Teresa Waligorska

Qualification of a Surrogate Matrix-Based Absolute Quantification Method for Amyloid-β42 in Human Cerebrospinal Fluid Using 2D UPLC-Tandem Mass Spectrometry

The primary aims of this work were to: 1) establish a calibrator surrogate matrix for quantification of amyloid-β (Aβ)42 in human cerebrospinal fluid (CSF) and preparation of quality control samples for LC-MS-MS methodology, 2) validate analytical performance of the assay, and 3) evaluate its diagnostic utility and compare it with the AlzBio3 immunoassay. The analytical methodology was based on a 2D-UPLC-MS-MS platform. Sample pretreatment used 5 M guanidine hydrochloride and extraction on μElution SPE columns as previously described. A column cleaning procedure involved gradual removal of aqueous solvents by acetonitrile assured consistent long-term chromatography performance. Receiver-operator characteristic (ROC) curve and correlation analyses evaluated the diagnostic utility of UPLC-MS-MS compared to AlzBio3 immunoassay for detection of Alzheimers disease (AD). The surrogate matrix, artificial CSF containing 4 mg/mL of BSA, provides linear and reproducible calibration comparable to human pooled CSF as calibration matrix. Appropriate cleaning of the trapping and analytical columns provided every-day, trouble-free runs. Analyses of CSF Aβ42 showed that UPLC-MS-MS distinguished neuropathologically-diagnosed AD subjects from healthy controls with at least equivalent diagnostic utility to AlzBio3. Comparison of ROC curves for these two assays showed no statistically significant difference (p = 0.2229). Linear regression analysis of Aβ42 concentrations measured by this mass spectrometry-based method compared to the AlzBio3 immunoassay showed significantly higher but highly correlated results. In conclusion, the newly established surrogate matrix for 2D-UPLC-MS-MS measurement of Aβ42 provides selective, reproducible, and accurate results. The documented analytical performance and diagnostic performance for AD versus controls supports consideration as a candidate reference method.

CSF biomarkers associated with disease heterogeneity in early Parkinson’s disease: the Parkinson’s Progression Markers Initiative study

The development of biomarkers to predict the progression of Parkinson’s disease (PD) from its earliest stage through its heterogeneous course is critical for research and therapeutic development. The Parkinson’s Progression Markers Initiative (PPMI) study is an ongoing international multicenter, prospective study to validate biomarkers in drug-naïve PD patients and matched healthy controls (HC). We quantified cerebrospinal fluid (CSF) alpha-synuclein (α-syn), amyloid-beta1-42 (Aβ1-42), total tau (t-tau), and tau phosphorylated at Thr181 (p-tau) in 660 PPMI subjects at baseline, and correlated these data with measures of the clinical features of these subjects. We found that CSF α-syn, t-tau and p-tau levels, but not Aβ1-42, were significantly lower in PD compared with HC, while the diagnostic value of the individual CSF biomarkers for PD diagnosis was limited due to large overlap. The level of α-syn, but not other biomarkers, was significantly lower in PD patients with non-tremor-dominant phenotype compared with tremor-dominant phenotype. In addition, in PD patients the lowest Aβ1-42, or highest t-tau/Aβ1-42 and t-tau/α-syn quintile in PD patients were associated with more severe non-motor dysfunction compared with the highest or lowest quintiles, respectively. In a multivariate regression model, lower α-syn was significantly associated with worse cognitive test performance. APOE ε4 genotype was associated with lower levels of Aβ1-42, but neither with PD diagnosis nor cognition. Our data suggest that the measurement of CSF biomarkers in early-stage PD patients may relate to disease heterogeneity seen in PD. Longitudinal observations in PPMI subjects are needed to define their prognostic performance.

Improved protocol for measurement of plasma β-amyloid in longitudinal evaluation of Alzheimer’s Disease Neuroimaging Initiative study patients

The interassay variability and inconsistency of plasma β‐amyloid (Aβ) measurements among centers are major factors precluding the interpretation of results and a substantial obstacle in the meta‐analysis across studies of this biomarker. The goal of this investigation was to address these problems by improving the performance of the bioanalytical method.

The Alzheimer’s Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans

We describe Alzheimers Disease Neuroimaging Initiative (ADNI) Biomarker Core progress including: the Biobank; cerebrospinal fluid (CSF) amyloid beta (Aβ1–42), t‐tau, and p‐tau181 analytical performance, definition of Alzheimers disease (AD) profile for plaque, and tangle burden detection and increased risk for progression to AD; AD disease heterogeneity; progress in standardization; and new studies using ADNI biofluids.

High-performance liquid chromatography-mass spectroscopy/mass spectroscopy method for simultaneous quantification of total or free fraction of mycophenolic acid and its glucuronide metabolites.

Measurement of unbound fractions of mycophenolic acid and its metabolites may prove useful in explaining the complicated pharmacokinetic and pharmacodynamic behavior of this drug as well as in therapeutic drug monitoring. We developed a reliable, accurate, and sensitive liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of mycophenolic acid (MPA), MPA glucuronide (MPAG), and MPA acyl-glucuronide (AcMPAG), total or unbound, in plasma, urine, and tissue extract. This method uses a single internal standard, carboxy-butoxy ether of mycophenolic acid (MPAC), and involves a simple sample preparation step. Aliquots of plasma, urine, or dissolved tissue extract (100 μL) or plasma ultrafiltrate for free analytes (50 μL) are treated with acetonitrile/formic acid mixture (99.5/0.5 v/v) followed by centrifugation and dilution with water. The prepared samples are then injected onto an extraction column (Eclipse XDB-C18 12.5 × 4.1 mm; Agilent Technologies, Palo Alto, CA) and washed with mobile phase composed of acetonitrile/water/formic acid (10/89.5/0.5 v/v/v) at a flow rate of 2.8 mL/min. A switching valve is activated 1 minute after sample injection. The analytes are eluted onto the analytical column (Eclipse XDB-C18 150 × 4.1 mm; Agilent Technologies) with a gradient of 0.5% aqueous formic acid, methanol, acetonitrile, and water. We used a tandem mass spectrometer with electrospray ion source, in which the tandem mass spectroscopy transitions were (m/z): 338→207 for MPA, 438→303 for MPAC, and 514→303 for MPAG and AcMPAG. The dynamic ranges (lower limit of quantitation and upper limit of quantitation) were as follows: 0.05 to 30 mg/L for total MPA and 1 to 300 μg/L for free MPA; 0.5 to 300 mg/L of total MPAG and 0.2 to 60 mg/L for free MPAG; and 0.025 to 15 mg/L of total AcMPAG and 1 to 60 μg/L for free AcMPAG. The precision at lower limit of quantitation was in the range of 8.0% to 11.9% for all three total analytes and 13.8 to 18.7% for the free analytes. Accuracy at lower limit of quantitation was in the range of 100% to 105% for total and 97% to 99% for free analytes. Between-day precision of quality control samples was 4.0% to 6.3% for human plasma spiked with total analytes and 4.5% to 14.4% for spiked plasma ultrafiltrate for free analytes. Mean absolute recovery ranged from 98.5% to 101.7% for MPA (both total and free), from 78.1% to 103.4% for MPAG and from 91.5% to 110.4% for AcMPAG. No significant ion suppression was found under these conditions for any of the analytes. Carryover effect was found to be at a maximum level of 0.02%. This method was successfully applied to analyze over 11,000 samples for total analytes, and over 8000 samples for free analytes in plasma, and has been in operation for nearly 3 years without loss of performance.

METHOD COMPARISON OF AB(1-42) MEASURED IN HUMAN CEREBROSPINAL FLUID SAMPLES BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY, THE INNO-BIA ALZBIO3 ASSAY, AND THE ELECSYS® B-AMYLOID(1-42) ASSAY

INHUMANCEREBROSPINALFLUID SAMPLESBY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY, THE INNO-BIA ALZBIO3 ASSAY, AND THE ELECSYS B-AMYLOID(1-42) ASSAY LeslieM. Shaw, Leona Fields, Magdalena Korecka, TeresaWalig orska, John Q. Trojanowski, Deirdre Allegranza, Tobias Bittner, Ying He, Kelly Morgan, Christina Rabe, University of Pennsylvania Hospital, Philadelphia, PA, USA; 2 Roche Diagnostics International AG, Rotkreuz, Switzerland; 3 Roche Diagnostics GmbH, Penzberg, Germany; 4 Roche Diagnostics Corporation, Indianapolis, IN, USA. Contact e-mail: Les. Shaw@uphs.upenn.edu

Prospective quality-control monitoring in the context of a clinical trial

enforce boundary smoothness. The technique’s validity was tested by comparing our results to the manual HarP reference segmentations on a sample of 100 subjects from the ADNeuroimaging Initiative (ADNI) database. We used a leave-one-out strategy to train and then test the segmentations. We used Dice similarity index for objects and correlation coefficients for volumes to verify compliance of automated to manual segmentations. Results: The results show that our method is bias-free with an average Dice similarity coefficient value of 0.803 and overall correlation coefficient of r1⁄40.95. Figure 1 compares the HC volumes computed from the segmentations made by anatomists (reference volumes) with the corresponding volumes computed from the automated segmentations (estimated segmentations). Two fits are shown, with and without intercept (fit parameters listed in Table 1). Both fits are virtually indistinguishable. Overlaid to a MRI image, the new contours often outperform those made by the anatomists in delineating the HC tissues in all three planes, due to the increased smoothness. Conclusions:Our automated segmentation algorithm was able to generate accurate HC volume measurements on a sample of the ADNI cohort that is heterogeneous in terms of ages, cognitive status, manufacturers, and atrophy levels. To our knowledge, this is the first study to assess the accuracy of an automated algorithm with the official release of HarP segmentation labels. Our results hold promise for the utilisation of automated segmentation in large clinical trials and in clinical practice where there is a growing need for biomarkers to support diagnosis and monitor progression of AD.

DIFFERENTIAL EFFECT OF PROSPECTIVE ESCITALOPRAM DECREASE ON LONGITUDINAL CSF AB CONCENTRATION IN AMYLOID (+) VERSUS AMYLOID (-) COGNITIVELY NORMAL ELDERLY INDIVIDUALS

P3-026 DIFFERENTIAL EFFECT OF PROSPECTIVE ESCITALOPRAM DECREASE ON LONGITUDINAL CSFAB CONCENTRATION IN AMYLOID (+) VERSUS AMYLOID (-) COGNITIVELY NORMAL ELDERLY INDIVIDUALS Yvette I. Sheline, B. Joy Snider, Russell T. Shinohara, Anne M. Fagan, Teresa Waligorska, Irem Aselcioglu, Linn Kristin, John R. Cirrito, Leslie M. Shaw, University of Pennsylvania, Philadelphia, PA, USA; Washington University, Saint Louis, MO, USA; Knight Alzheimer’s Disease Research Center, Saint Louis, MO, USA; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Washington University School of Medicine, Saint Louis, MO, USA. Contact e-mail: sheline@mail.med.upenn.edu

Derivation of cutoffs for the Elecsys® amyloid β (1–42) assay in Alzheimer's disease

An Elecsys® Amyloid β (Aβ [1–42]) immunoassay cutoff for classification of patients with Alzheimers disease was investigated.

PROSPECTIVE ESCITALOPRAM DECREASES LONGITUDINAL CSF Aβ CONCENTRATION IN COGNITIVELY NORMAL ELDERLY SUBJECTS: A RANDOMIZED DOSE FINDING STUDY

Susan Catalano, Michael Grundman, Lon S. Schneider, Steven DeKosky, Roger Morgan, Robert Guttendorf, Michelle Higgin, Julie Pribyl, Kelsie Mozzoni, Nicholas J. Izzo, Hank Safferstein, Cognition Therapeutics Inc., Pittsburgh, PA, USA; Global R&D Partners, San Diego, CA, USA; Keck School of Medicine of USC, Los Angeles, CA, USA; University of Florida, Gainesville, FL, USA; MedSurgPI, LLC, Raleigh, NC, USA; Aclairo Pharmaceutical Development Group, Vienna, VA, USA. Contact e-mail: scatalano@cogrx.com