Dennis Peterson (Presenter)
Thomson Instrument Company
Authorship: Nadine Koenig (1), Crystal Xander (1), Melanie Stauffer (1), Dean Fritch (2), Lisa Wanders (3), Sam Ellis (3)
(1) Health Network Laboratories, Allentown, PA (2) Analytical Associates, Greenville, PA (3) Thomson Instrument Company, Oceanside, CA
| Short Abstract This improved sample preparation method allows for the quantitative measurement of opiates in urine. The urine samples were prepared using the eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable urine analysis are the reduction of interferences from the sample matrix and analyte recovery. eXtreme|FV®, were compared to an existing SPE sample preparation method to reduce the sample matrix causing interference prior to analysis. SPE is time consuming, adversely impacts recovery, uses large amounts of solvent and are expensive. The improved sample preparation method using the Thomson eXtreme|FV® allows for the analysis of 12 Opiates. |
Long Abstract
Introduction:
This improved sample preparation method allows for the quantitative measurement of opiates in urine. The urine samples were prepared using the eXtreme|FV®, followed by LC/MS/MS analysis. The most critical aspects of reliable urine analysis are the reduction of interferences from the sample matrix and analyte recovery. eXtreme|FV®, were compared to an existing SPE sample preparation method to reduce the sample matrix causing interference prior to analysis. SPE is time consuming, adversely impacts recovery, uses large amounts of solvent and are expensive. The improved sample preparation method using the Thomson eXtreme|FV® allows for the analysis of 12 Opiates.
Equipment:
Vortex Mixer
Dry Block Heater set at 55ºC±2ºC
Bulk Liquid Nitrogen
MLA pipette or equivalent
Finnpipette
Eppendorf MixMate
ABI 4500 Mass Spectrometer
ABI 4500MD Mass Spectrometer
Shimadzu Prominence HPLC
Autosampler: SIL-20AC HT
Pumps A, B and C: LC-20AD
Communication Bus Module: CBM-20A
Column Oven: CTO-20AC
Degasser: DGU-20A5
Analyst / Multiquant Software/MPX Software
Thomson 48 Position Thomson Vial Press – Thomson Instrument Company #35010 or equivalent
Eppendorf Microcentrifuge 5430
Reagents
Methanol (HPLC Grade)
Water (HPLC Grade)
Drug Free Urine
≥96% Formic Acid (ACS Grade)
β-Glucuronidase - IMCSzymeTM,
Rapid Hydrolysis Buffer
0.1% Formic Acid in Water
Add 100 mL of HPLC Grade Water to a 1L volumetric flask.
Add 1 mL of Formic Acid.
Bring to volume with HPLC Grade Water.
Store at room temperature.
Expires four weeks from date of preparation.
2% Mobile Phase B
Add 20 mL of Methanol to a 1L volumetric flask.
Add 1 mL of Formic Acid.
Bring to volume with HPLC Grade Water.
Store at room temperature.
Expires four weeks from date of preparation.
2% Methanol in HPLC Grade Water
Add 2 mL of Methanol to a 100 mL volumetric flask.
Bring to volume with HPLC Grade Water.
Store at room temperature.
Expires six months from date of preparation.
Improved Sample Preparation:
Allow standards, specimens and controls to come to room temperature.
Label one 1.5 mL Safe-Lock Tube and one Thomson vial for each blank, standard, control and client specimen.
Place 300 µL 2% Methanol into the 12 x 75 glass tube for the LC Check.
To each 1.5 mL Safe-Lock Tube add 50 µL of Rapid Hydrolysis Buffer.
Prepare 1.5 mL Safe-Lock Tubes for analysis according to the following table:
Tube Working
UTO
I.S.
(µL) Working
UTO
STD #1
(µL) Working
UTO
STD #2
(µL) Urine
(µL)
Blank 20 - - 200
Level 1 20 5 - 200
Level 2 20 20 - 200
Level 3 20 - 2.5 200
Level 4 20 - 12.5 200
Level 5 20 - 25 200
Level 6 20 - 50 200
Blank 20 - - 200
Controls* 20 - - 200
Specimens 20 - - 200
Add 200 µL of 2% Methanol to each Thomson Vial.
Add 100 µL of the hydrolyzed urine sample to its respective Thomson Vial.
Place Thomson Filter Plunger on top of Thomson Vial.
Press filter plunger down approximately ¼ of the way into each of the Thomson Vials.
Vortex for 2 minutes at 1750 rpm using the Eppendorf Mix Mate.
Press the filter plunger the rest of the way down using the Thomson 48 position press.
Samples are now ready for LC/MS/MS analysis using the following conditions. Refer to the Standard Operating Procedure for Applied Biosystems LC/MS/MS.
Results:
This improved sample preparation method allows for the quantitative measurement of the following opiates in urine, Table 1 using the Thomson eXtreme|FV® for sample preparation significantly reducing the cost and time of per sample analysis. This method was validated for all 12 drugs in the supplemental pain management panel over 3 days using 6 point calibration curves. Final concentrations of the drugs analyzed can be seen in Table 3. The R2 achieved for all the Level 1 drugs was greater than > 0.99.
Codeine (CODE) Oxycodone (OCOD) Dihydrocodeine/Hydrocodol (DIHC)
Morphine (MORP) Noroxycodone (NOCOD) Naltrexone (NALTX)
Hydrocodone (HCOD) Norhydrocodone (NHCOD) Β-Naltrexone ( BNALT)
Hydromorphone (HMOR) 6-Monoacetylmorphine (6MAM) Oxymorphone (OMOR)
Table 2. Stock Solutions
Working
UTO
I.S.
(µL) Working
UTO
STD #1
(µL) Working
UTO
STD #2
(µL) Urine
(µL)
Blank 20 - - 200
Level 1 20 5 - 200
Level 2 20 20 - 200
Level 3 20 - 2.5 200
Level 4 20 - 12.5 200
Level 5 20 - 25 200
Level 6 20 - 50 200
Controls 20 200
Specimens 20 - - 200
Table 3. Final concentrations for the various analytes are as follows:
Final Concentration
(ng/mL)
Opiates Final Concentration
(ng/mL)
6MAM
Level 1 50 5
Level 2 200 20
Level 3 1000 50
Level 4 5000 250
Level 5 10000 500
Level 6 20000 1000
Conclusion:
This validated method alleviates the need for sample clean-up by SPE thereby reducing the amount of equipment required, solvent usage and sample preparation time. Samples are simply filtered by pipetting the sample into the filter vial shell, inserting the plunger into the shell, and then pushing the plunger into the shell. The filtration process from sample pipetting to autosampler ready only requires 15 seconds. Benefits to the use of Thomson eXtreme|FV® include lower cost, faster sample preparation time, less use and disposal of organic solvents.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |