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Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine M. I. Walash, F. Belal, N. El-Enany* and M. H. El-Maghrabey ABSTRACT: A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin (PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluo- rescence intensity concentration plot was rectilinear over the range of 0.01–0.3 mg mL -1 with a lower detection limit of 0.0017 mg mL -1 and limit of quantitation of 0.005 mg mL -1 . The developed method was successfully applied to the analysis of the drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.931.24 (n = 3). Statistical comparison of the results with those of the comparison method revealed good agreement and proved that there was no significant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated. Copyright © 2010 John Wiley & Sons, Ltd. Keywords: pregabalin; fluorescamine; spectrofluorimetry; capsules Introduction Pregabalin (PG) Fig. 1, (S)-3-(amino methyl)-5-methylhexanoic acid (1), is a lipophilic GABA (g-aminobutyric acid) analog but it is inactive at GABAA and GABAB receptors. Its main site of action appears to be on the a2d subunit of presynaptic, voltage- dependent calcium channels that are widely distributed throughout the peripheral and central nervous system. PG binds potently to the a2d subunit and modulates calcium influx at nerve terminals, thereby reducing the release of several neu- rotransmitters, including glutamate, noradrenaline, serotonin, dopamine and substance P. These activities and effects result in the anticonvulsant, analgesic and anxiolytic activity exhibited by PG (2). More recently, pregabalin has been approved by the FDA for the treatment of spinal cord injury and as the first drug indi- cated for the treatment of fibromyalgia (3,4). There is no official method for the analysis of pregabalin. A literature survey revealed that some analytical methods have been published concerning the analysis of pregabalin in pure and dosage forms or from biological matrices, including spectro- photometric, specrtofluorimetric (5) and chromatographic (6–13) methods. All the reported methods need expensive solvents and reagents with tedious extraction procedures. Therefore our target was to develop a rapid, simple, efficient and selective method for the analysis of PG in pharmaceutical formulations. The method is based on the reaction of PG through its primary amino group with fluorescamine at pH 10.0. The advantages of the proposed method over other existing methods are that it is simpler, quicker and has higher sensitivity (concentrations down to 10 ng mL -1 can be determined with suf- ficient accuracy) than the reported spectrophotometric and spectrofluorimetric methods and does not require expensive instrumentation (in contrast to reported chromatographic methods) or critical analytical reagents. Experimental Apparatus The fluorescence spectra and measurements were carried out using a Perkin–Elmer UK model LS 45 luminescence spectrom- eter, equipped with a 150 W xenon arc lamp and grating excita- tion and emission monochromators for all measurements and a Perkin–Elmer recorder. Slit widths for both monochromators were set at 10 nm. A 1 cm quartz cell was used. A Hanna poten- tiometer, Model pH equipped with glass–calomel electrode com- bination was used. Materials and reagents All the reagents used were of analytical reagent grade. Pure pre- gabalin was kindly provided by Pfizer (Sandwich, UK). Lyrica® * Correspondence to: N. El-Enany, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. E-mail: [email protected] Department of Analytical Chemistry, Faculty of Pharmacy, University of Man- soura, 35516, Mansoura, Egypt Research articles Received: 05 May 2010, Revised: 09 June 2010, Accepted: 15 June 2010, Published online in Wiley Online Library: 25 August 2010 (wileyonlinelibrary.com) DOI 10.1002/bio.1235 342 Luminescence 2011; 26: 342–348 Copyright © 2010 John Wiley & Sons, Ltd.

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Page 1: Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine

Simple and sensitive spectrofluorimetricmethod for the determination of pregabalin incapsules through derivatization withfluorescamineM. I. Walash, F. Belal, N. El-Enany* and M. H. El-Maghrabey

ABSTRACT: A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin(PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimentalparameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluo-rescence intensity concentration plot was rectilinear over the range of 0.01–0.3 mg mL-1 with a lower detection limit of0.0017 mg mL-1 and limit of quantitation of 0.005 mg mL-1. The developed method was successfully applied to the analysis ofthe drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.93�1.24 (n = 3). Statisticalcomparison of the results with those of the comparison method revealed good agreement and proved that there was nosignificant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated.Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: pregabalin; fluorescamine; spectrofluorimetry; capsules

IntroductionPregabalin (PG) Fig. 1, (S)-3-(amino methyl)-5-methylhexanoicacid (1), is a lipophilic GABA (g-aminobutyric acid) analog but it isinactive at GABAA and GABAB receptors. Its main site of actionappears to be on the a2d subunit of presynaptic, voltage-dependent calcium channels that are widely distributedthroughout the peripheral and central nervous system. PG bindspotently to the a2d subunit and modulates calcium influx atnerve terminals, thereby reducing the release of several neu-rotransmitters, including glutamate, noradrenaline, serotonin,dopamine and substance P. These activities and effects result inthe anticonvulsant, analgesic and anxiolytic activity exhibited byPG (2). More recently, pregabalin has been approved by the FDAfor the treatment of spinal cord injury and as the first drug indi-cated for the treatment of fibromyalgia (3,4).

There is no official method for the analysis of pregabalin. Aliterature survey revealed that some analytical methods havebeen published concerning the analysis of pregabalin in pureand dosage forms or from biological matrices, including spectro-photometric, specrtofluorimetric (5) and chromatographic (6–13)methods. All the reported methods need expensive solvents andreagents with tedious extraction procedures. Therefore ourtarget was to develop a rapid, simple, efficient and selectivemethod for the analysis of PG in pharmaceutical formulations.The method is based on the reaction of PG through its primaryamino group with fluorescamine at pH 10.0.

The advantages of the proposed method over other existingmethods are that it is simpler, quicker and has higher sensitivity(concentrations down to 10 ng mL-1 can be determined with suf-

ficient accuracy) than the reported spectrophotometric andspectrofluorimetric methods and does not require expensiveinstrumentation (in contrast to reported chromatographicmethods) or critical analytical reagents.

Experimental

Apparatus

The fluorescence spectra and measurements were carried outusing a Perkin–Elmer UK model LS 45 luminescence spectrom-eter, equipped with a 150 W xenon arc lamp and grating excita-tion and emission monochromators for all measurements and aPerkin–Elmer recorder. Slit widths for both monochromatorswere set at 10 nm. A 1 cm quartz cell was used. A Hanna poten-tiometer, Model pH equipped with glass–calomel electrode com-bination was used.

Materials and reagents

All the reagents used were of analytical reagent grade. Pure pre-gabalin was kindly provided by Pfizer (Sandwich, UK). Lyrica®

* Correspondence to: N. El-Enany, Department of Analytical Chemistry,Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt.E-mail: [email protected]

Department of Analytical Chemistry, Faculty of Pharmacy, University of Man-soura, 35516, Mansoura, Egypt

Research articles

Received: 05 May 2010, Revised: 09 June 2010, Accepted: 15 June 2010, Published online in Wiley Online Library: 25 August 2010

(wileyonlinelibrary.com) DOI 10.1002/bio.1235

342

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capsules (labeled to contain 75 mg PG per capsule), batch no.0744019, product of Pfizer, were obtained from commercialsources in the local pharmacy. Fluorescamine was purchasedfrom Sigma (St Louis, MO, USA). A stock solution containing0.04% (w/v) of fluorescamine was freshly prepared in acetone.Sodium hydroxide (BDH, UK) was prepared in a 0.02 M aqueoussolution. Acetone was obtained from Sigma. Aqueous boratebuffer solution (0.02 M, pH = 10) was prepared by mixing appro-priate volumes of 0.02 M boric acid with 0.02 M sodium hydroxideand adjusting the pH to pH 10.0 with a pH meter.

Standard sample preparation

Stock solution of PG was prepared by dissolving 10.0 mg of thedrug in 100 mL of distilled water. This solution was further dilutedwith the same solvent as appropriate to obtain the working con-centration range. The stock solution was stable for 15 days whenkept in the refrigerator.

General recommended procedures

Procedure for calibration graph. Aliquots of PG solution con-taining a final drug concentration of 0.01–0.30 mg mL-1 weretransferred into a series of 10.0 mL volumetric flasks. To each flask,1 mL of 0.02 M borate buffer of pH 10 was added followed by0.8 mL of 0.04% (w/v) fluorescamine solution and mixed well.Then the solution was completed to the volume with distilledwater.

The fluorescence of the resulting solutions was measured at487 nm after excitation at 390 nm. The blank experiment wascarried out simultaneously. The corrected fluorescence intensitywas plotted vs the final drug concentrations (mg mL-1) to obtainthe calibration graph; alternatively the regression equation wasderived.

Procedure for determination of the studied drug in capsules.The contents of the 10 Lyrica® capsules were emptied, mixed welland weighed. A weighed quantity of the powder from capsulesequivalent to 40.0 mg pregabalin was transferred into a 100 mLvolumetric flask, extracted with 3 ¥ 30 mL of distilled water andfiltered if needed into a 100 mL measuring flask, then the volumewas completed to 100 mL with distilled water to prepare a stocksolution of 400 mg mL-1. This solution was further diluted with thesame solvent as appropriate to obtain the working concentra-tion. Aliquots covering the working concentration range (cited inTable 1) were transferred into a series of 10 mL volumetric flasksand the procedure was performed as described under the ‘Pro-

Figure 1. Structural formula of pregabalin.

200 300 400 500 6000

200

400

600

800

1000

B B`

A'A

Wavelength(nm)

Flu

ore

scen

ce in

ten

sity

Figure 2. Fluorescence spectra of: (A, A′) blank fluorescamine at pH 10.0; (B, B′) pregabalin(0.3 mg mL-1) with fluorescamine at pH 10.0; (A, B) excitation spectra; (A′, B′) emission spectra.

Table 1. Performance data for the proposed method

Parameter Pregabalin

Concentration range (mg mL-1) 0.01–0.30Limit of detection (LOD) (mg mL-1) 0.0017Limit of quantification (LOQ) (mg mL-1) 0.005Correlation coefficient (r) 0.9999Slope 1513.6Intercept 26.7Sy/x = Standard deviation of the residual 1.3Sa =Standard deviation of the intercept 0.78Sb = Standard deviation of the slope 5.05%RSD = relative standard deviation 0.85%error = Percentage error 0.35

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Effect of pH

7 8 9 10 11 12 130

250

500

750

pH

RF

I

Figure 3. Effect of pH on the relative fluorescence intensity of the reactionproduct of pregabalin (0.3 mg mL-1) with fluorescamine.

0.0 0.5 1.0 1.5 2.0 2.50

250

500

750

volume of 0.04% fluorescamine

RF

I

Figure 4. Effect of volume of 0.04% fluorescamine on the relative fluorescenceintensity of the reaction product of pregabalin (0.3 mg mL-1) at pH 10.0.

Table 2. Application of the proposed and comparison methods to the determination of the pregabalin in pure form

Parameters The proposed method Comparison method (5)Concentration

taken (mg mL-1)Concentration

found (mg mL-1)Percentage

recoveryConcentration

taken (mg mL-1)Percentage

recovery

0.01 0.0099 99.000.02 0.0200 100.00 0.5 100.890.05 0.0493 98.60 1.0 100.680.10 0.1010 101.00 2.0 98.620.20 0.2003 100.15 3.0 100.510.30 0.2995 99.83

x ± S.D. 99.76 � 0.86 100.17 � 1.05t 0.68 (2.31)*F 1.49 (5.41)*

Each result is the average of three separate determinations.*Values in parentheses are the tabulated t and F values, at p = 0.05.

Table 3. Validation of the proposed method for the determination of pregabalin in pure form

Parameter Pregabalin concentration (mg mL-1)0.05 0.10 0.30

Intraday % Found 100.73 99.86 100.10100.82 100.31 101.03

99.50 99.82 100.41Mean ( x ) 100.5 99.99 100.51SD 0.74 0.27 0.47%RSD 0.73 0.27 0.47%error 0.42 0.16 0.27

Interday % Found 99.6 100.05 99.1101.35 99.88 101.38

99.03 100.50 99.48Mean ( x ) 99.99 100.14 99.99SD 1.21 0.32 1.22%RSD 1.21 0.32 1.22%error 0.70 0.18 0.71

Each result is the average of three separate determinations.344

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cedure for calibration graph’ section. The nominal content of thecapsules was determined using the corresponding regressionequation or the calibration graph.

Results and discussionFluorescamine, a heterocyclic dione, was chosen as a derivatizingagent because it is intrinsically weakly fluorescent but reactsrapidly with primary amines, amino acids, peptides and proteinsto yield a highly fluorescent derivatives. Excess reagent is rapidlyconverted to a nonfluorescent product by reaction with water.The fluorescence of a solution containing amines plus fluores-camine is proportional to the quantity of free amine groupspresent (14,15).

Fluorescamine is a useful derivatizing agent for determinationof many compounds of pharmaceutical interest, such as oselta-mavir (16), penicillamine (17), sulfonamide residues (18,19), lisi-nopril (20), methotrexate (21), vigabatrin and gabapentin (22).

Several trials were performed to measure the absorbance ofPG, and showed that it has no specific absorbance. This problemis aggravated when it is necessary to determine the drug spe-cially in pharmaceutical preparation.

The aliphatic nature of PG and the presence of a primary aminogroup which is susceptible to derivitazation with fluorescamineinitiated the present study. PG was found to form a highly fluo-rescent derivative with a wavelength of maximum emission at487 nm after excitation at 390 nm (Fig. 2).

Optimization of experimental conditions

The derivatization reaction between PG and fluorescamine pro-ceeded in a borate buffer at room temperature instantaneously.The spectrofluorimetric properties of the reaction product as wellas the different experimental parameters affecting the develop-ment of the reaction product and its stability were carefully inves-tigated and optimized. Such factors were changed individually

Table 4. Application of the proposed and comparison methods to the determination of the studied drug in dosage forms

Pharmaceutical preparation The proposed method Comparison method (5)Concentration

taken (mg mL-1)Concentration

found (mg mL-1)Percentage

recoveryConcentration

taken (mg mL-1)Percentage

recovery

Lyrica® capsule (75 mgpregabalin/capsule)a

0.05 0.0505 101.01 0.50 101.010.20 0.2004 100.20 1.00 99.430.30 0.2957 98.57 2.00 99.3

x ± SD 99.93 � 1.24 100.87� 1.23t 0.015 (2.776)*F 1.704 (19.00)*Nominal content of capsule (75 mg) 74.46 � 0.645

Each result is the average of three separate determinations.*Values in parentheses are the tabulated t and F values, at p = 0.05.aProduct of Pfizer (batch no. 0744019; expiry date October 2010).

Table 5. Validation of the proposed method for the determination of pregabalin in capsule form

Parameter Pregabalin concentration (mg mL-1)0.05 0.3

Intraday % Found 98.25 99.04100.34 99.23101.39 101.74

Mean ( x ) 99.99 100SD 1.6 1.5%RSD 1.6 1.5%error 0.92 0.87

Interday % Found 99.6 99.1101.35 101.38101.01 98.56

Mean ( x ) 100.65 99.68SD 0.93 1.5%RSD 0.92 1.5%error 0.53 0.87

Each result is the average of three separate determinations.

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while the others were kept constant. The factors include pH, con-centration of the reagent, reaction time and temperature.

Effect of pH. Reactions of amines with fluorescamine have beenshown to be pH-dependent (23). It was found that the fluores-cence developed only in alkaline medium and completely disap-peared in acidic medium. Therefore, the study pH was restrictedto the range 7.5–12 using 0.02 M borate buffers. It was found thatincreasing the pH resulted in a subsequent increase in the fluo-rescence intensity of the reaction product up to pH 9.5, remain-ing constant until pH 10.5, as shown in Fig. 3. Therefore, pH 10 �0.5 was chosen as the optimum pH for the study.

Effect of fluorescamine concentration. The influence of theconcentration of fluorescamine was studied using differentvolumes of 0.04% (w/v) of the reagent solution. It was found thatthe reaction of fluorescamine with PG was detectable using0.1 mL of the reagent. Increasing the volume of the reagent pro-duced a proportional increase in the fluorescence intensity of thereaction product up to 0.6 mL and remains constant up to 1 mL.Therefore, 0.8 � 0.2 mL of 0.04% of fluorescamine solution waschosen as the optimal volume of the reagent (Fig. 4).

Effect of reaction time. Different time intervals were tested toascertain the time after which the solution attained its highestfluorescence intensity. It was found that the reaction took placeinstantaneously after mixing PG with the reagent.

Effect of heating. Increasing the reaction temperature higherthan room temperature resulted in a subsequent decrease in thefluorescence intensity of the reaction product.Therefore, the reac-tion was carried out at room temperature (24).

Effect of the time on stability of the product. The fluores-cence of the reaction product reached the maximum intensityimmediately and remained constant at room temperature for atleast 2 h.

Analytical Performance

The validity of the proposed method was tested regarding linear-ity, specificity, accuracy, repeatability and precision according toICH Q2B recommendations (25).

Linearity. Using the above procedure, a linear regression equa-tion was obtained. The regression plot showed that there was alinear dependence of the fluorescence intensity on the concen-tration of the drug over the range cited in Table 1. Linear regres-sion analysis of the data gave the following equation:

F C r= + =26 7 1513 6 0 9999. . ( . )

where F is the fluorescence intensity, C is the concentration of thedrug in mg mL-1 and r is the correlation coefficient. The limit ofquantification (LOQ) and limit of detection (LOD) were calculatedaccording to ICH Q2B (25). The results are shown in Table 1. Thevalues of LOQ and LOD were calculated according to the follow-ing equation (25):

LOQ LOD= =10 3 3σ σS S; .

where s is the standard deviation of the intercept of regressionline and S is the slope of the calibration curve.

The proposed method was evaluated for the accuracy as per-centage relative error (%error) and the precision as percentagerelative standard deviation (%RSD) (Tables 1 and 2).

Accuracy. To test the validity of the proposed method it wasapplied to the determination of pure samples of PG over theworking concentration range. The results obtained were ingood agreement with those obtained using the comparisonmethod (5). Using Student’s t-test and variance ratio F-test (26)revealed no significant difference between the performance ofthe two methods regarding the accuracy and precision, respec-tively (Table 2). The comparison method was based onthe derivitization of PG with 7-chloro-4-nitrobenzofurazon (5).The absorbance of the reaction product was measured at460 nm and the concentration was rectilinear over the range0.5–7.0 mg mL-1.

The validity of the method was proven by statistical evaluationof the regression lines regarding the standard deviation of theresiduals (Sy/x), the standard deviation of the intercept (Sa) andstandard deviation of the slope (Sb). The results are summarized inTable 1. The small values for these figures point to the low degreeof scattering of the points around the calibration line and highprecision.

Precision. The repeatability was determined by applying theproposed method for the determination of three concentrationsof PG in the pure form three successive times, and the results are

-5.50 -5.25 -5.00 -4.75 -4.50 -4.25 -4.00

1.5

2.0

2.5

3.0

slope = 0.675

Log [fluorescamine]

Lo

g R

FI

-7.50 -7.25 -7.00 -6.75 -6.50 -6.25 -6.00 -5.75 -5.501.5

2.0

2.5

3.0

Log [pregabalin]

Lo

g R

FI

Slope= 0.69

A

B

Figure 5. Stoichiometry of the reaction between pregabalin and fluorescamineadopting limiting logarithmic method. (A) Log fluorescence vs log [fluorescamine].(B) Log fluorescence vs Log [pregabalin].

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listed in Table 3. Low percentage error and low percentage RSDindicate high accuracy and high precision of the proposedmethod.

Interday precision was determined through repeated analysisof PG in the pure form, using the concentrations 0.05, 0.10 and0.30 mg ml-1 for a period of three successive days. The results aresummarized in Table 3.

Robustness of the method. The robustness of the methodadopted is demonstrated by the constancy of the fluorescenceintensity with minor changes in the experimental parameterssuch as the volume of fluorescamine (0.04%), 0.8 � 0.2 mL, andchange in the pH, 10 � 0.5. These minor changes that may takeplace during the experimental operation did not affect the fluo-rescence intensity of the reaction product.

Application of the proposed method to the analysis ofcommercial capsules

The proposed method was applied to the determination of PG incapsules. The method was tested for linearity, specificity, accu-racy, repeatability and precision according to ICH Q2B recom-mendations (25).

Selectivity. The selectivity of the method was investigated byobserving any interference encountered from the commoncapsule excipients, such as lactose monohydrate, corn starch andtalc. These excipients did not interfere with the proposedmethod.

Accuracy. The results of the proposed method were statisticallycompared with those obtained using the comparison method (5).Statistical analysis (26) of the results, using Student’s t-test andvariance ratio F-test revealed no significant difference betweenthe performance of the proposed and comparison methodregarding the accuracy and precision (Table 4).

Precision. The repeatability was performed by applying the pro-posed methods for the determination of two concentrations ofPG in capsule three successive times, and the results are listed inTable 5.

The Interday precision was determined through repeatedanalysis of PG in capsules, using the concentrations shown inTable 5 for a period of three successive days. The results are sum-marized in Table 5.

Mechanism of the reaction

The stoichiometry of the reaction was studied adopting the lim-iting logarithmic method (27). The fluorescence intensity of thereaction product was alternatively measured in the presence ofexcess of either fluorescamine or PG. A plot of log fluorescence vslog [fluorescamine] and log [PG] gave straight lines, the values ofthe slopes are 0.67 and 0.69 respectively (Fig. 5). Hence, it is con-cluded that, the molar reactivity of the reaction is 0.67/0.69, i.e.the reaction proceeds in the ratio of 1:1. Based on the observedmolar ratio, depending on the presence of one primary aminogroup and by analogy to previous similar reports (22), the reac-tion pathway is postulated to proceed as shown in Scheme 1.

ConclusionThe proposed method has the advantage of being rapid, simpleand sensitive, with low cost and with no need for prior extractionprocedure. So it is suitable for routine analysis of PG in qualitycontrol laboratories. Also, it could be applied for the determina-tion of pregabalin in pharmaceutical preparations.

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Scheme 1. Proposed pathway of the reaction between pregabalin and fluorescamine in a borate buffer of pH 10.0.

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