, w/v).Figure 5
Effect of pH on the fluorescence intensity of the reduced reaction product of AMD-NQS. RFI
is the relative fluorescence intensity.


## 3.2.1. Optimization of Derivatization Reaction and Spectrophotometric Procedure

The factors affecting the derivatization reaction (the concentrations of
NQS and NaOH, reaction time, temperature, diluting solvent, and the extracting
solvent) were investigated by altering each variable in a turn while keeping
the others constant. The studying of NQS concentrations revealed that the reaction
was dependent on NQS reagent (Figure2). The highest absorption intensity was
attained when the concentration of NQS was 0.06–0.075% (w/v) in
the final solution; further experiments were carried out at NQS concentration
of 0.06% (w/v). The results of investigating the effect of NaOH concentration
on the reaction revealed that the optimum NaOH concentration was 0.01 M (Figure
2). The effect of temperature on the derivatization reaction was studied by carrying
out the reaction at different temperatures (25–100°C) and the
maximum readings were obtained at 70–100°C (Figure 3).
For more precise readings, further experiments were carried out at 80±5°C. The
effect of heating time on the formation of the reaction product was
investigated by carrying out the reaction at different times. The maximum absorbance
intensity was attained after 40 minutes, and longer reaction time did not
affect the absorbance intensity (Figure 3).
For more precise results, further experiments were carried out at 45 minutes.Figure 2
Effect of NaOH (∘) and NQS(•) concentrations on the reaction of
AMD (45 μg mL−1) with NQS.Figure 3
Effect of heating temperature (∘) and time (•) on
the reaction of AMD (45 μg mL−1)
with NQS.It was found that the colored AMD-NQS product is insoluble in the aqueous
reaction medium. For measurements, the reaction product might be either dissolved
in a miscible organic solvent of lower polarity than water or extracted with an
immiscible extractive solvent. Different solvents were tested for dilution;
methanol, ethanol, acetonitrile, dimethylsulphoxide, isopropanol, 1,4-dioxane,
and acetone. The highest readings were obtained when dioxane was used for
dilution (data not shown). As well, different nonmiscible solvents were tested
for the extraction of the AMD-NQS product: carbon tetrachloride, chloroform, dichloromethane,
ethyl acetate,
toluene, and benzene. The highest readings were obtained when chloroform was
used for extraction (Table1). The results revealed that the extractive
procedure is more sensitive (1.5 fold) than the nonextractive procedure. This
was attributed to the effective decrease in the blank readings and consequently
enhanced the sensitivity of the assay.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of AMD with NQS
(0.06%, w/v). Values for all
solvents are mean of three determinations; the RSDs for the readings were<3.
Spectrophotometric
methodSpectrofluorometric
methodExtracting
solventAbsorbanceDiluting solventFluorescence
intensityCarbon tetrachloride0.487Methanol65.66Chloroform0.718Ethanol78.38Dichloromethane0.616Isopropanol63.02Ethyl-acetate0.362Acetone2.00Toluene0.267Acetonitrile16.18Benzene0.239Dimethylformamide3.941,4-Dioxane76.11


## 3.2.2. Optimization of Spectrofluorometric Procedure

For developing the spectrofluorometric procedure, a reduction step for AMD-NQS
product was necessary. However, the reduced NQS reagent itself is also
fluorescent and it had the same excitation and emission maxima of the AMD-NQS product,
therefore, a selective extraction step for the AMD-NQS product from the
remaining NQS reagent was necessary before carrying out the reduction step. Furthermore,
the extraction step is essential to provide the required selectivity for
analyzing the plasma samples as the NQS products with endogenous amines were
water soluble. Based on the reported efficiency [30], potassium borohydride as
a reducing reagent was selected for NQS-derivatives. In order to investigate
the effect of potassium borohydride concentration on the reduction, the reaction
was performed using varying concentrations (0.0005–0.01%, w/v). The highest fluorescence
intensity was obtained when the concentration was 0.003% in the final solution
(1 mL of 0.03%, w/v). Concentrations more than 0.003% did not affect the
fluorescence intensity (Figure 4). The effect of pH on the fluorescence
intensity was also studied and the results showed that the highest fluorescence
intensity was obtained at pH 2.0 (Figure 5). This pH could be attained by
diluting the reaction mixture with 0.025 M ethanolic HCl solution.Figure 4
Effect ofKBH4 concentration on the fluorescence intensity of the reaction product
of AMD (1 μg mL−1) with NQS (0.06%, w/v).Figure 5
Effect of pH on the fluorescence intensity of the reduced reaction product of AMD-NQS. RFI
is the relative fluorescence intensity.


## 3.3. Stoichiometry of Derivatization Reaction

Under
the optimum conditions, the stoichiometry of the reaction between AMD and NQS was investigated by Job’s method [29] and was found
to be 1:1 because AMD molecule contains only one center (primary amino group)
available for this condensation reaction. Based on this ratio, the reaction
pathway was postulated to be proceeded as shown in Scheme 2.


## 3.4. Method Validation

### 3.4.1. Linearity, Limits of Detection and Quantitation

In the proposed methods, linear plots (n=6) with good correlation coefficients (0.9974 and 0.9972) were obtained in the
concentration ranges of 5–80 μg mL−1 for and 0.05–10 μg mL−1 for the
spectrophotometric and the spectrofluorometric methods, respectively (Table 2).
The limits of detection (LOD) and quantitation (LOQ) were determined [31] using
the formula LOD or LOQ = κSDa/b, where κ= 3.3
for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b
is the slope. The LOD values were 1.39 and 0.013 μg mL−1 for the
spectrophotometric and spectrofluorometric methods, respectively (Table 2).Table 2
Quantitative parameters and statistical data
for determination of amantadine hydrochloride by the proposed
spectrophotometric and spectrofluorometric methods.
ParameterSpectrophotometric methodSpectrofluorometric methodRange (μg mL−1)5.00–80.00.05–10.0Intercept (a)0.0759±0.0041−2.457±0.332Slope (b)0.00974±0.0003581.969±2.154Correlation coefficient (r)0.99740.9972ε×103(L mol−1cm−1)2.058—LOD (μg mL−1)1.390.013LOQ (μg mL−1)4.210.041


### 3.4.2. Precision and Accuracy

The precision of the proposed methods was determined by replicate
analysis of five separate sample solutions at three concentration levels of AMD.
The relative standard deviations (RSDs) were 0.83–0.96 and 0.46–1.01% for the spectrophotometric
and spectrofluorometric methods, respectively (Table3), indicating the good
reproducibility of the proposed methods. Furthermore, the inter- and intra-assay precisions
of the proposed spectrofluorometric method were determined from the recovery
studies of spiked human plasma samples. The RSD values of the recovery were
0.57–2.04 and 0.72–4.20% for the intra-
and inter-assay determinations, respectively (Table 4). The accuracy of the proposed methods
was evaluated by standard addition method. The obtained recovery values were 98.8–100.2±1.04–1.54% indicating the
high accuracy of the proposed methods. Moreover, the accuracy of the proposed spectrofluorometric
method was evaluated by the recovery studies of spiked human plasma samples.
The obtained recovery values were 96.3–101.2±0.57–4.2% (Table 4).
These recovery results of the spiked human plasma indicate the suitability of
the proposed spectrofluorometric method for the analysis of AMD in human
plasma.Table 3
The precision of the proposed methods at three
concentration levels of AMD.
MethodNominal concentration (μg mL−1)RSD%(a)Spectrophotometric8.00.9640.00.8360.00.87Spectrofluorometric0.11.014.00.588.00.46Values(a) are mean of five determinations.Table 4
Recovery studies for the proposed spectrofluorometric method for the determination
of AMD in spiked human plasma.
Spiked concentration (μg mL−1)Recovery(%±RSD)(a)Intra-assayInter-assay0.0598.2±2.0496.3±4.200.1099.3±1.01101.1±2.00.20100.5±0.9999.1±1.500.40100.9±0.7498.5±1.520.80100.1±0.75101.2±1.441.6098.6±0.82100.3±1.123.2098.9±0.5799.7±0.72Values(a) are mean of three and five determinations for intra-
and inter-assay, respectively.


### 3.4.3. Interference Studies

The results of the interferences study showed that no interferences were
found from any of the excipients studied; lactose, sucrose, starch, talc, gum
acacia, glucose, and magnesium stearate; the recovery of AMD was 98.15–100.72%. This indicated the absence of interferences
from these excipients. Moreover, the interferences from the amino acids with
the assay procedures were also studied using glycine as an example for the
amino acids. The results of this study revealed that the
amino acids could interfere with the spectrophotometric procedures. However, there
is no any interference coming from the amino acids after an extraction step for
the derivatized AMD product because the derivatized amino acids products are
water soluble. Therefore, the extraction step increased both the sensitivity
and selectivity by removing the interferences caused by both amino acids and
proteins in the plasma samples.


### 3.4.4. Robustness and Ruggedness

Robustness was examined
by evaluating the influence of small variation of method variables, including
concentration of analytical reagents and reaction time on the performance of
the proposed methods. In these experiments, one parameter was changed whereas
the others were kept unchanged, and the recovery percentage was calculated each
time. It was found that small variation of method variables did not significantly
affect the procedures. This provided an indication for the reliability of the
proposed method during its routine application for the analysis of AMD.
Ruggedness was also tested by applying the proposed methods to the assay of AMD
using the same operational conditions but using two different instruments at
two different laboratories and different elapsed time. Results obtained from
lab-to-lab and day-to-day variations were found to be reproducible, the full
range of recovery values was 98.4–101.3% and the
RSD was 0.73 and 1.06% for the spectrophotometric and spectrofluorometric methods,
respectively.


## 3.4.1. Linearity, Limits of Detection and Quantitation

In the proposed methods, linear plots (n=6) with good correlation coefficients (0.9974 and 0.9972) were obtained in the
concentration ranges of 5–80 μg mL−1 for and 0.05–10 μg mL−1 for the
spectrophotometric and the spectrofluorometric methods, respectively (Table 2).
The limits of detection (LOD) and quantitation (LOQ) were determined [31] using
the formula LOD or LOQ = κSDa/b, where κ= 3.3
for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b
is the slope. The LOD values were 1.39 and 0.013 μg mL−1 for the
spectrophotometric and spectrofluorometric methods, respectively (Table 2).Table 2
Quantitative parameters and statistical data
for determination of amantadine hydrochloride by the proposed
spectrophotometric and spectrofluorometric methods.
ParameterSpectrophotometric methodSpectrofluorometric methodRange (μg mL−1)5.00–80.00.05–10.0Intercept (a)0.0759±0.0041−2.457±0.332Slope (b)0.00974±0.0003581.969±2.154Correlation coefficient (r)0.99740.9972ε×103(L mol−1cm−1)2.058—LOD (μg mL−1)1.390.013LOQ (μg mL−1)4.210.041


## 3.4.2. Precision and Accuracy

The precision of the proposed methods was determined by replicate
analysis of five separate sample solutions at three concentration levels of AMD.
The relative standard deviations (RSDs) were 0.83–0.96 and 0.46–1.01% for the spectrophotometric
and spectrofluorometric methods, respectively (Table3), indicating the good
reproducibility of the proposed methods. Furthermore, the inter- and intra-assay precisions
of the proposed spectrofluorometric method were determined from the recovery
studies of spiked human plasma samples. The RSD values of the recovery were
0.57–2.04 and 0.72–4.20% for the intra-
and inter-assay determinations, respectively (Table 4). The accuracy of the proposed methods
was evaluated by standard addition method. The obtained recovery values were 98.8–100.2±1.04–1.54% indicating the
high accuracy of the proposed methods. Moreover, the accuracy of the proposed spectrofluorometric
method was evaluated by the recovery studies of spiked human plasma samples.
The obtained recovery values were 96.3–101.2±0.57–4.2% (Table 4).
These recovery results of the spiked human plasma indicate the suitability of
the proposed spectrofluorometric method for the analysis of AMD in human
plasma.Table 3
The precision of the proposed methods at three
concentration levels of AMD.
MethodNominal concentration (μg mL−1)RSD%(a)Spectrophotometric8.00.9640.00.8360.00.87Spectrofluorometric0.11.014.00.588.00.46Values(a) are mean of five determinations.Table 4
Recovery studies for the proposed spectrofluorometric method for the determination
of AMD in spiked human plasma.
Spiked concentration (μg mL−1)Recovery(%±RSD)(a)Intra-assayInter-assay0.0598.2±2.0496.3±4.200.1099.3±1.01101.1±2.00.20100.5±0.9999.1±1.500.40100.9±0.7498.5±1.520.80100.1±0.75101.2±1.441.6098.6±0.82100.3±1.123.2098.9±0.5799.7±0.72Values(a) are mean of three and five determinations for intra-
and inter-assay, respectively.


## 3.4.3. Interference Studies

The results of the interferences study showed that no interferences were
found from any of the excipients studied; lactose, sucrose, starch, talc, gum
acacia, glucose, and magnesium stearate; the recovery of AMD was 98.15–100.72%. This indicated the absence of interferences
from these excipients. Moreover, the interferences from the amino acids with
the assay procedures were also studied using glycine as an example for the
amino acids. The results of this study revealed that the
amino acids could interfere with the spectrophotometric procedures. However, there
is no any interference coming from the amino acids after an extraction step for
the derivatized AMD product because the derivatized amino acids products are
water soluble. Therefore, the extraction step increased both the sensitivity
and selectivity by removing the interferences caused by both amino acids and
proteins in the plasma samples.


## 3.4.4. Robustness and Ruggedness

Robustness was examined
by evaluating the influence of small variation of method variables, including
concentration of analytical reagents and reaction time on the performance of
the proposed methods. In these experiments, one parameter was changed whereas
the others were kept unchanged, and the recovery percentage was calculated each
time. It was found that small variation of method variables did not significantly
affect the procedures. This provided an indication for the reliability of the
proposed method during its routine application for the analysis of AMD.
Ruggedness was also tested by applying the proposed methods to the assay of AMD
using the same operational conditions but using two different instruments at
two different laboratories and different elapsed time. Results obtained from
lab-to-lab and day-to-day variations were found to be reproducible, the full
range of recovery values was 98.4–101.3% and the
RSD was 0.73 and 1.06% for the spectrophotometric and spectrofluorometric methods,
respectively.


## 3.5. Application of the Proposed Method to Analysis of AMD in Capsules

It is evident from the above-mentioned results that the proposed methods
gave satisfactory results with AMD in bulk. Thus, its capsules were subjected
to the analysis of their contents from the active ingredient by the proposed
methods and the official (nonaqueous titration) method [32]. The capsule contents, as percentage, were
98.70±1.79
and 98.91±1.93%
for the spectrophotometric and spectrofluorometric methods, respectively (Table 5). These results were compared with those obtained from the official method by
statistical analysis with respect to the accuracy (t-test) and precision
(F-test). No significant differences were found between the calculated and
theoretical values of t- and F-tests at 95% confidence
limit proving similar accuracy and precision in the analysis of AMD in its
dosage form.Table 5
Analysis of AMD in capsules by the proposed
and official methods.
MethodRecovery(%±SD)(a)t-value(b)F-value(b)Spectrophotometric98.70±1.790.421.64Spectrofluorometric98.91±1.910.191.85OfficialHPLC(c)99.13±1.41——Values(a) are mean of five determinations.
Theoretical(b) values for t and F at 95% confidence limit and n=5 were 2.31 and 6.39, respectively.
Reference(c) [32].


## 4. Conclusions

The present study described the use of NQS
reagent for the development of selective, sensitive, and accurate
spectrophotometric and spectrofluorometric methods for the determination of AMD
in bulk, capsules, and plasma. The described methods are superior to the
previously reported spectrophotometric or spectrofluorometric methods for
analysis of AMD in terms of their selectivity and sensitivity. The linear ranges
of the proposed spectrophotometric and spectrofluorometric methods were 5–80 and 0.05–10μg/mL,
respectively, which are much less than some reported methods 100–1300, 15–90, 25–75, 94–940 μg/mL [11, 14, 15] which were based on either the nonselective oxidation or
charge-transfer complex formation of AMD base. Although the sensitivity of the
proposed spectrofluorimetric method is comparable to that described by Darwish et al. [7], which was based on
the nonselective oxidation of AMD with cerric sulphate, however, our proposed methods
are more selective. Also, The proposed methods involved spectrophotometric and spectrofluorometric
measurements with comparable analytical performance devoid from any potential
interference. This gives the advantage of flexibility in performing the
analysis on any available instrument. Furthermore, all the analytical reagents
are inexpensive, have excellent shelf life, and are available in any analytical
laboratory. Therefore, these methods can be recommended for the routine
analysis of AMD in quality control and clinical laboratories.



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*Source: 810104-2009-01-25.xml*