The detection of recurrent breast cancer is limited by poorly performing CA tests that are both insensitive and late markers. Because of their sensitivity to biological status, metabolite markers may provide better diagnostic performance and earlier detection. Combining MS and NMR methods improves global metabolomics, resulting in a serum based metabolite profile that is twice as sensitive as the CA 27.29 assay, and detects recurrence 12 months earlier. The profile has been ported to a single MS platform and validated using an independent set of ~100 patient samples. Assay performance, and an outlook for the approach will be discussed.

The evolution of drug screen/confirmation assays has been driven by the replacement of low-throughput technologies (LC-UV, GC-MS) with open-access multiplexed TFC-LC-MS/MS and HILIC-MS/MS in our laboratory. Enhancements in multiplexing technology, mass spectrometric sensitivity and chromatographic system design have been the primary drivers for such advancements. The combination of analytical cassetting, generic automated sample preparation and tuned methodologies using generic LC formats have allowed for the analysis of greater than 2000 samples per day in a walk-up platform for a high throughput clinical diagnostic laboratory. This paper describes the systems and methodologies applied in the quantification of targeted clinical toxicology analytes.

The measurement of the standard curve for every batch of clinical samples comprises a major source of labor and materials costs for LC-MS/MS quantitative analysis. This work focused on the use of more efficient calibration strategies and their effects, if any, on assay quality of clinical nortriptyline serum concentration measurements. The strategies included: the use of the so-called “one-point” and “internal” calibration strategies. Retrospective (n=212 patients) and prospective (n=190 patients) analyses demonstrated excellent agreement with method versus method slopes of .97 to 1.03 (R > 0.99) for this patient cohort. Larger validation sets are in progress.

Gluten is one of eight allergens that FDA has identified in the consumer and food protection act. Dietary gluten (a class of proteins present in wheat, barley and rye) detection and quantification is important because some gluten proteins are implicated in a variety of immune diseases, food allergies and intolerances. This presentation will discuss the development of an analytical LC-MS based methodology for the identification and quantification of physiologically relevant gluten marker peptides that can serve as markers for gluten in food and consumer products. This approach utilized LC/ion trap, LC/QQQ and LC/Q-TOF accurate mass analysis.

When chronic pain patients are suspected of being non-compliant, their therapy can be withdrawn. Therefore, sensitive and specific confirmatory testing is important in identifying diversion and adherence. In this work, we aimed to develop a liquid chromatography tandem mass spectrometry method to detect 14 opioids and 6 opioid glucuronide metabolites. Detecting glucuronides increases the detection window and adds confidence. Internal standards were not available for every analyte to critically evaluate for ion suppression. Instead, we designed a novel approach to achieve the most rigorous quality control possible in which we evaluated the recovery of each analyte in each negative sample.

Mass spectrometry is increasing used in the clinical laboratory, primarily for toxicology and endocrinology testing. Validation can be considered as a series of analytical and statistical procedures designed to objectively demonstrate the testing procedures applicability for the intended purpose. Validation of GC-MS methods is included in a number of the CAP Checklists, particularly for toxicology. These validations have included both qualitative and quantitative procedures, for example specificity and sensitivity (limit of detection). Specific checklist questions will be used to frame this discussion. The second part of the discussion will focus on the newer areas of mass spectrometry.

In response to numerous reported poisonings annually from α-amanitin, a fungal toxin found in mushrooms including the Death Cap and Destroying Angel species, an LC/MS/MS analytical method has been developed to quantitate human exposure by laboratory analysis of urine. Symptoms of α-amanitin poisoning can be delayed up to 24 hours and may be initially dismissed as food poisoning. An early definitive test for α-amanitin could lead to more rapid diagnoses and treatment of poisoning cases. To address the need for a rapid, sensitive and robust analytical test, we have developed a novel, high throughput LC/MS/MS urinary assay.

LC-MS/MS has emerged as the gold standard for confirmatory and quantitative drug analysis. More recently, the high resolution and mass accuracy of LC- TOF/MS has raised the possibility of having a good alternative to LC-MS/MS in targeted drug screening and confirmation. Coupled with retention time matching, the high mass accuracy of LC-TOF/MS allows targeted drug identification without the need for parent compound fragmentation. We have assessed and demonstrated the suitability of a comprehensive LC-TOF/MS serum drug of abuse panel to screen and deliver semi-quantitative data for 214 drugs that may cause intoxication in cases seen at the emergency department.

The benefit of LC-MS technology for clinical assays is now recognized, but laboratory-developed tests require rigorous validation. FDA-approved reagent kits can ease this burden. One such kit for determination of tacrolimus has achieved FDA approval following performance evaluations by the manufacturer and an industry guideline-based validation performed by independent clinical labs. The validation experiments demonstrated that, by analyzing actual clinical specimens with the kit, predetermined goals for linearity, limits of detection/quantitation, precision, and accuracy as well as comparison to established LC-MS procedures were achieved. The ability to dilute and freeze/thaw specimens was demonstrated as were sample stability, and freedom from interference.

This abstract describes a highly efficient, robust proteomic workflow for routine quantitative proteomic analysis of Laser Microdissected tissues. Quantitative proteomics was performed with as few as 500 cells from frozen or archival FFPE biopsies. The approach wa compatible with spectral count quantitation. Comparative proteomic analysis of microdissected 1) glomeruli from frozen and FFPE needle biopsies from patients with glomerular nephritis and 2) tumor microenvironment (tumor vs. stroma) from murine breast cancer models. Validation of differentially expressed proteins by immunohistochemistry will be presented. These examples illustrate the capabilities of tissue proteomics for biomarker discovery from limited human tissues.

We evaluated the ability of an ultra-fast SPE/MS/MS system to analyze small molecule analytes in plasma with much faster sample cycle times and similar analytical results compared to LC/MS/MS methods. Critical bioanalytical parameters were systematically investigated using small molecule analytes (i.e. hydroxymidazolam) spiked into plasma. Comparable accuracy, precision, linearity, and sensitivity were achieved at rates 20-30 fold faster than LC/MS/MS. SPE/MS/MS may be useful for the fast and efficient analysis of similar analytes in biological matrices.

Proper method validation is required in forensic toxicology in order to ensure that results are correct, and that they will stand up under the scrutiny that may be encountered in a court of law. Method validation requirements depend on the type of method being performed. Qualitative or screening methods may require only the determination of limit of detection, selectivity, processed sample stability, and matrix effects. Quantitative methods may require further validation of accuracy, precision, calibration model, carryover, limit of quantitation, and recovery.

The new unique micro-fluidic H/DX technology will enable researchers to study structure, conformation, dynamics and molecular interactions in the liquid phase without tethering. The use of our latest“ Find the needle in the Haystack” invention coupled to API-MS as a novel analytical tool for on-line pI protein separation, digestion and quantitative analysis of peptides will be discussed. The HX-IA Instrument™ (prototype) could potentially become a unique tool for diagnosing patients with Alzheimers, Parkinsons and other neurodegenerative diseases in the early stage.

This presentation is designed to give an introduction to the use of supported liquid extraction in a clinical environment prior to LC-MS/MS analysis. Extract cleanliness is a major factor when investigating sample preparation strategies. Data will be presented with respect to serum/plasma in terms of endogenous matrix components; specifically proteins and phospholipids. pH control and extraction solvent selection can significantly influence extract cleanliness. We will then briefly discuss three applications where extraction optimization has been performed; corticosteroids, vitamin D and steroid hormones.

Spectra from multiple species may yield high-scoring matches for a single isolate in the Bruker MALDI Biotyper. Some laboratories resolve this issue with relative score cutoff such as the “10% rule,” which excludes species scoring >10% below the top match. We analyzed relative cutoffs ranging from 0-35% on a diverse set of isolates (170 species) to identify optimal values for excluding additional species. We concluded there is no universal cutoff, and further showed that “consistency categories” in the newest Biotyper software may obviate the need for arbitrary cutoffs. However, some consistency calls may be too conservative and require critical evaluation.

In this paper, we present a new way to dispense 100% (!!!) of liquid samples and liquid sample contents into mass spectrometers including: drugs of abuse; metals; peptides; proteins and cell or cell fragments using an induction base fluidic dispenser. We discuss and we show the technology that can also provide excellent MALDI depositions for bacteria characterizations, along with the specific physics of discrete droplet production and ion generation. We speculate as to why the integral of the 100% liquid sample introduction IBF approach could become the most efficient way to find biomarkers and how it might replace ESI.

For a given protein it is common for a single amino acid variation to change biological activity. Differentiating between isoforms is an essential part of quantification and establishing structure-function relationships. Mass spectrometers are capable of resolving these differences. Through use of a Perfinity Workstation, it is possible to couple extraction, buffer exchange, digestion, desalting and RPC such that proteins are ready for MS based detection in approximately 10 minutes. Preparation and analysis of samples containing hemoglobin variants was used to demonstrate this method. This workflow couples automated sample preparation to MS detection allowing for highly reproducible results (CV's < 10%).

Rapid identification of bacteria in cultures can aid in therapeutic decision-making. In this study, the Bruker Biotyper MALDI-TOF was used at 2 Infectious Disease laboratories at Quest Diagnostics to identify various bacterial isolates whose identifications were obtained using standard laboratory methods. Overall, 374 of 450 total isolates (83.1%) were correctly identified to the genus and species level, and 40 isolates (8.9%) were correctly identified to the genus level only. Using MALDI-TOF, 27 (6.0%) isolates were not identified and only 9 isolates (2.0%) were mis-identified with 16S rRNA sequencing to resolve discrepancies. This MALDI-TOF assay thus accurately identifies bacterial isolates.

Diagnosis of hemoglobin disorders such as sickle cell disease is complex, time consuming and requires multiple laboratory equipment. We developed a mass spectrometry-based assay in an ion trap instrument combining the specificity of selected reaction monitoring and the protein activation capabilities of electron-transfer dissociation. We showed that this method allows the unambiguous detection of hemoglobin S and C only hours after blood collection, including confirmation of the precise point-mutation sites. The presented ETD-based assay shows capabilities to be automated with GxP-compatible software. We conclude that the assay can be used in a clinical environment for the diagnosis of hemoglobinopathies.

Established methods for screening haemoglobin variants, such as sickle cell disease, in newborn screening programmes are laborious: They involve the resolubilisation of the dried blood spot (DBS) followed by analysis using high pressure liquid chromatography and/or isoelectric focusing, with the results being presumptive rather than absolute. Direct sampling of DBS by liquid extraction surface sampling eliminates the need for any sample preparation and the subsequent analysis by high resolution tandem mass spectrometry rapidly and unequivocally determines haemoglobin variants, including variants with mass shifts of <-1 Da (such as HbC and HbD) and those uncharacterised by standard clinical laboratory screening methods.

We evaluated a MALDi-TOF MS-based Biotyper system for identifying the bacterial pathogens in comparison a Phoenix system. A total of 1,024 urine bacterial isolates were identified and the Biotyper and Phoenix correctly identified 99.9% and 99.5% to the genus level (P>0.05) and 99.1% and 98.5% to the species level (P>0.05), respectively. A protein extraction processing was required for 85 (8.3%) isolates by Biotyper in which 28.1% were Gram-positive cocci and 2.8% were Gram-negative bacilli (P<0.0001). Both systems provide reliable identification while the Biotyper system offers an evolutionarily rapid tool for bacterial pathogen identification in urine.

Insulin measurement is primarily used by immunoassays in the clinical laboratory for the diagnosis and management of glycemic disorders and insulin resistant syndromes. However, differences in results between antibody-based platforms are well known. We have successfully developed a quantitative LC/MS/MS method to analyze the insulin concentrations. After off-line extraction, the serum sample underwent a reduction to free the insulin B chain, and then followed by online extraction and analytical separation before triple quadrupole mass spectrometry. Internal standards were applied and the validation has been completed in a high throughput clinical laboratory.

Towards a total automation of LC-MS/MS analysis of whole blood samples we developed two simple and reliable procedures which convert whole blood into a novel blood matrix, i.e. cell-disintegrated blood (CDB). For production of CDB a blood sample first is spiked with an Internal Standard and thereafter perfused through a heated (LC)-capillary or aspirated into a syringe-needle and snap-frozen in liquid nitrogen . Both processes are fully automated, highly efficient and highly reproducible using a dedicated instrument. After in-line generation of CDB by heat-shock or cryogenic treatment CDB can be further subjected to on-line SPE prior to LC-MS/MS analysis.

The goal of this study is to identify Aspergillus fumigatus proteins that are specifically present in patients with invasive aspergillosis, a potentially fatal pulmonary infection caused by A. fumigatus. Mice were used as the model organism and were divided into three groups: naïve control, asthma-model and IA-model. Plasma samples were analyzed via a bottom-up proteomics approach on an LTQ Orbitrap Velos. We identified one potential protein target that is exclusive to IA, and the expressed recombinant protein is used as a standard for MRM-type analysis. Several IA-exclusive pan-aspergillus and pan-fungal proteins were also identified and can be used as potential targets.