Lisa Wanders (Presenter)
Technicals Sales
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 psychoactive drugs, Benzodiazepines 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 SPE for sample preparation 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 9 Benzodiazepines. |
Long Abstract
This improved sample preparation method allows for the quantitative measurement of psychoactive drugs, Benzodiazepines 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 SPE for sample preparation 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 9 Benzodiazepines.
Equipment:
Vortex Mixer
Dry Block Heater set at 55ºC±2ºC
Bulk Liquid Nitrogen
MLA pipette or equivalent
Finnpipette
Eppendorf MixMate
ABI 3200 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
Thomson 48 Position Thomson Vial Press – Thomson Instrument Company #35010 or equivalent
Reagents
Methanol (HPLC Grade)
Water (HPLC Grade)
Drug Free Urine
≥96% Formic Acid (ACS Grade)
β-Glucuronidase - IMCSzymeTM, a purified beta glucuronidase #04-E1F-010 (IMCS-Integrated Micro-Chromatography Systems)
Refrigerate at 2-8ºC.
Expires one month after opening.
Rapid Hydrolysis Buffer – #04-EZ-RHB-020 (IMCS-Integrated Micro-Chromatography Systems)
Refrigerate at 2-8ºC.
Expires one month after opening.
0.1% Formic Acid in HPLC Grade Water (Mobile Phase A)
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.
0.1% Formic Acid in Methanol (Mobile Phase B)
Add 100 mL of Methanol to a 1L volumetric flask.
Add 1 mL of Formic Acid.
Bring to volume with Methanol.
Store at room temperature.
Expires four weeks from date of preparation.
40% Methanol in HPLC Grade Water
Add 40mL 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:
1. Allow standards, specimens and controls to come to room temperature.
Turn Block Heater on to 55ºC±2ºC.
Label one 1.5 mL Safe-Lock Tube and one Thomson vial for each blank, standard, control and client specimen.
For samples falling outside the calibration range, make appropriate dilutions using HPLC water and record on the run sheet. The goal is to prevent mass spectral distortion (failing ion ratios) that occurs in a sample that is too concentrated while keeping the concentration of the diluted sample above the cutoff (or a least the limit of quantitation).
Place 350 µL 40% Methanol into the 12 x 75 glass tube for the LC Check.
Add 5 µL of UBNZO Working STD #1 and 25 µL of UBNZO Working I.S. This is equivalent to a Level 1.
Vortex
Transfer to a conical insert autosampler vial.
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:
Cap and vortex for 30 seconds using the Eppendorf Mix Mate.
Uncap and add 40 µL IMSC β-glucuronidase to each tube.
Cap and vortex for 2 minutes at 1750 rpm to ensure sample is mixed.
UNCAP and Incubate at 55ºC±2ºC for 30 minutes.
Allow tubes to come to room temperature.
Add 300 µL of 40% Methanol to each Thomson Vial.
Give each Eppendorf tube a quick vortex and add 50 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.
SLOWLY press the filter plunger the rest of the way down using the Thomson 48 position press.
Results:
This improved sample preparation method allows for the quantitative measurement of the following pain management drugs in urine, Table 1. The improved method utilizes the Thomson eXtreme|FV® for sample clean-up significantly reducing the cost and time of per sample analysis. This method was validated for all 17 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 2. The R2 achieved for all the Level 1 drugs was greater than > 0.99. Mass spectrum of the Level 1 drugs included in the Supplemental Pain Management Panel in Urine can be seen in Fig. 1.
Table 1. Drugs analyzed as part of the Psychoactive Drug Panel in Urine.
Diazepam (DIAZ) 7-Aminoclonazepam (7AMINO) Zolpidem (ZOLP)
Oxazepam (OXAZ) Nordiazepam (NDIAZ)
Lorazepam (LOR) Temazepam (TEM)
α-hydroxy-Alprazolam (OH-AL) Hydroxy-Midazolam (OH-MID)
Table 2. Stock Solutions
Working
UBENZO
I.S.
(µL) Working
UBENZO
STD #1
(µL) Working
UBENZO
STD #2
(µL) Urine
(µL)
Blank 25 - - 200
Level 1 25 5 - 200
Level 2 25 20 - 200
Level 3 25 - 5 200
Level 4 25 - 25 200
Level 5 25 - 50 200
Controls 25 - - 200
Specimens 25 - - 200
Orasure Urine
Sample 25 - - 400 (Urine)
Fig.1. Mass spectrum of a Positive Result in Urine
Final concentrations for the various analytes are as follows:
Final Concentration
All other analytes (ng/mL) Final
Zolpidem
Concentration
(ng/mL)
Level 1 75 75
Level 2 300 300
Level 3 1000 500
Level 4 5000 2500
Level 5 10000 5000
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 |