UJIAN BACTERIAL ENDOTOXIN
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1 UJIAN BACTERIAL ENDOTOXIN MENGGUNAKAN KAEDAH KUANTITATIF BACTERIAL ENDOTOXIN TEST USING QUANTITATIVE METHOD Yahaya Talib dan Muhamad Syazwan Zulkifli Bahagian Teknologi Perubatan Abstrak Ujian bacterial endotoxin merupakan salah satu ujian kawalan kualiti dalam pembuatan produk perubatan penjana Techetium-99m (Tc-99m). Satu daripada kaedah yang dinyatakan dalam standard antarabangsa ialah ujian penentuan secara kuantitatif yang melibatkan penggunaan alat microplate reader bersama komputer untuk analisis data. Percubaan telah dijalankan ke atas beberapa sampel penjana bertujuan untuk menentukan kesesuaian kaedah ini. Kesemua sampel pada pencairan berbeza telah menunjukkan kesan positif terhadap kehadiran endotoxin standard. Ujian yang sama juga dilakukan ke atas sampel saline. Katakunci : ujian bacterial endotoxin, Technetium-99m Abstract Bacterial endotoxin test is one of quality control tests to be performed in manufacturing of Technetium- 99m (Tc-99m) generator. One of the test methods stated in international standards is quantitative method where microplate reader is used together with computer to analyze data. Experiment was done on several samples of Tc-99m generator product to find out the suitability of this method. All samples diluted at different concentration has shown positive respond on standard endotoxin. The same test was performed on saline PENDAHULUAN Endotoxin ialah lipopolysacchrides (LPS) yang terdapat di dalam dinding luar sel bakteria gram negatif. Contoh bakteria jenis ini ialah Escherichia coli. Endotoxin telah dikenalpasti sebagai bahan yang menyebabkan demam apabila bahan ini memasuki tubuh manusia. Maka adalah penting dipastikan supaya parenteral drug atau medical devices tidak mengandungi endotoxin melebihi had yang ditetapkan sebelum bahan tersebut dimasukkan ke dalam tubuh sesaorang. Maka ujian penentuan endotoxin ini menjadi kemestian sepertimana yang dinyatakan dalam rujukan standard. Pensterilan ialah proses membunuh bakteria pada suhu tinggi iaitu 121 C, namun endotoxin tidak terhapus dengan cara begini kerana sifatnya yang tahan haba. Ini bermakna endotoxin tidak bergantung kepada sel yang hidup. Oleh itu sel bakteria gram negatif, samada yang masih hidup atau mati adalah punca endotoxin. Ujian bacterial endotoxin atau dinamakan juga bacterial endotoxin limit test atau ujian Limulus Amoebocyte Lysate (LAL) mampu mengesan kandungan endotoxin dalam sesuatu sampel. LAL ialah ekstrak sel darah dari horseshoe crab, nama saintifiknya ialah Limilus polyphemus. Apabila LAL bertindak dengan endotoxin, berlakulah proses tindakbalas bersiri dan akhirnya terjadi pembentukan gumpalan atau kentalan (clot) sebagai respon terhadap endotoxin bakteria. Terdapat beberapa kaedah dalam ujian bacterial endotoxin, iaitu kaedah gel-clot, turbidimetric dan chromogenic. Kaedah gel clot ialah kaedah asas yang mana pembentukan gumpalan dapat dilihat dalam tabung uji. Kaedah ini mengukur kehadiran endotoxin secara kualitatif. Manakala kaedah yang lain boleh mengukur endotoxin secara kuantitatif iaitu tindakbalas kinetik yang diukur ada hubungkait dengan [1]
2 kepekatan endotoxin. Prinsipnya ialah apabila jumlah endotoxin tinggi maka tindakbalas yang berlaku akan mengambil masa lebih singkat. Kaedah ini memerlukan alat microplate reader dan perisian komputer. Microplate reader berfungsi menyediakan suhu eraman 37 C dan mengukur tindakbalas kinetik iaitu perubahan pada kekeruhan larutan ujian berbanding masa. Perisian komputer pula menganalisa data tindakbalas dan seterusnya menyimpan data serta menyedia laporan ujian. Setiap kali ujian sampel dilakukan, ujian terhadap SCE (standard control endotoxin) perlu dilakukan serentak supaya lengkuk piawai dibangunkan dan ini membolehkan kepekatan endotoxin dalam sampel ditentukan. Ujian bacterial endotoxin merupakan salah satu ujian kawalan kualiti yang perlu dilakukan terhadap sampel penjana Technetium-99m (Tc-99m) yang dikeluarkan di Bahagian Teknologi Perubatan (BTP) mengikut prosedur operasi standard (SOP). Pada masa ini kaedah yang digunapakai ialah penentuan secara kualitatif, iaitu kaedah kaedah gel-clot dengan kepekaan endotoxin ialah 0.125EU/ml. Penggunaan kaedah kuantitaif belum pernah dilakukan ke atas produk ini. Sebagai usaha untuk memperolehi maklumat awal mengenai kesesuaian kaedah kuantitatif, satu ujikaji telah dilakukan terhadap sampel produk tersebut. KAEDAH 1. Penentuan had endotoxin dan MVD (maximum valid dilution) khusus untuk produk Tc-99m (sampel diperolehi dari penjana Tc-99m). MVD ialah pencairan maksima yang dibenarkan untuk sampel yang mana had endotoxin boleh ditentukan. i. Had endotoxin untuk radiofarmaseutikal ialah 175/V, V ialah dos maksima. Untuk produk penjana Tc-99m, jumlah maksima yang dibenarkan disuntik kepada pesakit bersamaan dos maksima, iaitu 10 ml. Oleh itu, had endotoxin ialah 175/10 EU/ml = 17.5 EU/ml. ii. MVD dikira menggunakan formula; MVD = had endotoxin/λ (λ ialah lysate sensitivity) Ddalam ujikaji ini λ=0.01 EU/ml, maka MVD = 17.5/0.01= Ini bermakna, sampel penjana Tc- 99m boleh dicairkan tidak melebihi 1750 kali. 3. Ujian kualifikasi (initial qualification assay) dilakukan menggunakan larutan standard,sce sebelum ujian dilakukan terhadap sampel. i. SCE disediakan pada kepekatan; 0.01, 0.05, 0.5 dan 5 EU/ml (4 replicate untuk setiap kepekatan). ii. Sebanyak 0.1 ml SCE dimasukkan ke dalam microplate dan ujian dimulakan menggunakan kendalian perisian komputer. iii. Kemudian LAL (lysate) ditambah kepada SCE dan alat akan mengukur tindakbalas kinetik dan merekod data-data selama 1 jam sebelum laporan dihasilkan. 4. Ujian terhadap sampel i. Sampel Tc-99m Sebanyak 6 sampel diuji. Setiap sampel dicairkan sebanyak 10, 50 dan 100 kali dengan LAL water. Untuk setiap sampel (bagi setiap pencairan), ujian dilakukan untuk 2 keadaan; (i) sampel sahaja dan (ii) sampel yang mengandungi 0.5Eu/ml (SCE). Ujian dijalankan sepertimana dinyatakan dalam langkah 3. Ujian standard secara serentak yang mana kepekatan SCE ialah 0.01, 0.05, 0.5 dan 5 EU/ml. ii. Sampel saline Cara yang sama dilakukan untuk sampel saline dengan pencairan 2 kali. Saline ialah larutan yang digunakan semasa elution penjana Tc-99m. [2]
3 5. Keputusan ujian dipaparkan dalam laporan yang dijana oleh komputer dan boleh dicetak apabila diperlukan. KEPUTUSAN Data ujian kualifikasi ditunjukkan dalam Jadual 1. Rajah 1 pula menunjukkan masa yang diambil untuk indakbalas kinetik mengurang apabila kepekatan endotoksin meningkat. Keputusan akhir yang ditunjukkan dalam laporan ujian (Jadual 2) mengesahkan bahawa SCE dan lysate yang digunakan adalah menepati spesifikasi dan lulus ujian, bermakna kedua-dua bahan tersebut boleh digunakan dalam ujian ke atas sampel. Jadual 1: Data ujian kualifikasi Kepekatan Endotoxin Masa tindakbalas (saat) Blank * * * * 0.01 Eu/ml EU/ml EU/ml EU/ml (* bacaan terlampau tinggi atau di luar julat) Jadual 2: Laporan keputusan ujian Rajah 1: Graf untuk ujian kualifikasi Curve Parameters Parameter Value Specification Status Correlation Coefficient to PASS Slope to PASS Y Intercept to PASS Standards Concentration (EU/ml) CV Specification Status % <10% PASS % <10% PASS % <10% PASS % <10% PASS [3]
4 Ujian terhadap sampel Tc-99m Dari graf lengkuk piawai (Rajah 2), kepekatan endotoxin sampel boleh ditentukan. Perisian komputer berfungsi melakukan analisa data dan terus menjana laporan ujian. Rajah 2: Graf lengkuk piawai untuk ujian sampel Kandungan endotoxin (EU/ml) dipaparkan dalam Jadual 3. Kandungan endotoxin untuk kesemua sampel Tc-99m yang diuji didapati kurang dari had yang ditetapkan (17.5 EU/ml). Peratus perolehan kembali endotoxin dalam sampel ialah antara 71% dan 133%. Keputusan ini memenuhi spesifikasi iaitu masih dalam julat yang dibenarkan, antara 50% dan 200%. Ini bermakna kaedah ini sesuai digunakan untuk menentukan kehadiran endotoxin dalam sampel Tc-99m. Jadual 3 : Data ujian untuk sampel Tc-99m (tanpa SCE) dan sampel Tc-99m mengandungi SCE [(a) Kepekatan endotoxin (EU/ml) pada pencairan berbeza, (b) Peratus perolehan kembali endotoxin standard (CSE) untuk sampel pada pencairan berbeza] Pencairan 10x 50x 100x Pencairan 10x 50x 100x Sample 1 < 0.1 EU/ml <0.5EU/ml <1.0EU/ml Sample 1 +SCE 71% 93% 84% Sample EU/ml <0.5EU/ml <1.0EU/ml Sample 2 +SCE 100% 133% 118% Sample EU/ml 0.810EU/ml <1.0EU/ml Sample 5 +SCE 90% 103% 101% Sample EU/ml 0.675EU/ml 1.53EU/ml Sample 6 +SCE 94% 107% 92% Sample EU/ml <0.5EU/ml <1.0EU/ml (b) Sample 6 <0.1EU/ml <0.5EU/ml 4.4EU/ml (a) [4]
5 Ujian terhadap sampel saline Ujian saline juga telah dilakukan serentak dan keputusannya ialah; kepekatan endotoxin : <0.02 EU/ml (had yang diterima ialah tidak melebihi 0.25 EU/ml), peratus perolehan kembali: 63% (julat yang dibenarkan ialah 50% - 200%). Keputusan ini menunjukkan kaedah ujian ini juga sesuai dilakukan terhadap saline. KESIMPULAN Penentuan kandungan endotoxin menggunakan kaedah kuantitatif (tindakbalas kinetik) sesuai digunakan terhadap sampel Tc-99m yang dihasilkan di Bahagian Teknologi Perubatan. Kehadiran endotoxin dalam sampel tersebut pada pencairan 10, 50 dan 100 kali memberikan respon positif dengan peratus perolehan kembali menepati spesifikasi. Kaedah ini juga sesuai dilakukan terhadap sampel saline yang digunakan untuk mengeluarkan larutan Tc-99m semasa elution penjana. RUJUKAN USP24 NF29 (2006):<85> Bacterial Endotoxin Test. Page BP 2005, Vol IV Appendix XIV C: Test for Bacterial Endotoxin Test. Procedure for Bacterial Endotoxin Limit Test, SOP/QC/TP14-05, [5]
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