Int. J. Ion Mobil. Spec. (2008) 11:61–69 DOI 10.1007/s12127-008-0006-5

ORIGINAL RESEARCH

A rapid analytical method for hair analysis using ambient pressure ion mobility mass spectrometry with electrospray ionization (ESI-IMMS) Prabha Dwivedi & Herbert H. Hill Jr

Received: 13 September 2007 / Revised: 8 May 2008 / Accepted: 9 May 2008 / Published online: 10 June 2008 # Springer-Verlag 2008

Abstract Analysis of hair is often applied to assess drug abuse history, exposure to environmental and industrial pollutants, heavy metals, gestational drug exposure and various other screening purposes. This manuscript reports the application of ambient pressure ion mobility spectrometry mass spectrometry (IMMS) with electrospray ionization (ESI) source as a rapid analytical tool for hair analysis. The study demonstrated that ion mobility spectrometry (IMS) as a pre-separation technique prior to analysis by mass spectrometry (MS) provides detection and determination of compounds of interest present in hair at nano-molar concentration level. After extraction of analytes from hair, the ESI-IMMS method of analysis does not require the derivatization or sample treatment that is often required for other separation methods such as gas chromatography. One advantage of IMS over chromatography separation is that resolving powers are similar to those in GC and much greater than those possible by liquid chromatography. In addition, separation speed is faster than both gas and liquid chromatographic methods. Four of the nine hair samples anonymously donated by customers at a local hair salon tested positive for caffeine and two of the four samples that tested positive for caffeine also tested positive for nicotine. A positive response based on mass analysis for methamphetamine was obtained for one of the hair samples. Further investigation using the mobility data demonstrated that the response was a false positive and that it may have occurred from the use of a hair gel. This article reports the potential of IMMS as an analytical technique for rapid and routine screening of hair samples, cosmetics, and pharmaceuticals. P. Dwivedi : H. H. Hill Jr (*) Department of Chemistry, Washington State University, Pullman, WA 99164, USA e-mail: [email protected]

Keywords Hair analysis . Ion mobility spectrometry-mass spectrometry . Metabolomics . Drugs . Cosmetics . Pharmaceuticals

Introduction Analysis of hair is used in various toxicological, clinical, or forensic investigations. The advantages of performing hair analysis over other biological samples are that hair analysis (1) is a less intrusive method, (2) provides a history of drug use, (3) is more resistant to adulteration, (4) can be easily collected and stored, (5) has long shelf life and (6) can be easily decontaminated [1]. Determination of cause of death, or assessment of drug abuse and exposure levels to environmental and industrial pollutants [2–4] or monitoring therapeutic compliance and treatment [5, 6] are achieved by performing analysis of hair samples. Identification, detection and/or determination of metabolites present in hair can also be used to predict, detect, and treat various diseases [7–14]. In addition, segmental analysis of hair yields information that can be related or associated with chronology of diseases, poisonings, or drug use [15, 16]. The use of hair for forensic analysis is not a new concept. In 1858, hair obtained from a dead body, exhumed after 11 years was analyzed for arsenic and cause of death by arsenic poisoning was reported [17]. Hair analysis as a means to determine histories of drug abuse gained popularity only in 1970s and 1980s when methods to detect opiates in digested hair samples were reported [18, 19]. Currently, analysis of hair to determine drugs of abuse is performed by separation techniques such as gas chromatography [20, 21], liquid chromatography [22–24] and capillary electrophoresis [25–27] with or without mass spectrometry. However, the steps required for these analyses such as

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extraction, concentration, derivatization, separation and detection, of targeted analytes reduce sample throughput. Derivatization procedures are generally applicable to certain chemical classes and often render chromatographic methods suitable only for a specific class of analytes. For example, preparation of hair sample for drug analysis by gas chromatography-mass spectrometry (GC-MS) involved acid hydrolysis of hair, followed by a three-step liquid–liquid extraction, and derivatization with N,O-bis (trimethylsilyl) trifluoroacetamide plus 1% trimethylchlorosilane [28]. Similarly, for sensitive determination of histamine HA in hair by high pressure liquid chromatography– electrospray ionization mass spectrometry (HPLC–ESI-MS), HA was first extracted from hair by acid hydrolysis, followed by fluorescence labeling for HPLC separation and then detected by ESI-MS [29]. Thus, there is a need for development of innovative approaches for hair analysis in order to simplify the measurement, increase sample throughput and minimize diversity in sample preparation. Ion mobility spectrometry (IMS) with its millisecond separation time provides 105 times greater number of plates per second when compared to chromatographic separation methods such as GC, LC, and CE [30]. Currently, IMS is widely used as a rapid detection method for vapor phase analytes such as chemical warfare agents, explosives, drugs and air contaminants [31]. Pyrolysis-IMS and thermal desorption-IMS have also been applied for analysis of hair for drugs [32–35]. However, determination of non-volatiles present in hair is not feasible using pyrolysis-IMS and thermal desorption-IMS. With the introduction and development of electrospray ionization (ESI) as the ion source for IMS [36, 37], application of IMS have been extended to the analysis of biological samples and structural studies of non-volatile ionic species [38–41]. Thus, ESI-IMMS seems to be viable analytical option that can be used for rapid determination of volatile and non-volatile species present in hair. Two dimensional information (mobility and m/z) acquired through an IMMS experiment allows the analysis of complex biological samples. In a typical IMMS experiment, ions produced at atmospheric pressure by an ion source are injected in the drift region of an IMS where ions are separated based on their size-to-charge ratio. These ions are then introduced into the MS through a pin-hole leak, guided by einzel lenses to the mass analyzer at pressure of ∼10−5 torr, and are separated based on their mass-to-charge ratio. Due to high velocity of ions in vacuum compared to that at ambient pressures, time spent by an ion in the mass analyzer is considered negligible with respect to the time that the ion spends in the drift region of the ambient pressure IMS. Hair, as any other biological sample, is expected to be complex in nature with multiple constituents. The objective of this study was to investigate if ion mobility spectrometry

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coupled to ESI as ion source and mass spectrometry (ESIIMMS) can be applied for rapid analysis of hair samples. As a “proof of concept study” the study focused on determination of caffeine and nicotine present in hair samples that were obtained as donations from customers at a local hair salon. These two drugs were selected as the target compounds for this study because caffeine and nicotine are the most common drugs that can be found in hair samples from the general population.

Materials and methods Samples and solvents High performance liquid chromatography grade solvents (methanol, water and acetic acid) were purchased from J. T. Baker (Phillipsburg, NJ, USA). Caffeine and nicotine were purchased from Sigma-Aldrich (Sigma Aldrich Chemical Co., St. Louis, MO, USA). Hair samples were anonymously donated by customers at a local hair salon. Sample preparation Hair samples collected at a local hair salon from nine customers were cut into about 5 mm lengths, washed three times with 50:50 methanol water solution, and left to dry overnight at room temperature. After the sample was evaporated to dryness, 50 mg of each hair sample was extracted with 4 ml of methanol in a screw cap culture tube by heating in a water bath for 8 h at ∼80 °C and then cooled to room temperature. The extract were then filtered using a 0.2 μm size syringe filter, and diluted with HPLC grade water and acetic acid to make solutions of composition 47.5:47.5:5 (methanol–hair extract/water/acetic acid). The extract was diluted to approximately half its original concentration (110% dilution). Samples were stored at −4 °C until analysis. ESI-IMMS analyzer The ESI-IMMS analyzer comprised of an electrospray ionization source (ESI), an ion mobility analyzer (IMS) and a quadrupole mass analyzer (MS), the schematic of which is shown in Fig. 1.

ESI Source For the generation of electrospray ions, sample was infused into a 30 cm long, 50 μm inner diameter silica capillary by a KD Scientific 210 syringe pump (New Hope, PA, USA) at a flow rate of 5 μl/min. The capillary was then inserted into a water-cooled 22-gauge stainless steel needle, the tip of which was centered ∼1.0 cm from the target screen of the IMS

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Fig. 1 Schematics of the ion mobility mass spectrometer with electrospray ionization source. The mass spectrometer was a quadrupole mass analyzer with electron multiplier as detector. The ESI and the IMS were operated at ambient pressure and the MS was at a pressure of 10−5 torr. The IMS and MS were coupled through a 40 μm pinhole interface

analyzer and the capillary protruded ∼2 mm out of the stainless steel needle tip. The ESI needle was maintained at a potential of +3.0 kV with respect to that on the target screen of the IMS.

(Keithley Instruments, Cleveland, OH, USA). The amplified signal was then sent to either the MS data acquisition system or IMS data acquisition system.

IMS analyzer The IMS analyzer was constructed at Washington State University, and used a stacked-ring design that has been described in previous publications [30]. In summary, the IMS tube was divided into a desolvation region (7.2 cm in length) and a drift region (21.8 cm in length) separated by a Bradbury–Nielsen-style ion gate. Both regions consisted of alternating alumina spacers and stainless steel rings with high temperature resistors connecting the stainless steel rings (Caddock Electronics, Riverside, CA, USA; 500 kΩ resisters for the desolvation region, 1 MΩ resisters for the drift region, 250 °C, 0.1% tolerance). The temperature of the drift region, desolvation region, and the drift gas was maintained at 250 °C, the ion gate held at a potential of 7.51 kV, and the instrument was operated at ambient pressure (696 to 703 torr in Pullman, WA, USA). With a potential of 9.5 kV at the IMS target screen, electric field in the desolvation region was ∼276 V/cm and ∼344 V/cm in the drift region of the IMS (E/N between 2–3 Td). Preheated counter flowing drift gas (nitrogen) at a flow rate of ∼1,300 ml/min was introduced at the end of the drift region (preheated to the temperature of the IMS). The ions were injected into the IMS drift region in pulses of 0.2 ms using an ion gate operating at a frequency of 25 Hz.

Instrument control, data acquisition and processing Merlin software (ABB Extrel, Pittsburgh, PA, USA) was utilized for all MS data acquisition, analysis and control. For the IMS gating and data acquisition, the electronic controls were built at Washington State University. Labview (National Instruments, Austin, TX, USA) based software developed at Washington State University, Pullman WA, USA; was used for IMS data acquisition and IMS gate-control. Two types of ion mobility spectrum were generated through ESI-IMMS experiments: non selected ion mobility (NSIM) spectrum and selected ion mobility (SIM) spectrum. In the first case, the MS was operated in the “scan mode” with the DC voltage turned off and served as an ion transfer line between the IMS and the detector and provided a non-selected ion mobility (NSIM) spectrum for the analyte(s). In the second case, the MS was operated in the “single ion monitoring mode” and served as a filter that allowed transfer of ions with a preselected m/z value reach the detector. Under these conditions, a selected ion mobility spectrum (SIM) was obtained. Operation of IMMS in SIM mode allows one to monitor the drift time(s) of ions associated with preselected m/z values.

Calculations MS analyzer The IMS analyzer was interfaced to a model 150-QC ABB Extrel (Pittsburgh, PA, USA) quadrupole analyzer (m/z range of 0–4,000 amu) via a 40-μm pinhole interface. Earlier publications from our research group document the detailed description and schematics of the IMMS analyzer [30, 42]. The output signal from the multiplier was amplified by a Keithley model 427 amplifier

Reduced mobility value for ions (Ko in the units of square centimeter per volt second) was calculated using the following equation:    2  L 273:15 P K0 ¼ T 760 Vtd

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where, L is the length of the drift region in centimeter, V is the voltage applied to the ion-gate in volts, td is the ion drift time in seconds, T is the drift region temperature in Kelvin, and P is the ambient pressure in torr.

Results and discussion Analysis of hair samples Non selective ion mobility (NSIM) spectra of hair samples identified as A, B, C, D, E, F, and G were acquired by operating the MS in the “scan mode”. Figure 2 shows the superimposed NSIM spectra of (1) negative control sample (m/z 10–400 Da), (2) hair sample A (m/z 100–400 Da, and (3) hair sample E (m/z 100–400 Da). Since most of the ions detected in the negative control sample were of m/z values less than 100 Da, the cutoff m/z value for NSIM spectra of hair samples was set at 100 Da to exclude background ion peaks from the NSIM spectra. Depending on the identity of the hair sample, number of ions detected in the m/z range of 100–400 Da varied between 30 and 55. Peak patterns (NSIM spectra) and number of peaks detected were reproducible for each hair sample in two replicate measurements. Qualitative differences of the hair samples were observed by comparing their respective NSIM spectra. For example, Fig. 2 shows the difference in peak pattern/number

of ions detected between hair samples A and E. Absence of IMS peaks in the drift time range of 15–30 ms in the NSIM spectrum of the control and their presence in the NSIM spectra of hair extracts shows that various analytes were extracted from the hair samples and were detected by ESIIMS. Incomplete resolution of IMS response peaks corresponding to 30–55 m/z ions that were detected by MS resulted in a broad “hump” like peak shape in the NSIM spectra of each hair sample. Selected ion mobility (SIM) spectrum provided specific details (m/z, mobility, and intensity) for each of the detected analytes. SIM spectra of few target analytes detected in the extracts of hair samples are discussed in the following sections. Ability to rapidly analyze hair samples without any further sample treatment after extraction, allows one to use ESI-IMS as a screening tool for rapid analysis of hair samples. ESI-IMMS can be used to rapidly characterize complex biological samples such as hair and determine the identity of each constituent using two dimensional IMMS data. Targeted component analysis of hair samples Since the hair samples collected were anonymous and thus provided no prior information about the subjects, the experiments were directed towards detection of most common drugs that can be potentially present in hair samples

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Fig. 2 Non selective ion mobility (NSIM) spectra of negative control, hair sample A and hair sample E. Each of the hair samples produced characteristic and reproducible IMS spectrum as illustrated for hair samples A and E. The IMS spectra were obtained in the scanning mode of the MS. The negative control NSIM spectrum was obtained in the mass range of 10–400 Da and the NSIM spectra of hair samples were obtained in the mass range of 100–400 Da

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Fig. 3 Selected ion mobility (SIM) spectra obtained at m/z 195 of (a) negative control, (b) hair sample G, (c) hair sample B, (d) hair sample E, and (e) hair sample A. The drift time of ion at m/z 195, the (MH)+ ion of caffeine, was monitored in each sample. Variation in intensity of the IMS peak at m/z 195 in each hair sample was observed

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collected from general population. The focus of the study was to investigate if drugs such as nicotine (m/z=162) and or caffeine (m/z=194) in the collected hair samples could be extracted and detected by ESI-IMMS. Out of nine hair samples analyzed, based on m/z values of protonated   ions of nicotine ðMHÞþ
ðm=z ¼ 163Þ and caffeine ðMHÞþ
ðm=z ¼ 195Þg, peaks corresponding to caffeine were detected in hair samples identified as A, B, E, and G and peaks corresponding to nicotine were detected in hair samples identified as A and E. The peaks detected at m/z values of 163 and 195 were identified as that of nicotine and caffeine extracted from hair because (1) the m/z values and reduced mobility values (Ko) of these peaks matched those measured for standard solutions of nicotine and caffeine analyzed by ESI-IMMS and (2) these peaks were not detected in the negative control. The Ko values of the ions at m/z 163 and m/z 195 in hair sample A also matched those measured in hair sample E.

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Fig. 4 Ion mobility spectra illustrating the detection of nicotine in hair samples. On the left panel are shown the NSIM spectra of hair samples A, and E with highlighted nicotine peak at m/z value of 163. Also shown are the peaks at 150, 195, and 231 Da for reference. The right hand panel shows the SIM spectra obtained at m/z 163 with drift times of 19.24 and 19.26 ms in hair samples A and E, respectively

Figure 3 shows selected ion mobility (SIM) spectra of hair samples A, B, E, and G at m/z value of 195 acquired by operating the MS in the single ion monitoring mode. The drift times of the IMS peak at m/z 195 was measured to be 19.84, 19.81, 19.88, and 19.86 ms in samples A, B, E, and G, respectively, with Ko values of 1.53±0.02 cm2 V−1 s−1. With three consecutive measurements, the standard deviation in drift time for all samples was measured to be less than ±0.06 ms. The Ko for standard solution of caffeine at m/z 195 was measured to be 1.53±0.01 cm2 V−1 s−1 which compared well with the reduced mobility measured for the detected ion of m/z 195 in each of the hair samples. SIM spectra for the negative control at m/z 195 is also shown. Hair samples containing both caffeine and nicotine also produced a peak at m/z value of 231 Da and drift time of 21.26±0.04 ms. The ion was tentatively identified as water adduct peak of caffeine fM 2ðH2 OÞHgþ
ðm=z ¼ 231Þ. By comparing mobility values measured for the standard

drift time 19.26 ms 0.03 0.4

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IMS Response

A Hair Sample A; m/z 150 Drift time 18.14 ms

B Methamphetamine standard m/z 150; Drift time 18.66 ms

C Methamphetamine in Hair Sample A; m/z 150 Drift time 18.11 and 18.65 ms

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Fig. 5 SIM spectra at m/z 150 showing the separation of isobaric ions. SIM spectra of hair sample A (a), standard solution of methamphetamine (b) and hair sample A spiked with methamphetamine (c) are shown

Fig. 6 NSIM spectra of vitamin C (a), vitamin B (b), aspirin (c), hair gel (d), and hair sample A (e). IMS peak with a drift time of 18 ms at m/z 150 observed in hair sample A is aligned with that observed in the NSIM spectrum of the hair gel

caffeine solution and the peak observed in the hair samples A, B, E, and G at m/z 195 it was concluded that the samples contained caffeine and can be determined by ESI-IMMS method. NSIM spectra of hair sample extracts A and E in the drift time range of 17–22 ms are shown in Figure 4, left hand side panel. The IMS peaks at m/z values of 150, 163, 195 and 231 were identified in the two spectra for reference. As shown, a peak at m/z value of 163 was detected in both samples. From the acquired SIM spectra the drift times of the peak at m/z 163 was measured to be 19.24 and 19.26 ms in the samples A and E respectively as shown in the right hand panel of Fig. 4. The reduced mobility for nicotine in the standard solution was determined as 1.60±0.02 cm2 V−1 s−1. This peak was tentatively identified as that of nicotine by comparing the mobility of the m/z 163 peak detected in hair samples to the m/z 163 peak detected for a standard solution of nicotine. A Ko value of 1.61±0.01 cm2 V−1 s−1 was measured for the standard nicotine solution. Figure 4, left hand side panel, also shows an intense peak at m/z value of 150 that was detected in hair sample A. Since methamphetamine (m/z=149), a commonly used drug of abuse, produces a peak at m/z 150 Da ðMHÞþ
ðm=z ¼ 150Þg, IMMS measurements were performed to tentatively identify the peak. Figure 5 shows the SIM spectra at m/z 150 obtained for (a) hair sample A, (b) standard methamphetamine solution, and (c) hair sample A spiked with methamphetamine. The reduced mobility value

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Determination of concentration of extractible target compounds in hair samples To determine the extractible concentration of caffeine and nicotine incorporated in hair, calibration curves for both were constructed by spiking hair samples in which peaks for caffeine and/or nicotine were not detected with known amount of caffeine and nicotine. Standard solutions of caffeine and nicotine in mixing ratio range of 0.01–5.0 and 0.01–10.0 ppm respectively were analyzed by ESI-IMMS. Intensity of IMS spectra obtained from an average of 2,500 IMS spectra was measured. Average signal intensity of three consecutive measurements was plotted against the concentration of the standard caffeine and nicotine solutions (Fig. 7). In the concentration range studied, linear relationship between the concentration and IMS signal intensity was observed. Concentration of caffeine in hair sample A was measured to be ∼8.2 ng of caffeine in 1 mg of hair. Caffeine concentration in hair sample G was

Calibration curve for caffeine 4.0 IMS Response (nA)

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Mixing Ratio of Caffiene in ppm Calibration curve for nicotine 1.6 1.4 IMS Response (nA)

of the m/z 150 peak in the hair sample was measured to be 1.63±0.02 cm2 V−1 s−1. However, the standard solution of methamphetamine produced a peak at m/z 150 with a reduced mobility of 1.69±0.01 cm2 V−1 s−1 was produced. Mass-mobility comparison between standard and hair sample A suggested that the m/z 150 peak detected in the hair sample A was not methamphetamine. The separation of isobaric ions based on mobility measurements also demonstrates one of the advantages of using IMS as a rapid separation method prior to MS analysis. In an effort to identify the m/z 150 peak detected in hair extract, various commonly used hair cosmetics such as gels, shampoos and creams, and over the counter drugs and health supplements that were suspected to be the source of the peak at m/z 150, were analyzed by ESI-IMMS. Although all samples produced distinguishable ion mobility patterns, the drift time of the m/z 150 peak observed in the hair sample A matched only with a peak observed in one of the hair gels analyzed. Non selective ion mobility spectra of vitamin B, vitamin C, aspirin and hair gel are shown in Fig. 6. On the right hand side panel of the figure is shown the NSIM spectrum of the hair sample A aligned with the NSIM spectrum of hair gel. The presence of a m/z 150 peak in the hair gel at a drift time that matched with that measured in the hair sample suggested that the peak observed in the hair sample at m/z 150 could have originated from the hair gel either as adsorbed or absorbed hair gel component. In the absence of MS-MS capability the identity of the m/z 150 peak was not confirmed. However, the basic inference drawn from the experiment was that IMS can rapidly provide a confirmatory piece of data to distinguish ions with same m/z values present in complex samples.

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y = 0.138x + 0.0231 R2 = 0.996

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Fig. 7 Calibration curve for caffeine and nicotine. Standard solutions of caffeine and nicotine in the mixing ratio range of 0.01–5.0 ppm for caffeine and 0.05–10.0 ppm for nicotine were used. Measured concentration of caffeine in the hair samples A, B, E, and G varied between ∼8.2 to ∼0.5 ng/mg. Nicotine concentration was measured to be ∼12 and ∼ 6 ng/mg in hair samples E and A respectively

measured to be 0.5 ng/mg of hair sample. Concentration of nicotine in hair sample E was measured to be ∼12 ng of nicotine in 1 mg of hair whereas concentration of nicotine in hair sample A was measured to be ∼6 ng/mg of hair. For more accurate quantification of analytes, calibration curves should be generated using control hair samples with known amount of target compounds, and recovery data. The study illustrates the basic concept that ESI-IMMS method of analysis has the ability to rapidly analyze hair samples for either target compound measurement or comprehensive determination.

Conclusions Ion mobility spectrometry in tandem with mass spectrometry and electrospray ionization (ESI-IMMS) can be used as a rapid analytical tool for hair analysis. The ability of ion mobility spectrometry to rapidly provide confirmatory information in a second dimension allows one to exclude false positives and increase confidence in the identification of ions of interest. Also, the method does not require derivatization or extensive sample preparation. This method can be used either for measurement of target compound or

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for the determination of all constituents extracted from hair samples. ESI-IMMS method can thus be extended to target metabolite analysis or studies investigating the whole hair metabolome. With ability to analyze hair as a biological matrix and detect caffeine and nicotine at low nano-molar concentrations demonstrated, further modifications in sample preparation methods and/or improvement in instrument sensitivity can facilitate analysis of hair for determination of constituents (metabolites, drugs) present in sub nanomolar concentrations. Additionally, ESI-IMMS or ESI-IMS as a stand-alone method can be exploited for rapid screening of hair samples, cosmetics and pharmaceuticals. Acknowledgements The authors would like to thank Fantastic Sams of Pullman, Washington for providing hair samples. Also, the authors would like to thank the Alcohol and Drug Abuse Research Program (ADARP) of Washington State University (WSU) and National Institutes of Health (NIH) for providing financial support. This work was supported by an ADARP grant and a Road Map Grant from NIH the (R21 DK 070274).

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