A lot of useful information regarding medication taken and metabolism of drugs in the human organism can be extracted from urine. The technique most often used is enzymatic hydrolysis of metabolites, followed by solid-phase extraction (SPE) cleanup and high performance liquid chromatography tandem mass spectrometry (HPLC–MS–MS) determination. When performed manually, sample preparation is labour intensive and time-consuming. If hydrolysis and cleanup are automated and dispersive SPE (dSPE) is chosen instead of standard SPE, the process can be accelerated.
Disposable Pipette Extraction (DPX)When he was initially searching for a more efficient SPE method, William E. Brewer from the University of South Carolina developed disposable pipette extraction (DPX), a dispersive SPE (dSPE) technique. Instead of the sorbent being present as a packed bed, in dSPE the sorbent is a loose powder contained inside a standard disposable pipette tip by fixed screens at the top and bottom of the tip. The sample is aspirated into the tip only, eliminating both the risk of sample-to-sample carryover and the need for extensive washing of syringes used in systems based on standard SPE cartridges. With the sample-powder mix inside the tip, air is aspirated, leading to turbulent mixing of the phases and a highly efficient extraction. Typically, the remaining sample is discharged and the concentrated analytes are eluted with a small volume of solvent into a clean autosampler vial followed by liquid chromatography–mass spectrometry (LC–MS) or gas chromatography–mass spectrometry (GC–MS) analysis. Key differentiators of dSPE are: Fast extraction, high recovery rates, and very small volumes of solvent. Reducing solvent use in the laboratory brings many benefits, ranging from improved work environment to reduced cost for purchasing and disposing of often toxic solvents.
Brewer also wanted to automate this last key step to achieve a completely automated solution: This last key step involved the hydrolysis of the conjugates formed during drug metabolism to quantify the total amount of drug taken.
A Dual Head MultiPurpose Sampler (MPS) (Gerstel) was used for this application. One head performs the DPX-based sample extraction and clean-up, while the second head performs the injection into the LC–MS system.
A 1 mL sample of urine is manually pipetted into an autosampler vial. The vial is capped and placed in the autosampler tray. The sample preparation and introduction process were controlled by software (Maestro, Gerstel).
Separation of the Target Analytes: Separation of three target analytes and their corresponding glucuronide conjugates (morphine and morphine-3-glucoronide; oxazepam and oxazepamglucoronide; oxymorphone and oxymorphoneglucoronide) was performed using a 3.0 × 50 mm, 2.7-µm Poroshell 120, EC-C18 column (Agilent Technologies). The detection system used was a 6460 Triple Quadrupole MS (Agilent) with Jetstream electrospray source (Agilent). Analyte quantification was performed using deuterated isotope labelled standards.
Hydrolysis and DPX Extraction: Mobile phase: A: 5 mM ammonium formate in water with 0.05% formic acid, B: 0.05% formic acid in methanol; LC pump conditions: Isocratic, 50:50 (A:B) at a flow of 0.300 mL/min; Run time: 10 min; Injection volume: 2 µL (loop overfill); Column temperature: 55 °C 6460 MS–MS; Operating mode: Electrospray, positive mode + Agilent Jet stream; Gas temperature: 350 °C; Gas flow (N2): 5 L/min; Nebulizer gas pressure: 35 psi; Sheath gas temperature: 250 °C; Sheath gas flow: 11 L/min; Capillary voltage: 4000 V; Nozzle voltage: 500 V.