VALIDATION OF FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY FOR QUANTITATIVE ANALYSIS OF SIMETHICONE IN ANTACID SUSPENSION PREPARATIONS

By: Arini Musfiroh 09/282241/FA/08319

FACULTY OF PHARMACY UNIVERSITAS GADJAH MADA YOGYAKARTA 2013

APPROVAL SHEET

VALIDATION OF FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY FOR QUANTITATIVE ANALYSIS OF SIMETHICONE IN ANTACID SUSPENSION PREPARATIONS ABSTRACT Fourier transform infrared (FTIR) spectroscopy has been employed for the quantitative analysis of simethicone based on several compendium such as USP, Farmakope Indonesia, and Indian pharmacopeia. Analysis of simethicone is achieved by quantify polydimethylsiloxane (PDMS) component of the active ingredients. However, analysis using FTIR method for simethicone-containing preparations especially suspension preparation is difficult to get reproducible result due to many variations of the same samples measurement. It has been known previously that the extraction procedure used for sample preparation was causing loss of simethicone content. A sample preparation procedure for FTIR spectroscopy has been developed. The modified FTIR method has been validated. Linearity, sensitivity, accuracy and precision are presented for the modified method. These method has been also applied to quantify simethicone in suspension and chewable tablet antacid preparations and gave simethicone content within the range of 85-115% based on USP 32. Keywords: FTIR spectroscopy; simethicone; antacid; validation absence of a significant chromophore. This method quantifies PDMS component of simethicone by comparing the typical spectral band for PDMS with an external standard of known concentration. Because of the small silicon dioxide content of simethicone, it is sufficient to only quantify PDMS component although the silicone dioxide content can also be quantified through another testing procedure. However, analysis of simethicone using FTIR method is difficult to get reproducible results due to many variations of the measurements of the same samples. Several procedures for the analysis of simethicone in antacid and anti-flatulence preparations [3] , cosmetics preparation such as lotion and cream [9] do not provide satisfactory results. A study showed that the extraction procedure for sample preparation causing loss material. PDMS material sticking to the jar walls due to the hydrophobic nature of PDMS [3]. The variations of the measurements may be attributed to the

INTRODUCTION Antacids usually contain aluminum and magnesium compounds that can often relieve the symptoms of heartburn and reflux of gastric fluid [5]. Many antacid preparations use a combination with simethicone to reduce side effects of carbon dioxide as a byproduct from the gastric fluid neutralization reaction [6]. Simethicone is a mixture of polydimethylsiloxane (PDMS) and silicon dioxide (SiO2) which is used to relieve flatulence, abdominal discomfort due to excessive gas, colic in infants and ulcer disease [10]. Simethicone constitutes approximately 90.5 to 99.0 % of PDMS and 4 to 7% of SiO2 [11]. Fourier transform infrared (FTIR) spectroscopy has been employed for the quantitative analysis of simethicone based on several compendium such as USP, Farmakope Indonesia, and Indian pharmacopeia. Simethicone analysis is carried out by FTIR spectroscopy due to the

extraction procedure, so it is necessary to modify the extraction procedure from FTIR method.

MATERIAL AND METHODS 2.1 Materials Simethicone reference standard and placebo sample was purchased from Graha Farma (Indonesia). Toluene, hydrochloric acid, anhydrous sodium sulphate were from E. Merck (Darmstadt, Germany) and distilled water was purchased from Integrated Research and Testing Laboratory of Universitas Gadjah Mada (IRTL UGM, Indonesia). All of materials used for experiment were on analytical grade. Antacid suspension containing aluminium hydroxide, magnesium hydroxide, and simethicone as active ingridients was purchased from UGM Pharmacy (Yogyakarta, Indonesia). 2.2 Infrared spectroscopy ABB MB3000 FTIR spectrophotometer (Montreal, Canada) was used in the 650 to 4000 cm-1 scan range. Spectra were collected using Horizon MB (Version 3.1.24.2, ABB Analytical Measurement, Canada). For each spectra, 32 scans were performed with a resolution of 4 cm-1. 2.3 Sample preparation 2.3.1 Reagents and solutions A 10 % simethicone standard stock solution was prepared by diluting 5 mL of simethicone reference standard to a final volume of 50.00 mL with toluene. A 6 N hydrochloric acid was prepared by diluting 50 mL of concentrated hydrochloric acid to a final volume of 100.00 mL with distilled water.

2.3.2 Extraction Amount of sample preparations whose formulation contained 66.67 mg of simethicone was accurately weighed and transferred to a capped conical flask. Then 20.0 mL of 6 N hydrochloric acid was added to the jar and swirled until the preparations dissolved completely. Next, 25 mL of toluene was added for the extraction and the jar was mechanically shaken at a frequency 200 rpm for 10 minutes. After allowing the layers to separate, toluene layer was pipetted sufficiently and transferred to screw-capped tube. Any residual water in the organic layer was then removed using anhydrous sodium sulfate. Standards were prepared by the same extraction procedure using known quantities of simethicone. 2.4 Sample assay Toluene layer was then analyzed by FTIR. The height of the band (absorbance) at 1261, 1091 and 1014 cm-1 were used to determine simethicone content in preparations. All spectra were background corrected which consists of infrared-active atmospheric gases, carbon dioxide and water vapor. 2.5 Determination of analytical parameters 2.5.1 Linearity Linearity was evaluated by preparing simethicone standard in six different concentrations in the range from 0.05 to 1.00 % through the same preparation procedure used for sample. Each measurement in FTIR was performed 3 times. Linearity was evaluated by calculation of the correlation coefficient (r) from the regression of the relationship between the concentration of simethicone (x) and the average absorbance from each wavenumber used (y). Additionally,

wavenumber which shows the biggest r value is used for subsequent measurement. 2.5.2 Sensitivity Sensitivity was determined by testing 10 pieces of blank samples added with a concentration of simethicone standard that gives smallest measurable response. Standard deviation (SD) of the responses (Ysamp) was then calculated (RSD Ysamp 33.3%). LOD was evaluated by calculation with the following equation: YLOD = Yblank + 3SD Y blank is the response for blank sample, in this case Y blank = 0. SD is the standard deviation obtained from 10 absorbance of samples. YLOD value was used in order to obtain the value of x (concentration) from calibration curve equation obtained in linearity determination. The value of x is the LOD. LOQ value can be calculated with formula: LOQ = 3.33 x LOD. 2.5.3 Precision Repeatability was performed by testing 10 blank samples added with simethicone standard under the same conditions, including the analysts, laboratory equipment and the same time. Absorbance measurement of each sample was performed twice. Repeatability was evaluated by calculation of the relative standard deviation (RSD) according to the following equation: RSD (%) = (SD / ) x 100% SD is the standard deviation and is the mean of concentration of samples. Interday precision was calculated by comparing the

results obtained from 3 different test of repeatability in 3 different days. 2.5.4 Accuracy Accuracy was evaluated by standard addition method. Three groups of sample in triplicate were spiked with simethicone standard at 50, 100 and 150% of the sample solution concentration, while the other group without the addition of simethicone standard. Recovery was calculated according to the following equation: Recovery = (C1-C2)/C3 x 100 % C1 is concentration measured of spiked sample, C2 is concentration measured of sample without spiking and C3 is actual concentration added to sample.

RESULTS AND DISCUSSION 3.1 Extraction procedure Extraction was performed by dissolving antacid preparations firstly in hydrochloric acid (HCl) 6 N as the aqueous phase to neutralize the acid contents. The presence of aluminum and magnesium will be absorbed PDMS in solution [10]. Toluene was used as the organic phase. The use of 6 N HCl as the aqueous phase was a modification of USP method [11] which uses sodium hydroxide (NaOH) 0.1 N as the aqueous phase. Simethicone extraction using the modified method had better effectiveness than the USP method with the same extraction conditions (Tab. I). Extraction procedure was performed for 10 minutes by extraction effectiveness because after 10 minutes shaking, simethicone was extracted entirely to the toluene phase (Fig. 1).

Table I. Comparison of simethicone absorbance from USP extraction method and modified method Simethicone absorbance at 1261 cm-1 Extraction method Solvents Concentration of Concentration of 0,2 % b/v 0,5 % b/v USP 32 method (2009) NaOH 0,1 N + Toluene 0,01822 0,02933 Modified method HCl 6 N + Toluene 0,02055 0,03276

0.025

Absorbansi

0.02 0.015 0.01 0.005 0 0 10 20 30 40

Waktu (menit) Figure 1. Extraction time vs simethicone (0.25% w/v) absorbance at wavenumber 1261 cm-1

3.2 FTIR analysis of simethicone and linearity Infrared spectrum for simethicone dissolved in toluene (Fig. 2) showed a doublet at wavenumbers 1097 and 1014 cm1 due to asymmetric and symmetric stretching vibrations of Si-O-Si bond, [10] respectively , sharp bands at -1 wavenumbers around 1261 cm due to the symmetric stretching vibrations of CH3-Si and its asymmetric stretching vibrations at 1412 cm-1, CH3 rocking at 806 cm-1 [8], stretching vibrations of C-H from CH3 at 2965 cm-1 (symmetric) and 2906 cm-1 (asymmetric) [4].

PDMS spectra was quantified using the height of the band at wavenumber ~1261, 1097, and 1014 cm-1. These bands are typical bands for PDMS. Quantitative analysis using double bands at 1097 and 1014 cm-1 has the advantage because it may determine whether interfering substances are present. The absorption spectrum of a pure PDMS should show a symmetrical doublet with equal intensity [9]. The wavenumber 1261 cm-1 shows spectrum with the least interference from other compounds than the others [3]. The linearity of the method for simethicone assay was evaluated in the range of concentrations between 0.05-1.00 %w/v at 3 different wavenumbers, 1261, 1097, and 1014 cm-1 (Fig. 3). The correlation coefficient (r) for each wavenumbers are 0.9989, 0.9858, 0.9922, respectively. Based upon these result, peak at 1261 cm-1 was used for quantitative analysis for simethicone since it showed the best linearity than other wavenumbers. The slope at 1261 cm-1 was 0.0437 A.U. (Absorbance Units) and the intercept value was 0.0111 AU.

Figure 2. Simethicone standard spectrum, 100 %

Absorbance

0.13 0.08 0.03 0.00 0.20 0.40 0.60 0.80 1.00 concentration (% w/v) (a)

method was sensitive which can detect and quantify simethicone well, both in the range of the calibration curve made and in antacid suspension preparations.
Table II. Analysis of 10 blank samples spiked with 0,01 %w/v of simethicone standard to determine LOD and LOQ value

Absorbance

0.15 0.10 0.05 0.00 0.00 0.20 0.40 0.60 0.80 1.00 concentration (% w/v) (b)

Absorbance

0.070 0.050 0.030 0.010 0.00 0.20 0.40 0.60 0.80 1.00

concentration (% w/v) (c) Figure 3. Calibration curve of simethicone at wavenumber ~1014 cm-1 (a); ~1097 cm-1 (b); ~1261 cm-1 (c)

Samples 1 2 3 4 5 6 7 8 9 10 Average % RSD LOD (%w/v) LOQ (%w/v)

Absorbansi 0.01319 0.00706 0.00696 0.00673 0.01499 0.00381 0.01497 0.01162 0.01183 0.01019 0.01013 37.86 0.0094 0.0314

3.3 Sensitivity Sensitivity was determined by evaluating the limit of detection (LOD) and limit of quantitation (LOQ). LOD is the smallest concentration of analyte that can still be detected with its significantly different response [7] compared to noise , while LOQ is the lowest analyte level that can be determined quantitatively with [2] acceptable precision . LOD and LOQ were evaluated by measuring the absorbance of 10 pieces of blank samples which spiked with the simethicone standard that still gives a measurable response. LOD value was 0.0094% w/v and the LOQ value was 0.0314% w/v (Tab. II). The FTIR

3.4 Precision Precision is the degree of closeness of various sampling process in a series of measurements, which often expressed with the standard deviation (SD) or relative standard deviation (RSD) of the response [1]. This value is used to determine the constancy of the instrument response to an analyte from time to time. Precision assay was performed with repeatability assay and intermediate precision by spiking known concentrations of simethicone standard into 10 blank samples. In this study, samples were spiked with 0.6 % w/v of simethicone. The results for precision assay were shown in Table III. Repeatability assay at day 1, 3, and 8 gave RSD value 3.09, 2.32, 2.52 %,

respectively. Intermediate precision was evaluated from three different days of measurement and gave RSD value 2.71 %. Based on the range of RSD according to Horwitz, RSD value allowed for a 0.6% level of analyte is less than or equal to 4.3% [2]. It can be concluded that the FTIR method had a good precision. 3.5 Accuracy Accuracy is the degree of closeness between the value found with the true or accepted value [1]. Accuracy of the method was performed by spiking known concentrations of simethicone into solutions containing the preparation. Recovery was assessed from 3 different concentrations, 50, 100, and 150% of the simethicone concentration in the preparation and the results are shown in Table IV. The recovery values for preparations spiked with 0.1 %, 0.2 %, and 0.3 % were

100.95 1.36 %, 98.44 1.55 %, 101.64 2.32 %, respectively. The recovery value allowed for a concentration range of 0.1-1.0% w/v is between 95-105% [2]. From this study, it can be concluded that the FTIR method has good accuracy. 3.6 Quantitative analysis of simethicone in commercial antacid suspension FTIR spectroscopy was carried out for quantitiative analysis of simethicone in antacid suspension. The preparations showed similar spectrum with simethicone standard, as can be seen in Fig. 4. The mean concentrations of simethicone obtained from the study was 45.43 mg/5 mL antacid suspension or as much as 90.86% of the label claims. The predicted concentration of simethicone in suspension was in the range of concentration allowed, which is between 85-115% [11].

Table III. Analysis of 10 blank samples to determine repeatability and intermediate precision

Day 1 3 8

Actual concentrations (%w/v) 0.60 0.60 0.60

Predicted concentrations (%b/v) (n=10) 0.580.040 0.580.030 0.570.032

Repeatability (% RSD) 3.09 2.32 2.52

Intermediate precision (% RSD) 2.71

Table IV. Analysis of 10 blank samples to determine accuracy

Spiked Actual content concentrations (%w/v) (%w/v) None 0,20 0,10 0,30 0,20 0,40 0,30 0,50 Range of recovery (%)

Predicted concentrations (%w/v) (n=3) 0,150,0073 0,250,0014 0,350,0031 0,460,0070

Recoveries (%)

RSD (%)

100,951,36 0,42 98,441,55 0,50 101,642,32 0,72 98,44-101,64

[3]

Figure 4. Comparison of simethicone-containing antacid suspension spectrum (a) and standard simethicone (0.5 % w/v) spectrum (b) at wavenumber ~ 1261 cm-1

CONCLUSION For Simethicone-containing antacid suspensions, a modified method has been validated for analysis of simethicone with FTIR spectroscopy. The method had good validation parameters according to ICH. Method had linearity in the range of 0.51.0% w/v, good sensitivity with low level of LOD and LOQ, good precision (repeatability and intermediate precision), and showed acceptable accuracy in the analysis of simethicone.

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Hargis, A.D.; Whittall, L.B. Improvements in soft gelatin capsule sample preparation for USP-based simethicone FTIR analysis. J. Pharm. Biomed. Anal. 2013, 74, 223 226. [4] Hartzell, A.L.; Shea, H.R. Mems Reliability; Springer: Berlin, 2011. [5] Joint Formulary Committee. British National Formulary (BNF) 64; Pharmaceutical Press: Great Britain, 2012; Vol. 64, pp 45-48. [6] Kasture, A.V. Pharmaceutical Chemistry I; Pragati Books Pvt. Ltd.: Pune, 2008. [7] Miller, J.C.; Miller, J.N. Statistics and Chemometrics for Analytical Chemistry, 5th ed.; Pearson Education Limited: England, 2005. [8] OLenick (Jr.), A.J. Silicone for Personal Care, 2nd ed.; Allured Business Media: Carol Stream, IL, 2008. [9] Sabo, M.; Gross, J.; Rosenberg, I.E. Quantitation of dimethicone in lotions using Fourier Transform infrared spectral substraction. J. Soc. Cosmet. Chem. 1984, 35, 273-281. [10] Torrado, G.; Garcia-Arieta, A.; de los Ros, F.; Menendez J. C.; Torrado, S. Quantitative determination of dimethicone in commercial tablets and capsules by Fourier transform infrared spectroscopy and antifoaming activity test. J. Pharm. Biomed. Anal. 1999, 19, 285292. [11] United States Pharmacopeial Convention.United States Pharmacopeia 32 and National Formulary 27 (USP 32-NF 27); United States Pharmacopeial Convention: Rockville, MD, 2009; pp 107-117.