MSACL 2016 US Abstract

Characterizing Matrix Depletion Using a Novel 96-well Format Extraction Media - Tecan® AC Extraction Plate (AC Plate).

Judy Stone (Presenter)
Univ. of Calif. San Diego Health System

Bio: Judy Stone works as Senior Technical Specialist at the Univ. of California San Diego Health System Toxicology/Mass Spectrometry Laboratory. Her research interests are in small molecule method development and automation using LC-MSMS in the clinical laboratory and online education/training.

Authorship: Stone JA1, van Staveren DR3, Maisenhölder B3, Fitzgerald R 1,2.
1Univ. of Calif. San Diego Health System, 2Univ. of Calif. San Diego, Dept. of Pathology, 3Tecan Schweiz AG.

Short Abstract We interrogated the AC Plate to characterize serum phospholipid (PL) depletion. Extraction recovery, matrix effect, and chromatography were evaluated for testosterone, 13C3-testosterone, and eight PL MRMs. We compared the effects of varying pH, organic content (% methanol or acetonitrile) and two protein-binding releasing agents (ZnSO4 or LiCl/ammonium formate) in the AC Plate extraction reagent versus simple acetonitrile or ZnSO4/methanol protein precipitation with and without filtration through a Sigma HybridSPE plate. The AC Plate reduces background PL by 4 orders of magnitude compared with simple acetonitrile precipitation while providing sufficient sensitivity to measure 1 ng/dL of testosterone in 100 uL of serum.

Long Abstract

Introduction

Automation of sample preparation for LC-MSMS is desirable to reduce labor costs, sample identification errors and imprecision. The AC Plate is a novel, 96-well format extraction media with properties similar to liquid-liquid extraction but is more automation friendly. A proprietary extraction phase is coated in the plate wells. Non-polar analytes will partition from biological samples into the coating with mixing on an orbital shaker. After discarding the sample/extraction reagent residue, the wells are washed, wash residue is discarded, and analytes are partitioned back into a non-polar elution solvent. The elution solvent can be injected directly into an LC-MSMS. This media can be useful to clinical laboratories for adding robustness to their methods, due to ease of automation, simple protocols, no problems with clogging of a stationary phase, and the capability to concentrate analytes in order to achieve lower limits of quantitation (LLOQ).

LC-MSMS methods for 25-hydroxy-Vitamin D and clozapine/norclozapine using the AC Plate have been published (1-2). We wanted to characterize matrix depletion by the AC Plate, specifically serum phospholipids, as well as analyte recovery (3-4). We analyzed testosterone as it has an appropriate log P (3.3) for efficient AC Plate extraction and a challenging target LLOQ of 1-5 ng/dL.

Materials and Methods

The internal standard (I.S.) was 13C3-testosterone, 50 ng/dL in methanol. Delipidized human serum (Mass Spect Gold®, Female_Ultra-low Hormones- Golden West Biologicals) and Cerilliant testosterone standard, 1 mg/mL in methanol, were used for calibrators. The LC-MSMS was a Waters Acquity LC with a XEVO TQS triple quadrupole mass spectrometer. Solvents, water, and modifiers were LC-MS grade. Extraction reagent chemicals (ZnSO4, LiCl) were ACS grade. A Waters C18 HSS T3, 2.1x150mm, 2.7 um column was used with mobile phase A of 2 mmol/L ammonium acetate/0.1% formic acid in H2O, mobile phase B of acetonitrile/0.1% formic acid. AC Plate wash reagent was 0.2% formic acid, elution reagent was 35:65 H2O:acetonitrile. Extraction media (AC Plates, Sigma HybridSPE plates) were supplied by vendors at no charge. Organic/aqueous ratios for extraction reagents are vol:vol. ZnSO4 in extraction reagents was present at 10 g/dL, LiCl and ammonium formate at 0.33 mol/L, with 0.5% formic acid. Extraction reagent pH was adjusted with formic acid or NH4OH.

AC Plate extractions were performed on a Tecan Freedom EVO® 100 automated liquid handler. The AC Plate protocol was: pipet 100 uL of serum and 25 uL of I.S. to plate wells, shake for 2 min, add 175 uL of extraction reagent, shake for 10 min. Discard residual sample/extraction reagent, then perform two wash steps (200 uL wash reagent, shaking for 5 min, discard wash residue). Add elution reagent (100 uL), shake for 5 min and transfer the entire elution volume to a 1.0 mL, conical bottom, polypropylene injection plate. Experiments with a HybridSPE or no plate were manually pipetted using 100 uL of serum, 25 uL of I.S. and 200 uL (total) of solvent. We injected 10-20 uL on the LC-MSMS with a gradient of 10-90% MP-B over 7 min. Recovery and matrix effect experiments were performed in triplicate with male and female serum samples.

Results

We saw a 4 order of magnitude decrease in the total ion current (TIC) of 8 PL MRMs using the AC Plate protocol with a ZnSO4/30% acetonitrile extraction reagent as compared to simple acetonitrile protein precipitation (no plate). The PL TIC signal was 20-fold lower for the AC Plate using ZnSO4/ acetonitrile versus LiCl/acetonitrile extraction reagent. PL MRM signals were similar for serum extracted with an AC Plate whether the ZnSO4 extraction reagent contained 10-30% methanol or 10-30% acetonitrile, except for a peak near the testosterone retention time for a lysoglycerophosphatidyl choline MRM (m/z 524-184). Serum precipitated with either acetonitrile only or with equal volumes of ZnSO4/10% methanol and acetonitrile and processed through a HybridSPE plate had a similar PL TIC signal to serum processed with ZnSO4/acetonitrile extraction reagent - AC Plate. Ion suppression evaluated with post-column infusion of testosterone and I.S. was maximal for simple acetonitrile precipitation and minimal for serum processed through the HybridSPE plate. Ion suppression with the AC Plate was similar to the HybridSPE plate except where lysoglycerophosphatidyl choline MRM peaks were associated with a negative spike in the testosterone signal.

Testosterone recovery increased 2 fold with increasing methanol (0% to 30%) and 4 fold with increasing acetonitrile (0% to 30%) content in the ZnSO4 extraction reagent for the AC Plate. Similar, maximum recoveries were seen with ZnSO4/30% acetonitrile and LiCl/ammonium formate/formic acid/40% acetonitrile. No differences were seen in either testosterone or PL recovery with pH 4 versus pH 7 of the ZnSO4/10% methanol extraction reagent.

The testosterone calibration curve was linear from 1–1,000 ng/dL with signal-to-noise of at least 20:1 at the LLOQ, using 100 uL of sample extracted with the AC Plate-ZnSO4/30% acetonitrile extraction reagent.

Conclusions

We found effective depletion of PL using the AC Plate with optimal recovery for testosterone and reduction of PL content using ZnSO4/30% acetonitrile extraction reagent at pH 4. Additional studies are needed to characterize testosterone, I.S. and PL recovery at higher concentrations of acetonitrile and a wider range of pH, and to evaluate the effects of changing pH and acetonitrile/methanol content of wash and elution reagents.

References & Acknowledgements:

Acknowledgments

We thank Tecan Scheiz AG for use of a Freedom EVO 100 automated liquid handler, consumables and AC plates. We thank Sigma-Aldrich for consumables and HybridSPE plates.

References

1. Couchman L, Subramaniam K, Fisher DS, Belsey SL, Handley SA, Flanagan RJ. (2015). Automated analysis of clozapine and norclozapine in human plasma using novel extraction plate technology and flow-injection tandem mass spectrometry. Ther Drug Monit. Sep 2 [Epub ahead of print].

2. Baecher S, Geyer R, Lehmann C, Vogeser M. (2014). Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids - an application for 25-hydroxyvitamin D.

Clin Chem Lab Med. Mar;52(3):363-71.

3. Rappold B, Holland P, Grant R. (2010) The Phospholipid Fix: Quantitative Measurement and Analytical Solutions for Phospholipid Depletion. Conference abstracts and Proceedings of the 58th American Society for Mass Spectrometry. Salt Lake City, UT, Poster 411/2018.

4. Neveille D, Houghton R, Garrett S. (2012) Efficacy of plasma phospholipid removal during sample preparation and subsequent retention under typical UHPLC conditions. Bioanalysis 4:795-807.

Financial Disclosure

IP Royalty: no

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