Development and Estimation of Pyridoxine HCl in Bulk and Formulation by Second Order Derivative Area under Curve UV-Spectrophotometric Methods.

 

Audumbar Mali1*, Rahul Gorad2, Aamer Quazi2, Ritesh Bathe1, Ashpak Tamboli3

1Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

2Department of Pharmaceutics, ASPM’S K. T. Patil College of Pharmacy, Osmanabad-413501, Maharashtra, India.

1Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

*Corresponding Author E-mail: maliaudu442@gmail.com

 

 

 

Received on 22.03.2016       Accepted on 09.04.2016     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2016; 6(2): 106-112.

DOI: 10.5958/2231-5713.2016.00015.5

 

 

ABSTRACT:

Simple, novel, fast, accurate and reliable spectrophotometric methods were developed for determination of Pyridoxine HCl in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in Dist. water. The quantitative determination of the drug was carried out using the second order Derivative Area under Curve method values measured at 301-319nm. Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of Pyridoxine HCl using 5-25μg/ml (r²=0.9966) for second order derivative Area under Curve spectrophotometric method. The proposed methods have been extensively validated as per ICH guidelines. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations. The developed methods were successfully applied to estimate the amount of Pyridoxine HCl in pharmaceutical formulations.

 

KEY WORDS: Pyridoxine HCl, Second order derivative, Area under Curve (AUC), Precision, Accuracy.

 

 

1. INTRODUCTION:

Pyridoxine HCl is chemically described as 4, 5-Bis (hydroxyl methyl)-2-methylpyridine-3-ol Hydrochloride salt assists in the balancing of sodium and potassium as well as promoting red blood cell production. Pyridoxine is one of the compounds that can be called vitamin B6, along with pyridoxal and pyridoxamine. It is often used as pyridoxine hydrochloride. [1, 2]

 

 

In our Literature survey reveals that for Pyridoxine HCl Spectrophotometric [3-5] methods and HPLC [6, 7] methods have been reported for its determination in commercial formulation. To our notice, no UV- spectrophotometric method using Second Order Derivative Area under Curve has been reported for the determination of Pyridoxine HCl in bulk and tablets.

 

Hence an attempt has been made to develop new Second Order Derivative Area under Curve spectrophotometric method for estimation of Pyridoxine HCl in bulk and pharmaceutical formulations with good accuracy simplicity, precision and economy.

 

 

Fig. 1: Structure of Pyridoxine HCl

 

2. MATERIALS AND METHODS: [8-11]

2.1. Derivative Spectrophotometric Methods:-

The Second derivative spectrophotometry was used in the wavelength ranges from 301 and 319 nm.

 

[d2A/dλ2]= f (λ): second order

 

The Second derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a cross-over point at the λ max of the absorption band.

 

2.2. Area under curve (Area calculation):-

In this study area was integrated between wavelength ranges from 301-319 nm.

 

Area calculation: (α+β) =

 

Where, α is area of portion bounded by curve data and a straight line connecting the start and end  point, β is  the  area  of  portion  bounded  by  a  straight  line  connecting  the  start  and  end point on curve data and horizontal axis, λ1 and λ2  are wavelength range start and end point of curve region.

 

2.3. Apparatus and instrumentation:-

A Shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (Contech, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated volumetric glassware (Borosil®) was used for the validation study.

 

2.4. Materials:-

Reference standard of Pyridoxine HCl API was supplied as gift sample by FDA Mumbai. Tablets sample with label claim 50 mg per tablet were purchased from local market Pune.

 

2.5. Method development: [12-15]

2.5.1. Preparation of Standard and Sample Solutions:-

Stock solution of 10μg/ml of Pyridoxine HCl was prepared in Dist. water, for Second Order Derivative Area under Curve spectrophotometric analysis. The standard solutions were prepared by dilution of the stock solution with Dist. water in a concentration range of 5, 10, 15, 20 and 25μg/ml with Dist. water for Second Order Derivative Area under Curve spectrophotometric methods. Dist. water was used as a blank solution.

 

 

 

 

 

 

 

Fig. 2: Second order derivative Area under Curve spectrum of Pyridoxine HCl in Dist. water (25µg/ml).

 

 

Fig. 3: Second order derivative spectrum of Pyridoxine HCl in Dist. water (25µg/ml).

Conc.
 
 


Fig. 4: Linearity of Pyridoxine HCl.

 

 

 

2.5.2. Calibration curve for Pyridoxine HCl:-

The dilutions were made from Standard Stock solution to get concentration of 5, 10, 15, 20, and 25µg/ml respectively. These solutions were scanned from 400 to 200 nm and Second Order Derivative Area under Curve values was integrated in the range of 301-319 nm. The calibration curve was plotted between areas under curve values against concentration.

 

 

 

 

 

2.5.3. Assay of tablet formulation:-

Twenty tablets each containing 50mg of Pyridoxine HCl were weighed crushed to powder and average weight was calculated. Powder equivalent to 10 mg of Pyridoxine HCl was transferred in 100 ml of volumetric flask. A 50 ml of Dist. water was added and sonicated for 15 minutes. Then solution was further diluted up to the mark with Dist. water. The solution was filtered using Whatman filter paper no. 41, second 5 ml of filtrate was discarded. This solution was further diluted to obtain 10µg/mL solution with water, subjected for UV analysis using Dist. water as blank. This procedure was repeated three times.

 

 

 

 

Fig. 5: Second order derivative Area under Curve spectrum of Pyridoxine HCl of dosage form in Dist. water (25µg/ml).

 

 

 

 

Fig. 6: Second order derivative spectrum of Pyridoxine HCl of dosage form in Dist. water (25µg/ml).

 

Fig. 7: Second order derivative overlay of Pyridoxine HCl at different Concentration.

 

 

 

Table 1: Assay of tablet dosage form

Sr. No.

Sample Solution Concentration (µg/ml)

Amount found (%)

Mean % found*

% RSD*

1

25

99.05

 

 

2

25

101.28

99.63

0.5480

3

25

98.57

 

 

*n=3, % RSD = % Relative Standard Deviation.

 

 

 

Table 2: Accuracy results for Pyridoxine HCl

Accuracy level

Sample conc (µg/ml)

Std. conc

Total amount. Added (µg/ml)

% Recovery

Mean % Recovery

% RSD

80

25

12

22

98.61

 

 

100

25

15

25

102.12

99.99

0.5214

120

25

18

28

99.25

 

 

 

 

3. Method Validation: [16-18]

The above method was validated for various parameters such as Accuracy, Linearity, Precision, Limit of detection (LOD) and Limit of Quantitation (LOQ) according to ICH guideline.

 

3.1. Accuracy:

The  accuracy  for  the  analytical  method  was  evaluated  at  80%,  100%  and  120%  levels  of 25µg/ml Sample solution. Second Order Derivative Area under curve (AUC) was measured in wavelength range 301-319 nm and results were obtained in terms of percent recovery. Three determinations at each level were performed and % RSD was calculated for each level.

 

3.2. Precision:

The precision of an analytical procedure expresses the closeness of an agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions intraday precision was studied by integrating area of standard solution of 25µg/ml concentration at six independent series in the same day. Interday precision studies were performed by integrating area of standard solution of 25µg/ml concentration on three consequent days. The % RSD was calculated.

 

Table 3: Precision Study

Parameter

Intra day

Inter-day

Sample sol conc.µg/ml

25

25

AUC (mean)

0.7436

0.7982

%RSD

0.5248

0.5761

 

3.3. Limit of Detection and Limit of Quantification:-

The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula

 

LOD = 3.3 σ /S

 

The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula

 

LOQ = 10 σ/S

 

Where, σ is standard deviation of the response and

             S is the slope of the calibration curve.

LOD and LOQ of Pyridoxine HCl was found to be 0.4259µg/ml and1.2772µg/ml respectively.

 

 

Table 4: Summary of validation parameters

Parameter

Result

λ range

301-319

Regression Equation (Y=mx+c)

Y=0.0009x + 0.0013

Linearity range

5-25µg/ml

Slope

0.0009

Intercept

0.0013

Correlation coefficient (R2)

0.9966

Limit of Detection (LOD) µg/ml

0.4259

Limit of Quantitation (LOQ) µg/ml

1.2772

Accuracy (Mean % Recovery)

99.99

Precision (% RSD)

0.5214

 

 

 

4. RESULTS AND DISCUSSION:

The UV visible spectroscopic method for the Pyridoxine HCl by Second order derivative Area under Curve was found to be simple, accurate, economical and reproducible. The  drug  concentrations  were  found  to  be linear  in  the  range  of  05-25 µg/ml and the correlation coefficient value of 0.9966 indicates that  developed  method  was  linear.  For  Precision  the  percent  relative  standard  deviation  (% RSD) was found to be 0.5214 while, intra-day  and  inter-day  precision  results  in  terms  of  percent relative standard deviation values  were found to be 0.5248 and 0.5761 respectively thus the method is observed as precise. The accuracy of the method was assessed by recovery studies at three different levels i.e. 80%, 100%, 120%. The values of standard deviation were satisfactory and the recovery studies were close to 100%. The % RSD value is ≤ 2 indicates the accuracy of the method.  The  Limit  of  Detection  and  Limit  of  Quantitation  values  were found  to  be  0.4259µg/ml and 1.2772µg/ml respectively. The result of the analysis for pharmaceutical  formulation  by  the  developed  method  was  consistent  with  the  label  claim, highly reproducible and reliable. The method  can  be  used  for  routine  quality  control  analysis of Pyridoxine HCl in  bulk  and pharmaceutical formulations.

 

5. CONCLUSION:-

The UV spectroscopic AUC method for the analysis of Pyridoxine HCl by Second order derivative Area under Curve was found to be simple, precise, and accurate; can be used for assay of bulk drug and pharmaceutical dosage formulations.

 

6. ACKNOWLEDGEMENT:

The authors are highly thankful to the Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for proving all the facilities to carry out the research work.

 

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