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. 2017 Feb;38(1):13–34.

Table.

Sweat induction and collection methods for metabolomics.*

Study Aims n Sweat Induction Mode Methods Timing Amount Storage Sweat Preparation Protocols Analytical Chemistry Platforms
Adewole et al., 201672 Identify diagnostic biomarkers of active tuberculosis in eccrine sweat 83 Webster Sweat Inducer – pilocarpine iontophoresis × 5 min Macroduct® Sweat Collector – part of Macroduct® Sweat Analysis System – covers volar forearm × 15–35 min; sweat transferred to micro-centrifuge tube 5 min induction + 15–35 min collection ∼10–30 µL Samples placed on dry ice immediately, stored at −70°C until analysed Solubilised, reduced, alkylated, and digested with protease; then dried, desalted, dried again, then resuspended in ACN, formic acid LC – MS/MS, untargeted proteomics; FT mode for MS detection, ion trap mode for MS/MS detection
Jia et al., 201694 Assess feasibility of using HPLC-MS/MS for accurate quantifying of cortisol in human eccrine sweat 4 Hot room set at 41°C and ∼55% humidity Leg skin cleansed with alcohol pads, followed by dH2O and drying; sweat collected off skin into Eppendorf LoBind micro-centrifuge tubes 25–30 min collection times >200 µL Samples placed on dry ice immediately, stored at −80°C until analysed Sample mixed with ACN/ammonium acetate; addition of internal standard (in ACN); ethyl acetate extraction repeated twice, evaporated to dryness, re-constituted in ACN HPLC-MS/MS, SRM mode, targeted
Sheng et al., 201693 Monitor elimination of bioaccumulated heavy metals in humans with exercise 17 Exercise; no specific instruction as to type or location Direct collection of sweat from any part of the body into glass bottle with cover; then transferred into 50 mL glass vials with lid. Referenced methods from Genuis et al. 2011 utilised Same day as urine sample collection >20 mL Stored at −20°C until analysis Samples dried in oven for standardised weight, ashed in furnace, cooled in dryer; residue reconstituted in HNO3 with heat Flame atomic absorption spectrophotometry, targeted
Tang et al., 201695 Compare levels of 5 heavy metals (Cr, Cu, Zn, Cd, Pb) in human sweat and urine after physical exercise 9 Exercise; playing badminton × 2 h Upper bodies cleansed with ultrapure H2O before exercise; sweat scraped into polyethylene sample bottles. Samples allowed to stand for 30 min, then filtered using 9-mm filter paper into test tube ∼2 h >20 mL Stored at 4°C until testing 3 methods:

  1. direct dilution with HNO3

  2. wet digestion with HNO3 + HClO4, heated to 200°C; cooled, with HNO3 re-added, final dilution with ultrapure H2O

  3. microwave digestion with HNO3 added, microwaved, cooled, diluted with ultrapure H2O

 ICP-MS, targeted
Delgado-Povedano, Calderon-Santiago et al., 201671 Develop and validate a method for metabolomic analysis of human sweat using GC-TOF/MS 6 Webster Sweat Inducer – Pilogel® Iontophoretic discs; 1.5 mA electric current × 5 min Macroduct® Sweat Collector – part of Macroduct® Sweat-Analysis System – covers forearm skin × 15 min; sample transferred into micro-Eppendorf tube 5 min induction + 15 min collection >70 µL each participant – pooled into one sample Frozen at −80°C Pooled sweat into each of 3 protocols:

  1. deproteinisation with methanol-ACN;

  2. extraction with dichloro-methane;

  3. extraction with ethyl acetate.


Each followed with methoxymation + silylation		 GC-TOF/MS, full scan mode, untargeted
Calderon-Santiago et al., 201538 Identify metabolic markers of lung cancer in sweat to develop screening tool for diagnosis of lung cancer 96 Webster Sweat Inducer – Pilogel® Iontophoretic discs; 1.5 mA electric current × 5 min Macroduct® Sweat Collector – part of Macroduct® Sweat Analysis System – covers forearm skin × 15 min; sample transferred into micro-Eppendorf tube 5 min induction + 15 min collection >10 µL Frozen at −80°C until analysed Diluted with formic acid and vortexed LC-QTOF MS/MS, untargeted
Porucznik et al., 201589 Targeted detection of BPA in sweat in comparison to urine for biomonitoring 50 Passive sampling – no artificial modes of sweat induction Sweat patches (PharmChek®) applied after skin cleansed with alcohol wipes, to either upper-outer arm or front/back midriff 7 days Not specified Sweat patches stored and transported in sterile, BPA- free 4-oz polypropylene sample cups; no temperature specified Sweat patches extracted with methanol; evaporated in Turbovap®; reconstituted with ammonium bicarbonate: ACN (mobile phase) UHPLC-MS-MS, targeted; using methods initially designed for urine samples
Dutkiewicz et al., 201459 Untargeted metabolomics profiling of human sweat to evaluate hydrogel micropatch collection linked with direct mass spectrometry 9 Passive sampling – in room temperature, ∼25°C, 45% relative humidity Skin pre-wiped with cellulose tissue soaked with isopropanol: H2O; fabricated agarose hydrogel micropatch embedded with PTFE probe attached to forearm area with adhesive bandage tape 1 min–1 h ‘single droplet’ – unable to estimate volume of sweat sample accurately Hydrogel micropatch probe covered with glass slide, stored at 4°C Direct coupling of hydrogel micropatch probe to nanospray desorption electrospray ionisation mass spectrometer ESI + IT + FT-ICR-MS
Calderon-Santiago et al., 201437 Untargeted global metabolomics profiling of human sweat to optimise laboratory methods and chemometrics 96 Webster Sweat Inducer – Pilogel® Iontophoretic discs; 1.5mA electric current × 5 min Macroduct® Sweat Collector – part of Macroduct® Sweat Analysis System – covers forearm skin × 15 min; sample transferred into micro-Eppendorf tube 5 min induction + 15 min collection >5μL Frozen at −80°C Pooled samples diluted with formic acid:H2O with additional protocols:

  1. hydrolysis with 0.1M NaOH or HCL in H2O, vortexed, evaporated to dryness, reconstituted in chromatographic mobile phase A;

  2. solid phase extraction using C18 and hydrophilic centrifugal Micro SpinColumn™

 LC-QTOF MS/MS, untargeted
Shetage et al., 201423 Identify collection methods for RSSC and evaluate effects of ethnicity, gender and age on amount and composition 315 Passive sampling at room temperature: 18–25°C, 50– 60% relative humidity Forehead pre-wiped with cotton soaked in diethyl ether, allowed to dry. Cigarette paper applied, held in place with elastic headband, in duplicate × 1 h, fresh cigarette paper replaced every hour for total 3 h 3 h Totals not specified: peak amounts 0.11–0.12 +/− 0.06–0.07 mg/ cm2 of RSSC collected in first hour None specified Cigarette papers dehydrated × 2 h; extracted with hexane; extract filtered through 0.2 micron PTFE membrane, concentrated by purging nitrogen GC/MS, untargeted
Mark et al., 201351 Detailed amino acid analysis of sweat to better understand key biological mechanisms governing its composition 12 Hot room; 40°C, 60% relative humidity × 15–40 min Sweat droplets removed from axilla with positive displacement pipette using polypropylene tips; sample transferred directly into ‘low binding’ Eppendorf tube kept at 4°C ∼20 min >500 μL Frozen at −70°C Two methods:

  1. ninhydrin derivatisation for amino acid automated analyser

  2. oximation and trimethyl-silylation for GC-TOF/MS

 Targeted amino acid analysis; automated amino acid analyser + GC-TOF/MS, targeted
Raiszadeh et al., 201229 Untargeted and targeted analysis of healthy control and schizophrenic patient sweat, to identify candidate biomarkers of disease 78 Webster Sweat Inducer – pilocarpine iontophoresis applied to volar forearm Macroduct™ Sweat Collector – Macroduct ™ Sweat Stimulation and Sweat Collection System (Elitech/WESCOR, Inc., Logan, UT, USA); sample transferred into micro-centrifuge tubes 30 min 50–60 μL Stored on dry ice Pooled samples:

  • reduction (dithio-threitol/urea), alkylation (iodo-acetamide), overnight enzymatic digestion (trypsin/ammonium bi-carbonate), quenching (glacial acetic acid, then angiotensin II), desalting (C-18 Zip Tips), drying in vacuum concentrator, reconstitution in 0.1% formic acid

 LC-MS/MS;
LC-MS/MS + spectral counting;
MRM-MS verification
Genuis et al., 201257 Targeted profiling of phthalate compounds in blood, sweat and urine 20 Self-determined by participants – infrared sauna, steam sauna, exercise Direct collection from any body site into 500 mL glass jar using stainless steel spatula; participant-delivered to commercial laboratory; transferred to 4 mL glass jars at laboratory No time parameters around sweat collection except conditional within 1 week of blood collection (before/after) 100 mL Stored at −20°C; shipped frozen on dry ice from Canada to Sweden for analysis Not specified HPLC/MS, targeted;
GC/MS, targeted
Genuis et al., 201255 Targeted profiling of BPA in blood, sweat and urine LC-MS-MS, targeted
Genuis et al., 201156 Targeted profiling of 120 compounds (toxicants) in blood, sweat and urine ICP-MS, targeted
Lee et al., 201191 Untargeted metabolomics analysis to determine biochemical composition of exercise sweat 48 Exercise on ergometer for 60 min Collection with skin patch placed on the lower back Sweat patches removed at 3 time-points: 10–20min, 30–40min, 50–60min of exercise; placed on dry ice Not specified Frozen at −80°C until analysis. Not specified GC/MS and LC/MS/MS, untargeted
Kutyshenko et al., 201110 Untargeted metabolomics analysis to determine biochemical composition of human sweat 10 Natural environmental heat Direct collection from forehead, upper chest, upper/lower back, arms using glass pipette or glass roller, rolled in tray with dH2 O and/or sterile spray gun filled with D2O sprayed 3–5 min collection + 7–10 min sample preparation >0.56 mL Sample storage not specified; analysis performed 10–15 min after sweat collection Diluted with D2O, centrifuged, transferred to standard NMR tube 1H NMR Spectroscopy – high resolution, both one dimensional and two dimensional, untargeted
Michael-Jubeli et al., 201169 Develop simple analytical protocol for qualitative characterisation of individual SSLs and quantitative evaluation of lipid classes 1 Passive sampling Lipid-free absorbent papers placed on 6 areas

  • – forehead, back, thorax, forearm, thigh, calf

  • – maintained for 30 min with medical tape; removed with tweezers and placed into closed vials. Collections repeated 4 times

 30 min collection Not specified Storage of unprocessed samples not specified Extracted with diethyl ether twice, concentrated with rotary evaporation, transferred into 2 mL vials, dried under nitrogen stream; dried extract stored at −20°C until analysis; extracts derivatised/trimethyl-silylated; rotary evaporated, residue dissolved in isooctane HTGC-MS, with electron impact and chemical ionisation
Penn et al., 200792 Test the validity of individual odour hypothesis by analysing VOCs in sweat, urine and saliva 197 Passive sampling Axillary sweat sampled with devised twister PDMS-coated stir bars, held by special rollers, placed directly on skin; samples transferred to glass vials Once each fortnight sampling over 10-week period; unspecified sweat collection timing Not specified Stored at ∼4°C; shipped in cooler each week from Austria to USA for analysis Samples directly analysed with SBSE in connection with thermal desorption GC-MS SBSE with thermal desorption GC-MS
Harker et al., 200628 Untargeted metabolomics analysis of human eccrine sweat 60 Hot room at 43.3°C, 65% relative humidity × 15–40 min Underarm area wiped, then sweat collected with plastic-tipped pipette, sample transferred into sealed glass vials 15 min collection >50µL Frozen at −20°C until analysis Samples diluted and deuterated phosphate buffer (pH 7.4, 0.1M); transferred into 5 mm OK NMR tubes 1NMR Spectroscopy – high resolution, one dimensional, untargeted

BPA – Bisphenol A; PTFE – polytetra-fluoro-ethylene; PDMS – polydimethyl-siloxane; RSCC – residual skin surface components; SSLs – surface skin lipids; VOCs – volatile organic compounds; ACN – acetonitrile; SBSE – stir bar sorptive extraction; LC-MS/MS – Liquid Chromatography-Tandem Mass Spectrometry; HPLC-MS/MS – High Performance Liquid Chromatography-Tandem Mass Spectrometry; SRM – selected reaction monitoring; ICP-MS – Inductively Coupled Plasma mass Spectrometry; GC-TOF/MS – Gas Chromatography-Time of Flight/ Mass Spectrometry; LC-QTOF MS/MS – Liquid Chromatography-Quadripole Time Of Flight-Tandem Mass Spectrometry; ESI – Electrospray Ionisation; IT – Ion Trap; FT-ICR-MS – Fourier Transform Ion Cyclotron Resonance mass spectrometry; MRM-MS – Multiple Reaction Monitoring-Mass Spectrometry; UHPLC-MS-MS – Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry; GC / MS – Gas Chromatography / Mass Spectrometry; HTGC-MS – High Temperature Gas Chromatography-Mass Spectrometry; 1H NMR – Proton (Hydrogen -1 nuclei) Nuclear Magnetic Resonance Spectroscopy; ICP-MS – Inductively Coupled Plasma Mass Spectrometry; PTFE – polytetrafluoroethylene; dH20 – distilled water; -D2O – deuterated, heavy water

*

See Appendix (online supplement) for an expanded version of this table including more detailed information of sweat preparation protocols, chemometrics, databases and key findings pertaining to studies.