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Rapid Quantification of Endogenous Cholesterol in Human Serum on Paper Using Direct Analysis in Real Time Mass Spectrometry (pDART-MS) Hua-Yi Hsieh, Li-Hua Li, Ren-Yu Hsu, Wei-Fong Kao, Ying-Chen Huang, and Cheng-Chih Hsu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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Analytical Chemistry

Rapid Quantification of Endogenous Cholesterol in Human Serum on Paper Using Direct Analysis in Real Time Mass Spectrometry (pDART-MS) Hua-Yi Hsieh,† Li-Hua Li,‡§ Ren-Yu Hsu,† Wei-Fong Kao,┴¶ Ying-Chen Huang,† Cheng-Chih Hsu†* †

‡

Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan; Department of Pathology and La§ boratory Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan; School of Medical Laboratory Science ┴ and Biotechnology, Taipei Medical University, Taipei, 11031, Taiwan; Department of Emergency and Critical Care ¶ Medicine, Taipei Medical University Hospital, Taipei 11031, Taiwan. Department of Emergency Medicine, School of Medicine, College of Medicine, Taipei Medical University

ABSTRACT: Blood testing for endogenous small metabolites to determine physiological and biochemical states is routine for laboratory analysis. Here we demonstrate that by combining the commercial direct analysis in real time (DART) ion source with an ion trap mass spectrometer, native cholesterol in its free alcohol form is readily detected from a few hundred nanoliters of human serum loaded onto chromatography paper. Deuterium-labeled cholesterol was used as the internal standard to obtain the absolute quantity of the endogenous cholesterol. The amount of the cholesterol measured by this paper-loaded DART mass spectrometry (pDART-MS) is statistically comparable with that obtained by using commercially available fluorometric-enzymatic assay and liquid chromatography mass spectrometry. Furthermore, sera from twenty-one participants at three different time points in an ultra-marathon were collected to obtain their cholesterol levels. The test requires only very minimal sample preparation, and the concentrations of cholesterol in each sample were acquired within a minute.

Biochemical analysis of blood and serum tests such as metabolic panels are routine in modern clinical science. The analytical methods for such analysis are usually based on immunochemical probes or biochemical assays. Alternatively, mass spectrometry (MS) has become a powerful tool for targeted biochemical analysis. It provides information on the molecular weights and chemical structures of biomolecules without the need for biochemical probes specifically designed for the target compounds. Biological samples, however, most of which are complex mixtures, require tedious preparation and separation procedures before they are introduced into mass spectrometers.1 These procedures, including liquid-liquid extraction, chemical derivatization, and chromatographic separation, are implemented to ensure MS performance.2,3 Methods that could simplify these procedures offer great potential for translating mass spectrometric interrogation into high throughput biochemical analysis for clinical laboratories.4 In the past decade, a strategy known as ambient ionization mass spectrometry (AIMS) has been developed, aiming for rapid mass spectrometric detection, in which the time and resources spent on sample pretreatment and separation are largely reduced.5-9 These techniques include desorption electrospray ionization (DESI), paper spray ionization (PSI), and direct analysis in real time

(DART). One of the key features of AIMS is that it allows ionization of analytes under open-air conditions for the subsequent MS analysis and can be easily interfaced with many commercial mass spectrometers.5-9 As such, AIMS greatly simplifies the work that needs to be done prior to MS measurement and makes clinical diagnosis possible.1013 Electrospray ionization (ESI)-based AIMS methods like DESI and PSI utilize a high voltage (2-5 kV) to generate charged micro-droplets to mobilize molecules of high-tomedium polarity from the substrate, e.g. glass slides and filter paper.14 PSI in particular has attracted a lot of attention in recent years, as it can rapidly obtain molecular information from liquid biopsy.15 Liquid samples of only few microliters are loaded onto porous filter paper, which is usually held by a metallic clip and positioned in front of the MS ion inlet for subsequent analysis. Similar to other ESI-based methods, an eluting solvent system should be chosen to obtain an optimal MS readout for each molecular species. Meanwhile, compounds of low polarity are rarely detected by PSI-MS. Unlike the ESI-based methods, DART is a plasma ion source developed in 2005 by Cody et al.6 DART utilizes an excited helium or argon gas stream to generate metastable species, which are then carried by the heated gas stream to react with the analytes and form gaseous ions.16-

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Analytical Chemistry 18

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DART is featured with its atmospheric pressure chemical ionization (APCI)-like MS patterns and mainly used for relatively non-polar compounds with molecular weight lower than 1,500 Da.16 Since its commercial inception, DART-MS has become commonplace across the world and is arguably one of the most adopted AIMS methods available for biochemical analysis.1,19,20 The ability to detect endogenous biomolecules is crucial, as altered metabolomic profiles of serum and other body fluids are potential indicators of abnormal conditions or disease states.21-23 It has been shown that DART is capable of detecting metabolites in human blood. Amino acids of the hydrophobic side chain such as tyrosine in human plasma can be quantified by DART-MS.24 In addition, metabolomics fingerprinting of serum using DART was achieved when compounds in the serum were derivatized to become easily ionizable.25 However, the samples used in these studies were stored and analyzed in their liquid forms. In 1963, Guthrie and Susi reported that dried blood spots (DBS) on filter papers could be used for rapid metabolite screening in a large population of newborn infants.26 Since then, the utility of liquid biopsy spotted on filter papers has been explored for economical screening of large numbers of samples for biochemical analysis using liquid chromatography mass spectrometry (LCMS).27,28 Recently, such paper-biopsy concepts were combined with ESI-based AIMS, like PSI, and flowprobe, for the analysis of polar compounds.29-31 In this study, we propose a novel platform combining commercial DARTMS with paper-loaded dried serum samples for rapid mass spectrometric screening (see Figure 1 and the supporting information for a demonstration video). For ease of discussion, we will refer to this integrated platform as pDART-MS as follows. ■

MATERIALS AND METHODS

Materials and Reagents. Sera from five individual human volunteers (100 μL of each) were collected and pooled into a single vial as the standard serum used for pDART-MS demonstration and to construct the calibration curves for cholesterol quantification in pDART-MS, high performance liquid chromatography (HPLC)-MS, and fluorometric-enzymatic array experiments. Collection of serum samples from volunteers participating in an ultramarathon were described elsewhere.32 All human serum samples were obtained from Taipei Veterans General Hospital under Institutional Review Board (VGHIRB No: 2011-01-060IC) protocol and approved by the Ethics Committee of Taipei Veterans General Hospital. All the serum samples were stored in a −80 °C freezer before the experiments. Except for vortexing and centrifugation, no further sample-pretreatments were conducted to the serum. Standards compounds of cholesterol (≥ 99%) and isotopically labelled cholesterol-26,26,26,27,27,27-d6 (hereafter to referred as d6-cholesterol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). TOYO No. 51A chromatography paper (Advantec, TOYO, Tokyo) was used as the sample substrate. Ultra-pure water (18.2

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MΩ·cm) used in the HPLC system was prepared by a Milli-Q system (Millipore, Milford, MA, USA). MS grade methanol, HPLC grade 2-propanol, and reagent grade chloroform were purchased from JT Baker (Phillipsburg, NJ, USA). Trifluoroacetic acid (TFA) was purchased from Panreac (Barcelona, Spain). Samples Preparations. The cholesterol (600 μg/mL) and d6-cholesterol (300 μg/mL) standard stock solutions were prepared by dissolving the compounds in a mixture of chloroform/methanol (1:1, v/v). Serial dilution of the cholesterol stock solutions in CHCl3/MeOH provided calibration solutions with concentrations of 0, 100, 200, 300, 400, 600 μg/mL. Equal volumes of cholesterol calibration solutions and d6-cholesterol stock solution were mixed to obtain calibration solutions containing concentrations of cholesterol of 0, 50, 100, 150, 200, 300 µg/mL, and 150 µg/mL d6-cholesterol in the quantitative measurements of each solution. Chromatography papers were cut into equilateral triangles with side lengths of 1.0-cm (see Figure 1a). For cholesterol quantification using pDART-MS, 0.5 μL of human serum was pipetted and carefully loaded onto the front tip of the paper triangle (Figure 1b) and then left untouched for ~1 minute till it dried. A secondary aliquot of 0.5 μL of d6-cholesterol solution (150 μg/mL) was loaded onto the same serum sample spot of the paper tip. For the marathon samples, similarly, 0.5 μL of human serum from marathon participants were loaded onto the paper tips and left untouched ~1 minute. And then 0.5-μL standard solutions containing 150 μg/mL of d6cholesterol alone were added to the paper surface the same way. Thus, for the pDART-MS measurement of the marathon samples, all of the unlabeled cholesterol is endogenous to the human serum. DART Mass Spectrometry Analysis. A DART SVP ion source (IonSense, Saugus, MA, USA) interfaced to the mass spectrometer via a VAPUR interface (IonSense, Saugus, MA, USA) was utilized throughout all the pDART-MS experiments in this study.16 Ultra-high purity nitrogen (99.999%) was used as the standby gas, and ultra-high purity helium (99.999%) served as running gas. The output pressures of both nitrogen and helium were set at 0.5 MPa. The gas heater temperature of the helium reagent gas was set at 350 °C. A vacuum pump was connected to the VAPUR interface to reduce the excess amount of discharging gas flowing into the inlet of MS. An automatic transmission module (IonSense, Saugus, MA, USA) was used as a paper holder to align the paper to the DART gas stream. Details of the configuration of the transmission module have been described elsewhere.16 In short, it is a stainless steel frame with ten orifices that allow DART gas and desorbed analytes to pass through. Paper triangles were attached to the transmission module by double-sided tape (Figure 1c). A one-dimensional (in xaxis) motor is interfaced with the transmission module to move the samples across the DART gas stream (in z-axis). The planes of the paper triangles (x-y) are perpendicular to the flow of DART gas stream. The optimal MS signal is


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usually obtained when the DART source, transmission module, VAPUR interface are aligned in a straight line along the z-axis (Figure 1d). The distance between the DART source-transmission module and transmission module-VAPUR interface was about 2.0 mm. The velocity of the transmission module was set at 0.2 mm/sec. It takes about ten minutes to complete the scanning of a row of samples (ten pieces of paper). A Finnigan LTQ linear ion trap mass spectrometer (Thermo Scientiﬁc, San Jose, CA, USA) was used to acquire all the mass spectra for pDART-MS. The mass spectra were obtained in the positive mode. The LTQ tune file was optimized for m/z 369.5 and 375.5 by using commercial cholesterol and d6-cholesterol standards. The mass spectral analysis and peak integrations were processed by Xcalibur Qual-Browser (Thermo Scientiﬁc, San Jose, CA, USA) software. The TIC peak integrations were processed at m/z 369.0-370.0 and m/z 375.0-376.0 for cholesterol and d6-cholesterol respectively. Fluorometric-enzymatic Assay. Total Cholesterol Assay Kit (Fluorometric) purchased from Cell Biolabs, Inc. (San Diego, CA) was used to determine the concentration of cholesterol.33 The serum was diluted to 1:200 with 1X assay diluent, which contains Triton X-100 0.1% and sodium hydroxide 2 N. Preparations of cholesterol standards were carried out according to the manufacturer’s instructions to construct the calibration curve of cholesterol. In each well of the plates, 25 μL of diluted cholesterol standards/ serum samples as well as 25 μL of cholesterol reaction reagent containing cholesterol oxidase, fluorometric probe, and horseradish peroxidase (HRP) were added and fully mixed with 50 μL of 1X assay diluent. Cholesterol

oxidase was used to oxidize cholesterol and produce hydrogen peroxide (H2O2) as the side product. H2O2 was subsequently detected by a fluorometric probe, in which the reaction was catalyzed by horseradish peroxidase. Cholesterol esterase was omitted from the cholesterol reaction reagent so as to determine the concentration of free cholesterol only. The fluorescence microplate reader (Synergy H1, BioTek, Winooski, VT, USA) was set at an excitation of 550 nm and emission of 595 nm. All the samples were tested in triplicate and reported in average with standard deviations. Liquid Chromatography Mass Spectrometry for Quantification. 100 μL of pooled standard serum was mixed with 100 μL of 2-propanol, and centrifuged for 10 minutes at 12,000 rpm. After centrifugation, the supernatant was passed through a 0.22-μm membrane filter. The serum solution was then mixed with an equal volume of d6-cholesterol solution (300 μg/mL in MeOH/CHCl3) prior to the HPLC-MS analysis. All the LC-MS measurements were performed using a Shimadzu LC-20AD LC (Kyoto, Japan) interfaced with an Orbitrap Elite (Thermo Scientific). The flow rate of the LC system was set at 0.20 mL/min; mobile phase A was ultra-pure water; mobile phase B was composed of 70% MeOH and 30% 2propanol (v/v), both of the mobile phase solutions were added with 0.1% TFA. A ZORBAX XDB-C18 column, 3.5 μm, 50 mm × 2.1 mm i.d. (Palo Alto, CA, USA) was used as the LC separation column. The MS spectra were collected in FT mode with 120,000 resolving power. The mass spectral analysis and peak integrations were processed by Xcalibur QualBrowser.

Figure 1. Workflow of pDART-MS for serum cholesterol: (A) cut chromatography papers into small triangular pieces by paper punch; (B) load serum and internal standard (0.5 μL each) onto the paper tip; (C) fix the paper-based samples on the transmis-



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sion module and install them to the x-axial translational motor; (D) scan across the samples one row at a time using the DART ion source. The demonstration video and instrument setup figures are available in the supporting information. ■ RESULTS

AND DISCUSSION

Cholesterol is one of the major constituents of cell membranes and a major precursor for the synthesis of various steroid hormones.34 Meanwhile, the alteration of cholesterol in blood is associated with atherosclerosis,35,36 noninsulin-dependent diabetes,37 and brain diseases such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD).38 In addition, cholesterol level is widely used as a mean to predict an individual's risk of coronary heart disease.39 As a result, cholesterol measurement is one of the most common laboratory tests performed in clinical laboratories and plays a crucial role in preventive healthcare. A variety of methods has been developed for the quantification of cholesterol, including fluorometric-enzymatic assays, gas chromatography mass spectrometry (GC/MS), and liquid chromatography mass spectrometry (LC/MS).33,40-42 Direct quantitative cholesterol measurements using AIMS has been achieved with DESI and DART ion sources.43,44 With its low proton affinity and acidity, free cholesterol has to be derivatized by charge labelling in DESI, so-called reactive DESI, before MS analysis.43 On the contrary, the ability of DART to ionize nonpolar compounds allows it to readily detect cholesterol in biological samples.44 This is also true for pDART-MS. As shown in Figure 2, endogenous cholesterol in serum spotted onto the chromatography papers were desorbed and rapidly reacted with the plasma-induced metastable species of the DART gas stream to generate dehydrated cholesterol (m/z 369.5, [M-H2O+H+]), which becomes the dominant ion product in the mass range of 50-1,000 m/z. The tandem MS experiment confirms that m/z 369.5 is the reaction product of endogenous cholesterol with the DART ion source. The fragmentation spectrum of m/z 369.5 observed in serum is consistent with that obtained with the authentic cholesterol standard (Figure 3).

Figure 2. (A) Typical positive ion mode mass spectrum of serum obtained by the pDART-MS platform from a mixture of 0.5-μL serum sample and 0.5-μL d6-cholesterol solution. (B) Mass spectral details of (A) at m/z 350-400. Dehydrated forms of cholesterols are dominant in this region.

Figure 3. MS/MS spectra of commercial (A) and endogenous (B) cholesterol using pDART-MS.

The concentration of total cholesterol, occurring in the free form and cholesteryl esters (esterfied to long-chain fatty acids), is highly variable and fluctuates within a wide range of 1,200 to 2,500 µg/mL in human serum, whereas the concentration of the free cholesterol is usually within the range of 200 to 500 µg/mL.45-47 In this regard, we attempted to determine the concentration of free choles-


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terol using the isotopically labeled d6-cholesterol as the internal standard. The labeled cholesterol was spiked into the pooled human serum from five healthy volunteers without any prior extraction or purification. The spiked standard (d6-cholesterol) gave a signal at m/z 375.5, where the mass shifted as the result of deuterium labeling, and can be differentiated from the endogenous form by a common ion trap mass spectrometer (Figure 2b). The tandem MS of the internal standard d6-cholesterol at m/z 375.5 shows a similar fragmentation pattern to that of free cholesterol (See supporting information Figure S2). To evaluate the capability of the quantitative analysis of free cholesterol in serum using pDART-MS, calibration solutions of serial concentrations of standard cholesterol (0, 50, 100, 150, 200, 300 µg/mL in sample 1 to 6, respectively) and d6-cholesterol (kept constant at 150 µg/mL) mixtures were prepared. Aliquots of 0.5 µL of each calibration solution were spotted onto the chromatography papers loaded with 0.5 µL of the pooled serum before DART-MS interrogation. These paper samples containing equal amounts of endogenous cholesterol were placed on the sample carrier of the transmission module for DARTMS analysis. As shown in Figure 1 and the demonstration video in the supporting information, the paper tips containing the analytes were positioned close to the center of each orifice of the module to ensure the exposure of the analytes to the DART gas stream. The tip-to-tip distance was about 12 mm.

Figure 4a shows the extracted ion chromatograms (EIC) of free cholesterol (m/z 369.5) and d6-cholesterol (m/z 375.5) obtained by scanning across the six paper samples for calibration with the DART ion source. The constant cycling times (~60 seconds) between any two adjacent peaks in both m/z 369.5 and 375.5 were consistent with the time required to move from one tip to another, where the speed of the transmission module was set at 0.2 mm/sec. Notably, although d6-cholesterol and free cholesterol, consisting of the endogenous and the spiked ones, were spotted homogeneously around the paper tips, the ion chromatograms were dynamically different. As shown in Figure 4b, t1/2 (the time point when ion intensity rises to its half maximum) of both m/z 369.5 and m/z 375.5 in sample 4 are ~5.47 min, indicating unanimous onsets of ion chromatograms. Meanwhile, the ion signal of m/z 369.5 lasts much longer, in which the full width at half maximum (FWHM) of spiked d6-cholesterol (m/z 375.5) is only ~0.05 minute, and that of cholesterol (m/z 369.5) is about 0.10 minute. Furthermore, the entailed ion signal of m/z 369.5 continues for about half a minute (Figure 4b). This result implies that matrix effects from the other components in the serum are obstructive to the desorption/ionization of endogenous cholesterol when using pDART. Compared to the spiked d6-cholesterol, free cholesterol from serum required more time to be desorbed from the substrate, leading to a prolonged response.

Figure 4. (A) Extracted ion chromatogram of cholesterol and isotopically labeled standard d6-cholesterol obtained by pDARTMS. Each paper was loaded with 0.5 μL of human serum and 0.5 μL of calibration solutions containing 150 μg/mL of d6cholesterol and 0 (sample 1), 50 (sample 2), 100 (sample 3), 150 (sample 4), 200 (sample 5), and 300 μg/mL (sample 6) of unlabelled cholesterol standard. Signals of the blank paper were obtained as blank. The peak labeled with * was the signal of the paper loaded with only standard serum, which was inserted as a quality control to ensure that the analytes were not carried over to the next sample test. (B) The overlaid EICs of paper sample 4 show that although their t1/2 are of about the same value, EIC of m/z 369 (in red) has much larger FWHM than that of m/z 375 (in black). (C) The EIC of sample 1 (in black) and the de-



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convoluted EIC of endogenous cholesterol in sample 4 (in red). The scatterplot of the correlation between the two EICs is exhibited in the supporting information (Figure S4).

To gain more insights into the mechanism of desorption/ionization of serum metabolites using pDART, we turn to de-convolute the EIC of m/z 369.5 to obtain the component of endogenous cholesterol in the mixed samples. For example, in sample 4, in addition to the endogenous cholesterol, 150 µg/mL of the spiked cholesterol standard also contributed an ion intensity of m/z 369.5. On the contrary, 150 µg/mL of d6-cholesterol rendered the ion intensity of m/z 375.5 solely, which was recorded simultaneously. As the ionization efficiency of both of the spiked standard compounds are identical and they were fully mixed in a single stock before being loaded onto the chromatography papers, the pDART-MS of these two standards should retain similar elution patterns. Thus, the unadulterated EIC of the endogenous cholesterol in sample 4 could be obtained by subtracting EIC of m/z 369.5 with that of m/z 375.5. As shown in Figure 4c, the subtracted curve reveals the EIC of endogenous free cholesterol in the serum of sample 4. It is noteworthy that the EIC pattern of a subtracted curve overlaps nicely with sample 1, whose m/z 369.5 ion intensity was solely contributed from endogenous cholesterol. This experimental result of highly consistent EIC profiles provides us with a conclusive piece of evidence suggesting that the endogenous cholesterol in the serum and the spiked cholesterol standards performed different desorption/ionization procedures. The carry-over signal of m/z 375.5 was not observed in the control experiment with serum-only paper (Figure 4a). Figure 5 shows the standard addition calibration curve obtained by the triplicates of the six samples containing endogenous and spiked cholesterols. The fitted curve exhibits a good linearity from (x+0) to (x+300) µg/mL for free cholesterol, where x represents the concentration of endogenous cholesterol to be determined in the pooled serum samples. By extrapolating the fitted curve, the concentration x of the endogenous cholesterol was estimated to be 250±16 µg/mL with a relative standard deviation (RSD) of 6.2%. To evaluate the performance using pDART-MS, complementary LC-MS, and fluorometricenzymatic assay tests were also carried out. The cholesterol concentrations of the calibration samples were determined to be 263±12 and 265±16 μg/mL by using LC-MS and fluorometric-enzymatic assays, respectively. These experiments were all carried out in triplicate and the results show no statistical significance among each (Figure 6). The relative standard deviations are 4.4% for LC-MS, and 6.0% for fluorometric-enzymatic assay, revealing that the stability of pDART-MS is comparable to those of traditional LC-MS and commercialized fluorescence-based assay. The limit of detection (LOD) was determined as the amount of serum cholesterol giving a signal-to-noise ratio (S/N) of 3.0. The S/N was estimated by comparing the m/z 369.5 peak height of pooled serum with blank paper (which served as the baseline noise). As the S/N was ~34.8 with 250 µg/mL of serum cholesterol (see sample 1 in Figure 4 and Figure S5), the LOD for endogenous

free cholesterol in the serum was thereby estimated to be ~21.5 µg/mL in this study (Figure S5).

Figure 5. Calibration curve of peak area ratio of ion m/z 369.5 to m/z 375.5 obtained from sample 1 to 6 in Figure 4. The bars represent the standard deviations of the analysis from three replicates at each cholesterol concentration. The endogenous cholesterol concentration in the pooled serum (250±16 μg/mL) was estimated by extrapolating the fitted curve.

Figure 6. The statistical comparison of the level of cholesterol in the pooled standard serum analyzed by pDART-MS, LC-MS, and fluorometric-enzymatic assay. Each measurement was performed in triplicate. The relative standard deviations (RSD) are 4.4% (LC-MS), 6.2% (pDART-MS), and 6.0% (fluorometric-enzymatic assay). This result shows no statistical significance among three methodologies. Since cholesterol has been largely accepted as a screening test in adult healthcare, the ability of pDART-MS in cholesterol quantification offers great potential as a new platform for performing metabolic panels in clinical laboratories. In


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this regard, we collected sixty-three serum samples from twenty-one ultramarathon runners at three different time points, including (1) one-week before (pre-), (2) immediately post-race (post-), and (3) one-day after race (post-24h) for cholesterol pDART-MS analysis. In order to assess the performance of pDART-MS, paralleled fluorometric-enzymatic assay tests were also implemented for all of the sixty-three serum samples. As shown in Figure 7, the mean cholesterol levels at all three time points obtained by both methods lie around 330 µg/mL. The cholesterol levels in the post-race groups measured by both methods all show a narrower topto-bottom range, implying that ultramarathon may result in a regulatory effect on cholesterol metabolism. In fact, previous studies reported that ultramarathons are associated with 48 49 certain degrees of injury of the liver, kidney, and cardio50 myocytes. Further investigation is needed to explore the correlation. However, more importantly, our results demonstrated that pDART-MS serves as an effective platform for cholesterol screening tests that is comparable with the fluorometric-enzymatic approach, which has been routinely used in clinical laboratories.

terol. According to quantitative measurements, the performance of pDART-MS is comparable with conventional methods including LC-MS and fluorometric-enzymatic assays. The optimized pDART-MS enables the detection of cholesterol at the level >20 µg/mL of serum, which is an order of magnitude lower than the level in adult human serum. The time required for the pDART-MS of each sample is ~1 min and the cost is ~1 United State Dollar (USD), which was mostly spent on the purchase of internal standards. The MS result was acquired by a common ion trap mass spectrometer throughout the experiment. This platform represents a potential complementary tool for cholesterol tests in clinical laboratories and, with a larger amount of verification in the future, could possibly be translated to the use for point-of-care testing (POCT).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures of the experiment setup of pDART-MS, MS/MS spectra of cholesterol and d6-cholesterol, optimization of the DART gas heater temperature to the intensity of cholesterol, correlation plot of EICs in Figure 4c, and the LOD estimation of pDART-MS for cholesterol in serum (PDF). Demonstration video of pDART-MS (wmv).

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Cheng-Chih Hsu: 0000-0002-2892-5326

Notes Figure 7. Box-and-whisker plot of sera from twenty-one ultramarathon participants collected at three time points: (1) one-week before (pre-), (2) immediately post-race (post-), and (3) one-day after race (post-24h). Every box chart contains 21 data points. The upper, middle and lower edges of each box represent the 75%, 50%, and 25% distribution values of the data set, respectively. The upper- and lower-end of each line represent the 95% and 5% distribution value of the data set, respectively.

■CONCLUSION

In the present work, a method for rapid and cost-effective screening of endogenous cholesterol was demonstrated on dried human serum spots at ambient environment using DART-MS. We have shown that free cholesterol loaded onto the porous chromatography paper is able to readily interact with the DART gas stream in a heterogeneous phase reaction to form dehydrated ionic species. Quantitative analysis of endogenous cholesterol in human serum was achieved by a secondary aliquot of the internal standard solution containing deuterium labeled choles-

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by Ministry of Science and Technology (MOST), R.O.C. (Grant #: MOST 105-2113-M-002-004MY2) and Taipei Veterans General Hospital, Taipei, Taiwan (Grant #: V106C-130). We gratefully acknowledge the laboratory of Hwan-Ching Tai in National Taiwan University and the laboratory of Chien-Chen Lai in National Chung Hsing University for the access to mass spectrometers. We also acknowledge ASPEC Technology and IonSense Inc. for the access to the DART ion source.

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