Urine Adulteration Research Articles

Systematic web monitoring of drug test subversion strategies in the United States.

As negative drug tests are frequently a condition for employment, some people who use drugs will try to subvert the testing. In this study, systematic web monitoring was used to investigate how drug test subversion is discussed online. Posts pertaining to drug test subversion were obtained from public websites and the dark web (n= 634, July-December 2021). Most information from public websites came from Twitter (65%), and 94% of dark web posts were from Reddit. The posts were manually coded to extract quantitative and qualitative information about drug test subversion tactics. Most posts discussed urine drug tests (85%), followed by hair (11%) and oral fluid (2%), and the most discussed drugs were marijuana (72%) and cocaine (7.3%). Urine drug test subversion mainly pertained to specimen substitution, with synthetic urine or urine from another person. Another strategy was to mask diluted urine by ingesting creatine. Urine adulteration was rarely discussed. Hair test subversion involved harsh treatments with products such as bleach, baking soda, and/or detergent. Hair removal was also discussed. Oral fluid test subversion focused on removing drugs from the oral cavity through vigorous brushing of teeth and tongue as well as the use of mouthwash, hydrogen peroxide, gum, and commercial detox products. This study highlights subversion strategies used by donors. Although little evidence was provided as to the effectiveness of these strategies, this information may help guide future studies and development of specimen validity testing to minimize the impact of drug test subversion attempts.

Buprenorphine, Norbuprenorphine, and Naloxone Levels in Adulterated Urine Samples: Can They be Detected When Buprenorphine/Naloxone Film is Dipped into Urine or Water?

Reportedly, various urine manipulations can be performed by opioid use disorder (OUD) patients who are on buprenorphine/naloxone medications to disguise their non-compliance to the treatment. One type of manipulation is known as "spiking" adulteration, directly dipping a buprenorphine/naloxone film into urine. Identifying this type of urine manipulation has been the aim of many previous studies. These studies have revealed urine adulterations through inappropriately high levels of "buprenorphine" and "naloxone" and a very small amount of "norbuprenorphine." So, does the small amount of "norbuprenorphine" in the adulterated urine samples result from dipped buprenorphine/naloxone film, or is it a residual metabolite of buprenorphine in the patient's system? This pilot study utilized 12 urine samples from 12 participants, as well as water samples as a control. The samples were subdivided by the dipping area and time, as well as the temperature and concentration of urine samples, and each sublingual generic buprenorphine/naloxone film was dipped directly into the samples. Then, the levels of "buprenorphine," "norbuprenorphine," "naloxone," "buprenorphine-glucuronide" and "norbuprenorphine-glucuronide" were examined by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). The results of this study showed that high levels of "buprenorphine" and "naloxone" and a small amount of "norbuprenorphine" were detected in both urine and water samples when the buprenorphine/naloxone film was dipped directly into these samples. However, no "buprenorphine-glucuronide" or "norbuprenorphine-glucuronide" were detected in any of the samples. In addition, the area and timing of dipping altered "buprenorphine" and "naloxone" levels, but concentration and temperature did not. This study's findings could help providers interpret their patients' urine drug test results more accurately, which then allows them to monitor patient compliance and help them identify manipulation by examining patient urine test results.

B-317 Poor Standardization of Specimen Validity Tests Leads to Significant Variation of Urine Adulteration Positivity Rates

Abstract Background Drugs of abuse testing is performed in multiple settings including pain management, pre-transplant evaluations, and employment screening. It is important to be able to accurately assess the integrity of urine specimens to detect efforts to mask the presence of drugs through dilution, substitution, or other means of adulteration (e.g., adding bleach or other chemicals). These detection methods, however, are not standardized and therefore adulteration detection performance may vary widely. The purpose of this study was to compare several specimen validity testing (SVT) methods. Methods Results for SVT testing performed in January 2023 were retrospectively reviewed for urine specimens submitted for occupational drug testing (ODT) or for which comprehensive drug screening (CDS) was ordered. Specimens screening positive or near the cut-offs for adulteration/dilution by UrineCheck®7 (UC7) test strips (creatinine, specific gravity (SG), pH, bleach, pyridinium chlorochromate (PC), nitrites, glutaraldehyde) or the Thermo DRI® (SG, pH, oxidants) and Beckman creatinine assays were included in the study. Additional testing by the Roche Cobas Pro SVT assays (creatinine, SG, pH, oxidants, nitrites, chromate), pH meter (Fisher accumet AB315) and SG refractometer (Reichert® Goldberg TS Meter) was performed (n = 7 ODT, 22 CDS). Nitrite-positive specimens submitted for routine urine analysis (UA) (n = 6) were also included to assess each assay’s interference potential (i.e., false positive) from nitrites produced from urinary tract infections. Results Of the 244 ODT results, 5 were “dilute” and 7 were positive for bleach, PC and/or nitrites by UC7 test strips. There were 603 CDS results, of which 23 had a urine creatinine <20 mg/dL but only 6 of these also had a low SG (<1.003) by DRI assays. Only one specimen was flagged for low pH by all methods except the DRI assay. Of the 5 ODT specimens that were positive for bleach and PC by UC7, none exceeded the cutoffs when tested on the DRI oxidant (100 µg/mL), Roche oxidants (200 mg/L), or Roche nitrites (500 mg/L) assays, however these specimens did have higher values than the other specimens in the ODT group for all assays. The UC7 assay was the most sensitive for oxidant detection although this study was not designed to differentiate true from false positive results. The Roche assay was the most sensitive to urinary tract infection-related nitrites although values were still well below the positive cutoff (Mean ± SD = 46.7 ± 11.1 mg/L). Surprisingly, pH had poor agreement among methods but good overall concordance. For the assessment of specimen dilution, creatinine values correlated well but specific gravity generally did not. Additionally, overall concordance was poor (14/35 specimens) when manufacturer-specific cutoffs were applied. Notably, cutoffs varied greatly between assays which likely contributed to the poor concordance more so than the variation in measured values. Conclusion These findings suggest that the choice of urine SVTs can have a significant impact on positivity rate due to the lack of standardization. While we have determined that sensitivity and specificity varies significantly between assays, further studies are needed to evaluate method accuracy and overall adulteration detection performance.

B-166 Effect of Urine Adulteration on Quantifying both Delta 8- and Delta 9-carboxy tetrahydrocannabinol using a New UPLC-tandem Mass Spectrometry Method

Abstract Background The legality and use of marijuana is a controversial topic. With increased prevalence of usage, cannabis detection for legal, medical, and workforce drug testing is on the rise. LC-MS/MS quantification of delta 9-carboxy tetrahydrocannabinol (delta 9-THC-COOH) in urine is accepted as the standard to detect marijuana use. One challenge faced by testing laboratories has been identifying adulterated samples. Many products/reagents can be used to manipulate the urine to avoid positive drug testing results. In this study, we developed a dilute-and-shoot strategy to separate and quantify both the delta 8- and 9-THC-COOH in urine samples and tested the effects of adulteration using a combination of nitrite and/or acids which can be used to mask cannabinoid use. Methods Five authentic delta 8- and 9-THC-COOH positive specimens, and one spiked sample were treated with strong acids (1N HCL or 10% TCA), 500 µg/mL nitrite, or a combination. After hydrolysis with sodium hydroxide, the analytes were quantified using a 6495 Mass Spectrometer coupled to a StreamSelect LC system (Agilent). LC separation was performed with a CORTECS T3 column utilizing a shallow gradient. In addition, twelve urine specimens with varying concentrations of delta 8- and 9-THC-COOH were treated with nitrite and screened using a delta 9-THC-COOH immunoassay on the Roche Cobas C501 and analyzed by LC-MS/MS. Results Nitrite did not change the physical appearance or the pH of the urine samples. Strong acid treatment also did not alter the appearance but did drop the urine pH ≤2. Pre-treated specimens with 1N HCL, 10% TCA, or 500 µg/mL of nitrite did not affect the delta 8- and 9-THC-COOH concentrations up to 6 days (refrigerated); however, specimens treated with strong acid combined with 500 µg/mL of nitrite for 30 min made both analytes undetectable. Additional experiments confirmed that 0.5 and 1 mol/L of nitrite lowered the concentration of all analytes, including internal standard, to undetectable levels in 48 h. Twelve additional specimens were collected, 3 with delta 8-THC-COOH, 3 with delta 9-THC-COOH, and 6 with both. All 12 samples screened positive using the Roche immunoassay and were quantified using the LC-MS/MS method. After treatment with 0.5 mol/L of nitrite, 10 of the 12 samples were negative using the immunoassay, but 2 samples remained positive. However, all nitrite treated specimens were negative (<LLOQ) using the LC-MS/MS method and the internal standard was eliminated. Later experiments confirmed that the delta 8- and 9-THC-COOH could be eliminated with as little as 0.1 mol/L nitrite for 30 min. Conclusion The unique dilute-and-shoot method can separate and quantify delta 8- and 9-THC-COOH. In addition, high concentrations of nitrite were shown to degrade both analytes along with the isotopically labeled internal standard. As a result, labs can distinguish true negative delta 8- and 9-THC-COOH (no analyte peaks) from nitrite adulterated specimens since the adulterated samples have no peaks for both the analytes and internal standard. A limitation of the study is that only sodium nitrite was tested. Other adulterants, such as potassium nitrite, pyridinium chlorochromate, peroxide etc. require further investigation.

Creatinine determination by origami paper-based device for identifying urine adulteration

The attendees will receive detailed information on the use of a novel tech to control the integrity of urine samples using paper-based microfluidics technology. The problem of sample tampering is well-known in forensic toxicology being sample dilution the most used method to cheat toxicological controls. Although the phenomenon is known for a long time in analytical toxicology, it resulted to be reported also in recent papers. Among the possible strategies to exclude urine dilution, the quantification of creatinine represents probably the most adopted method. Currently, creatinine determination is performed at analytical laboratories hindering the immediate check of the integrity of the sample. On these grounds, the goal of the present work was to develop a low-cost method able to provide on-site determination of creatinine in urine samples using paper-based microfluidic technology [Martinez AW et al, Angew. Chem. Int. Ed. Engl. 46 (2007) 1318–20]. The procedure for the production of origami paper-based microfluidic devices (μPADs) included two steps: (i) patterning chromatography paper into hydrophilic channels by fabricating hydrophobic barriers; (ii) addition of the reagents specific for creatinine (picric acid, 3,5-dinitrobenzoic acid and Nessler's reagent) to the hydrophilic portion of the paper support. The analysis was performed transferring 30 μL urine without any pre-treatment onto the hydrophilic portion of the paper in order to permit the interaction of creatine with the reagents with consequent formation of colored products. The colorimetric reactions occurred in few minutes and were measured in terms of “RGB distance” by using a simple and free software for smartphone cameras. The device was validated for quantitative determinations in terms of accuracy and precision, resulting both within 20%. The optimized method was then tested on real urine samples ( n = 53) using as reference a clinical chemistry method performed on immunoassay instrument. The comparison between the two methods showed a significant correlation (R = 0.92). The proposed alternative analytical strategy to identify urine adulteration looks particularly attractive since it is low-cost and easy to use. In addition, the possibility of performing the test on-site prevents claims of post-collection counterfeiting.

Uncovering urine adulteration attempts in forensic toxicology: A LC-MS based proteomics approach

Sample adulteration aims at circumventing positive drug-testing results in a clinical, forensic or doping context by means of dilution, sample replacement or chemical alteration. Approaches employing liquid-chromatography mass-spectrometry (LC-MS) based metabolomics analysis showed promising results to identify manipulated samples, although identification and verification of actual biomarkers remains a challenging task. With this study we wanted to expand further to detect sample adulteration with hydrogen peroxide (H2O2) in human urine by LC-MS based protein-analysis. Urine samples from ten donors were split into two groups. One group was left as is, the other was treated with 10% H2O2 for 30 minutes. Urinary proteins were isolated, purified and enzymatically digested overnight with trypsin. 5 μg of digested protein were injected and separated on a Dionex LC system equipped with an Acclaim PepMap column (1.0 mm × 150 mm, 2 μm) running a gradient from 3% ACN, 3% DMSO and 0.1% formic acid to 28% ACN, 3% DMSO and 0.1% formic acid over 60 minutes at a flow-rate of 50 μl/min and measured by high-resolution quadrupole time-of-flight mass spectrometry (Sciex 6600). The acquired data was analysed using FragPipe (v17.1), where retention-time alignment, normalization, identification and label-free quantification of peptides and proteins was performed. Protein and peptide abundances were further evaluated and visualized with Prism. Across all urine samples, we were able to reliably identify and quantify a total of 1250 peptides corresponding to 180 different proteins. Protein and peptide abundances showed distinct differences in samples treated with 10% H2O2 compared to untreated urine samples. Several proteins and peptides were significantly affected by the H2O2 treatment and either increased or decreased in quantity. For instance, uromodulin (UROM_HUMAN), cytoskeletal keratin (K2C1_HUMAN) and immunoglobulin lambda (IGLC7_HUMAN) showed a more than 5-fold decrease after oxidative treatment ( P < 0.005) and alpha-1-acid glycoprotein (ORM1) could not be detected anymore after treatment with H2O2 in all measured samples. Beyond that, alterations to the primary amino-acid sequences could also be observed. H2O2 should mainly introduce oxidative changes to the primary amino-acid sequence of proteins and peptides. Methionine, tryptophan, lysine and tyrosine could be found in either oxidized (15.9949 Da) or dioxidized (+31.9898 Da) variants. These modified peptides, such as QM_dioxPGKGLEWM_oxGR (HV5X1_HUMAN) or VSLKTALQPM_dioxVSALNIR (UROM_HUMAN) could proof themselves as ideal biomarker peptides for exposing oxidative adulteration, as they could only be observed after aggressive oxidative treatment and are not present under normal physiological conditions in human urine. Further, the analysis of the human urinary proteome bears another advantage: It is likely very hard to fool the forensic toxicologist using synthetic urine as a substitute in urine drug-testing by using the approach presented here. The inherent complexity of the human physiology that ultimately results in the secreted urinary proteome is too complex to be recreated by any means other than substituting it with the urine of another, preferably drug-negative, healthy person. Consequentially, analysing the urinary proteome can already expose two common adulteration approaches: Using synthetic urine and chemical adulteration with H2O2. Sample adulteration aims at producing false-negative drug-testing results. With the study presented here, we explored the possibility of using LC-MS based proteomics analysis to reveal chemically altered urine samples and ultimately avoid false-negative results. We found distinct quantitative changes as well as possible marker peptides that are only present in oxidatively treated urine samples. Even though by no means standard in a forensic context, a method such as this could be employed when in doubt about the integrity of an authentic urine sample prior or after routine toxicological analysis and might, eventually, extent the toolbox that is available to the forensic toxicologist in routine casework.

Using Machine Learning to Predict Treatment Adherence in Patients on Medication for Opioid Use Disorder.

Patients receiving medication for opioid use disorder (MOUD) may continue using nonprescribed drugs or have trouble with medication adherence, and it is difficult to predict which patients will continue to do so. In this study, we develop and validate an automated risk-modeling framework to predict opioid abstinence and medication adherence at a patient's next attended appointment and evaluate the predictive performance of machine-learning algorithms versus logistic regression. Urine drug screen and attendance records from 40,005 appointments drawn from 2742 patients at a multilocation office-based MOUD program were used to train logistic regression, logistic ridge regression, and XGBoost models to predict a composite indicator of treatment adherence (opioid-negative and norbuprenorphine-positive urine, no evidence of urine adulteration) at next attended appointment. The XGBoost model had similar accuracy and discriminative ability (accuracy, 88%; area under the receiver operating curve, 0.87) to the two logistic regression models (accuracy, 88%; area under the receiver operating curve, 0.87). The XGBoost model had nearly perfect calibration in independent validation data; the logistic and ridge regression models slightly overestimated adherence likelihood. Historical treatment adherence, attendance rate, and fentanyl-positive urine at current appointment were the strongest contributors to treatment adherence at next attended appointment. There is a need for risk prediction tools to improve delivery of MOUD. This study presents an automated and portable risk-modeling framework to predict treatment adherence at each patient's next attended appointment. The XGBoost algorithm appears to provide similar classification accuracy to logistic regression models; however, XGBoost may offer improved calibration of risk estimates compared with logistic regression.

Update on Urine Adulterants and Synthetic Urine Samples to Subvert Urine Drug Testing.

To avoid a positive urine drug test, donors might try to subvert the test, either by adulterating the specimen with a product designed to interfere with testing or by substituting the specimen for a synthetic urine. A market search conducted in December of 2020 identified 3 adulterants and 32 synthetic urines, and a selection was procured based on specific criteria. Samples prepared with the 3 adulterants and 10 synthetic urines were submitted for testing at five forensic drug testing laboratories to perform immunoassay screening, chromatographic confirmation analysis and specimen validity testing (SVT). One adulterant determined to contain iodate reduced THC-COOH concentrations by 65% and the concentrations of 6-acetylmorphine, morphine, oxycodone, oxymorphone, hydrocodone and hydromorphone by 6-27%. Another adulterant determined to contain nitrite reduced THC-COOH concentrations by 22%, while the third did not affect drug screening or confirmatory testing. Both active adulterants could be identified through positive oxidant screens as well as through signal suppression in cloned enzyme donor immunoassay (CEDIA). The synthetic urines could not be identified either through traditional SVT or by the AdultaCheck10 dipstick. The Synthetic UrineCheck dipstick produced a difference in response between the authentic urine specimen and the synthetic urine samples, but the difference was small and difficult to observe. While most synthetic urines now contain uric acid, magnesium and caffeine, the results indicated that a biomarker panel including endogenous and exogenous markers of authentic urine performed well and clearly demonstrated the absence of biomarkers in the synthetic urines. The SVT assay also offers potential targets for future screening assays.

Interpretable machine learning model to detect chemically adulterated urine samples analyzed by high resolution mass spectrometry.

Urine sample manipulation including substitution, dilution, and chemical adulteration is a continuing challenge for workplace drug testing, abstinence control, and doping control laboratories. The simultaneous detection of sample manipulation and prohibited drugs within one single analytical measurement would be highly advantageous. Machine learning algorithms are able to learn from existing datasets and predict outcomes of new data, which are unknown to the model. Authentic human urine samples were treated with pyridinium chlorochromate, potassium nitrite, hydrogen peroxide, iodine, sodium hypochlorite, and water as control. In total, 702 samples, measured with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, were used. After retention time alignment within Progenesis QI, an artificial neural network was trained with 500 samples, each featuring 33,448 values. The feature importance was analyzed with the local interpretable model-agnostic explanations approach. Following 10-fold cross-validation, the mean sensitivity, specificity, positive predictive value, and negative predictive value was 88.9, 92.0, 91.9, and 89.2%, respectively. A diverse test set (n=202) containing treated and untreated urine samples could be correctly classified with an accuracy of 95.4%. In addition, 14 important features and four potential biomarkers were extracted. With interpretable retention time aligned liquid chromatography high-resolution mass spectrometry data, a reliable machine learning model could be established that rapidly uncovers chemical urine manipulation. The incorporation of our model into routine clinical or forensic analysis allows simultaneous LC-MS analysis and sample integrity testing in one run, thus revolutionizing this field of drug testing.

Effect of some new urine adulterants on tramadol positive samples using screening tests

Effect of some new urine adulterants on tramadol positive samples using screening tests

Review on The Effects of Adulterants on Drugabuse Testing in Urine Samples

Introduction: A growing concern over the use of illicit drugs in the work place has led to an interest in urine analysis as a way to detect drug abuse. Sample adulteration is a serious potential problem in forensic urine drug testing. Federal guidelines define an adulterated specimen as a urine specimen containing a substance that is not a normal constituent or containing an endogenous substance at a concentration that is not a normal physiologic concentration. Adulterants act by either interfering with immunoassay procedures or by converting the target drugs to other compounds. Once the adulterants are converted to other compounds they do not bind to the antibodies used in immunoassay. In some cases these converted compound produce false negative results in confirmatory testing. Adulterants can be classified into two categories. The first category includes in vivo adulteration comprising intentional ingestion of fluids, substances or drugs designed to dilute urine. The second category includes in vitro adulteration such as common household chemicals and nitrite containing agents. Methods of detection of urine adulterants include urine integrity tests, color tests and spectrophotometric methods.

Effect of urine adulterants on commercial drug abuse screening test strip results.

Immunochromatographic strips for urine drug screening tests (UDSTs) are common and very suitable for drug abuse monitoring, but are also highly susceptible to adulterants kept in the household, which can significantly alter test results. The aim of this study was to see how some of these common adulterants affect UDST results in practice and whether they can be detected by sample validity tests with pH and URIT 11G test strips. To this end we added household chemicals (acids, alkalis, oxidizing agents, surfactants, and miscellaneous substances) to urine samples positive for amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), tetrahydrocannabinol, heroin, cocaine, or benzodiazepines (diazepam or alprazolam) and tested them with one-component immunochromatographic UDST strips. The UDST for cocaine resisted adulteration the most, while the cannabis test produced the most false negative results. The most potent adulterant that barely changed the physiological properties of urine specimens and therefore escaped adulteration detection was vinegar. Besides lemon juice, it produced the most false negative test results. In conclusion, some urine adulterants, such as vinegar, could pass urine specimen validity test and remain undetected by laboratory testing. Our findings raise concern about this issue of preventing urine tampering and call for better control at sampling, privacy concerns notwithstanding, and better sample validity tests.

Influence of zinc and some commercial products on tramadol and apetryl detection in human urine samples

Urine drug testing plays an important role in detecting licit and illicit drug use. Adulteration of urine samples have been employed to disrupt these drug tests. Recently, zinc sulfate was used as an effective adulterant to bypass drug testing. Aim of the work: The aim of the current study was to evaluate the effect of zinc sulfate and some adulteration methods on detection of tramadol and apetryl in urine and their effects on the validity tests. Method: Tramadol and apetryl were added to urine sample that were obtained from a healthy, drug-free subject to yield samples of urine containing tramadol and apetryl with concentrations (200 ng/ ml & 400 ng/ ml). Visine eye drop, zinc sulfate and lemon were added to these samples as well as dilution was done. These samples were tested for their ability to generate false negative results for the immunoassay test screen. Results: All adulterated urine samples as regard both drugs generated false negatives results. PH paper test showed more acidic PH with lemon juice while visine, zinc sulfate and dilution gave light green coloration indicating slightly acidic PH. Specific gravity showed increase of specific gravity for urine adulterated with lemon but decrease in diluted urine. Conclusion: There are needs to a more effective and efficient approach to urinalysis due to the false negatives that can result from adulteration of urine samples.

Determination of cocaine adulterants in human urine by dispersive liquid-liquid microextraction and high-performance liquid chromatography.

This study aimed to determine simultaneously five major street cocaine adulterants (caffeine, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine by dispersive liquid-liquid microextraction (DLLME) and high-performance liquid chromatography. The chromatographic separation was obtained in gradient elution mode using methanol:water plus trifluoroacetic acid 0.15% (v/v) (pH = 1.9) at 1mLmin-1 as mobile phase, at 25°C, detection at 235nm, and analysis time of 20min. The effect of major DLLME operating parameters on extraction efficiency was explored using the multifactorial experimental design approach. The optimum extraction condition was set as 4mL human urine sample alkalized with 0.5M sodium phosphate buffer (pH 12), NaCl (15%, m/v), 300μL acetonitrile (dispersive solvent), and 800μL chloroform (extraction solvent). Linear response (r2 ≥ 0.99) was obtained in the range of 180-1500ngmL-1 with suitable selectivity, quantification limit (180ngmL-1), mean recoveries (33.43-76.63%), and showing relative standard deviation and error (within and between-day assays) ≤15%. The analytes were stable after a freeze-thaw cycle and a short-term room temperature stability test. This method was successfully applied in real samples of cocaine users, suggesting that our study may contribute to the appropriate treatment of cocaine dependence or with the cases of cocaine acute intoxication.

Evaluation of endogenous urinary biomarkers for indirect detection of urine adulteration attempts by five different chemical adulterants in mass spectrometry methods.

Reliable detection of urine adulteration attempts to circumvent positive drug testing represents a critical step for laboratories in abstinence control settings. An ideal workflow for high-throughput testing would involve simultaneous detection of adulteration attempts in the same run with drug detection. Monitoring of degraded or oxidized endogenous urinary compounds as indirect markers has been previously evaluated for that purpose exemplified for the adulterant potassium nitrite (KNO2 ). Fifteen, previously identified endogenous markers should now be evaluated for their general applicability to detect adulteration attempts for the adulterants hypochlorite-based bleach (NaOCl), peroxidase and peroxide (H2 O2 ), pyridinium chlorochromate (PCC), and iodine (I2 ). Initial experiments revealed similar results for the tested adulterants regarding degradation of indolylacryloylglycine (IAG), uric acid (UA), or UA derivatives. 5-Hydroxyisourate (HIU), the oxidation product of UA, was however only formed by KNO2 , PCC, and H2 O2 . Amino acids showed larger adulterant-dependent differences. All reactions were shown to be influenced by the adulterant concentration and the urinary pH with large variances depending on compound and adulterant. Except for HIU/PCC, all markers were stable within +/- 30% variation for all adulterants at -20°C. Receiver operating characteristics indicated best sensitivity and specificity over all adulterants for IAG (specificity 0.9, sensitivity 1.0) and UA (specificity 1.0, sensitivity 0.9). HIU gave best results for KNO2 , PCC, and H2 O2 and N-acetylneuraminic acid for PCC and H2 O2 , respectively. When integrating a limited number of targets into existing screening methods, monitoring of UA, IAG, N-acetylneuraminic acid, and HIU is recommended.

Suitability evaluation of new endogenous biomarkers for the identification of nitrite-based urine adulteration in mass spectrometry methods.

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5-hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO2 ). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO2 concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and-20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after biomarker quantification (n=100 per group). Blinded measurements (n=50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over onemonth. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO2 and are ready for implementation into routine MS procedures.

Systematic investigations of novel validity parameters in urine drug testing and prevalence of urine adulteration in a two-year cohort.

Urinalysis is well established for drug screening. Various methods of urine adulteration such as dilution, addition of oxidative/reductive chemicals or detergents, and handing over urine-like fluids are used to circumvent a positive screen. Validity parameters such as determination of pH, gravidity, urine temperature, or testing for oxidative/reductive chemicals are therefore used to uncover adulterated urine specimens. However, synthetic urine ("fake urine") has nowadays been used for manipulations, leading to inconspicuous results with common validity test systems. Therefore, the aims of the study were (a) to evaluate additional validity parameters, (b) to evaluate the prevalence of urine adulteration, (c) to identify adulteration markers in purchased fake urine samples. Urine samples (n=550) submitted for drug abstinence testing were analyzed by a standard urine liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach using library-assisted identification of 10 different endogenous biomolecules. The detection rates of biomolecules in authentic samples were phenylalanine (93.4%), tryptophan (97.1%), propionyl-carnitine (67.1%), butyryl-carnitine (99.6%), isovaleryl-carnitine (92.8%), hexanoyl-carnitine (91.0%), heptanoyl-carnitine (97.1%), octanoyl-carnitine (98.9%), and indoleacetylglutamine (98,2%). Phenylacetylglutamine was detected in each authentic sample. Based on the detection rates and measured creatinine levels, six manipulated samples were identified in this study. In two cases, fake urine was handed over, one time fake urine was most likely used for dilution. Once dilution with other fluids was used as adulteration method, while in another sample a detergent solution was handed over. Additionaly, one sample contained reactive chemicals. All fake urine samples were additionally identified by the detection of unique polyglycole patterns, which were observed in purchased fake urine samples.

Addressing discordant quantitative urine buprenorphine and norbuprenorphine levels: Case examples in opioid use disorder

IntroductionUrine adulteration is a concern among patients treated for opioid use disorder. Quantitative urine testing for buprenorphine (B) and norbuprenorphine (NB), and the appropriate interpretation of B and NB levels, can facilitate constructive conversations with patients that may lead to modifications in the treatment plan, and strengthening of the patient-provider relationship. Case summaryThree cases are presented in which discordant urine B and NB levels were recognized. Each patient was submerging buprenorphine/naloxone strips in their urine to mask ongoing illicit drug use. The authors used an approach to addressing intentional adulteration of urine samples that adheres to the principles of harm-reduction, the centrality of the patient-provider relationship, and the acknowledgment that ongoing illicit drug use and subsequent dishonesty about disclosure may be common among persons with substance use disorders. Each of the three patients ultimately endorsed diluting their urine, which allowed for strengthening of the patient-provider relationship and modifications to their treatment plans. Two of the three patients stabilized and achieved abstinence, while the third was eventually referred to a methadone treatment program. ConclusionProviders should routinely monitor B and NB levels, rather than qualitative screening alone, and discordant levels should elicit a timely conversation with the patient. The authors use of a nonjudgmental approach to address urine adulteration, including giving patients an opportunity to reflect on potential solutions, has been effective at helping patients and providers to reestablish a therapeutic alliance and maintain retention in treatment.

Interpreting quantitative urine buprenorphine and norbuprenorphine levels in office-based clinical practice

BackgroundQuantitative urine buprenorphine testing is used to monitor patients receiving buprenorphine for the treatment of opioid use disorder (OUD), however the interpretation of urine buprenorphine testing is complex. Currently, interpretation of quantitative buprenorphine testing is guided by data from drug assay development studies and forensic labs rather than clinical treatment cohorts. MethodsIn this retrospective study, we describe the patterns of urine buprenorphine and norbuprenorphine levels in patients prescribed sublingual buprenorphine for OUD in an office-based addiction treatment clinic. Urine buprenorphine and norbuprenorphine levels were analyzed in patients who reported having adulterated their urine, patients clinically suspected of adulterating their urine, and patients without concern for urine adulteration. Finally, we tested the accuracy of urine buprenorphine, norbuprenorphine, and norbuprenorphine: buprenorphine ratio (Norbup:Bup) to identify adulterated urine samples. ResultsPatients without suspicion for urine adulteration rarely provided specimens with buprenorphine >=1000ng/ml (4.4%), while the proportion provided by those who endorsed or were suspected of urine adulteration was higher (42.9%, 40.6%, respectively). Compared to patients without reported urine adulteration, specimens from patients who reported or were suspected of urine adulteration had significantly higher buprenorphine (p=0.0001) and lower norbuprenorphine (<0.0001) levels, and significantly lower Norbup:Bup ratios (p=0.04). Buprenorphine >=700ng/ml offered the best accuracy for discriminating between adulterated and non-adulterated specimens. ConclusionThis study describes the patterns of urine buprenorphine and norbuprenorphine levels from patients with OUD receiving buprenorphine treatment in an office-based addiction treatment clinic. Parameters for identifying urine adulterated by submerging buprenorphine medication in the urine specimen are discussed.

A new metabolomics-based strategy for identification of endogenous markers of urine adulteration attempts exemplified for potassium nitrite.

Urine adulteration to circumvent positive drug testing represents a problem for toxicological laboratories. While creatinine is a suitable marker for dilution, detection of chemicals is often performed by dipstick tests associated with high rates of false positives. Several methods would be necessary to check for all possible adulterants. Untargeted mass spectrometry (MS) methods used in metabolomics should theoretically allow detecting concentration changes of any endogenous urinary metabolite or presence of new biomarkers produced by chemical adulteration. As a proof of concept study, urine samples from 10 volunteers were treated with KNO2 and analyzed by high-resolution MS. For statistical data evaluation, XCMSplus and MetaboAnalyst were used. Compound identification was performed by database searches using an in-house database, Chemspider, METLIN, HMDB, and NIST. Principle component analysis revealed clear separation between treated and untreated urine samples. In detail, 307 features showed significant concentration changes with fold changes greater than 2 (79 decreased; 228 increased). Mainly amino acids (e.g., histidine, methylhistidine, di- and trimethyllysine) and purines (uric acid) were detected in lower amounts. 5-HO-isourate was found to be formed as a new compound from uric acid and, e.g., imidazole lactate concentrations increased due to the breakdown of histidine. This metabolomics-based strategy allowed for a broad identification range of markers of urinary adulteration. More studies will be needed to investigate routine applicability of identified potential markers exploring urinary conditions of their formation and stability. Selected markers might then be integrated into routine MS screening procedures allowing for detection of adulteration within routine MS analysis. Graphical Abstract ᅟ.