Graphical representation of the automated DPX urine cleanup process.
MultiPurpose Sampler (MPS XL) with GERSTEL DPX Option used for the automated hydrolysis and DPX-LC/MS/MS method shown mounted on top of an Agilent 6460 LC-MS/MS system without adding to the lab bench footprint.

Automated DPX Prep Sequence
(Cleanup procedure).

1. Aspirate 750 μL acetonitrile from the fast wash station using the 2.5 mL DPX syringe.
2. Pick up DPX tip (patented sorbent: DPX-RPS) from the DPX Tray.
3. Add 500 μL acetonitrile through the DPX tip into the urine sample on the MPS tray.
4. Wait for 6 seconds to ensure complete wetting of the DPX sorbent with acetonitrile.
5. Aspirate the complete sample; mix sample and sorbent turbulently by aspirating 1400 μL of air into the DPX tip.
6. After an equilibration period of 5 sec: Discharge the content of the DPX tip back into the vial on the tray.
7. Bring the DPX tip to the “PipWaste“ position and discard it.
8. Transfer 250 μL of the upper liquid layer in the vial to a clean 2 mL autosampler vial; Cap the vial.
9. Dilute the extract by adding 250 μL of formic acid 0.05% in water into the sample vial.
10. Inject 25 μL of the diluted sample into the HPLC injection valve.
Method parameters

Analysis conditions LC

  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 Minutes
  Injection volume: 2 μL (loop overflow)
  Column temperature: 55 °C

Analysis conditions MS

  Operating mode: Electrospray, positive mode + Agilent Jet stream
  Gas temperature: 350 °C
  Gas flow (N2): 5 L/min
  Nebulizer pressure: 35 psi
  Sheath Gas Temp: 250 °C
  Sheath Gas Flow: 11 L/min
  Capillary voltage: 4000 V
  Nozzle voltage: 500 V
Calibration curves for Morphine and Oxazepam resulting from automated hydrolysis of Morphine-3-glucuronide and Oxazepam glucuronide, DPX clean-up, and LC/MS/MS determination.
Stacked view of Oxazepam peaks resulting from pH variation experiments. No significant difference was found when the 0.66M acetate buffer pH was varied between 4.0 (A), 4.5 (B), and 5.0 (C).


Pain Management (PM) drugs in urine – can the analysis be fully automated?

A lot of useful information can be extracted from urine regarding medication taken and metabolism of drugs in the human body. The technique most often used is enzymatic hydrolysis of metabolites followed by SPE clean-up and HPLC-MS/MS determination. When performed manually, the sample preparation is labor intensive and time consuming. If hydrolysis and clean-up are automated and dispersive SPE (DPX) is chosen instead of standard SPE, the process is accelerated as shown in the following example.

The amount of work required is significant whenever toxicologists and clinical chemists want to determine the concentration of active pharmaceutical ingredients (APIs) - or of their metabolites - in urine. One of the time-intensive steps required is the hydrolysis of conjugated analytes to their original form. This transformation is typically performed enzymatically, for example, using β-glucuronidase (GUSB). To ensure that the hydrolysis reaction is complete and reproducible, control, monitoring and optimization of various parameters is needed. Among these are the pH value, the temperature and the length of the hydrolysis period, which varies 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 urine. In order to reach the very low limits of detection required for 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 also widely used in forensic analysis. Performing standard, cartridge based SPE has a number of drawbacks when used for this type analysis. It uses relatively large amounts of costly solvent, the solvent and sample elution need to be precisely controlled, increasing the risk of error and making the process slow, and the resulting sample dilution increases detection limits.

Attractive alternative to standard SPE

When he was initially searching for a more efficient solid phase extraction 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 DPX the sorbent is a loose powder contained inside a standard disposable pipette tip by fixed screens at the top and bottom of the tip. Sample is aspirated into the DPX tip only, eliminating both the risk of sample to sample carry-over 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 resulting in highly efficient extraction. Typically, the remaining sample is discharged and the concentrated analytes eluted with a small volume of solvent into an empty clean autosampler vial followed by LC/MS or GC/MS determination. Key differentiators of DPX are: Fast extraction; high recovery rates, and the very small amounts of solvent used. 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. “A further key benefit is the ease of automation”, says Fred D. Foster, Application Scientist at GERSTEL, Inc. near Baltimore, Maryland, “and this includes automated introduction to the LC/MS analysis system.”

Wanted: Complete automation

At GERSTEL, Inc. Fred Foster and his colleagues have long been focused on automating the last key step on the way to a completely automated solution: Hydrolysis of the conjugates formed during drug metabolism in order to quantify the total amount of drug taken. They used a Dual Head version of the GERSTEL Multi-Purpose Sampler (MPS). This system uses one head to perform the DPX based sample extraction and clean-up, while the second head performs the injection into the LC/MS system. The Dual Head configuration enables optimum sample preparation and injection time of less than 7.5 minutes per sample following the automated hydrolysis [1]. Fred Foster arrives at the following conclusion after finishing and evaluating the project: “We can now offer a combined automated system, which performs high throughput urine analysis, including enzymatic hydrolysis, extraction and LC-MS/MS determination.” The manual work required is minimal: A 1 mL sample of urine is manually pipetted into an autosampler vial. The vial is capped and placed in the autosampler tray. All further steps in the sample preparation and introduction process are controlled by the MAESTRO software, fully automated.
Separation of the target analytes: Morphine and its hydrolyzed conjugate morphine- 3-glucoronide, as well as oxazepam (oxazepamglucoronide) and oxymorphone (oxymorphoneglucoronide) was performed using the following column: Poroshell 120, EC-C18 (3.0x50 mm, 2.7 μm). The detection system used was an Agilent 6460 Triple Quadrupole MS with Jetstream electrospray source. Analyte quantification was performed using deuterated isotopes.

Putting the automated system to the test

In order to establish that the enzymatic hydrolysis of urine samples using the typical β-glucuronidase procedure could be automated successfully, triplicate urine samples spiked at a concentration of 1000ng/mL with Oxazepam glucuronide were hydrolyzed both manually and using the automated hydrolysis procedure. Following hydrolysis, all samples were extracted and analyzed using the DPX-LC/MS/MS procedure. The results for the manual and automated procedures matched well with only a 4% difference between the two sets of results.
The automated β-glucuronidase hydrolysis procedure was then compared to a typical acid hydrolysis procedure in which equal parts concentrated hydrochloric acid were added to spiked urine samples containing either Oxymorphone-3--D-glucuronide or Oxazepam glucuronide at 1000ng/mL and then allowed to incubate at 100o C for 90 minutes. After cooling to room temperature, the pH of these samples was adjusted to 4 using dilute ammonium hydroxide prior to extraction along with the automated β-glucuronidase hydrolyzed sample group using the automated DPX-LC/MS/MS procedure. The final volumes of the samples being compared were adjusted prior to extraction, in order to ensure that the final concentrations would be equivalent. The native Oxymorphone and Oxazepam concentrations were found to be lower when the acid hydrolysis procedure was used. Since no response was observed when monitoring for Oxymorphone-3-β-D-glucuronide and Oxazepam glucuronide, it is believed that the lack of response may be due to either further degradation of the native analytes or interference with their ionization rather than incomplete hydrolysis when using the acid hydrolysis procedure.
One of the benefits of automation is the ease with which designed experiments can be performed in order to quickly optimize or compare various steps involved in the manual procedures being automated. An examination of the automated β-glucuronidase hydrolysis procedure using 0.66M acetate buffers of different pH was easily set up in MAESTRO and performed by the MPS, the MAESTRO Prep Sequence Scheduler for the experiment is shown in the box on this page. A stacked view of mass chromatograms resulting from Oxazepam determinations is also shown; as can be seen, no significant difference was found when changing the pH of the 0.66 M acetate buffer from 4.0 (A) to 4.5 (B) or to 5.0 (C).
In order to ensure that the automated hydrolysis procedure was complete and could be used within an automated DPX-LC/MS/ MS method for the quantitation of analytes, standards and QC samples in urine were prepared using the glucuronide conjugated analytes Morphine-3-β-D-glucuronide or Oxazepam glucuronide and then both the native and the conjugated forms of the analytes were monitored using the previously described LC/MS/MS method. In all cases, the absence of detected response for the glucuronide conjugated analytes proved the successful complete hydrolysis of the urine sample being analyzed. The accuracy and precision achieved for Morphine and Oxazepam using the complete automated hydrolysis-DPX-LC/MS/ MS method were determined by extracting replicate (n=6) QC samples at 75 ng/mL concentrations. Accuracy data averaged 102 % for Morphine and 96.3 % for Oxazepam and the precision (% CV) was 3.52 % for Morphine and 4.70 % for Oxazepam.
Representative calibration curves for Morphine and Oxazepam are shown on the preceding page. Regression analysis for both analytes resulted in R2 values of 0.99 or greater. The complete automated process resulted in linear calibration curves with R2 values 0.99 or greater achieved for glucuronide conjugated analytes with LOQs of 1 ng/mL for both Morphine and Oxazepam.

Conclusion and outlook

The automated system performed well; Fred Foster and his colleagues were able to demonstrate that the described enzymatic hydrolysis and subsequent DPX cleanup methods were successfully automated for glucuronide conjugated analytes in urine using the dual head GERSTEL MPS autosampler and sample preparation robot. Analytes were rapidly and reproducibly isolated from hydrolyzed urine samples and subsequently determined using the described automated DPX cleanup procedure coupled with introduction to LC/MS/MS based on the Agilent 6460 Triple Quadrupole Mass Spectrometer.
The automated hydrolysis method described here can be combined with other sample preparation techniques such as SPE, liquid/liquid extraction, centrifugation, and evaporative concentration etc.
This research is currently being expanded to facilitate the hydrolysis procedure in smaller liquid handling containers, such as microtiter and deepwell plates, with automated heating and shaking included. In addition to this, other enzymatic reaction possibilities (e.g., Tryptic Digestion) will be evaluated for full automation with the GERSTEL MPS system.



[1] F. D. Foster, J. R. Stuff, E. A. Pfannkoch, W. E. Brewer. Automated Hydrolysis, DPX Extraction and LC/MS/MS Analysis of Pain Management Drugs from Urine, GERSTEL AppNote 1/2014