т. 84, № 6 журнал прикладной спектроскопии

Т. 84, № 6 ЖУРНАЛ ПРИКЛАДНОЙ СПЕКТРОСКОПИИ НОЯБРЬ — ДЕКАБРЬ 2017

V. 84, N 6 JOURNAL OF APPLIED SPECTROSCOPY NOVEMBER — DECEMBER 2017

MODIFIED EXTRACTION-FREE ION-PAIR METHODS FOR THE DETERMINATION OF FLUNARIZINE DIHYDROCHLORIDE IN BULK DRUG, TABLETS, AND HUMAN URINE** K. N. Prashanth, K. Basavaiah *

University of Mysore, Manasagangotri, Mysore, 570006, Karnataka, India; e-mail: [email protected]

Two simple and sensitive extraction-free spectrophotometric methods are described for the determina-tion of flunarizine dihydrochloride. The methods are based on the ion-pair complex formation between the nitrogenous compound flunarizine (FNZ), converted from flunarizine dihydrochloride (FNH), and the acidic dye phenol red (PR), in which experimental variables were circumvented. The first method (method A) is based on the formation of a yellow-colored ion-pair complex (1:1 drug:dye) between FNZ and PR in chloro-form, which is measured at 415 nm. In the second method (method B), the formed drug-dye ion-pair complex is treated with ethanolic potassium hydroxide in an ethanolic medium, and the resulting base form of the dye is measured at 580 nm. The stoichiometry of the formed ion-pair complex between the drug and dye (1:1) is determined by Job’s continuous variations method, and the stability constant of the complex is also calcu-lated. These methods quantify FNZ over the concentration ranges 5.0–70.0 in method A and 0.5–7.0 µg/mL in method B. The calculated molar absorptivities are 6.17103 and 5.5104 L/molcm–1 for method A and method B, respectively, with corresponding Sandell sensitivity values of 0.0655 and 0.0074 µg/cm2. The methods are applied to the determination of FNZ in pure drug and human urine.

Keywords: extraction-free ion-pair complex, phenol red, flunarizine, pharmaceutical formulation, hu-man urine. МОДИФИЦИРОВАННЫЕ МЕТОДЫ ЭКСТРАКЦИИ СВОБОДНОЙ ИОННОЙ ПАРЫ ДЛЯ ОПРЕДЕЛЕНИЯ ДИГИДРОХЛОРАТА ФЛУНАРИЗИНА В ЛЕКАРСТВЕННЫХ ПРЕПАРАТАХ И ЧЕЛОВЕЧЕСКОЙ УРИНЕ K. N. Prashanth, K. Basavaiah *

УДК 535.243:615.45

Майсурский университет, Манасаганготри, Майсур, 570006, Карнатака, Индия; e-mail: [email protected]

(Поступила 25 января 2017)

Для определения дихлоргидрата флунаризина предложены два чувствительных экстракционных спектрофотометрических метода, основанных на образовании ионных комплексов между азоти-стым соединением флунаризином (FNZ), превращенным из дигидрохлорида флунаризина (FNH), и кислым красителем фенольным красным (PR). Метод А основан на образовании окрашенного в желтый цвет комплекса ионных пар (препарат:краситель 1:1) между FNZ и PR в хлороформе, исследуемого на длине волны 415 нм. В методе B образованный комплекс ионной пары краситель–препарат обработан этанольным гидроксидом калия в этанольной среде и полученная форма кра-сителя исследована при 580 нм. Методом вариаций Джоба определена стехиометрия образованного комплекса ионных пар между лекарством и красителем (1:1) и рассчитана константа его стабиль-ности. Методы позволяют давать количественную оценку содержания FNZ в диапазонах концен-траций 5.0–70.0 (метод А) и 0.5–7.0 мкг/мл (В). Для методов А и В рассчитаны молярные коэффици-

** Full text is published in JAS V. 84, No. 6 (http://springer.com/10812) and in electronic version of ZhPS V. 84, No. 6 (http://www.elibrary.ru/title_about.asp?id=7318; [email protected]).

ABSTRACTS ENGLISH-LANGUAGE ARTICLES

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енты поглощения 6.17103 и 5.5104 л/мольсм–1 и чувствительности 0.0655 и 0.0074 мкг/см2..Методы применены для определения FNZ в лекарственных средствах и моче человека.

Ключевые слова: бессывороточный ионно-парный комплекс, фенольный красный, флунаризин, фармацевтическая композиция, человеческая урина.

Introduction. Flunarizine dihydrochloride (FNH) is a piperazine derivative with antihistamine proper-ties and calcium channel blocking activity widely used for migraine treatment [1]. FNH prevents calcium ions from entering cells in the brain and muscles, which, in turn, prevents blood vessels from dilating, thus blocking the migraine process [2]. FNH is chemically known as [trans-1-cinnamyl-4-(4,4-difluorobenz-hydryl) piperazine dihydrochloride] [3]:

F

+

NH

NH+F

2Cl–

FNH

FNH has official monographs in the European Pharmacopeia [3] and the British Pharmacopeia [4], which describe potentiometric titration for its assay using sodium hydroxide as the titrant. Apart from this, a few researchers have dealt with the development of methods that quantify FNH in its pure form and in tab-lets. Methods using techniques such as high-performance liquid chromatography (HPLC) [5, 6], high-perfor-mance thin layer chromatography (HPTLC) [7], spectrofluorimetry [8], and UV-spectrophotometry [9–11] have been described for the determination of FNH in pharmaceuticals.

The reported official methods require a large quantity of the drug and an organic solvent. Most of the reported methods are time-consuming, poorly sensitive, and require an expensive instrumental set-up and skillful personnel. In contrast to this, visible spectrophotometry is the technique of choice even today be-cause of its inherent simplicity, sensitivity, reasonable selectivity, accuracy, precision, and cost-effective-ness. Visible spectrophotometric methods based on diverse reaction chemistries have been proposed for the assay of FNH in pharmaceuticals. Adapa et al. [12] reported two methods based on the oxidation of the drug with an excess of NBS and estimating the unreacted NBS either with celestine blue (CB) or p-methylamino-phenol sulfate (PMAP)-sulfanilamide (SA). The same authors [13] reported other methods for the assay of FNH. The first method is based on the reaction of potassium permanganate with the olefinic bond in FNH and the estimation of the unreacted permanganate with Fast Green FCF (FG FCF). The second method in-volves the treatment of the olefinic double bond in FNH with a Lemieux reagent (KMnO4 and NaIO4) and the estimation of the aldehyde formed with 3-methyl-2-benzothiazolinone hydrazone (MBTH). El-Maamli [14] developed a method based on the formation of a tris (o-phenanthroline) iron(II) complex (Ferroin) upon reaction of FNH with iron(III)-o-phenanthroline. A method reported by Kelani et al. [15] is based on the formation of the ternary complex of FNH with eosin and lead(II). Two methods reported by Badalani et al. [16] describe the complexation reaction between FNH and either carbol fuchsin or thorium nitrate-thoron.

Moreover, a few extractive spectrophotometric methods based on the ion-pair reaction have also been reported for FNH. The three methods reported by Elazazy et al. [17] are based on the formation of colored ion-pair complexes between the forging basic nitrogen of FNH and the inorganic complex: molybdenum(V) thiocyanate, Mo(V) (SCN) or acid dyes, orange G (OR.G), and alizarin red S (ARS). In another method, de-scribed by Adapa et al. [18], a colored coordination complex formed between FNH and cobalt-thiocyanate (CTC) is extracted into nitrobenzene and measured. The four methods reported by Zarapkar and Bapat [19] involve the formation of ion-pair complexes between FNH and bromocresol purple, bromocresol green, bromophenol blue, or bromothymol blue. Based on the formation of chloroform soluble ion-association complexes of FNH with Supracen violet 3B, tropacolin 000, or woolfast blue BL, Adapa et al. [20] reported three methods. El Walily et al. [21] reported a method based on the molecular interaction between FNH and iodine to form a charge-transfer complex. Based on the charge-transfer complexation reaction of the drug either with picric acid or 2,3-dichloro-5,6-dicyano-p-benzoquinone, Mohammad et al. [8] reported two methods.

Most of the above-mentioned visible spectrophotometric methods suffer from poor sensitivity [8, 16] and the need for expensive reagents and organic solvents [8, 13, 16, 18–20], a heating stage [15], or strict pH control [15, 17, 19, 20], as shown in Table 1.

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TABLE 1. Comparison of the Proposed and the Existing Visible Spectrophotometric Methods

Sl. No.

Reagent’s used Methodology λmax, nm Linear range,

µg/mL (ε, L/(molcm)

Remarks Refe-rence

1 NBS a) Celestine blue b) P-N-Me amino phe-nol sulfate-sulfanilamide

Unreacted NBS meas-ured by its color reac-tion

NA NA Use of NBS which require daily stan-

dardization

[12]

2 KMnO4 a) Fast Green-FCF b) NaIO4-MBTH

Unreacted KMnO4

measured

620

620

1.0–5.0 (5.77×104)

4.0–24.0

(1.22×104)

Use of expensive reagents and KMnO4 which requires daily

standardization

[13]

3 Iron(III)-o- phenan-throline mixture

Ferroin complex formed was measured

510 0.6–77.0

Require heating step [14]

4 Lead(II) and eosin Ternary complex measured

547.5 2.4–19.1 (3.2×104)

Time consuming and involve strict pH con-

trol, heating step

[15]

5 a) Carbol fuchsin b) Thorium nitrate-Thoron

Complexes formed were measured

285 555

10–200 50–300

Less sensitive and use of expensive reagents,

narrow linear range

[16]

6 a) Molybdenum(V) thiocyanate b) Orange G c) Alizarin red S

Extractable ion-pair complexes were measured

469–471

498–500 425–426

NA

Involve extraction step and strict pH

control

[17]

7 Cobalt thiocyanate Nitrobenzene soluble coordination complex was measured

620 NA Involve extraction step

[18]

8 a) Bromocresol green b) Bromocresol purple c) Bromophenol blue d) Bromothymol blue

Extractable ion-pair complexes were measured

NA NA Involve extraction step and strict pH

control

[19]

9 a) Supracen violet-3B b) Tropacolin 000 c) Woolfast blue-BL

Chloroform soluble ion-association com-plexes measured

560

480

580

4.0–24.0 (1.27×104)

1.0–6.0 (2.50×104)

1.0–6.0 (4.21×104)

Involve extraction step, strict pH control

and narrow linear range

[20]

10 Iodine Charge-transfer com-plex measured

355 8.0–13.0 (4.40×104)

Narrow linear range [21]

11 a) Picric acid b) DDQ

Charge-transfer com-plex measured

402.8 460

12.0–65.0 30.0–175.0

Less sensitive [8]

12 Phenol red a) FNZ-PR ion pair b) Ion-pair broken by ethanolic KOH

Yellow colored ion-pair measured break-ing of the yellow FNZ–PR ion-pair complex in alkaline medium followed by measurement of the red color free dye

415

580

5.0–70.0

(6.17×103) 0.5–7.0

(5.5×104)

Simple, economical, sensitive and no ex-haustive control of

experimental condi-tions like pH, and free from heating

and/or extraction step

This work

N o t e. DDQ: 2,3-dicloro-5,6-dicyano-1,4-benzoquinone, MBTH: 3-Methyl-2-benzothiazolinone hydrazone hydrochloride, NA: Not available.

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The reported ion-pair extraction methods [8, 17–21] require the optimization of various experimental variables, such as the pH, extraction efficiency of different solvents, contact time, volume of organic and aqueous phases, and extraction equilibration periods, which makes the analytical method cumbersome, tedi-ous, and time-consuming. In response to the problems resulting from the extraction of the ion-pair, a few articles have been published for the analysis of pharmaceutical compounds through ion-pair formation with-out extraction [22–25], but no reports can be found for the ion-pair breaking method in extraction-free tech-niques. The aim of the present study is to develop two simple, sensitive, and rapid extraction-free spectro-photometric methods based on the measurement of the drug: the dye ion-pair complex of the base form of the dye after breaking the complex with еthanolic KOH for the quantification of FNH in bulk drug, tablets, and human urine. The important advantage of these methods is that the ion-pair complex formed in a single-phase organic solvent is measured directly, eliminating the conventional biphasic extraction procedure.

Experimental. A Systronic model 106 digital spectrophotometer equipped with 1 cm quartz cells was employed for absorbance measurements. Pharmaceutical-grade flunarizine dihydrochloride certified to be 99.78% pure was received as a gift sample from Inga Pharmaceuticals, Mumbai, India, and was used as re-ceived. All the chemicals used were of analytical grade. The solvents used were of spectroscopic grade. Throughout the investigation, distilled water was used.

Standard flunarizine base (FNZ) solution. Into a 125 mL separating funnel, accurately weighed 23.6 mg of pure FNH was transferred and dissolved in about 20 ml of the methanol:water (1:1 v/v) mixture; the solu-tion was rendered alkaline with 1 M sodium hydroxide solution (5 mL), and 1 mL was added in excess and the whole shaken for 5 min. The free base (FNZ) formed was extracted with four 20.0 mL portions of chlo-roform, and the extract was passed over anhydrous sodium sulfate and collected in a 100 mL volumetric flask. The volume was made up to the mark with chloroform, and the resulting solution (200 µg/mL FNZ) was further diluted with chloroform to get a working concentration of 100 µg/mL FNZ for method A. In method B, the stock standard solution was prepared by adding 3 mL of PR to 10 mL of FNZ (100 µg/mL) in a 100 mL volumetric flask and made up to the mark with chloroform, which results in the formation of the FNZ:PR ion-pair complex (10 µg/mL in FNZ) solution.

Both tablets, Flunatrac-10 (Minova Life Sciences Ltd., Bangalore, India) and Flunarin-5 (FDC Ltd., Goa, India), were purchased from commercial sources. Human urine was collected from a healthy male, aged 31.

Method A. Different aliquots (0.25, 0.5, 1.0, 2.0, 3.0, and 3.5 mL) of FNZ standard solution (100 µg/mL) were measured accurately using a microburette and transferred into a number of 5 mL volumet-ric flasks, and the total volume was brought to 3.5 mL by adding chloroform. To each flask, 1 mL of 0.05 % PR solution was added, diluted to the mark with chloroform, and mixed well. The absorbance of the result-ing yellow colored chromogen was measured after 5 min at 415 nm against the reagent blank.

Method B. Various aliquots (0.25, 0.5, 1.0, 2.0, 3.0, and 3.5 mL) of the FNZ-PR ion-pair complex (10 µg/mL in FNZ), equivalent to 0.5–7.0 µg/mL with respect to FNZ, were transferred into a number of 5 mL standard flasks, and the total volume was brought to 3.5 mL by adding ethanol. To each flask, 1 mL of 1% еthanolic KOH was added, the content was then mixed and set aside for 5 min. Finally, the volume was made up to the mark with ethanol, and the absorbance was measured at 580 nm against the reagent blank.

Procedure for commercial tablets. Ten tablets, each containing 5 or 10 mg of FNH, were weighed and finely powdered. An accurately weighed amount of the powder, equivalent to 11.8 mg of FNH, was dis-solved in about 10 mL of methanol:water (1:1 v/v) mixture in a 125 mL separating funnel. The solution was rendered alkaline with 1 M sodium hydroxide solution (2 mL), and 1 mL was added in excess and shaken for 5 min. The base was extracted as described before and collected in a 50 mL volumetric flask. The base solu-tion (i.e., 200 μg/mL FNZ) was diluted appropriately to get 100 and 10 μg/mL and assayed by method A and method B, respectively.

Procedure for the analysis of the placebo blank and synthetic mixture. A placebo blank, containing starch (10 mg), acacia (15 mg), sodium citrate (30 mg), hydroxyl cellulose (20 mg), lactose (10 mg), talc (60 mg), acacia (30 mg), magnesium stearate (25 mg), and sodium alginate (30 mg), was prepared; 20 mg of it was extracted with chloroform, and the solution was made as described under Procedure for commercial tablets and then subjected to analysis.

A synthetic mixture was prepared by adding 11.8 mg of FNH to about 10 mg of the placebo blank pre-pared above and homogenized; then the solution of the free base was prepared as done under Procedure for commercial tablets. The filtrate was collected in a 50 mL flask. The synthetic mixture solution was then ap-propriately diluted with chloroform to get 100.0 and 10.0 µg/mL FNZ solutions, and the aliquots were sub-jected to analysis by method A and method B, separately.

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Procedure for the analysis of spiked human urine. Spiked human urine (15 ml), containing 5 mg equiva-lent FNZ of FNH, was transferred into a 125 mL separating funnel and extracted three times with 15 mL of chloroform; the lower organic layer was collected in a beaker containing anhydrous sodium sulfate. The wa-ter-free organic layer was transferred into a 50 mL calibrated flask and diluted up to the mark with chloro-form. The resulting solution, equivalent to 100 g/mL FNZ, was diluted to get 10 g/mL FNZ and analyzed by the procedures described earlier.

Results and discussion. Chemistry. FNZ contains two tertiary aliphatic amino groups (Scheme 1), be-tween which the amino group attached to the secondary carbon atom forms an ion-pair complex with the acidic dye (PR). As another amino group attached to the tertiary carbon atom experiences an electron-withdrawing effect from two fluorobenzenes, this amino group does not take part in the reaction. Since the ion pair reaction is carried in nonpolar solvents, the insolubility of FNH in any of the nonpolar solvents was overcome by neutralizing the hydrochloride by sodium hydroxide, and FNZ was extracted into chloroform. The solutions of FNZ in chloroform and dye in acetone have insignificant absorbance (Fig. 1) in the range analyzed. The more basic aliphatic tertiary amine, attached to the secondary carbon atom, gets protonated in preference to the other tertiary amine. Finally, protonated FNZ forms an ion-pair complex with the anionic form of the dye. This results in the formation of an intense yellow-colored product with an absorption maxi-mum at 415 nm (method A). The possible reaction pathway is shown in Scheme 1. In an ethanolic alkaline medium, this ion-pair complex gets disturbed, and it breaks to form a red-colored basic dye, the drug and the former peaking at 580 nm. This mechanism is shown in Scheme 2.

PR (lactoid ring) (quinoid ring)

H+

1:1 yellow ion-pair complex of FNZ-PR measured at 415 nm (method A)

Scheme 1. The proposed reaction pathway for ion-pair complex formation.

1:1 yellow ion-pair complex of FNZ-PR

Red colored base form of PR, measured at 580 nm (method B)

FNZ

Ethanolic KOH

Scheme 2. The proposed reaction pathway for the formation of anionic form of PR

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Absorbance Blank for method A Sample for method A Blank for method B Sample for method B

350 450 550 650 750 , nm

0.5

0.4

0.3

0.2

0.1

0

Fig. 1. Absorption spectra of the ion-pair complex of FNZ-PR (30 g/mL FNZ) in method A

and anionic form of the dye (3.0 g/mL FNZ) in method B. Absorption spectra. The absorption spectrum of the ion-pair complex formed between FNZ and PR was

recorded at 360–560 nm against the respective reagent blank (Fig. 1). The yellow ion-pair complex showed maximum absorbance at 415 nm. For the free dye obtained after breaking the ion pair, the absorption spec-trum was recorded at 500–740 nm against the respective reagent, and the red-colored free dye showed maximum absorbance at 580 nm. The measurements were thus made at these wavelengths.

Selection of the reaction medium (choice of organic solvent). Several organic solvents, such as di-chloromethane, chloroform, and 1,2-dichloroethane, were used for the extraction of the base form of flu-narizine. Only chloroform favored the extraction of the drug to its base form. A few organic solvents, such as dichloromethane, chloroform, acetone, methanol, ethanol, and 1,4-dioxane, were tried to dissolve the dye. Among them, acetone was preferred to dissolve the dye, and chloroform was the most suitable as the me-dium in method A. In method B, ethanol was found to be the optimum medium for the experiment.

Effect of volume of dye, reaction time, and stability of the ion-pair complex. In order to determine the optimum amount of the dye required to obtain maximum absorbance, an experiment was performed sepa-rately by measuring the absorbance of the final solution resulting from the reaction mixture containing a fixed concentration of FNZ and various amounts of the dye. It was found that 1 mL of the dye solution (0.05% PR in method A) was sufficient to produce maximum and reproducible absorbance at 415 nm (Fig. 2). In method B, the concentration of FNZ:PR ion-pair was kept constant and various amounts of еthanolic KOH were added. One mL of еthanolic KOH was found to be sufficient to produce maximum ab-sorbance at 580 nm, as shown in Fig. 2. A 5 min standing time was sufficient for the complete formation of the ion-pair complex (method A) and for breaking the ion-pair complex (method B). The absorbance of the resulting products was found to be stable for at least 24 h in method A and 3 h in method B at room tempera-ture (28±2°C).

Absorbance

1

2

0 0.5 1.0 1.5 2.0 Vreagent, mL

0.6

0.5

0.4

0.3

Fig. 2. Effect of volume of reagent 30 g/mL FNZ at 415 nm in method A (1) and 3.0 g/mL FNZ at 580 nm in method B (2).

Composition of the ion-pair complex and its conditional stability constant. The composition and condi-tional stability constant of the FNZ-PR complex formed were evaluated by applying Job’s method of con-tinuous variations [26], using equimolar concentrations of FNZ (prepared by following the general proce-dure) and the dye. The FNZ and dye concentration were 6.92×10–4 M each. The experiments were performed by mixing equimolar solutions of the drug and the dye while maintaining the total volume at 5.0 mL.

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The plot reached its maximum value at a mole fraction of 0.5, which indicated the formation of the 1:1 (FNZ:dye) complex (Fig. 3), and the results revealed that the formation of the ion-pair complex between the drug and the reagent followed a 1:1 reaction stoichiometry.

Absorbance

0.2 0.4 0.6 0.8 1.0 VFNZ/(VFNZ – VPR)

0.4

0.3

0.2

0.1

0

Fig. 3. Job’s continuous-variations plot for FNZ-PR complex.

The conditional stability constant (Kf) of the ion-association complex was calculated from the continu-ous variation data using the following equation [27]:

2

/

1 / ( )m

f n nm M

A AK

A A C n

,

where A and Am are the observed maximum absorbance and the absorbance value when all the drug present is associated, respectively, CM is the mole concentration of the drug at its maximum absorbance, and n is the stoichiometry when the PR ion associates with FNZ. The log Kf value was calculated to be 4.431.

Linearity, sensitivity, limits of detection and quantification. A linear correlation was found between the absorbance at max and the concentration of FNZ in the ranges given in Table 2. Regression analysis of the Beer’s law data using the method of least squares was made to evaluate the slope (b), intercept (a) and corre-lation coefficient (r) for each system, and the data obtained from this investigation are presented in Table 2. Sensitivity parameters, such as apparent molar absorptivity and Sandell sensitivity values [28], and the limits of detection and quantification, calculated in accordance with the current ICH guidelines [27], are compiled in the same table and demonstrate the excellent sensitivity of the proposed method. The limits of detection (LOD) and quantification (LOQ) were calculated using the formulas

LOD = 3.3σ/s, LOQ = 10σ/s,

where σ is the standard deviation of five reagent blank determinations and s is the slope of the calibration curve.

TABLE 2. Regression and Analytical Parameters

Parameter Method A Method B max, nm 415 580 Beer’s law limits, µg/mL) 5.0–70.0 0.5–7.0 Molar absorptivity, L/(mol cm) 6.17×103 5.5×104 Sandell sensitivity*, µg/cm2 0.0655 0.0074 Limit of detection, µg/mL 0.93 0.08 Limit of quantification, µg/mL 2.81 0.23 Regression equation Y = a + bX** Intercept a

–0.0046

0.0008

Slope b 0.0154 0.1354 Correlation coefficient r 0.9995 0.9996 Standard deviation of intercept Sa 0.0101 0.0030 Standard deviation of slope Sb 0.0002 0.0007

*Limit of determination is the weight in µg/mL of solution that corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. ** Y is the absorbance, a is the intercept, b is the slope, and X is the concentration in µg/mL.

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The high values of and low values of Sandell’s sensitivity and LOD indicate the high sensitivity of the proposed methods.

Intra-day and inter-day precision and accuracy. The precision and accuracy of the proposed methods were studied by repeating the experiment seven times within one day to determine the repeatability (intra-day precision) and five times on different days to determine the intermediate precision (inter-day precision) of the methods. These assays were performed for three levels of the analyte. The results of this study are summarized in Table 3. The relative standard deviation (%RSD) values were ≤1.47% (intra-day) and ≤1.76% (inter-day), indicating the good precision of the proposed methods. Their accuracy was assessed as the percentage relative error (%RE) between the measured mean concentrations and taken concentrations of FNZ, and it was ≤1.61%, which demonstrates the high accuracy of the proposed methods.

TABLE 3. Evaluation of Intra-day and Inter-day Precision and Accuracy

Intra-day (n = 7) Inter-day (n = 5) FNZ taken, µg/mL FNZ found, µg/mL %RSD %RE FNZ found, µg/mL %RSD %RE

Method A 20.0 19.71 1.31 1.44 19.68 1.66 1.60 40.0 40.45 1.10 1.13 40.53 1.45 1.32 60.0 60.87 1.47 1.44 60.97 1.76 1.61

Method B 2.00 2.02 1.26 1.10 2.03 1.74 1.35 4.00 3.97 1.10 0.82 3.96 1.50 1.04 6.00 5.94 0.83 0.98 5.93 1.66 1.17

Selectivity. A systematic study was performed to ascertain the effect of the matrix on the absorbance by

analyzing the placebo blank. In the analysis of the placebo blank solution, the absorbance in both cases was equal to the absorbance of the blank, which revealed no interference. To assess the role of the inactive ingre-dients on the assay of FNZ, the general procedure was followed by taking three different concentrations of FNZ prepared by using a synthetic mixture. The percentage recovery values were in the range 97.9–102.2%, with RSD < 2.4%, and clearly indicated the noninterference by the inactive ingredients in the assay of FNZ.

Robustness and ruggedness. The robustness of the methods was evaluated by making small deliberate changes in the volume (1±0.1 mL) of the dye in method A, and еthanolic KOH in method B, and the effect of the changes was studied on the absorbance of the ion-pair complex or the free dye in method A and method B. The changes had a negligible impact on the results, as revealed by the small intermediate preci-sion values expressed as % RSD (≤1.82%). The method ruggedness was demonstrated by the analysis done by three different analysts, and also by a single analyst performing analysis on three different instruments in the same laboratory. Intermediate precision values (%RSD) in both instances were in the range 1.40–2.24%, indicating acceptable ruggedness. These results are presented in Table 4.

TABLE 4. Robustness and Ruggedness

Robustness Ruggedness Method

FNZ taken, µg/mL Volume of

dye/ethanolic KOH*Inter-analysts

(%RSD), n = 3 Inter-instruments (%RSD), n = 3

20.0 1.41 1.91 1.77 40.0 1.38 2.09 1.90 A 60.0 1.07 1.56 2.05 2.00 1.82 1.40 1.81 4.00 1.50 1.73 2.00 B 6.00 0.99 2.12 2.24

* In method A, the volumes of phenol red dye were 1.4, 1.5, and 1.6 mL, and in method B the volumes of ethanolic KOH added were 0.9, 1.0, and 1.1 mL.

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Application to commercial tablets. The proposed methods were applied to the quantification of FNZ in commercial tablets. The same batch tablets were assayed by the official method [3], which describes potenti-ometric titration for its assay using sodium hydroxide as the titrant. The results obtained by the proposed methods agreed well with the claim and are also in agreement with those by the published reference method. The statistical analysis of the results did not find any significant difference between the performance of the proposed methods and the reference method with respect to the accuracy and precision as revealed by the Student’s t-value and the variance ratio F-value. The results of the assay are given in Table 5.

TABLE 5. Results (Percent of label claim ±SD)a of Analysis of Tablets by the Proposed Methods

Tablet Label claim, mg/tablet Reference method Method A Method B

Flunarin-5 5.0 100.1±0.46 99.02±1.01

t = 2.18, F = 4.82101.2±0.89

t = 2.46, F = 3.74

Flunaract-10 10.0 99.86±0.57 100.9±1.13

t = 1.84, F = 3.9398.76±1.04

t = 2.07, F = 3.33 a Mean value of five determinations. Tabulated t-value at the 95% confidence level is 2.78. Tabulated F-value at the 95% confidence level is 6.39.

Recovery study (standard addition method). To further assess the accuracy of the methods, recovery experiments were performed by applying the standard-addition technique. The recovery was evaluated by determining the agreement between the measured standard concentration and the added known concentration to the sample. The test was done by spiking the pre-analyzed tablet with pure FNZ at three different levels (50, 100, and 150 % of the content present in the tablet taken), and the total was found by the proposed methods. Each test was repeated three times. In both methods, the recovery percentage values ranged be-tween 98.80% and 102.1%, with a standard deviation of 0.82–2.05%. The closeness of the results to 100% showed the fairly good accuracy of the methods. The results are shown in Table 6.

TABLE 6. Results of Recovery Study by Standard Addition Method

Method A Method B FNZ in tablets, µg/mL

Pure FNZ added, µg/mL

Total found, µg/mL

Pure FNZ recovered*,

%±SD

FNZ in tablets, µg/mL

Pure EPR added, µg/mL

Total found, µg/mL

Pure FNZ recovered*,

%±SD Flunarin-5

19.80 10.0 29.68 98.80±1.23 2.02 1.0 3.03 101.4±0.82 19.80 20.0 40.07 101.4±1.21 2.02 2.0 4.00 99.00±1.16 19.80 30.0 50.35 101.8±0.85 2.02 3.0 5.05 101.0±1.03

Flunaract-10 20.18 10.0 30.38 102.0±2.05 1.98 1.0 2.99 101.1±1.25 20.18 20.0 39.99 99.05±0.87 1.98 2.0 4.00 101.2±1.17 20.18 30.0 50.62 101.5±1.11 1.98 3.0 5.04 102.1±1.04

* Mean value of three determinations.

Application to the analysis of spiked human urine sample. The proposed methods were successfully ap-plied to the determination of FNZ in spiked human urine, with a mean percent recovery of 98.12±1.87 (n = 5) and 103.2±2.19 (n = 5) for method A and method B (Table 7). The results indicate that there is no interference from other biological materials present in urine.

TABLE 7. Determination of FNZ in spiked urine sample

Method Spiked concentration, g/ml

Concentration found*, g/ml

%Recovery ±SD*

A B

40.0 4.00

39.2 4.13

98.12±1.87 103.2±2.19

*Mean value of five determinations of FNZ; n = 5.

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Conclusion. The proposed methods require only dye or еthanolic KOH as reagents, which is cheaper; no pH adjustment is required, and the procedures do not involve any critical reaction conditions or tedious sample preparation. Both methods are simple, fast, accurate, extraction-free, adequately sensitive, and free from interference by common additives and excipients. Furthermore, they use very accessible instruments and do not require a skillful operator. The proposed methods can be applied for the assay of FNZ in tablets without any interference. The statistical parameters and the recovery data reveal good accuracy and high precision. Hence, the methods can be used in a routine analysis of drugs in quality-control laboratories and are also useful for the analysis of urine. REFERENCES 1. P. A. Todd, P. Benfield, Drugs, 38, 481 (1989). 2. M. R. Alan, D. S. Fred, J. T. Stewart, Conquering Headache, 4th edn., Decker DTC, Hamilton, London (2003). 3. European Pharmacopoeia, Vol. II, European Department for the Quality of Medicines, Council of Europe, Stranbourg, France (2005). 4. British Pharmacopoeia, Vol. II, Her Majesty’s Stationary Office, London (2009). 5. A. A. M. Wahbi, M. E. Abdel-Fattah, E. M. Hassan, F. G. Saliman, El-Gendi, J. Pharm. Biomed. Anal., 13, 777 (1995). 6. W. F. Kartinasari, H. Chufianty, G. Indrayanto, J. Liq. Chromatogr. Related Technol., 26, 1059 (2003). 7. S. P. Amod, A. A. Shirkhedkar, J. S. Surana, S. P. Nawale, J. Chil. Chem. Soc., 57, 1033 (2012). 8. M. Abdul-Azim Mohammad, Bull Fac. Pharm. Cairo Univ., 42, 27 (2004). 9. S. A. Patil, A. A. Shirkhedkar, J. S. Surana, S. P. Nawale, Pharma Chem., 3, 404 (2011). 10. A. K. Doshi, B. N. Patel, C. N. Patel, Int. J. Pharm. Sci. Res., 3, 1741 (2012). 11. K. Busaranon, W. Suntornsuk, L. Suntornsuk, J. Pharm. Biomed. Anal., 41, 158 (2006). 12. V. S. S. P. Adapa, C. Ravi, C. S. P. Sastry, U. V. Prasad, Acta Cienc. Indica, Chem., 27, 105 (2001). 13. V. S. S. P. Adapa, C. S. R. Lakshmi, C. S. P. Sastry, V. P. Uppuleti, J. Anal. Chem., 58, 937 (2003). 14. M. Y. El-Maamli, Egypt. J. Biomed. Sci., 11, 122 (2003). 15. K. Kelani, I. L. Bebawy, L. A. Fattah, J. Pharm. Biomed. Anal., 18, 985 (1999). 16. M. J. Badalani, K. R. Mahadik, H. N. More, S. S. Kadam, East Pharm., 38, 133 (1995). 17. M. S. Elazazy, M. El-Mammli, A. Shalaby, M. A. Magda, Biosci., Biotechnol. Res. Asia, 5, 107 (2008). 18. V. S. S. P. Adapa, G. P. V. Mallikarjuna Rao, C. S. P. Sastry, U. V. Prasad, J. Ind. Council Chem., 18, 36 (2001). 19. S. S. Zarapkar, R. K. Bapat, Indian Drugs, 31, 170 (1994). 20. V. S. S. P. Adapa, C. S. P. Sastry, Sci. Asia, 33, 119 (2007). 21. A. F. M. E. Walily, A. E. Gindy, A. A. M. Wahbi, J. Pharm. Biomed. Anal., 13, 53 (1995). 22. K. N. Prashanth, K. Basavaiah, M. S. Raghu, Chem. Sci. J., 80, 1–14 (2012). 23. K. N. Prashanth, K. Basavaiah, K. B. Vinay, Arab. J. Chem., (2011). doi:10.1016/j.arabjc.2011.10.006. 24. M. S. Raghu, K. Basavaiah, Proc. Natl. Acad. Sci., India A: Phys. Sci., 82, 187 (2012). 25. K. B. Vinay, H. D. Revannasiddappa, N. Rajendraprasad, P. J. Ramesh, M. X. Cijo, K. Basavaiah, Drug Test Anal., 4, 116 (2012). 26. H. Zavis, D. Ludvik, K. Milan, S. Ladislaw, V. Frantisck, Handbook of Organic Reagents in Inorganic Analysis, Chichester, A Division of John Wiley & Sons IC, New York, London, Sydney, Toronto (1976). 27. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuti-cals for Human Use, ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology, Q2 (R 1), Complementary Guideline on Methodology dated 06 November 1996, London, in-corporated in November 2005. 28. J. Inczedy, T. Lengyel, A. M. Ure, IUPAC Compendium of Analytical Nomenclature: Definitive Rules, Blackwell Science Inc., Boston (1998).

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