Courtney Heideloff

A novel HPLC method for quantification of 10 antiepileptic drugs or metabolites in serum/plasma using a monolithic column.

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important in maximizing the therapeutic response while minimizing the adverse effects. High-performance liquid chromatography (HPLC) is the most commonly used technique for this purpose. Recently, commercial monolithic columns were introduced, which consist of a single rod of fused silica or polymer. The objective of this work was to develop a simple and fast method to quantify 10 commonly measured AEDs or metabolites [carbamazepine, carbamazepine-10,11-epoxide, felbamate, lamotrigine, 10,11-dihydro-10-hydroxy-carbamazepine (active metabolite of oxcarbazepine), pentobarbital, phenobarbital, phenytoin, primidone, and zonisamide] in serum/plasma by HPLC using a reverse-phase monolithic column. Serum/heparin plasma (100 μL) was mixed with an internal standard solution (5-ethyl-5-p-tolylbarbituric acid in methanol, 250 μL). After centrifugation at 15,500g for 10 minutes, 15 μL of supernatant was injected into a monolithic column. The analytes were eluted with an isocratic solution of 0.1 M, pH 6.5, phosphate buffer:methanol:acetonitrile (77:20:3), monitored at 210 nm. The chromatography time was 16 minutes. The method was linear from 0.4-4.9 to 21.2-190.9 μg/mL depending on the analytes with analytical recovery of 80%-114%. The inter- and intra-assay coefficients of variation were <8% in 3 levels of serum-based controls for all the analytes. No significant carryover was observed. Commercial controls containing >100 therapeutic drugs and common endogenous substances were tested and showed no interference. Comparison studies for 6 AEDs or metabolites were performed against commercial HPLC methods. Three AEDs were compared with Food and Drug Administration-approved immunoassays. All comparisons had R > 0.96 with slope ranging from 0.86 to 1.20. This is a simple and fast HPLC method suitable for measuring the 10 AEDs or metabolites. The use of the monolithic column resulted in increased sensitivity, better resolution, and a shorter analytical time compared with a regular C18 column.

A fast and simple assay for busulfan in serum or plasma by liquid chromatography–tandem mass spectrometry using turbulent flow online extraction technology

Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d(8)-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0min. The method was linear from 0.15 to 41.90μmol/L with an accuracy of 87.9-103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1-7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC-MS/MS method using patient specimens (n=45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept -0.02μmol/L, and a linear correlation coefficient 0.9883. The LC-MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.

Simultaneous quantification of 19 drugs/metabolites in urine important for pain management by liquid chromatography-tandem mass spectrometry

Abstract Background: Monitoring pain management drugs and frequently abused drugs is important for physicians to assess patient compliance. Liquid chromatography-tandem mass spectrometry offers high specificity needed for this purpose. In this report, a novel liquid chromatography-tandem mass spectrometry method for simultaneously monitoring 19 drugs/metabolites in urine was developed and validated. Methods: Sample preparation included hydrolysis, dilution and turbulent flow online extraction. Analysis was achieved by reverse phase liquid chromatography and triple-quadruple tandem mass spectrometry. Two fragment ions, one for quantification and the other for assuring identification, were monitored for each analyte. Results: No matrix effect or interference was observed. Lower limits of quantification ranged from 5 to 25 ng/mL. Within the linear range, analytical recovery was between 85.8% and 119.4%. Intra-assay and total coefficient of variations were between 0.2% and 12.7%. This method was compared with mass spectrometry methods offered by two other laboratories using 82 patient samples and 60 spiked urine samples showing 60%–100% agreement. The current method identified more positive samples for all analytes except THCA. The discrepancy in detection rates was primarily due to the different cut-offs used by the other laboratories. Conclusions: A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed for measuring 19 drugs/metabolites in urine important for pain management clinics.

Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography-tandem mass spectrometry method.

Background: Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography–mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC–tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards. Objectives: To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method. Methods: Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory. Results: The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%–108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%–7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2–12.7 ng/mL. Conclusions: Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.

Sensitive, Simple, and Robust Nano-Liquid Chromatography-Mass Spectrometry Method for Amyloid Protein Subtyping.

Amyloidosis is a rare condition characterized by deposits of insoluble proteins in the form of β-pleated sheets. These deposits interfere with the normal structure and function of varying tissues. Thirty-one amyloid proteins have been identified, and the correct identification is critical due to the varying treatments. Immunohistochemistry, the most routine method for identification of amyloid proteins, suffers from limitations. Mass spectrometry (MS)-based methods offer better sensitivity and specificity. We describe here a sensitive, simple, and robust MS-based method for the identification of amyloid proteins. Amyloid deposits are excised from formalin-fixed tissue by laser microdissection and is put through protein extraction followed by trypsin digestion. The resulting peptides are separated by nano-liquid chromatography and analyzed by high-resolution Orbitrap mass spectrometry. The mass spectrometry data are then searched against a human protein database for identification and semi-quantification.

Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Plasma metanephrines are measured to aid in the diagnosis of pheochromocytomas. In patients with pheochromocytomas there is excessive production of catecholamines and metanephrines. Measurement of plasma free metanephrines is one of the first-line clinical tests that are used for the diagnosis and follow-up of pheochromocytoma. We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 m/z for d3-normetanephrine) with positive electrospray ionization. Quantitation is based on peak area ratio of the analyte to its respective deuterated internal standard. The assay is linear from 5.9 to 4090.0 pg/mL for metanephrine and 22.0 to 4386.7 pg/mL for normetanephrine with precision of <6 % over the ranges.