BIOANALISIS 1A

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1 BIOANALISIS (analisis BIOANALISIS (analisis biomedik, bioanalytical biomedik, bioanalytical chemistry) chemistry) TERDIRI DARI: TERDIRI DARI: 1. ANALISIS HAYATI (BIOASSAY) 1. ANALISIS HAYATI (BIOASSAY) 2. ANALISIS OBAT DLM CAIRAN HAYATI 2. ANALISIS OBAT DLM CAIRAN HAYATI 3. ANALISIS BIO MOLEKUL 3. ANALISIS BIO MOLEKUL

Transcript of BIOANALISIS 1A

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BIOANALISIS (analisis BIOANALISIS (analisis biomedik, bioanalytical biomedik, bioanalytical chemistry)chemistry)

TERDIRI DARI:TERDIRI DARI:1. ANALISIS HAYATI (BIOASSAY)1. ANALISIS HAYATI (BIOASSAY)2. ANALISIS OBAT DLM CAIRAN HAYATI2. ANALISIS OBAT DLM CAIRAN HAYATI3. ANALISIS BIO MOLEKUL3. ANALISIS BIO MOLEKUL

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Analisis Hayati Analisis Hayati (Bioassay)(Bioassay) Adalah analisis dengan Adalah analisis dengan

menggunakan media dan menggunakan media dan parameter hayati.parameter hayati.– Analisis hayati kuantitatifAnalisis hayati kuantitatif– Analisis hayati kualitatifAnalisis hayati kualitatif

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Analisis Hayati Analisis Hayati (bioassay)(bioassay) Quantitatif: penetapan potensi/kadar obat dengan Quantitatif: penetapan potensi/kadar obat dengan

media hayatimedia hayatiPenetapan potensi hormon insulinPenetapan potensi hormon insulin

Human, CHuman, C257257HH383383NN6565 O O7777SS66 (5808) (5808)Porcine, CPorcine, C256256HH381381NN6565OO7676SS66 (5778) (5778)Bovine, CBovine, C254254HH377377NN6565OO7575SS66 (5734) (5734)

Penetapan potensi antibiotikPenetapan potensi antibiotik Qualitatif: analisis kualitatif sifat-sifat tertentu untuk Qualitatif: analisis kualitatif sifat-sifat tertentu untuk

produk-produk biologis maupun wadah atau produk-produk biologis maupun wadah atau peralatan pendukungnya (transfusion assemblies peralatan pendukungnya (transfusion assemblies mis)mis)

Penetapan angka bakteriPenetapan angka bakteriUji sterilitas sediaan injeksiUji sterilitas sediaan injeksiUji pirogen preparat parenteralUji pirogen preparat parenteral

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Analisis obat dlm bahan Analisis obat dlm bahan baku, dlm tablet, dalam baku, dlm tablet, dalam cairan hayaticairan hayati Pd. Divisi QC industri farmasi:Pd. Divisi QC industri farmasi:

Flavouring agent benzaldehyde to Flavouring agent benzaldehyde to confirm the identity and stated purity confirm the identity and stated purity

• Asam asetil salisilat dalam tablet:Asam asetil salisilat dalam tablet:Asetosal tercampur dengan laktosa Asetosal tercampur dengan laktosa (pengisi), amilum (desintegrant) dan (pengisi), amilum (desintegrant) dan mg stearat (pelicin)mg stearat (pelicin)

• Analisis mefenitoin dalam darah/urine:Analisis mefenitoin dalam darah/urine:Kadar obat dalam darah, komposisi Kadar obat dalam darah, komposisi kimia darah, metabolit yang ada, kimia darah, metabolit yang ada, stabillitas obat dan metabolitnyastabillitas obat dan metabolitnya

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Keterkaitan dengan Keterkaitan dengan Ilmu lainIlmu lain FarmakologiFarmakologi Fisiologi/Ilmu FaalFisiologi/Ilmu Faal BiokimiaBiokimia Kimia AnalisisKimia Analisis Kimia OrganikKimia Organik

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ARTI PENTINGARTI PENTING DRUGS IN USEDRUGS IN USE

– Forensic ToxicologyForensic Toxicology– OverdosageOverdosage– Drugs in SportDrugs in Sport– Therapeutic Drug MonitoringTherapeutic Drug Monitoring– PharmacogeneticsPharmacogenetics

DRUGS IN RESEARCH AND DEVELOPMENTDRUGS IN RESEARCH AND DEVELOPMENT– PharmacologyPharmacology– ToxicologyToxicology– Phase I Clinical TestingPhase I Clinical Testing– MetabolismMetabolism– PharmacokineticsPharmacokinetics– Formulation DevelopmentFormulation Development– PharmacodynamicsPharmacodynamics

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BibliografiBibliografi

DAFTAR PUSTAKA UTAMADAFTAR PUSTAKA UTAMA Chamberlain, J.,1995, Chamberlain, J.,1995, The Analysis of Drugs in Biological The Analysis of Drugs in Biological

FluidsFluids 2nd Ed, CRC Press, Boca Raton 2nd Ed, CRC Press, Boca Raton Departemen Kesehatan RI, 1972, Departemen Kesehatan RI, 1972, Farmakope IndonesiaFarmakope Indonesia, Ed. , Ed.

II, III (1979) dan IV JakartaII, III (1979) dan IV Jakarta Mikkelsen, S.R. and Corton, E., 2004, Mikkelsen, S.R. and Corton, E., 2004, Bio-analytical Bio-analytical

Chemistry,Chemistry, Wiley-Interscience, A John Wiley & Sons Inc. Wiley-Interscience, A John Wiley & Sons Inc. Publication, Hoboken, NYPublication, Hoboken, NY

Rossi G.V., 1980, Rossi G.V., 1980, Biological TestingBiological Testing, in Remington’s , in Remington’s Pharmaceutical Sciences 16th edition, Mack Publishing Co, Pharmaceutical Sciences 16th edition, Mack Publishing Co, Easton, 563Easton, 563

Smith, R.V. & Stewart, J.T., 1981, Smith, R.V. & Stewart, J.T., 1981, Textbook of Textbook of Biopharmaceutic Analysis: A description of methods for the Biopharmaceutic Analysis: A description of methods for the determination of drugs in biological fluidsdetermination of drugs in biological fluids, Lea & Febiger, , Lea & Febiger, PhiladelphiaPhiladelphia

Berbagai Journal IlmiahBerbagai Journal Ilmiah

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BibliografiBibliografi

Buku Bacaan Pendukung:Buku Bacaan Pendukung: Tallarida, R.J., Jacob, L.S., 1979, Tallarida, R.J., Jacob, L.S., 1979, The Dose-The Dose-

Response Relation in PharmacologyResponse Relation in Pharmacology, S-V, Berlin, S-V, Berlin Daniel, W.W., 1999, Daniel, W.W., 1999, Biostatistics, A Foundation Biostatistics, A Foundation

for Analysis in The Health Sciences,for Analysis in The Health Sciences, 7th 7th edition, John Wiley & Sons Inc, New Yorkedition, John Wiley & Sons Inc, New York

Hugo, W.B., and Russel, A.D., 1987, Hugo, W.B., and Russel, A.D., 1987, Pharmaceutical MicrobiologyPharmaceutical Microbiology 4th ed, BSP- 4th ed, BSP- LondonLondon

Robyt, J.F., and White, B.J., 1987, Robyt, J.F., and White, B.J., 1987, Biochemical Biochemical Techniques, Theory and Practice,Techniques, Theory and Practice, Brooks/Cole Brooks/Cole Publishing Company, CaliforniaPublishing Company, California

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Mengapa bioassayMengapa bioassay

If the chemical identity of the active principle has not If the chemical identity of the active principle has not been fully elucidated, e.g., new drugbeen fully elucidated, e.g., new drug

If no adequate chemical assay has been devised for If no adequate chemical assay has been devised for the active principle, although its chemical structure the active principle, although its chemical structure has been established, e.g., insulinhas been established, e.g., insulin

If the drug is composed of a complex mixture of If the drug is composed of a complex mixture of substances of varying structure and activity, e.g., substances of varying structure and activity, e.g., digitalis, posterior pituitary hormonedigitalis, posterior pituitary hormone

If purification of the crude drug, sufficient for the If purification of the crude drug, sufficient for the performance of a chemical assay, is not possible or performance of a chemical assay, is not possible or practical, e.g., separation of Vitamin D from certain practical, e.g., separation of Vitamin D from certain irradiated oilsirradiated oils

If the chemical assay is not a valid indication of If the chemical assay is not a valid indication of biological activity, due, for example, to lack of biological activity, due, for example, to lack of defereentiation between active and inactive isomers, defereentiation between active and inactive isomers, e.g., panthotenic acid e.g., panthotenic acid

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Analisis hayati: media Analisis hayati: media hayatihayati Animal assayAnimal assay

– Hewan UtuhHewan Utuh– Organ, Jaringan, sel, organel, Organ, Jaringan, sel, organel,

makromolekul (antibodi, enzim dll)makromolekul (antibodi, enzim dll) Microbial assayMicrobial assay

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Analisis hayati: efek Analisis hayati: efek yang diamatiyang diamati

Efek Terukur (dapat diukur): graded Efek Terukur (dapat diukur): graded effect, gradual effect, misalnya kadar effect, gradual effect, misalnya kadar gula darah, luas daerah hambatan gula darah, luas daerah hambatan pertumbuhan bakteri dllpertumbuhan bakteri dll

Efek Tidak terukur (tidak dapat Efek Tidak terukur (tidak dapat diukur): quantal effect, all or none diukur): quantal effect, all or none effect, go or no go effect, misalnya effect, go or no go effect, misalnya kematian, ada-tidaknya kematian, ada-tidaknya pertumbuhan bakteri, pertumbuhan bakteri, kelumpuhan/paralisis dllkelumpuhan/paralisis dll

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Efek Farmakologik: efek Efek Farmakologik: efek hipoglikemik karena insulin, hipoglikemik karena insulin, bronkhodilatasi oleh salbutamolbronkhodilatasi oleh salbutamol

Efek Biologik: penghambatan Efek Biologik: penghambatan pertumbuhan bakteripertumbuhan bakteri

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Laboratory Animal Laboratory Animal CareCare1.1. All animals used for experimental purposes must be All animals used for experimental purposes must be

law-fully acquired. Standards for their care shall be in law-fully acquired. Standards for their care shall be in strict compliance with the national laws, and with strict compliance with the national laws, and with governmental and institutional regulationsgovernmental and institutional regulations

2.2. Scientific institutions shall maintain a standing Scientific institutions shall maintain a standing committee, or other appropriate administrative body committee, or other appropriate administrative body to set policies and guidelines for the use and care of to set policies and guidelines for the use and care of animals in experiments conducted under their animals in experiments conducted under their auspicesauspices

3.3. Experiments involving live animals must be Experiments involving live animals must be performed by, or under the immediate supervision of, performed by, or under the immediate supervision of, a qualified biological scientist.a qualified biological scientist.

4.4. The housing, care, and feeding of all experimental The housing, care, and feeding of all experimental animals shall be supervised by a properly qualified animals shall be supervised by a properly qualified veterinarian or other biological scientist competent in veterinarian or other biological scientist competent in such matterssuch matters

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Laboratory Animal Laboratory Animal CareCare5. All laboratory animals must received every 5. All laboratory animals must received every

consideration for their comfort; they must be kindly consideration for their comfort; they must be kindly treated, properly fed, and their surroundings kept treated, properly fed, and their surroundings kept in a sanitary condition.in a sanitary condition.

6. In any operation likely to cause greater discomfort 6. In any operation likely to cause greater discomfort than that attending anesthetization, the animal than that attending anesthetization, the animal shall first be rendered incapable of perceiving pain shall first be rendered incapable of perceiving pain and must be maintained in that condition until the and must be maintained in that condition until the operation is ended.operation is ended.

7. If an acute study does not require survival, the 7. If an acute study does not require survival, the animal must be killed in a humane manner at the animal must be killed in a humane manner at the conclusion of the experiment by a procedure that conclusion of the experiment by a procedure that insures immediate death, in accordance with insures immediate death, in accordance with practices established by the institutional practices established by the institutional committee. The animal is not to be discarded until committee. The animal is not to be discarded until its death is certain.its death is certain.

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Struktur parktikum Struktur parktikum bioanalisisbioanalisis Percobaan I: evaluasi efek terukur dan Percobaan I: evaluasi efek terukur dan

tidak terukurtidak terukur Percobaan II: analisis hayati qualitatif: Percobaan II: analisis hayati qualitatif:

Pyrogen testPyrogen test Percobaan III: Analisis obat dan Percobaan III: Analisis obat dan

metabolitnya di dalam darahmetabolitnya di dalam darah Percobaan IV: analisis obat di dalam Percobaan IV: analisis obat di dalam

berbagai sampel hayatiberbagai sampel hayati Percobaan V: Pemanfaatan efek Percobaan V: Pemanfaatan efek

terukur untuk penetapan potensi obatterukur untuk penetapan potensi obat

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Analisis obat dalam Analisis obat dalam Sampel hayatiSampel hayati Dg cara fisiko kimiawi:Dg cara fisiko kimiawi:

Kolorimetri, spektrofotometri, Kolorimetri, spektrofotometri, kromatografi, elektrokimiawi, dllkromatografi, elektrokimiawi, dll

Dg cara hayati:Dg cara hayati:Menggunakan hewan utuh (Menggunakan hewan utuh (in in

vivo, in situvivo, in situ, organ terisolasi, fraksi , organ terisolasi, fraksi subseluler (ribosom, subseluler (ribosom, reseptor/protein, enzim, antibodi reseptor/protein, enzim, antibodi dll)dll)

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Sampel hayatiSampel hayati Liquids:Liquids:

Cerebrospinal FluidCerebrospinal Fluid TearsTears SweatSweat SalivaSaliva UrineUrine BileBile

MixedMixed PlasmaPlasma SerumSerum BloodBlood Gastric FluidGastric Fluid FecesFeces

SolidsSolids BrainBrain Heart, Kidney, LiverHeart, Kidney, Liver Lung, MuscleLung, Muscle Hair, BoneHair, Bone

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Sampel HayatiSampel Hayati

Pertanyaan yang harus dijawab dalam Pertanyaan yang harus dijawab dalam analisis adalah Apa? (What ?) dan Berapa analisis adalah Apa? (What ?) dan Berapa banyak ? (How much ?)banyak ? (How much ?)

Cairan Hayati/sampel hayati bukan Cairan Hayati/sampel hayati bukan campuran sederhana tetapi campuran campuran sederhana tetapi campuran komplek terdiri dari banyak komponen komplek terdiri dari banyak komponen berbeda yang mungkin bereaksi satu berbeda yang mungkin bereaksi satu dengan lainnyadengan lainnya

Sehingga menyebabkan beberapa Sehingga menyebabkan beberapa pengaruh, memperbesar atau menutupi pengaruh, memperbesar atau menutupi respons analisis atau mengubah nilai respons analisis atau mengubah nilai sebenarnya karena degradasi (pH, enzim).sebenarnya karena degradasi (pH, enzim).

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Sampel hayatiSampel hayati

Yang paling umum dianalisis kandungan Yang paling umum dianalisis kandungan obat/metabolitnya adalah darah utuh, plasma, obat/metabolitnya adalah darah utuh, plasma, serum atau urin.serum atau urin.

Karena preparasi plasma atau serum sangat Karena preparasi plasma atau serum sangat mudah didapat dari darah utuh dan mudah didapat dari darah utuh dan memberikan sampel yang berkurang nyata memberikan sampel yang berkurang nyata komponen penggangunya maka darah utuh komponen penggangunya maka darah utuh biasanya lebih jarang digunakan, kecuali untuk biasanya lebih jarang digunakan, kecuali untuk binatang kecil atau pada forensic toksikologi.binatang kecil atau pada forensic toksikologi.

Sampel hayati yang jarang dianalisis Sampel hayati yang jarang dianalisis kandungan obat/metabolitnya adalah empedu, kandungan obat/metabolitnya adalah empedu, keringat, air susu, air mata, dan saliva.keringat, air susu, air mata, dan saliva.

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Sampel HayatiSampel Hayati

Saliva perlu dipertimbangkan sebagai Saliva perlu dipertimbangkan sebagai sampel yg ideal mengingat sampel yg ideal mengingat pengambilannya tidak invasiv dengan pengambilannya tidak invasiv dengan syarat apabila kadar obat dalam saliva syarat apabila kadar obat dalam saliva menggambarkan kadar obat dalam menggambarkan kadar obat dalam darah.darah.

Cairan cerebrospinal juga dapat Cairan cerebrospinal juga dapat digunakan sbg sampel, utamanya untuk digunakan sbg sampel, utamanya untuk obat-obat yang bekerja di CNS, obat-obat yang bekerja di CNS, mengingat kedekatan fisiologis dengan mengingat kedekatan fisiologis dengan organ target, yaitu otak.organ target, yaitu otak.

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Beberapa Beberapa permasalahan dengan permasalahan dengan sampel hayatisampel hayati Pemisahan/isolasi analit: mudah-Pemisahan/isolasi analit: mudah-

tidaknya sampel dianalisis tidaknya sampel dianalisis sebanding dengan fluiditasnya.sebanding dengan fluiditasnya.

Cairan cerebrospinal paling mudah Cairan cerebrospinal paling mudah ditangani, sedang darah utuh dan ditangani, sedang darah utuh dan jaringan organ/paling sulitjaringan organ/paling sulit

Untuk meningkatkan fluiditas, Untuk meningkatkan fluiditas, sampel hayati yg solid atau semi sampel hayati yg solid atau semi solid sering kali mengalami proses solid sering kali mengalami proses mekanis, misalnya homogenisasi dllmekanis, misalnya homogenisasi dll

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Prosedur untuk Prosedur untuk merusak merusak jaringan/organ sampel jaringan/organ sampel hayatihayatiMortar-Pestle Blades Others

Characteristic

Potter-Elvejehen

Teflon-Glass

Waring blender

Virtis Sorval

Sonication

Chemical

Mechanism

Shear Shear Cut/shear Cut/shear Vibration Hydrolysis

Cool Possb.

Easily Easily No Easily Perhaps Not normally

Speed Var.

Continous

Continous

Continous/Stepped

Continous

Metal Cont

None Nominal Severe Severe Minimal None

Foaming Slight Slight Moderate Moderate Moderate Severe ?

Concistency

Poor Poor Good Good Good Good

Limitation Hard tissues

Hard tissues

Container size

Minimal Type of sampel

Harsh condition

Safety Poor Fair Good Excellent Good Fair

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Prosedur mekanis….Prosedur mekanis….

Prosedur mekanis tersebut dapat Prosedur mekanis tersebut dapat menyebabkan hal-hal yg tidak menyebabkan hal-hal yg tidak diinginkan, mis. Naiknya suhu, khelasi diinginkan, mis. Naiknya suhu, khelasi metal, hidrolisis konjugat yang dapat metal, hidrolisis konjugat yang dapat mengubah kadar aktual analitmengubah kadar aktual analit

Pengaruh yang lain adalah Pengaruh yang lain adalah mengakibatkan penangan sampel mengakibatkan penangan sampel berikutnya menjadi lebih berikutnya menjadi lebih sulit( foaming, emulsions, rupture of sulit( foaming, emulsions, rupture of red cells)red cells)

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Solvent untuk media proses Solvent untuk media proses mekanik juga menentukanmekanik juga menentukan

Solvent Advantages Disadvantages

Distilled Water Relatively good, do not destroy tissue const, final pH 7.0

Degree of ionisation may vary, do not denature enymes consistently

Dilute acid (<0.5 N)

Relatively good, denatures many enzymes, final pH < 7.0, foaming is minimal

Final pH may vary with tissue, Considerable protein denaturation, compounds may be acid sensitive

Strong acid (>0.5 N)

Good solvent, denatures all enzymes, precipitate proteins, final pH < 4.0

Clumping & aggregation may occur, compounds may be acid sensitive, tissue constituent may break down

Dilute alkali (< 0.5 N)

Relatively good, denatures many enzymes, final pH > 7.0

Considerable protein denaturation, compounds may be alkali sensitive, may be foaming

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Ekstraksi dengan Ekstraksi dengan solvet organiksolvet organik Derajat kemudahan proses ekstraksi Derajat kemudahan proses ekstraksi

obat/metabolit dari lingkungan berair ke obat/metabolit dari lingkungan berair ke solvent organik berikutnya tergantung solvent organik berikutnya tergantung pada solvent yang digunakanpada solvent yang digunakan

Secara umum tujuannya menyekat Secara umum tujuannya menyekat sebanyak-banyaknya analit dan sebanyak-banyaknya analit dan membuang sebanyak mungkin membuang sebanyak mungkin pengganngunyapengganngunya

Biasanya dilakukan dengan beberapa Biasanya dilakukan dengan beberapa kombinasi langkah dimana polaritas solvet kombinasi langkah dimana polaritas solvet pengekstraksi atau pH fase air disesuaikan.pengekstraksi atau pH fase air disesuaikan.

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Urutan polaritas Urutan polaritas solvent pengekstraksisolvent pengekstraksiSolvent UV limit

(nm)Boiling point Polarity

N-Hexane 210 69 Least polar

Cyclohexane 210 81

CCL4 265 77

Benzene 280 80

Toluen 285 111

Di-isopropil ether

220 68

Diethyl ether 220 35

Amyl acetate 285 149

Chloroform 245 61

Ethyl acetate 260 77

N-Butanol 215 118 Most polar

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Prinsip analisisPrinsip analisis

AccuracyAccuracy

– Precise and UnbiasedPrecise and Unbiased

SensitivitySensitivity

SelectivitySelectivity

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Prinsip-prinsip analisisPrinsip-prinsip analisis

Accuracy: precise and unbiasedAccuracy: precise and unbiased Random errorRandom error Systematic errorSystematic error

Biological VariableBiological Variable

Experimental design: simple, Experimental design: simple, cross over and Latin squarecross over and Latin square

Statistical analysisStatistical analysis

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SENSITIVITY & SENSITIVITY & SELECTIVITYSELECTIVITY Sensitivitas: jumlah minimum Sensitivitas: jumlah minimum

obat/metabolit yang masih bisa obat/metabolit yang masih bisa terdeteksiterdeteksi

Selektivitas: kemampuan metoda Selektivitas: kemampuan metoda untuk membedakan obat asal dg untuk membedakan obat asal dg metabolit dan atau impuritiesmetabolit dan atau impurities

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Accurate = precise & Accurate = precise & unbiasedunbiased

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Sample handlingSample handling

Sample collectionSample collection StorageStorage Protein denaturationProtein denaturation LiophylisationLiophylisation Hydrolisis of conjugate(s)Hydrolisis of conjugate(s) Derivatisation of analyte(s) (if Derivatisation of analyte(s) (if

needed)needed) Isolation of the Analyte(s) Isolation of the Analyte(s)

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Beberapa contohBeberapa contoh

Mirfazaehian, A., Goudarzi, M., Tabatbaiuefar, Mirfazaehian, A., Goudarzi, M., Tabatbaiuefar, M. and Mahuaudian, M. (2002) A Quantitative M. and Mahuaudian, M. (2002) A Quantitative Thin Layer Chromatography Method for Thin Layer Chromatography Method for Determination of Theophylline in Plasma, Determination of Theophylline in Plasma, Journal of Pharmacy and Pharmaceutical Journal of Pharmacy and Pharmaceutical Sciences, 5Sciences, 5(2), 131-134(2), 131-134

Mi-Kyeong Seo, Ji-Na Jeoy, Hoon-Joo Kim, In-Mi-Kyeong Seo, Ji-Na Jeoy, Hoon-Joo Kim, In-Chull Kim and Yong-Hee Lee (1996) High Chull Kim and Yong-Hee Lee (1996) High Performance Liquid Chromatographic Assay Performance Liquid Chromatographic Assay of New Fluoroquinolone, LB20304, in The of New Fluoroquinolone, LB20304, in The Plasma of Rats and Dogs, Plasma of Rats and Dogs, Archieves of Archieves of Pharmacal Research, 19Pharmacal Research, 19(6), 554-558(6), 554-558

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Beberapa contohBeberapa contoh

Cook, D.S., Braithwaithe, R.A. and Hale, K.A. Cook, D.S., Braithwaithe, R.A. and Hale, K.A. (2000) Estimating Antemortem Drug (2000) Estimating Antemortem Drug Concentrations from Postmortem Blood Concentrations from Postmortem Blood Samples: The Influence of Postmortem Samples: The Influence of Postmortem Redistribution,Redistribution, Journal of Clinical Pathology, Journal of Clinical Pathology, 53,53, 282-285 282-285

JunShik Choi and Jin Pil Burn (2002) JunShik Choi and Jin Pil Burn (2002) Pharmacokinetics of Acebutolol and Its Main Pharmacokinetics of Acebutolol and Its Main Metabolite, Diacetolol, After Oral Metabolite, Diacetolol, After Oral Administration of Acebutolol in Rabbits with Administration of Acebutolol in Rabbits with Carbon Tetrachloride-Induced Hepatic Carbon Tetrachloride-Induced Hepatic Failure, Failure, Arch Pharm Res, 25Arch Pharm Res, 25(4), 541-545(4), 541-545

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Beberapa contohBeberapa contoh

Lelievre, E., Cardona, H., Brillanceau, M.H., Lelievre, E., Cardona, H., Brillanceau, M.H., Piraube, C., and Sauveur, C. (1992) Piraube, C., and Sauveur, C. (1992) Radioimmunoassay for a New Angiotensin-Radioimmunoassay for a New Angiotensin-Converting Enzyme Inhibitor, Zabicipril, and Its Converting Enzyme Inhibitor, Zabicipril, and Its Active Metabolite, Zibiciprilat, in Human Active Metabolite, Zibiciprilat, in Human Plasma, Plasma, Journal of Pharmaceutical Sciences, 81Journal of Pharmaceutical Sciences, 81 (11), 1065(11), 1065

Mei-xia Zhou, Cha-ying Guan, Guang Chen, Xin-Mei-xia Zhou, Cha-ying Guan, Guang Chen, Xin-you Xie and Sheng-hai Wu (2005) you Xie and Sheng-hai Wu (2005) Determination of Theophylline Concentration in Determination of Theophylline Concentration in serum By Chemiluminescent Immunoassay, serum By Chemiluminescent Immunoassay, Journal of Zhejiang University of SienceJournal of Zhejiang University of Sience, 6(12), , 6(12), 1148-11521148-1152

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Beberapa contohBeberapa contoh

Rossi, S. and Yaksh, T (2003) Rapid Rossi, S. and Yaksh, T (2003) Rapid Quantification of The Non-Competitive NMDA Quantification of The Non-Competitive NMDA Antagonist MK-801 in Canine Cerebrospinal Antagonist MK-801 in Canine Cerebrospinal Fluid and Plasma By Capillary Gas Fluid and Plasma By Capillary Gas Chromatography-Nitrogen Phosphorus Chromatography-Nitrogen Phosphorus Detection,Detection, Journal Pharmaceutical and Journal Pharmaceutical and Biomedical Analysis, 31Biomedical Analysis, 31, 243-250, 243-250

Kenichiro Nakashima (2005) High-Kenichiro Nakashima (2005) High-Performance Liquid Chromatographic Analysis Performance Liquid Chromatographic Analysis of Drugs of Abuse in Biologic Samples, of Drugs of Abuse in Biologic Samples, Journal Journal of Health Science, 51of Health Science, 51 (3), 272-277 (3), 272-277

Bioassay of Pituitary Posterior HormoneBioassay of Pituitary Posterior Hormone

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Pituitary Posterior Pituitary Posterior HormoneHormone Pharmacodynamic effect:Pharmacodynamic effect:

– Rise in blood pressureRise in blood pressure– Contraction of uterine smooth muscleContraction of uterine smooth muscle– Renal tubular re-absorption of water Renal tubular re-absorption of water

(antidiuretic effect)(antidiuretic effect)– Milk ejection (galactokinetic effect)Milk ejection (galactokinetic effect)

Extract from posterior lobe of Extract from posterior lobe of hypophysis glandhypophysis gland

How many hormones in this extract ?How many hormones in this extract ?

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At least 2 active principles:At least 2 active principles:– Oxytocin: exhibit oxytosic & galactokinetic Oxytocin: exhibit oxytosic & galactokinetic

effecteffect– Vasopressin: vasopressor & antidiuretic effectVasopressin: vasopressor & antidiuretic effect– Both are octapeptides (isolated & event Both are octapeptides (isolated & event

synthesized)synthesized) Posterior Pituitary Powder & Injection Posterior Pituitary Powder & Injection

(USP) prepared from posterior lobe of the (USP) prepared from posterior lobe of the neurohypophysis posses the two neurohypophysis posses the two activities with variable proportionsactivities with variable proportions

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Bioassay of oxytocic Bioassay of oxytocic activity of PPHactivity of PPH Assayed using the method based on Assayed using the method based on

contraction of isolated guinea-pig uterinecontraction of isolated guinea-pig uterine Alternatifly using Chicken Vasodepressor Alternatifly using Chicken Vasodepressor

methods (simpler, more reproducible & methods (simpler, more reproducible & specific) e.g. Posterior Pituitary Injection specific) e.g. Posterior Pituitary Injection USP, Oxytocin Injection USPUSP, Oxytocin Injection USP– This method does not represent the clinical This method does not represent the clinical

use of oxytocin, however the assay of use of oxytocin, however the assay of oxytosic activity present in vasopressin oxytosic activity present in vasopressin injection must be performed by this guinea-injection must be performed by this guinea-pig uterine methodpig uterine method

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Bioassay of Bioassay of vasopressor activity of vasopressor activity of PPHPPH Vasopressin Injection USP is Vasopressin Injection USP is

asssayed by Rat Vasopressor asssayed by Rat Vasopressor Method:Method:– IV injection on anesthetized male IV injection on anesthetized male

rats which has been given rats which has been given phenoxybenzamine previouslyphenoxybenzamine previously

– Vasopressin presents in oxytocin Vasopressin presents in oxytocin injection is assayed by this methodinjection is assayed by this method

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(Biological) Sample (Biological) Sample collectioncollection Darah, plasma dan serum:Darah, plasma dan serum:

Vol 5 s.d 15 mlVol 5 s.d 15 ml

Serum: 30-50% darah, Plasma: Serum: 30-50% darah, Plasma: 50% darah50% darah

Komposisi hampir sama, secara Komposisi hampir sama, secara kuantitatif protein utama (albumin kuantitatif protein utama (albumin dan globulin dan globulin ++ 2%) tdpt dalm 2%) tdpt dalm serum maupun plasmaserum maupun plasma

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Sample collectionSample collection

Urine:Urine:

*jumlah cukup banyak*jumlah cukup banyak

*individu sehat tidak mengandung *individu sehat tidak mengandung

protein, shg tak perlu protein, shg tak perlu deproteinisasideproteinisasi

*studi metabolisme*studi metabolisme

*dapat dilakukan pemekatan*dapat dilakukan pemekatan

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Sample collectionSample collection

Feces:Feces:

* ditampung menggunakan * ditampung menggunakan alluminium foilalluminium foil

*Liofilisasi*Liofilisasi

*Banyak protein*Banyak protein

*Material padat banyak*Material padat banyak

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Noninvasive Methode of Noninvasive Methode of CollectionCollection

Dlm farmakokinetik klinik: monitoring dan Dlm farmakokinetik klinik: monitoring dan penetapan aturan dosis theofilin dilakukan penetapan aturan dosis theofilin dilakukan monitoring kadar obat dalam darah dengan monitoring kadar obat dalam darah dengan menggunakan sample saliva. Rasio kadar menggunakan sample saliva. Rasio kadar saliva/plasma =1/2, konstant pada dosis saliva/plasma =1/2, konstant pada dosis terapetikterapetik

Saliva: harus diketahui rasio kadar saliva/plasmaSaliva: harus diketahui rasio kadar saliva/plasma Bbrp obat diketahui ratio kadar saliva/plasma Bbrp obat diketahui ratio kadar saliva/plasma

konstankonstan Jumlah obat yg diekskresikan ke saliva Jumlah obat yg diekskresikan ke saliva

berhubungan dengan derajad ionisasi dlm pH berhubungan dengan derajad ionisasi dlm pH fisiologis dan protein-bindingfisiologis dan protein-binding

Tak terionkan akan terdapat paling banyakTak terionkan akan terdapat paling banyak Terapetik indek teofilin sangat sempitTerapetik indek teofilin sangat sempit

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Noninvasive….Noninvasive….

Nafas: obat dengan bobot molekul Nafas: obat dengan bobot molekul titik didih yang rendahtitik didih yang rendah

Oswaltd solubility coefficient (OSC):Oswaltd solubility coefficient (OSC): OSC etanol 2000: 2000ml alveolar OSC etanol 2000: 2000ml alveolar

breath = 1ml plasmabreath = 1ml plasma OSC aseton dan CHClOSC aseton dan CHCl33 masing2: 333 masing2: 333

dan 10.3 < dari etanol dan terdapat dan 10.3 < dari etanol dan terdapat dlm konsentrasi yang tinggi dlm nafasdlm konsentrasi yang tinggi dlm nafas

Untuk kasus2 intoksikasi Untuk kasus2 intoksikasi

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Storage for Biological Storage for Biological SamplesSamples Untuk menghindari dekomposisi dan perubahan kimia Untuk menghindari dekomposisi dan perubahan kimia

lainlain

Bila obat/metabolit sensitif thd esterase: pendinginan Bila obat/metabolit sensitif thd esterase: pendinginan pd -60pd -6000 C dan atau penambahan NaF pada sample yg C dan atau penambahan NaF pada sample yg mengandung eritromosisin propionatmengandung eritromosisin propionat

Apomorfin: peka thd oksidasi oleh OApomorfin: peka thd oksidasi oleh O22 di udara. di udara. Pendinginan pada -15Pendinginan pada -1500 C mencegah terbentuknya C mencegah terbentuknya quinon sd 4 minggu, pendinginan selanjutnya tdk quinon sd 4 minggu, pendinginan selanjutnya tdk menjamin terjadinya dekomposisi kecuali dengan menjamin terjadinya dekomposisi kecuali dengan penambahan as.askorbatpenambahan as.askorbat

Pendinginan/pembekuan: mempertahankan stabilitas Pendinginan/pembekuan: mempertahankan stabilitas L-DOPA sampai 16 hariL-DOPA sampai 16 hari

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Storage…..Storage…..

Plasma dan serum memerlukan sentrifugasi dalam Plasma dan serum memerlukan sentrifugasi dalam proses perolehannya, Vacutainer tube mengandung proses perolehannya, Vacutainer tube mengandung silikon yang memudahkan memisahkan serum/plasma silikon yang memudahkan memisahkan serum/plasma dari corpus culidari corpus culi

Tabung palstic (Ependorf) biasanya mengandung ester Tabung palstic (Ependorf) biasanya mengandung ester ftalat sbg plasticizer dan ester fosfat: dapat ftalat sbg plasticizer dan ester fosfat: dapat mengganggu analisismengganggu analisis

Antikoagulant bila diperlukan: sitrat, heparin, EDTA dll Antikoagulant bila diperlukan: sitrat, heparin, EDTA dll (selama tidak terjadi interaksi dengan analit)(selama tidak terjadi interaksi dengan analit)

Pembekuan/frozen segera setelah pengambilan sampel Pembekuan/frozen segera setelah pengambilan sampel seringkali tidak dimungkinkan; pendinginan sementara seringkali tidak dimungkinkan; pendinginan sementara dengan es dianggap cukup sebelum dipindah ke tempat dengan es dianggap cukup sebelum dipindah ke tempat penyimpanan dalam suhu bekupenyimpanan dalam suhu beku

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Storage…..Storage…..

Kontrol temperatur penyimpanan: K klorazepat akan Kontrol temperatur penyimpanan: K klorazepat akan mengalami dekarboksilasi menjadi nordiazepam yang mengalami dekarboksilasi menjadi nordiazepam yang dipengaruhi oleh suhu dan pHdipengaruhi oleh suhu dan pH

Wadah penyimpanan: wadah gelas lebih disukai sebab Wadah penyimpanan: wadah gelas lebih disukai sebab kemungkinan berkurangngnya analit dan adanya plasticiser kemungkinan berkurangngnya analit dan adanya plasticiser bila dg plastikbila dg plastik

Nitrogliserin terabsorpsi oleh wadah plastik, ada bbrp obat Nitrogliserin terabsorpsi oleh wadah plastik, ada bbrp obat yg dapat terabsorpsi oleh wadah gelas: dpt diatasi dengan yg dapat terabsorpsi oleh wadah gelas: dpt diatasi dengan sililasisililasi

Plastik punya kelebihan tidak mudah pecah, dalam Plastik punya kelebihan tidak mudah pecah, dalam pengangkutan lebih disukaipengangkutan lebih disukai

Dalam banyak hal perlu verifikasi dengan Dalam banyak hal perlu verifikasi dengan spiked biological spiked biological samplesample utk mengetahui pengaruh wadah dan penyimpanan utk mengetahui pengaruh wadah dan penyimpanan

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ISOLATION OF THE ISOLATION OF THE ANALYTE(s)ANALYTE(s) Solvent extraction :Solvent extraction :

Liquid-liquidLiquid-liquid

Solid-liquidSolid-liquid Chromatography:Chromatography:

Thin layer chromatographyThin layer chromatography

High-performance Liquid High-performance Liquid ChromatographyChromatography

Gas chromatographyGas chromatography

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A quantitative thin layer chromatography method for determination of Theophylline in Plasma 2 ml plasma + 20 2 ml plasma + 20 μμl acetaminophen (1 l acetaminophen (1

mg/ml) sbg standard internal + 1 ml NHmg/ml) sbg standard internal + 1 ml NH44SO4 SO4 dlm tabung bertutup di campur dg vortex dlm tabung bertutup di campur dg vortex mixermixer

Diekstraksi dg 5 ml CHClDiekstraksi dg 5 ml CHCl33-Isopropanol (75:25) -Isopropanol (75:25) dg digojog selama 10 menit pd rotary mixer dg digojog selama 10 menit pd rotary mixer kmd disentrifuge 10 min pd 8000 rpmkmd disentrifuge 10 min pd 8000 rpm

Fase organik dipisah dan diuapkan dibawah Fase organik dipisah dan diuapkan dibawah aliran gas Naliran gas N22

Residu kering dilarutkan kembali dlm 0.2 ml Residu kering dilarutkan kembali dlm 0.2 ml MetOH, kmd ditotolkan pd TLC platesMetOH, kmd ditotolkan pd TLC plates

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A quantitative TLC for A quantitative TLC for Theophylline………….Theophylline…………. Samples were spotted on pre-coated TLC Samples were spotted on pre-coated TLC

plates (silica gel 60 F254, 10x20cm, 0.25mm plates (silica gel 60 F254, 10x20cm, 0.25mm thickness, Merck)thickness, Merck)

Mobile phase consisted of AcetOH, isopropanol Mobile phase consisted of AcetOH, isopropanol and toluene (1:12:6). A Camag Linomat IV and toluene (1:12:6). A Camag Linomat IV (Switzerland) was used for spotting. Sample (Switzerland) was used for spotting. Sample volume was 50 ml, was spotted at a speed of volume was 50 ml, was spotted at a speed of 10 ml/sec on a band of 4mm width10 ml/sec on a band of 4mm width

A Camag TLC Scanner II equipped with a A Camag TLC Scanner II equipped with a deuterium lamp set at 277nm in the reflection deuterium lamp set at 277nm in the reflection mode was used for scanning. The peak mode was used for scanning. The peak heights and areas of chromatograms were heights and areas of chromatograms were determined using Camag version 3.11 determined using Camag version 3.11 softwaresoftware