The Effect of the Physical Morphology of Dried Biofluids on the Chemical Stability of Analytes Stored in Paper and Direct Analysis by Mass Spectrometry.

Three-dimensional (3D) dried blood spheroids formed on hydrophobic paper are a new microsampling platform that can stabilize labile molecules in whole blood stored in ambient air at room temperature. In this study, we define the ideal conditions for preparing the dried blood spheroids. The physical morphology of 3D dried blood spheroids is found to be largely impacted by the unregulated relative humidity of the surrounding environment. A solution of KOH placed in an enclosed chamber offers a facile way to control humidity. We also report a general polymer coating strategy that serves to stabilize dried biofluids when prepared under ordinary ambient conditions without regulation of humidity. Dried blood spheroids coated in xanthan gum polymer exhibited enhanced chemical and physical stability. The same xanthan gum polymer provided chemical stability for 2D dried blood spots when compared with the conventional noncoated samples. We have expanded the application of xanthan gum to less viscous biofluids such as urine to induce an artificial protective barrier that also provides enhanced stability for labile performance-enhancing drugs stored at room temperature. The impact of polymer coating on direct biofluid analysis via paper spray mass spectrometry was determined by comparing the relative ionization efficiency, percent difference of ionization efficiency, and matrix effects of performance-enhancing drugs that were spiked in undiluted raw urine. Acceptable analytical performance was recorded for all three criteria, including high ionization efficiencies that ranged from 77 to 93% in the presence of the xanthan gum polymer.

Embossed Paper Platform for Whole Blood Collection, Room Temperature Storage, and Direct Analysis by Pinhole Paper Spray Mass Spectrometry.

Dry-state microsampling techniques are convenient and advantageous for sample collection in resource-limited settings, including healthcare systems designed for the underserved population. In this work, a microsampling platform based on an embossed hydrophobic paper substrate is introduced together with three-dimensional (3D) printed cartridges that offer opportunities for rapid (<30 min) drying of the collected samples while also preserving sample integrity when the embossed paper chip is shipped at room temperature. More importantly, a new pinhole paper spray ionization method was developed that facilitates direct mass spectrometry (MS) analysis of the dried blood samples without prior sample preparation. We compared the direct pinhole paper spray MS method with a liquid chromatographic (LC) MS approach that relied upon electrospray ionization (ESI) after analytes present in the blood sample were extracted through liquid-liquid extraction. Limits of detection as low as 0.12 and 0.49 ng/mL were calculated for cocaine and its metabolite benzoylecgonine, respectively, when using the direct pinhole paper spray MS method. Analytical merits such as precision and accuracy, recovery, carryover effects, and analyte stability were all quantified for this new paper spray method and compared to the traditional LC-ESI-MS. Although LC-ESI-MS was observed to be 10× more sensitive, the linear dynamic range for both methods was determined to be the same, in the range of 1-500 ng/mL for both cocaine and benzoylecgonine analytes. When fully developed, the current microsampling strategy could offer an easy-to-use kit that can enable a more effective MS analysis of 20 µL dried blood samples delivered by mail. Both sensitivity (10×) and sample stability are found to be more superior for blood prepared in the embossed hydrophobic paper compared to samples prepared in the planar hydrophilic paper.

Emerging trends in paper spray mass spectrometry: Microsampling, storage, direct analysis, and applications.

Recent advancements in the sensitivity of chemical instrumentation have led to increased interest in the use of microsamples for translational and biomedical research. Paper substrates are by far the most widely used media for biofluid collection, and mass spectrometry is the preferred method of analysis of the resultant dried blood spot (DBS) samples. Although there have been a variety of review papers published on DBS, there has been no attempt to unify the century old DBS methodology with modern applications utilizing modified paper and paper-based microfluidics for sampling, storage, processing, and analysis. This critical review will discuss how mass spectrometry has expanded the utility of paper substrates from sample collection and storage, to direct complex mixture analysis to on-surface reaction monitoring.

Protective mechanism of dried blood spheroids: stabilization of labile analytes in whole blood, plasma, and serum.

Three-dimensional (3D) dried blood spheroids form when whole blood is deposited onto hydrophobic paper and allowed to dry in ambient air. The adsorbed 3D dried blood spheroid present at the surface of the hydrophobic paper is observed to offer enhanced stability for labile analytes that would otherwise degrade if stored in the traditional two-dimensional (2D) dried blood spot method. The protective mechanism for the dried blood spheroid microsampling platform was studied using scanning electron microscopy (SEM), which revealed the presence of a passivation thin film at the surface of the spheroid that serves to stabilize the interior of the spheroid against environmental stressors. Through time-course experiments based on sequential SEM analyses, we discovered that the surface protective thin film forms through the self-assembly of red blood cells following the evaporation of water from the blood sample. The bridging mechanism of red blood cell aggregation is evident in our experiments, which leads to the distinct rouleau conformation of stacked red blood cells in less than 60 min after creating the blood spheroid. The stack of self-assembled red blood cells at the exterior of the spheroid subsequently lyse to afford the surface protective layer detected to be approximately 30 µm in thickness after three weeks of storage in ambient air. We applied this mechanistic insight to plasma and serum to enhance stability when stored under ambient conditions. In addition to physical characterization of these thin biofilms, we also used paper spray (PS) mass spectrometry (MS) to examine chemical changes that occur in the stored biofluid. For example, we present stability data for cocaine spiked in whole blood, plasma, and serum when stored under ambient conditions on hydrophilic and hydrophobic paper substrates.

Direct differentiation of whole blood for forensic serology analysis by thread spray mass spectrometry.

Direct analysis of whole blood on bloodstained textiles is achieved with thread spray mass spectrometry (MS). This capability satisfies investigators' first priority in crime scene investigations, which is determining if a stain is blood. This thread spray method explores the use of evidentiary fabric threads for rapid determination of hemoglobin directly from whole blood within textiles without prior extraction steps. The multiplicity of information that can be derived from the thread spray MS method distinguishes it from the current presumptive Bluestar® method, by enabling the detection of hemoglobin (both α- and ß-chains), the heme co-factor and lipids all from a single blood sample. Lipid composition was found to differ for blood samples originating from human, canine, and horse species. The robustness of the thread spray MS method as a forensic analytical platform was evaluated in three ways: (1) its successful applicability to samples previously tested by the Bluestar® presumptive method, offering a confirmatory test without prior sample pre-treatment, (2) successful detection of heme from previously washed fabrics, which demonstrated the unprecedented sensitivity of the thread spray method, and (3) the ability to analyze samples stored under ambient conditions for up to 30 days. These results attest to the potential capabilities of the thread spray MS platform in forensic serology, and its application for direct analysis of evidentiary garments, which confer the advantages of rapid analysis and the reduction of the false positive and negative identification rates for blood on textiles.