Table 1. Experimental Conditions of Each DBS Method Development Study^a.

Study	Target compounds	Exposure (biomarker)	Instrument	Punch size and/or sample volumed	Reconstituted volume for LC analyses	Reconstituted quantityf (mm/μL)	Sample type and size:  case/control	Locations of sample collected	Published country
Cramer et al.34	OTA	OTA	HPLC-MS/MS	Spot corresponding to 100 μL DBS (∼ 20 mm)	100 μLe (W:M:F = 60:40:0.1)	0.2	50 participants:	Műnster, Germany	Germany
		2’R-OTA					29 male, 21 female
Osteresch et al.48		OTA	HPLC-MS/MS	• 8.8 mm punch (∼20 μL of blood) •		8.8 mm punch: 0.088	34 coffee drinkers, 16 noncoffee drinkers
		2’R-OTA		Spot corresponding to 100 μL DBS (∼20 mm)		100 μL DBS: 0.2	Age: 18 and >60 years.
Osteresch et al.46	OTA, FB1	27 mycotoxins and mycotoxin metabolitesb	HPLC-MS/MS	Spot corresponding to 100 μL DBS or DSS (∼20 mm)	Volume not specifiede (W:A:AC = 95:5:0.1)	-	50 German cohorts: for applying developed 27-mycotoxins-method		Germany/Portugal
Xue et al.35	AFB1	AFB1 (AFB1-lysine)	HPLC-FDSc	• 12.7 mm punch (50 μL whole blood)	150 μL (25% MeOH)	8.47 × 10–2	36 participants (Kenyan mother and children): low, medium, and high dietary AFB1 exposure (no case/control)	Kenya	United States
			LC-MS/MS
Riley et al.41	FB1	Sa-1-P	HPLC-MS/MS	• 8 mm punch (∼17 μL whole blood)	Volume not specified	-	Volunteers who consumed maize-based foodsg(n = 186)	• Athens, Georgia, USA; Chimaltenango and Escuintla, Guatemala
		So-1-P
Riley et al.42	FB1	Sa-1-P	HPLC-MS	• 6- or 8 mm punch (∼17 μL whole blood)	Volume not specified	-	Large field study: 1240 women	Guatemala	United States
							low exposure: n = 841
							high exposure: n = 399
							Confirmatory field study: 299 women
		So-1-P					low exposure: n = 100
							high exposure: n = 199
Renaud et al.47	AFB1	AFB1 (AFB1-lysine)	LC-MS/MS HPLC-FLDc ELISA	Not specifiedh	380 μLi	-	Not specified	jN/A	Canada
Vidal et al.30	OTA, FB1, AFB1	24 mycotoxins	UPLC-MS/MSk	10.4 μLl	50 μL	0.126	mN/A

^aAflatoxin B₁ (AFB₁), Enniatin B (EnB), fumonisin B₁ (FB₁), ochratoxin A (OTA), sphinganine 1-phosphate (Sa-1-P), sphingosine 1-phosphate (So-1-P), and 2’R- ochratoxin A (2’R-OTA).

^bThe list of these 27 mycotoxins and mycotoxin metabolites are described in Table 3.

^cFDS: fluorescence detection system; FLD: fluorescence detector.

^dBlood volume estimates for each disc size were derived using blood applied to a blank filter paper spot.

^eW: water; M: methanol; F: formic acid; A: acetonitrile, AC: acetic acid.

^fReconstituted quantity (mm/μL) = disk area (mm)/reconstituted volume (μL).

^gFrom cohorts in Athens, Georgia: n = 10; From cohorts in Guatemala: n = 76 women and 100 men.

^hThe DBS paper was carefully excised and cut into 8 pieces with a scalpel.

ⁱ111.5 μL PBS buffer + 40 μL H₂O + 20 μL Pronase + 8.5 μL 2nd internal standard +200 μL MeOH = 380 μL

^jAFB₁-serum albumin (SA) reference material was used.

^kUltraperformance liquid chromatography - tandem mass spectrometry.

^lCorresponds to 6.3 mm of DBS punch.

^mBlood was purchased instead of collected from human participants.