Comprehensive pain panels for clinical research often include such commonly used substances such as opioids, benzodiazepines and stimulants. Other classes of compounds can include muscle relaxants, anti-epileptic drugs such as gabapentin, synthetic cathinones (“bath salts”) and other substances. Often, multiple methods are used to obtain a comprehensive view of the multiple drug classes Some of the key workflow considerations for these labs include the use of fewer, more comprehensive analyte panels and rapid sample preparation and analytical techniques, all of which must be balanced against the need for sample integrity and data quality. We have developed a comprehensive analysis strategy using a simplified solid phase extraction (SPE) protocol incorporating in-well hydrolysis and pretreatment, eliminating the need for any sample transfer steps. This is combined with UPLC-MS/MS analysis resulting in a rapid method that accurately quantifies 80 compounds from 22 drug classes in 3 minutes.
Urine samples were extracted using mixed-mode cation exchange polymeric SPE plates. All hydrolysis and pretreatment steps were conducted within the SPE plate wells. Analytes were then extracted using a modified procedure designed to extract basic, neutral and acidic components in a single protocol. LC-MS/MS analysis was conducted using a Waters ACQUITY I-Class UPLC system coupled to a Waters Xevo TQ-S micro mass spectrometer under reversed-phase conditions.
Results and Conclusions:
The method was evaluated for extraction recovery, matrix effects, linearity, accuracy, precision, and limits of quantification (LOQ). All analytes eluted within 3.1 minutes while maintaining baseline separation of all isobaric compounds. Recoveries were high and consistent for all compounds, averaging >80% with %RSDs under 15% for all analytes. Calibration curves were tailored to individual compounds, and ranged from 2 ng/mL for 6-MAM and fentanyl, to 2,500 ng/mL for many opiates and amines. Calibration curves and QC results were accurate and precise for all compounds over the entire calibration ranges, with LOQs confirmed for the lowest calibrators.
This method enables the rapid extraction and analysis of a diverse panel of drugs for clinical research. A single, rapid SPE method is used to extract 80 compounds with high efficiency. The use of 96-well plates, combined with in-well sample pre-treatment eliminates sample transfer steps, minimizing the risk of cross-contamination or sample transfer errors. The rapid chromatography maintains baseline separation of all relevant compounds, enabling accurate and precise quantification with an analysis time of just over 3 minutes. The combination of sample preparation, chromatography and tandem MS analysis results in a complete, comprehensive workflow for a wide variety of relevant compounds.