Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection.

BACKGROUND: High-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. RESULT: Automatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 µL droplet on MALDI plate, high recovery (76-116%) and rapid evaporation in 2 min. The analytes were concentrated 4-8 times, forming even crystallization, reaching the detection limit at the concentration of 50 µg L-1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%-114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SIGNIFICANCE: The ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

Rapid and sensitive dilute-and-shoot analysis using LC-MS-MS for identification of multi-class psychoactive substances in human urine.

The emergence of new psychoactive substances currently exceeding a thousand is rapidly changing substance prevalence patterns and straining the methods used for detection, most of which are suitable only for a single class of substances. This study presents a rapid and facile dilute-and-shoot system operated in conjunction with an optimized liquid chromatographic separation system for the high-sensitivity detection of substances across a range of substance classes with 3 isotopes used only. The proposed method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is able to identify 68 substance and their metabolites in urine samples as small as 50 µL. Optimal chromatographic conditions including 95% water/methanol ratio with 0.1% added formic acid and a prolonged LC gradient run-time (15 min) improved the peak shape of polar compounds and enhanced signal strength by 5%. Under 4-fold dilution, all analytes were within 80-120% of tolerance response levels, indicating that the matrix effect was insignificant. In experiments, the limit of detection (LOD) ranged from 0.05 to 0.5 ng mL-1, while the coefficient of determination (R2) was > 0.9950. The retention time shift of each peak remained at < 2% with an inter-day relative standard deviation (RSD) of 0.9-14.9% and intra-day RSD of 1.1%- 13.8%. The rapid dilute-and-shoot presents a high-sensitivity, significant stability, robustness and reproducibility without serious interference. To demonstrate the effectiveness of the system, 532 urine samples were collected from suspected drug abusers, and the proposed method was used for rapid analysis. Of these samples, 79.5% contained between one and twelve analytes, and 12.4% tested positive for new psychoactive substances, mostly derivatives of amphetamine and synthetic cathinones. The study presents a high-sensitivity analytic system that is capable of detecting substances from multiple classes and can be used for effective monitoring of substance prevalence in urine.

A proposed approach to confirm heroin administration - Regional differences in heroin purity is a major factor.

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use.

Transcription factor-based biosensor for detection of phenylalanine and tyrosine in urine for diagnosis of phenylketonuria.

Clinicians require a simple quantitative method for the detection of both phenylalanine and tyrosine to facilitate the diagnosis of phenylketonuria, a common inherited disorder of amino acid metabolism. In this study, we developed a novel whole-cell biosensor for the quantification of phenylalanine and tyrosine through the expression of red and green fluorescent proteins. The proposed system responds specifically and sensitively to phenylalanine/tyrosine without interferences from other amino acids. Furthermore, the precision of the biosensor was evaluated using specimens of normal human urine by LC-MS.