Protocol for quantifying amino acids in small volumes of human sweat samples

Summary

Sweat, a biofluid rich in water, ions, and amino acids, is emerging as a valuable, non-invasive source for continuous health monitoring and disease diagnostics. Here, we present a protocol for amino acid analysis in small volumes of sweat samples. We describe steps for sweat collection, fluorescence derivatization of amino acids, and liquid chromatography analysis, which can be applied to the analysis of very small volumes of sweat samples.

For complete details on the use and execution of this protocol, please refer to Tsunoda et al.¹ and Kuroki et al.²

Graphical abstract

Highlights

• •Collection of sweat samples from the fingertip
• •Steps for fluorescence derivatization of amino acids
• •Instructions for the LC-fluorescence method for amino acid analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Sweat, a biofluid rich in water, ions, and amino acids, is emerging as a valuable, non-invasive source for continuous health monitoring and disease diagnostics. Here, we present a protocol for amino acid analysis in small volumes of sweat samples. We describe steps for sweat collection, fluorescence derivatization of amino acids, and liquid chromatography analysis, which can be applied to the analysis of very small volumes of sweat samples.

Before you begin

Sweat, a biofluid produced by sweat glands primarily for temperature regulation and skin hydration, has emerged as a valuable non-invasive source for continuous and real-time health monitoring.^3,4,5 Unlike blood and urine, sweat can be collected easily without the risk of infection, making it an attractive alternative for medical diagnostics.^6,7 This fluid contains water, essential ions like chloride, sodium, and potassium, and trace amounts of various metabolites, including amino acids. Amino acids, which are essential for protein synthesis and numerous physiological processes, have drawn significant attention for their potential to provide insights into metabolic health, exercise-induced stress, and disease markers.^8,9,10,11

Analyzing sweat for amino acid concentrations can reveal information about an individual’s health status and provide non-invasive biomarkers for various conditions. For example, differences in amino acid levels have been observed based on sex and exercise levels, and there is potential to use sweat analysis for diagnosing conditions like atopic dermatitis.^12,13,14 With its non-invasive nature, sweat collection offers a comfortable and efficient alternative to traditional diagnostic procedures, such as blood sampling. As research progresses, sweat analysis, particularly for amino acid profiling, holds great potential for advancing personalized healthcare and enhancing disease diagnostics.

Innovation

This protocol presents a significant advancement in sweat analysis by enabling the precise quantification of amino acids from extremely small sweat volumes, collected under resting conditions. Traditionally, sweat analysis required large volumes obtained through exercise-induced perspiration, limiting its practical and non-invasive applications. The innovation lies in combining a highly sensitive HPLC-fluorescence detection method with sweat volume measurements from a micro sweat sensor, ensuring accurate quantification even at femtomole detection levels. Sixteen amino acids were successfully separated and quantified using this method, with robust validation demonstrating excellent linearity, precision, and accuracy. Notably, this approach reveals distinct amino acid profiles in sweat compared to plasma, offering new insights into localized skin metabolism and the unique molecular signature of eccrine gland secretions. The method’s sensitivity, minimal sample requirement, and non-invasive nature make it a promising tool for biomarker discovery and potential clinical applications, including disease screening and metabolic monitoring without the need for induced sweating.

Institutional permissions

This research was approved by the Ethical Review Committee, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (4-1), and experiments were conducted in agreement with the Declaration of Helsinki. All participants provided written informed consent. Data obtained for this study, using this protocol, has been published in Heliyon by Tsunoda and Tsuda (2024) and in J. Pharm. Biomed. Anal. by Kuroki and Tsunoda (2025). Herewith, readers are reminded to acquire permission from their relevant institutions.

Preparation 1: Sweat sample collection

Timing: 30 min

• 1.
  Preparation of sweat collection solution (1% ethanol solution).

  • a.
    Add 297 μL of Milli-Q water and 3 μL of ethanol to a 1.5 mL tube.
  • b.
    Mix well using vortex mixer.
• 2.
  Preparation of micro sweat sensor.

  • a.
    Open the lid of the micro sweat sensor. (Figure 1A).
  • b.
    Fill the sweat sensor with silica gel (ca. 4 g). (Figure 1B).
  • c.
    Fill with silica gel and close the lid. (Figure 1C).
  • d.
    Connect the sweat sensor to the PC. (Figure 1D).
  • e.
    Start the software for sweat volume measurement.
  • f.
    Start recording and allow to stand for 5 min.
  • g.
    After 5 min, when it can be confirmed that the sweat rate value is within ±0.1, the preparation for sweat rate measurement is complete.

Figure 1.

Preparation of micro sweat sensor

(A) Open the lid of the micro sweat sensor, (B) fill the sweat sensor with silica gel, (C) close the lid, and (D) connect the sweat sensor to the PC.

Preparation 2: Preparation of derivatization reaction solutions and liquid chromatography mobile phases

Timing: 1 h

• 3.
  Preparation of 400 mM borate buffer (pH 8.5) solution.

  • a.
    Weigh 2.47 g of boric acid.
  • b.
    Add 90 mL of Milli-Q water.
  • c.
    Add NaOH solution and adjust pH to 8.5.
  • d.
    Add Milli-Q to make the total volume 100 mL.
• 4.
  Preparation of 20 mM NBD-F solution.

  • a.
    Weigh 1.83 mg of NBD-F reagent.
  • b.
    Add 500 μL of MeCN.
  • c.
    Mix well using vortex mixer for 30 s.

20 mM NBD-F solution

Reagent	Final concentration	Amount
NBD-F	20 mM	1.83 mg
MeCN	N/A	500 μL
Total	N/A	500 μL

Note: NBD-F solution is not stable and must be prepared on an as-needed basis. But, the solution can be used for 1 week when stored at −20°C.

• 5.
  Preparation of 100 mM HCl solution.

  • a.
    Add 200 μL of HCl reagent (11.65 M) to a volume of 23.1 mL of Milli-Q water.

100 mM HCl solution

Reagent	Final concentration	Amount
HCl	100 mM	200 μL
Milli-Q water	N/A	23.1 mL
Total	N/A	23.3 mL

• 6.
  Preparation of mobile phase A: 10 mM citrate buffer containing.

  • a.
    Weigh 160 mg of citric acid monohydrate, 2.7 g of trisodium citrate dihydrate, and 9.2 g of sodium perchlorate, respectively.
  • b.
    Put them into a beaker, and mix them with 1 L of Milli-Q water.
• 7.
  Preparation of mobile phase B: water/acetonitrile (50/50, v/v).

  • a.
    Weigh out 500 mL of MeCN.
  • b.
    Add 500 mL of Milli-Q water.

Mobile phase A

Reagent	Final concentration	Amount
Citric Acid Monohydrate	10 mM	0.16 g
Trisodium Citrate Dihydrate		2.72 g
Sodium Perchlorate	75 mM	9.2 g
Milli-Q water	N/A	1000 mL
Total	N/A	1000 mL

Mobile phase B

Reagent	Final concentration	Amount
MeCN	N/A	500 mL
Milli-Q water	N/A	500 mL
Total	N/A	1000 mL

Note: The mobile phases used during the analysis are given as 1 L volumes (sufficient for at least 50 and 75 injections, for mobile phase A and B, respectively). However, always prepare fresh solvents by calculating the required amount.

CRITICAL: If large volumes need to be stored for extended periods of time, we recommend to store them at 4°C.

CRITICAL: Use only LC-grade solvents.

• 8.Program the LC gradient in the analytical method.

Column	Inertsil ODS-4V
Column temperature	40°C
Flow rate	0.6 mL/min
Fluorescence Detection	Ex. 479 nm, Em. 530 nm
LC gradient
Time (min)	Solution B (%)
0	10
3	10
23	50
33	100
38	100
38.1	10

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Aspartic acid	Sigma-Aldrich	A9006
Asparagine monohydrate	Sigma-Aldrich	A8131
Glutamic acid	Sigma-Aldrich	G1001
Glutamine	Sigma-Aldrich	G3126
Serine	Sigma-Aldrich	S4375
Glycine	Sigma-Aldrich	G7251
Histidine hydrochloride	Sigma-Aldrich	H7625
Citrulline	Sigma-Aldrich	C7629
Threonine	Sigma-Aldrich	T8441
Alanine	Sigma-Aldrich	A7627
Arginine hydrochloride	Sigma-Aldrich	A6757
Proline	Sigma-Aldrich	P4266
Valine	Sigma-Aldrich	V0500
Methionine	Sigma-Aldrich	M1126
Isoleucine	Sigma-Aldrich	I7634
Leucine	Sigma-Aldrich	L8000
Phenylalanine	Sigma-Aldrich	P1751
Lysine	Sigma-Aldrich	L5501
Citric acid monohydrate	FUJIFILM Wako Pure Chemical	032-08385
Trisodium citrate dihydrate	FUJIFILM Wako Pure Chemical	190-05535
Sodium perchlorate	FUJIFILM Wako Pure Chemical	192-09255
Acetonitrile, gradient grade for liquid chromatography	Merck	1.00030
Methanol, gradient grade for liquid chromatography	Merck	1.06007
Ethanol (99.5%)	FUJIFILM Wako Pure Chemical	054-00466
4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)	FUJIFILM Wako Pure Chemical	348-04753
Hydrochloric acid	FUJIFILM Wako Pure Chemical	086-03925
Sodium hydroxide	FUJIFILM Wako Pure Chemical	198-13765
Boric acid	FUJIFILM Wako Pure Chemical	020-07305
6-Aminocaproic acid	Sigma-Aldrich	01-3540
Software and algorithms
LC-20AD Pump	SHIMADZU	–
DGU-20A5R Degassing Unit	SHIMADZU	–
SIL-20AC Autosampler	SHIMADZU	–
CTO-20AC Column Oven	SHIMADZU	–
RF-20A Fluorescence Detector	SHIMADZU	–
Lab Solutions	SHIMADZU	–
CBM-20A Communication Bus Module	SHIMADZU	–
Other
1.5 mL light shield tube, amber	Eppendorf SE	0030 120.191
1.5 mL, microtube, flat bottom	WATSON BIO LAB	131-415C
TORAST PP vial	SHIMADZU GLC	MK-LG-20-0025A
Delta mixer	TAITEC	Se-08
CHIBITAN-R	Merck	–
Thermal Robo	AS ONE	TR-2α
Micro sweat sensor	TECHNO NEXT	TPL3520
Silica gel, medium granular, green	FUJIFILM Wako Pure Chemical	190-16645
Kimwipe wiper S-200	NIPPON CRECIA	62011
Milli-Q purification system	Merck Millipore	–

Note: Cystine, tryptophan, and tyrosine were excluded in this analysis.

Step-by-step method details

Sweat collection

Timing: 20–30 min

This section provides step-by-step guidelines for sweat collection.

• 1.Wash the sampling site (index finger) with tap water for 15 seconds (Figure 2A).
• 2.Wash with aqueous ethanol solution for another 15 seconds (Figure 2B).
• 3.Wipe off the water with a Kim wipe (Figure 2C).
• 4.Immediately after wiping with a kim wipe, place the index finger of the right hand on the probe of the micro sweat sensor (Figure 2D).
• 5.Place the tube containing the ethanol solution between the thumb and index finger so that the mouth of the vial is on the top and touching the index finger of your left hand (Figure 2E).
• 6.Turn the wrist and flip the vial upside down to bring the aqueous ethanol solution into contact with the fingertips (Figure 2F).
• 7.Invert the vial upside down so that the mouth of the tube is at the top, and collect the aqueous ethanol solution that was in contact with the fingertip into the tube.

Note: Sweat samples can be stored at −20°C for short durations without significant stability issues. For extended storage, it is recommended to store at −80°C to maintain sample quality more effectively.

CRITICAL: Participants must be in a resting state during sweat collection.

Pause point: The assay would be paused at step 7 and the collected sweat samples should be stored at −20°C or −80°C.

Figure 2.

Sweat collection

(A) Wash the index finger with tap water, (B) wash with aqueous ethanol solution, (C) wipe off the water with a Kim wipe, (D) place the index finger on the probe of the micro sweat sensor, (E) place the tube containing the ethanol solution between the thumb and index finger, and (F) turn the wrist and flip the vial upside down.

Fluorescence derivatization of amino acids

Timing: 10–20 min

This step provides guidance on how to derivatize amino acids with NBD-F.

• 8.Label the tube with the sample information, including the standards and sample number.
• 9.Add 70 μL of 400 mM borate buffer (pH 8.5) to a 1.5 mL light-shielded tube.
• 10.Add 10 μL of 10 μM 6-aminocaproic acid solution.
• 11.Add 10 μL of sweat sample or amino acid standards solution or water.
• 12.Add 10 μL of 20 mM NBD-F solution.
• 13.Vortex well for 30 seconds (Figure 3A).
• 14.Place the tube in a 60°C hot water bath for 5 min (Figure 3B).
• 15.Add 100 μL of 100 mM HCl solution.
• 16.Vortex well for 30 seconds (Figure 3C).
• 17.Put in an ice bath until the analysis.

Note: Hot water baths should be prepared in advance of the reaction.

CRITICAL: Pipette tips are replaced each time another reagent is added.

Figure 3.

Fluorescence derivatization of amino acids

Note: Although pictures using transparent tubes are shown to make it easier to see the volume and color of the solution, in actual practice, light-shielded tubes should be used.

(A) Before derivatization, (B) during derivatization, and (C) after derivatization.

Measurement of sweat amino acids by LC-fluorescence detection method

Timing: 1 day

This step provides guidance on how to set up the LC system ahead of analysis.

Note: Before the analysis of sweat samples, the LC system performance needs to be verified.

• 18.For the calibration, prepare the blank sample and amino acid standards solution (each 10 μM) in LC vials.
• 19.Inject 10 μL of each sample into the LC system.
• 20.First, verify the performance check results by confirming that there is no signal for the blank sample. Minor peaks from derivatization by-products are expected, but there should be no other signals present in the blank sample. This ensures that the system is functioning properly and that no contaminants are present in the sample.
• 21.Then, examine the QC mix result and check that the tested compounds elute at the expected RT with the expected intensities.

Note: Expected RT of each amino acid.

Amino acid	RT (min)
Asp	3.04
Glu	3.84
Ser	7.92
Gly	10.04
His	10.86
Cit	11.50
Thr	12.13
Ala	12.89
Arg	13.76
Pro	14.13
Val	20.53
Met	21.51
Ile	24.76
Leu	25.08
Phe	27.30
Lys	28.36

• 22.If the performance check results are satisfactory, proceed to the actual sample analysis.
• 23.Transfer samples in vials for LC.
• 24.Inject 10 μL of each sample into the LC system.

Expected outcomes

The representative chromatograms of the blank sample, standard reference, and sweat sample are shown in Figure 4. Linearity can be obtained in the range of 0.01–10 μM. Both intra-day and inter-day precision were less than 8.8%, except at LLOQ, where the precision was within 9.4% and 11.2%, respectively.

Figure 4.

Representative chromatograms

Chromatograms of (A) blank sample, (B) NBD-amino acids standards, and (C) sweat sample. Peaks; 1 = NBD-Asp; 2 = NBD-Glu; 3 = NBD-Ser; 4 = NBD-Gly; 5 = NBD-His; 6 = NBD-Cit; 7 = NBD-Thr; 8 = NBD-Ala; 9 = NBD-Arg; 10 = NBD-Pro; 11 = NBD-Val; 12 = NBD-Met; 13 = NBD-Ile; 14 = NBD-Leu; 15 = NBD-Phe; 16 = NBD-Lys.

Limitations

In blood sampling, the results are not easily affected under normal circumstances because a certain homeostasis is maintained in the body. However, in the case of sweat, room temperature and humidity greatly affect the amount of perspiration, and if the environmental conditions are not constant, the measurement results will fluctuate.

Blood, which is collected directly from the body, has almost no risk of contamination by external factors. In contrast, sweat is collected on the surface of the skin, and if the skin is not thoroughly washed before collection, amino acids and other substances derived from keratin and dirt may be contaminated in the sample. In addition, if cosmetics or creams have been used beforehand, their components may contaminate the sweat sample or interfere with measurements.

There are many reports on blood amino acid concentrations, and reference values have been clarified.^15,16 On the other hand, little is known about amino acids in sweat, and much data acquisition is still needed to realize disease screening by sweat amino acid analysis.

Troubleshooting

Problem 1

Contamination during sweat sampling (related to steps 1, 2, and 3).

Potential solution

Contamination of sebum or external factor components is possible. The sweat collection site should be properly cleaned before collection, and the participant should be informed in advance not to touch the collection site after cleaning.

Problem 2

Sweating fluctuation due to mental stress (related to step Sweat collection).

Potential solution

Mental stress in the participant may cause abnormal sweating. It is advisable to provide sufficient rest time before collection and to create an environment in which participants can relax.

Problem 3

Adverse effects of ethanol sanitation on the skin (related to step 2).

Potential solution

In persons with low alcohol tolerance or sensitive skin, ethanol antiseptic may cause redness or irritation. Prior to sweat collection, participants will be asked if they have ever experienced reactions such as redness or itching of the skin with ethanol antiseptic. Participants who respond “yes” will be excluded from the study.

Problem 4

Leakage of sampling solution (related to step 5).

Potential solution

As the tube containing the solution is pressed against the sweat collection area, the solution in the tube may leak. It is necessary to make sure that the edges of the opening of the tube are in close contact with the skin, and to provide assistance, such as a pair of persons to collect the sweat.

Problem 5

Variations in amino acid concentrations during storage (related to step Sweat collection).

Potential solution

Inappropriate storage temperatures or improper tube storage conditions may result in altered amino acid concentrations. Collected samples should be refrigerated immediately. It is stable for 1 week if stored at 4°C or below. Confirm that the lids of the tubes are tightly closed during storage to prevent sample evaporation.

Problem 6

Broadening of peaks and separation deterioration (related to step 22).

Potential solution

Column degradation causes broad peaks and poor separation. When this occurs, it is necessary to replace the column with a new one.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Makoto Tsunoda (makotot@mol.f.u-tokyo.ac.jp).

Technical contact

Questions about the technical specifics related to performing the protocol should be directed to and will be answered by the technical contact, Makoto Tsunoda (makotot@mol.f.u-tokyo.ac.jp).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data in this study are derived from the same experiments as reported by Tsunoda et al.¹ and Kuroki et al.²

Acknowledgments

This work was partly supported by a grant from the Koyanagi-Foundation.

Author contributions

Conceptualization, writing, and project administration, M.T.

Declaration of interests

The authors declare no competing interests.