DOSE-RESPONSE OF PORCINE OVARIAN GRANULOSA CELLS TO AMYGDALIN TREATMENT COMBINED WITH DEOXYNIVALENOL Marek Halenár*, Marína Medveďová, Nora Maruniaková, Dagmara Packová, Adriana Kolesárová Address(es): Ing. Marek Haklenár, Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic. *Corresponding author: [email protected]
Pocd.co.nzPoint of Care Diagnostics
DIPSCAN 10 PLUS
with extra opiates
(AMP, BUP, BZD, COC, MET, MTD, OPI, OXY, TRA, THC)
Multi Drug Screen teSt
Dipscan 10 Plus is an immunochromatography based one step in vitro The appearance of a line next to the Control "C" and the absence of a line
test. It is designed for qualitative determination of drug substances in human next to the Test "T" indicates a positive result. This is an indication the urine specimens. This assay may be used in the point of care setting. Below level of tested drug(s) in the specimen is above the cut-off level.
is a list of drug groups and their cut-off concentrations used in this test:- The absence of a line in the Control "C" arean is an invalid result. Retest AMP Amphetamines 300 ng/ml of d-amphetamine the sample with a new device. Follow the test procedure closely. BZD Benzodiazepines 200 ng/ml of oxazepam BUP Buprenorphine 10 ng/ml of Buprenorphine-3-β-d-glucoronide 300 ng/ml of benzoylecgonine 300 ng/ml of methadone MET Methamphetamines 300 ng/ml of (+)methamphetamine Each component strip of Dipscan 10 Plus is based on the principle of
specific immunochemical reaction between antibodies and antigen to 300 ng/ml of morphine analyze particular compound in human urine specimen. The assay relies 100 ng/ml of oxycodone on the competition for binding antibody. When drug is present in the THC Cannabinoid (THC) 50 ng/ml of 11-nor-9-THC-9-COOH urine specimen, it competes with drug conjugate for the limited amount of 200 ng/ml of Tramadol antibody-dye conjugate. When the amount of drug is equal or more than the cut-off, it will prevent the binding of drug conjugate to the antibody. NB. Dipscan 10 Plus is set to the AS/NZS 4308 Australian/New Zealand
Therefore, a positive urine specimen will not show a coloured band on the Standards cut-off levels for AMP, BZD, COC, MET, OPI, THC. Cut-off test line zone, indicating a positive result, while the presence of a coloured levels for BUP, MTD, OXY, TRA are not specified in the Standards. band indicates a negative result.
A control line is present in the test window to work as procedural control. 1. Collect at least 50ml of urine. This coloured band should always appear on the control line zone if the test device is stored in good condition and the test is performed appropriately. 2. Remove the test card from sealed foil pouch.
The Control line serves to validate the results. 3. Remove the protective cap and place the revealed strips into the urine STORAGE AND STABILITY
4. Hold there until the urine is seen to visibly migrate up each of the ten test The test device should be stored at 2 to 30oC and will be effective until the windows. Do not allow the urine to touch the plastic section of the device. expiration date stated on the package. The product is humidity-sensitive and should be used immediately after being open. 5. Remove from the urine, replace the cap and place the test on a flat dry 6. Read the results at 5-8 minutes after adding the sample.
For in vitro diagnostic and forensic use only.
Do not interpret the result after 10 minutes. Do not use the product beyond the expiration date.
Handle all specimens as potentially infectious.
INTERPRETATION OF RESULTS
Do not open foil pouch until it is ready to be tested.
Use a new urine specimen cup for each sample to avoid cross The appearance of a line next to the Control "C" and Test "T" indicates a negative result. The negative result does not indicate the absence of drug SPECIMEN COLLECTION AND PREPARATION
in the specimen; it only indicates the level of tested drug in the specimen is Samples greater than 50ml are required for this test. Fresh urine does not less than the cut-off level. Please note that the colour intensity of Test "T" require any special handling or pretreatment. Specimen should be collected lines can vary.
in a clean, dry, plastic or glass container. If the assay is not performed Free Call Aust : 1800 640 075
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Point of Care Diagnostics
immediately, urine specimen may be refrigerated at 2-8 °C or frozen up to systems to produce respiratory and circulatory depression. Methadone 7 days. Specimens should be brought to room temperature before testing. also produces miosis and increases the tone of smooth muscle in the lower Urine specimens exhibiting a large amount of precipitate or turbidity should gastrointestinal tract while decreasing the amplitude of contractions. Acute be centrifuged or allowed to settle before testing. higher doses induce analgesia, sedation, respiratory depression and coma. After methadone administration, the major urinary excretion products are methadone and its metabolites, EDDP and EMDP. Large individual The control band is an internal reagent and procedural control. It will appear variations in the urine excretion of methadone are output of methadone in each of the test's ten windows if the test has been performed correctly and from 5-22%. Typically, following a 5 mg oral dose, methadone and EDDP the reagents are reactive. account for 5% of the dose in the 24-hour urine. In those individuals on Control standards can be used to validate reagent performance and maintenance therapy, methadone may account for 5 to 50% of the dose in the establish test reliability. Controls, which are not provided with this test, are 24-hour urine and EDDP may account for 3 to 25% of the dose.
commercially available. DRUG GROUPS TESTED:-
Methamphetamine is the most popular synthetic derivative of the amphetamines. It is a potent sympathomimetic agent with therapeutic Amphetamines are a class of potent sympathominetic agents with therapeutic applications. Acute large doses lead to enhanced stimulation of the central applications. The most common amphetamines are d-amphetamine and d,l- nervous system and induce euphoria, alertness, reduced appetite, and a sense amphetamine. Amphetamines are central nervous stimulants that cause the of increased energy and power. More acute response produces anxiety, neutrotransmitters epinephrine, norepinephrine and dopamine to be released paranoia, psychotic behavior, and cardiac dysrhythmias. Methamphetamine into the brain and body giving users feelings of euphoria, alertness, and is excreted in the urine as amphetamine and oxized and deaminated increased energy. Chronic abuse of amphetamines leads to tolerance and drug derivatives. However, 10-40% of methamphetamine is excreted unchanged. reinforcement effect. Cardiovascular responses to amphetamine include increased Methamphetamine is generally detectable in the urine for 3 to 5 days after blood pressure and cardiac arrhythmias. More acute responses produce anxiety, paranoia, hallucinations and psychotic behavior. Amphetamines are metabolised Opiate (OPI)
by a number of pathways. In general, acid urine promotes excretion whereas alkaline urine retards it. In 24 hours, approximately 79% of the amphetamine Opioid analgesics comprised of a large group of substances that control pain dose is excreted in acid urine and about 45% in alkaline urine. Typically, about by depressing the central nervous system. Acute high dose used by abusers 20% is excreted as unchanged amphetamine. Unchanged amphetamine can be or addicts can cause depressed coordination, disrupted decision, decreased detected up to 10 days after use.
respiration, hypothermia and coma. Morphine is excreted unmetabolized and is the marker metabolic product of opiates. Morphine and morphine glucuronide is detectable in urine for several days after opiates dose.
Benzodiazepines are a class of widely prescribed central nervous system depressants which have anxiolytic, hypnotic, anticonvulsant and muscle relaxant effects. Chronic abuse can result in addiction and tardive Oxycodone is known as Oxycontin, Roxicodone and is an ingredient of dyskinnesia. Acute higher doses lead to drowsiness, dizziness, muscle Percodan, Percocet, Roxicet and Tylox. Oxycodone is a semi-synthetic relaxation, lethargy, coma and possible death. The effects of benzodiazepines opiate derived from opium. Like other opiates, oxycodone is characterized use last 4 – 8 hours. Many of the benzodiazepines share a common metabolic by its analegestic properties, and the tendency for users to form a physical route, and are excreted as oxazepam and its glucuronide in urine. Oxazepam dependency and develop tolerance with extended use. Oxycodone is is detectable in the urine for up to 7 days after drug use.
usually administered in combination with non-opiate analegesics such as acetaminophen and salicylates for the relief of moderate to severe pain. Oxycodone is a central nervous system depressant that may cause drowsiness, A derivative of thebaine, buprenorphine is an opioid that resembles dizziness, lethargy, weakness and confusion. Toxicity in an overdose of morphine structurally but has a longer duration of action than morphine oxycodone can lead to stupor, coma, muscle flaccidity, severe respiratory and can be administrated sublingually as an analgesic. In October 2002, depression, hypotension, and stripiac arrest. Oxycodone is metabolized by FDA approved the use of a buprenorphine monotherapy product, Subutex, N- and O-demethylation. One of the metabolites, oxymorphone, is a potent and a buprenorphine/naloxone combination product, Suboxone, for the narcotic analgesic, while the other, noroxycodone, is relatively inactive. treatment of opioid addiction. It has been shown that buprenorphine has Between 33 to 61% of a single dose of oxycodone is excreted in a 24 hour abuse potential and may itself cause dependency. In addition, a number urine collection and consists of 13-19% free oxycodone, 7-29% glucuronide of deaths have been recorded as a result of overdose with intravenously conjugated oxycodone, 13-14% glucuronide conjugated oxymorphone injected buprenorphine in conjunction with other psychotropic drugs such as and an unknown amount of noroxycodone. The detection time window of benzodiazepines. Buprenorphine is metabolized primarily by n-dealkylation oxycodone is 1-3 days following use.
to form glucuronide-buprenorphine and glucuronide-norbuprenorphine.
Marijuana (THC )
The agents of Marijuana that cause various biological effects in humans are Derived from the leaves of cocoa plant, cocaine is a potent central nervous called cannabinoid. Cannabinoid is a central nervous stimulant that alters system stimulant as well as a local anesthetic. Some of the psychological mood and sensory perceptions, produces loss of coordination, impairs short effects induced by cocaine are: euphoria, confidence and a sense of increased term memory, and produces symptoms of anxiety, paranoia, depression, energy, accompanied by increased heart rate, dilation of the pupils, fever, confusion, hallucination, and increased heart rate. Large doses of cannabinoid tremors and sweating. Continued ingestion of cocaine could induce could cause the development of tolerances and physiological dependency tolerances and physiological dependency which leads to its abuse. Cocaine is and lead to abuse. A tolerance to the cardiac and psychotropic effects can used by smoking, intravenous, intranasal or oral administration and excreted occur and withdrawal syndrome produces restlessness, insomnia, anorexia in the urine primarily as benzoylecgonine in a short period. Benzoylecgonine and nausea. Δ9-THC is the primary active ingredient in cannabinoids. The has a biological half-life of 5 – 8 hours, which is much longer than that of main metabolite excreted in the urine is 11-nor- Δ 9-THC-9-COOH, which cocaine (0.5 – 1.5 hours), and can be generally detected for 12 – 72 hours are found within hours of exposure and remain detectable in the urine for after cocaine use or exposure. 10-30 days after smoking, longer for chronic users.
Methadone is a synthetic opioid, clinically available. It is used clinically for Tramadol is a quasi-narcotic analgesic used in the treatment of moderate the treatment of severe pain and in maintenance programs for morphine and to severe pain. It is a synthetic analog of codeine, but has a low binding heroine addicts. Methadone acts on the central nervous and cardiovascular affinity to the mu-opioid receptors. Large doses of tramadol can develop tolerance and physiological dependency and lead to its abuse. Tramadol is GC/MS at a cut-off of 300 ng/ml of benzoylecgonine. Eighty one (81) urine extensively metabolized after oral administration. Approximately 30% of the specimens with GC/MS confirmed benzoylecgonine concentration were dose is excreted in the urine as unchanged drug, whereas 60% is excreted evaluated in this study. The results are summarized and presented below: as metabolites. The major pathways appear to be N- and O- demethylation, Positive % agreement: 94, Negative % agreement: 100 glucoronidation or sulfation in the liver. Two specimens were found discrepant between the RapidCOC and GC/MS LIMITATION OF PROCEDURE
method. When compared those data, 100% (2 out of 2 ) of the discrepancy The assay is designed for use with human urine only. A positive result specimens were found between –25% and +25% cut-off concentration (225 with any of the tests indicates only the presence of a drug/metabolite and does not indicate or measure intoxication. There is a possibility that technical or procedural error as well other substances in certain foods and 5. Methadone The accuracy of the methadone test was evaluated in
medicines may interfere with the test and cause false results. Please refer comparison to GC/MS at a cut-off of 300 ng/ml of methadone. One hundred "SPECIFICITY" section for lists of substances that will produce either and nineteen urine specimens with confirmed methadone concentrations positive results, or that do not interfere with test performance. If a drug/ were evaluated in this study. The results are summarized and presented metabolite is found present in the urine specimen, the assay does not indicate frequency of drug use or distinguish between drug of abuse and Positive % agreement: 98.3, Negative % agreement: 98.3.
certain foods and medicines.
Two specimens were found discrepant between the RapidMTD and GC/MS method. When compared those data, 100% (2 out of 2) of the discrepancy Dipscan 10 Plus is a qualitative assay. It identifies the drug(s) in human
specimens were found between –25% and +25% cut-off concentration (225 urine at its cut-off concentration or higher. The concentration of the drug(s) can not be determined by this assay. The test is intended to distinguish 6. Methamphetamine The accuracy of the methamphetamine test
negative result from presumptive positive result. All positive results must be was evaluated in comparison to GC/MS at a cut-off of 300 ng/ml of (+) confirmed using an alternate method, preferably GC/MS as outlined in the methamphetamine. Eighty (80) urine specimens with GC/MS confirmed (+) AS/NZS 4308 Standards.
methamphetamine concentration were evaluated in this study. The results are summarized and presented below: A. Accuracy
Positive % agreement: 95, Negative % agreement: 100 The accuracy of the Dipscan 10 Plus were evaluated in each component
Two specimens were found discrepant between the RapidMET and GC/MS strip and in comparison to GC/MS method at the following concentration: method. When compared those data, 100% (2 out of 2 ) of the discrepancy d-amphetamine 300ng/ml (AMP), oxazepam, 200 ng/ml (BZD), specimens were found between –25% and cut-off concentration (225-375). buprenorphine-3-β-d-glucoronide 10ng/ml (BUP), benzoylecgonine 300ng/ ml (COC), methadone 300 ng/ml (MTD), (+)methamphetamine 300ng/ml 7. Opiate The accuracy of the opiates test was evaluated in comparison
(MET), morphine 300 ng/ml (OPI), oxycodone 100ng/ml (OXY), 11-nor- to GC/MS at a cut-off of 300 ng/ml of morphine. One hundred and twenty Δ9-THC-9-COOH 50ng/ml (THC), Tramadol 200 ng/ml (TRA). The results three urine specimens with GC/MS confirmed morphine and codeine of each component strip are listed below: concentrations were evaluated in this study. The results are summarized and 1. Amphetamine The accuracy of the amphetamine test was evaluated in
comparison to GC/MS method at a cut-off of 300 ng/ml. Eighty one (81) Positive % agreement: 97.4, Negative % agreement: 91.3 urine specimens with GC/MS confirmed d-amphetamine concentration were Six specimens were found discrepant between the RapidOPI and GC/MS evaluated in this study. The results are summarized and presented below: method. When compared those data, 50% (3 out of 6 ) of the discrepancy Positive % agreement: 92, Negative % agreement: 98 specimens were found between –25% and +25% cut-off concentration Four specimens were found discrepant between the RapidAMP and the ( 225 – 375 ng/ml ). GC/MS method. When compared those data, 50% (2 out of 4) of the 8. Oxycodone The accuracy of the oxycodone test was evaluated in
discrepancy specimens were found between +25% to –25% of cutoff comparison to GC/MS method at a cut-off of 100 ng/ml. One hundred and forty urine specimens with GC/MS confirmed oxycodone concentration were 2. Benzodiazepine The accuracy of the benzodiazepine test was evaluated
evaluated in this study. The results are summarized and presented below: in comparison to GC/MS at a cut-off of 200 ng/ml of oxazepam. Seventy Positive % agreement: 100, Negative % agreement: 95 nine (79) urine specimens with GC/MS confirmed oxazepam concentration were evaluated in this study. The results are summarized and presented Four specimens were found discrepant between RapidOXY and the GC/MS method. When compared those data, 75% (3 out of 4) of the discrepancy specimens were found between cut-off and +25% of cutoff concentration Positive % agreement: 97, Negative % agreement: 100 (100-125 ng/ml). One specimen was found discrepant between the RapidBZD and GC/MS method. When compared those data, it was found to be between –25% and 9. THC The accuracy of the THC test was evaluated in comparison to
+25% cut-off concentration (150-250).
GC/MS at a cut-off of 50 ng/ml of 11-nor-Δ9-THC-9-COOH. Eighty eight (88) urine specimens with GC/MS confirmed 11-nor-Δ9-THC-9-COOH 3. Buprenorphine The accuracy of the Buprenorphine test was evaluated
concentration were evaluated in this study. The results are summarized and in comparison to GC/MS at a cut-off of 10 ng/ml of buprenorphine-3-β-d- glucoronide. One hundred and one (101) urine specimens with confirmed buprenorphine-3-β-d-glucoronide concentrations were evaluated in this Positive % agreement: 95, Negative % agreement: 100 study. Borderline readings were recorded as negative. The results are Two specimens were found discrepant between the RapidTHC and GC/MS summarized and presented below: method. When compared those data, 50% (1 out of 2 ) of the discrepancy Positive % agreement: 96, Negative % agreement: 100.
specimens were found between –25% and cut-off concentration ( 37.5 – 50 Two specimens were found discrepant between the RapidBUP and GC/MS method. When compared those data, 50% (1out of 2) of the discrepancy specimens were found between -25% cut-off and cut-off concentration 10. Tramadol 30 urine samples collected from non-users were tested. All
(7.5 – 10 ng/ml).
30 samles were negative at 200 ng/ml cut-off of tramadol. 40 urine tramadol positive samples at a cut-off of 200 ng/ml were tested and all showed 4. Cocaine The accuracy of the cocaine test was evaluated in comparison to
positive. Positive % agreement: 100, Negative % agreement: 100.
1. Interference testing
The cut-off concentrations (sensitivity level) of DOA panel test are Dipscan 10 Plus performance at cut-off level is not affected when pH
determined to be: AMP 300 ng/ml, BZD 200 ng/ml, BUP 10 ng/ml, COC and Specific Gravity ranges of urine specimen are at 4.5 to 9.0 and 1.005 300 ng/ml, MTD 300 ng/ml, MET 300 ng/ml, OPI 300 ng/ml, OXY 100 ng/ to 1.035 respectively.
ml, THC 50 ng/ml, 200ng/ml of TRA.
The following substances were tested and confirmed did not interfere with C. Precision
DOA panel tests at the listed concentrations.
The precision of DOA panel tests were determined by conducting the test with spiked controls and interpreted the results by three individuals to verify the random error of visual interpretation. The results of 40 samples each Human hemoglobin of 50% above and 50% below cut-off specimens are 100% agreed by three The precision study of was performed by three individuals observing the test result to determine the random error of visual interpretation. The test results The following table lists compounds that are detected by Dipscan 10 Plus
were found to have no significant differences between the three observers.
which produced positive results when tested at levels equal or greater than the cut-off levels.
The specificity for Dipscan 10 Plus were tested by adding various drugs,
drug metabolites, and other compounds that are likely to be present in urine. All compounds were prepared in drug-free normal human urine. 11-nor-Δ9-THC-9-COOH 11-nor- Δ 8-THC-9-COOH 11-hydroxy- Δ 9-THC Δ 8-Tetrahydrocannabino (+)methamphetamine Chloradiazepoxide HCI Desmethyldiazepam (+)Methamphetamine β-Phenylethylamine d-Pseudoephedrine Buprenorphine-3-β-d- The following compounds show no cross-reactivity at concentration up to 100 g/ml unless specified.
4-Acetamidophenol Acetylsalicylic acid Dextromethorphan Ecgonine methyl ester Ephedrine Guaiacol glycer ester Hydrochlorothiazide Phenylethylamine- Phenylpropanolamine Promethazine Quinine antidine Tetrahydrozoline Urine testing for drugs of abuse, NIDA Research Monograph 73 (1986) Steven B. Karch, Drugs of abuse hand book, CRC Press, 1st. Ed. (1998) Ray H. Liu and Bruce A. Goldberger, Handbook of workplace drug testing, AACC Press, Washington DC (1995) Free Call Aust : 1800 640 075
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