2-Nitro-6-monoacetylmorphine: potential marker for monitoring the presence of 6-monoacetylmorphine in urine adulterated with potassium nitrite

Abstract

6-Monoacetylmorphine (6-MAM), being a unique metabolite of heroin, is routinely tested in urine samples to monitor heroin use. However, detection of 6-MAM-related opiates such as morphine is known to be affected by in vitro urine adulteration using oxidizing adulterants such as potassium nitrite. This study aimed to investigate the fate of 6-MAM after exposure to nitrite and to identify any formed oxidation products that may potentially be used for monitoring heroin abuse despite nitrite adulteration. Potassium nitrite (0.05 M and 0.6 M) was reacted with 6-MAM (5-10,000 ng/mL) in both water and blank urine with pH adjusted to range from 3 to 8. Following reaction at room temperature for varying periods, the reaction mixtures were monitored by both the CEDIA® Heroin Metabolite (6-AM) immunoassay and liquid chromatography-mass spectrometry (LC-MS) methods. Structural elucidation of the isolated oxidation products was based on mass spectrometry and nuclear magnetic resonance spectroscopic evidence. Nitrite, under acidic environment (pH<7), was shown to be effective in masking the detection of 6-MAM by both the CEDIA® immunoassay and the LC-MS methods. 2-Nitro-6-monoacetylmorphine (2-nitro-MAM) was identified as the sole oxidation product, which remained detectable in urine for at least 11 days under the experimental conditions investigated. 2-Nitro-MAM was detectable in a urine sample of a heroin user after nitrite exposure. 2-Nitro-MAM has shown potential to serve as a marker for monitoring heroin abuse when urine is adulterated with nitrite. Certification of 2-nitro-MAM reference standard for further development of its quantitative testing methods is thus warranted.

Detection and identification of 2‐nitro‐morphine and 2‐nitro‐morphine‐6‐glucuronide in nitrite adulterated urine specimens containing morphine and its glucuronides

“…The MS fragmentation patterns of the morphine starting material (Figure 2a) and the reaction product ( Figure 2b) were obtained. Based on the fragmentation and accurate mass data obtained and the reaction mechanism proposed previously for the formation of 2-nitro-MAM upon exposure of 6-MAM to KNO 2 in aqueous matrices, [12] it was hypothesised that the morphine reaction product was 2-nitro-morphine ( Figure 3). Furthermore, the loss of a -NO 2 group would result in a radical cation of morphine, consistent with the major fragment ion observed at m/z 285.1326.…”

“…[1,5] According to the 2012 World Anti-Doping Code (International Standard) released by the World Anti-Doping Agency (WADA), heroin and morphine are listed under class S7 in the prohibited list, with its use banned in-competition. [12] Although urine collection protocols are quite strict, particularly in sports drug testing, the act of adulteration is not impossible. The Australian/New Zealand Standard 4308: 2008 [7] lists 6-MAM, morphine and codeine as some of the drugs of abuse that are monitored in urine specimens submitted for workplace drug testing.…”

“…[8][9][10][11] A previous study conducted by the authors has shown that exposure of 6-MAM to nitrite (active ingredient of commercial oxidizing adulterants 'Klear' and 'Whizzies') results in the formation of 2-nitro-6monoacetylmorphine (2-nitro-MAM), rendering the initial 6-MAM analyte in the urine specimen undetectable. [12] Although urine collection protocols are quite strict, particularly in sports drug testing, the act of adulteration is not impossible. Individuals motivated to circumvent a drug test can be very innovative, as demonstrated by the use of 'rice grains' that contain proteases placed into the urethra prior to urination to mask the use of certain peptide hormones such as EPO.…”

Oxidation of testosterone by permanganate and its implication in sports drug testing

“…1 H, 13 C, COSY, HSQC, HMBC and NOESY experiments were performed. P1 and P2 were dissolved in deuterated methanol and chloroform, respectively, and spectra were recorded at room temperature.…”

“…[6][7][8] These adulterants act by either interfering with immunoassay procedures or by converting the target drugs to other compounds. [11][12][13][14] In the present study we report isolation and characterisation of new oxidation products formed from the reaction of potassium permanganate with testosterone applying normal phase chromatography, nuclear magnetic resonance (NMR) and mass spectrometry (MS). We have reported that some popular oxidizing adulterants can selectively and significantly reduce the absolute concentrations of certain endogenous steroids in human urine and yet maintain steroid ratios within the acceptable population-based ranges.…”