Article pubs.acs.org/ac
Direct Biofluid Analysis Using Hydrophobic Paper Spray Mass Spectrometry Deidre E. Damon, Kathryn M. Davis, Camila R. Moreira, Patricia Capone, Riley Cruttenden, and Abraham K. Badu-Tawiah* Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43110, United States S Supporting Information *
ABSTRACT: Ambient electrostatic paper spray ionization from a hydrophobic paper occurs when a DC potential is applied to the dry paper triangle. Online liquid/liquid extraction of small organic compounds from a drop of biological fluid present on the dry hydrophobic paper is achieved with an organic spray solvent in under 1 min and utilizes in situ electrostatic-spray ionization for more efficient detection of extracted molecules. Direct analysis of small volumes of biofluids with no sample pretreatment is possible, which is applicable in point-of-care analyses. High sensitivity and quantitative accuracy was achieved for the direct analysis of illicit drugs in 4 μL of raw blood, serum, and whole urine. The study was extended to monitor the activity of alanine transaminase enzyme, a key biomarker for the detection of liver injury in patients (with HIV and tuberculosis) who typically take several medications at once.
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using portable mass spectrometers and in the analysis of small sample volumes. In this work, we present a liquid/liquid extraction approach performed on a hydrophobic paper substrate. The hydrophobic paper was prepared in-house through self-assembly chemistry of silanization (Figure 1A)13,14 by exposing laser-cut filter paper triangles to the vapor of trichloro(3,3,3-trifluoropropyl)silane reagent under vacuum (∼20 Torr) for approximately 4 h. We chose to perform slow silanization (with no heating) to ensure only exposed fabric surface hydroxyl (OH) groups are derivatized while leaving most of the OH groups involved in intermolecular hydrogen bonding within the fiber core unreacted. This prevents significant alteration of mechanical properties of the paper. (Figure S1, Supporting in formation). The surface energy of the 3,3,3-trifluoropropyl functionalized paper was estimated to be 44 mN/m (Figure S2, Supporting Information). Unlike a previous report that used hydrophobic surfaces to differentially extract analytes from a matrix,15 the underlying principle for the current experiment is to prevent wetting by creating a mismatch in surface energies, where aqueous-based samples (surface tension ∼72 mN/m) can be organized as spherical droplets (Figure 1B) on the low-energy (hydrophobic) paper surface for enhanced sampling. For complex biological samples, MS analysis of the sample droplet present
stablished protocols exist for biological sample loading, storage, and extraction from paper,1 but the ability to perform in situ chemical detection directly from the inexpensive paper substrate has profound implications on clinical analyses, especially in resource-limited settings.2 The relatively new paper spray (PS) ambient ionization technique enables handheld mass spectrometers to directly characterize untreated biofluid samples with no need for sample preparation.3 In PS mass spectrometry (MS), sample is simply placed on a paper triangle, and a high DC voltage (∼3−5 kV) is applied to the wet paper triangle. This process releases charged microdroplets that contain the analyte of interest, which are then transported to the mass spectrometer for characterization. PS-MS has simplified and expanded the utility of mass spectrometric analysis to include the quantitative detection of drugs and their metabolites directly from biofluids,4,5 detection of food contaminants,6 lipid profiling from tissues and bacteria samples,7,8 and analysis of noncovalent protein complexes,9 all requiring minimal sample pretreatment. Unfortunately, detection limits of PS analyses from complex samples such as blood and urine are often inadequate. These higher limits of detection have been attributed to inefficient analyte extraction from the sample matrix,10 and recent efforts have focused on developing solid-phase extraction (SPE) methods to enhance the sensitivity of the PS experiment by concentrating the analyte onto the SPE surface (e.g., a delrin plastic cartridge and C18-coated metal blade).11,12 Although lower detection limits are obtained, the separate extraction and cleaning steps required for these SPE methods can limit field analysis when © XXXX American Chemical Society
Received: November 11, 2015 Accepted: January 5, 2016
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DOI: 10.1021/acs.analchem.5b04278 Anal. Chem. XXXX, XXX, XXX−XXX
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transferase from human liver was purchased from Lee Biosolutions (Maryland Heights, MO). Whatman filter paper (24 cm, grade 1) was purchased from Whatman (Little Chalfont, England). Hydrophobic Paper Preparation. Using a digital template, paper triangles were cut from filter paper with an Epilog Legend 36EXT laser with 15% power at 1000 Hz. Typically, 0.5 mL of silanization reagent (Trichloro(3,3,3trifluoropropyl) silane) was used for 4−5 sheets of filter paper. For all experiments, the required paper size was cut before silanization. Paper size was approximately 80 mm2 (base width of 9.5 mm, height of 16.6 mm). Mass Spectrometry and Paper Spray Video. Samples were analyzed by a Thermo Fisher Scientific Velos Pro LTQ linear ion trap mass spectrometer (San Jose, CA, U.S.A.). MS parameters used were as follows: 150 °C capillary temperature, 3 microscans, and 60% Slens voltage. Thermo Fisher Scientific Xcalibur 2.2 SP1 software was applied for MS data collecting and processing. Tandem MS with collision-induced dissociation (CID) was utilized for analyte identification. Dry hydrophobic PS spray plume was observed using a Watec camera (WAT704R). Enzyme Preparation. Lyophilized alanine aminotransferase was prepared in 1× PBS solution with 2% BSA in DI water. Samples were directly spiked into 30 μL human blood with 2 μL each of L-alanine (1.75 M) and α-ketoglutarate (60 mM) solutions. Triplicate solutions were prepared in a temporally staggered manner such that each sample was analyzed after the reaction was allowed to occur for 10 min.
Figure 1. (A) Functionalization of paper by modification of fiber surface using trichloro(3,3,3-trifluoropropyl) silane vapor to create a hydrophobic oligomeric silylated layer. Gaseous HCl released as a byproduct of the reaction is removed by vacuum in situ. (B) Photograph showing interaction of a dye solution on untreated versus treated (hydrophobic) paper. (C) Hydrophobic paper spray in which the biological sample (e.g., blood) prevents the spreading of the organic solvent beyond the point where the sample was deposited.
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on the hydrophobic paper triangle is achieved by adding an organic solvent to the paper and applying a DC potential (3 kV) (Figure 1C). The organic solvents suitable for in situ analyte extraction from the sample droplet must have the following properties: (i) surface tension less than that of the hydrophobic paper to allow wetting, (ii) immiscible with the biofluid sample, (iii) good solubilizing power for the target analytes, and (iv) suitable for electrospray. Ethyl acetate (surface tension 20 mN/m) satisfied these criteria16 and facilitated the analysis of both wet and dried biological samples. Contact with the organic solvent causes the fresh 4 μL sample droplet to collapse into a thin film, which allows several extraction cycles to be performed. This alternative paper spray ionization approach to biological sample analysis requires no sample preparation, where small organic molecules are extracted directly from undiluted biofluids; enzyme analysis is possible via direct detection of product without coupled reactions.
RESULTS AND DISCUSSION Analysis of Illicit Drugs. The liquid/liquid extraction process occurring on the hydrophobic paper surface after application of 10 μL of ethyl acetate and 3 kV is very efficient, as demonstrated for the extraction of cocaine, methamphetamine, amphetamine, and benzoylecgonine from whole human blood, serum, and urine samples (Table 1). Limits of detection Table 1. Limits of Detection (LODs) of Analytes in Blood, Serum, and Urine Using In Situ Liquid/Liquid during Hydrophobic Paper Spray LODs (ng/mL)a in biological matrices
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EXPERIMENTAL SECTION Chemicals and Reagents. Standard solutions (1.0 mg/ mL) of benzoylecgonine, cocaine, amphetamine, and (±)-methamphetamine were obtained from Cerilliant (Round Rock, TX). Human blood was purchased from Innovative Research (Novi, MI). Phosphate-buffered saline (PBS) tablets were purchased from AMRESCO (Solon, OH). Glacial acetic acid was purchased from Thermo Fischer Scientific (Waltham, MA). Methanol (99.9%, HPLC grade), (3,3,3-trifluoropropyl)silane, bovine serum albumin (BSA) (10%) in PBS, myoglobin from equine skeletal muscle, human serum, α-ketoglutaric acid disodium salt hydrate, and L-Alanine were purchased from Sigma-Aldrich (St. Louis, MO), including standards (hexadecane, tridecane, dodecane, decane, octane, benzene, toluene, pxylene, nitrobenzene, glycerol, ethylene glycol) used in the estimation of surface energy. Lyophilized alanine amino-
analyte
logP
blood
serum
urine
cocaine amphetamine methamphetamine benzoylecgonine
2.28 1.80 2.24 −0.59
0.17 0.06 0.18 0.13
0.06 0.02 0.09 0.08
0.04 0.08 0.05 0.2
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LODs were calculated from respective calibration curves using signal corresponding to Sblank + 3 × σblank; where Sblank is the average blank signal and σblank is the standard deviation of the signal from three replicates
(LODs) as low as 0.06 ng/mL for amphetamine were recorded for undiluted human blood samples using the hydrophobic paper spray experiment. Analyte signal for LOD measurements were based on tandem MS (MS/MS) characteristic transitions. Representative product ion spectra for methamphetamine and benzoylecgonine at 3.9 ng/mL concentrations are shown in panels A and B of Figure 2, respectively. For urine samples, extraction efficiency correlated with hydrophobicity (log P) of the drugs (Figure 2C). LOD data, however, reveal that the B
DOI: 10.1021/acs.analchem.5b04278 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 3. Quantitative analysis of (A) undiluted human serum spiked with methamphetamine, and its internal standard methamphetamined5 (50 ng/mL) and (B) dried blood spot spiked with cocaine, and it internal standard cocaine-d3. Analyte concentration of 0.24−500 ng/ mL was used. Error bars represent the standard deviation of analyses for three replicates with independent hydrophobic paper triangles. Ethyl acetate (10 μL for wet samples and 20 μL for dried samples) was used as the extraction/spray solvent.
Figure 2. Tandem MS analysis of drugs extracted from urine and blood. Representative product ion spectra illustrate (A) the methamphetamine transitions 150 → 119 (primary product ion) as well as 150 → 91 (benzylic cation) and (B) the benzoylecgonine transitions 290 → 168 (primary product ion) as well as 290 → 272 ([M + H − H2O]+). (C) Effect of the number of liquid/liquid extraction cycles on ion signal. MS/MS product ion intensities were monitored for cocaine (m/z 304 → 182), amphetamine (m/z 136 → 119), and benzoylecgonine (m/z 290 →168), each at 125 ng/mL spiked into urine (6 μL sample volume was used). MS/MS product ion intensities for corresponding internal standards at 50 ng/mL were also monitored: cocaine-d3, amphetamine-d5, and benzoylecgonined3.
have been removed (Table 1). Thus, accurate quantitation of pharmaceutical drugs or illicit compounds in biological samples can only be achieved after the free and bound drugs have been determined in a separate preparatory experiment.18,19 Another factor identified to influence the sensitivity of our liquid/liquid extraction approach is matrix effect. Direct MS analysis of raw urine using either electrospray ionization or atmospheric pressure chemical ionization (APCI) methods typically do not provide sufficient sensitivity because of strong interference by the high concentration of salts and other chemicals in the mixture. The use of organic spray solvent allows the extraction of only small organic compounds while leaving behind the majority of proteins and salt components in the biological sample. This reduces ion suppression effects due to endogenous matrices and significantly enhances the ionization efficiency of the extracted analytes. Viscosity of the sample was also observed to influence drug extraction efficiency and hence the analytical sensitivity of the method. For example, recorded LODs are comparable for drugs spiked into serum and urine (Table 1), although more drug molecules are expected to be available for sampling when spiked into urine. This result may be attributed to a biofluid thin film that is formed on the hydrophobic paper for more viscous samples (e.g., serum and blood). This thin film prevents further spreading of the 10 μL spray solvent and enables efficient and extended analyte extraction. Under this condition of limited solvent spreading, much of the area of the hydrophobic paper triangle is dry, and yet, ion signal is observed when DC potential is applied (Videos S1 and S2, Supporting Information). During the period of the applied voltage, electrostatic charging of the dry paper causes charges to accumulate at the solution−air interface. Progeny of charged
absolute analyte amount in the organic solvent is not the limiting factor in MS sensitivity determination. For example, benzoylecgonine is a hydrophilic metabolite from cocaine with limited solubility in organic solvents (logP = −0.6) but it is detected with high sensitivity (Table 1). Limit of quantification ranged from 0.2−6.4 ng/mL for blood samples to 0.1−12.3 ng/ mL for serum samples to 0.1−18 ng/mL for urine samples, with benzoylecgonine registering the highest quantities in all cases. For all biofluids tested, relative standard deviations less than 10% were obtained for samples with concentrations higher than 3.9 ng/mL. This performance was achieved via the use of internal standards, and monitoring analyte-to-internal standard ratios (A/IS) as a function of analyte concentration; this yielded good linearity for both wet and dried biological samples (Figure 3). We observed that the main determining factor for drug LOD is its protein binding capacity.17 In our experiment, only free drug molecules are detected, which are in equilibrium with protein-bound drug and drug that has diffused into red blood cells in the matrix. LOD was significantly improved for serum samples where the cellular and fibrinogen contents of blood C
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Figure 5. Signal time compared to sample volume of methamphetamine (125 ng/mL) spiked in serum. All samples were extracted with 10 μL of ethyl acetate. Sample sizes of greater than 4 μL showed a significant increase in analysis lifetime. Inset is MS/MS product ion mass spectrum recorded by using 1 μL of human serum spiked with methamphetamine (125 ng/mL).
Figure 4. Photographs showing various stages during electrostaticspray ionization from dry hydrophobic paper: (A) voltage off, (B) onset of applied voltage, (C) on-set of spray, (D) stable spray formed just before 0.02 s, (E) stable spray after 0.02 s, and (F) after 2 min of voltage application. DC voltage of 4000 V was applied across a distance of 5 mm.
cocaine were analyzed on untreated paper, a larger LOD of 3.5 ng/mL was achieved, which is 2 orders of magnitude higher than LOD recorded from treated hydrophobic paper. Our analysis is synergistic with portable, minimally invasive, and high-throughput biochemical analysis systems because small sample volumes do not spread on the hydrophobic paper and are able to be stored for later analysis. Analysis of Alanine Transaminase Enzyme Activity. Recent interest in paper-based sensors will also benefit from the capacity to monitor enzymatic activities directly from biological samples for disease diagnosis.22,23 For example, liver transaminases (e.g., alanine transaminase (ALT)) are biomarkers for liver injury and are regularly used in laboratory tests to monitor patients under treatment for HIV and tuberculosis.24,25 With the exception of chromatographic methods, all liver function tests rely on complex and coupled reactions to enable colorimetric, spectrophotometric, chemiluminscence, and radiochemical detection of the transaminase enzyme activity.26 We have used the hydrophobic liquid/liquid extraction method to monitor ALT levels in whole human blood via direct MS detection of pyruvate, one of the reaction products formed after ALT catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate (Figure 6A). Specifically, 30 μL blood samples previously spiked with various amounts of ALT (0−400 U/L) were mixed with 2 μL each of L-alanine (1.75 M) and α-ketoglutarate (60 mM) solutions. The mixtures were incubated for 10 min at 37 °C, and 4 μL blood aliquots were deposited on the hydrophobic paper triangle for MS analysis. Resultant pyruvate product in the blood aliquots was analyzed after applying 10 μL ethyl acetate and 3.5 kV DC voltage; representative tandem mass spectra are shown for control and 400 U/L ATL-spiked blood samples in Figure 6B,C, respectively. Unlike chromatography, separation of the four closely related compounds (L-alanine, pyruvate, α-ketoglutarate, and L-glutamate) is not required. Instead, the use of molecular weight information and MS/MS fragmentation of pyruvate enables direct detection of ALT
between the interior and exterior of the droplet (i.e., Laplace pressure).20 Further experiments with optical microscope (Videos S3 and S4, Supporting Information) have shown strong vibrations within the droplet when the potential is applied.21 We believe this vibration further enhances the extraction process, which contributes to the high sensitivity observed for serum and blood samples. For blood and serum, sample volumes of greater than 4 μL are required to enable the formation of the solvent bridge (involving the thin film of the biofluid), which produces an ion signal over a longer period of time (Figure 5). (This required sample volume is dependent on paper geometry and the position of the loaded sample with respect to the paper tip; smaller droplet sizes can be used with narrower paper triangles or when samples are loaded nearer the tip and still form a solvent bridge.) In the current experiment, the 15 s signal generated from 3kV (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: (614) 929-4276. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Ohio State University startup funds. C.R.M. thanks the Brazilian Scientific Mobility Program for funding.
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