# UHPLC‐QTOFMS Urine Drug Screening With Dilute‐and‐Shoot Sample Preparation and Vacuum‐Insulated Probe‐Heated Electrospray Ionization

## TL;DR

A new urine drug screening method uses simplified sample prep and a novel ionization technique to improve detection of certain drugs.

## Contribution

The novel VIP-HESI ionization technique enables efficient dilute-and-shoot urine drug screening with improved detection of amphoteric drugs.

## Key findings

- The DS-UHPLC-VIP-HESI-QTOFMS method achieved a median limit of identification of 5 ng/mL for 56 drugs.
- The method showed improved detectability for pregabalin, gabapentin, and ritalinic acid compared to conventional methods.
- The new method offers fast turnaround, reduced manual workload, and cost efficiency for routine urine drug screening.

## Abstract

We developed a method for comprehensive urine drug screening by applying dilute‐and‐shoot extraction and vacuum‐insulated probe‐heated electrospray ionization with ultra‐high performance liquid chromatography high‐resolution quadrupole time‐of‐flight mass spectrometry (DS‐UHPLC‐VIP‐HESI‐QTOFMS). The method involved five‐fold post‐hydrolysis dilution of urine samples and chromatography on a C18 UHPLC column prior to QTOFMS analysis. The recently introduced VIP‐HESI ion source was chosen due to its enhanced ionization efficiency and compatibility with UHPLC‐QTOFMS. Extensive data was acquired in positive ion mode with a low collision energy (7 eV) and an elevated collision energy (30 eV), using the broadband collision‐induced dissociation data acquisition scan mode that continuously generated high‐resolution and accurate mass for parent and fragment qualifier ions, and parent ion isotopic patterns. Compound identification was performed against an in‐house database with 1263 compound entries, using an automated post‐run reverse target database search with preset identification criteria. Method validation with 56 different drugs showed acceptable results for the limit of identification (median 5 ng/mL), matrix effects (70–130%), repeatability of retention times (< 1%), mass accuracy (< 1 mDa), as well as for specificity and stability. As compared with an established UHPLC‐QTOFMS method relying on solid‐phase extraction and conventional electrospray ionization, DS‐UHPLC‐VIP‐HESI‐QTOFMS produced comparable results from authentic clinical urine samples for most drugs, but showed clearly improved detectability for pregabalin, gabapentin, and ritalinic acid. We anticipate that the new method will be a step forward for laboratories performing routine urine drug screening due to its fast turnaround time, reduced manual workload, cost efficiency, and broad substance coverage.

The new ionization technique, vacuum‐insulated probe‐heated electrospray, allows for a simple dilute‐and‐shoot sample preparation to be used with liquid chromatography time‐of‐flight mass spectrometry in urine drug screening. Comparison with a method relying on solid‐phase extraction and conventional electrospray ionization shows improved performance with amphoteric hydrophilic drugs such as pregabalin.