452

SummaryA high-performance thin-layer chromatographic (HPTLC) meth-od for the simultaneous quantitative determination of caffeic acid, vanillic acid, syringic acid and kaempferol in flowers and buds of three different Bauhinia species was developed for the first time in the case of these species. Methanol was found to be the best for the highest possible recovery of target analytes. For achieving good sep-aration, a mobile phase of toluene–ethyl acetate–formic acid (5:4:1, v/v) was used. The densitometric determination was carried out at 350 nm in reflection–absorption mode. The calibration curves were linear in the range of 100–700 ng per spot for caffeic acid, vanillic acid, syringic acid, and kaempferol. The methanolic frac-tions of Bauhinia variegata L. flowers (BVFM) showed the highest amount of caffeic acid (0.08%), B. variegata L. buds (BVBM) and Bauhinia purpurea L. flowers (BPFM) showed the highest amount of kaempferol (1.53%), Bauhinia acuminata L. flowers showed the highest amount of vanillic acid (0.40%), and B. acuminata L. buds showed the highest amount of syringic acid (0.08%). The proposed method is simple, precise, specific, accurate, less time-consuming, and cost-effective. The statistical analysis of data obtained proves that the method is reproducible and selective and can be used for routine analysis of reported phenolic compounds in crude drug and extracts. The simultaneous quantification of these phenolic com-pounds has not yet been reported in the case of these species which may be utilized for the proper standardization of the drug.

1 Introduction

Genus Bauhinia (family: Caesalpiniaceae) consists of over 250 species of trees and shrubs [1]. They are distributed in vari-ous geographic locations in tropical climates. Plants in genus Bauhinia have characteristic butterfly-shaped leaves. Certain Bauhinia species have a long history of traditional medicinal applications for the treatment of different kinds of ailments,

A. Gupta, S. Verma, S. Khatoon, and A.K.S. Rawat, Pharmacognosy and Ethnopharmacology Division, CSIR – National Botanical Research Institute, Lucknow – 226001, India; and A. Gupta and H. Dwivedi, School of Pharmacy, Babu Banarsi Das University, Faizabad Road, Lucknow, India.E-mail: rawataks@rediffmail.com

particularly diabetes, infections as well as pain and inflamma-tion [2, 3].

Bauhinia purpurea L., Bauhinia variegata L., and Bauhinia acuminata L., known as Kanchanar in Hindi, are flowering plant species commonly found in Southeast Asia [4, 18, 26]. The aerial parts of the plant are reported to contain amino ac-ids, flavonoids, steroids, terpenoids, tannins, lactones, glyco-lipids, glycosyl steroids, quinines, phenyl fatty ester, pacharin, bauhiniastatins, and b-sitosterol. Certain flavanones, dihydrod-ibenzoxepins, and bibenzyls were reported from the root of this plant [5–9]. A wide range of chemical compounds including mixture of phytol fatty esters, lutein, isoquercitin, astragalin, etc. were also isolated [10].

Five flavonoids isolated from the different parts of Bauhinia have been identified as quercetin, rutin, apigenin, and apigenin 7-O-glucoside [22]. The chemical constituents isolated so far from the plant are b-sitosterol, lupeol, kaempferol-3-glucoside, tannins [23], quercitrin, heptatriacontan-12,13-diol, and do-tetracont-15-en-9-ol [24]. Simultaneous determination of major flavonoids (apigenin, quercetin, rutin, luteolin, and quercitrin) in B. variegata has been performed earlier [25].

Different parts of these plants have been used in traditional medicine to cure a host of illness such as body pain, rheuma-tism, fever, dropsy, skin diseases, septicemia, diarrhea, tonic, astringent, dysentery, hemorrhoids, piles, laxative, anthelmint-ic, antileprotic, antigoitrogenic, obesity, stomatitis, antidote for snake-poisoning, dyspepsia, flatulence, and as carminative [11, 19–21]. Several activities like antidiabetic, antibacterial, wound-healing, antioxidant, antimalarial, antimycobacterial, antifungal, and anticancerous activities have been reported [8, 12–15]. The decoction of its bark and leaves is given in biliousness, stone in bladder, venereal diseases, leprosy, and asthma [27, 28]. Bark is used for the treatment of leucorrhoea and shyphilis [29].

Phytochemical studies on Bauhinia racemosa Lam., B. pur-purea Linn., involving preliminary phytochemical studies, physicochemical studies, quantitative estimation of primary and secondary metabolites, thin-layer chromatographic (TLC) study, and high-performance liquid chromatographic (HPLC)

High-Performance Thin-Layer Chromatographic Analysis for the Simultaneous Quantification of Four Phenolics in Flowers and Flower Buds of Bauhinia purpurea L., Bauhinia variegata L., and Bauhinia acuminata L.

Abhishek Gupta, Shikhar Verma, Sayyada Khatoon, Harinath Dwivedi, and Ajay K.S. Rawat*

Key Words:

Bauhinia speciesCaffeic acidKaempferolSyringic acidVanillic acid

Journal of Planar Chromatography 28 (2015) 6, 452–457 DOI: 10.1556/1006.2015.28.6.60933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

HPTLC Analysis for Simultaneous Quantifications of Four Phenolics

Journal of Planar Chromatography 28 (2015) 6 453

fingerprint have been performed earlier [16]. Effect of extraction techniques on phenolic content, antioxidant, and antimicro-bial activity of B. purpurea and high-performance thin-layer chromatography (HPTLC) determination of antioxidants was performed by Annegowda et al. [17]. Further, B. acumina-ta L. crude extract showed hemolytic activity against human erythrocytes in a dose-dependent manner [30]. Paper chro-matography of flavonoids showed the presence of kaempferol, quercetin, and apigenin in B. acuminata L. [31]. Phytol, ses-quiterpenoids, b-caroyphyllene, and caryophyllene oxide were identified as major constituents in B. acuminata L. leaf oil [32]. Several chemical compounds including palmitic acid and three phthalic acid esters, phthalic acid, gallic acid, and ursolic acid, were identified from the leaves of B. acuminata [33]. The simul-taneous quantification with method validation of caffeic acid, vanillic acid, syringic acid, and kaempferol has not yet been reported in these species which may be utilized for the proper standardization of these drugs.

2 Experimental

2.1 Chemicals and Reagents

HPTLC analyses were performed on Merck 20 cm × 10 cm HPTLC silica gel 60 F254 (0.25 mm) plates. Caffeic acid, van-illic acid, syringic acid, and kaempferol were supplied by Sig-ma, Aldrich, Germany. All the reagents used in the experiment were of analytical grade and were supplied by Merck (Darm-stadt, Germany).

2.2 Preparation of Standard Solutions

Stock solutions of caffeic acid, vanillic acid, syringic acid, and kaempferol, respectively, were prepared separately by dissolv-ing 0.1 mg mL−1 in methanol.

2.3 Plant Material

The plant specimens, i.e., flowers and flower buds of B. pur-purea L., B. variegata L., and B. acuminata L. (Figure 1), were collected from the National Botanical Research Institute, Lucknow, India, in the month of December 2014. The plant was identified and authenticated by Dr. Tariq Hussain, CSIR–NBRI. A voucher specimen has been submitted to LWG herbarium.

2.4 Sample Preparation

The fresh plant was collected and thoroughly washed with wa-ter to remove all debris. The plants were shade-dried. The dried plants were powdered by using electric grinder at 100 mesh size. Extraction was performed by cold percolation and sox-hlation. Firstly, the powdered plants are defatted under soxhlet assembly using 250 mL of 98% petroleum ether for 6 h. This was followed by 9 h soxhlation of defatted powder by using 250 mL of chloroform followed by methanol and water. The final extracts obtained were passed through Whatman No. 1 filter paper. The filtrates obtained were concentrated under vac-uum in a rotary evaporator at 40°C and stored at 4°C for further use. The crude extracts were obtained by dissolving a known amount of dry extract in 98% methanol to obtain a stock solu-tion of 1000 μg mL−1.

2.5 Development of HPTLC Fingerprinting of Vanillic Acid, Caffeic Acid, Syringic Acid, and Kaempferol

2.5.1 Instrumentation and Chromatographic Conditions

The following were the instruments and chromatographic con-ditions used: spotting device: Linomat V automatic sample spot-ter, CAMAG (Muttenz, Switzerland); syringe: 100 μL Hamil-ton (Bonaduz, Switzerland); TLC chamber: glass twin-trough chamber (20 × 10 × 4 cm), CAMAG; densitometer: TLC Scanner 3 linked to winCATS software V.4.06, CAMAG; HPTLC plates: 10 × 10 cm, 0.2 mm thickness precoated with silica gel 60 F254, E. Merck (Darmstadt, Germany). Experimental conditions: temperature, 25 ± 2°C; relative humidity, 40%. Solvent system: toluene–ethyl acetate–formic acid (5:4:1). Detection wavelength: 350 nm for caffeic acid, vanillic acid, syringic acid, and kaemp-ferol. Slit dimension: 6.00 × 0.20 mm. Scanning speed: 20 mm s–1 and source of radiation: deuterium lamp.

2.5.2 Calibration Curve of Caffeic Acid, Vanillic Acid, Syringic Acid, and Kaempferol

A stock solution of caffeic acid, vanillic acid, syringic acid, and kaempferol (100 μg mL–1) was prepared in methanol. Differ-ent volumes of stock solution were spotted on the TLC plate to obtain concentrations of 100–700 ng per band of caffeic acid, vanillic acid, syringic acid, and kaempferol, respectively. The data of peak areas plotted against the corresponding concentra-tions were treated by least square regression analysis method validation.

2.6 Method Validation

The method was validated according to the International Con-ference on Harmonization (ICH) guidelines [34, 35], and statis-tical analysis was done using Excel 2000 (MS Office®).

Figure 1

Bauhinia purpurea (A), Bauhinia variegata (B), Bauhinia acuminata (C), and chemical structures of phenolic compounds identified and quantified.

A B C

HPTLC Analysis for Simultaneous Quantifications of Four Phenolics

454 Journal of Planar Chromatography 28 (2015) 6

2.6.1 Precision

Repeatability of sample application and measurement of peak area were carried out using nine determinants (3 concentra-tions/3 replicates) covering the specified range for the proce-dure (200, 400, and 600 ng per band of caffeic acid, vanillic acid, syringic acid, and kaempferol) and expressed in terms of relative standard deviation (RSD). Intra- and inter-day varia-tion for the determination of caffeic acid, vanillic acid, syringic acid, and kaempferol were carried out at three different con-centration levels of 200, 400, and 600 ng per band. Acceptance criteria for a procedure’s repeatability or intermediate precision are based on the intended use of the analytical method.

2.6.2 Robustness of the Method

By introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation, and activation of prewashed TLC plates with methanol, the effects on the results were examined. Robustness of the method was determined 3 times at a concentration level of 200 ng per band for caffeic acid, vanillic acid, syringic acid, and kaempferol, and the RSD and SD of peak areas were calculated.

2.6.3 Limit of Detection and Limit of Quantitation

In order to estimate the limit of detection (LOD) and limit of quantitation (LOQ), blank methanol was spotted six times and the signal-to-noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting the known concentrations of caffeic acid, vanillic acid, syringic acid, and kaempferol until the average responses were approximately 3 or 10 times the responses for six replicate determinations.

2.6.4 Recovery

The preanalyzed samples were spiked with extra 50, 100, and 150% of the standard caffeic acid, vanillic acid, syringic acid, and kaempferol, and the mixtures were reanalyzed by the pro-posed method. The experiment was conducted six times. This was done to check the recovery of the targeted analytes at dif-ferent levels in the formulations.

2.6.5 Ruggedness

Caffeic acid, vanillic acid, syringic acid, and kaempferol solu-tion of concentration 200 ng per band was prepared and an-alyzed on day 0 and after 6, 12, 24, 48, and 72 h. Data were treated for % RSD to assess the ruggedness of the method.

2.6.6 Specificity

The specificity of the method was confirmed by analyzing the standard drugs and the extract. The band for caffeic acid, vanillic acid, syringic acid, and kaempferol in the sample was confirmed by comparing the Rf values and spectra of the band with those of the standard. The peak purity of the caffeic acid, vanillic acid, syringic acid, and kaempferol was assessed by comparing the spectra at three different levels, viz., peak start (S), peak apex (M), and peak end (E) positions of the band.

3 Results and Discussion

Standardization of suitable mobile phase equally plays a cru-cial role in chromatographic method development. As far as in-dividual estimation of caffeic acid, vanillic acid, syringic acid, and kaempferol by chromatographic methods is concerned, a

Table 1

Summary of validation parameters.

Parameters Caffeic acid Vanillic acid Syringic acid Kaempferol

Rf 0.54 ± 0.00 0.61 ± 0.005 0.57 ± 0.00 0.64 ± 0.005

Linearity range 100–700 ng 100–700 ng 100–700 ng 100–700 ng

Regression via area y = 164.551 + 3.504*x y = 638.777 + 6.375*x y = 164.551 + 3.504*x y = 436.218 + 6.020*x

r 0.998 0.992 0.998 0.999

Slope 3.504 6.375 3.504 6.020

Intercept 164.551 638.777 164.551 436.218

LOD (ng) 40 40 40 40

LOQ (ng) 100 100 100 100

Table 2

Quantification of caffeic, vanillic, syringic acid, and kaempferol compounds in different Bauhinia species methanolic fraction.

Bauhinia species Caffeic acid (%) Vanillic acid (%) Syringic acid (%) Kaempferol (%)

B. purpurea L. flowers (BPFM)

B. purpurea L. buds (BPBM)

B. variegata L. flowers (BVFM)

B. variegata L. buds (BVBM)

B. acuminata L. flowers (BAFM)

B. acuminata L. buds (BABM)

0.02

0.03

0.08

0.04

0.06

0.02

0.13

0.16

0.26

0.35

0.40

0.18

0.07

0.03

0.02

0.01

0.02

0.08

1.14

1.53

0.33

1.53

0.51

0.26

HPTLC Analysis for Simultaneous Quantifications of Four Phenolics

Journal of Planar Chromatography 28 (2015) 6 455

number of solvent systems have been reported. However, there has not been cited a single report for the separation of these phenolics simultaneously in a single solvent system. Therefore, in this study, several solvent systems used for individual es-timation of these phenolics were investigated to evaluate the combinatorial separation of these compounds in a single sol-vent system and between different components of the extract. Among the different solvent systems investigated, the mobile phase consisting of toluene–ethyl acetate–formic acid in the ratio of 5:4:1 v/v demonstrated compact spots with typical Gaussian shaped peaks with good resolution between other peaks of the extract.

The procedure for the separation and determination of dif-ferent phenolic compounds in methanolic fraction of three different Bauhinia species using HPTLC–densitometry is reported at seven-point calibration curve in which caffeic acid, vanillic acid, syringic acid, and kaempferol were ob-served and quantified with method validation (Tables 1 and 2). HPTLC chromatogram and densitograms were obtained from methanolic fractions (Figures 2 (a), (b) and 3). Each targeted compound was identified by retardation factor (Rf), peak purity, and overlaid ultraviolet (UV) spectra (Figure 4). Precision studies have been performed by analyzing intra- and inter-day variation for the determination of these pheno-lics compounds, and they were carried out at three different concentration levels of 200, 400, and 600 ng per band; mean percentage relative standard deviation values were found to be 0.533, 0.396, 0.163, and 0.22 for caffeic acid, vanillic acid, syringic acid, and kaempferol, respectively, in intra-day anal-ysis while inter-day analysis showed mean percentage rela-tive standard deviation values of 1.4, 1.653, 1.05, and 1.586 for caffeic acid, vanillic acid, syringic acid, and kaempferol, respectively, showing good precision (Table 3). For recov-ery studies, preanalyzed samples of Bauhinia species were spiked with extra 50, 100, and 150% of the standard phenolic compounds, and the mixtures were reanalyzed which showed a good recovery ranging from 97.073 to 98.770% for caffeic

acid, 97.386 to 99.606% for vanillic acid, 98.883 to 99.813% for syringic acid, and 98.430 to 99.033% for kaempferol. The experimental data are expressed as mean percentages of re-covered analyte, and standard deviation and relative standard deviation are also presented (Table 4). The methanolic frac-tions of B. variegata L. flowers (BVFM) showed the highest amount of caffeic acid (0.08%), B. variegata L. buds (BVBM), and B. purpurea L. buds (BPBM) showed the highest amount of kaempferol (1.53%), B. acuminata L. flowers (BAFM) showed the highest amount of vanillic acid (0.4%), whereas B. acuminata L. buds (BABM) showed the highest amount of syringic acid (0.08%).

Figure 2

HPTLC chromatogram at 366 nm (a) and HPTLC chromatogram at 254 nm (b). HPTLC chromatogram of plants extracts and pheno-lic standards. (1) B. purpurea L. flowers (BPFM), (2) B. purpurea L. buds (BPBM), (3) B. variegata L. flowers (BVFM), (4) caffeic acid, (5) vanillic acid, (6) syringic acid, (7) kaempferol, (8) B. variegata L. buds (BVBM), (9) B. acuminata L. flowers (BAFM), (10) B. acuminata L. buds (BABM).

Figure 3

HPTLC densitogram of plants extracts and phenolic standards. (1) B. purpurea L. flowers (BPFM), (2) B. purpurea L. buds (BPBM), (3) B. variegata L. flowers (BVFM), (4) caffeic acid, (5) vanillic acid, (6) syringic acid, (7) kaempferol, (8) B. variegata L. buds (BVBM), (9) B. acuminata L. flowers (BAFM), (10) B. acuminata L. buds (BABM).

Figure 4

Overlaid UV absorption spectra of standards overlaid with the cor-responding band in the samples track. (A) Syringic acid, (B) vanillic acid, (C) caffeic acid, (D) kaempferol.

(a) (b)

B

D

A

C

AA

HPTLC Analysis for Simultaneous Quantifications of Four Phenolics

456 Journal of Planar Chromatography 28 (2015) 6

4 Conclusion

A validated HPTLC analytical method has been developed for the simultaneous determination of caffeic acid, vanillic acid, syringic acid, and kaempferol in flowers and buds of three different Bauhinia species. The proposed method is simple, precise, specific, accurate, less time-consuming, and cost-ef-fective. The statistical analysis of data obtained proves that the method is reproducible and selective and can be used for rou-tine analysis of reported phenolic compounds in crude drug and extracts. The method can be used to determine the purity of the drug available from various sources by detecting the related

impurities as well as for quality control of herbal formulations containing Bauhinia species as an ingredient. HPTLC analysis has indicated the presence of optimum amount of caffeic acid, vanillic acid, syringic acid, and kaempferol in the samples.

Acknowledgment

The authors are thankful to the Director of CSIR–NBRI for providing all the facilities to conduct this research work.

Funding

This work was supported by the Council of Science and Tech-nology, Uttar Pradesh, India.

References

[1] A. Cronquist, An Integrated System of Classification of Flower-ing Plants, Columbia University Press, New York, 1981, p. 597.

[2] C.F. Valdir, Phytother. Res. 23 (2009) 1347–1354.

[3] J.M. Watt, M.G. Bryer-Bradwick, The Medicinal and Poisonous Plants of Southern and Eastern Africa, E. & S. Livingstone, Edin-burgh, 1962, p. 560.

[4] L.V. Asolker, K.K. Kakkar, O.J Chakre, Supplement to glossary of Indian medicinal plants. Part-I (A–K) National Institute of Sci-ence Communication, New Delhi, 2000, p. 116–117.

[5] A. Salatino, C.T.T. Blatt, D.Y.A.C. Dos Santos, A.M.S.F. Vaz, Rev. Bras. Bot. 22 (1999) 17–20.

[6] R.N. Yadava, P. Tripathi, Fitoterapia 71 (2000) 88–90.

[7] Y.H. Kuo, M.H. Yeh, S.L Huang, Phytochemistry 49 (1998) 2529–2530.

Table 3

Intra-day and inter day precisions.

Standard markers Conc. (ng band−1)Intra-day Inter-day

% RSD Mean RSD % RSD Mean RSD

Caffeic acid

200 0.63 1.73

400 0.51 0.533 1.36 1.4

600 0.46 1.11

Vanillic acid

200 0.52 1.61

400 0.39 0.396 1.53 1.653

600 0.28 1.82

Syringic acid

200 0.18 0.92

400 0.15 0.163 1.04 1.05

600 0.16 1.19

Kaempferol

200 0.26 1.62

400 0.36 0.22 1.65 1.586

600 0.04 1.49

Table 4

Recovery analysis of caffeic, vanillic, syringic acid, and kaempferol.

Standard Amount added (%)

Amount recovered (%) Mean SD RSD

Caffeic acid

50 97.073 0.545 0.562

100 97.560 1.706 1.748

150 98.770 1.359 1.376

Vanillic acid

50 97.386 1.646 1.690

100 99.606 1.694 1.701

150 98.333 1.950 1.983

Syringic acid

50 99.813 1.938 1.942

100 98.936 1.878 1.898

150 98.883 1.416 1.432

Kaempferol

50 98.430 1.461 1.484

100 99.033 1.908 1.927

150 98.626 1.087 1.102

HPTLC Analysis for Simultaneous Quantifications of Four Phenolics

Journal of Planar Chromatography 28 (2015) 6 457

[8] G.R. Pettit, A. Numata, C. Iwamoto, Y. Usami, T. Yamada, H. Ohi-shi, G.M. Cragg, J. Nat. Prod. 69 (2006) 323–327.

[9] R. Ramachandran, B.C. Joshi, Current Sci. 36 (1967) 5774–5775.

[10] T. Kumar, K.S. Chandrashekar, Res. J. Med. Plant 5 (2011) 420–431.

[11] K.R. Kirthikar, D.B. Basu, Indian Medicinal Plants, vol. 4, 2nd edn., Oriental Enterprises, Dehradun, 2001, p. 1255–1257.

[12] K.S. Muralikrishna, K.P. Latha, C.S. Shreedhara, V.P. Vaidya, A.M. Krupanidhi, Int. J. Green Pharm. 2 (2008) 83–86.

[13] S. Boonphong, P. puangsombat, A. Barmee, C. Mahidol, S. Ruchirawat, P. Kittakoop, J. Nat. Prod. 70 (2007) 795–801.

[14] K.V. Ananth, M. Asad, N.P. Kumar, S.M.B. Asdaq, G.S. Rao, Indi-an J. Pharm. Sci. 72 (2010) 122–127.

[15] V.D. Joshi, T. Verma, P.R. Shetty, Int. J. Pharm. Res. 1 (2009) 51–55.

[16] G.K. Sharanabasappa, M.K.D. Santosh, Shaila, Y.N. Seetharam, I. Sanjeevarao, E-J. Chem. 4 (2007) 21–31.

[17] H.V. Annegowda, M.N. Mordi, S. Ramanathan, M.R. Hamdan, S.M. Mansor, Food Anal. Methods 5 (2012) 226–233.

[18] Anonymous, The Wealth of India; A Dictionary of Indian Raw Materials and Industrial Products, CSIR, New Delhi, 1998, 2 (B), p. 49–52.

[19] P.D. Badhe, V.K. Pandey, Bull. Med. Ethnobot. Res. 11 (1990) 1–39.

[20] N.S. Balajirao, D. Rajasekhar, R. Narayana, D. Raju, Biosci. Res. Bull. 11 (1995) 81–85.

[21] L.S. Bhatnagar, V.K. Singh, G. Pande, J. Res. Ind. Med. 8 (1973) 67–100.

[22] G.P. Rajani, A. Purnima, Indian J. Pharmacol. 41 (2009) 227–232.

[23] R.N. Chopra, S.L. Nayer, I.C. Chopra, Glossary of Indian Medic-inal Plants, Council of Industrial and Scientific Research, New Delhi, 1956, p. 35.

[24] S.L. Kapoor, L.D. Kapoor, Bull. Med. Ethnobot. Res. 1 (1980) 120–144.

[25] P. Bhandari, N. Kumar, A.P. Gupta, B. Singh, V.K. Kaul, J. Sep. Sci. 30 (2007) 2092 – 2096.

[26] D.A. Dhale, J. Ecobiotechnol. 3 (2011) 4–7.

[27] Anonymous, The Wealth of India. A Dictionary of Indian Raw Materials and Industrial products, CSIR, New Delhi, 1959, p. 56–57.

[28] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, Volume II, 2nd Edition, Bio-Green Books, New Delhi, 2006, p. 892–894.

[29] K. Mohan, A.K. Singh, Adv. Plant Sci. 9 (1996) 1–16.

[30] K. Phansri, R. Sarnthima, S. Thammasirirak, P. Boonchalee, S. Khammuang, Chiang Mai J. Sci. 38 (2011) 242–251.

[31] K.N. Sinha, T. Singh, Int. J. Recent Sci. Res. 4 (2013) 1812–1816.

[32] V. Vasudevan, J. Mathew, B. Sabulal, Asian J. Chem. 25 (2013) 2329–2330.

[33] S. Nag, A. Paul, R.P. Datta, Int. J. Sci. Res. Publ. 3 (2013) 1648.

[34] ICH-Q2A, Text on Validation of Analytical Procedures, Har-monized Tripartite Guideline prepared within the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Geneva, 1994.

[35] ICH-Q2B, Validation of Analytical Procedures: Methodology, Harmonized Tripartite Guideline prepared within the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Geneva, 1996.

Ms received: June 2, 2015Accepted: September 1, 2015