Novel analytical method development for some amide group containing drugs using Bougainvillea spectabilis bract extracts

Novel analytical method development for some amide group containing drugs using Bougainvillea spectabilis bract extracts

S560 Asian Pac J Trop Med 2014; 7(Suppl 1): S560-S567 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal ...

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S560

Asian Pac J Trop Med 2014; 7(Suppl 1): S560-S567

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm

Document heading

Novel

doi: 10.1016/S1995-7645(14)60290-X

analytical method development for some amide group containing drugs using Bougainvillea spectabilis bract extracts 1*

Suresh Killedar

, Anuja Pawar2, Sameer Nadaf3, Ashwini Nale2, Umarfarukh Tamboli3, Sachin Pishawikar4

1

Department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416013, India [M.S.]

2

Department of Quality Assurance, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416013, India [M.S.]

3

Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416013, India [M.S.]

4

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416013, India [M.S.]

ARTICLE INFO

ABSTRACT

Article history: Received 27 Apr 2014 Received in revised form 15 May 2014 Accepted 20 Jun 2014 Available online 1 Sep 2014

Objective: To develop and validate a simple, accurate and precise colorimetric method using Bougainvillea spectabilis (B. spectabilis) bract color previously not exploited for estimation of amide group containing drugs i.e. lidocaine and ranolazine in pharmaceutical formulations. Methods: Methanolic extract of B. spectabilis was prepared and evaluated for stability of its color at different pH and temperature for a period of 3 weeks. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity, precision, limit of detection, limit of quantitation and specificity according to International Conference on Harmonization guidelines. About 0.5% of B. spectabilis bract color was added to the working standard solutions of the drugs separately and after formation of color complex, and absorbances were noted at 418 nm. Results: For color complexes of lidocaine and ranolazine, linearity was found to be in the range of 4 to 24 and 5 to 25 µg/mL respectively. The % relative standard deviation was found to be within specification limits. Presence of lone pair of electron on nitrogen of amide group of both drugs shows basic nature, contributed in formation of color complex between amide and the color pigment obtained from B. spectabilis bracts. Conclusions: It can be concluded that the method is simple, accurate, economic, and rapid hence can be employed for routine analysis.

Keywords: Colorimetric method

Bougainvillea spectabilis

Lidocaine Ranolazine Validation

1. Introduction Colorimetry is concerned about the measurement of the intensity of electromagnetic radiation in the visible spectrum transmitted through a solution or transparent solid. Passing of electromagnetic radiation through object or solution leads to absorption of certain wavelength, leaving unabsorbed wavelength to be transmitted or reflected. This observed as color which is complementary to absorbed color[1]. Bougainvillea spectabilis (B. spectabilis) belonging to N yctaginaceae family is a popular ornamental plant,

*Corresponding author: Dr. Suresh Killedar, Department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416013, Maharashtra State, India. Tel: 91-231-2637286 Fax: 91-231-2638833 E-mail: sureshgk64_rediffmail.com

commonly known as “paper flower” due to the bracts are thin and papery. B. spectabilis is used in several countries to prepare extracts with antibacterial activity[2-4]. I ts aqueous and methanolic extracts show good oral glucose tolerance and significantly reduce the intestinal glucosidase activity. Study on leaves revealed its potential as anti-inflammatory, anti-diabetic, anti-fertility, antiviral, antibacterial and so on[5-11]. Lidocaine [2-(diethylamino)-N-(2,6-dimethylphenyl) acetamide] is a first amino amide-type local anaesthetic, white or slightly yellowish, crystalline powder, having molecular formula C14H22N2O and molecular weight 234.34 g/ mol (Figure 1). Lidocaine is used topically to relieve itching, burning and pain from skin inflammations, injected as a dental anesthetic or as a local anesthetic for minor surgery. It acts by blocking the fast voltage gated sodium (Na+) channels

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HO HO

HO

O OH

O

OH

I

OH OH O

O

O

N

HO HO

CH3

O

O N H OH

O

H N

CH3

N O

N

N H

CH3

II

OH N

CH3

O

O O

III

Figure 1. Structure of bougainvillein-V (I), lidocaine (II) and ranolazine (III).

Literature survey reveals that previously work has been

carried out on estimation of lidocaine either single or in combination using UV spectrophotometer[16-18]. Similar type of work has also been reported for estimation of ranolazine in bulk and tablet dosage form[19,20]. Colorimetric method for estimation of both lidocaine and ranolazine using synthetic chemicals has been already reported [21,22]. H owever, no attempt has been previously made for colorimetric estimation of aforesaid drugs using a natural color pigment. So in present work, colorimetric method using extract of B. spectabilis bracts was developed for estimation of lidocaine and ranolazine based on principle that the amide group due to presence of lone pair of electron on nitrogen is going to show basic nature which contributes in formation of color complex between amide and the color pigment obtained from B. spectabilis bracts. 2. Materials and methods

Principal, Shri Vijaysinha Yadav Arts and Science College, Peth Vadgaon. Further, the herbarium sheet was submitted to the Department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur.

2.2. Chemical L idocaine and ranolazine were procured from pharmaceutical companies. Sodium hydroxide, hydrochloric acid, ascorbic acid, methanol and other chemical used were of analytical grade. Further dilutions were made as per the requirement.

2.3. Extraction The extracts were obtained by immersing fresh bracts, dried bracts and dried bracts powder of B. spectabilis in 1% methanolic HCl for 24 h at room temperature. The extract was further concentrated under reduced pressure. To the concentrated solution, a mixture of diethyl ether and light petrol in 2:1 ratio was added when the colouring matter separates. The supernatant liquid was decanted after a couple of hours and lastly the deep blue-red coloured viscous residue was dried in a vacuum dessicator over anhydrous calcium chloride.

2.4. Spectrophotometric analysis of extract solution A Jasco spectrophotometer (model: UV-630) was used to measure the λmax and absorbance of extracts of B. spectabilis bracts solutions at pH values of 3, 5 and 8. For this sample, 0.06 g each of various extracts of B. spectabilis bracts were dissolved in 10 mL of methanol and pH were adjusted using 0.1 mol/L HCl and NaOH. The λmax and absorbances of the solutions were measured using the spectrophotometer in the visible light spectra (400-800 nm) (Figure 2). Values of λmax and absorbance at different pH are reported in Table 1. 0.9

0.8

pH 3 pH 5

0.6

pH 7

Absorbance

in the neuronal cell membranes that are responsible for signal propagation and stabilizes the membrane[12,13]. R anolazine [ ( RS ) - N - ( 2 , 6 -dimethylphenyl ) - 2 -[ 4 -[ 2 hydroxy-3-(2-methoxyphenoxy)-propyl] piperazin-1-yl] acetamide] is an antianginal medication, white or slightly yellow, crystalline powder having molecular formula C 24H 33N 3O 4 and molecular weight 427 . 537 g/mol ( F igure 1 ) . R anolazine affects the sodium-dependent calcium channels during myocardial ischemia in rabbits and alters the transcellular late sodium current. It is useful in the treatment of neuropathic pain[14,15].

0.4

2.1. Plant material B. spectabilis bracts were collected from local area (NH4 highway between Kolhapur and Peth Vadgaon) during the flowering season in the month of April to July. The collected material was air dried and packed in airtight bags. Identification and authentication of plant was done with the help of herbarium sheet by Dr. M. Y. Cholekar-Bachulkar,

0.2

0

400

500

600

Wavelength (nm)

700

800

Figure 2. UV overlain spectra of B. spectabilis colour at different pH solution.

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Table 1 Absorbance and λmax of 0.12% (w/v) solutions of B. spectabilis bracts colour at different pH values. pH 3

5 8

λ (nm)

Absorbance

516.2依0.2

0.420依0.050

516.0依0.2

515.5依0.3

Values are expressed as mean依SD, n=3.

0.290依0.032 0.440依0.100

2.5. Stability study of B. spectabilis bracts colour Reversibility of the colour change was also tested for this natural colour. The extract was subjected to various treatments including thermal treatment, pH, antioxidant additives and light exposure. C olour changes of the treated samples were monitored from Week 0 to 3 with a UV-visible spectrophotometer. The stability study of B. spectabilis bracts colour was carried out by considering the following factors.

2.5.1. Additional of antioxidant A scorbic acid of various concentrations, 0 . 1 % ( w/v ) , 0 . 5 % ( w/v ) and 1 . 0 % ( w/v ) was added into the extracts separately. Extracts along with ascorbic acid were then subjected to pH adjustment. Control was prepared without addition of ascorbic acid. 2.5.2. pH treatment Samples added with different percentages of ascorbic acid were subjected to pH adjustment using 1 mol/L HCl or 1 mol/ L N a OH . E ach set of samples in section was adjusted to pH 3.0, pH 5.0 (original pH of B. spectabilis bracts ) and p H 7 . 0 respectively. T hese samples were further treated at different temperature. 2.5.3. Heat treatment Each set of the sample was finally kept at 4 °C, 25 °C in dark and 25 °C with light exposure. 2.5.4. Spectrophotometric analysis Colour changes of the treated samples were monitored weekly at 516 nm with a UV-visible spectrophotometer from Week 0 to 3. 2.6. Colorimetric estimation of lidocaine and ranolazine formulation 2.6.1. Selection of drug Phytochemical investigation of extract of B. spectabilis bract shows presence of betalain group of compounds which have polyphenolic nature similar to synthetic dyes like methyl orange, bromocresol green, etc. The synthetic dyes generally show reactivity with amide linkage whenever colorimetric method has to be developed. A s lidocaine and ranolazine contain amide linkage, the selection of drug was done for colorimetric method development.

2.6.2. Preparation of standard stock solutions 2.6.2.1. Lidocaine Standard stock solution of lidocaine was prepared by dissolving 50 mg of lidocaine in few mL of methanol by sonication and the final volume was made up to 100 mL using methanol to get a stock solution having concentration 500 µg/mL. 2.6.2.2. Ranolazine Standard stock solution of ranolazine was prepared by dissolving 20 mg of ranolazine in few mL of methanol by sonication and the final volume was made up to 100 mL with methanol to get a stock solution having concentration 200 µg/ mL. 2.6.3. Preparation of standard working solutions 2.6.3.1. Lidocaine A volume of 0.4 mL of stock solution was pipetted out in 10 mL graduated volumetric flask. Volume was made up to 10 mL with methanol to give standard working solution of 20 µg/mL of lidocaine. 2.6.3.2. Ranolazine A volume of 1 mL of stock solution was pipetted out in 10 mL graduated volumetric flask. Volume was made up to 10 mL with methanol to give standard working solution of 20 µg/mL of ranolazine. 2.6.4. Optimization of reagents and reaction condition 2.6.4.1. Lidocaine Temperature of reaction, quantity, concentration and sequence of addition of reagents were optimized after several experimental trails. Using extract, solutions of different concentration like 0.5%, 1%, 1.5% and 2% were prepared in methanol. About 1 mL of standard working solution of lidocaine having concentration 20 µg/mL was pipetted out in different 10 mL graduated volumetric flasks. To this 1 mL of extract, solution having different concentrations was added. All the solutions were screened at identified λmax (418 nm). It was found that 0.5% solution of extract gives excellent color complex. The optimization of volume of 0.5% solution was the next step. About 0.48 mL volume of 0.5% extract of B. spectabilis bract solution was found to be optimum for completion of reaction. The intensity of the colour formed, reached maximum in 10 min after mixing the reagent at room temperature and was stable up to 2 h. 2.6.4.2. Ranolazine Using extract, solutions of different concentration like 0.5%, 1%, 1.5% and 2% were prepared in methanol. Stock solution of 1 000 µg/mL of ranolazine was prepared. From this, 0.1 mL was pipetted out in 10 mL volumetric flask. To this 1 mL of above extract, solution with different concentration was added.

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Identification of λmax for developed complex was carried out, which was found to be 418 nm.

2.6.5. Procedure for plotting calibration curve 2.6.5.1. Lidocaine S erial volumes of standard working solution were pipetted out into a series of 10 mL volumetric flasks. To each volumetric flask, 1 mL of 0.5% extract solution of B. spectabilis bract was added and the volume was then made up to 10 mL with methanol to get final concentrations of 4 to 24 µg/mL. Flasks were kept aside for 5 to 10 min for completion of formation of reaction complex in dark. The absorbance of the red colored complex was measured at 418 nm against reagent blank. 2.6.5.2. Ranolazine A ppropriate aliquots of standard drug solution were taken into a series of 10 mL volumetric flasks. To each volumetric flask, freshly prepared 0.5% solution of extract of B. spectabilis bracts was added and the volume was then made up to 10 mL with methanol to get final concentrations of 5 to 25 µg/mL and kept aside for 10 min for completion of formation of color complex. The absorbance of the pale yellow colored complex was measured at 418 nm against reagent blank. 2.6.6. Spectrophotometric analysis of drug-extract complexes A Jasco spectrophotometer (model: UV-630) was used to measure the λmax and absorbance of lidocaine-extract color complex and ranolazine-extract color complex. 2.6.7. Fourier transform infrared spectroscopy (FTIR) study Study of possible interaction between B. spectabilis extract and drug i.e. lidocaine and ranolazine has also been carried out using an infrared spectrophotometer ( J asco- V - 730 model). 2.7. Method validation 2.7.1. Analysis of formulation An accurately measured volume equivalent to 10 mg of lidocaine was dissolved in 10 mL of methanol by sonication. The solution was then filtered through Whatman filter paper No. 41. Appropriate aliquots within the Beer’s law limit were analyzed by the proposed method using the procedure described earlier. The concentration of lidocaine present in the sample solution was calculated by using the formula: Absorbances=A+B伊C Where, A=-0.005 32, B=0.000 327 8 and C=concentration of lidocaine. 2.7.2. Accuracy Accuracy of the proposed method was determined using recovery studies. The recovery studies were conducted by adding different amounts (80%, 100% and 120%) of the pure drug to the pre-analysed formulation. The solutions were

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prepared in triplicates and the % recovery was calculated.

2.7.3. Precision P recision studies were carried out to determine the reproducibility of the proposed method. Repeatability was determined by preparing nine replicates of three different concentrations of the sample and the absorbance was measured. For lidocaine sample, concentration was 4, 12 and 20 µg/mL whilst for ranolazine it was 5, 10 and 25 µg/mL. Intraday precision study was conducted by preparing drug solution of aforesaid concentration and analyzing it at three different times in a day. To determine interday precision, the same procedure was followed for three different days. P recision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample, it may be investigated using artificially prepared samples or a sample solution. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. 2.7.4. Specificity A nalytical techniques that can measure the analyte response in the presence of all potential sample components should be used for specificity validation. 2.7.5. Limit of detection (LOD) LOD is the lowest amount of analyte in the sample that can be detected. LOD may also be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula: LOD=3.3(SD/S) 2.7.6. Limit of quantitation (LOQ) LOQ is the least possible quantity of analyte in the sample that can be quantitatively determined by suitable precision and accuracy. The calculation method is again based on the standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula: LOQ=10(SD/S) 2.7.7. Linearity The linearity of the analytical method was its capability to bring forth test results which are directly proportional to analyte concentration in samples within a given range. I t may be demonstrated directly on the test substance (by dilution of a standard stock solution) or by separately weighing synthetic mixtures of the test product components. The response for the complex was strictly linear in the investigated concentration range. 3. Results Extract was successfully isolated from B. spectabilis

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Table 2 Effect of pH, temperature and antioxidant on the degradation of extract of B. spectabilis colour at 25 °C. pH 3

5

7

Concentration of

ascorbic acid (%) 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0

25 °C (light)

Weak 0

Weak 1

0.7依0.5

8.5依1.2

0.7依0.3 0.5依0.3 0.6依0.4

Weak 1

68.2依0.4

0.2依0.4

6.6依1.4

60.8依0.6

8.7依0.3

32.5依1.2

66.9依0.5

0.3依0.5 0.3依0.2

10.0依1.6

48.1依0.3

72.7依0.5

0.2依0.8

9.3依1.0

30.0依1.0

68.4依0.3

0.3依0.1

10.5依0.5

0.7依0.4

25.0依0.4

0.5依0.2

21.6依0.3

0.6依0.3

Weak 0

45.9依0.4 40.4依0.2

39.3依0.5 54.6依0.9

22.4依2.5

43.3依0.6 35.5依2.5

70.3依1.5 80.1依0.6 74.6依0.8 71.1依1.4

3.1. Stability study of B. spectabilis bracts colour 3.1.1. Additional antioxidant Addition of 0.1% ascorbic acid to B. spectabilis bracts colour managed to preserve the colour in all dark storage conditions. U nfortunately, 0 . 1 % ascorbic acid was not enough to stop colour degradation in sample exposed to light. Contrast to this, addition of 1.0% ascorbic acid managed to preserve the colour of the pigment, though the extract was exposed to light. Results are shown in Tables 2 and 3. 3.1.2. pH treatment S ample at p H 7 , stored in the dark at 4 ° C showed approximately 25% degradation from the initial reading after 3 weeks. In contrast, sample at pH 3 managed to maintain 80 % of the colour at the end of W eek 3 . T he results indicated that betacyanin seems to be in favor of acidic pH region (Tables 2 and 3). Table 3 Effect of pH, temperature and antioxidant on the degradation of extract of B. spectabilis colour at 4 °C. pH Concentration of ascorbic acid (%) 3 0.1 0.5 1.0 5 0.1 0.5 1.0 7 0.1 0.5 1.0

Week 0

4 °C (dark)

Week 1

Week 2

Week 3

0.3依0.1

11.0依1.2

16.3依0.6

20.2依0.8

0.3依0.2

9.5依1.4

15.8依0.2

19.2依2.3

0.2依0.3

10.9依1.5

14.7依0.3

0.4依0.9

12.0依0.5

17.1依0.7

0.4依0.2

10.3依0.3

16.5依1.0

0.5依0.4 0.6依0.4 0.5依0.6 0.4依0.5

11.8依0.6 13.5依0.5 13.2依0.4 12.6依1.0

16.1依1.0 19.8依0.4 19.3依0.6 18.9依0.3

19.6依1.1 22.1依1.4 21.6依0.6 21.1依0.4 25.0依0.2 24.7依0.6

21.7依2.1

D ata are expressed as mean依 SD , n= 3 . D egradation ( % ) = ( original

concentration−determined concentration)×100.

Weak 2

6.8依0.5

43.6依1.4

6.2依0.3

27.5依1.0

9.7依0.3

22.7依1.0

59.1依0.2

0.6依0.3

19.4依1.2

48.0依0.4

0.1依0.8

17.3依0.6

19.0依0.9

50.3依1.1

64.3依0.5

30.5依0.8

0.3依0.4

56.7依1.3

36.4依0.5

10.0依0.4

9.1依1.1

Weak 3

60.5依0.6

38.7依0.7

0.4依0.6

Data are expressed as mean依SD, n=3. Degradation (%)=(original concentration−determined concentration)×100.

bracts and percentage yield was found to be in the range of 18.3%-22.5% in methanol.

25 °C (dark)

Weak 3

7.0依0.1

0.4依0.5 0.4依0.9

Weak 2

61.4依0.8 48.0依0.4

36.2依0.7

65.8依0.9

30.6依0.3

66.0依1.3

3.1.3. Heat treatment Results revealed light as the major factor of betalain pigment degradation. Refrigeration storage (4 °C) condition without light exposure managed to preserve the colour up to 3 weeks than storage at 25 °C with exposure to light (Tables 2 and 3). 3.2. Optimization of reagents and reaction condition F or lidocaine, the method employs 0 . 5 % solution of

B. spectabilis bracts prepared by making use of freshly prepared extract. The absorption peak of complex was located at 418 nm. T emperature of reaction, quantity, concentration and sequence of addition of reagents were optimized after several experimental trials. For lidocaine, the optimum quantity and concentration of B. spectabilis bracts dried extract solution was found to be 0.48 mL of 0.5% solution. The concentration of lidocaine over a range of 4 to 24 µg/mL was found to obey Beer’s law (Table 4). Table 4 Absorbance values for calibration curve of lidocaine colour complex and ranolazine color complex. Complex

Lidocaine

Concentration (µg/mL) 4 8

0.017 0.042

12

0.065

20

0.114

16

Ranolazine

Absorbance

24 5

0.090 0.142 0.053

10

0.069

20

0.095

15 25

-

0.082 0.111

-

For ranolazine, the optimum quantity and concentration of B. spectabilis extract solution was found to be 0.48 mL of 0.5% solution. The concentration of ranolazine over a range of 1-25 µg/mL was found to obey Beer’s law in the

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[D

D+CAA (Donar) (Acceptor)

CAA]

0.12

0.400

0.1

0.300 0.200

0.06

A

0 400

500

0.09

0.08

600

Wavelength (nm)

700

Absorbance

0.100

Absorbance

stated range employed for analysis (Table 4). The correlation coefficient value for this curve was 0.998. The intensity of the colour formed, reached maximum in 1 min after mixing the reagent at room temperature and was stable up to 1 h. The results showed that optimal condition for the reaction of B. spectabilis bracts extract solution with ranolazine was in an alkaline medium. General mechanism of complex formation is reported in Figure 3.

800

0.000 240.00

272.28

300.00

350.00

Wavelength (nm)

B

400.00

0.06 0.04

n-π Complex

0.02 Absorbance

C

0

-0.01

400

500

600

Wavelength (nm)

700

800

Figure 5. UV spectra of B. spectabilis extract ( A ) , ranolazine ( B ) and ranolazine–color complex (C) . 0.017

D++CAARadical ions

0.016

Absorbance

0.01

3.3. Spectrophotometric analysis of drug-extract complexes UV spectra of B. spectabilis color, lidocaine and lidocaine-color complex has been reported in Figure 4. While UV spectra of B. spectabilis color, ranolazine and ranolazine-color complex has been reported in Figure 5. UV overlain spectra of lidocaine color complex and ranolazinecolor complex has shown in Figure 6 A and B respectively. 0.12

0.8

0.1

0.4 0.2

0.09

0.08

A 500

600

Wavelength (nm)

700

B

Absorbance

Absorbance

0 400

0

800

220 240 260 280 300 320 340 360 380 400 420

Wavelength (nm)

0.06 0.04

Absorbance

0.02

C

0

-0.01

400

500

600

Wavelength (nm)

0

-0.001

0.04

700

800

Figure 4. UV spectra of B. spectabilis extract (A), lidocaine (B) and lidocaine– color complex (C).

A 400

500 Wavelength (nm) 600

700

400

500

700

800

0.00 0.02 0.0 0

-0.0

0.6

0.06

0.005

Absorbance

Figure 3. Possible mechanism action of colorimetric reaction.

Wavelength (nm)

600

B

800

Figure 6. UV overlain spectra of lidocaine - colour complex ( A ) and ranolazine-colour complex (B) .

3.4. FTIR study FTIR study of pure drugs ( lidocaine and ranolazine ) , extract of B. spectabilis and complex of extract and drug indicated there were no incompatibilities between extract and drugs. B. spectabilis color showed major peak was at -1 3 375.89 wavenumber cm which corresponds to phenols, -OH stretching. Even as peak was found to be retained in B. spectabilis color-lidocaine complex was at 3 322.17 wavenumber cm-1 which also corresponds to phenols, -OH stretching. While in case of B. spectabilis color-ranolazine complex peak was at 3 222 . 15 wavenumber cm - 1 and

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corresponded the same. So the major peak has found be retained both in color and complexes (Figures 7 and 8).

Table 5 Results of accuracy. Level

Lidocaine

Concentration

80%

100% 120%

Ranolazine 80%

100%

Transmitance (%)

A

120%

Concentration

added (mg) 34.16 42.70 51.24 8.00 10.00 12.00

found (mg) 34.02 41.83 50.98 7.87 9.82 11.94

RSD: relative standard deviation.

%

Recovery

%

RSD

99.60

0.253

98.33

0.592

97.95 98.33 98.73 99.50

0.266 0.898 1.386 0.971

3.5.2. Precision I ntraday and interday precision was found to be

B

3000

2000

C

Wavenumber (cm ) -1

approximately 99 % for both lidocaine and ranolazine. Results of repeatability have reported as % relative standard deviation in Table 6. 1000

Figure 7. FTIR spectra of B. spectabilis extract (A ), lidocaine (B) and lidocaine-colour complex (C).

Table 6 Results of repeatability (intraday and interday). Analyte

Lidocaine

Concentration

Intraday Interday

Ranolazine Intraday Transmitance (%)

A

Interday

(µg/mL)

4

12 20 4

12 20 5

10 25 5

10 25

Data are expressed as mean依SD, n=9.

3000

2000

Wavenumber (cm ) -1

99.410依0.102

99.720依0.160 99.010依0.225 96.750依0.212

99.080依0.110 99.410依0.189 98.730依0.060

99.590依0.062 99.620依0.086 98.600依0.014

99.480依0.066 99.440依0.040

3.5.3. LOD LOD was found to be 0.088 and 0.26 µg/mL for lidocaine and ranolazine respectively.

B

C

Concentration estimated (%)

1000

Figure 8. FTIR spectra of B. spectabilis extract (A), ranolazine (B) and ranolazine-colour complex (C) .

3.5. Method validation 3.5.1. Accuracy The results indicated excellent recoveries ranging from 98.33% to 99.60% and 98.33% to 99.50% for lidocaine and ranolazine respectively and reliability of the method ( T able 5 ) . R ecoveries obtained for the drug did not differ significantly from 100 % showing that there was no interference from common excipients used in the formulation and thus indicating accuracy and reliability of the method.

3.5.4. LOQ LOQ was found to be 0.266 and 0.80 µg/mL lidocaine and ranolazine respectively. 4. Discussion Formation of color complex between drugs and extract is contributed to the keto-enol tautomerism. Electrons on the phenolics oxygen get simply delocalized in aromatic ring, making it acidic due to easy removal of proton (H+). While amide due to presence of lone pair of electron on nitrogen is going to show basic nature which contributes in formation of color complex between amide and the color pigment obtained from B. spectabilis. Study revealed that increased concentration of ascorbic acid directly contributed in

Suresh Killedar et al./Asian Pac J Trop Med 2014; 7(Suppl 1): S560-S567

stability of color. Extract mainly contains betalains, stable at acidic pH so it is essential to store up this extract at acidic pH to maintain its activity. B etalains are the compound mainly responsible for color of B. spectabilis bract which mainly contains bougainvillein-V. B. spectabilis bracts color was found to be less stable at 25 °C exposed to light and at pH 7. While, refrigeration storage (4 °C) condition without light exposure managed to preserve the color up to 3 weeks than storage at 25 °C with exposure to light. The proposed method for colorimetric estimation of lidocaine and ranolazine is accurate, precise, economical and convenient, yielding reproducible results. Moreover, the method is economic, simple and rapid, hence can be employed for routine analysis in quality control laboratories as it is specific without interference of excipients. If the B. spectabilis bract extact is stored properly, this could be useful in colorimetric estimation of aforesaid amide containing drugs. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements Authors are thankful to Dr. M. Y. Cholekar-Bachulkar, Principal, Shri Vijaysinha Yadav Arts and Science College, Peth Vadgaon, India (M.S.) for their valuable guidance.

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