Rapid fabrication of hydrophobic/hydrophilic patterns on paper substrates for paper spray mass spectrometry.

A simple, rapid chemical coating and patterning method was developed and optimized for paper-based substrates for use in paper spray mass spectrometry (PS-MS). A variety of chlorosilanes were explored for coating paper substrates, and their effectiveness in forming hydrophobic surfaces was characterized via contact angle goniometry, scanning electron microscopy, and energy dispersive X-ray spectroscopy. Trichloromethylsilane was selected as the primary coating agent because of the short time required to produce a hydrophobic surface (contact angle > 130°), as well as the ease of patterning. Patterning was performed using 3D-printed masks and an oxygen/plasma cleaner. Optimal mask thickness and oxygen/plasma cleaning parameters were determined to produce channels varying from 0.5 to 2.5 mm in width. The effectiveness of the patterned substrates for PS-MS was determined via analysis of four antiretrovirals: emtricitabine, lamivudine, efavirenz, and dolutegravir. Calibration curves were made for each antiretroviral at varying channel widths, and the limits of detection and limits of quantification for each drug were determined. These results show that this patterning method results in an average 7.2-fold improvement in sensitivity and an average 190-fold improvement in limits of detection over uncoated paper substrates in a neat matrix. In a proof-of-concept experiment, calibration curves were generated for each antiretroviral in urine. A patterned paper substrate with a 2-mm channel resulted in an average 7.4-fold improvement in sensitivity and an average 18-fold improvement in limits of detection over uncoated paper substrates.

Mass spectrometry imaging of hair identifies daily maraviroc adherence in HPTN 069/ACTG A5305.

Objective measures of adherence for antiretrovirals used as pre-exposure prophylaxis (PrEP) are critical for improving preventative efficacy in both clinical trials and real-world application. Current objective adherence measures either reflect only recent behavior (eg days for plasma or urine) or cumulative behavior (eg months for dried blood spots). Here, we measured the accumulation of the antiretroviral drug maraviroc (MVC) in hair strands by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) to evaluate adherence behavior longitudinally at high temporal resolution. An MSI threshold for classifying daily adherence was established using clinical samples from healthy volunteers following directly observed dosing of 1 to 7 doses MVC/week. We then used the benchmarked MSI assay to classify adherence to MVC-based PrEP regimens in hair samples collected throughout the 48-week HPTN069/ACTGA5305 study. We found that only ~32% of investigated hair samples collected during the study's active dosing period showed consistent daily PrEP adherence throughout a retrospective period of 30 days, and also found that profiles of daily individual adherence from MSI hair analysis could identify when patients were and were not taking study drug. The assessment of adherence from MSI hair strand analysis was 62% lower than adherence classified using paired plasma samples, the latter of which may be influenced by white-coat adherence. These findings demonstrate the ability of MSI hair analysis to examine daily variability of adherence behavior over a longer-term measurement and offer the potential for longitudinal comparison with risk behavior to target patient-specific adherence interventions and improve outcomes.

Mass Spectroscopy Imaging of Hair Strands Captures Short-Term and Long-Term Changes in Emtricitabine Adherence.

Most measures of adherence to antiretroviral therapy require a blood sample, and none capture longitudinal daily adherence. A new noninvasive method for measuring daily adherence to antiretroviral regimens containing emtricitabine (FTC) was developed for intact hair strands using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI). A directly observed therapy study of daily and intermittent (3, 1, and 0 doses/week) FTC dosing (n = 12) benchmarked adherence in hair, revealing distinct accumulation patterns and median FTC signal abundance (1,702, 495, 352, and 0, respectively) with each dosing frequency. A threshold value of FTCsignal abundance of 500 differentiated daily dosing from 3 or fewer doses/week (specificity, 100%; sensitivity, 100% over 30 days and 80% over 60 days). Using these criteria, daily FTC hair adherence was classified in young men (n = 8) who have sex with men (YMSM) engaged in or initiating preexposure prophylaxis (PrEP). Four types of adherence profiles were observed in sequential 30-day periods: consistently high, occasional missed doses, improvement following study initiation, and intermittent. Discrete days of nonadherence were identified across the 60-day window, with the average number of consecutive days classified as nonadherent increasing across the four profile types (1, 2, 19, and 58 days, respectively). Additionally, cumulative FTC response in hair (60-day average) significantly correlated with dried blood spot tenofovir diphosphate concentrations collected simultaneously (rs = 0.79, P = 0.03). Based on these data, IR-MALDESI FTC adherence classification in hair strands can better delineate short-term changes in adherence behaviors over a long retrospective window, offering great potential for noninvasive adherence monitoring and quick supportive interventions.

Influence of hair treatments on detection of antiretrovirals by mass spectrometry imaging.

Analysis of drugs in hair by mass spectrometry imaging (MSI) has great potential as an objective, long-term measure of medication adherence. However, the fidelity of the chemical record in hair may be compromised by any cosmetic hair treatments. Here, we investigate infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI response to multiple antiretrovirals (ARVs) in cosmetically treated hair. Hair strands from patients on different ARV regimens were mechanically treated with dye, bleach, and relaxer. The treatments had little or no effect relative to untreated controls for cobicistat, abacavir, dolutegravir, maraviroc, efavirenz, and darunavir, but all three treatments removed emtricitabine (FTC) to undetectable levels from patient hair strands. We also evaluated hair strands by IR-MALDESI MSI from 8 patients on FTC-based regimens who reported a range of hair treatments at varying recency prior to hair collection. While FTC was undetectable in the treated portion of these hair strands, ARVs coadministered with FTC remained detectable in hair strands after treatment. We conclude that IR-MALDESI MSI can be used when measuring adherence to ARV therapy, provided that ARVs other than FTC are targeted in people using hair treatments.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging of Human Hair to Characterize Longitudinal Profiles of the Antiretroviral Maraviroc for Adherence Monitoring.

Here, we assess infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) analysis of hair as a clinical tool for monitoring patient adherence to the antiretroviral maraviroc (MVC). A custom MATLAB-based algorithm has been developed to streamline data analysis and generate longitudinal profiles of drug incorporation along the length of hair strands. Hair strands from volunteers enrolled in a directly observed therapy study were analyzed by IR-MALDESI MSI and processed using this tool to characterize the profiles of single doses and a daily dose regimen of MVC. Single dose responses were 1.7 [1.1, 2.5] mm (median [range]) wide along the length of the hair and were detected in 8 out of 12 volunteers. Daily dose profiles capturing 28 days of continuous dosing were approximately 5 times the intensity of single dose profiles and 10.5 [7.0, 13] mm wide, corresponding to 1 month of hair growth. MVC ion abundance was observed in all 12 volunteers for the daily dosing period. Daily dosing profiles were consistent with a model of MVC accumulation in hair based on linear superposition of a single dose response, indicating the potential for prediction of daily drug-taking behavior based on deconvolution of a complex longitudinal profile in hair.

Antiretroviral concentrations and surrogate measures of efficacy in the brain tissue and CSF of preclinical species.

1. Antiretroviral concentrations in cerebrospinal fluid (CSF) are used as surrogate for brain tissue, although sparse data support this. We quantified antiretrovirals in brain tissue across preclinical models, compared them to CSF, and calculated 90% inhibitory quotients (IQ90) for nonhuman primate (NHP) brain tissue. Spatial distribution of efavirenz was performed by mass-spectrometry imaging (MSI). 2. HIV or RT-SHIV-infected and uninfected animals from two humanized mouse models (hemopoietic-stem cell/RAG2-, n = 36; bone marrow-liver-thymus/BLT, n =13) and an NHP model (rhesus macaque, n =18) were dosed with six antiretrovirals. Brain tissue, CSF (NHPs), and plasma were collected at necropsy. Drug concentrations were measured by LC-MS/MS. Rapid equilibrium dialysis determined protein binding in NHP brain. 3. Brain tissue penetration of most antiretrovirals were >10-fold lower (p < 0.02) in humanized mice than NHPs. NHP CSF concentrations were >13-fold lower (p <0.02) than brain tissue with poor agreement except for efavirenz (r = 0.91, p = 0.001). Despite 97% brain tissue protein binding, efavirenz achieved IQ90>1 in all animals and 2-fold greater white versus gray matter concentration. 4. Brain tissue penetration varied across animal models for all antiretrovirals except raltegravir, and extrapolating brain tissue concentrations between models should be avoided. With the exception of efavirenz, CSF is not a surrogate for brain tissue concentrations.

Development of a Microchip CE-HPMS Platform for Cell Growth Monitoring.

Improvements were made to a previously developed platform coupling microchip capillary electrophoresis (CE) with high pressure mass spectrometry (HPMS). The RF drive frequency was increased to over 30 MHz from less than 10 MHz, and the ion trap was scaled down to 100 µm critical dimensions. A stretched length ion trap was used to improve sensitivity, and a tube lens was used to improve ion transmission. Detection of the 20 common amino acids was demonstrated, resulting in an average improvement of signal-to-noise of 28 times and an average improvement in peak width of 2.6 times over those obtained in previous work. Consumption of amino acids by cells in growth media was monitored over time using the improved CE-HPMS platform, and several amino acids were shown to be consumed at different rates, demonstrating the potential for real-time bioreactor monitoring.

N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides.

Herein we describe a synthetically simple strategy for increasing the lifetime of unstructured peptides in cytosolic environment via dimerization at the N-terminus to block threading into the catalytic cleft of cytosolic proteases. We establish this approach with kinase substrates, allowing for phosphorylation in cells as a demonstration of protease resistance.

Coupling Microchip Electrospray Ionization Devices with High Pressure Mass Spectrometry.

A microchip electrospray ionization source was coupled with high pressure mass spectrometry (HPMS). A continuous atmospheric inlet consisting of a stainless steel capillary and DC ion optics was designed to conduct ions into the mass spectrometer. Infusions of amino acids and peptides were performed and detected with a miniature cylindrical ion trap (mini-CIT)-based mass spectrometer operated at ≥1 Torr with air as the buffer gas. Detection of glycine and thymopentin (separately) demonstrated the mass range of the mini-CIT detector could span from m/z 75 to 681. A microchip capillary electrophoresis (CE) separation with mini-CIT detection was performed, and the results were compared with detection using a commercial instrument (Waters Synapt G2). Comparable separation efficiencies were observed with both mass spectrometers as detectors, with about 6 times better signal-to-noise observed on the Synapt G2. Comparison of mass spectra in the two systems reveals similar features observed, but with wider peak widths in the mini-CIT than on the Synapt G2 as expected due to high-pressure operation.

High pressure mass spectrometry of volatile organic compounds with ambient air buffer gas.

RATIONALE: There are many chemical measurement scenarios that would benefit from hand portable mass spectrometry tools including forensics, environmental monitoring, and safety and security. High pressure mass spectrometry (HPMS) facilitates miniaturization by significantly reducing vacuum system requirements. Previous work demonstrated HPMS using helium buffer gas, but HPMS conducted using ambient air would further reduce the size and weight of a portable instrument while also reducing logistical demands by eliminating the need for a helium supply. METHODS: Mass spectrometry was performed at pressures exceeding 1 Torr with ambient air as the buffer gas. A glow discharge electron ionization source and a miniature cylindrical ion trap mass analyzer with a radius of 0.5 mm were used. Mass analysis was possible at these pressures with increased radiofrequency (RF) drive frequencies (10 MHz) compared with commercial ion traps (~1 MHz). A differentially pumped chamber was used so that mass spectrometry could be performed at high pressures and detection performed at low pressures with an electron multiplier. RESULTS: HPMS with air buffer gas was demonstrated using a suite of volatile organic compounds (VOCs). The glow discharge ionization source was optimized for operation using air. Mass spectral peak widths increased a factor of 8 compared with helium, as expected, but useful chemical information was still acquired. A mixture of VOCs was detected with ambient air as the buffer gas, showing that valuable mass information can be gained using HPMS without the requirement of an onboard buffer gas source. CONCLUSIONS: HPMS significantly reduces the pumping requirements required for miniature mass spectrometers and the use of ambient air buffer gas further reduces size, weight, and logistics requirements. Mass analysis at high pressures of ambient air is another important step for the development of hand portable mass spectrometers. Copyright © 2016 John Wiley & Sons, Ltd.