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Journal of Pharmaceutical and Biomedical Analysis 60 (2012) 86– 90

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j ourna l ho me p a ge: www.elsev ier .com/ locate / jpba

hort communication

uantification of potential impurities by a stability indicating UV-HPLC methodn niacinamide active pharmaceutical ingredient

aji Thomas ∗, Amber Bharti, Kalsang Tharpa, Ashutosh Agarwalubilant Life Sciences Ltd., Analytical Research Department R&D Centre, C-26, Sector-59, Noida, Uttar Pradesh 201301, India

r t i c l e i n f o

rticle history:eceived 29 August 2011eceived in revised form 28 October 2011ccepted 29 October 2011vailable online 6 November 2011

a b s t r a c t

A sensitive, stability indicating reverse phase UV-HPLC method has been developed for the quantitativedetermination of potential impurities of niacinamide active pharmaceutical ingredient. Efficient chro-matographic separation was achieved on C18 stationary phase in isocratic mode using simple mobilephase. Forced degradation study confirmed that the newly developed method was specific and selectiveto the degradation products. Major degradation of the drug substance was found to occur under oxidative

eywords:iacinamide

mpurityorced degradationalidation

stress conditions to form niacinamide N-oxide. The method was validated according to ICH guidelineswith respect to specificity, precision, linearity and accuracy. Regression analysis showed correlation coef-ficient value greater than 0.999 for niacinamide and its six impurities. Detection limit of impurities wasin the range of 0.003–0.005% indicating the high sensitivity of the newly developed method. Accuracy ofthe method was established based on the recovery obtained between 93.3% and 113.3% for all impurities.

. Introduction

Niacinamide (nicotinamide or nicotinic acid amide) is chem-cally known as pyridine-3-carboxamide was first isolated fromorse erythrocytes in 1935. It is a form of vitamin B3, and itseficiency and adverse effects are well documented [1]. Both niaci-amide and niacin are converted into NAD and NADP, in vivo [2].iacinamide inhibits ADP-ribosyl transferring enzymes modulat-

ng immune cell function and cell [3].There are quite a number of reports on a simultaneous assay

etermination of niacinamide and niacin in samples such as foodnd multi-vitamin tablets by LC-UV [4–7]; and stability indicat-ng LC-UV method for the determination of niacinamide in theresence of riboflavin, pyridoxine and thiamine is also reported8]. Current United States Pharmacopoeia [9] and European Phar-

acopoeia [10] employ assay procedures without concerninghe related substance of niacinamide. There is no report onhromatographic separation of related substances (process ortressed) in niacinamide partly because niacinamide is classi-ed as food additive rather than pharmaceutical agent thereby

ts formal safety evaluation remained less important. The con-

ern is that since niacinamide is given in high doses, its purityeeds to be scrutinized. Therefore it was felt necessary to developn accurate, specific and stability indicating method for the

∗ 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.2011.10.033

© 2011 Elsevier B.V. All rights reserved.

determination of potential impurities of niacinamide. The presentICH drug stability test guideline [11] suggests that stress stud-ies should be carried out on a drug substance to establish itsinherent stability characteristics, leading to separation of degra-dation impurities and hence supporting the suitability of theproposed analytical procedure, which must be fully validated[12].

The present work deals with method development, methodvalidation and forced degradation study of niacinamide. Niaci-namide is commercially manufactured by the catalytic hydrol-ysis of 3-cyanopyridine. Therefore, the positional isomers of3-cyanopyridine and undesired hydrolyzed products were con-sidered as potential impurities for method development. To thebest of our knowledge, this is the first report on identification ofstress related impurities of niacinamide and method for quantita-tive determination of process related impurities.

2. Experimental

2.1. Materials and reagents

Sample of niacinamide API and standards of niacin, isonico-tinic acid, niacinamide-N-oxide, isonicotinamide, picolinamide and3-cyanopyridine were obtained from Chemical Research and Devel-

opment Department, Jubilant Life Sciences Limited (Noida, India).Deionized water was prepared using a Milli-Q plus water purifi-cation system from Millipore (Bedford, MA, USA). HPLC gradeacetonitrile, analytical reagent grade of ammonium acetate, acetic

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S. Thomas et al. / Journal of Pharmaceut

cid, sodium hydroxide, hydrogen peroxide and hydrochloric acidere purchased from Merck India Limited (Mumbai, India).

.2. High performance liquid chromatography

Samples were analyzed on Waters alliance 2690 separationodule equipped with 2487 UV detector and 2996 PDA detector

or stability study (Waters Corporation, Milford, MA, USA) using annertsil ODS 3V (250 mm × 4.6 mm, 5.0 �m, GL Sciences Inc., Tokyo,apan). The mobile phase consisted of 20 mM ammonium acetatedjusted to pH 5.00 ± 0.05 with acetic acid–acetonitrile (970:30,/v). Mobile phase was used as diluent for sample preparation. Thenjection volume was 10 �L (1.50 mg/mL sample concentration)nd the wavelength of detector was set to 254 nm. The column wasaintained at 30 ◦C throughout the analysis.

.3. Sample preparation for forced degradation studies

About 55 mg each of niacinamide sample were weighed intoour different 20 mL volumetric flasks and labeled them as A, B, Cnd D. Two milliliters each of 1 N hydrochloric acid, 1 N sodiumydroxide and Milli-Q water (pH 7.01), and 5 mL of 1% (v/v)ydrogen peroxide were added in volumetric flask A, B, C and D,espectively. Volumetric flasks A, B and C were kept in a water batht 80 ◦C for 3 h and volumetric flask D at room temperature for 3 h.he excess of acid or base in volumetric flask A and B were neutral-zed and made up to the volume with diluent. Corresponding blankolutions were prepared following the sample procedure withoutiacinamide sample. Thermal degradation was done at 105◦ C for4 h on solid sample. Photolytic degradation was performed byeeping 500 mg of each sample in two separate loss on drying (LOD)ottles in a photo stability chamber model TP 0000090G (Thermoab equipments Pvt. Ltd., Mumbai, India). One bottle was cov-red with lid and then with aluminum foil (dark control) whereasnother bottle (photolytic exposed sample) was covered with lido get a minimum exposure of 1.2 million lux hours for light and00 Wh/m2 for ultraviolet region. A 1.50 mg/mL sample was pre-ared for thermal degradation and photolytic degradation samples.

. Results and discussion

.1. HPLC method development

Initially niacinamide sample was analyzed by pharmacopoeialethods [9,10] and found that most of the impurities were co-

luting with the main peak. The main objective of the methodevelopment was to achieve efficient separation of impurities

isted in Table 1. Using the literature report, the niacin pKa of 4.8113] and niacinamide pKa of 3.35 [14], formed the basis of choicesver the pH of the mobile phase. At pH 5.00, the protonation ofmide moiety of niacinamide, Imp-3 and Imp-4 is suppressed andhe carboxylate ion of Imp-1 and Imp-2 predominates. Therefore,oth Imp-1 and Imp-2 elute earlier on the hydrophobic columnC18) with less protonated or relatively more non-polar com-ounds such as niacinamide, Imp-3 and Imp-4 eluting later. Finallyfter conducting serious of experiments for method development,nd routine optimization experiments, using an Intersil ODS 3V-18 column with ammonium acetate buffer at pH adjusted to.00 ± 0.05 with acetic acid, in combination with 3% acetonitrile,ielded optimum resolution under isocratic condition (Fig. 1A).

.2. Forced degradation studies and identification of major

egradants

Niacinamide molecule was found to be susceptible to acid,ase and oxidative conditions. In oxidative degradation condition,

d Biomedical Analysis 60 (2012) 86– 90 87

significant degradation was observed and the impurity formed atRRT 0.42 was identified as niacinamide N-oxide. In acidic and alka-line conditions niacin was the major degradant. Chromatogramsof forced degradation study have been depicted in Fig. 1B. Degra-dation studies and peak purity test results derived from PDAdetector confirmed that the spectral purity of niacinamide peakwas homogenous thus confirmed the stability indicating power ofthe newly developed method.

4. Method validation

Validation study was carried out for the analysis of Imp-A,Imp-1, Imp-2, Imp-3, Imp-4 and Imp-5. The system suitability andselectivity were checked by injecting 1.50 mg/mL of niacinamidesolution containing 0.15% of all impurities (Fig. 1A). Method vali-dation results are summarized in Table 2.

4.1. Accuracy and precision

Accuracy of the method was evaluated in triplicate at threeconcentration levels, i.e. QL, 0.015% and 0.226% of the analyteconcentration (1.5 mg/mL). The percentage of recovery for eachimpurity was calculated at each level and found in the range of88.89–113.33% (Table 2). The precision of the related substancesmethod was checked by injecting six individual preparations of(1.5 mg/mL) niacinamide spiked with 0.15% of each impurity.Percentage RSD for peak areas of each impurity was calculated(Table 2). Precision was also determined by performing the sameprocedures on a different day (inter-day precision).

4.2. Sensitivity

The sensitivity was determined by establishing the detectionlimit (DL) and quantitation limit (QL) for all the impurities by inject-ing a series of dilute solutions with known concentration. The DLand QL for Imp-1, Imp-2, Imp-3, Imp-4, Imp-5 and Imp-A wereabout 0.005% and 0.015%, respectively indicating high sensitivityof the method.

4.3. Linearity and range

Linearity was established between ranges of QL to 0.225% of theanalyte concentration (1.50 mg/mL) using least squares method.The correlation coefficient obtained was not less than 0.999 for allimpurities. Standard deviation of peak area was significantly lowand %RSD was below 4.1%.

4.4. Robustness and ruggedness

Close observation of analysis results of the deliberately changedin chromatographic conditions (flow rate, pH, mobile phase com-position and column temperature) revealed that the resolutionbetween niacinamide and Imp-3 was greater than 2.0, illustratingthe robustness of the method. The intermediate precision (rugged-ness) of the method was also evaluated by a different analyst anddifferent instrument in the same laboratory with %RSD areas of eachimpurity within 4.0.

4.5. Solution stability

The solution stability of niacinamide and its related substanceswas established by spiked and unspiked sample solution in tightlycapped HPLC vials at 30 ◦C in auto sampler. Content of each impuritywas determined after every 4 h against freshly prepared standard

88 S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 60 (2012) 86– 90

Table 1Niacinamide and potential impurities.

N

NH2

O

Niacinamide

Sl. no. Name Structure Mol. wt. Code Origin

1 Isonicotinic acid N

OHO

123.03 Imp-1 Process impurity

2 Niacin N

COOH

123.03 Imp-2 Process and degradation impurity

3 Isonicotinamide N

NH2O

122.05 Imp-3 Process impurity (positional isomer)

4 Picolinamide

NNH2

O 122.05 Imp-4 Process impurity (positional isomer)

5 3-Cyanopyridine N

CN

104.04 Imp-5 Process impurity (starting material)

6 Niacinamide N-oxide

N

NH2

O

O 138.04 Imp-A Oxidative degradation impurity

Table 2System suitability, response factor, DL, QL, linearity, precision and accuracy.

Parameter Imp-1 Imp-A Imp-2 Imp-3 Niacinamide Imp-4 Imp-5

System suitabilityRT 4.04 4.56 5.03 9.98 11.02 24.90 26.87RRT 0.37 0.41 0.46 0.91 1.00 2.26 2.46Rs – 2.80 2.47 18.85 3.06 24.97 2.54N 7487 10304 10428 15735 15647 17583 18370T 1.11 1.11 1.08 1.01 1.07 1.04 1.01

Linearity (�g/mL) 15.0–226 9.00–226 15.0–226 15.0–226 15.0–226 15.0–226 15.0–226r 0.9999 0.9994 0.9996 0.9992 0.9995 0.9990 0.9984Slope 183,784 635,629 219,994 151,863 202,434 229,253 182,407Confidence interval of slopea ±2002.9 ±12957.7 ±1783.9 ±1427.6 ±1777.4 ±2757.8 ±1968.8Intercept 274 −613 −468 126 140 296 419

Detection limit 0.005% 0.003% 0.005% 0.005% 0.005% 0.005% 0.005%Quantitation limit 0.015% 0.009% 0.015% 0.015% 0.015% 0.015% 0.015%Precision %RSD (n = 6) 1.75 4.01 1.66 0.94 2.95 3.88 1.29Accuracy at QL level (n = 3)

Amount added (%) 0.015 0.009 0.015 0.015 0.015 0.015 0.015Amount recovered (%) 0.014 0.008 0.017 0.015 0.014 0.014 0.015% Recovery 93.33 88.89 113.33 100.00 93.33 93.33 100.00

Accuracy at 100% level (n = 3)Amount added (%) 0.151 0.150 0.149 0.149 0.151 0.150 0.150Amount recovered (%) 0.150 0.147 0.158 0.157 0.149 0.148 0.149% Recovery 99.34 98.00 106.04 105.37 98.68 98.67 99.33

Accuracy at 150% level (n = 3)Amount added (%) 0.226 0.225 0.227 0.224 0.226 0.227 0.225Amount recovered (%) 0.224 0.224 0.240 0.232 0.222 0.221 0.220% Recovery 99.12 99.56 105.73 103.57 98.23 97.36 97.78

n, number of determinations; RT, retention time; RRT, relative retention time; Rs , USP resolution; N, number of theoretical plates; T, USP tailing factor; r, correlation coefficient.a At 95% confidence level.

S. Thomas et al. / Journal of Pharmaceutical and Biomedical Analysis 60 (2012) 86– 90 89

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. Conclusions

The newly developed RP-LC method for quantitative determi-ation of niacinamide and related substances was found to beensitive, precise, accurate, specific and stability indicating. Theajor oxidative degradant was identified as niacinamide N-oxide.

his newly developed method has been validated as per regula-ory requirements and can be used for routine and stability studiesor the quantitative determination of potential impurities in niaci-amide drug substance.

cknowledgements

The authors are thankful to the management of Jubilant Lifeciences Limited for providing necessary facilities. Authors would

gradation.

like to thank Mr. Sanjeev Shandilya, Dr. Hawaldar Maurya for theirco-operation in carrying out this work.

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[

[

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