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Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 136–140

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.e lsev ier .com/ locate / jpba

hort communication

stability indicating simultaneous dual wavelength UV–HPLC method for theetermination of potential impurities in fampridine active pharmaceutical

ngredient

aji Thomas ∗, Sanjeev Shandilya, Amber Bharti, Ashutosh Agarwalubilant Life Sciences Ltd., Analytical Research Department, R&D Centre, C-26, Sector-59, Noida, Uttar Pradesh 201 301, India

r t i c l e i n f o

rticle history:eceived 2 July 2011eceived in revised form 17 August 2011ccepted 15 September 2011vailable online 19 September 2011

eywords:

a b s t r a c t

A novel, sensitive, stability indicating simultaneous dual wavelength reverse phase UV–HPLC methodhas been developed for the quantitative determination of potential impurities of fampridine active phar-maceutical ingredient. Efficient chromatographic separation was achieved on a C18 stationary phase ingradient mode and quantitation by ultraviolet dual wavelength detection. The method was validatedaccording to ICH guidelines with respect to specificity, precision, linearity and accuracy. Regression anal-ysis showed correlation coefficient value greater than 0.999 for fampridine and its seven impurities.

ampridinempuritytress studyalidation

Detection limit as low as 0.003% was achieved for fampridine N-oxide and 0.01% for other impurities.Accuracy of the method was established based on the recovery obtained between 93.3% and 110.0%for all impurities. The method was found to be specific, selective to the degradation products androbust. Peak purity analysis by PDA detector confirmed the specificity of the method. Major degrada-tion of the drug substance was found to occur under oxidative stress conditions to form fampridineN-oxide.

. Introduction

Dalfampridine is the first drug approved in the United States bySFDA to improve walking in patients with multiple sclerosis and

s chemically known as 4-aminopyridine or fampridine. Ampyra®

s an extended release tablet formulation of dalfampridine whichas previously called Fampridine-SR. Fampridine is a potassium

hannel-blocker that enhances conduction in focally demyeli-ated axons, improves synaptic transmission and potentiatesuscle contraction. It has shown efficacy in patients with all fiveajor types of multiple sclerosis namely relapsing, remitting, sec-

ndary progressive, progressive relapsing and primary progressive1–4].

A HPLC method has been reported in literature for the quantita-ive determination of fampridine in pharmaceutical formulations5]. However extensive survey revealed that no stability indicatingPLC method has been reported including major pharmacopoeias

uch as USP, EP, JP and BP for the quantitative determination of

otential impurities of fampridine active pharmaceutical ingredi-nt. Therefore it was felt necessary to develop an accurate, rapid,pecific and stability indicating method for the determination of

∗ Corresponding author. Tel.: +91 120 4362210; fax: +91 120 2580033.E-mail address: saji thomas@jubl.com (S. Thomas).

731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2011.09.009

© 2011 Elsevier B.V. All rights reserved.

potential impurities of fampridine. The present ICH drug stabilitytest guideline [6] suggests that stress studies should be carried outon a drug substance to establish its inherent stability character-istics, leading to separation of degradation impurities and hencesupporting the suitability of the proposed analytical procedure,which must be fully validated [7,8].

The presence of impurities in active pharmaceutical ingredient(API) can have a significant impact on the quality, safety and effi-cacy of drug products. Therefore, it is important to have a stabilityindicating validated method for the quantitative determination ofpotential impurities in the drug substance.

The present work deals with method development, method val-idation and forced degradation study of fampridine. One of theimpurities was found to have absorbance at higher wavelengthhence simultaneous dual wavelength detection was proposed. Tothe best of our knowledge, no LC methods have been reported so farfor the impurity profile study of fampridine active pharmaceuticalingredient.

2. Experimental

2.1. Materials and chemicals

Sample of fampridine API (Batch No. FAM/11001), standards ofImp-6, Imp-7 were obtained from Chemical Research Department

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 136–140 137

Table 1

List of potential impurities in fampridine.

N

NH2

Fampridine

S. no. Name Structure Mol. wt Assigned code Source

1 Isoniacin

N

COOH

123.03 Imp-1 Process impurity

2 NiacinN

COOH

123.03 Imp-2 Process impurity

3 Isonicotinamide

N

CONH2

122.05 Imp-3 Process impurity

4 3-AminopyridineN

NH2

94.05 Imp-4 Isomeric impurity

5 2-AminopyridineN NH2

94.05 Imp-5 Isomeric impurity

6 Fampridine-N-oxide N

NH2

O

110.05 Imp-6 Oxidative degradant

7 3-Hydroxy-4-aminopyridine

N

NH2OH

110.05 Imp-7 Metabolite

oI(pHstb

2

mMcpa0pATwm

f Jubilant Life Sciences Limited (Noida, India). Imp-1, Imp-2, Imp-3,mp-4 and Imp-5 were purchased from Sigma Aldrich CorporationSt. Louis, MO, USA). Deionized water was prepared using a Milli-Qlus water purification system from Millipore (Bedford, MA, USA).PLC grade methanol, acetonitrile, 1-octane sulphonic acid sodium

alt monohydrate, ammonium acetate, orthophosphoric acid, andriethylamine were purchased from Qualigens India Limited (Mum-ai, India).

.2. High performance liquid chromatography

Samples were analysed on a Waters alliance 2690 separationodule equipped with 2487 UV detector (Waters Corporation,ilford, MA, USA) using a Xterra RP18 (250 mm × 4.6 mm, parti-

le size 5 �m, Waters Corporation, Milford, MA, USA). The mobilehase consisted of a mixture of A, 10 mM 1-octane sulphoniccid sodium salt monohydrate, 10 mM ammonium acetate and.1% triethylamine adjusted to pH 4.00 ± 0.05 with ortho phos-horic acid–methanol (95:5, v/v) and B, methanol–mobile phase

(80:20, v/v) with a timed gradient programme (TminA:B): T097:3,

1590:10, T3070:30, T3597:3, and T6097:3. The injection volumeas 20 �L for a sample concentration of 0.75 mg/mL prepared inobile phase-A. Detector wavelength was fixed at 240 nm and

282 nm and the column was maintained at 40 ◦C throughout theanalysis.

2.3. Photo stability

Photo stability studies were carried out using a photo stabilitychamber model TP 0000090G (Thermo Lab equipments Pvt. Ltd.,Mumbai, India). Study was performed on dark control and pho-tolytic exposed sample in a way to get the minimum exposureof 1.2 million lux hours for light and 200 W h/m2 for ultravioletregion.

2.4. Preparation of stock solution for method validation

A test preparation of 750 �g/mL of fampridine API sample wasprepared by dissolving the appropriate amount in mobile phase-A.A stock solution of impurities was prepared by dissolving 7.5 mgeach of Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7 and7.5 mg of fampridine in 20 mL of mobile phase-A and furtherdiluted 5–100 mL with mobile phase-A. From this stock solu-

tion a standard solution containing 1.125 �g/mL of each impurityand 1.125 �g/mL of fampridine was prepared. This standard solu-tion was also used for checking solution stability and robustnessparameters.

138 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 136–140

Table 2Method validation summary report.

Parameter Impurity-1 Impurity-2 Impurity-3 Impurity-4 Impurity-5 Impurity-6 Impurity-7 Fampridine

System suitabilityRT 2.77 2.99 4.97 10.65 14.69 5.99 18.49 11.97RRT 0.23 0.25 0.42 0.89 1.23 0.50 1.54 1.00Rs – 1.31 9.85 14.71 5.30 3.89 8.27 2.92N 4469 5278 8509 17,642 17,950 6159 25,179 6927T 1.03 1.18 1.09 1.05 1.09 1.03 1.02 1.76RF 1.92 2.79 2.29 0.67 1.13 0.23 1.25 1.00

Linearityr 0.9999 0.9999 0.9999 0.9998 0.9999 1.0000 0.9998 0.9999Slope 181,191 124,531 151,834 521,853 308,426 956,989 278,843 347,526Intercept −45 −224 63 −443 −277 −142 −1126 −159Detection limit (%) 0.01 0.01 0.01 0.01 0.01 0.003 0.01 0.01Quantitation limit (%) 0.03 0.03 0.03 0.03 0.03 0.01 0.03 0.03Precision % RSD (n = 6) 1.17 1.89 2.73 0.77 3.42 0.81 2.39 1.41

Accuracy at QL level (n = 3)Amount added (%) 0.030 0.033 0.030 0.030 0.030 0.010 0.030 0.030Amount recovered (%) 0.029 0.033 0.029 0.028 0.028 0.011 0.028 0.030% recovery 96.67 100.00 96.67 93.33 93.33 110.00 93.33 100.00

Accuracy at 100% level (n = 3)Amount added (%) 0.152 0.151 0.150 0.150 0.149 0.151 0.149 0.150Amount recovered (%) 0.144 0.148 0.144 0.144 0.142 0.145 0.141 0.150% recovery 94.74 98.01 96.00 96.00 95.30 96.03 94.63 100.00

Accuracy at 150% level (n = 3)Amount added (%) 0.228 0.226 0.224 0.225 0.224 0.226 0.224 0.225Amount recovered (%) 0.216 0.221 0.216 0.216 0.214 0.219 0.212 0.230

n SP resc

3

3

wIr

% recovery 94.74 97.79 96.43

, number of determinations; RT, retention time; RRT, relative retention time; Rs , Uorrelation coefficient.

. Results and discussion

.1. Method development

The main aim of the chromatographic method developmentas to achieve the separation of closely eluting impurities namely

mp-1, Imp-2, Imp-4, Imp-5 and to retain fampridine peak. Impu-ities were named based on their elution pattern. The details of

Impu

rity

-6 -

5.9

92

Fam

prid

ine

- 11

.96

9

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

M

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

Impu

rity

-1 -

2.7

75Im

puri

ty-2

- 2

.995

Impu

rity

-3 -

4.9

71

Impu

rity

-6 -

5.9

93

Impu

rity

-4 -

10.

648

Fam

prid

ine

- 11

.96

9

Impu

rity

-5 -

14.

688

mV

mV

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

M0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

Fig. 1. (a) Chromatogram of fampridine spiked with impurities at 240 nm.

96.00 95.54 96.90 94.64 102.22

olution; RF, response factor; N, number of theoretical plates; T, USP tailing factor; r,

potential impurities of fampridine were listed in Table 1. Fampri-dine, Imp-4 and Imp-5 were co-eluted when different stationeryphases like C8, C18, and phenyl were used in different mobilephases containing phosphate, ammonium and combination of

these two buffers along with acetonitrile, methanol, with pH rang-ing from 3 to 6. Tailing was observed to be more at higher pH.Use of Xterra-RP18 column and introduction of 1-octane sul-phonic acid sodium salt ion pair reagent was the major factor

inutes

18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

Impu

rity

-7 -

18.

491

inutes18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

a

b

(b) Chromatogram of fampridine spiked with impurities at 282 nm.

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 58 (2012) 136–140 139

Fig. 2. (a) Typical chromatogram of fampridine under stress conditions at 240 nm: (a) oxidative degradation, (b) acid hydrolysis, (c) base hydrolysis, (d) thermal degradation,and (e) photolytic degradation. (b) Typical chromatogram of fampridine under stress conditions at 282 nm: (a) oxidative degradation, (b) acid hydrolysis, (c) base hydrolysis,(d) thermal degradation, and (e) photolytic degradation.

iactflm2

4

fisc5cis

n achieving the desired resolution of closely eluting impuritiesnd retention of fampridine peak. Use of triethylamine and 40 ◦Column temperature reduced the tailing of fampridine peak. Inhe new method, resolution between fampridine and Imp-4 wasound to be more than 2.0 (Table 2) and the peak tailing wasess than 2.0. After several experiments for gradient profile, chro-

atographic conditions were optimized as described under Section.2.

. Method validation

The newly developed method was validated to establish speci-city, precision, linearity, accuracy, sensitivity, robustness andystem suitability according to ICH guidelines. Validation study wasarried out for the analysis of Imp-1, Imp-2, Imp-3, Imp-4, Imp-

, Imp-6 and Imp-7. The system suitability and selectivity werehecked by injecting 750 �g/mL of fampridine solution contain-ng 0.15% of all impurities (Fig. 1). Method validation results areummarized in Table 2.

4.1. Specificity

Specificity is the ability of the method to unequivocally assessthe analyte response in the presence of its potential impurities.Specificity was established by injecting fampridine co-spiked withpotential impurities. Forced degradation study was performed todemonstrate the stability indicating power and specificity of theproposed method. The stress conditions employed for degradationstudy included acid (1 N HCl, 80 ◦C, for 3 h), base (1 N NaOH, 80 ◦C,for 3 h), thermal (105 ◦C, for 24 h), oxidation (30% H2O2, 30 ◦C, for15 min), photolytic (1.2 million lux h/and 200 W h/m2) and waterhydrolysis (80 ◦C, for 3 h).

4.2. Results of forced degradation and identification of majordegradant impurity

Fampridine molecule was found to be very stable during stressconditions in acid, base, thermal, photolytic and water hydroly-sis. In oxidative degradation condition, significant degradation wasobserved and the impurity formed at RRT of 0.51 and was identified

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[6] ICH Guidelines, Stability testing of new drug substances and drug products: text

40 S. Thomas et al. / Journal of Pharmaceutic

s fampridine N-oxide. Chromatograms of forced degradation studyave been depicted in Fig. 2. Peak purity of stressed samples of

ampridine was checked using a SPD M20A photo diode arrayetector (Shimadzu Corporation, Japan). Degradation studies andeak purity test results derived from PDA detector confirmed thathe fampridine peak was homogenous and pure in all the stressamples. The developed LC method was found to be specific inhe presence of Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-and their degradation products confirmed the stability indicatingower of the newly developed method.

.3. Precision

The precision of the related substances method was checkedy injecting six individual preparations of (750 �g/mL) fampridinepiked with 0.15% of each impurity. Percentage RSD for peak areasf each impurity was calculated. Precision was also determinedy performing the same procedures on a different day (inter-dayrecision). The intermediate precision (ruggedness) of the methodas also evaluated by a different analyst and different instrument

n the same laboratory. Percentage RSD of areas of each impurityas within 4.0, confirming the good precision at low level of theeveloped analytical method.

.4. Sensitivity

The sensitivity was determined by establishing the detectionimit (DL) and quantitation limit (QL) for all impurities by injectingseries of dilute solutions with known concentration. The detec-

ion limit and quantitation limit for Imp-1, Imp-2, Imp-3, Imp-4,mp-5 and Imp-7 were about 0.01% and 0.03% respectively. DL andL for Imp-6 were 0.003% and 0.01% respectively, indicating high

ensitivity of the method. The precision at QL concentration for allmpurities was below 4%.

.5. Linearity and range

A linearity test solution for related substance method was pre-ared by diluting the impurity stock solution to the requiredoncentrations. The solutions were prepared at six concentra-ion levels. From QL to 150% of the permitted maximum levelf impurity (i.e. QL, 0.075%, 0.1125%, 0.15%, 0.1875% and 0.225%)as subjected to linear regression analysis with the least squaresethod. Calibration equation obtained from regression analysisas used to calculate the corresponding predicted responses. The

esiduals and sum of the residual squares were calculated fromhe corresponding predicted responses. The correlation coefficientbtained was greater than 0.999 for all impurities. The resulthowed an excellent correlation between the peak area and con-entration of all impurities. Standard deviation of peak area wasignificantly low and RSD was below 4.0%. Linearity was estab-ished between ranges of QL to 0.225% of the analyte concentration750 �g/mL).

.6. Accuracy

The accuracy of the method was evaluated in triplicate athree concentration levels, i.e. QL, 100% level (0.15% of the drugubstance) and 150% level (0.225% of the drug substance). The per-entage of recovery for each impurity was calculated at each level.

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Biomedical Analysis 58 (2012) 136–140

The percentage recovery of all impurities ranged from 93.3% to110.0% indicating the accuracy of the method.

4.7. Robustness

To determine the robustness of the developed method, exper-imental conditions were deliberately changed and the resolutionbetween fampridine and Imp-4 was evaluated. Close observa-tion of analysis results of deliberately changed chromatographicconditions (flow rate, pH, mobile phase composition and columntemperature) revealed that the resolution between Imp-4 and fam-pridine was greater than 2.0, illustrating the robustness of themethod.

4.8. Solution stability

The solution stability of fampridine and its related impuritieswas demonstrated by leaving both spiked and unspiked samplesolution in tightly capped HPLC vials at 25 ◦C in auto sampler. Con-tent of each impurity was determined every 4 h against freshlyprepared standard solution. The solution stability experiments dataconfirmed that sample solutions were stable up to 24 h.

5. Conclusion

The newly developed RP-LC method for quantitative determi-nation of fampridine related substances was found to be sensitive,precise, accurate specific and stability indicating. The major oxida-tive degradant was identified as fampridine N-oxide. This newlydeveloped method has been validated as per regulatory require-ments and can be used for routine and stability studies for thequantitative determination of potential impurities in fampridinedrug substance.

Acknowledgements

The authors are thankful to the management of Jubilant Life Sci-ences Limited for providing necessary facilities. Authors would liketo thank Dr. Hawaldar Maurya, Mr. Dinesh Vishwakarma, Mr. SarojKumar Paul, Mr. Rameshwar Tripathi and Ms. Samreen Siddiqui fortheir co-operation in carrying out this work.

References

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3] T. Vollmer, H.R. Henney, Pharmacokinetics and tolerability of single escalatingdoses of fampridine sustained-release tablets in patients with multiple sclerosis:a phase I–II, open-label trial, Clin. Ther. 31 (2009) 2206–2210.

4] T. Vollmer, A.R. Blight, H.R. Henney III, Steady-state pharmacokinetics a tolerabil-ity of orally administered fampridine sustained-release 10-mg tablets in patientswith multiple sclerosis: a 2-week, open-label, follow-up study, Clin. Ther. 31(2009) 2215–2223.

5] R.F. Donnelly, Chemical stability of 4-aminopyridine capsules, Can. J. HospitalPharm. 57 (2004) 284–287.

and methodology Q1 A (R2), February 2003.7] ICH Guidelines, Validation of analytical procedures stability testing of new drug

substances and drug products: text and methodology Q2 (R1), November 2005.8] ICH Guidelines, Impurities in new drug substances Q3A (R2), October 2006.