Minimally invasive point-of-care diagnostic devices are of great interest for rapid detection of biomarkers in diverse settings. Although blood is the most common source of biomarkers, interstitial fluid (ISF) is an alternate body fluid that does not clot or contain red blood cells that often complicate analysis. However, ISF is difficult to collect. In this study, we assessed the utility of a microneedle patch to sample microliter volumes of ISF in a simple and minimally invasive manner. We demonstrated the use of ISF collected in this way for therapeutic drug monitoring by showing similar vancomycin pharmacokinetic profiles in ISF and serum from rats. We also measured polio-specific neutralizing antibodies and anti-polio IgG in ISF similar to serum in rats immunized with polio vaccine. These studies demonstrate the potential utility of ISF collected by microneedle patch in therapeutic drug monitoring and immunodiagnostic applications.

An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL.

We have developed a novel immunomagnetic scavenging technique for extracting cholinesterase inhibitors from aqueous matrixes using biological targeting and antibody-based extraction. The technique was characterized using the organophosphorus nerve agent VX. The limit of detection for VX in high-performance liquid chromatography (HPLC)-grade water, defined as the lowest calibrator concentration, was 25 pg/mL in a small, 500 μL sample. The method was characterized over the course of 22 sample sets containing calibrators, blanks, and quality control samples. Method precision, expressed as the mean relative standard deviation, was less than 9.2% for all calibrators. Quality control sample accuracy was 102% and 100% of the mean for VX spiked into HPLC-grade water at concentrations of 2.0 and 0.25 ng/mL, respectively. This method successfully was applied to aqueous extracts from soil, hamburger, and finished tap water spiked with VX. Recovery was 65%, 81%, and 100% from these matrixes, respectively. Biologically based extractions of organophosphorus compounds represent a new technique for sample extraction that provides an increase in extraction specificity and sensitivity.

Clinical cases of paralytic shellfish poisoning (PSP) are common in Alaska, and result from human consumption of shellfish contaminated with saxitoxin (STX) and its analogues. Diagnosis of PSP is presumptive and based on recent ingestion of shellfish and presence of manifestations consistent with symptoms of PSP; diagnosis is confirmed by detection of paralytic shellfish toxins in a clinical specimen or food sample. A clinical diagnostic analytical method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to evaluate the diagnosis of saxitoxin-induced PSP (STX-PSP) in 11 Alaskan patients using urine specimens collected between June 2010 and November 2011. Concentrations of urinary STX were corrected for creatinine concentrations to account for dilution or concentration of urine from water intake or restriction, respectively. Of the 11 patients with suspected PSP, four patients were confirmed to have STX-PSP by urine testing (24-364ng STX/g creatinine). Five patients had clinical manifestations of PSP though no STX was detected in their urine. Two patients were ruled out for STX-PSP based on non-detected urinary STX and the absence of clinical findings. Results revealed that dysphagia and dysarthria may be stronger indicators of PSP than paresthesia and nausea, which are commonly used to clinically diagnose patients with PSP. PSP can also occur from exposure to a number of STX congeners, such as gonyautoxins, however their presence in urine was not assessed in this investigation. In addition, meal remnants obtained from six presumptive PSP cases were analyzed using the Association of Official Analytical Chemists' mouse bioassay. All six samples tested positive for PSP toxins. In the future, the clinical diagnostic method can be used in conjunction with the mouse bioassay or HPLC-MS/MS to assess the extent of STX-PSP in Alaska where it has been suggested that PSP is underreported.

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VX (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) is a highly toxic organophosphorus nerve agent, and even low levels of contamination in water can be harmful. Measurement of low concentrations of VX in aqueous matrixes is possible using an immunomagnetic scavenging technique and detection using liquid chromatography/tandem-mass spectrometry. Performance of the method was characterized in high-performance liquid chromatography (HPLC)-grade water preserved with sodium omadine, an antimicrobial agent, and sodium thiosulfate, a dechlorinating agent, over eight analytical batches with quality control samples analyzed over 10 days. The minimum reportable level was 25 ng/L with a linear dynamic range up to 4.0 μg/L. The mean accuracies for two quality control samples containing VX at concentrations of 0.250 and 2.00 μg/L were 102 ± 3% and 103 ± 6%, respectively. The stability of VX was determined in five tap water samples representing a range of water quality parameters and disinfection practices over a 91 day period. In preserved tap water samples, VX recovery was between 81 and 92% of the fortified amount, 2.0 μg/L, when analyzed immediately after preparation. Recovery of VX decreased to between 31 and 45% of the fortified amount after 91 days, indicating hydrolysis of VX. However, the preservatives minimized the hydrolysis rate to close to the theoretical limit. The ability to detect low concentrations of VX in preserved tap water 91 days after spiking suggests applicability of this method for determining water contamination with VX and utility during environmental remediation.