Polfa Kutno S.A., Poland, 93040031, (05 2004); diamicron gliclazide 80 mg, Servier, France, 0217011 O l, 9 H 0715, (08. 2002); diamicron gliclazide 80 mg, ...
ldentification and Determination of Gliclazide and its lmpurities in Various Medicines by Thin-Layer Chromałography and Densitometry Jan Krzek*, Monika
Dąbrowska,
and Urszula Hubicka
Key Words: Pharmaceutical research Gliclazide Determination Densitometry
Summary A thin-Iayer chromatographic and densitometric metbod bas been developed for the identiłication and q uantitation of głicłazide and its impurities on siłica geJ, using chloroform-methanol, 19 +l (v/v), as mobile phase. UV densitometric measurements we re mad e at,\= 226 nm. The metbod is characterized by high sensitivity (15 ng), Iinearity over a wide concentration range (0.06 to 0.30 mg mL-'), and high recovery (100.23%). lt was found that the estabłished conditions can be ałso used for determination of impurities in medicines.
1 lntroduction Gliclazide belongs to a new group of second-generation hypoglycemic dmgs, thiourea derivatives, used in medicine in the form of oral drugs with variable release of the active substance. In contrast with the first generation dmgs, gliclazide preparations are of very high efficiency, and so treatment doses are several times !ower. lt is, therefore, necessary to seek new sensitive and accurate analytical methods [l]. Many methods for quantitative analysis of gliclazide in pharmaceuticals have been described in the literature. An important group is the UV and Vis spectrophotometric methods used after formation of a colored derivative [2-5] . Chromatographic methods, mainly HPLC [6-8], TLC, often in combination with densitometry [9, l 0], and gas chromałography with flame ionization detection (FID) and packed columns (in particular OY-1, OV-17, and XE-60) [ 11 , 12] have also proven to be useful. Electrochemical methods such as voltammetry [ 13] Dąbrowska, and U.l-lubicka, Departmentoflnorganic andAna lytical Chem1stry, Colleg1um Medicum of Jagiellonian University, Medyczna 9 30-688
.l. Krzek, M.
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Journal of Planar Chromatography
'
and coulometry [14] have also been developed for analysis of gliclazide in pharmaceuticals. According to the British Pharmacopoeia the HPLC method is recommended for assessment of gliclazide purity [ 15]. Because of considerable advances in detection methods for thin-layer chromałography and its wid e range of application, an attempt was made to develop a chromatographic and densitometric method for the identification and quantitation of gliclazide in dmgs of different origin. The purity of the medicines has also been investigated by determination of the impurities, if any. Because no similar study has been found in the available literature, the problem seems to be worthy of investigation both for scientific and practical reasons.
2 Experimental
2.1 Solutions
Standard solutions at concentrations from 1.5 f.lg mL-1 to 1.5 mg mL- 1 were prepared in methanol (Merck, Germany) from gliclazide substance confonning to British Pharmacopoeia BP [15]. Sample solutions at concentrations of 0.1 and l. Omg mL -l were also prepared in methanol. Ten tablets ofan appropriate preparation were weighed to determine the mean tablet weight. The tablets were then ground and weighed with an accuracy of 0.1 mg to obtain an amount corresponding to 100 mg of gliclazide. Methanoi (100 mL) was added to the weighed preparation and the mixture was shaken for 15 min and filtered. The filtra te was diluted to l 00 mL. Developing reagent was prepared by dissolving ninhydrin (0.2%) in methanol.
VOL. 14. MAY/JUNE 2001
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TLC ot Gliclazide in Medicine
2.2 Preparations
2.3.3 Linearity
The study encompasses the following randomly selected gliclazide preparations, available commercially from a variety of manufacturers: diabezidum 80 mg gliclazidum, Jelfa (Poland), 20999, (09 200 l); glinormax gliclazidum 0.08 g, Polfa Kutno S.A., Poland, 93040031 , (05 2004); diamicron gliclazide 80 mg, Servier, France, 0217011 Ol , 9 H 0715, (08 2002); diamicron gliclazide 80 mg, Servier, Belgium, B02FS02 GS/427, 96Lll , (EXP 12 200); diabrezide gliclazide 80 mg, Molteni Farmaceutici, Italy, 331 98, (09 2003); diamicron gliclazide Inn 80 mg, PL0093/0024 POM, P a 68/211, 8G 0802, Servier, UK, 2 170 1198, 6A 0791 , (O l 200 l) ; and diaprel gliclazidum 80 mg, Les Laborataires Servier, France, 021511194, 9 F 0764, (06 2004).
To establish the linearity, five solutions (5 f!L) at concentrations from 0.06 to 0.30 mg mL- 1 were applied to the plates. The results were analyzed by linear regression (Figure 4).
2.3
Chromałography
2.3.4 Precision
The consistency of the results was checked for five sampies o f model mixtures prepared as above ( sarople solutions) oflmown gliclazide concentration ranging from 50 to 150%. The determination was performed three times for each sample. The fina! result was the mean gliclazide concentration [%]for n= 5. The random errors were defined by standard deviation (Sx), the coeff}cient of variability-standard deviation of the arithrnetic mean (SX), and the confidence interval (jl) at a probability of 95%. The results obtained were [%]: 93.90, 95.00, 95.69, 97.25 , 97.48; X. = 95 .85%; S = 1.52; Sx = 0.68; p = ± 1.89.
2.3. 1 Establishing Test Conditions
2.3.5 Accuracy
Standard and sarople solutions (2-60 f!L) were applied in bands to 10 cm x 10 cm (cut from 20 x 20 cm) aluminum-backed precoated s ilica geJ TLC plates (M erek, Germany; # 1.05553). Chromatograms were developed to a clistance of 9 cm in a variety of mobile phases-ethyl acetate-methanol-25% ammonia, 8.5 + l + 0.5 (vlv); chloroform-methanol, 9 + l (v!v); chloroform-ethyl acetate, 6 + 4 (v/v); cyclohexane-chloroform-methanol, 12 + 6 + l (v/v); dioxane-xylene-tolueneisopropanol-25% ammonia, l + 2 + l + 65 + l (vlv) ; and chloroform-methanol, 19 + l (v/v)-in a 20 cm x 5 cm x 11 cm Merck TLC chamber. Chromatograms were dried in air at room temperature, sprayed with 0.2% ninhydrio solution to identify the spots in the chromatograms, and heated at 105°C for 3 min. The absorption spectra were recorded for individual spots and peaks registered by densitometry at A = 226 run.
The accuracy of the method was expressed as percentage recovery of analyte added compared with the known sarople s ize. For this purpose 80-120% of the reference standard s we re added to weighed samples, after the determination of gliclazide, and the determination was performed six times. Every fina] result was the mean value of three measurements. The results obtained were [%] : 96.63, 98.00, 98.75, 102.50, 105.25, x = 100.23%; S = 3.55 ; SX. = 1.59; p = ±4.41.
lt was found that the mobile phase chloroform-methanoll9 + l (v/v), enabled separation of gliclazide from additional spots of different RF (Figure l) and with different absorption spectra (Figure 2); chloroform was obtained from Merck. The presence of additional spots not attributed to gliclazide was confirmed by applying solutions (50 ].lL) of relevant preparations (concentration l mg mL- 1) to the plates. Densitograms revealed the presence ofan additional peak (RF :::: 0.95) that was ignored because it originated from the mobile phase.
Standard (0.100 mg mL- 1) and sample solutions (both 5 f!L) were applied as bands l cm wid e to l Ocm x l Ocm TLC plates, by means of a Camag (Muttenz, Switzerland) Linomat IV sample applicator. The chromatograms were developed to clistance of 9 cm with chloroform-methanol, 19 + l (v/v), as mobile phase and then dried at room temperature. Peak areas for relevant standard and sarople solutions were recorded by densitometry in the UV at A = 226 run with a Camag TLC Scanner 3 with Cats 4 software, and the spo ts in chromatograms were quantified by comparing peak areas from the standard and sarople solutions. The results were means from three measurements . Calculations were performed with a Pentium MMX computer with 16 MB RAM (Tajwan) and a Hewlett-Packard LaserJet 6L printer (USA) was used for documentation. This procedure was used for the detennination of gliclazide in the medicines under examination. The results are presented in Tabłe l.
In preliminary analysis the conditions for determination of gliclazide and impurities, if any, were established, while performing validation ofthe method [ 16, 17]. 2.3.2 Limits ot Detection and Quantitation
The limits of detection and determination were eonsiciered together because densitometry enables well developed peaks to be obtained at a low noise level (Figure 3). The signal of a sam p le with an appropriate amount of analyte added was at least three times larger than that ofblind sample. Solutions (5 f!L) of decreasing concentration-0.250, 0.125, 0.0625, 0.00315, and 0.0015 mg mL-1 were applied to the plates.
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VOL. 14. MAY/JUNE 2001
The fina] result was the procedure established for the determination of gliclazide and its impurities resulting from active substance decomposition, manufacturing process, or medicine formulation. 2.4 Procedurefor Analytical Determination of Gliclazide
2.5 Determination of Gliclazide and Totallmpurities
The amount of gliclazide and total impurities in preparations were determined by the interna! normalization method. The
Journal ot Planar
Chromałography
TLC of Gliclazide in Medicine
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