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Abstract INTRODUCTION:
In North America, there is mounting evidence from various sources such as drug checking services, law enforcement drug seizures, and coroners’ reports that unregulated fentanyl supply increasingly contains various fentanyl analogues, animal tranquilizers, and designer benzodiazepines. These mixes, of which people who use drugs are often not aware, can increase the toxicity and risk of overdose and overdose-related death. In our clinical laboratory, we aim to regularly update our targeted LC-MS/MS urine broad spectrum toxicology screen to include new drugs of potential clinical relevance. As previously published, we use Toronto Drug Checking Services bi-weekly reports as an important surveillance tool to learn about new drugs emerging and taking hold in the regional drug supply. In 2024, we updated our urine drug screen method to include Flubromazepam, HO-Flubromazepam, Bromazolam, HO-Bromazolam, Fluorofentanyl, Xylazine, Phenacetin, Isotonitazene, N-Desethylisotonitazene, Protonitazene, N-Desethylprotonitazene, N-Desethylmetonitazene, and HO-Nitazene. The assay was validated for accuracy, precision, carryover, sample and extract stability. Here we describe the method development and validation protocol for the update as well as the positivity of new drugs in urine samples.
METHOD:
Standards were purchased from Sigma Aldrich and Cayman Chemicals. Target solutions (100 ng/mL) were prepared in 0.1% formic acid (FA) in water – methanol (MeOH) (1:1) and infused at a flow rate of 10uL/min into the mass spectrometer to identify the most abundant transitions per analyte and optimize the mass spectrometric parameters.
The sample preparation was adopted from the validated and in use LifeLabs Broad Spectrum Toxicology method. In brief, 100µL of sample containing internal standards underwent enzymatic hydrolysis by treatment with KURA BG100 glucuronidase (0.1M acetate buffer, pH=4.8) at 60 °C for 30min. The samples were then acidified with 2% FA and underwent cation exchange solid phase extraction (SPE). The analytes were recovered with 5% NH4OH in ACN/MeOH (1:1, v/v) and the eluents were transferred for evaporation. Following SPE and dry-down, samples were reconstituted in 20% MeOH with 0.1% FA and submitted for analysis by LC-MS/MS (Shimadzu Nexera - Sciex Triple Quad 4500).
The HPLC system was equipped with a 2-position/10-port switching valve and two columns. Separation of the analytes was achieved under gradient elution on a 2.1 × 50 mm Kinetex Biphenyl column and at a flow rate of 0.5ml/min. Mobile phases consisted of 0.1% FA in water and 0.1% FA in MeOH. Analytes were detected by monitoring at least two transitions for each compound.
RESULTS:
Positive identification of each compound was based on several criteria including signal/noise, retention times (between two ion transitions and relative to the standard material), ion ratio, and the presence of parent/metabolite pairs, where applicable. It was achieved by comparison of a sample’s LC/MS-MS data with data from a quality control supermix, containing all the target compounds present at the reporting cut-off concentration. The reporting cut-off was considered the lowest concentration at which each compound can be reproducibly detected in various urine matrices. The reporting cut-off concentration was validated at 2 ng/mL for Fluorofentanyl, Bromazolam, HO- Bromazolam, Flubromazepam, HO-Flubromazepam and Xylazine and 5 ng/mL for the nitazene drug targets.
The intra and inter day precision of the assay was validated by analyzing quality control standards for four days. Coefficient variation of ion ratio and area ratio of the quantifier ion to the area of the Internal standard were calculated. The acceptance criteria for this study were the following. Intra-day precision: %CV of both area ratio and ion ratio <20% (n=8). Inter-day precision: %CV of both area ratio and ion ratio <30% (n=32). Our results showed that all acceptance criteria were met for the new compounds.
47 positive urine samples and 3 standard-spiked urine samples were used for a method comparison study with a High-Resolution Mass Spectrometry method and a second LC-MS/MS method. Patient correlation showed 100% concordance, considering differences in reporting cut-offs and testing menu between the methods.
Stability studies showed that all analytes were stable after 21 days of storage (8 days at 4°C, then transferred at -20°C up to 21 days) and after 2 freeze-thaw cycles. Extracted samples were stable after 4 days at 4°C.
Autosampler and column carryover were validated by injections of a high concentration sample (concentration of targets: 25,000 ng/mL) followed by injections of urine blanks. No autosampler carryover was detected for any of the compounds. Column carryover was detected for Flubromazepam and Bromazolam, but it is unlikely that such high concentration will be observed in urine samples.
Data review of drug positivity from March to September 2024 (47297 samples) show that, on average, 18% of urine samples screened contained a fentanyl analogue, Fluorofentanyl. 49% of Fluorofentanyl samples are co-positive with a tranquilizer, Xylazine, and 86% are co-positive with a designer benzodiazepine, Bromazolam. The nitazene drugs included in our menu were detected in less than 0.5% of samples screened in this period, where they were present in samples co-positive for Fentanyl, and in samples co-positive for Oxycodone.
CONCLUSION:
Here we describe a protocol that can be used to update an existing targeted LC-MS/MS drug screen with new compounds as they emerge in the local drug supply. The total time for the completion of method development and validation was two months, including literature search, procurement of materials and completion of all the stability studies. Urine screen positivity for the new compounds correlated with the trends observed by the Toronto Drug Checking services.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
