Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In ...this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography–tandem mass spectrometry (LC–MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3
min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection.
13C
3 isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400
μmol
L
−1 for both theobromine (average
R
2 0.9968) and caffeine (average
R
2 0.9997) respectively. Analyte peak area variations for 2.5
μmol
L
−1 caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day,
N
=
10) to 9 and 13% (inter-day,
N
=
25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300
μmol
L
−1. This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.
...biomarker discovery researchers find themselves using "samples of convenience" of unknown integrity. To address these issues, we recommend that the NIH develop a list of important clinical ...diagnostic questions in a variety of disease areas that have been prioritized by healthcare impact and that this list also indicate the corresponding medical centers or consortium studies that can provide the appropriate specimens, along with all needed contact information.
Abstract
A continual trend of annual growth can be seen within research devoted to the discovery and validation of disease biomarkers within both the natural and clinical sciences. This expansion of ...intellectual endeavours was quantified through database searches of (a) research grant awards provided by the various branches of the National Institutes of Health (NIH) and (b) academic publications. A search of awards presented between 1986 and 2009 revealed a total of 28,856 grants awarded by the NIH containing the term "biomarker". The total funds for these awards in 2008 and 2009 alone were over $2.5 billion. During the same respective timeframes, searches of "biomarker" and either "discovery", "genomics", "proteomics" or "metabolomics" yielded a total of 4,928 NIH grants whose combined funding exceeded $1.2 billion. The derived trend in NIH awards paralleled the annual expansion in "biomarker" literature. A PubMed search for the term, between 1990 and 2009, revealed a total of 441,510 published articles, with 38,457 published in 2008. These enormous investments and academic outputs however have not translated into the expected integration of new biomarkers for patient care. For example no proteomics derived biomarkers are currently being utilized in routine clinical management. This translational chasm necessitates a review of the previously proposed biomarker definitions and evaluation schema. A subsequent discussion of both the analytical and pre-analytical considerations for such research is also presented within. This required knowledge should aid scientists in their pursuit and validation of new biological markers of disease.
Patients treated for chronic pain may frequently undergo urine drug testing to monitor medication compliance and detect undisclosed prescribed or illicit drug use. Due to the increasing use and abuse ...of benzodiazepines, this class of medications is often included in drug screening panels. However, immunoassay-based methods lack the requisite sensitivity for detecting benzodiazepine use in this population primarily due to their poor cross-reactivity with several major urinary benzodiazepine metabolites. A High Sensitivity Cloned Enzyme Donor Immunoassay (HS-CEDIA), in which beta-glucuronidase is added to the reagent, has been shown to perform better than traditional assays, but its performance in patients treated for chronic pain is not well characterized.
To determine the diagnostic accuracy of HS-CEDIA, as compared to the Cloned Enzyme Donor Immunoassay (CEDIA) and Kinetic Interaction of Microparticles in Solution (KIMS) screening immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS), for monitoring benzodiazepine use in patients treated for chronic pain.
A study of the diagnostic accuracy of urine benzodiazepine immunoassays.
The study was conducted at an academic tertiary care hospital with a clinical laboratory that performs urine drug testing for monitoring medication compliance in pain management.
A total of 299 urine specimens from patients treated for chronic pain were screened for the presence of benzodiazepines using the HS-CEDIA, CEDIA, and KIMS assays. The sensitivity and specificity of the screening assays were determined using the LC-MS/MS results as the reference method.
Of the 299 urine specimens tested, 141 (47%) confirmed positive for one or more of the benzodiazepines/metabolites by LC-MS/MS. All 3 screens were 100% specific with no false-positive results. The CEDIA and KIMS sensitivities were 55% (78/141) and 47% (66/141), respectively. Despite the relatively higher sensitivity of the HS-CEDIA screening assay (78%; 110/141), primarily due to increased detection of lorazepam, it still missed 22% (31/141) of benzodiazepine-positive urines. The KIMS, CEDIA, and HS-CEDIA assays yielded accuracies of 75%, 79%, and 90%, respectively, in comparison with LC-MS/MS.
This study was limited by its single-site location and the modest size of the urine samples utilized.
While the HS-CEDIA provides higher sensitivity than the KIMS and CEDIA assays, it still missed an unacceptably high percentage of benzodiazepine-positive samples from patients treated for chronic pain. LC-MS/MS quantification with enzymatic sample pretreatment offers superior sensitivity and specificity for monitoring benzodiazepines in patients treated for chronic pain.
The small injection volumes and narrow dimensions characteristic of microseparation techniques place constraints on concentration sensitivity that is required for trace chemical analyses. On-line ...sample preconcentration techniques using dynamic pH junction and its variants have emerged as simple yet effective strategies for enhancing concentration sensitivity of weakly ionic species by capillary electrophoresis (CE). Dynamic pH junction offers a convenient format for electrokinetic focusing of dilute sample plugs directly in-capillary for improved detection without off-line sample pretreatment. In this report, we highlight new advances in dynamic pH junction which have been reported to enhance method performance while discussing challenges for future research.
Whey protein intake reduces CVD risk, but little is known whether whey-derived bioactive peptides regulate vascular endothelial function (VEF). We determined the impact of a whey-derived extract ...(NOP-47) on VEF in individuals with an increased cardiovascular risk profile. Men and women with impaired brachial artery flow-mediated dilation (FMD) (n 21, age 55 (sem 1·3) years, BMI 27·8 (sem 0·6) kg/m2, FMD 3·7 (sem 0·4) %) completed a randomised, cross-over study to examine whether ingestion of NOP-47 (5 g) improves postprandial VEF. Brachial artery FMD, plasma amino acids, insulin, and endothelium-derived vasodilators and vasoconstrictors were measured for 2 h after ingestion of NOP-47 or placebo. Acute NOP-47 ingestion increased FMD at 30 min (4·6 (sem 0·5) %) and 120 min (5·1 (sem 0·5) %) post-ingestion (P< 0·05, time × trial interaction), and FMD responses at 120 min were significantly greater in the NOP-47 trial compared with placebo (4·3 (sem 0·5) %). Plasma amino acids increased at 30 min following NOP-47 ingestion (P< 0·05). Serum insulin increased at 15, 30 and 60 min (P< 0·001) following NOP-47 ingestion. No changes were observed between the trials for plasma NO∙ and prostacyclin metabolites or endothelin-1. Ingestion of a rapidly absorbed extract derived from whey protein improved endothelium-dependent dilation in older adults by a mechanism independent of changes in circulating vasoactive compounds. Future investigation is warranted in individuals at an increased CVD risk to further elucidate potential health benefits and the underlying mechanisms of extracts derived from whey.
Characterization of unknown low-abundance metabolites in biological samples is one the most significant challenges in metabolomic research. In this report, an integrative strategy based on capillary ...electrophoresis-electrospray ionization-ion trap mass spectrometry (CE-ESI-ITMS) with computer simulations is examined as a multiplexed approach for studying the selective nutrient uptake behavior of E. coli within a complex broth medium. On-line sample preconcentration with desalting by CE-ESI-ITMS was performed directly without off-line sample pretreatment in order to improve detector sensitivity over 50-fold for cationic metabolites with nanomolar detection limits. The migration behavior of charged metabolites were also modeled in CE as a qualitative tool to support MS characterization based on two fundamental analyte physicochemical properties, namely, absolute mobility (μo) and acid dissociation constant (pK a). Computer simulations using Simul 5.0 were used to better understand the dynamics of analyte electromigration, as well as aiding de novo identification of unknown nutrients. There was excellent agreement between computer-simulated and experimental electropherograms for several classes of cationic metabolites as reflected by their relative migration times with an average error of <2.0%. Our studies revealed differential uptake of specific amino acids and nucleoside nutrients associated with distinct stages of bacterial growth. Herein, we demonstrate that CE can serve as an effective preconcentrator, desalter, and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing unknown yet biologically relevant metabolites that are not readily synthesized or commercially available.
Background
It can be challenging to successfully monitor medication compliance in pain management. Clinicians and laboratorians need to collaborate to optimize patient care and maximize operational ...efficiency. The test menu, assay cutoffs, and testing algorithms utilized in the urine drug testing panels should be periodically reviewed and tailored to the patient population to effectively assess compliance and avoid unnecessary testing and cost to the patient.
Objective
Pain management and pathology collaborated on an important quality improvement initiative to optimize urine drug testing for monitoring medication compliance in pain management.
Methods
We retrospectively reviewed 18 months of data from our pain management center. We gathered data on test volumes, positivity rates, and the frequency of false positive results. We also reviewed the clinical utility of our testing algorithms, assay cutoffs, and adulterant panel. In addition, the cost of each component was calculated.
Results
The positivity rate for ethanol and 3,4‐methylenedioxymethamphetamine were <1% so we eliminated this testing from our panel. We also lowered the screening cutoff for cocaine to meet the clinical needs of the pain management center. In addition, we changed our testing algorithm for 6‐acetylmorphine, benzodiazepines, and methadone. For example, due the high rate of false negative results using our immunoassay‐based benzodiazepine screen, we removed the screening portion of the algorithm and now perform benzodiazepine confirmation up front in all specimens by liquid chromatography‐tandem mass spectrometry.
Conclusion
Conducting an interdisciplinary quality improvement project allowed us to optimize our testing panel for monitoring medication compliance in pain management and reduce cost.
TheORCIDgroup has proposed the creation of a system in which each individual researcher is assigned a specific alphanumeric identifier, which in turn is tethered to each of the researcher's ...scientific contributions. ... ORCID could directly link researcherspecific end points to their respective producers via the corresponding author-specific identifier.
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