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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Metabolomic Strategies in Biomarker Research-New Approach for Indirect Identification of Drug Consumption and Sample Manipulation in Clinical and Forensic Toxicology?

Drug of abuse (DOA) consumption is a growing problem worldwide, particularly with increasing numbers of new psychoactive substances (NPS) entering the drug market. Generally, little information on their adverse effects and toxicity are available. The direct detection and identification of NPS is an analytical challenge due to their ephemerality on the drug scene. An approach that does not directly focus on the structural detection of an analyte or its metabolites, would be beneficial for this complex analytical scenario and the development of alternative screening methods could help to provide fast response on suspected NPS consumption. A metabolomics approach might represent such an alternative strategy for the identification of biomarkers for different questions in DOA testing. Metabolomics is the monitoring of changes in small (endogenous) molecules (<1,000 Da) in response to a certain stimulus, e.g., DOA consumption. For this review, a literature search targeting "metabolomics" and different DOAs or NPS was conducted. Thereby, different applications of metabolomic strategies in biomarker research for DOA identification were identified: (a) as an additional tool for metabolism studies bearing the major advantage that particularly a priori unknown or unexpected metabolites can be identified; and (b) for identification of endogenous biomarker or metabolite patterns, e.g., for synthetic cannabinoids or also to indirectly detect urine manipulation attempts by chemical adulteration or replacement with artificial urine samples. The majority of the currently available literature in that field, however, deals with metabolomic studies for DOAs to better assess their acute or chronic effects or to find biomarkers for drug addiction and tolerance. Certain changes in endogenous compounds are detected for all studied DOAs, but often similar compounds/pathways are influenced. When evaluating these studies with regard to possible biomarkers for drug consumption, the observed changes appear, albeit statistically significant, too small to reliably work as biomarker for drug consumption. Further, different drugs were shown to affect the same pathways. In conclusion, metabolomic approaches possess potential for detection of biomarkers indicating drug consumption. More studies, including more sensitive targeted analyses, multi-variant statistical models or deep-learning approaches are needed to fully explore the potential of omics science in DOA testing.

Can synthetic urine replace authentic urine to "beat" workplace drug testing?

Synthetic urine (SU), which was primarily utilized by drug testing laboratories as a matrix for quality control preparations, is now commercially sold and can be used to "fool" a positive drug test. To determine if SU can pass as authentic urine, we challenged Army urine drug testing specimen accessioning and testing procedures using eight different commercial SU products. Adulteration (Sciteck AdultaCheck® 6) and Onsite SU (Synthetic UrineCheck™) test strips were also evaluated. Five of the eight SU were identified by physical observation. All SU products screened negative in the drug immunoassay and additionally passed the specimen validity testing (SVT) as authentic urine. Furthermore, SU was not detected as adulterated with the adulteration test strips (Sciteck AdultaCheck® 6) but was successfully detected as SU with the On-site synthetic urine (Synthetic UrineCheck™). To deter SU use, direct observation, as utilized by the military, may be recommended during the collection process.

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 (pH 4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over one month. 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.

Determining the effects of adulterants on drug detection via enzyme-linked immunosorbent assay and adulterant tests strips.

The use of immunoassays for drug screening has increased due to their sensitivity towards target analytes, specifically the enzyme-linked immunosorbent assay (ELISA) technique. Adulterant test strips are commonly used in conjunction with immunoassay tests to ensure the integrity of the specimen has not been altered prior to drug screening. This research focuses on determining which adulterants can produce a false negative result, not only with 1 of the screening tests, but throughout the entire screening process. Seven adulterants were tested for their ability to generate false negative results for the ELISA by decreasing the detectable antigen concentration to below industry-used cut-off levels. Each adulterant was added to a urine sample containing 1 of 5 different drugs at 5 different concentrations (0, 5, 10, 25, and 50%). Five different urine samples that contained drugs and drug metabolites of benzoylecgonine, THC-COOH, α-PVP, D-amphetamine, and Diazepam, were treated with each of the 7 adulterants and analyzed on the ELISA and subsequently by 2 different test strips. The results indicated that 4 adulterants (ie, bleach, Drano®, vinegar, and sodium nitrite) generated the most false negatives for both test strips and the ELISA at surprisingly low concentrations, ~5% v/v. Thus indicating that there are still ways that a urine sample that contains drugs could be analyzed and labeled "clean and free of drugs" after going through a common screening process. These results suggest that new drug screening techniques need to be developed to detect adulterants in urine samples for drug screening.

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.

Quantitative testing of buprenorphine and norbuprenorphine to identify urine sample spiking during office-based opioid treatment.

BACKGROUND: Patients may spike urine samples with buprenorphine during office-based opioid treatment to simulate adherence to prescribed buprenorphine, potentially to conceal diversion of medications. However, routine immunoassay screens do not detect instances of spiking, as these would simply result in a positive result. The aim of this study was to report on the experience of using quantitative urine testing for buprenorphine and norbuprenorphine to facilitate the identification of urine spiking. METHODS: This is a retrospective chart review of 168 consecutive patients enrolled in outpatient buprenorphine treatment at an urban academic medical setting between May 2013 and August 2014. All urine samples submitted were subjected to quantitative urine toxicology testing for buprenorphine and norbuprenorphine. Norbuprenorphine-to-buprenorphine ratio of less than 0.02 were further examined for possible spiking. Demographic and clinical variables were also extracted from medical records. Clinical and demographic variables of those who did and did not spike their urines were compared. Statistically significant variables from the univariate testing were entered as predictors of spiking in a regression analysis. RESULTS: A total of 168 patients were included, submitting a total of 2275 urine samples. Patients provided on average 13.6 (SD = 9.9) samples, and were in treatment for an average 153.1 days (SD = 142.2). In total, 8 samples (0.35%) from 8 patients (4.8%) were deemed to be spiked. All of the samples suspected of spiking contained buprenorphine levels greater than 2000 ng/mL, with a mean norbuprenorphine level of 11.9 ng/mL. Spiked samples were submitted by 6 patients (75.0%) during the intensive outpatient (IOP) phase of treatment, 2 patients (25.0%) during the weekly phase, and none from the monthly phase. Regression analysis indicated that history of intravenous drug use and submission of cocaine-positive urine samples at baseline were significant predictors of urine spiking. CONCLUSIONS: Even though only a small number of patients were identified to have spiked their urine samples, quantitative testing may help identify urine spiking during office-based opioid treatment with buprenorphine.

Adulterants in Urine Drug Testing.

Urine drug testing plays an important role in monitoring licit and illicit drug use for both medico-legal and clinical purposes. One of the major challenges of urine drug testing is adulteration, a practice involving manipulation of a urine specimen with chemical adulterants to produce a false negative test result. This problem is compounded by the number of easily obtained chemicals that can effectively adulterate a urine specimen. Common adulterants include some household chemicals such as hypochlorite bleach, laundry detergent, table salt, and toilet bowl cleaner and many commercial products such as UrinAid (glutaraldehyde), Stealth® (containing peroxidase and peroxide), Urine Luck (pyridinium chlorochromate, PCC), and Klear® (potassium nitrite) available through the Internet. These adulterants can invalidate a screening test result, a confirmatory test result, or both. To counteract urine adulteration, drug testing laboratories have developed a number of analytical methods to detect adulterants in a urine specimen. While these methods are useful in detecting urine adulteration when such activities are suspected, they do not reveal what types of drugs are being concealed. This is particularly the case when oxidizing urine adulterants are involved as these oxidants are capable of destroying drugs and their metabolites in urine, rendering the drug analytes undetectable by any testing technology. One promising approach to address this current limitation has been the use of unique oxidation products formed from reaction of drug analytes with oxidizing adulterants as markers for monitoring drug misuse and urine adulteration. This novel approach will ultimately improve the effectiveness of the current urine drug testing programs.

Evaluation and analytical validation of a handheld digital refractometer for urine specific gravity measurement.

OBJECTIVES: Refractometers are commonly used to determine urine specific gravity (SG) in the assessment of hydration status and urine specimen validity testing. Few comprehensive performance evaluations are available demonstrating refractometer capability from a clinical laboratory perspective. The objective of this study was therefore to conduct an analytical validation of a handheld digital refractometer used for human urine SG testing. DESIGN AND METHODS: A MISCO Palm Abbe™ refractometer was used for all experiments, including device familiarization, carryover, precision, accuracy, linearity, analytical sensitivity, evaluation of potential substances which contribute to SG (i.e. "interference"), and reference interval evaluation. A manual refractometer, urine osmometer, and a solute score (sum of urine chloride, creatinine, glucose, potassium, sodium, total protein, and urea nitrogen; all in mg/dL) were used as comparative methods for accuracy assessment. RESULTS: Significant carryover was not observed. A wash step was still included as good laboratory practice. Low imprecision (%CV, <0.01) was demonstrated using low and high QC material. Accuracy studies showed strong correlation to manual refractometry. Linear correlation was also demonstrated between SG, osmolality, and solute score. Linearity of Palm Abbe performance was verified with observed error of ≤0.1%. Increases in SG were observed with increasing concentrations of albumin, creatinine, glucose, hemoglobin, sodium chloride, and urea. Transference of a previously published urine SG reference interval of 1.0020-1.0300 was validated. CONCLUSIONS: The Palm Abbe digital refractometer was a fast, simple, and accurate way to measure urine SG. Analytical validity was confirmed by the present experiments.

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

In vitro urine adulteration is a well-documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine-3-glucuronide and morphine-6-glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography-mass spectrometry (LC-MS) when morphine and morphine-6-glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2-nitro-morphine and 2-nitro-morphine-6-glucuronide, respectively. These reaction products were also formed when an authentic morphine-positive urine specimen was fortified with nitrite. 2-Nitro-morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography-mass spectrometry (GC-MS) after forming a trimethylsilyl derivative. On the contrary, morphine-3-glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC-MS and GC-MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide as markers for the indirect monitoring of morphine and morphine-6-glucuronide in urine specimens adulterated with nitrite.