Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Issue 4, 2013

Research Article

METHOD DEVELOPMENT FOR SIMULTANEOUS ANALYSIS OF STEROID AND NON STEROID
ANTIINFLAMATORY SUBSTANCES IN JAMU PEGAL LINU USING TLC-
SPECTROPHOTODENSITOMETRY

HILDA APRILIA WISNUWARDHANI, IRDA FIDRIANNY, SLAMET IBRAHIM
School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia. Email: hilda.aprilia@gmail.com
Received: 28 Aug 2013, Revised and Accepted: 25 Sep 2013

ABSTRACT
Objective: Illegally added chemical adulteration of jamu become a problem that more difficult to stop. Therefore method development of
simultaneous detection of chemical adulteration must be conducted. The aim of this research is to develop analytical method to detect
simultaneously chemical adulteration in jamu pegal linu.
Methods: Stationary phase, mobile phase, sample preparation and instrument condition were optimized.
Results: The result of optimization was validated for specificity according to International Conference on Harmonization. The validated methods
were applied to analyze of three jamu pegal linu products in the market for the qualitative analysis of chemical adulteration. Analytical method
optimization showed that the best separation of five substances, acetaminophen, dexamethasone, prednisone, mefenamic acid and piroxicam was
performed on silica gel GF254, using chloroform – methanol (9:1) as a mobile phase. Samples were prepared by extraction using ethanol as a solvent.
Extraction was done for 30 minutes using 3D shaker. Chamber saturation was done for 60 minutes. Detection of chemical adulteration was
performed using CAMAG TLC (Thin Layer Chromatography) Scanner at λ 254 nm for simultaneous detection of acetaminophen, dexamethasone,
prednisone, mefenamic acid, piroxicam.
Conclusion: A new validated methods for qualitative analysis (acetaminophen, dexamethasone, prednisone, mefenamic acid and piroxicam was
deeloped) by thin layer chromatography (TLC) at λ 254 nm. The separation was performed on silica gel GF254, using chloroform – methanol (9:1) as
mobile phase. The methods can be used to analyze jamu pegal linu in the market.
Keywords: TLC-spectrophotodensitometry, Jamu pegal linu, Simultaneous detection.

INTRODUCTION white to white off crystalline powder, melting point 230 ⁰C, with
decomposition. Solubility: soluble in alkali hydroxide solution,
Jamu is a Indonesian traditional medicine. According to Regulation of sparingly soluble in chloroform, slightly soluble in alcohol and
Ministry of Health Indonesia No. 007, 2012, jamu is prohibited for methanol, and practically insoluble in water [5].
chemical adulteration including isolate from natural product, and
chemical substances from synthesis, ethanol more than 1.0%.
Nowadays, illegally added chemical adulteration in jamu becomes a
problem that difficult to stop. According to public release data from
NADFC, there always chemical adulteration finding in Indonesia. From
2001 to 2007, illegally added chemical adulteration in jamu, include
antiinfamatory drug substances like acetaminophen, dexamethasone,
prednisone, mefenamic acid and phenylbutazone [1,2]. Since 2007,
chemical adulteration included sildenafil, tadalafil, and sibutramin HCl
[1,2]. In 2012, NADFC report there were more than one chemical Fig. 2: Molecular structure of dexamethasone
substances were added to jamu pegal linu. The chemical substance
trends were back to antiiflamatory substances like acetaminophen,
dexamethasone, prednisone, mefenamic acid and piroxicam [3].
Acetaminophen, C8H9NO2, chemical name: 4-hydroxyacetanilide; p-
hydroxyacetanilide; p-acetamidophenol; p-acetaminophenol; p-
acetylaminophenol; N-acetyl-p-aminophenol [4]. Description: white
crystalline powder, odorless, slightly bitter. Solubility: freely soluble
in alcohol, soluble in boiling water and NaOH 1 N [5].
Fig. 3: Molecular structure of mefenamic acid
Piroxicam, C15H13N3O4S, chemical name 4-hidroxy-2-methyl-N-(2-
pyridyl)-2H-1,2-benzotiazine-3-carboxamide-1,1-dioxid.
Description: crystalline powder, colorless, odorless, bitter. Solubility:
insoluble in water and chloroform, sparingly soluble in toluene and
diisopropyl ether, slightly soluble in aliphatic alcohol, such as
Fig. 1: Molecular structure of acetaminophen methanol, ethanol, isopropanol [6].

Dexamethasone, C22H29FO5, description: white to almost white
powder, odorless, crystalline powder, stabile at room temperature,
decompose at 250 ⁰C. Solubility: sparingly soluble at acetone,
alcohol, dioxane and methanol, slightly soluble in chloroform, very
slightly soluble in ether, practically insoluble in water [5].
Mefenamic acid, C15H15NO2, chemical name benzoic acid, 2-(2,3-
dimethylphenyl) amino, N-2,3-cylylantranilate acid. Description: Fig. 4: Molecular structure of piroxicam

operated by WinCATS software (CAMAG). Curcuma Microscopic examination of crude drug domesticae rhizome.75 gram Curcuma domesticae chloroform-methanol (9:1). Then.20.12. Solubility: slightly soluble in alcohol. 240 µL of prednisone. 0. it was dissolved and add to 5 mL Analytical reference standard of acetaminophen. scanning speed was 20 mm/s.5 gram Zingiberis rhizome.2)[7]. Indonesia. crystalline powder. the optimum mobile phase to separate and analyze five substances. in jamu pegal linu. methanol. The slit dimension were 6. Based on mobile phase optimization. Determination of scan wavelength Instrumentation Each of substance was scanned using CAMAG TLC Scanner III to CAMAG TLC Scanner III in reflectance-absorbance mode and determine the wavelength which gave the maximum absorbance. so substances both in standard mixture and also in sample mixture. and Zingiberis rhizome. Issue 4. if analyzed for marker characteristic using Microscope Olympus DP we used glycerin as a microscopic reagent. 40. all of crude drug that were used in this research are Curcumae rhizome. dioxane. dichlormethane-ether-methanol-water (77: 15:8:1. dexamethasone. Curcuma domesticae mL of ethanol was used for the extraction. Source of radiation was Then. description: white to almost white. 6: Microscopic examination of crude drugs. 0. reagents. ethyl acetate-methanol-ammonia (85:10:5).16. Preparation of Jamu Pegal Linu Matrices prednisone. 80. ethanol was added to a volume. The crude drug was purchased as No. a powder.00 x 3. RESULT AND DISCUSSION Methods Based on microscopic analysis of crude drugs. precoated TLC plates with silica gel GF254 (Merck) Sample preparation Crude drug Samples of jamu pegal linu were weighed approximately 500 mg.00 mm. The crude drug was Before using as a matrix of jamu.04.08. using ethanol on a volumetric flasks. mefenamic acid and piroxicam. 10 The crude drug were Curcumae rhizome. The activated plates were equilibrated and stored in dessicators. TLC plates were washed with methanol. 749-753 Prednisone. 0. It we had a homogenous mixture of jamu pegal linu matrices. can be seen in Figure 7.1. (a) Curcuma domesticae rhizome. 0. wavelength 200 nm and 400 nm. such as starch. both for preparation of mobile phase and also for the other standard solution with concentration 0.24 mg/mL were obtained. were analytical grades. Fig. crude drugs were microscopically showed marker characteristic for each crude drug. 21.75 gram of Curcumae rhizome. mefenamic acid and piroxicam were purchased from the solution was transferred to 5 mL volumetric flasks using NADFC (The National Agency of Drug and Food Control). Bandung. dried in oven soluble in water [5]. (b) Curcumae rhizome. After that optimizations were continued with addition of methanol in every ratio. were mixed in mortar. Int J Pharm Pharm Sci. 120. dichlormethane. 160. Mobile phase optimization Mobile phase optimizations were started using medium strength solvent. This mobile phase can separate each rhizome and 1. and filtered through Whatman in Pasar Baru. 200. and Zingiberis rhizome. 120 ºC for 30 minutes. acetaminophen. TLC analysis was performed on 0. dexamethasone. very slightly Before use. The sample was shaking rhizome. 5: Molecular structure of prednisone and heptanes-2-propanol-acetic acid (15:5:1)[8]. Plate activation odorless. Then the filtrate was ready for application on TLC plates. Wisnuwardhani et al. chloroform. MATERIALS AND METHODS Standard preparation Materials 10 mg of acetaminophen was weighed as an analytical standard by using an analytical balance. using glycerin 750 . After that. 1. Vol 5. were purchased from herbal store for 30 minutes by using 3D shaker. The filtrate was diluted to 1/5 proportion. dan (c) Zingiberis rhizome. All volumetric pipette. the wavelength for simultaneous analysis of five substances deuteurium lamp emitting a continuous UV spectrum between was determined too. (a) (b) (c) Fig. melt at 230 ⁰C with decomposition. was 1. Mobile phase from solvent combinations were also tested such as ethyl acetate 100%. It can be seen in Figure 6. Optimizations were continued by replacing dichlormethane with ethyl acetate and chloroform. procedure.

(b) dexamethasone. The overlaid spectrum showed that no difference between separation (Figure 8).63 dexamethasone. one samples were illegally added by piroxicam. 7: Thin layer chromatogram of analytical standard. was showed good matrices.15].23 (9:1) as a mobile phase. detect those substances in jamu pegal linu sample. All of five substances can had a good specificity [12]. means that the two spots is spectrum was similar with sample spectrum (Figure 9).47 simulation of jamu and chemical adulterance.13. Issue 4. It can be seen on the resolution. C. 8: Densitogram of standard solution mixture Table 1: Resolution of the substances Fig. The resolution for each peak to the other was showed in Table 1. So. (e) piroxicam [ This method was used to analyze sample of jamu pegal linu in the acetaminophen. densitogram and were produced by different kind of jamu industry. because all of the five could analyze it simultaneously in one way procedure.0.methanol Acetaminophen-Dexamethasone 1. Wisnuwardhani et al. This can be assured by thin layer chromatogram.14. samples that were analyzed. using UV 254 nm as a derivatization agent Each of substance spectrums were overlaid with the standard spectrum to assure the identity of the substance in jamu pegal linu The resolution of each peak in densitogram. Vol 5. 11. 14). samples. B. (3) prednisone. and market. (9) mixture of standard. From three spectrum overlaid between standard and sample (Figure 10. This method substances had a good solubility in ethanol. Sample preparation using even in a jamu matrices environment. 12. 9: Overlaid spectrum between standard and sample. well separated [9. which was standard spectrum and sample spectrum. Those three jamu pegal linu. 10. one samples were illegally added by 13. 749-753 Fig. Fig. this method was valid to ethanol also considered efficiency of the methods. (a) acetaminophen. (4) phenylbutazon. (5) Prednisone-Mefenamic Acid 1. (2) Dexamethasone-Prednisone 1. (8) methampiron. (d) mefenamic acid. Jamu pegal linu sample was taken from two different herbal one samples were illegally added by acetaminophen and piroxicam. because all of the substances gave a good absorbance in this wavelength. A.0. 751 . Simultaneous analysis was done at wavelength 254 nm. because it still could detect a substance be extracted in one way using ethanol.11. Chemical Adulterance Resolution (Rs) stationary phase silica gel GF254 using chloroform. we Sample preparation was chose using ethanol. (1) acetaminophen. (7) piroxicam.79 acid. stores in Bandung. It means that the standard more than 1. (6) mefenamic Mefenamic Acid-Piroxicam 1. Resolution more than 1. So. (c) prednisone. Int J Pharm Pharm Sci. Rs.

(2) to (4) sample A Fig. 11: Overlaid spectrum between standard and sample. (1) to (3) sample D. Vol 5. 13: Overlaid spectrum between acetaminophen standard and sample B 752 . dexamethasone. (a) acetaminophen. mefenamic acid. (1) standard mixture of acetaminophen. (b) densitogram. (b) densitogram. mefenamic acid and piroxicam. Int J Pharm Pharm Sci. (a) using UV λ 254 nm. prednisone. (a) using UV λ 254 nm. Issue 4. (4) standard mixture of acetaminophen. dexamethasone. 10:Thin layer chromatogram of sample A and standard mixture. prednisone. 749-753 Fig. 12:Thin layer chromatogram for sample B and standard mixture. and piroxicam Fig. (b) piroxicam Fig. Wisnuwardhani et al.

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