Analyte Names and Structures:
Morphine and Glucuronide Metabolites
morphine1.jpgmorphine2.jpg

Synonyms:
morphinum
morphia

MS contin
duramorph
oramorph


Relevant Physicochemical Data:
MW: 285.3 morphine, 461.5 morphine glucuronide
Formula: C17H19NO3 morphine, C23H27NO9 morphine glucuronide
Chemical Name:
(5a,6a)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol
Solubility: drug usually contains 5 molecules of water, soluble in water and organic solvents
Appearance: white, crystalline powder


Images:
See //drugs.com// for additional images
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General Relevancy:
Morphine is a primary constituent of opium and is used primarily in the relief of severe pain. Morphine may also be present as a metabolite after heroin administration. It is metabolized in the body to morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and other minor metabolites [1]. The metabolites, M3G and M6G, possess pharmacological activity [2]. Research has indicated that M6G is effective as an analgesic and M3G possesses neuroexcitatory activity [3-5]. Studies suggest M3G may play a role in the development of tolerance in individuals chronically exposed to morphine [4]. Both metabolites are eliminated primarily via the kidney. Changes in renal function can result in the accumulation of M6G and M3G with little change in the blood concentration of free morphine. The resultant increase in M6G may be contributory to toxicity in patients.

Mechanism of Action:

Morphine produces its analgesic and sedating effects primarily through stimulation of the m opioid receptor (MOR). Receptor sites throughout the central nervous system and gastrointestinal tract mediate pain, respiratory rate, euphoria, constipation and other effects associated with morphine and the opioid drugs. Glucuronidation at the 6-hydroxyl site of morphine and codeine does not interfere with binding to MOR [6, 7]. This explains why M6G possesses activity comparable to morphine. On the other hand, glucuronidation at the 3-hydroxyl site interferes with MOR recognition of opioids including morphine and hydromorphone [8, 9]. Despite less activity at MOR, these metabolites are not devoid of physiological effects. Instead, M3G and hydromorphone-3-glucuronide produce neuro-excitatory effects, possibly through activation of N-methyl-D-aspartic acid (NMDA) receptors [10, 11].

Metabolism and Pharmacokinetics:

t1/2 morphine: 1.3-6.7 hr [1]
t1/2 M6G: 2.5-4.7 hr [12]
t1/2 M3G: 2.4-3.8 hr [12]

The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates. Both glucuronides are formed by the 2B7 isoform of uridine-5’-diphosphate glucuronosyltransferase (UGT2B7) in the liver, with M3G predominating [2]. About 5% of a dose is demethylated to normorphine, which is also mostly glucuronidated [1]. Normorphine and its conjugate are readily detected in urine [1]. The excretion of M3G is through bile, feces, and urine. Extensive enterohepatic recirculation causes much of the biliary M3G to be excreted in urine. In fact, the 72-hour urine includes about 87% of a morphine dose, excreted as free morphine (10% of dose) and various metabolites [1]. The figure below describes the relationship between morphine and its glucuronide metabolites in plasma after iv administration [12].

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There does not appear to be any genetic variation in glucuronide formation by UGT2B7. However, ethanol, naltrexone, naloxone and ranitidine may interfere with UGT2B7 activity [13, 14]. In addition, steroid hormones and bilirubin in newborn infants are metabolized by UGT2B7 [15]. Genetic differences in other glucuronosyl transferase enzymes (e.g., Gilbert’s syndrome) do not appear to alter morphine pharmacokinetics [16]. The ratios of free morphine, M6G and M3G have been investigated as predictive markers for a variety of physiological outcomes including effectiveness of analgesia and acute overdose with heroin (see Critical Concentrations below). Levels of M3G peak around 2 hours after morphine administration, exceeding the concentration of free morphine remaining in serum [1, 17, 18]. M6G is slow to cross the blood brain barrier during both the absorptive and elimination phases [2].
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Adverse Effects
:

Morphine is available in a variety of dosage forms including oral (immediate and controlled release), rectal, intravenous, epidural, and intramuscular [20]. Morphine is well known for its ability to induce sedation and euphoria, often described as a dreamlike state. The euphoric effects are believed to contribute to reinforcement and abuse of morphine. Morphine is currently a schedule II controlled substance and package inserts include a warning that the drug may be habit forming. Morphine and M6G are central nervous system (CNS) depressants and both inhibit respiration. Patients are also warned that morphine may impair abilities necessary for the safe operation of a motor vehicle.

The most common adverse effects in patients treated with morphine are sedation, dizziness, nausea, vomiting, sweating, and constipation. Morphine may also cause visual disturbances, transient hallucinations, circulatory depression and allergic reactions. Drugs that act as CNS depressants (including alcohol, benzodiazepines, anesthetics, antipsychotics and tricyclic antidepressants) may produce at least additive effects in combination with morphine.


Proper Specimen Types:

B, S/P, U

Collection Tubes:

Special requirements for container type or preservatives are not indicated in the literature. However, it may be advisable to use a preservative that inhibits enzymatic activity (NaF, gray top tube) to prevent in vitro hydrolysis.

Source of Standards:

Morphine sulfate is available from Mallinckrodt (1521), Sigma (M-8777) and USP (1448005)
Morphine (1 mg/mL) is available from Cerilliant (M-005) and Alltech (018033)
D6-morphine is available from Cerilliant (M-085)
M3G is available from Sigma (M-6886) and Alltech (01390)
M3G (1 mg/mL) is available from Cerilliant (M-031) and Lipomed (M-10-HY)
D3-M3G is available from Cerilliant (M-017) and Lipomed (M-42-HY)
M6G is available from Sigma (M-3528)
D3-M6G is available from Cerilliant (M-120) and Lipomed (M-48-HY)
M6G (0.1 mg/mL) is available from Cerilliant (M-096) and Lipomed (1 mg/mL, M-57-HY)


Analyte(s) to be Determined:
Morphine, M3G, M6G

Methods of Analysis:

There are published GC, HPLC, GC/MS, LC/MS and LC/MS/MS methods for the determination of morphine and its glucuronide metabolites [25, 28, 32]. Morphine and its glucuronide metabolites may be collected during solid phase extraction from the same column, however differences in polarity of these compounds may require separate elution solvents. Hydrolysis may be performed with heat and acidic conditions or enzymatically (b-glucuronidase). Glucuronidated metabolites are likely to elute as a group separate from their free opioid counterparts depending on the type of column used for chromatography.

Measurable Range (Upper/Lower Detection Limits):


Critical Concentrations:
Intravenous administration of 10 mg morphine produced reported peak therapeutic blood levels of 60 ng/mL, which declined to 3 ng/mL after 36 hr [21]. A single therapeutic 10 mg intramuscular injection produced peak blood levels of 70 ng/mL after 10 to 20 min, which declined to 10 ng/mL after 4 hr [17]. In two reported fatalities, free morphine blood levels were reported as 70 and 350 ng/mL [22]. A report of 10 fatalities after intravenous morphine reported a range of morphine in blood from 200 to 2,300 ng/mL [23].

Antemortem Levels – Chronic Pain Patients, Serum/Plasma
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In one reported case study, three patients with renal failure exhibited respiratory failure following morphine administration. M6G plasma levels ranged from 130 to 1,100 ng/mL with morphine levels less than 4 ng/mL [26]. Following ingestion of 5 grams of sustained release morphine, M3G, M6G and morphine plasma levels at 60 hours post ingestion were 6,200 ng/mL, 11,000 ng/mL and 620 ng/mL respectively [27].

Based on observations that M3G concentrations exceed free morphine within 2 hours post-dose, several methods have been proposed to estimate survival time by comparing morphine and metabolite concentrations. This technique often uses the free/total ratio, comparing morphine levels before and after hydrolysis. If the free morphine is less than some percentage of the total, it is assumed that the individual had time to metabolize the dose. However, if the free morphine constitutes the majority of the total, the death was probably acute and the individual did not have time for metabolism to take place. Since hydrolysis introduces an additional source of uncertainty to the analysis, many recent reports are using specific measurements of morphine, M3G and M6G to determine total morphine. In two cases of heroin overdose, blood levels were 360 ng/mL morphine, 82 ng/mL M3G and 5 ng/mL M6G in an acute death and 110 ng/mL morphine, 1900 ng/mL M3G and 120 ng/mL M6G in a delayed death [28]. The guidelines proposed by the authors include the following:


Immediate death

–Free morphine >500 ng/mL
–Free ~80% of total morphine


Subacute death (<3h)

–Free morphine <500 ng/mL
–Free >50% of total morphine


Delayed death

–Free <50% of total morphine [28]

Care should be taken with interpretation, since it is also possible for hydrolysis to occur during a prolonged postmortem interval, particularly if the sample is exposed to heat. In addition, the ratios of the morphine glucuronide metabolites appear to be different for heroin versus morphine administration. Heroin abusers given morphine had higher ratios of M6G to M3G than nonheroin users [29]. Since heroin is metabolized to morphine there is no ready explanation for these apparent differences in glucuronidation.


Potential Interfering Compounds:


Stability Data:
NMS data for free morphine stability in serum
Room Temperature: 10 days
Refrigerated: 20 days
Frozen (-20oC): 12 months

Standards should be protected from light and stored at 15-30oC.
Specimens stored without preservatives will show a decrease in concentrations of total morphine and total codeine, (increases in free morphine and codeine), at room, refrigerator and freezer temperatures over an 11 month period [30]. One study reports that morphine, M3G and M6G are stable in blood stored at 4oC and protected from light for up to 181 days [31]. It recommends storage of postmortem specimens at –20oC for opiate analysis.


Interfering Substances:

Drugs with chemical structures similar to morphine: heroin, 6-acetylmorphine, codeine, hydrocodone, hydromorphone, naloxone, other opiates.

Drugs that may be coadministered with morphine: NSAIDs, cyclooxygenase inhibitors (-coxibs), fentanyl and other analgesics; diltiazem, gabapentin or amitriptyline (for neuropathic pain), nifedipine and other calcium channel blockers (these have been shown to enhance the analgesic effect of morphine), metoclopramide, ondansetron and other antiemetics, senna, lactulose and other laxatives.


Concentration Range:

For the purposes of therapeutic monitoring, morphine concentrations should be accurately determined within a range that includes 10-100 ng/mL. Fatalities have reported free morphine blood levels above 200 ng/mL [23], although one fatality was only 70 ng/mL [22]. While these latter concentrations are reference points, as with all postmortem findings, the concentration should be viewed in respect to the totality of information.

Plasma (published articles – LC/MS/MS):
LOQ morphine 0.5ng/mL
LOQ M3G, M6G 1.0 ng/mL
Calibration range – Morphine, 0.5 to 50 ng/mL
Calibration range – M6G, 1.0 to 100 ng/mL
Calibration range – M3G, 10 to 1000 ng/mL


Maximum Acceptable Error:

The interpretation of morphine and glucuronide concentrations in biological specimens is facilitated by analytical methods with <20% total error.

Proposed Reference Comments:
Morphine
B/SP - Usual range following therapeutic doses: 10 - 70 ng/mL.

U - Up to 90% of a parenteral dose will be excreted in the urine within 24 hours and up to 60% of an oral dose will be excreted in the urine within 24 hours.

Forensic

Peak serum concentrations occur within 10-20 minutes of a 10 mg/kg intramuscular dose, with an average concentration of 60 ng/mL 30 minutes following administration. IV administration of the same dose resulted in an average concentration of 80 ng/mL after 30 minutes. Cancer patients (N=151) being treated with 10-2540 mg/day oral morphine had free morphine concentrations of <1-1000 ng/mL. In 87 cases of fatal heroin overdose where opioids were solely responsible for intoxication, free morphine concentrations were 0-2800 ng/mL. In comparison, five survivors intoxicated by only morphine had free morphine concentrations of 28-93 ng/mL. In 15 cases where cause of death was attributed to opiate toxicity (heroin, morphine or both), free morphine concentrations were 0-3700 ng/mL (mean = 420 ± 940). In comparison, in cases where COD was unrelated to opiates (n=20) free morphine was 0 – 850 ng/mL (mean = 90 ± 200). The reported blood to plasma concentration of free morphine is approximately unity.

TI/U/FL - In general, free morphine is the active biologic agent. Morphine has diverse effects that may include analgesia, drowsiness, nausea and respiratory depression. 6-monoacetylmorphine (6-MAM) is the 6-monoacetylated form of morphine, which is pharmacologically active. It is commonly found as the result of heroin use.


M3G

Clinical
B/SP - Chronic pain patients receiving an average of 90 mg (range 20-1460) daily oral morphine had average serum concentrations of 73 ng/mL (range 13-710) morphine, 2,900 ng/mL (range 800-14,000) M3G and 530 ng/mL (range 69-3,600) M6G.

U - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating.

Forensic
B/SP - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating. The excretion of M3G is through bile and feces, but extensive enterohepatic recirculation causes much of the biliary M3G to be excreted in urine. Although devoid of opioid activity, M3G may produce neuro-excitatory effects, possibly through activation of N-methyl-D-aspartic acid (NMDA) receptors. Levels of M3G peak around 2 hours after morphine administration, exceeding the concentration of free morphine remaining in serum. Chronic pain patients receiving an average of 90 mg (range 20-1460) daily oral morphine had average serum concentrations of 73 ng/mL (range 13-710) morphine, 2,900 ng/mL (range 800-14,000) M3G and 530 ng/mL (range 69-3,600) M6G. In two cases of heroin overdose, blood levels were 360 ng/mL morphine, 82 ng/mL M3G and 5 ng/mL M6G in an acute death and 110 ng/mL morphine, 1900 ng/mL M3G and 120 ng/mL M6G in a delayed death.

U - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating. The excretion of M3G is through bile and feces, but extensive enterohepatic recirculation causes much of the biliary M3G to be excreted in urine. Although devoid of opioid activity, M3G may produce neuro-excitatory effects, possibly through activation of N-methyl-D-aspartic acid (NMDA) receptors.


M6G

Clinical
B/SP - Chronic pain patients receiving an average of 90 mg (range 20-1460) daily oral morphine had average serum concentrations of 73 ng/mL (range 13-710) morphine, 2,900 ng/mL (range 800-14,000) M3G and 530 ng/mL (range 69-3,600) M6G.

U - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating.

Forensic
B/SP - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating. Research has indicated that M6G is effective as an analgesic. M6G is slow to cross the blood brain barrier during both the absorptive and elimination phases. Changes in renal function can result in the accumulation of M6G and M3G with little change in the blood concentration of free morphine. The resultant increase in M6G may be contributory to toxicity in patients. Chronic pain patients receiving an average of 90 mg (range 20-1460) daily oral morphine had average serum concentrations of 73 ng/mL (range 13-710) morphine, 2,900 ng/mL (range 800-14,000) M3G and 530 ng/mL (range 69-3,600) M6G. In two cases of heroin overdose, blood levels were 360 ng/mL morphine, 82 ng/mL M3G and 5 ng/mL M6G in an acute death and 110 ng/mL morphine, 1900 ng/mL M3G and 120 ng/mL M6G in a delayed death.

U - The majority (~65%) of a dose of morphine is excreted as glucuronide conjugates, with M3G predominating. Research has indicated that M6G is effective as an analgesic. M6G is slow to cross the blood brain barrier during both the absorptive and elimination phases. Changes in renal function can result in the accumulation of M6G and M3G with little change in the blood concentration of free morphine. The resultant increase in M6G may be contributory to toxicity in patients.


References:

1. Baselt, R., C., The Disposition of Toxic Drugs and Chemicals in Man. 2008. 8th Edition.

2. Wittwer, E. and S.E. Kern, Role of morphine's metabolites in analgesia: concepts and controversies. Aaps J, 2006. 8(2): p. E348-52.

3. Smith, T.W., A.R. Binning, and A. Dahan, Efficacy and safety of morphine-6-glucuronide (M6G) for postoperative pain relief: a randomized, double-blind study. Eur J Pain, 2009. 13(3): p. 293-9.

4. Smith, M.T., Neuroexcitatory effects of morphine and hydromorphone: evidence implicating the 3-glucuronide metabolites. Clin Exp Pharmacol Physiol, 2000. 27(7): p. 524-8.

5. Quigley, C., et al., Plasma concentrations of morphine, morphine-6-glucuronide and morphine-3-glucuronide and their relationship with analgesia and side effects in patients with cancer-related pain. Palliat Med, 2003. 17(2): p. 185-90.

6. Brown, G.P., et al., 3H-morphine-6beta-glucuronide binding in brain membranes and an MOR-1-transfected cell line. J Pharmacol Exp Ther, 1997. 282(3): p. 1291-7.

7. Kilpatrick, G.J. and T.W. Smith, Morphine-6-glucuronide: actions and mechanisms. Med Res Rev, 2005. 25(5): p. 521-44.

8. Ulens, C., et al., Morphine-6beta-glucuronide and morphine-3-glucuronide, opioid receptor agonists with different potencies. Biochem Pharmacol, 2001. 62(9): p. 1273-82.

9. Bartlett, S.E. and M.T. Smith, The apparent affinity of morphine-3-glucuronide at mu1-opioid receptors results from morphine contamination: demonstration using HPLC and radioligand binding. Life Sci, 1995. 57(6): p. 609-15.

10. Halliday, A.J., et al., Brain region-specific studies of the excitatory behavioral effects of morphine-3-glucuronide. Life Sci, 1999. 65(2): p. 225-36.

11. Hemstapat, K., et al., Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: mechanistic studies in embryonic cultured hippocampal neurones. Anesth Analg, 2003. 97(2): p. 494-505, table of contents.

12. Stuart-Harris, R., et al., The pharmacokinetics of morphine and morphine glucuronide metabolites after subcutaneous bolus injection and subcutaneous infusion of morphine. Br J Clin Pharmacol, 2000. 49(3): p. 207-14.

13. Aasmundstad, T.A. and P. Storset, Influence of ranitidine on the morphine-3-glucuronide to morphine-6-glucuronide ratio after oral administration of morphine in humans. Hum Exp Toxicol, 1998. 17(6): p. 347-52.

14. Faura, C.C., et al., Systematic review of factors affecting the ratios of morphine and its major metabolites. Pain, 1998. 74(1): p. 43-53.

15. Coffman, B.L., et al., The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268). Drug Metab Dispos, 1998. 26(1): p. 73-7.

16. Skarke, C., et al., Pharmacokinetics of morphine are not altered in subjects with Gilbert's syndrome. Br J Clin Pharmacol, 2003. 56(2): p. 228-31.

17. Berkowitz, B.A., et al., The diposi tion of morphine in surgical patients. Clin Pharmacol Ther, 1975. 17(6): p. 629-35.

18. Drost, R.H., et al., Pharmacokinetics of morphine after epidural administration in man. Arzneimittelforschung, 1986. 36(7): p. 1096-100.

19. Karch, S., B., Karch's Pathology of Drug Abuse. 2001. 3rd Edition.

20. Healthcare, T., Physician's Desk Reference. 2006.

21. Aitkenhead, A.R., et al., Pharmacokinetics of single-dose i.v. morphine in normal volunteers and patients with end-stage renal failure. Br J Anaesth, 1984. 56(8): p. 813-9.

22. Chan, S.C., E.M. Chan, and H.A. Kaliciak, Distribution of morphine in body fluids and tissues in fatal overdose. J Forensic Sci, 1986. 31(4): p. 1487-91.

23. Felby, S., H. Christensen, and A. Lund, Morphine concentrations in blood and organs in cases of fatal poisoning. Forensic Sci, 1974. 3(1): p. 77-81.

24. Peterson, G.M., C.T. Randall, and J. Paterson, Plasma levels of morphine and morphine glucuronides in the treatment of cancer pain: relationship to renal function and route of administration. Eur J Clin Pharmacol, 1990. 38(2): p. 121-4.

25. Klepstad, P., et al., Day-to-day variations during clinical drug monitoring of morphine, morphine-3-glucuronide and morphine-6-glucuronide serum concentrations in cancer patients. A prospective observational study. BMC Clin Pharmacol, 2004. 4: p. 7.

26. Osborne, R., S. Joel, and M. Slevin, Morphine intoxication in renal failure; the role of morphine-6-glucuronide. Br Med J (Clin Res Ed), 1986. 293(6554): p. 1101.

27. Westerling, D., J. Sawe, and G. Eklundh, Near fatal intoxication with controlled-release morphine tablets in a depressed woman. Acta Anaesthesiol Scand, 1998. 42(5): p. 586-9.

28. Al-Asmari, A.I. and R.A. Anderson, Method for quantification of opioids and their metabolites in autopsy blood by liquid chromatography-tandem mass spectrometry. J Anal Toxicol, 2007. 31(7): p. 394-408.

29. Antonilli, L., et al., High levels of morphine-6-glucuronide in street heroin addicts. Psychopharmacology (Berl), 2003. 170(2): p. 200-4.

30. Lin, D.L., H. Liu, and C.Y. Chen, Storage temperature effect on the stability of morphine and codeine in urine. J Anal Toxicol, 1995. 19(5): p. 275-80.

31. Skopp, G., et al., Stability of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in fresh blood and plasma and postmortem blood samples. J Anal Toxicol, 2001. 25(1): p. 2-7.

32. Bogusz, M.J., R.D. Maier, and S. Driessen, Morphine, morphine-3-glucuronide, morphine-6-glucuronide, and 6-monoacetylmorphine determined by means of atmospheric pressure chemical ionization-mass spectrometry-liquid chromatography in body fluids of heroin victims. J Anal Toxicol, 1997. 21(5): p. 346-55.


General Questions:

1.
What is the application of the test?
Reference Laboratories/Medical Examiner/Pain Management

2.
Who would use this application and why?
Reference Labs/ Physicians/ ME/Coroners

3. Is testing currently available / new to the industry by some other means?
Testing is available in selected independent laboratories utilizing HPLC and LC/MS methods. Availability of test is limited to a few medical examiner laboratories.

4. What are the features / benefits of this test to the user?
Ability to detect and quantitate morphine, M3G and M6G in diverse biological matrices.

5.
What is the scientific significance of this test?

a. Ability to quantitate & confirm morphine and its glucuronide metabolites in diverse biological matrices
b. Avoids the uncertainty introduced by enzymatic hydrolysis step
c. Interpretative aid to forensic toxicologists and pathologist

d. Postmortem determination for morphine-3-glucuronide and morphine-6-glucuronide.