We have explored the relative merits of employing DBS and dried matrix spots (biological samples other than whole blood) in the recent past. We employed manual punching of DBS spots from both fortified and incurred guanfacine to develop and partially validate a method with an LLOQ of 5 pg/mL based upon 20 uL of whole blood. To circumvent the difficulties involved with manual punching of DBS cards, we have reported the use of semi-automated direct elution of the DBS spot for the bioanalysis of guanfacine from whole blood spots. The latter results were sufficiently encouraging that we have now investigated a fully-automated, commercially-available, direct elution device for the bioanalytical determination of selected drugs in whole rat blood with varying hematocrit levels. This presentation will describe these results employing 2D HPLC coupled with high resolution TOF MS.

Trypsin is an endoprotease commonly used for sample preparation for mass spectrometry based proteomics and is typically either porcine or bovine sequence in origin. While the general conditions for optimum trypsin activity are well understood, less is known about it's reproducibility and specificity from the various suppliers. Further characterizing trypsin activity would improve the reliability of peptide detection and quantitation for both SRM-based and complex sample biomarker discovery studies. The results identified significant and reproducible differences between the bovine and porcine trypsin digests of HSA. Furthermore, the detected abundance profiles and reproducibility of these peptide intensities revealed the importance for careful selection of candidate peptides for later assay development.

This presentation demonstrates how to use biological variation to develop performance criteria for clinical assays using testosterone as a model. The technical advisory subcommittee of the Partnership for the Accurate Testing of Hormones reviewed the available literature and compiled a database of articles associated with analytical and biological variability of testosterone. This data was used to specify analytical performance goals based on within and between subject variability of testosterone. The allowable limits of desirable imprecision and bias based on currently available biological variation data are 5.3% and 6.4%, respectively. The total error goal is 16.7%.

Dilute&shoot (D&S) methods are widely used in clinical/bioanalytical practice – mainly for time- and cost-saving benefits. Using our opioid LC-MS/MS method remodeling/expansion as an example, we show the importance of performing a signal suppression experiment early in the method validation process and on a large number of specimens to determine whether D&S sample preparation is adequate for the developed LC-MS/MS method or whether more extensive sample cleanup is necessary. While analytical sensitivity using D&S on Triple Quad 5500TM was sufficient even at 20-fold specimen dilution, oxycodone was underestimated as much as 60% compared to the existing method with SPE sample preparation.

Glycoforms were analyzed on a nanospray-Velos/Orbitrap instrument with identification by fragmentation in the ion trap by resonant collision-induced dissociation (CID) and in the collision cell (HCD) over a range of energies. A clustering algorithm was used to obtain hundreds of quality consensus spectra for each glycan. To identify glycopeptides in MS/MS spectra, ProMS Glyco was developed to search for the presence of oxonium ions and oligosaccharide neutral losses in the HCD MS2 spectra. Several complex and high mannose glycans were observed from the glycan profiles. Glycosylation sites and their glycan heterogeneity were determined from the C18 LC-MS/MS tryptic digest runs.

Steroid hormone measurements are increasingly used in patient care and public health. However, the accuracy and reliability of hormone tests prevent appropriate detection, treatment and prevention of diseases. The aim of the CDC effort is to standardize steroid hormone measurements such that accurate and comparable measurements are obtained regardless of the measurement procedure. The CDC Hormone Standardization Program collaborates closely with its partners such as the Partnership for Accuracy in Hormone Testing (PATH) to assure clinical and public health needs are met. The programs and services available will be discussed and impact of these programs will be presented.

Instrument Top Sample Preparation (ITSP) coupled to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) has proven itself a viable option for urine toxicology. In this study, post mortem blood samples from death investigation cases were analyzed with a fully automated system performing ITSP on a CTC DLW autosampler with an integrated centrifuge coupled to an Agilent LC/MS/MS. Results from this automated system were compared with results from immunoassay followed by standard solid phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) or LC/MS/MS.

In clinical studies, protein dynamics (or kinetics) measurements are undertaken primarily in order to understand method of action. Where an observed change in protein concentration could be attributable to either modulation of protein synthesis or protein clearance, dynamics measurements can be used to parse these processes. Traditionally, analyses of protein dynamics have required laborious sample preparation techniques followed by GC-MS on the hydrolyzed protein. However, recent advances in LC-MS and associated techniques have facilitated the analysis of protein dynamics by removing the requirement for extensive protein purification. Here, we describe dynamics measurements of low abundant plasma proteins with a method where proteins are separated from the majority of plasma proteins and enriched in concentration by immuno-affinity and analyzed by micro-flow LC-MS.

Standardization is achieved by setting criteria for assay accuracy and certifying tests that can consistently achieve the accuracy standard. Programs have been ongoing for several years, notably cholesterol, hemoglobin A1c, and c-peptide. CDC has been involved in cholesterol standardization for over 50 years, allowing creation of cholesterol-concentration goals and treatment standards without regard to assay d. Hormone standardization impacts everyday results for patient care in endocrinology. The presentation will give examples of how recent standardization achievements help laboratories, patients and physicians with confidence, portability, and interpretation.

Directly measuring different mercury compounds in blood can provide valuable information about the source of mercury exposure. To this end, a biomonitoring method has been developed for the quantification of inorganic (InHg), methyl (MeHg), and ethyl (EtHg) mercury in whole blood using a species-specific isotope dilution (SSID) method employing capillary GC and ICP-DRC-MS. Particularly, the GC autosampler features twin solid phase microextraction (SPME) fibers that reduce sample analysis cycle times making this method very suitable for high sample throughput, a requirement for large public health biomonitoring studies. As such, this method is currently being utilized for the National Health and Nutrition Examination Survey (NHANES), a unique study designed to explore the health and nutritional status of adults and children in the United States.

Accurate detection of amino acid mutations is critical for amyloid subtyping and subsequent therapy. We present a tandem mass spectrometry-based (MS/MS) informatics workbench for detecting mutations present in amyloidogenic proteins. Proteins in the plaques are captured with laser microdissection, denatured, and digested with trypsin. Resulting peptides are subjected to MS/MS. Spectra of known variants are identified with database searches, whereas novel variants are detected with error-tolerant sequence tag searches. Application of this workflow on transthyretin and serum amyloid A4 subtypes revealed several mutations, which were validated by Sanger sequencing. This study demonstrates the power of bioinformatics in detecting amyloidogenic variants.

Personalized healthcare of the future is going to rely on the analytical technologies and metabolomics as one of the essential tools. Here we demonstrate that longitudinal study design allows extracting complimentary information from metabolic data and focusing on the one of interest: either specific for individuals or shared between them and corresponding to the time trend. Moreover, we demonstrate that different analytical platforms give the data with different content of person-specific information, LC-MS being highly ‘personalized’. This fact is of importance for designing future clinical studies and choosing the methods appropriate for the research question.

Iodine is an essential element in the synthesis of thyroid hormone and plays a crucial role in brain development. To accurately measure iodine in urine and serum specimens, an inductively coupled plasma-mass spectrometry (ICP-MS) method was developed and validated using an Agilent 7700x ICP-MS. 100 uL sample was extracted with 4.9 mL of basic diluent containing Indium and Rhodium internal standards. Serum or synthetic urine was added to all calibrators (5-1000 ug/L) to achieve a matrix matched method. The analytical measurement range was established as 5 ug/L to 1000 ug/L with inter and intra-assay imprecision equal to 5.1% or less.

Blood and cerebral spinal fluid (CSF) sample collection, processing, handling and storage protocols are based on accepted practices rather than careful testing. We examined variables intrinsic to each step in the process of obtaining and storing clinical samples, beginning with electronically monitored collection of samples in controlled studies. Various blood collection tubes, times on bench, incubation temperatures, freeze-thaw cycle and freezer storage effects were compared. Seated or recumbent CSF collection and fasted or fed conditions were compared. Sample analysis was performed by high resolution mass spectrometry, leading to the identification of specific proteins that are affected by the various parameters tested. A multiplexed MRM assay is being assembled in order to determine sample integrity and utility for use of stored samples in clinical research.

In this study, global metabolomic profiling and transcriptomic analysis were combined to identify early noninvasive biomarkers of colorectal cancer as well as to elucidate the underlying mechanism using mouse model. Analysis of longitudinal evolution of urinary metabolic profiles of naïve (wild-type) and spontaneously tumor-developing (ApcMin/+) mice using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry revealed early modulation in excretion of amino acid metabolites during tumorigenesis. These changes in metabolite abundances were found to be linearly dependent on intestinal tumor load. Gene expression analysis indicated these metabolic derangements to be associated with c-MYC activation.

Metal on metal total hip replacement devices having a high failure rate are being recalled by the manufacturer. We report the case of a 69 year old female with previous bilateral hip replacement, presenting with neurological deterioration with progressive vision and hearing loss. On admission, the blood Cobalt level was approximately 385 times the company action level. The patient was immediately admitted and treated by chelation with Succimer and renal dialysis. ICPMS proved valuable for rapid analysis of samples taken regularly during intensive treatment reduced blood cobalt to 16% of the level at presentation within 14 days.

Translation of proteomics discovery data from research into clinical practice is constrained due to the lack of efficient biomarker verification and validation methods. Here, we present a biomarker verification workflow based on mass spectrometry and multiplex selected reaction monitoring (SRM) assays. We used SRM workflow to verify a panel of 30 biomarker candidates in seminal plasma and identify two protein markers (TEX101 and ECM1) that facilitate highly specific and sensitive differential diagnosis of male infertility. Pending the development of a routine clinical assay, proposed markers will eliminate the need for testicular biopsy to diagnose multiple categories of male infertility.

We present a triple quadrupole-based approach for profiling metabolite classes which utilizes a modification of the widely used XCMS software. Advantages of our method include rapid identifications without subsequent experimentation and approximate molar quantitation using representative class standards. This semi-targeted approach can significantly accelerate discovery profiling of many compounds, but is limited to metabolite classes having characteristic fragmentation patterns. Importantly, unknown compounds (i.e., are not in any metabolite database) in the class of metabolites screened will also be monitored effectively with this method. We demonstrate the utility of this platform by identifying metabolites unique to different cell types.

The management of pain has grown rapidly in recent years and common classes of drugs prescribed for the therapeutic management of pain include opioids, barbiturates and benzodiazepines. While immunoassay-based techniques have been used to identify general drug classes, analytical selectivity and sensitivity is dependent on antibody cross-reactivities for structurally similar analytes. Due to analyte recognition based on a specific mass to charge ratio, mass spectrometry allows for the screening of many analytes from a single specimen. Using high resolution accurate mass spectrometry (HRAM), a qualitative method for screening 46 pain management and illicit substances in urine was developed and validated.

Analyses of cerebrospinal fluid (CSF) biomarkers, Amyloid beta1-42, t-tau and p-tau181, provides for reliable measures of plaque burden and neurodegeneration. The data supporting this are based on correlations with clinical diagnosis, autopsy diagnosis in large multicenter and single center studies and in-vivo measures of plaque burden. A current limitation of these tests is the differences obtained in the concentration values that differ across studies due to inherent differences in analytical platforms for the available immunoassays and there is clearly a need for development and implementation of a reference method tied to a standard reference human CSF pool.

Rapid identification of blood pathogens leads to better patient outcomes and cost reduction. We compared the Sepsityper specimen processing (Bruker) and MALDI-TOF-MS Biotyper (Bruker) to MicroScan (Siemens) for bacterial identification using 535 positive BC. Of 498 BC with single bacteria, MALDI-TOF-MS produced 81% and 63% correct genus and species identifications, respectively, for Gram positive bacteria (n=365), and 90% and 85% correct genus and species identifications, respectively, for Gram negative bacteria (n=133). Of 37 BC with multiple species, MALDI-TOF-MS identified only one. MALDI-TOF-MS allows rapid and accurate bacterial identification from BC in a short time and cost-effective manner, especially for Gram-negative bacteria.

Drug testing to evaluate adherence is becoming standard of care in pain management settings. Screen with Reflex to Confirmation is the current ‘gold-standard’ for drug testing. The evolving role of the clinical laboratory in supporting pain management testing is one key example that challenges the utility of the gold standard paradigm as a one-size-fits-all solution. Here, a qualitative time-of-flight mass spectrometry-based method (LC-TOF) was developed as an alternative with initial application towards serum/plasma testing due to specificity concerns regarding immunoassay-based screening. We compared drug detection rates between LC-TOF and traditional immunoassay with reflexive LC-MS/MS confirmation for 493 authentic patient samples.

Alzheimer’s disease research relies on the accurate and precise quantitation of amyloid peptides in cerebrospinal fluid. While measurement of these peptides has historically relied on immunoassays, these tests may lack specificity owing to matrix interferences, cross reactivity and non-specific binding. This work focuses on the development of a selective SPE-LC-MS/MS method for the 1-38, 1-40, and 1-42 fragments of amyloid precursor protein (APP). This method provides significant benefits over immunoassay and should aid in the elucidation of the natural history of the disease, the diagnosis of disease risk and progression, and evaluation of pharmacologic intervention.

MALDI-TOF is a mass-spectrometry-based method that promises to greatly reduce both the operating cost and turn-around-time of organism identification in the clinical microbiology laboratory. This study describes the results of a verification process which included >300, >200, and >100 distinct Gram negative, Gram positive and yeast isolates, respectively. This study will also look at post-implementation data to measure the impact of MALDI on resource utilization, turn-around-time and laboratory workflow. Particular attention will be paid to changes in staffing needs and time to reporting a definitive identification.

We have validated a new gas chromatography-mass spectrometry (GC-MS) platform that facilitates routine screening and automated reporting of 212 drugs, in any shift, by laboratory technologists without the need for sign-out by an onsite mass spectrometry-trained toxicologist. The platform uses a programmable temperature vaporizer (PTV) injector for large sample volume injection and free software for data reduction and spectral matching that facilitates rapid library searching and analyte identification. Validation with 118 patient samples demonstrated that this platform and data searching algorithm independently provide improvements in sensitivity compared to an established GC-MS platform.

Several protein biomarkers are known to be associated with Alzheimer’s Disease (AD). The leading theory of AD pathophysiology is the Amyloid Cascade Hypothesis, which was originally focused on the extracellular deposition of beta amyloid peptides (Aβ) in large fibrillar aggregates. To date, several variants of Aβ have been found in brain and cerebrospinal fluid (CSF) while data from blood and plasma are more ambigous. Another possible biomarker of AD, apolipoprotein E (Apo E) may also play a central role in the AD amyloid cascade leading to cognitive decline.In order to further investigate the relationship of these markers to AD, we developed multiplexed, MSIA-SRM assays that allow quantification of CP, APOE monitoring and isoform- specific peptides and several Aβ peptides.

Increasing rates of resistance has become a global concern, particularly among multidrug resistant (MDR) Gram-negative pathogens. Using two examples of MDR pathogens, the isolates were grown, harvested and lysed, then digested with trypsin and analysed by LC-MS/MS. The results revealed not only antibiotic resistance proteins, but also proteins involved in previously unknown changes in cellular physiology caused by resistance acquisition. This approach demonstrates that mechanisms of resistance could also be characterised using LC-MS/MS