Academy of Diagnostics & Laboratory Medicine - Scientific Short

Can High Concentrations of Morphine in Urine specimens form 6-monoacetylmorphine (6-MAM) During Enzymatic Hydrolysis?

Courtney Heideloff, Jessica Gabler, Chao Yuan, Lin Zhang, Sihe Wang

There is a high prevalence of chronic pain in the United States. Effective pain management including prescribing pain medication significantly improves life quality of the individuals suffering from pain. However, pain medication can be diverted and abused. To ensure patient adherence to prescribed pain medication, urine drug testing is frequently performed. This provides objective information to assist the physician in determining compliance with prescribed treatment medications or to detect drug misuse. Drug misuse can be the absence of the prescribed drug, the presence of legal medications not prescribed to the patient, or the presence of illicit drugs in the urine. Patients may be denied further pain medication if they are found to be misusing drugs. Therefore, it is extremely important that the correct medications are reported during urine drug testing so as not to wrongfully accuse the patient of drug misuse. An example of an illicit drug is heroin, which is commonly detected in urine as its unique metabolite 6-monoacetylmorphine (6-MAM) and its major metabolite morphine. Detection of 6-MAM has been considered the definitive evidence of heroin use. However this metabolite frequently presents in low concentrations due to its short in vivo half-life.

Immunoassay drug testing is commonly used for screening, however these tests are not considered highly accurate. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are preferred because they are highly specific and sensitive, and can identify specific drugs and their metabolites with high confidence. Due to variable glucuronide conjugation rates within and between individuals, laboratories often perform hydrolysis using glucuronidase during sample preparation to improve detection of drug metabolites. Acetate buffer is a common choice for preparing enzymatic hydrolysis solutions.

In our routine LC-MS/MS analysis [1, 2], a number of low-level 6-MAM results were observed in patient urine specimens with high concentrations of morphine. We hypothesized that the acetate buffer used for enzymatic hydrolysis could serve as an acetylating agent, leading to the formation of 6-MAM in urine specimens with high morphine concentrations. To test this, we investigated formation of 6-MAM in urine with various spiked morphine (10,000-200,000 ng/mL) or morphine-3-glucuronide (M3G, 16,250-325,000 ng/mL) concentrations, incubation times (0, 2, 4, 6, 12, 18 hours), and buffer solutions (acetate buffer or citrate buffer) when incubated with β-glucuronidase at 60 °C. We demonstrated that if the morphine concentration in a patient’s urine is >100,000 ng/mL, 6-MAM could be formed during hydrolysis and low amounts of 6-MAM would be present. We showed that false positive identification of heroin is possible when extremely high levels of morphine are present in urine specimens when acetate buffer is used for enzymatic hydrolysis. Because of this, we suggest that caution be taken in result interpretation to avoid wrongful accusation of patients when a low 6-MAM is detected with the presence of a very high morphine level (>100,000 ng/mL). An alternate buffer with no acetate, such as citrate buffer, may be considered for this application to prevent false-positive detection of 6-MAM.


  1. Yuan C, Heideloff C, Kozak M, Wang S. Quantification of urine pain management drugs by LC-MS/MS. Clin Chem Lab Med 2012;50:95–103.
  2. Yuan C, Lembright K, Heideloff C, Wang S.  Quantification of buprenorphine, norbuprenorphine and 6-monoacetylmorphine in urine by liquid chromatography-tandem mass spectrometry. J Chromat Separation Techniq 2013;4:174.

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Fellows of the Academy use the designation of FADLM. This designation is equivalent to FACB and FAACC, the previous designations used by fellows of the National Academy of Clinical Biochemistry and AACC Academy. Those groups were rebranded as Academy of Diagnostics & Laboratory Medicine in 2023.