6th Annual Conference & Exhibits of The Association for Mass Spectrometry: Applications to the Clinical Laboratory, Inc.

Restek at MSACL EU 2019
Restek at MSACL EU 2019


Topic: Small Molecules / Tox / TDM
Title: Simultaneous Quantitation of Controlled Substances/Pain Management Drugs in Oral Fluids via Coated Blade Spray–Tandem Mass Spectrometry
German A. Gómez-Ríos, Frances A. Carroll (presenter), Shane Stevens, Dave S. Bell
Restek Corporation
For more information, email Frances Carroll.
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INTRODUCTION: Coated blade spray (CBS) is a technology that combines sample preparation and direct coupling to mass spectrometry (MS) on a single device. CBS is a coated stainless-steel sheet with the shape of a small sword with an ultrathin SPME coating that permits rapid enrichment of small molecules present in complex samples via free-concentration. As a substrate spray technology, it can generate instrumental signal on a mass spectrometer by supplying a small amount of organic solvent to the coated area of the device and then applying a strong electrical field to the non-coated area of the device, which in turn generates gaseous ions from the tip of the CBS via electrospray ionization (ESI).

OBJECTIVES: In this work, we explore for the first time its application towards the determination of several controlled-substances/pain-management drugs in oral fluids. As a proof-of-concept, we investigate its applicability to large (>100 µL) and small (<20 µL) sample volumes of OF.

METHODS: Hydrophobic-lipophilic balance (HLB) particles were selected as the extraction phase and CBS devices. The CBS analytical protocol comprised three steps: 1.) Analyte-enrichment, by extracting from a 96-well plate containing the samples; 2.) Coating-cleaning, which involved immersing the CBS device in water for fast removal of any loosely attached; and 3.) Instrumental analysis, which is performed by applying 5 µL of a methanol/water onto the coated area. After 10 s, 4 kV were applied to the non-coated area of blade for 5 s, inducing electrospray ionization.

RESULTS: The optimized CBS-MS/MS method enabled outstanding limits of quantitation (≤ 5 ng/mL) for most of the target molecules (e.g., cocaine, fentanyl, EDDP, fluoxetine) in oral fluids. A comparison between the CBS-MS/MS method and the confirmatory test (via LC-MS/MS) showed good agreement (R2 ≥ 0.95) between both approaches for all analytes. As a proof-of-concept, the method was also examined for the analysis of droplets of oral fluid.

CONCLUSIONS: The optimized CBS-MS/MS method demonstrated outstanding precision (RSD <10%), LOQs equal to or below 5 ng/mL and excellent linearity (R2 > 0.99) for each of the selected compounds over the range evaluated. Furthermore, it was proven that the methodology could also be applied to small sample volumes such as oral fluid droplets. Our work is currently focused on providing a fully automated platform that can aid in decreasing the total analysis time for 96 samples to under 10 min.

Topic: Small Molecules / Tox / TDM
Title: Phospholipid Removal from Protein Precipitated Plasma Using Inline Sample Preparation (ILSP)
Sharon Lupo, Randy Romesberg, Xiaoning Lu, Christian Weyer (presenter)
Restek Corporation
For more information, email Christian Weyer.
Download a PDF of the full presentation.
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INTRODUCTION: Phospholipids are major constituents of plasma membranes and have been shown to cause severe ion suppression or enhancement in the analysis of biological samples by liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS). Additionally, phospholipids tend to accumulate on reversed-phase columns, causing a decrease in column performance and reduced lifetime.

OBJECTIVE: In this study, we have developed an innovative inline sample preparation (ILSP) technique to remove phospholipids from protein precipitated plasma samples.

METHODS: Protein precipitation of human plasma was performed by adding acetonitrile containing 1% formic acid to blank or fortified plasma in a (3:1) ratio, respectively. After vortexing for 30 s at 3000 rpm, samples were centrifuged for 10 min at 4000 rpm/10 °C. All data was collected on a Shimadzu Nexera UHPLC equipped with an additional binary pump and 6-port switching valve. Analysis was conducted using a 5 x 2.1 mm ILSP cartridge followed by a Raptor Biphenyl 2.7 µm, 100 x 2.1 mm analytical column. Phospholipids and target analytes were monitored in SIM and scan modes using a Shimadzu 2020 MS with electrospray ionization in positive ion mode.

RESULTS: Accurately timed valve switching is critical for a successful inline phospholipid removal method to facilitate washing of phospholipids from the ILSP cartridge while maintaining target analytes. Timing is dependent on the degree of phospholipid retention on the ILSP cartridge in conjunction with the hydrophilic or hydrophobic properties of the analytes. Once the timing of the valve switch is determined, an analytical column is added and the gradient is optimized for speed. For initial experiments, the Raptor Biphenyl 2.7 µm, 100 x 2.1 mm column was chosen. The time program was optimized to allow for simultaneous flushing of phospholipids from the ILSP cartridge, while gradient analysis proceeds on the analytical column. The final analysis with phospholipid removal was completed in <7 minutes. Utilizing this method, lifetime of the ILSP cartridge was demonstrated by successfully performing 500 consecutive injections of protein precipitated fortified human plasma with consistent analyte response (≤2.2% RSD) and retention time (≤0.24% RSD).

CONCLUSIONS: Phospholipid removal by ILSP results in very clean sample extracts and improved signal to noise. A comparison study was performed where plasma samples were fortified with ketoprofen, sulfadiazine, amphetamine, methadone, nortriptyline, and prednisolone. Samples were extracted using 36 wells of a representative 96-well plate offline phospholipid removal product and compared to 36 ILSP injections following protein precipitation. Average analyte signal to noise ratios increased by 15–189% on average for the ILSP technique, with the exception of amphetamine, which showed a marginal decrease of 23%.

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