toxicology exam opioids and opiates

what is the definition of opiate? opioid? opioid analgesics?

opiate: compounds isolated from the opium poppy (morphine and codeine) that act at opioid receptors; opiates are natural occurring

opioids: compounds of any structural type that interact with the opioid receptors. natural or synthetic

opioid analgesics: natural and semisynthetic alkaloids prepared from opium, as well as synthetic surrogates with similar pharmacological effects. structures may vary.

how are opioids related to the limbic system? what are their mechanism of action? agonists/antagonists?

limbic system: rich in opioid binding sites and is primarily involved in the arousal of human emotions

mechanism of action: opioids produce an analgesic effect by blocking the transmission of painful stimuli. the interaction between the opioid and specific receptors as terminal nerve endings impedes the release of neurotransmitter, thus interrupting the pain

agonists/antagonists: full agonists, mixed agonists/antagonists, full antagonists

what receptors do opioids interact with and what is the effects of each?

mu receptor (primary receptor responsible for pain relief): binding of the opioid to the mu receptor yields effects of central depression which is clinically manifested as supra-spinal and spinal analgesia, respiratory depression, miosis (pinpoint pupils), euphoria, reduced GI motility (affects absorption of any co-admin drugs), hypothermia (lower body temp), bradycardia (slow heart rate), physical tolerance and dependence

kappa receptor: binding of the opioid to the kappa receptor yields effects of spinal analgesia, sedation, miosis, diuresis, mild respiratory depression, low addiction liability

delta receptor (binding sites for most endogenous peptides): binding of the opioid to the delta receptor yields effects of spinal analgesia, dysphoria, delusions, hallucinations, respiratory and vasomotor stimulation

sigma receptor: binding of the opioid to the sigma receptor yields effect of central excitation (tachycardia (increased heart rate), hypertension, tachypnea (breathing very quickly), mydriasis (dilated pupils), hallucinations)

→ respiratory depression is the primary cause of death for opioids → medical examiners commonly find pulmonary edema with opioid overdose

what are the classes of opioids based on structure?

opioid classes: phenanthrenes, benzomorphans, phenylpiperidines, diphenylheptanes, and phenylpropyl amines

→ the detection of metabolites is very important because the parent molecule may be metabolized so fast that it may not be detected in blood

what are the therapeutic uses of opioids and what is the pharmacological effects?

therapeutic uses: postoperative analgesia (to reduce pain), surgical and medical emergencies, management of chronic pain associated with cancer and terminal illness and use as anesthetic and sedative supplement, antitussive and antidiarrheal properties

pharmacological effects: exert their major effects on the CNS. provide analgesia without loss of consciousness. analgesia is associated with euphoria, sedation and mental clouding. may also produce pulmonary and GI effects, respiratory depression, nausea, vomiting, constipation

what are the disadvantages and withdrawal symptoms of opioids?

disadvantages: risk of respiratory failure. if a person continuously takes an opioid, tolerance is very likely to develop.

withdrawal symptoms (abstinence syndrome): increased heart rate, irritability, sweating, piloerection, uncontrollable muscle spasms and pain

what drug is used to reverse opioid intoxication and what is the active ingredient?

• reversal of opioid intoxication with naloxone is carried out at the risk of precipitating severe withdrawal symptoms

• active ingredient of narcan (drug) = naloxone (antidote)

  • only works in the case of an overdose; doesnt worm for fentanyl or xylezine overdose

• additional care is needed for opioids with longer half lives

what is the structure activity relationship of opioids?

structure activity relationship: opioid’s binding interactions with the receptor are determined not only by the structure but also by the stereospecificity. only the levoratory isomer of morphine is a potent analgesic.

in terms of pharmacokinetics, what is the absorption and distribution of opioids?

absorption and distribution:

• most opioids are absorbed readily after subcutaneous or IM injection

• lipid soluble opioids are effectively absorbed via the usual routes

• IV is common administration is opioids consumed for illegal purposes due to the rapid effect. however, because of HIV spread smoking, nasal insufflation, inhalation of vapors are also popular

what is the metabolism and excretion of opioids in terms of pharmacokinetics?

metabolism and excretion:

• metabolism takes place primarily in the liver to form mostly polar metabolites which are eliminated via enterohepatic or renal recirculation

• sharing of common metabolic pathways: oxidation, hydroxylation, o-demethylation, n-demethylation which underogoes conjugation

what is the metabolism and excretion in terms of codeine and morphine?

• codeine: 10-20% of the parent drug is excreted in the urine unchanged (this is why codeine is difficult to detect in urine; must look for metabolites). N- and O- demethylation following by glucuronidation and sulfation. 10% is metabolized as morphine accounting for most of the analgesic effect. further metabolism yields the active metabolite M6G which is more potent than morphine.

• morphine: major metabolic routes are 3- and 6- glucoronidation and sulfation, N- and O- demethylation, and N-oxide formation. both morphine-3-glucuronide and normorphine are pharmacologically inactive metabolites. the analgesic potency of M6G is approx twice that of morphine

what is the metabolic pathway of heroin and how can we tell if heroin was prescribed or not?

metabolic pathway:

heroin ((diacetyl morphine) heroin is hydrolyzed by blood enzymes: cholinesterase very quickly, makes detection window difficult) → 6-monoacetylmorphine ((6-MAM) unique metabolite, the presence of this indicates the person did use heroin, need confirmation test to identify metabolite) → morphine. codeine can be turned into morphine.

if morphine/codeine > or equal to 3 → person did use heroin

if morphine/codeine < 3 → was prescribed

what are the 2 definitive indicators of heroin use?

6-monoacetyl morphine and acetyl codeine

what are the methods of analysis for opioids?

• immunoassay for screening (class specific, but not analyte specific)

  • antibodies developed toward the morphine structure

  • commercial kits are available at cut-offs of 300 ng/mL (lab decides cut-off limit)

  • opiate immunoassay are only useful for opiates

  • GC MS with full scan and LC QTOF MS also used

    • LC QTOF MS: high res mass spec

    • LC MS or GC MS: both unit resolution; provide accuracy up to 1 decimal point

• mass spectral confirmation

  • all positive immunoassay screens should be confirmed to identify which opiate(s) is (are) present and to quantify the drug(s)

  • GCMS (SIM), LCMS, and LC MS/MS are all commonly used

what is the difference between SCAN and SIM mode?

a molecule is ionized by ion source then enters the MS → directed through a single quad to filter the ions by SCAN or SIM mode

SCAN mode: detects any substance in the sample with an m/z in the 0-500 range

SIM mode: set detector to exclude/filter all other ions than what we are selection, allows selected ions to pass through to detector, all other ions crash into sides of quadrupole

what does a triple quad MS do and how is MRM related to it?

triple quad: MS/MS or tandem MS; provides additional selectivity and sensitivity

• specific ions selected from quad 1 pass through quad 2 to further fragment → additional fragments pass through to quad 3 with MS/MS

• MRM: multiple reaction monitoring → you must identify at least 2 MRM transitions to identify the presence of a substance (transitions from parent to fragment)

• fragmentation potential is much higher in triple quad vs single quad = higher sensitivity and selectivity

• most abundant ions provide the best sensitivity (consider when selecting the 2 MRM transitions)

• most sensitive MRM is used for quantitative purposes (quantifier ion) and the second most sensitive is used for qualitative purposes (qualifier ion)

how are immunoassays used for screening of opioids?

commercial immunoassay kits available for:

1. phenanthrene - type drugs (opiates)

   1. not specific

   2. cross reactivity towards compounds structurally similar to morphine

2. methadone

3. buprenorphine

4. fenatnyl

5. tramadol

in terms of an ELISA opioid kit, what is cross reactivity and why is it important?

cross reactivity can occur in ELISA/immunoassays; antibodies must be specific for opiates, but may interact with structurally similar substances or the matrix

when a substance with a related structure to the analyte yields a significant response, this is important because of false/true positives and false/true negatives

• false positive: tests positive for drug, but not actually present, must use confirmatory technique

• false negative: preliminary screening is negative, but confirmatory test is positive

the extraction of opioids is based on what 2 different types of interactions?

the extraction of opioids is based on:

1. strong cation exchange (SCX)

2. nonpolar interactions

what is LLE?

LLE is partitioning of the sample between organic phase and aqueous phase depending on hydrophobicity and ionization of molecule in aquatic phase, it can be partitioned into organic phase by increasing the hydrophobicity

in LLE, pH is important to facilitate partition of sample into organic phase; the more substance in organic phase = higher sensitivity

what are the 3 basics of opioid extraction?

1. opioids are basic drugs: requires sample pH range between 8-10 for extraction

2. different extraction techniques

   1. LLE: easier

   2. SPE: takes 3-4 hr time

   3. protein precipitation with methanol or acetonitrile

3. enzymatic hydrolysis of urine samples: only for drugs that provide conjugates

   1. Glucuronide B: added into urine sample; the purpose is to break the bonds between glucuronic acids and the molecule itself (yields more free drug = improving method sensitivity)

what are the 3 different types of SPE?

3 different types of SPE:

1. carbon chains (R) → nonpolar

2. (-) strong cation exchange columns/cartridge

3. (+) strong anion exchange columns/cartridge

the SPE cartridge contains a silica based column; carbon chains or anionic groups to facilitate exchange of protonated molecules

what is SPE?

SPE:

1. mixed mode SPE most often used for opioid extraction

   1. mixed mode: different interactions combined on the same cartridge (ex: nonpolar and strong cation exchange)

2. mixed mode chromatography uses more than one separation mode

   1. mainly reversed phase combined with ion exhchange interactions

   2. cation exchange for opioids

→ allows for retention and separation of both polar and nonpolar analytes in a single analysis

what 2 substances are naturally found in the opium plant from which morphine is extracted?

papaverine and noscapine are the two natural substances found in the opium plant

→ these will show up if someone consumed poppy seeds

→ it is very likely to detect natural substances under heroin use

→ if natural substances are present, the person did use heroin (not prescribed)

→ if a person has been prescribed codeine, we will not find natural substances and also by the morphine/codeine ratio

what type of ionization source is typical for a GC MS?

a GC MS detector with an electron impact (EI) ionization source 70eV

→ this is the most widely used because it provides robust results compared to LCMS because of the unique ion source of electron impact

→ the molecule is ionized into the GC MS → molecule is bombarded with electrons to form the molecular ions

→ EI always uses the same voltage applied (70 eV) → sufficiently breaks down into fragments and yields reproducible mass specs at 70 eV

what are the autopsy findings of an opioid overdose?

• foam cone

• pulmonary/cerebral edema

• heavy organs

• distended bladder

• constipation

• track marks

what s the medical review officer?

the medical review officer (MRO): licensed physician responsible for receiving, reviewing, and interpreting laboratory drug test results for employers. acting an an independent gatekeeper, the MRO evaluates potential legitimate medical explanation for positive, adulterated, or invalid results, ensuring accuracy and confidentially in compliance with regulations

what is a drug recognition expert?

a drug recognition expert (DRE): certified law enforcement officer trained to identify impairment in drivers under the influence of drugs other than, or in addition to, alcohol. using a 12-step, scientifically validated evaluation process, DREs determine the category of drugs causing impairment, often testifying in court

what is the drug symptom matrix?

the drug symptom matrix (or drug category symptomology matrix): a tool used by MROs and DREs to correlate physical, mental, and behavioral symptoms with specific drug classes to determine the cause of impairment. the matrix categorizes drugs into seven categories and outlines expected indicators such as pulse, blood pressure, body temp, and pupil size

→ the factors/symptoms are evaluated to determine if someone has prior use of one or more of these substances. according to each class, symptoms are evaluated to be further tested for impairment

what are the 7 drug categories of the DSM?

CNS depressants, CNS stimulants, hallucinogens, dissociative anesthetics, narcotic analgesics, inhalants, cannabis