Dried Blood Spot Test for Glycated Hemoglobin Measurement in Pediatric Diabetes Care.

BACKGROUND: The dried blood spot (DBS) card is a novel collection method for measuring glycated hemoglobin (A1C) in individuals with diabetes mellitus. The potential benefits of DBS specimens compared with traditional phlebotomy include a reduction in required total blood volume, reduced procedural pain, and an ability for self-initiated collection. DBS cards for A1C measurement have been validated in the adult population, but there is a paucity of pediatric data. METHODS: The aim of this study was to validate the use of A1C measurement by DBS cards in comparison to venous A1C and to identify potential barriers to implementing this novel approach. Venous and DBS card A1C samples were collected simultaneously from 62 patients at their local laboratory and transported to the central provincial lab for analysis. Correlation analyses compared venous and DBS A1C with data rescaling performed to account for the DBS-venous interassay difference. RESULTS: Mean venous A1C was 7.49% and DBS A1C was 7.26%, with an interassay difference of 0.23%. Data showed a strong, positive correlation between A1C collection methods (r=0.86, p<0.001); this was further strengthened at lower A1C values (A1C <7.5%, r=0.87, p<0.0001). A stronger relationship emerged when the data were rescaled to account for the DBS-venous interassay difference (r=0.8935, p<0.0001). CONCLUSIONS: Given the potential feasibility, practicality, accessibility, cost-effectiveness, and performance characteristics of the DBS A1C, especially at lower A1C values hovering around the diagnostic threshold for diabetes, this study provides supporting evidence for consideration of the use of DBS A1C testing in pediatric diabetes care.

Rising above helium: A hydrogen carrier gas chromatography flame ionization detection (GC-FID) method for the simultaneous quantification of toxic alcohols and ethylene glycol in human plasma specimens.

Here we validate a GC, Flame Ionization Detection (GC-FID), liquid injection method using hydrogen as a carrier gas combining analysis of toxic volatile alcohols (VA): methanol, ethanol, isopropanol, acetone, as well as glycols, ethylene glycol (EG) and propylene glycol (PG), in a single method. METHODOLOGY: 200 µL of calibrator, QC, or patient specimen were deproteinized with 400 µL of acetonitrile containing internal standards (10 mmol/L N-propyl alcohol for VA and 2.5 mmol/L 1,2-butanediol for glycols). GC-FID analysis using hydrogen carrier gas and nitrogen makeup gas utilized an Agilent 7890 system equipped with Agilent 7683 liquid autosampler on a 30 m × 530 µm RTX-200 fused silica column. Method validation included repeatability, recovery, carryover, linearity, lower limit of quantification (LLOQ), accuracy, selectivity and measurement uncertainty. RESULTS: The 8.3 min from injection to injection reduced time of analysis by 45% over a previously reported method using Helium carrier gas with no loss in resolution. Within-run and Between-run variability were ≤1.4% and ≤6.8% respectively. Recovery was 100% within a 95% confidence interval. Carryover was negligible for all but EG. LLOQ was <1 mmol/L for all analytes. The upper range of linearity was 120 mmol/L for methanol, ethanol and isopropanol, 100 mmol/L for acetone and 50 mmol/L for EG. Analytes demonstrated acceptable accuracy and measurement uncertainty using College of American Pathologists (CAP) criteria. Toluene can cause a false positive EG, while benzene, xylene and 1,3 butanediol can cause false negative EG. CONCLUSIONS: Converting from Helium to Hydrogen carrier gas benefits patient care through a reduction in turnaround time and provides a cost savings to the laboratory.

Intervention to Reduce Unnecessary Glucose Tolerance Testing in Pregnant Women.

BACKGROUND: Gestational diabetes mellitus (GDM) can be diagnosed in pregnant women by increased fasting plasma glucose alone, which eliminates the need for performing a 75 g oral glucose tolerance test (OGTT). If whole blood glucose meters are used to triage fasting samples in order to decide whether to give the glucose drink, a cutpoint with appropriate sensitivity and specificity for elevated fasting plasma glucose is needed. METHODS: The number of GDM diagnoses by increased fasting plasma glucose alone was determined from specimens collected and tested at core laboratories in urban hospitals, rural health centers, and from specimens collected at patient phlebotomy service centers (PSCs) for plasma testing at a central laboratory. The number of glucose drinks avoided was counted after implementing the diagnostic cutoff of ≥95 mg/dL (5.3 mmol/L) at urban hospitals and rural health centers, which have on-site plasma testing, and after selecting a PSC meter fasting venous whole blood glucose cutpoint after calculating sensitivity and specificity for plasma glucose ≥95 mg/dL (5.3 mmol/L) using logistic regression. RESULTS: Among 4850 OGTTs, there were 1315 GDM diagnoses annually, of which 409 were from increased fasting plasma glucose. Ninety-one percent of OGTTs were performed at PSCs. If a fasting plasma glucose cutpoint of ≥95 mg/dL (5.3 mmol/L) was implemented at urban hospitals and rural health centers and a meter fasting venous whole blood glucose cutpoint of ≥108 mg/dL (6.0 mmol/L) (25% sensitivity, 99.9% specificity) was implemented at PSCs, the drink would be appropriately avoided by 145 patients/year, and inappropriately avoided by 3 patients/year. After implementing these cutpoints, the drink was appropriately avoided in 91 patients during a 36-week period, with none inappropriately avoiding it. CONCLUSION: Modifying fasting glucose cutpoints reduced unnecessary diagnostic OGTTs in pregnant women.

Influence of diurnal variation and fasting on serum iron concentrations in a community-based population.

OBJECTIVES: Serum iron is an important clinical test to help identify cases of iron deficiency or overload. Fluctuations caused by diurnal variation and diet are thought to influence test results, which may affect clinical patient management. We examined the impact of these preanalytical factors on iron concentrations in a large community-based cohort. DESIGN AND METHODS: Serum iron concentration, blood collection time, fasting duration, patient age and sex were obtained for community-based clinical testing from the Laboratory Information Service at Calgary Laboratory Services for the period of January 2011 to December 2015. RESULTS: A total of 276,307 individual test results were obtained. Iron levels were relatively high over a long period from 8:00 to 15:00. Mean concentrations were highest at blood collection times of 11:00 for adult men and 12:00 for adult women and children, however iron levels peaked as late as 15:00 in teenagers. With regard to fasting, iron levels required approximately 5h post-prandial time to return to a baseline, except for children and teenage females where no significant variation was seen until after 11h fasting. After 10h fasting, iron concentrations in all patient groups gradually increased to higher levels compared to earlier fasting times. CONCLUSIONS: Serum iron concentrations remain reasonably stable during most daytime hours for testing purposes. In adults, blood collection after 5 to 9h fasting provides a representative estimate of a patient's iron levels. For patients who have fasted overnight, i.e. ≥12h fasting, clinicians should be aware that iron concentrations may be elevated beyond otherwise usual levels.

The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells.

Mass spec studio for integrative structural biology.

The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS and tandem MS (MS(2)) data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-MS(2) and data-independent HX-MS(2). The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions.

A general liquid chromatography tandem mass spectrometry method for the quantitative determination of diquaternary ammonium gemini surfactant drug delivery agents in mouse keratinocytes' cellular lysate.

Development of a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of diquaternary ammonium gemini surfactants, utilized as gene deliver agents, is required for the evaluation of their post transfection fate. The continued development of efficient and safe gene delivery agents will benefit directly from an understanding of their rate of uptake, metabolism and excretion. A method is reported that is specific for the quantification of twenty-nine individual diquaternary ammonium gemini surfactant molecules and was validated for N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cell lysate according to USFDA bioanalytical method validation guidelines. The 10min chromatographic separation procedure utilized an Agilent Zorbax CN column (100mm×2.1mm with 3µ particles) with LC-MS grade water and acetonitrile, both containing 0.3% (v:v) formic acid and 1mM triethylamine. Extraction of the gemini surfactant from PAM212 keratinocyte cell lysate was performed using octanol and 10µL aliquots were injected onto the column. The standard curve was linear from 0.30µg/mL to 220µg/mL (r(2)≥0.999) for G16-3 and precision and accuracy were within USFDA specified limits. G16-3 analyte was assessed as stable during storage in the auto-injector, bench-top, freeze-thaw cycling and long-term (60 days) storage at -20°C. Evaluation of the cellular uptake and fate of G16-3, during both the incubation and post incubation periods, has established the potential of the application of the LC-MS/MS quantification method for monitoring diquaternary ammonium gemini surfactants in transfection studies.

Tandem mass spectrometric analysis of novel diquaternary ammonium gemini surfactants and their bromide adducts in electrospray-positive ion mode ionization.

Gemini surfactants are cationic lipids which are utilized for both in vitro and in vivo gene delivery. Structurally, they are comprised of two hydrophobic tail regions with polar head termini that are attached to one another through a spacer region. Structural elucidation and characterization of 29 novel diquaternary ammonium gemini surfactant molecules were achieved using a quadrupole time-of-flight mass spectrometer (QqToF-MS) and a quadrupole-hexapole-quadrupole mass spectrometer (QhQ-MS). The tested compounds were categorized into four distinct structural families based upon the composition of the spacer region. Single stage (MS), tandem stage (MS/MS) and quasimulti-stage (quasi MS(3)) mass spectrometric analysis allowed for confirmation of each gemini surfactant's molecular composition and structure through the identification of common and unique product ions. Identification of similarities in the gemini surfactants' fragmentation behaviour resulted in the production of a universal fragmentation pathway that can assist in the future MS/MS analysis of novel quaternary ammonium gemini surfactants, with unique product ions being indicative of specific structural elements. Furthermore, evidence for the association of agemini surfactant with bromine counter ion was confirmed during MS analysis of tested gemini surfactants regardless of their chemical composition; previously, evidence for bromine and gemini surfactant association was only observed with compounds bearing short alkyl spacer regions. MS/MS analysis of the bromine adducts was also confirmatory to the molecular structure.Understanding the ionization and fragmentation behaviour of gemini surfactants, including bromine adducts, will allow for future qualitative and quantitative identification of these novel drug delivery agents within biological samples.

Properties, engineering and applications of lipid-based nanoparticle drug-delivery systems: current research and advances.

Lipid-based drug-delivery systems have evolved from micro- to nano-scale, enhancing the efficacy and therapeutic applications of these delivery systems. Production of lipid-based pharmaceutical nanoparticles is categorized into top-down (fragmentation of particulate material to reduce its average total dimensions) and bottom-up (amalgamation of molecules through chemical interactions creating particles of greater size) production methods. Selection of the appropriate method depends on the physiochemical properties of individual entities within the nanoparticles. The production method also influences the type of nanoparticle formulations being produced. Liposomal formulations and solid-core micelles are the most widely utilized lipid-based nanoparticles, with surface modifications improving their therapeutic outcomes through the production of long-circulating, tissue-targeted and/or pH-sensitive nanoparticles. More recently, solid lipid nanoparticles have been engineered to reduce toxicity toward mammalian cells, while multifunctional lipid-based nanoparticles (i.e., hybrid lipid nanoparticles) have been formulated to simultaneously perform therapeutic and diagnostic functions. This article will discuss novel lipid-based drug-delivery systems, outlining the properties and applications of lipid-based nanoparticles alongside their methods of production. In addition, a comparison between generations of the lipid-based nano-formulations is examined, providing insight into the current directions of lipid-based nanoparticle drug-delivery systems.