Bioanalisis EMIT Kualitatif Amfetamin dan Benzodiazepin

TUGAS MAKALAH BIOANALISIS ANALISIS KUALITATIF AMFETAMIN DAN BENZODIAZEPIN SECARA EMIT (Enzyme Multiplied Immunoassay Technique)

Disusun Oleh: Kenny Ryan Limanto

098114006

Bernadetta Arum Wijayanti

098114007

Rachelia Octavia

098114008

Jenny Marina

098114016

Ina Juni Natasia

098114023

Kristina Nety

098114034

FAKULTAS FARMASI UNIVERSITAS SANATA DHARMA YOGYAKARTA 2011

A. Immunoassay Immunoassay merupakan uji untuk mengidentifikasi keberadaan suatu obat maupun metabolitnya dalam sampel biologis. Tujuannya untuk memonitor penyalahgunaan obat maupun terapi suatu obat pada pasien (Stanley, 2002). Immunoassay merupakan metode analisis yang didasarkan pada interaksi antigen dan antibodi. Antibodi merupakan molekul protein (yang diproduksi oleh hewan) yang dapat mengenal antigen (obat) secara spesifik pada tingkat molekuler. Kemampuan antibodi untuk membentuk kompleks dengan antigen merupakan basis immunoassay. Immunoassay merupakan

teknik

yang

spesifik

dan

sensitif.

Konsentrasi

analit

diidentifikasi dengan cara membandingkan absorbansi suatu obat yang tidak diketahui konsentrasinya dengan absorbansi konsentrasi obat standar, dengan menggunakan spektrofotometer (Stripp, 2007). Kebanyakan teknik immunoassay didasarkan pada pemisahan imunospesies yang bebas dan yang terikat. Dalam teknik ini, immuno-agent (antigen atau antibodi) diam pada pembawa yang padat (solid carrier). Kemudian akan terjadi immuno-interaction yang akan menghasilkan kompleks antigen-antibodi pada sisi spesifik di permukaan solid carrier. Jumlah ikatan immuno-complex ditunjukkan dengan pemberian label yang sesuai dengan sifat spesifik immune-agent supaya dapat dideteksi. Label yang biasa digunakan adalah isotop radioaktif, enzim, atau label fluoresen. Hasil akhir immunoassay berupa kuantifikasi deteksi label (Law, 2002). Immunoassay lebih sering menggunakan sampel urin karena dibutuhkan sampel bebas protein. Pada sampel lain yang masih mengandung protein perlu dilakukan pemisahan terlebih dahulu karena protein dapat mengganggu pembacaan absorbansi. Beberapa jenis immunoassay adalah sebagai berikut: 1. Enzyme-multiplied immunoassay technique (EMIT) 2. Radioimmunoassay (RIA) 3. Fluorescent polarization immunoassay (FPIA) 4. Kinetic interaction of microparticles in solution immunoassay (KIMS) 1

5. Enzyme-linked immunosorbent assay (ELISA) (Stripp, 2007). Immunoassay mudah dilakukan, relatif murah untuk pengujian tiap sampel, dan dapat mengidentifikasi suatu golongan obat. Namun perlu diperhatikan adanya senyawa yang mirip dengan senyawa target dapat mengganggu pengukuran dan memberikan hasil positif yang tidak diharapkan atau hasil positif yang salah (Stripp, 2007).

B. Enzyme-Multiplied Immunoassay Technique Pengujian dengan menggunakan metode EMIT merupakan salah satu cara pengujian secara immunoassay yang menggunakan suatu enzim yang sama untuk menguji beberapa senyawa. EMIT sendiri merupakan teknik immunoassay untuk beberapa jenis obat yang reseptornya berupa enzim. Pengujian dengan menggunakan metode ini didasarkan dari adanya kompetisi antara obat pada sampel dan obat yang telah dilabeli dengan enzim glukosa-6-fosfat dehidrogenase (G6P-DH) dengan sisi aktif dari suatu

antibodi

(immunoassay

competitive).

Enzim

glukosa-6-fosfat

dehidrogenase (G6P-DH) diperoleh dari bakteri Leuconostoc mesenteroides yang digunakan dalam pengujian ini. Pengikatan dari antibodi terhadap obat yang telah dilabeli dengan suatu enzim akan menghambat aktivitas enzimatik dari obat tersebut. Obat yang terdapat di dalam urin pasien dapat mengikat antibodi dan menggantikan kompleks yang ada sehingga meningkatkan aktivitasnya. G6P-DH

dari bakteri

nicotinamide

adenine

Leuconostoc mesenteroides dinucleotide

(NAD+)

akan

menjadi

mereduksi

NADH

yang

menyebabkan perubahan absorbansi dari sampel. Perubahan absorbansi ini diukur secara spektrofotometri dengan panjang gelombang 340 nm. Penggunaan enzim pada metode ini tidak akan diganggu oleh G6P-DH yang terdapat di dalam tubuh (endogen) karena enzim G6P-DH endogen hanya mereduksi NADP menjadi NADPH, bukan NAD+. Maka dalam pengujian ini ditambahkan NADP sebagai koenzim.

2

Gambar 1. Skema Representasi dari Enzyme Multiplied Immunoassay Technique (EMIT). Antibodi dapat mengikat enzim konjugat obat (Gambar 1.b) atau obat yang terdapat dalam sampel (Gambar 1.c). Ikatan kompleks obat-enzim dengan antibodi akan menurunkan aktivitas enzim. Akibatnya konsentrasi obat di dalam sampel akan meningkat sehingga kompleks obat aktif-enzim akan meningkat. Jadi konsentrasi obat di dalam sampel proporsional dengan aktivitas enzim (Alain, 2011). EMIT dapat digunakan untuk mengidentifikasi, antara lain: 1. Pengujian untuk benzodiazepin dan metabolitnya pada urin manusia. Pengujian ini menggunakan larutan oxazepam dengan konsentrasi 200 ng/mL untuk mengidentifikasi hasil positif/ negatif. 2. Pengujian untuk amfetamin monoklonal/ metamfetamin pada urin manusia. Pengujian ini dapat digunakan untuk mendeteksi adanya damfetamin,

d,l-amfetamin,

metilen-dioksi-amfetamin

(MDA),

dan

metilen-dioksi-metamfetamin (MDMA) pada urin manusia. Pengujian ini menggunakan larutan d-metamfetamin dengan konsentrasi 1000 ng/mL untuk mengidentifikasi hasil positif/ negatif.

3

Komponen yang digunakan dalam metode pengujian EMIT adalah obat, antibodi, substrat dan enzim yang akan berikatan dengan obat. Prosedur umum pada pengujian EMIT adalah sebagai berikut: Mencampur sampel yang mengandung obat dengan sejumlah obat diketahui konsentrasinya yang terikat pada enzim dan antibodi. Kemudian substrat ditambahkan. Absorbansi diukur pada detik ke 15 dan 45 setelah penambahan substrat. Jumlah obat dapat diketahui dengan mengukur absorbansi hasil reaksi enzim dan substrat dengan spektrofotometri UV-Vis. Perubahan absorbansi hasil reaksi menggambarkan konsentrasi obat dalam sampel. Tidak ada hubungan yang linear antara perubahan absorbansi dan konsentrasi obat.

Sumber: Anonim, 2011 C.

Preparasi Sampel Sampel urin yang digunakan sekurang-kurangnya 20 mL, namun biasanya sampel dikumpulkan sebanyak 30 mL dan dikirimkan ke laboratorium (pengujian dengan metode EMIT hanya membutuhkan 0,5 mL sampel, namun hasil positif/ negatif tidak dilaporkan jika tidak ada data pendukung lainnya seperti TLC ataupun GC/MS yang membutuhkan jumlah sampel yang banyak). Urin diambil dengan menggunakan Rainin pipettor. Pada pengambilan sampel, tidak boleh digunakan pipet yang terbuat dari plastik yang lembut (soft plastic). Sampel yang telah diambil dimasukkan ke dalam wadah yang kemudian diberi label nama pasien, nomor, tanggal dan jam pengambilan, asal dan nama dokter. Jika spesimen yang dikirimkan lebih dari 1 tube, tiap tube minimal harus dilabeli dengan nama, nomor , dan tanggal. Jika sampel yang dikirimkan tidak langsung diuji, maka sampel tersebut harus disimpan pada tempat yang dingin (refrigerator). Sampel tidak dapat diuji jika 4

disimpan lebih dari 3 hari. Sampel dijaga pada pH 5-8 (pH normal manusia). Pengaturan pH dapat dilakukan dengan cara menambahkan NaOH 1M ataupun HCl 1M. Spesimen yang akan diuji sebelumnya harus disentrifuge agar larutan yang dihasilkan jernih sehingga tidak mengganggu hasil pengukuran. Sampel yang akan diuji kandungannya (meliputi amfetamin dan benzodiapin) harus dilaksanakan dalam kurun waktu kurang dari 24 jam.

D.

Senyawa Uji Senyawa

yang diuji

dalam

makalah

ini

adalah

amfetamin

dan

benzodiazepin.

Reagen Reagen yang digunakan dalam penelitian ini adalah: 1.

Reagen Amfetamine/Methamphetamine A.

Monoklonal Amphetamine/Methamphetamine Working reagen A, Reagen 1: 1. Rekonstitusi reagen A Amfetamin/Metamfetamin monoklonal dengan 6 mL air Tipe I. 2. Pindahkan 6 mL reagen A yang telah direkonstitusi ke dalam labu Erlenmeyer 1L. 3. Tambahkan 210 mL Working EMIT Buffer dengan NAD/G6P-DH. 4. Sumbat labu dan gojog labu beberapa kali. 5. Reagen disesuaikan pada suhu kamar selama 1 jam sebelum digunakan. 6. Simpan pada suhu 2-8°C dalam botol polietilen 250 mL sampai 4 minggu.

B.

Monoclonal Amphetamine/Methamphetamine Working reagent B, Reagen 2: 1. Rekonstitusi reagen B Amfetamin/Metamfetamin monoklonal dengan 6 mL air Tipe I 5

2. Pindahkan 6 mL Reagen B yang telah direkonstitusi ke dalam labu Erlenmeyer 250 mL. 3. Tambahkan 210 mL Working EMIT Buffer dengan NAD/G6P. 4. Bilas botol reagen B dengan larutan ini untuk memastikan pemindahan akurat. 5. Sumbat labu dan gojog labu beberapa kali. 6. Reagen disesuaikan pada suhu kamar selama ± 1 jam sebelum digunakan. 7. Simpan dalam tiga botol reagen modular 70 ml pada suhu 2-8oC sampai 12 minggu. 2.

Reagen Benzodiazepine A.

Benzodiazepine Working reagent A, Reagent 1: 1. Rekonstitusi Benzodiazepine pereaksi A dengan 6 mL air Tipe I. 2. Pindahkan 6 mL reagen A masukkan dalam labu erlenmeyer 1L. 3. Tambahkan 210 mL Working EMIT Buffer dengan NAD/G6P. 4. Sumbat labu dan gojog labu beberapa kali. 5. Reagen disesuaikan pada suhu kamar selama ± 1 jam sebelum digunakan. 6. Simpan dalam botol polietilen 250 mL pada suhu 2-8°C sampai 12 minggu.

B.

Benzodiazepine Working reagent B, Reagent 2: 1. Rekonstitusi reagen B Benzodiazepine dengan 6 mL air Tipe I. 2. Pindahkan 6 mL reagen B masukkan dalam labu erlenmeyer 250 mL. 3. Tambahkan 210 mL Working EMIT Buffer. 4. Bilas botol reagen B dengan larutan ini untuk memastikan pemindahan akurat 5. Sumbat labu dan gojog beberapa kali 6. Reagen disesuaikan pada suhu kamar selama ± 1 jam sebelum digunakan.

6

7. Simpan dalam tiga botol reagen modular 70 ml pada suhu 2-8oC sampai 12 minggu.

Hasil positif/ negatif Pengujian ini dikalibrasi menggunakan kalibrator EMIT

A

level 1, yang

dinyatakan 200 ng/ mL oxazepam untuk benzodiazepine, 1000 ng/ mL dmethamphetamine untuk amfetamin. Kalibrator ini digunakan sebagai penentu untuk membedakan hasil "positif" dan "negatif" dari sampel. 1.

Hasil positif a. Benzodiazepin: Sampel yang memberikan perubahan nilai absorbansi (A) sama atau lebih tinggi dari kalibrator EMIT level 1 dari Oxazepam dinyatakan positif untuk benzodiazepin. Sampel mengandung benzodiazepin dan metabolit benzodiazepine. b. Amphetamine: Sampel yang memberikan perubahan nilai absorbansi (A) sama atau lebih tinggi dari kalibrator EMIT level 1 dari amfetamin monoklonal harus dikonfirmasi pada AxSYM. Pengujian EMIT yang menggunakan antibodi monoklonal memiliki sedikit gangguan dari zat yang meyerupai amfetamin. Oleh karena itu, sampel yang memberikan hasil positif dalam pengujian EMIT dan positif pada AxSYM dinyatakan sebagai positif. Sampel mengandung amfetamin. Sampel yang memberikan hasil positif dalam pengujian

EMIT dan negatif pada AxSYM dinyatakan

sebagai negatif. 2.

Hasil negatif Setiap sampel yang memberikan perubahan nilai absorbansi (A) lebih rendah dari kalibrator EMIT A level 1 untuk amfetamin dan benzodiazepin diinterpretasikan sebagai hasil negatif. Pengujian ini dapat digunakan untuk sampel yang mengandung obat dalam konsentrasi kecil atau sama sekali tidak mengandung obat.

Catatan khusus mengenai pengujian ini antara lain: 1.

Pengujian ini dirancang untuk digunakan dengan urin manusia. 7

2.

Penggunaan sampel urin selain sampel urin manusia, dapat menyebabkan hasil yang salah. Jika dicurigai sampel yang digunakan bukan sampel urin manusia, maka perlu digunakan sampel lain atau metode alternatif (misalnya, kromatografi lapisan tipis) untuk mengkonfirmasi hasil. Periksa pH spesimen yang diduga palsu. PH urin normal adalah 5,0-8,0.

3.

Reaksi silang dapat terjadi oleh adanya obat tertentu. Akibatnya diperoleh hasil positif lemah yang tidak dapat dikonfirmasi oleh kromatografi lapisan tipis. Hal ini dapat diatasi dengan cara mengetahui pengobatan lain yang sedang dilakukan pasien.

4.

Amfetamin a. Amfetamin muncul dalam urin dalam waktu 3 jam setelah terjadinya semua jenis administrasi dan dapat dideteksi dengan uji EMIT selama 24 sampai 48 jam setelah pemberian dosis terakhir. b. Uji EMIT d.a.u. untuk amfetamin/metamfetamin monoklonal mendeteksi adanya

d-amfetamin;

d,l-amfetamin,

methylenedioxyamphetamine

(MDA),

d-metamfetamin, dan

metil-

enedioxymethamphetamine (MDMA) dalam urin manusia. c. Dosis terapi dari obat-obat berikut dapat menghasilkan hasil dengan tes ini: klorokuin

(Aralen),

klorpromazin

(Thorazine),

methoxyphenamine,

quinicrine, ranitidine (Zantac), Procainamide dan metabolitnya Nacetylprocainamide dimetabolisme

(NAPA).

menjadi

Karena

amphetamine

dan

benzphetamine

(Didrex)

methamphetamine,

dosis

terapeutik obat ini juga dapat menghasilkan hasil yang positif. 5. Benzodiazepin a. Uji EMIT d.a.u. untuk benzodiazepin mendeteksi metabolit benzodiazepin dan benzodiazepin dalam urin manusia. b. Dosis terapi oxaprozin (DAYPRO), obat non-benzodiazepine dapat memberikan hasil yang positif dengan tes ini. Hasil positif dari pemakaian tunggal oxaprozin harus ditafsirkan dengan hati-hati dan dikonfirmasi dengan metode lain.

8

Batas Deteksi Minimum Batas deteksi minimum senyawa amfetamin: d-Amphetamine

< 400 ng/mL

d,l-Amphetamine

1000 ng/mL

d- Methamphetamine

1000 ng/mL

Methylenedioxyamphetamine (MDA)

1000 ng/mL

Methylenedioxymethamphetamine (MDMA)

3000 ng/mL

Batas deteksi minimum senyawa Benzodiazepin: Oxazepam

200 ng/mL

Alprazolam

50 ng/mL

Bromazepam

400 ng/mL

Chlordiazepoxide

500 ng/mL

Clobazam

100 ng/mL

Clonazepam

500 ng/mL

Clotiazepam

100 ng/mL

Demoxepam

900 ng/mL

N-Desalkylflurazepam

100 ng/mL

Diazepam

40 ng/mL

Flunitrazepam

100 ng/mL

Flurazepam

100 ng/mL

Halazepam

80 ng/mL

Lorazepam

1000 ng/mL

Lormetazepam

200 ng/mL

Medazepam

100 ng/mL

Midazolam

120 ng/mL

Nitrazepam

200 ng/mL

Norchlordiazepoxide

1800 ng/mL

Nordiazepam

60 ng/mL

Prazepam

80 ng/mL

9

Kalibrasi dan pengisian sampel pasien 1. Sebuah kalibrasi Blanko dilakukan setidaknya sekali seminggu dengan menggunakan kalibrator EMIT Level 1, yang berisi konsentrasi dinyatakan dari 200 ng /mL Oxazepam untuk Benzodiazepine, dan 1000 ng/ mL dMetamphetamine

untuk

amfetamin.

Dalam

mentransfer

jangan

menggunakan pipet plastik yang lunak. 2.

Verifikasi kalibrasi dengan menjalankan Immunoassay Cair Kontrol Urin Detectabuse Seri V cutoff kontrol negatif -25% dan Detectabuse Immunoassay Kontrol Urin Cair Seri V cutoff kontrol positif +25%. Menggunakan pipettor MLA, transfer 300 µL dari kontrol negatif dan positif. Dalam mentransfer lunak salah satu kontrol jangan menggunakan pipet plastik yang lunak. Kedua kontrol harus jatuh dalam batas yang dapat diterima. Jika ada kontrol negatif lebih besar atau sama dengan nol atau kontrol positif kurang dari atau sama dengan nol, dapatkan kontrol yang terbaru kemudian dijalankan kembali. Jika kontrol masih di luar jangkauan, kalibrasi ulang dan jalankan kontrol lagi. Jalankan baik kontrol positif dan negatif sekali pergeseran.

3.

Setelah kalibrasi divalidasi, jalankan perlakuan dengan menempatkan sampel urin dalam tabung barcode. Transfer 500 µL dari sampel urin menggunakan Pipetman MLA.

Verifikasi Kalibrasi Kalibrasi verifikasi dari metode ini adalah tidak diperlukan sebagai uji yang dikalibrasi mingguan. Metode ini dikalibrasi menggunakan prosedur kalibrasi blanko. Kontrol harus berada dalam rentang yang didirikan setelah dan di antara kalibrasi. Ini adalah jaminan bahwa kalibrasi dapat diterima. Ini adalah tes kualitatif dengan kinerja yang diverifikasi oleh kontrol positif dan negatif. Sistem P modular juga memverifikasi bahwa hasil dari uji kalibrasi memenuhi spesifikasi ditugaskan untuk memilih parameter validitas. Pesan kesalahan terjadi ketika kalibrasi gagal memenuhi spesifikasi.

10

Gangguan: Hemolisis: Tidak ada gangguan dari hemoglobin sampai dengan 1,0 g/L (2 +). Lipemia: Semua spesimen dengan kekeruhan penting harus disentrifugasi sebelum analisis.

DAFTAR PUSTAKA Alain Verstraete, 2011, Workplace Drug Testing, 219, Pharmaceutical Press, USA. 11

Anonim, 2011, Immunoassay Basics, http://www.enzolifesciences.com/support/ immunoassay-kits/immunoassay-basics/, diakses tanggal 27 Oktober 2011. Law, W.T., 2002, Biomedical Disgnostic Science and Technology, 79-80 , Mrcel Dekker Inc., New York. Shizume, T, Donabedian, R., Hodson, M., 2004, Journal: Urine Drug of Abuse by Emit, Deparment of Laboratory Medicine Clinical Chemistry. Stanley, J., 2002, Essentials of Immunology and Serology, 162 , Thomson Learning Inc., USA. Stripp, R.A., 2007, The Forensic Aspect of Poisons, 90-92 , Infobase Publishing, New York.

12

TITLE:

URINE DRUGS OF ABUSE BY EMIT

DEPT OF LAB MEDICINE CLINICAL CHEMISTRY DOCUMENT NO: CC-110013

REVIEW BY PREP/SUPVR:

EFFECTIVE DATE: November 1, 2004

REVISION: H-7

Page 1 of 25

LAB MANAGER: Timothy Shizume, MT (ASCP)

DIRECTOR: Richard Donabedian, M.D.

DIRECTOR: Michael Hodsdon, M.D.

DIRECTOR: Herbert Malkus, PhD.

I.

PRINCIPLE: The EMIT assay is a homogenous enzyme immunoassay technique used for the analysis of specific compounds in biological fluids. EMIT stands for Enzyme Multiplied Immunoassay Technique. The assay is based on competition between drug in the sample and drug labeled with the enzyme glucose-6-phosphate dehydrogenase (G6P-DH) for antibody binding sites. Binding of the antibody to the drug-enzyme complex partially inhibits enzymic activity. Drug in the patient's urine can bind to the antibody and displace the complex leading to increased activity. Enzyme activity increases with increased drug concentrations, but in a nonlinear way. G6P-DH converts oxidized nicotinamide adenine dinucleotide (NAD+) to NADH resulting in an absorbance change that is measured spectrophotometrically. Endogenous G6P-DH does not interfere, because it uses NADP as a coenzyme. NAD+ functions only with the bacterial (Leuconostoc mesenteroides) enzyme employed in the assay. We have modified the EMIT dilution protocol of Neeley1 in which extra substrates, glucose-6-phosphate and NAD+, are added to the diluted reagent to enhance the enzymic response to drug concentration. We add sodium oxamate to the substrate to inhibit LD activity. LD greater than 1000 U/L in the presence of elevated pyruvate can convert NADH back to NAD+ and thus falsely lower the measured drug concentration. We perform this assay on the Modular DPP analyzer. The Emit D.A.U. Cocaine metabolite assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of benzoylecgonine (the metabolite of cocaine) in human urine. This assay uses a cutoff level of 300 ng/mL to distinguish positive from negative samples. The Emit D.A.U. Opiate assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of opiates in human urine. This assay detects morphine, morphine glucuronide (the major metabolites of heroin ) and codeine in the human urine and gives a positive result if any of these opiates are present. It also detects synthetic opiates related to morphine, such as hydromorphone and high concentrations of the analgesic meperidine. This assay uses the signal from 300 ng/mL of morphine as a cutoff to distinguish positive from negative samples. The Emit D.A.U. Barbiturate assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of barbiturates in human urine. This assay tests for both long and short acting barbiturates in human urine. This assay uses the signal from 200 ng/mL of secobarb as a cutoff to distinguish positive from negative samples. The Emit D.A.U. Methadone assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of methadone in human urine. This assay detects methadone in human urine. This assay uses the signal from 300 ng/mL of methadone as a cutoff to distinguish positive from negative samples. The Emit D.A.U. Benzodiazepine assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of benzodiazepines in human urine. This assay tests for benzodiazepines and

TITLE:


DEPT. OF LAB MEDICINE CLINICAL CHEMISTRY DOCUMENT NO: CC-110013


REVISION: H-7

Page 2 of 25

benzodiazepine metabolites in human urine. This assay uses the signal from 200 ng/mL of Oxazepam as a cutoff to distinguish positive from negative samples. The Emit D.A.U. Monoclonal Amphetamine/Methamphetamine assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of amphetamines in human urine. This assay detects d-amphetamine, d,l-amphetamine, methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA) in human urine. This assay uses the signal from 1000 ng/mL of d-Methamphetamine as a cutoff to distinguish positive from negative samples. Because the assay contains monoclonal antibodies, it is less subject to interference by amphetamine-like compounds than assays containing polyclonal antibodies. While interferences are reduced with this assay, like any immunological test, some interfering compounds do exist. The Emit II Plus Cannabinoid assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of cannabinoids in human urine. The EMIT II Plus Cannabinoid assay detects the major metabolite of ∆ 9 -THC,11-nor-∆ 9 –carboxylic acid in human urine. It also detects other ∆ 9 -THC metabolites. This assay uses the signal from 50 ng/mL of 11-nor- ∆ 9 - THC-9-carboxylic acid as a cutoff to distinguish positive from negative samples. The DRI Emit Phencyclidine assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of phencyclidine in human urine. This assay uses a cutoff level of 25 ng/mL Phencyclidine to distinguish positive from negative samples. The DRI Emit Oxycodone assay is a homogenous enzyme immunoassay intended for use in the qualitative analysis of Oxycodone in human urine. This assay detects oxycodone and oxymorphone in human urine. This assay uses the signal from 100 ng/mL of Oxycodone as a cutoff to distinguish positive from negative samples. The DRI Oxycodone assay uses specific antibodies that can detect oxycodone and oxymorphone without any significant crossreactivity to other opiate compounds. II.

SPECIMEN PREPARATION: Sample collection, Handling and Storage Spot urine samples of at least 20 mL, preferably 30 mL should be collected and sent to the chemistry lab (EMIT requires 0.5 mL of sample only, however positives can not be reported unless confirmed by TLC or GC/MS which needs the greater volume). YNHPH samples for DAU are done only by EMIT. The minimum amount of sample required from YNHPH patients is at least 1 mL of urine. Transfer at least 1 mL of urine using the Rainin pipettor. Do not use the soft plastic transfer pipettes when processing the YNHPH urine specimens. Do not QNS samples from pediatrics (NBSCU). Ask the TDM/TOX technologist first if the amount of urine received is acceptable. All specimen containers must be labeled with patient's name, unit number, date and time of collection, origin and doctor's name. If the specimen is sent in more than one tube, each tube must be labeled with at least name, unit # and date. Unlabeled tubes cannot be accepted, due to the sensitive legal aspects of drug testing. The floor cannot correct labeling errors. A new sample must be collected. If immediate transport is not available, refrigerate specimen. No sample will be accepted by the lab if older than 3 days. Samples should be

TITLE:




REVISION: H-7

Page 3 of 25

within the pH range 5-8. Add 1M HCl or 1M NaOH if adjustment is necessary. Turbid specimens should be centrifuged before doing the analysis. Samples requested for any of these 9 analytes ( cocaine metabolite, opiates, PCP, barbiturate, methadone, amphetamine, benzodiazepine, cannabinoid, and oxycodone) or for full urine toxicology screen or DAU requests from YNHPH should be brought to Modular DPP where they are done on request, 24 hours a day. Cannabinoid is for YNHPH panels only. When requested from other locations, it may be performed but it must also be discussed by the Laboratory Medicine resident only if the result is positive. Confirmation, when requested, is sent to an outside lab. (Note: Only EMIT portion of urine toxicology screen is done around the clock. The TLC portion is only done Mon, Wed, or Fri on the day shift). Samples received before 8:00 AM will be included in the Narcotics run for that day. Alert and deliver the urine to Narcotics tech so they can include that sample for the day’s run. A.

Spinning Procedure

NOTE: PROCESS ONE SAMPLE AT A TIME 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

III.

Check specimen identification Check amount of urine (minimum 20 mL, except for YNHPH) Check pH (5-8). Adjust with 1M HCl or 1M NaOH if necessary. Use a 1000 uL Rainin pipet to aliquot urine if cannabinoid is requested. Take 1 mL aliquot for Modular DPP. Spin Modular DPP aliquot Transfer 0.5 mL aliquot into labeled Oly tube. Put the rest of the spun urine in the general rack. Deliver to Modular DPP. Bring Narcotics portion (including requisition) to Narcotics bucket in refrigerator including YNHPH urine toxicology screens. YNHPH samples for individual tests go in YNHPH bucket. (Do not include requisition)

MATERIALS: A.

Equipment: 1. 2. 3. 4. 5. 6.

130 mL Modular Reagent bottles 70 mL Modular Reagent bottles Transport vials, 7 mL, Evergreen #240-30007-060, $53/pkg of 1000 75 x 100 test tubes ( Sarstedt Cat.# 55.476) Screw capped polyethylene vials, Sarstedt (Cat.# 72-694/007) Minimal (generic) reagent barcode labels

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B.

Page 4 of 25

Reagents: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

C.

REVISION: H-7

D-Glucose-6-Phosphate, monosodium salt; 10 g bottle (Cat.# 500116-2; Research Organics Inc.) NAD, 100 g bottle Cat.# 045517 Amresco EMIT d.a.u. Cocaine Metabolite Assay Cat.# 3H019 SYVA EMIT d.a.u. Opiate Assay Cat.# 3B019 SYVA EMIT d.a.u. Phencyclidine Assay Cat.# 3J019 SYVA EMIT d.a.u. BarbiturateAssay Cat. # 3D229UL Dade Behring EMIT d.a.u. Methadone Assay Cat. # 3E019 Dade Behring EMIT d.a.u. Monoclonal Amphetamine/Methamphetamine Assay Cat. # 3C549 Dade Behring EMIT d.a.u. Benzodiazepine Assay Cat. # 3F229 Dade Behring EMIT II Plus Cannabinoid Assay Cat. # 9N029UL Dade Behring EMIT Calibrator/Control Level 3 Dade Behring Cat. #. 9A569UL Emit Calibrator A Level 1 Dade Behring Cat. #. 9A169UL Emit Calibrator A Level 2 Dade Behring Cat. #. 9A189UL EMIT Calibrator B Level 1 Dade Behring Cat. #. 9A279 DRI Multidrug Calibrator 2 (PCP) Cat. # 1591 (10 mL) Microgenics. Oxycodone Assay, 500 mL Microgenics Cat. # 100249 Oxycodone 100 ng/mL Calibrator, 10 mL Microgenics Cat. # 100251 Oxycodone Control kit (225 ng/mL and 375 ng/mL), 2 x 10 mL, Microgenics Cat. # 100255 Sodium Oxamate, Sigma Cat# O 2751, $35.10/25g Detectabuse Immunoassay Liquid Control Urine Series V Cutoff –25% Biochemical Diagnostics,Inc Cat.# 1945825-2. Detectabuse Immunoassay Liquid Control Urine Series V Cutoff +25% Biochemical Diagnostics,Inc Cat.# 1945875-6. Tris, base, (Tris[hydroxy methyl]aminomethane). Amresco# 0497, $24.75/kg. Any Tris[hydroxy methyl]aminomethane may be used such as Fisher’s "THAM" or Sigma’s TRIZMA". (Do not use Tris Hydrochloride) Tris, hydrochloride, (Tris[hydroxy methyl]aminomethane hydrochloride) Research Organics #.3098T-2, $33.50/kg. (Use also THAM HCl. Do not use Tris) D-Mannitol, Sigma# M 9647 $21.80/500 g Sodium Azide, Practical OR. S/P# 1953-500*NY, $33.80/500 g

Reagent Preparation: 1.

EMIT Buffer with NAD/G6P (210 mL Aliquots): This is an augmented buffer used to dilute reagent A. a.

To a large mortar add: (1) (2)

43.4 g D-Glucose-6-Phosphate, monosodium salt 94.0 g Sodium oxamate

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(3) (4) (5) (6) b.

(4)

2.

21 g sodium azide 160 g sodium chloride 53.1 g Tris, base, (Tris[hydroxy methyl]aminomethane 112.6 g Tris, hydrochloride, (Tris[hydroxy methyl]aminomethanehydrochloride)

Add 110.3 g NAD Add 8 g D-Mannitol Grind mixture with a large pestle until homogeneous, approximately 15 minutes. Weigh 6.00 g aliquots into prelabeled polyethylene scintillation vials.

Store tightly capped at -20°C for up to 3 years.

Working EMIT Buffer with NAD/G6P (210 mL aliquots): a. b. c.

3.

Page 5 of 25

Place mortar in dry box and allow to equilibrate for 15 minutes: (1) (2) (3)

c.

REVISION: H-7

In a 500 mL Erlenmeyer flask dissolve 1 vial of EMIT Buffer with NAD/G6P with 210 mL of Type I water. Add 0.20 mL 10% Triton X-100 Store at 2-8°C in a 250 mL polyethylene bottle for up to 6 months.

EMIT Buffer with NAD/G6P ( one liter aliquots): This is an augmented buffer used to dilute reagent A. a.

To a large mortar add: (1) (2) (3) (4) (5) (6)

b.

103.3 g D-Glucose-6-Phosphate, monosodium salt 223.8 g Sodium oxamate 50 g sodium azide 381 g sodium chloride 126.4 g Tris, base, (Tris[hydroxy]aminomethane) 268 g Tris, hydrochloride, (Tris[hydroxy]aminomethane hydrochloride)

Place mortar in drybox and allow to equilibrate for 15 minutes: (1) (2) (3) (4)

Add 262.6 g NAD Add 39 g D-Mannitol Grind mixture with a large pestle until homogeneous, approximately 15 minutes. Weigh 29.00 g aliquots into prelabeled 50 mL polyethylene vials.

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c. 4.

b. c. d.

b. c. d.

f.

In a 500 mL Erlenmeyer flask, dilute 1 bottle of EMIT buffer concentrate (13.3 mL) with 200 mL of Type I water. Rinse the concentrate bottle several times with Type I water to ensure complete transfer of buffer. Add 200 uL of 10% Triton-X. Mix gently to avoid too much foaming. Store at 2-8°C in a 250 mL polyethylene bottle for up to 3 months.

Reconstitute Cocaine Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask. Add 210 mL of Working EMIT Buffer with NAD/G6P Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 12 weeks.

Cocaine working reagent B, Reagent 2: a. b. c. d. e. f. g.

8.

In a one liter volumetric flask dissolve 1 vial of EMIT Buffer with NAD/G6P with about 500 mL of Type I water. Fill to volume with Type I water. Add 1.00 mL 10% Triton X-100. Mix gently to avoid foaming. Store at 2-8°C in a 500 mL polyethylene bottle for up to 6 months.

Cocaine working reagent A, Reagent 1: a. b. c. d. e.

7.

Store at -20°C for up to 3 years.

Working EMIT Buffer: Each of the reagent kits contains a bottle of EMIT Buffer Concentrate. a.

6.

Page 6 of 25

Working EMIT Buffer with NAD/G6P (one liter aliquots): a.

5.

REVISION: H-7

Reconstitute Cocaine Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

Opiate working reagent A, Reagent 1: a. b.

Reconstitute Opiate Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask.

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c. d. e. f. 9.

g.

Add 210 mL of Working EMIT Buffer with NAD/G6P. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 12 weeks.

Reconstitute Opiate Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

DRI PCP reagent A (Antibody/Substrate Reagent), Reagent 1: a. b. c.

11.

Page 7 of 25

Opiate working reagent B, Reagent 2: a. b. c. d. e. f.

10.

REVISION: H-7

This reagent is ready for use. No reagent preparation is required. Transfer 60 mL of Reagent A into a 70 mL modular reagent bottle. Store at 2-8°C for up to expiration date indicated on the label.

DRI PCP reagent E (Enzyme Conjugate reagent), Reagent 2: a. b. g.

This reagent is ready for use. No reagent preparation is required. Transfer 60 mL of Reagent E into a 70 mL modular reagent bottle. Store at 2-8oC for up to expiration date indicated on the label.

12.

DRI Multidrug Calibrator 2 (PCP) is in liquid form (10 mL) – ready to use. Store at 2 - 8˚ C for up to the expiration date on the label.

13.

Barbiturate working reagent A, Reagent 1: a. b. c. d. e. f.

Reconstitute Barbiturate Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask. Add 210 mL of Working EMIT Buffer with NAD/G6P. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 12 weeks.

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14.

g.

f.

Reconstitute Methadone Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask. Add 210 mL of Working EMIT Buffer with NAD/G6P. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 12 weeks.

Methadone working reagent B, Reagent 2: a. b. c. d. e. f. g.

17.

Reconstitute Barbiturate Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

Methadone working reagent A, Reagent 1: a. b. c. d. e.

16.

Page 8 of 25

Barbiturate working reagent B, Reagent 2: a. b. c. d. e. f.

15.

REVISION: H-7

Reconstitute Methadone Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

Benzodiazepine working reagent A, Reagent 1: a. b. c. d. e. f.

Reconstitute Benzodiazepine Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask. Add 210 mL of Working EMIT Buffer with NAD/G6P. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 12 weeks.

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18.

g.

b. c. d. e. f.

Reconstitute Monoclonal Amphetamine/Methamphetamine Reagent A with 6 mL of Type I water. Transfer the 6 mL of Reagent A into a one liter Erlenmeyer flask. Add 210 mL of Working EMIT Buffer with NAD/G6P. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store at 2-8°C in 250 mL polyethylene bottles for up to 4 weeks.

Monoclonal Amphetamine/Methamphetamine working reagent B, Reagent 2: a. b. c. d. e. f. g.

21.

Reconstitute Benzodiazepine Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

Monoclonal Amphetamine/Methamphetamine working reagent A, Reagent 1: a.

20.

Page 9 of 25

Benzodiazepine working reagent B, Reagent 2: a. b. c. d. e. f.

19.

REVISION: H-7

Reconstitute Monoclonal Amphetamine/Methamphetamine Reagent B with 6 mL of Type I water. Transfer the 6 mL reconstituted reagent B into a 250 mL Erlenmeyer flask. Add 210 mL of Working EMIT Buffer. Rinse the reagent B bottle with this solution to ensure accurate transfer. Stopper and mix well by inverting the flask gently several times. Allow the reagent to equilibrate at room temperature for at least 1 hour before using. Store in three 70 mL modular reagent bottles at 2-8oC for up to 12 weeks.

EMIT II Plus Cannabinoid reagent A, Reagent 1: a. b. c. d.

This reagent is in liquid form (115 mL) – ready to use. No reconstitution is required. Transfer Reagent A into a 130 mL Modular reagent bottle. Reagent when stored covered with a screw cap tightly at 2-8°C is stable up to the expiration date on the label. Reagent on board the Modular P analyzer is stable for up to 4 weeks.

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22.

REVISION: H-7

Page 10 of 25

EMIT II Plus Cannabinoid reagent B, Reagent 2: a. b. c. d.

This reagent is in liquid form (50 mL) – ready to use. No reconstitution is required. Transfer Reagent B into a 70 mL Modular reagent bottle. Reagent when stored covered with a screw cap tightly at 2-8°C is stable up to the expiration date on the label. Reagent on board the Modular P analyzer is stable for up to 4 weeks.

NOTE: Reagents A and B are provided as a matched set. They should not be interchanged with components of kits with different lot numbers. 23.

DRI EMIT Oxycodone, R1, Antibody/Substrate Reagent: Contains mouse monoclonal anti-oxycodone derivative antibody, glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide (NAD) in Tris buffer with sodium azide as preservative. a. b. c.

24.

This reagent is ready for use. No reagent preparation is required. Transfer 60 mL of Reagent A into a 70 mL modular reagent bottle. Store at 2-8˚ C for up to the expiration date indicated on the label.

DRI EMIT Oxycodone, R2, Enzyme/Conjugate Reagent: Contains oxycodone derivative labeled with glucose-6-phosphate dehydrogenase (G6PDH) in Tris buffer with sodium azide as preservative. a. b. c.

This reagent is ready for use. No reagent preparation is required. Transfer 60 mL of Reagent E into a 70 mL modular reagent bottle. Store at 2-8˚ C for up to the expiration date indicated on the label.

25.

Oxycodone Calibrator is in liquid form (10 mL) – ready to use. Store at 2-8˚ C for up to the expiration date on the label.

26.

Emit Calibrator A Level 1: a. b. c. d.

Following the directions in the package insert, add 5.0 mL Type I water to the vial. Swirl the vial until the powder is dissolved. Allow the reconstituted calibrator to sit at room temperature (20-25oC) for at least one hour before using. The calibrator is stable for 12 weeks when stored at 2-8oC.

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27.

b. c. d.

V.

Following the directions in the package insert, add 5.0 mL Type I water to the vial. Swirl the vial until the powder is dissolved. Allow the reconstituted calibrator to sit at room temperature (20-25oC) for at least one hour before using. The calibrator is stable for 12 weeks when stored at 2-8oC.

EMIT Calibrator/Control Level 3 : a. b.

IV.

Page 11 of 25

Emit Calibrator A Level 2: a.

28

REVISION: H-7

The Cannabinoid calibrator comes in liquid form (14 mL) – ready to use. Store at 2-8°C for up to the expiration date on the label.

INSTRUMENT PARAMETERS: A.

Modular P Parameters: COCAINE- see Addendum 1

B.

Modular P Parameters: OPIATE- see Addendum 2

C.

Modular P Parameters: PCP- see Addendum 3

D.

Modular P Parameters: CANNABINOID- see Addendum 4

E.

Modular P Parameters: AMPHETAMINE- see Addendum 5

F.

Modular P Parameters: BENZODIAZEPINE- see Addendum 6

G.

Modular P Parameters: METHADONE- see Addendum 7

H.

Modular P Parameters: UBARB- see Addendum 8

I.

Modular P Parameters: OXYCODONE- see Addendum 9

CALCULATIONS AND METHODS OF REPORTING RESULTS: A.

Positive/Negative Results:

This assay is calibrated using the EMIT calibrator A Level 1, which contains a stated concentration of 300 ng/mL benzoylecgonine for cocaine, 300 ng/mL morphine for opiates, 200 ng/mL secobarb for Urine Barbiturate, 200 ng/mL Oxazepam for Benzodiazepine, 1000 ng/mL d-Metamphetamine for Amphetamine, DRI Multidrug Calibrator 2, which contains a stated concentration of 25 ng/mL phencyclidine for PCP, EMIT calibrator B Level 1 with a concentration of 300 ng/mL methadone for Methadone, EMIT Calibrator/Control Level 3 with a

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REVISION: H-7

Page 12 of 25

concentration of 50 ng/mL of 11-nor- ∆ 9 -THC-9-carboxylic acid for Cannabinoid, and Oxycodone Calibrator 100 ng/mL for oxycodone, all of which are set to zero. These calibrators are used as the cut-off and the reference for distinguishing "positive" from "negative" samples. 1.

Positive Results: a.

Cocaine: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator A Level 1 for cocaine is interpreted as positive. The sample contains benzoylecgonine.

b.

Opiates: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator A Level 1 for opiates is interpreted as positive. The sample contains opiates.

c.

PCP: Any sample that gives a change in absorbance ( A) value equal to or higher DRI multidrug Calibrator 2 for PCP (i.e. zero or higher) is interpreted as positive. The sample contains phencyclidine or phencyclidine metabolites or analogs.

d.

Urine Barbiturate: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator A Level 1 for urine barbiturate is interpreted as positive. The sample contains barbiturate.

e.

Benzodiazepine: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator A Level 1 for benzodiazepine is interpreted as positive. The sample contains benzodiazepine and benzodiazepine metabolites.

f.

Amphetamine: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator A Level 1 for monoclonal amphetamine must be confirmed on the AxSYM. The EMIT assay, using monoclonal antibodies is less subject to interference by amphetamine-like substances. However, there are still some interfering substances that interfere with the assay. Therefore, a sample that gives a positive result on the EMIT and positive result on the AxSYM is interpreted as positive. The sample contains amphetamine. A sample that gives a positive result on the EMIT and negative result on the AxSYM is interpreted as negative.

g.

Methadone: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator B Level 1 for Methadone is interpreted as positive. The sample contains methadone.

h.

Cannabinoid: Any sample that gives a change in absorbance ( A) value equal to or higher than EMIT Calibrator/Control Level 3 for cannabinoid is interpreted as positive. The sample contains cannabinoid.

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i.

REVISION: H-7

Page 13 of 25

Oxycodone: Any sample that gives a change in absorbance (A) value equal to or higher than Oxycodone Calibrator 100 ng/mL for oxycodone (i.e. zero or higher) is interpreted as positive. The sample contains oxycodone and/or oxymorphone.

The instrument output is a number, which reflects the rate of NADH production in the sample minus the rate of NADH production in the cutoff calibrator. Thus only specimens with higher drug concentrations than the calibrator will have a result which is a positive number. 2.

Repeat and confirm all positive results. Modular DPP is programmed to automatically rerun positive results of 1 and above. If the repeat comes out negative, run the sample again. If the 3rd result comes out negative again, report the test as negative.

3.

Negative Results: a.

Any sample that gives a change in absorbance ( A) value lower than EMIT Calibrator A Level 1 for Cocaine, Opiate, Urine Barbiturate, Amphetamine, and Benzodiazepine, EMITCalibrator B Level 1 for Methadone, DRI Multidrug Calibrator 2 for PCP, EMIT Calibrator/Control Level 3 for Cannabinoid, and Oxycodone Calibrator 100 ng/mL for Oxycodone is interpreted as negative. Either the sample does not contain the drug being assayed or drug is present in concentration below the cutoff level for this assay. (1)

NOTE: Any result that has a LIMIT 0, 1, or 2 flag is not acceptable and must be repeated on a manually diluted sample. The LIMIT 0, 1, or 2 flag means that the urine has a very high background absorbance (over 20,000 milliabsorbance units). This may be due to an interfering substance which can give a false negative result. (a)

(b)

Check absorbance by going to WORK PLACE, DATA REVIEW, highlight the Accession #, REACTION MONITOR, choose the TEST, touch the global button PRINT, choose REACTION MONITOR, then PRINT. The absorbance during the reaction (look at cycle 7 absorbance) must be less than or equal to 20,000. If the absorbance is >20,000 make a 1:2 dilution into a labelled 13 x 100 mm polystyrene test tube. i) Using the 200 uL MLA pipette dilute 200 uL of Type I water and 200 uL of sample. ii) Mix iii) Transfer to labeled Hitachi cup.

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REVISION: H-7

iv) (c)

(d)

(e)

(2)

Page 14 of 25

Repeat assay

If the absorbance of diluted sample (1:2) is <20,000, the result can be reported. Do not multiply the result by the dilution factor. This is a "qualitative test", only the rate is needed. If the absorbance of diluted sample is still >20,000 milliabsorbance units make a 1:5 dilution into a 13 x 100 mm polystyrene test tube. i) Dilute 800 uL (use 1000 uL pipetman) of Type I water with 200 uL (use 200 uL MLA Pipette) of sample. ii) Mix iii) Transfer to labeled Hitachi cup. iv) Repeat assay If the absorbance of diluted sample (1:5) is <20,000, the result can be reported. Do not multiply the result by the dilution factor. This is a "qualitative test", only the rate is used.

NOTE: Any result that has a Limit L (less than technical limit) for cocaine, opiate, and pcp must be repeated on a manually diluted sample. The high negative reading may be due to interfering substances or substrate depletion. (a)

(b)

(c)

If the cause is substrate depletion make a 1:10 dilution into a labelled 13 x 100 mm polystyrene test tube. i) Dilute 900 uL of Type I water (using 1000 uL pipetman) with 100 uL of sample (using 100 uL MLA pipette). ii) Mix iii) Transfer to labelled Hitachi cup. iv) Repeat assay If the result of diluted sample (1:10) is within technical limit, the result can be reported. Do not multiply the result by the dilution factor. This is a qualitative test, only the rate is needed. If the result of diluted sample (1:10) is still less than technical limit (Limit L) repeat on 1:20 dilution. i) Dilute 950 uL of Type I water (using 1000 uL pipetman) with 50 uL of sample (using 5-50uL Finnpipette). ii) Mix. iii) Transfer to labeled Hitachi cup. iv) Repeat assay.

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(d)

B.

REVISION: H-7

Page 15 of 25

If the result of diluted sample (1:20) is within technical limit, the result can be reported. Do not multiply the result by the dilution factor. This is a qualitative test, only the rate is needed.

Confirmation of Results: 1.

Repeat all positive results. Connecticut law requires that confirmation of positive drugs of abuse results be performed by a second, independent method before they may be officially reported. They must be confirmed by thin layer chromatography (TLC). Confirmation by gas chromatography mass spectroscopy (GC/MS) is also acceptable. Confirmation of positive results are required if the results are used for non medical purposes.

2.

Samples from YNHPH that have positive results are reported as positive with a comment "This result is positive by a screening method and is unconfirmed. False positives are known to occur by this screening method. Result is provided for medical management purposes only" (comment #480). These samples are not confirmed by TLC or GC/MS unless specifically requested. All results (both positive and negative) on YNHPH samples are final and are entered in the computer by typing DAUSCR. A comment indicating that the positive result is an unconfirmed result is automatically appended once you enter the result as positive. After direct entering the results, generate YNHPH green sheets by YPIREPT. Initial all copies. Save the office copy in the Autochem area . Give the physician’s copy and medical record copy to office. They will mail the reports.

3.

With patients for whom information is urgently needed for medical management, unconfirmed EMIT positives may be reported by telephone as follows: For those greater than zero, report as "provisionally positive, with confirmation pending". This result should not be used for non-medical purposes." A hard copy may be requested by the Emergency Department on patients that need to be transported to another facility. NOTE: Put report in envelope marked confidential. Someone from ED must come to pick it up. It must not be sent through the pneumatic tube system or FAX. Negative results are final and may be reported, verbally as "negative".

NOTE: Call all Emergency Department results that request only EMIT tests to Physician, PA, or nurse practitioner. Call positive Urine Tox Screen results to all floors (Physician, PA, or nurse practitioner) with the exception of YNHPH and outpatient clinics. 4.

Results affecting immediate patient management may be released as described above by telephone to a licensed physician caring for the patient. Before releasing results over the phone, you must confirm that you are speaking to a physician. Ask for the physician's name and pager number and indicate you will call the physician after retrieving the result. If you are given a phone number, confirm the appropriateness of the number if it is not a hospital number by checking the telephone book. Results

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REVISION: H-7

Page 16 of 25

reported to a physician outside the hospital may only be given to a number listed for that physician. All telephone requests for complete urine toxicology results may only be reported by a technologist trained in the urine toxicology procedure, supervisor, resident or attending physician. A patient unit number is always required before results may be released.

VI.

5.

To protect patient rights, non-confirmed results cannot be reported into the permanent record until confirmation is complete. Confirmed results may be reported only by means of the special report entered into the patient chart. They may not be entered into the computer.

6.

Results which are not confirmed by TLC should be tested by GC/MS.

7.

Results negative by GC/MS should be reported as "negative" without comment in all cases.

QUALITY CONTROL: Run the positive and negative controls once a shift. Using the MLA pipettor, transfer 300 uL of the negative and positive controls. Do not use the soft plastic transfer pipettes in transferring any of the controls. If these controls are outside their established range, get fresh vials and rerun the controls. If problem persists, do a BLANK calibration, then rerun the controls again. Notify the supervisor if the controls are still outside the established range.

VII.

SPECIAL NOTES: A.

Special Notes about the assays: 1.

These assays are designed for use with human urine.

2.

Adulteration of the urine sample may cause erroneous results. If adulteration is suspected, obtain another sample or use an alternate method (e.g., thin layer chromatography) to confirm results. Always check the pH of specimens suspected of adulteration. Normal urine pH is 5.0 - 8.0.

3.

Cross reactions do occur, especially with opiates, in the presence of certain medications. A weak positive that cannot be confirmed by thin layer chromatography may fall in this category. Even after hydrolysis, a specimen can be negative for morphine, yet the EMIT is consistently weakly positive. A detailed history of patient medication can clear this up.

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4.

5.

REVISION: H-7

Page 17 of 25

Opiates (Morphine): a.

A positive result from the assay indicates the presence of opiates but does not indicate or measure intoxication.

b.

Oxycodone (found in Percodan) is an opiate and high concentrations of this drug will cause a positive EMIT result, but cannot be seen by thin layer chromatography.

c.

This assay also detects high concentrations of the analgesic meperidine (Demerol) and the narcotic antagonist nalorphine.

d.

Floxin (ofloxacin) can interfere with the assay at concentrations greater than 226 ug/mL.

e.

Poppy seeds can contain opiates, and ingestion of products containing poppy seeds can cause a positive test result.

f.

EMIT Opiate positive but TLC negative specimens should be tested by GC/MS.

Phencyclidine: Various potentially interfering substances were tested by Micorgenics for cross-reactivity in the DRI Phencyclidine assay. The following compounds produced a negative result at the concentration tested:

COMPOUND

CONCENTRATION TESTED (ng/mL)

Acetaminophen Acetylsalicylic acid Amobarbital Amphetamine Brompheniramine Chlorpheniramine Dextromethorphan Diphenhydramine EMDP Imipramine Ketamine Meperidine Methaqualone Methadone Metronidazole Morphine Naltrexone Orphenadrine

1,000,000 1,000,000 1,000,000 1,000,000 50,000 50,000 1,000,000 100,000 100,000 500,000 100,000 50,000 100,000 1,000,000 1,000,000 200,000 10,000 200,000

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REVISION: H-7

Oxazepam Phenobarbital 1-Phenylcyclohexylamine 1-Piperidinocyclohexane Carbonitrile (PCC) Promethazine Thiroadazine Triprolidine 6.

7.

8.

Page 18 of 25

100,000 1,000,000 50,000 100,000 100,000 80,000 10,000

Urine Barbiturate: a.

The EMIT d.a.u. Barbiturate assay tests for both long and short acting barbiturates in human urine.

b.

A positive result from the assay indicates the presence of barbiturates but does not indicate or measure intoxication

c.

In rare instances, samples from patients on phenytoin therapy may give elevated negative rates or false positive results.

Methadone a.

Methadone is a synthetic narcotic/analgesic drug that is administered orally or intravenously. It is frequently used in maintenance programs as a substitute for heroin or other abused opioids while allowing the subject to successfully participate in drug rehabilitation.

b.

A positive result from the assay indicates the presence of methadone but does not indicate or measure intoxication

c.

The EMIT d.a.u. Methadone assay detects high concentration of the antihistamine doxylamine and gives a false positive result.

d.

This assay does not detect the metabolites of the long acting form of methadone, 1-α- acetylmethadol (LAAM), in concentrations that would be found in the urine of patients of LAAM therapy.

Amphetamine a.

Amphetamines appear in the urine within 3 hours after any type of administration and can be detected by EMIT assay for as long as 24 to 48 hours after the last dose.

b.

The EMIT d.a.u. monoclonal amphetamine/methamphetamine detects d-amphetamine, d,l-amphetamine, d-methamphetamine,

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REVISION: H-7

Page 19 of 25

methylenedioxyamphetamine (MDA), and methyl-enedioxymethamphetamine (MDMA) in human urine. c.

9.

10.

Therapeutic doses of the following drugs may produce results with this assay: chloroquine (Aralen), chlorpromazine (Thorazine), methoxyphenamine, quinicrine, phentermine, ranitidine (Zantac), Procainamide and its metabolite N-acetylprocainamide (NAPA). Because benzphetamine (Didrex) metabolizes to amphetamine and methamphetamine, therapeutic doses of this drug may also produce a positive result.

Benzodiazepine a.

The EMIT d.a.u. Benzodiazepine assay detects benzodiazepine and benzodiazepine metabolites in human urine.

b.

Therapeutic doses of oxaprozin (DAYPRO), a non-benzodiazepine may produce positive result with this assay. A positive result from an individual taking oxaprozin should be interpreted with caution and confirmed by another method.

Cannabinoid a.

The cannabinoid ∆ 9 –tetrahydrocannabinol (∆ 9 -THC) is the principal psychoactive ingredient in marijuana and hashish. The compound ∆ 9 -THC is quickly and effectively absorbed by inhalation or from the gastrointestinal tract and is almost completely metabolized by liver enzymes. Peak plasma levels of ∆ 9 –THC occur within 10 minutes of inhalation and approximately 1 hour after ingestion. Excretion of urinary metabolites and excretion by way of the feces begins within 72 hours after exposure.

b.

The EMIT II Plus Cannabinoid assay detects the major metabolite of ∆ 9 – THC, 11-nor- ∆ 9 -THC-9-carboxylic acid in human urine. It also detects other ∆ 9 –THC metabolites.

c.

Passive inhalation of marijuana smoke may produce positive results with low cutoff cannabinoid assays. Urine specimens from nonsmokers can test positive for cannabinoid metabolites, but only after exposure to high concentrations of marijuana smoke in a small unventilated area.

d.

A positive result from the assay indicates the presence of cannabinoids but does not indicate or measure intoxication.

TITLE:

DEPT. OF LAB MEDICINE CLINICAL CHEMISTRY


DOCUMENT NO: CC-110013 EFFECTIVE DATE: November 1, 2004

11.

REVISION: H-7

Page 20 of 25

Oxycodone:

Oxycodone is a semi-synthetic opioid prescribed for pain management on patients with moderate to severe pain. It is similar to codeine and morphine in its analgesic properties, but is more potent than morphine and has higher dependence potential. The drug oxycodone is supplied as OxyContin (oxycodone HCl) or in combination with aspirin (Percocet) or acetaminophen (Percodan). Drug abusers crush the pills into a powder and snort them for faster effect resulting in fatal outcome. According to Drug Abuse Warning Network (DAWN), there has been a dramatic increase in oxycodonerelated deaths. Oxymorphone and noroxycodone are the known metabolites of oxycodone. The metabolite, oxymorphone, is a potent narcotic analgesic, while the other metabolite, noroxycodone, is relatively inactive. From 33-61% of a single dose of oxycodone is excreted in urine within 24 hours as unconjugated oxycodone (13-19%), conjugated oxycodone (7-29%), and unconjugated oxymorphone (13-14%). B.

Minimum Detection Limit:

For Syva EMIT assay on the Modular P: generic name

common name

metabolite

sensitivity of method

cocaine*

coke, crack

Benzoylecgonine

300 ng/mL

phencyclidine

pcp, angel dust

N/A

25 ng/mL

glucuronides

300 ng/mL

opiates

*The antibodies essentially recognize the metabolite; the unchanged parent drug may go unrecognized unless present in extremely high concentrations or has undergone "natural" hydrolysis in part to the metabolite. 1.

Minimum detection limit of Opiate Compounds: Codeine Hydrocodone Hydromorphone Meperidine Morphine Morphine-3-Glucuronide Oxycodone

2.

12.5 ng/mL 400 ng/mL 700 ng/mL 160,000 ng/mL 300 ng/mL 1050 ng/mL 26,000 ng/mL

Minimum detection limit of Benzodiazepine Compounds: Oxazepam alprazolam Bromazepam

200 ng/mL 50 ng/mL 400 ng/mL

TITLE:




Chlordiazepoxide Clobazam Clonazepam Clotiazepam Demoxepam N-Desalkylflurazepam Diazepam Flunitrazepam Flurazepam Halazepam Lorazepam Lormetazepam Medazepam Midazolam Nitrazepam Norchlordiazepoxide Nordiazepam Prazepam Temazepam Tetrazepam Triazolam 3.

REVISION: H-7

500 ng/mL 100 ng/mL 500 ng/mL 100 ng/mL 900 ng/mL 100 ng/mL 40 ng/mL 100 ng/mL 100 ng/mL 80 ng/mL 1000 ng/mL 200 ng/mL 100 ng/mL 120 ng/mL 200 ng/mL 1800 ng/mL 60 ng/mL 80 ng/mL 70 ng/mL 100 ng/mL 70 ng/mL

Minimum detection limit of Amphetamine Compounds < 400 ng/mL d-Amphetamine d,l-Amphetamine 1000 ng/mL d- Methamphetamine 1000 ng/mL Methylenedioxyamphetamine (MDA) 1000 ng/mL Methylenedioxymethamphetamine (MDMA) 3000 ng/mL

4.

Minimum detection limit of Barbiturate Compounds Alphenal Amobarbital Aprobarbital Barbital Butabarbital Butalbital Cyclopentobarbital Pentobarbitol Phenobarbital Secobarbital Talbutal Thiopental

300 ng/mL 300 ng/mL 180 ng/mL 1000 ng/mL 300 ng/mL 150 ng/mL 200 ng/mL 300 ng/mL 700 ng/mL 200 ng/mL 200 ng/mL 10000 ng/mL

Page 21 of 25

TITLE:




5.

REVISION: H-7

Page 22 of 25

Minimum Detection Limit of Oxycodone Compounds: a.

The specificity of the assay was evaluated by Microgenics by testing parent drug and its metabolites using the 300 ng/mL cut off. The following compounds produced a positive result in the assay at the concentrations listed below:

Oxycodone Oxymorphone Noroxycodone Noroxymorphone b.

300 ng/mL 300 ng/mL 500 µg/mL 100 µg/mL

Various opiate compounds were tested by Microgenics using the 300 ng/mL cutoff. The following compounds produced a negative result at the concentrations listed below:

6-Acetyl morphine Codeine Dihydrocodeine Hydrocodone Hydromorphone Levorphanol Morphine-3-glucoronide Morphine

500 µg/mL 1250 µg/mL 500 µg/mL 150 µg/mL 100 µg/mL 750 µg/mL 500 µg/mL 1000 µg/mL

Non-Opiate compounds Naltrexone Ranitidine C.

3000 µg/mL 3500 µg/mL

Calibration and loading patient sample: 1.

A Blank calibration is performed at least once a week using EMIT calibrator A Level 1, which contains a stated concentration of 300 ng/mL benzoylecgonine for cocaine, 300 ng/mL morphine for opiates, 200 ng/mL secobarb for Urine Barbiturate, 200 ng/mL Oxazepam for Benzodiazepine, 1000 ng/mL d-Metamphetamine for Amphetamine, DRI multidrug Calibrator 2 with a concentration of 25 ng/mL phencyclidine for PCP, EMIT calibrator B Level 1 with a concentration of 300 ng/mL methadone for Methadone, EMIT Calibrator/Control Level 3 with a concentration of 50 ng/mL of 11-nor- 9 -THC-9-carboxylic acid for Cannabinoid, and DRI Oxycodone calibrator 100 ng/mL for oxycodone as Std. 1. Use the MLA pipettor in transferring the cannabinoid calibrator into the sample cup. Do not use the soft plastic transfer pipettes.

TITLE:

DEPT. OF LAB MEDICINE CLINICAL CHEMISTRY


DOCUMENT NO: CC-110013 EFFECTIVE DATE: November 1, 2004

D.

REVISION: H-7

Page 23 of 25

2.

Verify calibration by running the Detectabuse Immunoassay Liquid Control Urine Series V Cutoff –25% negative control and Detectabuse Immunoassay Liquid Control Urine Series V Cutoff +25% positive control. Using the MLA pipettor, transfer 300 uL of the negative and positive controls. Do not use the soft plastic transfer pipettes in transferring any of the controls. Both controls should fall within acceptable limits. If any negative control is greater or equal to zero or any positive control less than or equal to zero, get fresh controls and run them again. If controls are still out of range, recalibrate and run the controls again. Run both positive and negative controls once a shift.

3.

Once the calibration is validated, run the patients by putting the sample in barcoded urine tubes. Using the MLA Pipetman, transfer 500 uL of the urine sample. Do not use the soft plastic transfer pipettes on any of the urine samples for cannabinoid.

Calibration Verification:

Calibration verification of this method is not required as the assay is calibrated weekly. This method is calibrated using blank calibration procedure. Controls must be within the established ranges after and between calibrations. This is assurance that the calibration is acceptable. This is a qualitative assay with performance verified by positive and negative controls. The Modular P system also verifies that the results of an assay calibration meet the specifications assigned to selected validity parameters. An error message occurs when the calibration fails to meet a specification. E.

Interferences: Hemolysis: Lipemia: analysis.

VIII.

There is no interference from hemoglobin up to 1.0 g/L (2+). All specimens with significant turbidity must be centrifuged prior to

OVERVIEW Drugs of Abuse Testing: A.

Accessioning On accessioning, Drugs of Abuse testing can be ordered in three ways: (1)

Nonstat Urine Drug Screen - Test #95 includes:

Cocaine

Emit performed on Modular P

Amphetamine

Emit performed on Modular P

TITLE:




PCP Emit performed on Modular P Opiate Emit performed on Modular P Barbiturate Emit performed on Modular P

REVISION: H-7

Page 24 of 25

Methadone Emit performed on Modular P Benzodiazepines Emit performed on Modular P Oxycodone Emit performed on Modular P

Thin-layer Chromatography, hydrolysis, or GC/MS to identify or confirm drugs (2) Individual requests for specific EMIT tests. EMIT is performed on Modular DPP. These will be accessioned by test numbers. Barbiturate, urine - #93 Amphetamine, urine - #94 Cocaine, urine - #185 Methadone, urine - #186 Benzodiazepines, urine - #190 Phencyclidine (PCP), urine - #191 Opiates, urine - #194 Oxycodone, urine - # 198 Cannabinoid, urine - #245 – When requested from other locations, it may be performed, but must be discussed with the Laboratory Medicine Resident only if the result is positive. a.

YNHPH 4 drug - cocaine, urine; opiate, urine; benzodiazepines, urine; cannabinoid, urine

b. YNHPH 8 drug - all 8 tests c. Urine Drugs of Abuse Panel - includes 8 tests (cannabinoid by special request only) (3)

CCSS

YNHPH urine samples for DAU – done by EMIT only (Modular P). It will include Cocaine, Opiate, PCP, UBarbiturate, Amphetamine, Methadone, Benzodiazepine, Cannabinoid, Oxycodone B.

Availability (1)

Requests for Individual Drugs: (a) Modular DPP Tests:

Opiates, Cocaine, PCP, UBarbiturate, Amphetamine, Methadone, Benzodiazepine, Cannabinoid, Oxycodone are available on request. Any combination of the nine will be considered as 1 (one) request.

TITLE:




(2)

REVISION: H-7

Page 25 of 25

Requests for Nonstat Urine Drug Screen/Urine Toxicology Screen:

The EMIT tests for cocaine, PCP, and Opiates will be done in a manner to ensure the results of the EMIT tests are completed before the Modular DPP goes down for maintenance and in time for the TLC run. NOTE: We have targeted a 4 hour turn-around time for the results of the EMIT tests.

IX.

X.

REFERENCES: 1.

Sung E and Neeley, W, A Cost-effective system for the performing therapeutic drug assays. I. Optimization of the theophylline assay, Clin. Chem. 31, 1210-1215 (1985).

2. 3. 4.

Syva Emit Cocaine metabolite, Opiate, and Phencyclidine Assay reagent inserts. Microgenics DRI Phencyclidine Assay reagent insert. DRI Oxycodone Assay package insert.

HISTORY: H-1

Cocaine, Opiate, PCP methods adapted to the Hitachi 911 by F. Santos, June, 1996, placed in service August, 1996.

H-2

Urine Barbiturate, Amphetamine, Benzodiazepine, Methadone, and Cannabinoid methods adapted to the Hitachi 911 by F. Santos, June, 2000, placed in service June, 2000. Procedure written by F.Santos, June, 2000.

H-3

Method adapted on the Modular DPP and Modular P by F.Santos and H.Malkus, 4/03.

H-4

Procedure revised by F.Santos, 10/03.

H-5

Oxycodone procedure written by F. Santos, March, 2004. Adapted on Modular DPP by F. Santos and H. Malkus, March, 2004. Implemented April, 2004.

H-6

Microgenics DRI PCP using cut off of 25 ng/mL evaluated on the Modular P by F. Santos, March, 2005. Implemented May, 2005.

H-7

Procedure revised by F. Santos, May, 2005.

Kenny, Bernadetta, Rachel, Jenny, Ina, Nety

Bioanalisis

FST A-2009

ANALISIS KUALITATIF UNTUK AMFETAMIN DAN BENZODIAZEPIN SECARA EMIT (ENZYME-MULTIPLIED IMMUNOASSAY TECHNIQUE)

Bioanalisi

FST A-2009

Immunoassay Merupakan uji untuk mengidentifikasi keberadaan suatu obat maupun metabolitnya dalam sampel biologis . Tujuan: Memonitor penyalahgunaan obat Memonitor terapi suatu obat pada pasien antigen

γ

antibodi

Dibaca absorbansinya

Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Immunoassay

SAMPEL yang digunakan


Bioanalisi

FST A-2009

Jenis-jenis Immunoassay Enzyme-multiplied immunoassay technique (EMIT) Radioimmunoassay (RIA) Fluorescent polarization immunoassay (FPIA) Kinetic interaction of microparticles in solution immunoassay (KIMS)

Enzyme-linked immunosorbent assay (ELISA) Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Enzyme-Multiplied Immunoassay Technique (EMIT) EMIT


Bioanalisi

FST A-2009

ENZIM Enzim glukosa-6-fosfat dehidrogenase (G6P-DH) bakteri Leuconostoc mesenteroides Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009


Bioanalisi

FST A-2009

EMIT dapat digunakan untuk mengidentifikasi: Amfetamin monoklonal/metamfetamin pada Benzodiazepin dan metabolitnya pada urin manusia.

urinDapat manusia. mendeteksi Hasil +

d- amfetamin, d,l-amfetamin, metilen-dioksi/ amfetamin (MDA), dan metilen-dioksimetamfetamin (MDMA) pada urin manusia.

Hasil +/- menggunakan larutan oxazepam dengan konsentrasi menggunakan larutan d-metamfetamin 200 konsentrasi ng/mL. dengan 1000 ng/mL Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Komponen yang digunakan dalam metode pengujian EMIT Komponen yang digunakan dalam metode pengujian EMIT  Obat  Antibodi  Substrat  Enzim


Bioanalisi

FST A-2009


Bioanalisi

FST A-2009

Preparasi Sampel:

Sampel : urin

Rainin pipettor

Pemberian label

Sampel dijaga pada pH 5-8 (pH normal manusia)  dengan cara menambahkan NaOH 1M ataupun HCl 1M

Senyawa Uji :

amfetamin dan benzodiazepin Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Reagen: Reagen Amfetamine/Methamphetamine:  Monoklonal Amphetamine/ Methamphetamine Working reagen A  Monoclonal Amphetamine/ Methamphetamine Working reagent B Reagen Benzodiazepine:  Benzodiazepine Working reagent A  Benzodiazepine Working reagent B


Bioanalisi

Catatan khusus:

FST A-2009

1. Amfetamin a. Amfetamin dalam urin muncul dalam waktu 3 jam dan dapat dideteksi dengan uji EMIT selama 24 sampai 48 jam b. Uji EMIT d.a.u. untuk amfetamin/metamfetamin monoklonal mendeteksi adanya damfetamin; d,l-amfetamin, d-metamfetamin, methylenedioxyamphetamine (MDA), dan metil-enedioxy methamphetamine (MDMA) dalam urin manusia. c. Dosis terapi dari obat-obat berikut dapat menghasilkan hasil dengan tes ini: klorokuin (Aralen), klorpromazin (Thorazine), methoxyphenamine, quinicrine, ranitidine (Zantac), Procainamide dan metabolitnya N-acetylprocainamide (NAPA). Karena benzphetamine (Didrex) dimetabolisme menjadi amphetamine dan methamphetamine, dosis terapeutik obat ini juga dapat menghasilkan hasil yang positif. 2. Benzodiazepin a. Uji EMIT d.a.u. untuk benzodiazepin mendeteksi metabolit benzodiazepin dan benzodiazepin dalam urin manusia. b. Dosis terapi oxaprozin (DAYPRO), obat non-benzodiazepin dapat memberikan hasil yang positif dengan tes ini. Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Batas deteksi minimum senyawa Amfetamin: d-Amphetamine < 400 ng/mL d,l-Amphetamine 1000 ng/mL d- Methamphetamine 1000 ng/mL

Methylenedioxyamphetamine (MDA) 1000 ng/mL Methylenedioxymethamphetamine (MDMA) 3000 ng/mL Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Batas deteksi minimum senyawa Benzodiazepin: Oxazepam 200 ng/mL

Flurazepam 100 ng/mL

Alprazolam 50 ng/mL

Halazepam 80 ng/mL

Bromazepam 400 ng/mL

Lorazepam 1000 ng/mL

Chlordiazepoxide 500 ng/mL

Lormetazepam 200 ng/mL

Clobazam 100 ng/mL

Medazepam 100 ng/mL

Clonazepam 500 ng/mL

Midazolam 120 ng/mL

Clotiazepam 100 ng/mL

Nitrazepam 200 ng/mL

Demoxepam 900 ng/mL

Norchlordiazepoxide 1800 ng/mL

N-Desalkylflurazepam 100 ng/mL

Nordiazepam 60 ng/mL

Diazepam 40 ng/mL

Prazepam 80 ng/mL

Flunitrazepam 100 ng/mL Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Hasil positif/ negatif: Kalibrator: kalibrator EMIT A level 1  untuk membedakan hasil +/- dari sampel.  Benzodiazepin  200 ng/ mL oxazepam  Amfetamin  1000 ng/ mL d-methamphetamine Sampel

Standar

Hasil

Benzodiazepin

>

Oxazepam 200 ng/ mL

Positif

Amfetamin

>

Amfetamin 1000 ng/ mL

Positif


Bioanalisi

Kalibrasi dan pengisian sampel pasien

FST A-2009

Kalibrasi Blanko dilakukan setidaknya sekali seminggu dengan menggunakan kalibrator EMIT Level 1 Verifikasi kalibrasi dengan menjalankan Immunoassay Cair Kontrol Urin Detectabuse Seri V cutoff kontrol negatif 25% dan Detectabuse Immunoassay Kontrol Urin Cair Seri V cutoff kontrol positif +25%.

Transfer 300 μL dari kontrol negatif dan positif dengan menggunakan pipettor MLA. Dalam mentransfer salah satu kontrol jangan menggunakan pipet plastik yang lunak Kedua kontrol harus jatuh dalam batas yang dapat diterima. Jika ada kontrol negatif > 0 atau kontrol positif < nol, dapatkan kontrol yang terbaru kemudian dijalankan kembali. Jika kontrol masih di luar jangkauan, kalibrasi ulang dan jalankan kontrol lagi. Jalankan baik kontrol positif dan negatif sekali pergeseran. Setelah kalibrasi divalidasi, jalankan perlakuan dengan menempatkan sampel urin dalam tabung barcode. Transfer 500 μL dari sampel urin menggunakan MLA. and humanistic values Integrating Pipetman academic excellence

Bioanalisi

Verifikasi Kalibrasi

FST A-2009

 





Prosedur: kalibrasi blanko Kontrol harus berada dalam rentang kalibrasi  sebagai jaminan bahwa kalibrasi dapat diterima. Hal ini merupakan tes kualitatif dengan kinerja yang diverifikasi oleh kontrol positif dan negatif. Pesan kesalahan terjadi ketika kalibrasi gagal memenuhi spesifikasi. Gangguan Hemolisis: Tidak ada gangguan dari hemoglobin sampai dengan 1,0 g/L (2 +). Lipemia: Semua spesimen yang keruh harus disentrifugasi sebelum analisis. Integrating academic excellence and humanistic values

Bioanalisi

FST A-2009

Kesimpulan


Bioanalisi

FST A-2009


Bioanalisis EMIT Kualitatif Amfetamin dan Benzodiazepin

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