Table 1. Overview of the selected applications of cerumen analysis in laboratory diagnostics.

Approach	Population tested	Sample collectionn	Sample pretreatment	Method of analysis	Disorder	Biomarkers detected	Detected ranges	Conventional bio-fluids	Reference
Proteomics	2 female, 3 male	Small metal scoop	1D-PAGE pre-fractionation, online SCX-fractionation	LC-MS/MS	chronic otitis externa, otomycosis, benign or malignant ear tumors, systemic diseases as (diabetic nephropathy, breast cancer)	zinc-alpha-2-glycoprotein	Qualitative	Plasma, saliva, urine	Feig et al. (2011)
Genomics	40 patients (18 females, 23 males, 17-57 years)	Sterile swabs	Homogenization in saline, then DNA extraction	Real-time PCR	Diagnosis of Hepatitis B infection	Hepatitis B virus DNA	4.2 x 102 - 4.7 x 106 copies per sample	Serum	Kalcioglu et al (2004)
	50 patients (21 females, 29 males, 20-40 years)	Sterile spoon /sterile swabs	Homogenization in saline, then DNA extraction	Real-time PCR	Diagnosis of Hepatitis B infection	Hepatitis B virus DNA	ND	Serum, saliva	Parizad et al. (2016)
	35 patients (21 females, 29 males, 21-70 years)	Sterile swabs	Homogenization in saline, then DNA extraction	Real-time PCR	Transmission of Hepatitis C infection	Hepatitis C virus RNA	no copies per sample	Serum	Bayindir et al. (2005)
Metabolomics
Volatile organic compounds	8 patients (24-48 years)	ND	Desiccation by electric plate at 110oC	Change in weight	Allergic rhinitis	Total volatile compounds	28.4 ± 8.5 (43.8 ± 12.2 µg/g)*	Blood	Paiva et al. (1973)
	17 patients (7-61 years)	ND	Desiccation by electric plate at 110oC	Change in weight	Otoschlerosis	Total volatile compounds	31.1±10.4 (43.8±12.2 µg/g)*	-	Paiva et al. (1973)
	12 patients (46-72 years)	ND	Desiccation by electric plate at 110 oC	Change in weight	Cancer	Total volatile compounds	30.7 ± 10.3 (43.8 ± 12.2 µg/g)*	Blood, urine, saliva, sweat	Paiva et al. (1973)
	Neonates (< 5 days)	Metal scoop	Solvent extraction and derivatization	GC, GC-MS	Maple syrup urine syndrome	Sotolone (4,5-dimethyl-3-hydroxy-2[5H]-furanone), a metabolite of isoleucine or allo-isoleucine	Qualitative (burnt sugar odour)	Urine	Kataoka et al. (2013), Liebich et al. (1983)
	2 females patients (61 years)	ND	Solvent extraction	Paper chromatography, visualization with 5% ammonium silver nitrate	Alkaptonuria	Homogentisic acid	Qualitative	Urine	Frohlich et al. (1973)
	26 patients (13 males, 13 females)	Plastic curette	No previous extraction	HS/GC-MS	Diabetes mellitus (Types 1 and 2)	Acetone, methoxyacetone, ethanol, isobutyraldehyde, hydroxyurea, acetic acid	Chemometric treatment of quantitative data	Blood, plasma, urine	Shokry et al. (2017)
Approach	Population tested	Sample collectionn	Sample pretreatment	Method of analysis	Disorder	Biomarkers detected	Detected ranges	Conventional bio-fluids	Reference
Xenobiotics	ND	ND	Solvent extraction	GC, GC-MS	Exposure to toxic chemicals	Lindane, chlordane, DDT, DDE, dieldrin, HCB, HCH (long term) exposure)	Detected (0.0 µg/g)*	Breath, blood (short term exposure), adipose tissue, breast milk (long-term exposure)	Lauwerys et al. (1991), Wang et al. (1988)
	3800 (35-54 years)	ND	Solvent extraction	GC, GC-MS	DDT, HCH induced cancer mortality	DDT, HCH	Detected (0.0 µg/g)*	Breath, blood, adipose tissue	Wang et al. (1988)
	10 males and 7 females (≥ 18 years)	Plastic curette	Direct extraction with methanol	UPLC- MS/MS	Administration of drugs of abuse or drug facilitated crimes	lacosamide	13.2 ng/mg (0.0 ng/mg)*	Blood, plasma, urine, saliva (short term), hair (long term)	Shokry et al. (2017)
						lamotrigine	9.5 - 115.0 ng/mg (0.0 ng/mg)*
						carbamazepine	13.2 - 259.5 ng/mg (0.0 ng/mg)*
						phenytoin	8.7 - 243.3 ng/mg (0.0 ng/mg)*
						levitracetam	52.0 ng/mg (0.0 ng/mg)*
						oxcarbazepine	5.0 - 326.5 ng/mg (0.0 ng/mg)*
						valproic acid	186.5 - 4850.0 pg/mg (0.0 pg/mg)*
						topiramate	9.8 - 175.5 ng/mg (0.0 ng/mg)*
						clobazam	186.5 - 4850 pg/mg (8.0 - 175.5 ng/mg) (0.0 pg/mg)*
						clonazepam	5.6 - 8.4 ng/mg (0.0 ng/mg)*
						phenobarbital	5.6 - 6.3 ng/mg (0.0 ng/mg)*
						clozapine	31.7 ng/mg (0.0 ng/mg)*
	38 postmortem samples	Cotton swab	Drying at room temperature for 24 h, solvent extraction	LC-TOF MS, LC-MS/MS	Drug abuse	Opiates, cannabinoids	2-100 ng/ 0.42- 8.2 mg cerumen	Blood, urine, hair, bile	Meier et al. (2017)
	24 females, 37 males (18-35 years)	Plastic curette	No sample pre-treatment	HS- GC/MS	Tobacco use/exposure	Nicotine	1.2 ± 0.5 ng/mg, passive smoker; 16.8 ± 32.9 ng/ mg, active smoker	Blood, urine, saliva, sweat, hair, nails	Shokry et al. (2017)
						Cotinine	5.3 ± 2.9 ng/mg, passive smoker; 25.4 ± 26.4 ng/ mg, active smoker
						Anabasine	0.0 ng/mg, passive smoker; 326 ± 890 ng/ mg, active smoker
						o-nicotine	3.7 ± 4.4 ng/mg passive smoker; 12.4 ± 17.4 ng/ mg, active smoker
Approach	Population tested	Sample collectionn	Sample pretreatment	Method of analysis	Disorder	Biomarkers detected	Detected ranges	Conventional bio-fluids	Authors
Chemical elements	1 male, 1 female	clean Q tip	Desiccation, digestion in 10% nitric acid	Inductively coupled plasma atomic emission spectroscopy	Exposure to toxic elements	Lead	13.5 ± 0.71 μg/g (0.0 µg/g)*	Plasma, sweat, skin	Krishnan et al. (1992)
						Cadmium	1.14 ± 0.66 μg/g (0.0 µg/g)*
	10 males, 10 females	ND	Incineration (for 6 hours) at 600oC	Flame Photometry	Fungal infection in the ear	Copper	0 ng/100gm (0.942- 3.314 ng/100gm)*	-	Yassin et al. (1966)
	4 male, 2 female (11-17 years), 1 male adult	Curette	Desiccation under vacuum, digestion in nitric/perchloric acid mixture (5:1 V/V)	Atomic absorption spectroscopy	Cystic fibrosis	Zinc	118 ± 103 μg/g (1857 ± 1341 μg/g)*	Blood, urine, saliva, sweat	Brand-Auraban et al. (1972)
	17 patients (7- 61 years)	ND	Incineration in a muffle furnace at 550 °C for 12 hours, dissolving in HCl	Clark-Collip method	Allergic Rhinitis	Calcium	6.87 ± 1.69 mEq/100 g (8.0 9± 1.86 mEq/100 g)*	Blood	Paiva et al. (1973)
				Fiske-Subbarow method		Phosphorus	26.9 ± 8.7 mg/100 g (18.63 ± 6.52 mg/100 g)*
				Turbidimetric method		Sulfur	510.9 ± 152.7 mg/100 g (223.9 ± 35.1 mg/100 g)*
				Diethyldithio-carbamate method		Copper	1.62 ± 0.49 mg/100 g (2.43 ± 1.29 mg/100 g)*
	12 patients (46-72 years)	ND	Incineration in a muffle furnace at 550°C for 12 hours, dissolving in HCl	flame spectrophoto-metry	Cancer (prostate, mandible, tongue, tonsils, larynx)	Sodium	29.15 ± 8.11 mEq/100 g (39.5 ± 14.42 mEq/100 g)*	Blood, serum, urine, saliva, sweat	Paiva et al. (1973)
				Clark-CoUip method		Calcium	5.59 ± 1.76 mEq/100 g (8.09 ± 1.86 mEq/100 g)*
				Yellow titan		Magnesium	1.84 ± 0.49 mEq/100 g (4.9 6± 2.56 mEq/100 g)*
				Fiske-Subbarow method		Phosphorus	89.7 ± 15.1 mg/100 g (18.63 ± 6.52 mg/100 g)*
	1 female (16 years), 2 males (22 and 28 years)	ND	ND	Histochemical examination	Wilson’s disease (hepatolenticular degeneration)	Copper	7.17 (2.43 mg/100 g)*	Serum, urine, faeces, cerebrospinal fluid, bile, saliva	Canelas et al. (1963)
	8 patients (24- 48 years)	ND	Incineration in a muffle furnace at 550 °C for 12 hours, dissolving in HCl	Clark-Collip method	Otosclerosis	Calcium	9.43 ± 3.03 mEq/100 g (8.09 ± 1.86 mEq/100 g)*	-	Paiva et al. (1973)
				flame spectrophotometry		Potassium	32.24 ± 4.17 mEq/100 g (37.67 ± 17.99 mEq/100 g)*
1D-PAGE – one-dimensional polyacrylamide gel electrophoresis. LC-MS/MS – liquid chromatography/tandem mass spectrometry. DNA – deoxyribonucleic acid. PCR – polymerase chain reaction. RNA - ribonucleic acid. GC – gas chromatography. HS/GC-MS - headspace gas chromatography/mass spectrometry. DDT – dichlorodiphenyltrichloroethane. DDE – dichlorodiphenyldichlororthylene. HCB – hexachlorobenzene. HCH – hexachlorocyclohexane. UPLC-MS/MS - ultra performance liquid chromatography - tandem mass spectrometry. *Reference ranges for the biomarkers studied. ND - Not defined.