VALIDASI METODE ANALISIS DAN PENETAPAN KADAR PIRIDOKSIN, NIKOTINAMID, TAURIN, DAN KAFEIN DALAM MINUMAN BERENERGI SECARA KROMATOGRAFI LAPIS TIPIS-DENSITOMETRI

FLORENTINUS DIKA OCTA RISWANTO, Prof. Dr. Sudibyo Martono., MS., Apt.

2013 | Tesis | S2 Ilmu Farmasi

Minuman berenergi termasuk dalam suplemen yang dinyatakan sebagai minuman yang mengandung vitamin, mineral, serta stimulan seperti kafein dan taurin. Untuk menjamin kebenaran kadar komponen-komponen dalam minuman berenergi diperlukan suatu metode analisis yang sederhana, cepat dan efisien. Metode KLT-densitometri dipilih untuk menetapkan kadar piridoksin, nikotinamid, kafein, dan taurin dalam minuman berenergi. Senyawa taurin hanya memiliki gugus kromofor yang sangat pendek pada struktur molekulnya sehingga lemah menyerap radiasi UV/Visibel. Oleh karena itu perlu dilakukan metode derivatisasi taurin menggunakan reagen ninhidrin 10% menghasilkan senyawa yang berwarna ungu (Ruhemann purple). Panjang gelombang maksimum yang digunakan dalam deteksi kafein, nikotinamid, piridoksin, dan taurin terderivatisasi berturut-turut adalah 274 nm, 263 nm, 293 nm, dan 489 nm. Dalam penelitian ini digunakan fase diam lempeng KLT silika gel 60 F254 dan fase gerak metanol : etil asetat : amonia 25% (4 : 23 : 3). Validasi metode analisis yang dilakukan meliputi selektivitas, linieritas, batas deteksi, batas kuantitasi, presisi, akurasi, dan robustness. Diperoleh nilai kafein, nikotinamid, piridoksin, dan taurin terderivatisasi berturut-turut adalah 0,9966; 0,9997; 0,9982; dan 0,9993. Batas deteksi dan batas kuantitasi keempatnya berturut-turut 5,43 dan 18,11 μg/mL; 13,15 dan 43,83 μg/mL; 4,05 dan 13,51 μg/mL; serta 3,50 dan 11,67 μg/mL. Nilai recovery kafein, nikotinamid, dan piridoksin berada dalam rentang yang dipersyaratkan menurut Horwitz yaitu 95-105%. Persen RSD berada di bawah nilai yang dipersyaratkan menurut Horwitz yaitu 5,7% kafein dan nikotinamid serta 8% untuk piridoksin. Taurin yang dalam sampel belum dapat ditetapkan kadarnya. Kadar kafein dalam sampel 1 dan 2 berturut-turut 249,50 ± 5,080 dan 252,80 ± 2,640 μg/mL. Kadar nikotinamid dalam sampel 1 dan 2 berturut-turut 106,53 ± 3,521 dan 98,20 ± 3,648 μg/mL. Kadar piridoksin dalam sampel 1 dan 2 berturutturut 33,59 ± 0,981 dan 30,29 ± 2,061 μg/mL. Uji kekuatan metode yang dilakukan meliputi penggunaan fase gerak untuk elusi dua kalinya (re-use), pergeseran panjang gelombang deteksi, pemendekan jarak elusi, serta penggantian proporsi komponen fase gerak. Hasil uji kekuatan metode menunjukkan bahwa kondisi analisis paling optimal dihasilkan pada penggunaan fase gerak freshly prepared, deteksi pada panjang gelombang maksimumnya, jarak elusi 7,5 cm, dan fase gerak metanol : etil asetat : amonia 25% (4 : 23 : 3).

Energy drink is classified as a food supplement which contains vitamins, minerals, and stimulants, such as caffeine and taurine. It is necessary to develop an analytical method that is simple, rapid, and efficient in order to evaluate the compounds in energy drinks. TLC-densitometry method was chosen to evaluate the pyridoxine, nicotinamide, caffeine and taurine in the energy drink sample. Taurine has only a very short chromophore group in its molecular structure and very weak absorption to the UV/Visible radiation. Therefore it is necessary for taurine to be reacted with ninhydrin reagent 10% to yield a purple compound (Ruhemann purple) as the derivatization method. The maximum wavelengths used in the detection of caffeine, nicotinamide, pyridoxine, and taurine after derivatization were 274 nm, 263 nm, 293 nm, and 489 nm, respectively. In this study, TLC plates of silica gel 60 F254 was used as the stationary phase and methanol : ethyl acetate: ammonia 25% (4 : 23 : 3) was used as the mobile phase. Analytical method validation was performed to determine the selectivity, linearity, limit of detection, limit of quantitation, precision, accuracy, and robustness of the TLC-densitometry method. Resolution values obtained from the nicotinamide, pyridoxine, and taurine were 0.9966; 0.9997; 0.9982, and 0.9993. Limits of detection and limits of quantitation from the four analytes were 5.43 and 18.11 mg/mL; 13.15 and 43.83 mg/mL; 4.05 and 13.51 mg/mL, 3.50 and 11.67 ug/mL, respectively. The recovery of caffeine, nicotinamide, and pyridoxine were within the required limit range of 95-105% according to Horwitz. The percent of RSD were below the limit value of 5.7% for caffeine and nicotinamide and 8% for pyridoxine according to Horwitz. Taurine in the sample could not be determined. The content amount of caffeine in the sample 1 and 2 were 249,50 ± 5,080 and 252,80 ± 2,640 μg/mL, respectively. The content amount of nicotinamide in the sample 1 and 2 were 106,53 ± 3,521 and 98,20 ± 3,648 μg/mL, respectively. The content amount of pyridoxine in the sample 1 and 2 were 33,59 ± 0,981 and 30,29 ± 2,061 μg/mL, respectively. The robustness test was performed to acquire information of the effect of using of re-useing the mobile phases, shifting wavelength detection, shortening the elution distance, and changing of the proportion of the mobile phase components. Robustness test results showed that the most optimal method conditions for analysis was in the application of freshly prepared mobile phase, detection at the maximum wavelength, elution distance of 7.5 cm, and mobile phase of methanol: ethyl acetate: ammonia 25% (4: 23: 3).

Kata Kunci : KLT-densitometri, minuman berenergi, kafein, taurin, vitamin