Emergency Diagnosis Made Easy: Matrix Removal and Analyte Enrichment from Raw Saliva using Paper-Arrow Mass Spectrometry

Paracetamol overdose is a leading cause of acute liver failure that can prove fatal. Establishing paracetamol concentration accurately and quickly is critical. Current detection methods are invasive, time-consuming and/or expensive. Non-invasive, rapid and cost-effective techniques are urgently required. To address this challenge, a novel approach, called Paper-Arrow Mass Spectrometry (PA-MS) has been developed. This technique combines sample collection, extraction, enrichment, separation and ionisation onto a single paper strip, and the entire analysis process, from sample to result, can be carried out in less than 10 min requiring only 2 μL of raw human saliva. PA-MS achieved a LOQ of 185 ng mL-1, mean recovery of 107 ± 7%, mean accuracy of 11 ± 8% and precision ≤5% using four concentrations, and had excellent linearity (r2 = 0.9988) in the range of 0.2-200 μg mL-1 covering the treatment concentration range, surpassing the best-in-class methods currently available for paracetamol analysis. Furthermore, from a panel of human saliva samples, inter-individual variability was found to be <10% using this approach. This technique represents a promising tool for rapid and accurate emergency diagnosis.


Standard solution preparation
A standard stock solution of paracetamol (2 mg/mL) was prepared with pure methanol and stored at -20 °C. 100 μg/mL paracetamol diluted from 2 mg/mL stock solution using water or blank saliva was used as a working solution.Stock solutions of iron(III) chloride (0.2M), potassium ferricyanide(III) (0.02M) and ammonium formate (1M) were prepared with water and stored at 4°C.

Instruments and software for PS-MS/MS and PA-MS/MS
PS-MS/MS and PA-MS/MS were performed with a Thermo Scientific Orbitrap Exploris 240 mass spectrometer (Thermo Fisher, Waltham, MA, USA).For paper spray ionisation, the paper was held by a copper clip at its base to provide an electrical connection and placed 3 mm from the inlet of the mass spectrometer.The spray solvent, 9:1 methanol: water (v/v) with 0.5 % formic acid and 10 mM ammonium formate, was automatically pumped onto the centre of the paper at a rate of 500 μL/min during 0.01-0.09min using the instrument's syringe pump.The ion source conditions were set as: spray voltage, +3.5 kV; ion transfer tube temperature, 320 o C; without nebuliser gas supply.Nitrogen was used as the collision gas.Multiple reaction monitoring (MRM) transitions were: m/z 152.0706 → 110.060 (quantifier) and m/z 152.0706 → 65.071 (qualifier) for paracetamol, and m/z 156.0957 → 114.085 (quantifier) and m/z 156.0957 → 69.090 (qualifier) for paracetamol-D4.The normalised HCD collision energy was set at 70%.The spray voltage was applied to induce an electrospray event from 0.10 min and stopped at 0.45 min.The 0.35 min voltage application cycle was repeated three more times to generate a total of four peaks in one chromatogram.The total run time was 1.66 min.The data acquisition was under the control of Thermo Scientific Xcalibur software and data processing was completed using the Xcalibur Quan Browser.

Instruments and software for UPLC-MS/MS method
UPLC was carried out on a Waters ACQUITY UPLC system (Waters Corporation, Milford, MA).The injection volume was 3 µL.UPLC separation was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm, 1.7 µm) with a BEH C18 guard column (2.1 mm x 5 mm, 1.7 µm).The mobile phase consisted of combinations of A (0.1% formic acid in water, v/v) and B (0.1% formic acid in methanol, v/v) at a flow rate of 0.3 mL/min with an elution gradient as follows: 0 min, 5% B; 3 min, 30% B; 3.01-4.5min, 95% B. A 2.5-min post-run time was set to fully equilibrate the column.Column and sample chamber temperatures were 40 o C and 6 o C, respectively.Mass spectrometry analysis was conducted by a Waters Xevo triple quadrupole mass spectrometer (Waters, Milford, USA) with electrospray ionisation in positive mode.Desolvation and cone gases used nitrogen set at 1000 L/h and 150 L/h, respectively.The desolvation and source temperatures were kept at 600 o C and 150 o C, respectively.The source capillary voltage was 3.7 kV.Argon was used as the collision gas.The MRM transitions were: m/z 151.94 → 109.95 as a quantifier and m/z 151.94 → 92.84 as a qualifier for paracetamol and m/z 155.96 → 114.05 as quantifier and m/z 155.96 → 96.69 as qualifier for paracetamol-D4.The optimised parameters for the four MRM transitions were: cone voltage, 46, 46, 38 and 38V, respectively; collision energy: 16, 22, 15 and 21 eV, respectively.The dwell time was 0.1 s per transition.Peaks were integrated using MassLynx V4.1 SCN 901 (Waters, Milford, USA) and the peak area ratio of the quantifiers of paracetamol and paracetamol-D4 were used for quantification.

Chromatography paper preparation
Chromatography paper was cut into different shapes (Figure S1) with a digital template using a 40 W laser cutter at 20% speed and 10% cutting power (HPC Laser Ltd, UK), and then these pieces of paper were cleaned under sonication with methanol for 5 min, followed by water for 5 min and then methanol for 5 min.They were dried in air overnight before use.

Human Resting Saliva Collection
The collection and preparation of human saliva was conducted with requisite ethical approval (approval number: 10058) by the ethical committee of the University of Liverpool.Informed consent from participants was obtained.Volunteers were restricted from intake of any food or drinks for at least 1 hr prior to sample collection.Human whole saliva was spat into a vial after being passively pooled at the bottom of the mouth for 2 min. 1 Saliva samples were collected and analysed on the same day without any sample storage for re-usage.

Staining method for visualisation of paracetamol movement on arrow-shaped paper
The migration of paracetamol on arrow-shaped paper after PC was compared with that of traditional triangular paper.It was visualised by a colourimetric method based on a redox reaction reported in the literature [2][3][4] with slight modifications.20mM iron(III) chloride, 10mM potassium ferricyanide(III) (K3Fe(III)(CN)6) and 0.2M hydrochloric acid in water was sprayed onto the paper by a fine mist sprayer.The spray solution was freshly made from the stock solutions (Supplementary method 2) no longer than 2 hours in advance of the experiment.After drying under ambient  Intensities of [M+H] + in water or saliva with or without PC. 2 µL of 100 µg/mL paracetamol in water was applied on a separated piece of serrated paper, and then [M+H] + was detected without PC, which is indicated by the label of "Water-no PC". 2 µL of 100 µg/mL paracetamol in saliva was also applied on a separated piece of serrated paper, and then [M+H] + was detected without PC, which is shown with label of "Saliva-no PC". 2 µL of saliva spiked with 100 µg/mL paracetamol was applied at the origin.PC was conducted separately with three different mobile phases: mobile phase 1, 9:1(v/v) ethyl acetate: formic acid; mobile phase 2, 9:1(v/v) of ethyl acetate: formic acid with 10 mM ammonium formate; mobile phase 3, 9:1(v/v) of ethyl acetate: formic acid with 50 mM ammonium formate.The data are expressed as mean ± SD (n =3).Experiment parameter PA-MS/MS [a] UPLC-MS/MS [b] Sample volume per test 2 µL 50 µL

Solvent volume for MS analysis 40 µL ~2500 µL
[a] The sample was prepared by a 5-min process of paper chromatography before MS detection.

Figure S2 .
Figure S2.Dimensions of three shapes of chromatography paper used in this study.(a) Serrated paper strips used to determine the location of salivary components and paracetamol (for method development and paper arrow design purposes).(b) Triangular paper used for traditional PS-MS analysis.(c) Finalised arrow-shaped paper strips used for PA-MS.

Figure S3 .
Figure S3.Workflow and result of mobile phase screening for PA-MS.(a) Workflow of mobile phase screening for PA-MS, where 2 µL of saliva spiked with 100 µg/mL paracetamol was applied at the origin and subjected to 12 min of PC using different mobile phases.When the front of the mobile phase reached the 10 th region from the origin, the paper strip was taken out of the flask and dried in air for 1 min.Then individual regions, labelled 0-10, were cut apart manually and the signal intensities of the protonated paracetamol ion, [M+H] + , on each piece were acquired by MS analysis.(b) Intensity of [M+H] + after PC with pure ethyl acetate.(c) Full scan mass spectrum at the origin after PC with pure ethyl acetate.(d)

Figure S4 .
Figure S4.Average mass spectra of full scans from Regions 0-10.Blank saliva (2 µL) was applied at the origin and dried in air.After 12 min of PC with the optimised mobile phase [ 9:1 ethyl acetate: formic acid (v/v) with 50 mM ammonium formate], Regions 0-10 were cut apart.2uL of 100 µg/mL paracetamol in water was added onto each piece of paper.After air-drying, MS analysis was conducted.Region 4 gave the highest signal intensity of the paracetamol ion.

Figure S5 .
Figure S5.Selected ion monitoring (SIM) chromatograms of [M+H] + were obtained for 2µL of saliva and water spiked with 100 µg/mL paracetamol applied onto triangular paper for PS-MS and onto arrow-shaped paper for PA-MS.The chromatograms are shown with a mass tolerance of 5 ppm centred around the protonated molecular ion, m/z 152.0705.

Figure S7 .
Figure S7.Comparison of calibration curves of matrices spiked with paracetamol (5-1000 ng/mL) detected by different methods.The matrices and paracetamol detection methods were: (a) raw saliva detected by PA-MS/MS, (b) water detected by PA-MS/MS, (c) pre-treated saliva detected by UPLC-MS/MS, (d) pre-treated saliva detected by PS-MS/MS, and, (e) raw saliva detected by PS-MS/MS.The data points are expressed as mean ± SD (n =3).

Figure S8 .
Figure S8.Procedure for extraction recovery experiment of paracetamol in saliva by PA-MS/MS.

Table S2 . Precision and accuracy of paracetamol in raw saliva detected by PA-MS/MS (5-1000 ng/mL).
[a] Ratio of S/N is the signal intensity of the product ion m/z 110.06 over the signal intensity of the background noise.