Papain: A Novel Urine Adulterant*

Abstract

The estimated number of employees in the United Stated screened annually for illicit drugs is approximately 20 million, with marijuana being the most frequently abused drug. Urine adulterants provide an opportunity for illicit drug users to obtain a false-negative result on commonly used primary drug screening methods such as the enzyme multiplied immunoassay technique and the fluorescence polarized immunoassay technique (FPIA). Typical chemical adulterants such as nitrites are easily detected or render the urine specimen invalid as defined in the proposed SAMHSA guidelines for specimen validity testing based on creatinine, specific gravity, and pH. Papain is a cysteine protease with intrinsic ester hydrolysis capability. The primary metabolite of the psychoactive chemical in marijuana, 11-norcarboxy-Delta9-tetrahydrocannibinol (THC-COOH), was assayed by FPIA in concentrations ranging from 25 to 500 ng/mL, at pH values ranging from 4.5 to 8, over the course of 3 days with papain concentrations ranging from 0 to 10 mg/mL. FPIA analysis of other frequently abused drugs: amphetamines, barbiturates, benzodiazepines, cocaine, opiates, and phencyclidine, along with gas chromatography-mass spectrometry (GC-MS) of THC-COOH and high-pressure liquid chromatography-ultraviolet detection (HPLC-UV) of nordiazepam was performed in order to determine if the mechanism of urine adulteration by papain was analyte specific. Control and adulterated urine specimens (n = 30) were assayed for creatinine, specific gravity, and pH to determine if papain rendered the specimens invalid based on the proposed SAMHSA guidelines. There was a direct pH, temperature, and time-dependent correlate between the increase in papain concentration and the decrease in THC-COOH concentration from the untreated control groups (p < 0.01). The average 72-h THC-COOH concentration decrease at pH 6.2 with a papain concentration of 10 mg/mL was 50%. Papain did not significantly decrease the concentration of the other drugs analyzed with the exception of nordiazepam. GC-MS of THC-COOH and HPLC-UV of nordiazepam revealed a 66% and 24% decrease in concentration of the respective analyte with 10 mg/mL papain after 24 h at room temperature (approximately 23 degrees C). No adulterated specimens were rendered invalid based on the SAMHSA guidelines. Immediate FPIA analysis is suggested to minimize the interfering effects of papain with regards to primary drug screening.

Urine Samples Tampering Pattern for Drugs of Abuse Testing: Experience of the Saudi Arabia Poison Control Centers

“…Burrows et al [18] mentioned that urine should be reported as adulterated if the pH was <3 or >11. In this study, only one adulterated cases were found.…”

Adulteration of urine drug testing: Case report and review

“…In a study related to this method, it has been found that 84% of urine samples were recollected because of low creatinine level and 20% of samples had a pH level out of the normal range (17). In addition to water, urinary adulteration may be tried using sodium bicarbonate, diuretics, and drugs containing salicylates (5). (1,2,(5)(6)(7)9).…”

“…In addition to water, urinary adulteration may be tried using sodium bicarbonate, diuretics, and drugs containing salicylates (5). (1,2,(5)(6)(7)9). High demand in this field has led to development of commercial kits specific to the substance used by the individual.…”

Proteases in Doping Control Analysis

“…In contrast, those incubated in the presence of 20 -100 µg of protease per mL of urine did not allow the determination of neither endogenous nor recombinant EPO. These results demonstrate that the addition of small amounts of proteases during doping control sample collection, e.g., by introducing a water soluble "rice grain" containing proteases, can alter urine specimens and complicate or even impede the analysis for peptide hormones such as EPO as well as low molecular weight target analytes [2] for sports drug testing purposes.…”

“…As urine specimens are not fortified with protease inhibitor cocktails immediately after sampling, proteases can digest endogenous as well as recombinant EPO and other renally excreted peptide hormones and make them invisible for commonly employed drug testing assays [1,6,8,9,21]. The issue of urine manipulation has frequently been discussed in doping controls but also in workplace testing, and numerous agents and substitution tools are available via the Internet announcing options to pass drug tests for a variety of drugs of abuse [2,24,25]. In the present study, the possibility of manipulating doping control urine samples using proteases was investigated by analyzing EPO-spiked urine specimens analyzed using conventional electrophoretic EPO test assays.…”