Separation of Opiate Isomers using Electrospray Ionization and Paper Spray Coupled to High-Field Asymmetric Waveform Ion Mobility Spectrometry

One limitation in the growing field of ambient or direct analysis methods is reduced selectivity caused by the elimination of chromatographic separations prior to mass spectrometric analysis. We explored the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS), an ambient pressure ion mobility technique, to separate the closely related opiate isomers of morphine, hydromorphone, and norcodeine. These isomers cannot be distinguished by tandem mass spectrometry. Separation prior to MS analysis is, therefore, required to distinguish these compounds, which are important in clinical chemistry and toxicology. FAIMS was coupled to a triple quadrupole mass spectrometer, and ionization was performed using either a pneumatically assisted heated electrospray ionization source (H-ESI) or paper spray, a direct analysis method that has been applied to the direct analysis of dried blood spots and other complex samples. We found that FAIMS was capable of separating the three opiate structural isomers using both H-ESI and paper spray as the ionization source

Development of a prototype blood fractionation cartridge for plasma analysis by paper spray mass spectrometry

Drug monitoring of biofluids is often time consuming and prohibitively expensive. Analysis of dried blood spots offers advantages, such as reduced sample volume, but depends on extensive sample preparation and the presence of a trained lab technician. Paper spray mass spectrometry allows rapid analysis of small molecules from blood spots with minimal sample preparation, however, plasma is often the preferred matrix for bioanalysis. Plasma spots can be analyzed by paper spray MS, but a centrifugation step to isolate the plasma is required. We demonstrate here the development of a paper spray cartridge containing a plasma fractionation membrane to perform automatic on-cartridge plasma fractionation from whole blood samples. Three commercially available blood fractionation membranes were evaluated based on: 1) accuracy of drug concentration determination in plasma, and 2) extent of cell lysis and/or penetration. The accuracy of drug concentration determination was quantitatively determined using high performance liquid chromatography–mass spectrometry (HPLC–MS). While the fractionation membranes were capable of yielding plasma samples with low levels of cell lysis, the membranes did exhibit drug binding to varying degrees, as indicated by a decrease in the drug concentration relative to plasma obtained by centrifugation. Using the membrane exhibiting the lowest binding, we developed a composite paper spray cartridge incorporating the selected fractionation membrane. Quantitative analysis of the plasma samples by paper spray MS yielded results similar to those found with HPLC–MS, but without the need for offline extraction or chromatography

Targeted Protein Detection using an All-In-One Mass Spectrometry Cartridge

We developed a simple 3D printed cartridge for mass spectrometry (MS) targeted detection of plasma proteins, including post-translational modifications (PTMs). The cartridge uses an integrated antibody enrichment column to preconcentrate the protein target as well as a novel built-in substrate to ionize the protein targets for MS detection. We show several examples of using this cartridge to perform rapid detection of clinically significant proteoforms from plasma samples

Analysis of Biofluids by Paper Spray Mass Spectrometry: Advances and Challenges

Abstract Paper spray MS is part of a cohort of ambient ionization or direct analysis methods that seek to analyze complex samples without prior sample preparation. Extraction and electrospray ionization occur directly from the paper substrate upon which a dried matrix spot is stored. Paper spray MS is capable of detecting drugs directly from dried blood, plasma and urine spots at the low ng/ml to pg/ml levels without sample preparation. No front end separation is performed, so MS/MS or high-resolution MS is required. Here, we discuss paper spray methodology, give a comprehensive literature review of the use of paper spray MS for bioanalysis, discuss technological advancements and variations on this technique and discuss some of its limitations

Ionization Suppression and Recovery in Direct Biofluid Analysis using Paper Spray Mass Spectrometry

Paper spray mass spectrometry is a method for the direct analysis of biofluid samples in which extraction of analytes from dried biofluid spots and electrospray ionization occur from the paper on which the dried sample is stored. We examined matrix effects in the analysis of small molecule drugs from urine, plasma, and whole blood. The general method was to spike stable isotope labeled analogs of each analyte into the spray solvent, while the analyte itself was in the dried biofluid. Intensity of the labeled analog is proportional to ionization efficiency, whereas the ratio of the analyte intensity to the labeled analog in the spray solvent is proportional to recovery. Ion suppression and recovery were found to be compound- and matrix-dependent. Highest levels of ion suppression were obtained for poor ionizers (e.g., analytes lacking basic aliphatic amine groups) in urine and approached –90%. Ion suppression was much lower or even absent for good ionizers (analytes with aliphatic amines) in dried blood spots. Recovery was generally highest in urine and lowest in blood. We also examined the effect of two experimental parameters on ion suppression and recovery: the spray solvent and the sample position (how far away from the paper tip the dried sample was spotted). Finally, the change in ion suppression and analyte elution as a function of time was examined by carrying out a paper spray analysis of dried plasma spots for 5 min by continually replenishing the spray solvent

A statistical approach to optimizing paper spray mass spectrometry parameters

Rationale Paper spray mass spectrometry (PS‐MS) was used to analyze and quantify ampicillin, a hydrophilic compound and frequently utilized antibiotic. Hydrophilic molecules are difficult to analyze via PS‐MS due to their strong binding affinity to paper substrates and low ionization efficiency, among other reasons. Methods Solvent and paper parameters were optimized to increase the extraction of ampicillin from the paper substrate. After optimizing these key parameters, a Resolution IV 1/16 fractional factorial design with two center points was employed to screen eight different design parameters simultaneously. Results Pore size, sample volume, and solvent volume were the most significant factors affecting average peak area under the curve (AUC) and the signal‐to‐blank (S/B) ratio for the 1 μg/mL ampicillin calibrant. After optimizing the key parameters, a linear calibration curve with a range of 0.2 μg/mL to 100 μg/mL was generated (R2 = 0.98) and the limit of detection (LOD) and lower limit of quantification (LLOQ) were calculated to be 0.07 μg/mL and 0.25 μg/mL, respectively. Conclusions The statistical optimization procedure undertaken here increased the mass spectral signal intensity by more than a factor of 40. This statistical method of screening followed by optimization experiments proved faster and more efficient, and produced more drastic improvements than typical one‐factor‐at‐a‐time experiments

Rapid measurement of tacrolimus in whole blood by paper spray-tandem mass spectrometry (PS-MS/MS)

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory. Methods Whole blood sample was mixed with stable isotope labeled internal standard ([13C, 2H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3 min/sample. Results Analytical measurement range was 1.5–30 ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5 ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope = 1.0, intercept = − 0.02; r2 = 0.99), and to a conventional LC-MS/MS method (slope = 0.90, intercept = 0.4; r2 = 0.94). Conclusions PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols

Female Blow Flies As Vertebrate Resource Indicators

Rapid vertebrate diversity evaluation is invaluable for monitoring changing ecosystems worldwide. Wild blow flies naturally recover DNA and chemical signatures from animal carcasses and feces. We demonstrate the power of blow flies as biodiversity monitors through sampling of flies in three environments with varying human influences: Indianapolis, IN and two national parks (the Great Smoky Mountains and Yellowstone). Dissected fly guts underwent vertebrate DNA sequencing (12S and 16S rRNA genes) and fecal metabolite screening. Integrated Nested Laplace Approximation (INLA) was used to determine the most important abiotic factor influencing fly-derived vertebrate richness. In 720 min total sampling time, 28 vertebrate species were identified, with 42% of flies containing vertebrate resources: 23% DNA, 5% feces, and 14% contained both. The species of blow fly used was not important for vertebrate DNA recovery, however the use of female flies versus male flies directly influenced DNA detection. Temperature was statistically relevant across environments in maximizing vertebrate detection (mean = 0.098, sd = 0.048). This method will empower ecologists to test vertebrate community ecology theories previously out of reach due practical challenges associated with traditional sampling

Detection of chemical warfare agent simulants and hydrolysis products in biological samples by paper spray mass spectrometry

Paper spray ionization coupled to a high resolution tandem mass spectrometer (a quadrupole orbitrap) was used to identify and quantitate chemical warfare agent (CWA) simulants and their hydrolysis products in blood and urine. Three CWA simulants, dimethyl methylphosphonate (DMMP), trimethyl phosphate (TMP), and diisopropyl methylphosphonate (DIMP), and their isotopically labeled standards were analyzed in human whole blood and urine. Calibration curves were generated and tested with continuing calibration verification standards. Limits of detection for these three compounds were in the low ng mL−1 range for the direct analysis of both blood and urine samples. Five CWA hydrolysis products, ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (iBuMPA), cyclohexyl methylphosphonic acid (CHMPA), and pinacolyl methylphosphonic acid (PinMPA), were also analyzed. Calibration curves were generated in both positive and negative ion modes. Limits of detection in the negative ion mode ranged from 0.36 ng mL−1 to 1.25 ng mL−1 in both blood and urine for the hydrolysis products. These levels were well below those found in victims of the Tokyo subway attack of 2 to 135 ng mL−1. Improved stability and robustness of the paper spray technique in the negative ion mode was achieved by the addition of chlorinated solvents. These applications demonstrate that paper spray mass spectrometry (PS-MS) can be used for rapid, sample preparation-free detection of chemical warfare agents and their hydrolysis products at physiologically relevant concentrations in biological samples