Comparison of the Detection Limit and the Quantification Limit of Nicotinamide on Different Chromatographic Sorbents in TLC

Research Article

Austin Chromatogr. 2015;2(3): 1034.

Comparison of the Detection Limit and the Quantification Limit of Nicotinamide on Different Chromatographic Sorbents in TLC

Malgorzata Dolowy¹, Alina Pyka¹* and Milena Pluta²

¹Department of Analytical Chemistry, Medical University of Silesia in Katowice, Poland

²School of Pharmacy and the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Poland

*Corresponding author:Alina Pyka, Department of Analytical Chemistry, School of Pharmacy and the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, 4 Jagiellonska St., PL- 41-200, Sosnowiec, Poland

Received: April 09, 2015; Accepted: May 28, 2015; Published: June 04, 2015

Abstract

TLC combined with densitometry was successfully applied to determine the Detection Limit (LOD) and also Quantification Limit (LOQ) of nicotinamide. Average values of LODs and LOQs were determined on the basis of the standard deviation of the response and the slope of specific calibration curve according to the procedure recommended by ICH guideline. Influence of chromatographic conditions such as stationary phase (sorbent) and also mobile phase composition on determining of LOD and LOQ values of examined nicotinamide by NP-TLC and RP-TLC systems was estimated. Of all chromatographic plates and mobile phases used, those which give the best results (the lowest LOD and LOQ values) by NP-TLC system is silica gel 60F254 plates developed with methanol as mobile phase: LOD=0.088 μg/spot, LOQ=0.265 μg/spot. In the case of RP-TLC method the three of four applied mobile phases: dioxane, methanol, and ethanol enable detection of nicotinamide at the lowest level on chromatographic plates precoated with RP18F254: LOD ranges from 0.077 to 0.183 μg/spot. The LOQ value is placed in the range from 0.233 to 0.553 μg/spot.The results of LODs and LOQs indicate that the proposed TLC-densitometric method is suitable for the detection and also quantification of nicotinamide at different level.

Keywords: Nicotinamide; Vitamin PP; TLC-densitometry; LOD; LOQ

Introduction

Nicotinamide (known as Vitamin PP) is as member of vitamin B group (vitamin B3) [1]. This bioactive compound is commercially available in various pharmaceutical dosage forms (i.e., tablets, intramuscular and intravenous injection) and also diet supplements. It is widely used for the treatment of central nervous system disorders, cancer therapy and also in synthesis of some steroid hormones [2-6]. Different analytical methods, such High Performance Liquid Chromatography in Reversed Phase System (RP-HPLC), spectrophotometry, photochemical spectrofluorometry and also micellar chromatography were found to be satisfactory to assay of nicotinamide in different simple and complex tablets [7- 10]. These methods are useful for the determination of nicotinamide in pharmaceutical formulations but they are tedious or expensive because required large amount of solvents like for example HPLC. Therefore, it is very important to find a simple and sensitive analytical method for determining nicotinamide in appropriate amount, like for example Thin-Layer Chromatography combined with densitometry (TLC-densitometry). The measure of sensitivity of each analytical method including TLC-densitometry is Detection Limit (LOD) and also Quantification Limit (LOQ). Many of scientific papers and guidelines are focused on the procedure of calculations of the two parameters [11-16]. According to the guideline on Validation of analytical procedures (ICH Harmonised Tripartite Guidelines) numerous methods for determining of LOD and LOQ values are possible [11]. The choice of calculating procedure depends on the type of analytical method used: if is it instrumental or non-instrumental. Generally, the following approaches for determining of LODs and LOQs are acceptable [11]:

It is well known that the choice of each analytical method including TLC for assay a proper compound as active ingredient as well as impurity present in pharmaceutical or biological matrix depends strongly on its sensitivity, thus on LOD and LOD values. Therefore the aim of this study was to determine the LODs and LOQs of nicotinamide by the use of TLC-densitometry under different chromatographic conditions (various sorbents and mobile phases) in Normal Phase and Reversed Phase Systems (NP-TLC and RPTLC). Influence of all applied stationary phases and mobile phases on LODs and LOQs, so on sensitivity of proposed method for detection of this compound was estimated. Present study is continuation of previous researches by Pyka et al.[17-23] concerning the application of TLC-densitometry in the determination of chemical stability and also separation of various nicotinic acid derivatives. Our last paper confirms the applicability of RPHPTLC-densitometric method with the use of silica gel 60 RP18WF254 as stationary phase and mixture of methanol-water (3:7, v/v) for the quantification of nicotinamide in selected pharmaceutical formulations [23]. In the present study evaluation of the LOD and LOQ study of examined nicotinamide was conducted by the use of two TLC systems. NP-TLC method was performed on five chromatographic plates: silica gel 60 F254 (Art. 1.05554), silica gel 60 (Art. 1.05553), a mixture of silica gel 60 and kieselguhr F254 (Art. 1.05567), aluminum oxide 60 F254 (Art. 1.05550) and aluminum oxide 150 F254 (Art. 1.05551). The plates were developed using the mixtures containing methanol and acetone, respectively. In the case of RP system two different sorbents (silica gel RP18F254s, silica gel RP8F254s) and four mobile phases consisted of methanol, ethanol, propanol and dioxane was applied. Comparison of LODs and LOQs obtained in both chromatographic systems (NP-TLC and RP-TLC) was done. Present work can be a useful indicator for the choice of chromatographic conditions of all described enabling detection of nicotinamide by TLC-densitometry in RP-TLC and also NP-TLC with appropriate sensitivity (at needed level) which is enough for pharmaceutical analysis preparations containing this compound.

Experimental

Standard and chemicals

Reference standard of investigated nicotinamide (NAM) was procured from Sigma-Aldrich (St. Louis, MO, USA). Ethanol absolute (=99.8%) and other solvents which have been applied as mobile phase compositions, such as dioxane, propanol, methanol, acetone and methanol were obtained from POCh (Gliwice, Poland). All chemicals and reagents were analytical grade.

Materials

The following chromatographic plates were used in this study:

Chromatography

Preparing of standards solutions of nicotinamide for NP-TLC and RP-TLC analysis: Standard solution of examined nicotinamide was obtained by dissolving of 200mg of accurately weighed nicotinamide in 50 mL of ethanol (99.8%). This solution was applied for the preparation of appropriate dilutions. Samples solutions for study at the following concentrations: 0.20 mg/mL, 0.18 mg/mL, 0.16 mg/mL, 0.14 mg/mL, 0.12 mg/mL, 0.10 mg/mL, 0.08 mg/mL, 0.06 mg/mL, and 0.04 mg/mL were spotted on the chromatographic plates in quantity of 5 μL in each case.

NP-TLC analysis of nicotinamide: TLC analysis of nicotinamide in normal phase was performed on chromatographic plates for NPTLC precoated with 0.20 mm layers of various sorbents: aluminum oxide 60 F254 (Art. 1.05550), aluminum oxide 150 F254 (Art. 1.05551), silica gel 60 (Art. 1.05553), silica gel 60 F254 (Art. 1.05554) and also with a mixture of silica gel and kieselguhr 60 F254 (Art. 1.05567). The plates were procured from Merck (Darmstadt, Germany). The amount of 5 μL of respective standard solution was applied in form of spots on chromatographic plates of 10 cm x 20 cm in size which were previously activated at temperature of 120°C during 30 minutes before use. Plates were developed using two mobile phases: methanol and also acetone to a distance of approximately 7.5 cm from baseline in TLC chamber for 20 cm x 10 cm plates (Art. 0.222.5221) obtained from Camag (Muttenz, Switzerland). After developing, the plates were dried at room temperature (21±1°C) in a fume cupboard during 24 hours and next densitometrically analyzed.

RP-TLC analysis of nicotinamide: TLC analysis of nicotinamide in reversed phase was performed on chromatographic plates for RP-TLC precoated with 0.20 mm layers of silica gel RP18F254 (Art. 1.05559) and on glass plates precoated with 0.20 mm layer of silica gel RP8F254s(Art. 1.15424) supported from Merck (Darmstadt, Germany). The amount of 5 μL of standard solutions was spotted on chromatographic plates of 10 cm x 20 cm in size which were previously activated at temperature of 120°C during 30 minutes before use. Plates were developed with the four mobile phases consisted of methanol, ethanol, propanol and dioxane, respectively to a distance of approximately 7.5 cm from baseline in TLC chamber for 20 cm x 10 cm plates (Art. 0.222.5221) obtained from Camag (Muttenz, Switzerland). After developing, the plates were dried at room temperature (21±1°C) in a fume cupboard during 24 hours and next densitometrically analyzed.

Densitometric analysis of nicotinamide

Spectrodensitometric scanning was done by Camag TLC Scanner 3 (Muttenz, Switzerland) equipped with WinCATS 1.4.2 software. All spectrodensitometric measurements were conducted in reflectance absorbance mode at wavelength of 222 nm. This wavelength was found to be suitable for densitometric analysis of nicotinamide under applied chromatographic conditions. The source of radiation was a deuterium lamp. The scanning speed was 20 nm/s and the data resolution was 100 nm/step. The slit dimension was kept at 8.00 mm x 0.60 mm, Macro. The spot areas (AU) obtained under applied chromatographic systems (NP-TLC and also RP-TLC) were used in determination of LOD and LOQ values. Each analysis was repeated three times.

Determination of Detection Limit and Quantification Limit of nicotinamide (LOD and LOQ)

In order to estimate the LODs and LOQs of nicotinamide, the method based on standard deviation value of the response and also on the slope of a specific regression line was applied. The lowest amount of examined nicotinamide in a studied sample that can be detected by applied TLC-densitometry (designated as LOD) was calculated according to formula (1). The LOQ value of nicotinamide which is defined as the lowest amount of nicotinamide that might be quantitatively determined by the use of TLC-densitometry (under applied chromatographic conditions) with good accuracy was calculated according to the equation (2). It could be noted that in determining of LOD and LOQ values, the standard deviation (s) could be calculated in two ways using standard deviation of intercept of specific regression line (sa) and with the use of residual standard deviation of regression line (sxy), respectively. Statistical evaluation of the obtained results (i.e., regression plots) was prepared by Statistics v 10.0 PL (StatSoft, Kraków, Poland).

Additionally, the correctness of LOD values obtained in each case was checked with the use of the following relationships (Eqs. 3 and 4) which can be observed between calculated LOD value and the concentration of examined substance [16]:

10 × LOD > C (3)

LOD < C (4)

where:

LOD - Limit of Detection,

C - Concentration of analyzed substance in the reference samples.

Results and Discussion

In present study the limit of detection and quantification of nicotinamide by TLC-densitometry on various sorbents and using different mobile phases was determined. Average values of LOD and LOQ calculated on the basis of standard deviation of intercept of specific regression line (sa) and using residual standard deviation of regression line (sxy) - Eqs. 1 and 2 for NP-TLC systems are listed in Table 1. Comparison of all obtained results shows Figures 1&2. The results obtained using TLC-densitometry in normal phase system indicate that the lowest LOD and LOQ values, thus the best detection shows the chromatographic plates precoated with silica gel 60 F254 (Art. 1.05554) and developed by methanol (LOD=0.088 μg/ spot, LOQ=0.265 μg/spot). The highest LOD and LOQ values have been obtained using this mobile phase on the chromatographic plates precoated with aluminum oxide 60F254and 150 F25 (Art. 1.05550, Art. 1.05551): LOD=0.264÷0.328 μg/spot and LOQ=0.801÷0.994 μg/spot. This fact confirms the worst results of the detection and quantification limit of nicotinamide predicted with the use of methanol as mobile phase.