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.
A significant amount of work is required whenever toxicologists and clinical chemists want to determine the concentration
of active pharmaceutical ingredients (APIs) or of their metabolites in urine. One of the most time-intensive steps is the
hydrolysis of conjugated analytes to their original form, typically performed enzymatically, for example, using β-glucuronidase
(GUSB). Control, monitoring, and optimization of various parameters is needed to ensure that the hydrolysis reaction is complete
and reproducible. Among these are pH, temperature, and length of the hydrolysis period that vary from enzyme to enzyme. These
factors have a profound impact on the quality of the analysis results. The same is the case for matrix compounds, present
in significant amounts in uri ne. To reach the very low limits of detection required for the monitoring of API residues and
metabolites, interfering compounds from the matrix, or those generated during the hydrolysis process, must be eliminated.
Typically, this is done using a suitable extraction technique such as solid-phase extraction (SPE), which is widely used in
forensic chemistry. Performing standard, cartridge-based SPE has a number of drawbacks when used for this type of analysis.
It uses relatively large amounts of costly solvent; the solvent and sample elution need to be precisely controlled, therefore
increasing the risk of error and making the process slow; and the resulting sample dilution increases detection limits.
(PHOTO CREDIT: FRANKRAMSPOTT/GETTY IMAGES)
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.