Table 4.

Determination of total phytosterols in dietary supplements by chromatographic methods.

Method	Detector	Sample Preparation/Extraction	Analytical Methods	Target Compounds	Ref.
GC	FID	Addition internal standard solution Saponification: KOH (2 h/100 °C) Derivatization: BSTFA, and pyridine	Column: (25m × 0.25mm, 0,33 μm, Dimethylpolysiloxane stationary phase) Temperatures (°C): Detector: 325; Injector: 325 Gas (mL/min): Helium (0.5) Injection type: Split ratio, 1:40 Injection volume (μL): 1	campesterol stigmasterol SIT total sterols	[118]
GC	FID/MS	Addition internal standard solution (epicoprostanol) Acid hydrolysis HCl (4M/1 h/100 °C) Lipid extraction: EE: diethyl ether: petroleum ether (50:50/v:v) Saponification: ethanolic KOH (2 M/80 °C/60 min) Unsaponifiable extraction (LLE): EE (3 times) Derivatization: BSTFA, and pyridine	Column: (30 m × 0.32 mm, 0.25 μm, (poly (94%methyl/5% phenyl) silicone)) Temperatures (°C): column 250, injector 290, detector 290 Gas (mL/min): H2 (1) Injection type Split ratio, 1:25 Injection volume (μL): 1	campesterol stigmasterol SIT	[22]
GC	FID	Alkaline and acid/alkaline protocols Addition internal standard solution (epicoprostanol) Acid hydrolysis: HCl (3N) Saponification: NaOH 2.3 N in MeOH + HCl 3N + NaCl Derivatization: BSTFA, and pyridine	Column: (30 m × 0.32 mm, 0.25 μm) (5%-phenyl)-methylpolysiloxane stationary phase)/(30 m × 0.32 mm, 0.25 μm, (5%-phenyl) (1%-vinyl)-methylpolysiloxane stationary phase) Temperatures (°C): column 250, Injector 290, detector 290 Gas (mL/min): H2 (1) Injection type Split ratio, 1:25 Injection volume (μL): 1	17 phytosterols	[138]
GC	FID/MS	Addition internal standard solution (epicholesterol) Saponification: aqueous KOH (1.3 N/85–89 °C/20 min) Unsaponifiable extraction: cyclohexane Derivatization: (BSTFA/TMCS)	Column: (15 m × 0.32-mm,0.25 μm/5% diphenyl-95%-Dimethylpolysiloxane) Temperatures (°C): column 270, injector 280, detector 300. Gas (ml/min): helium (0.58) Injector type: split ratio, 17:1 Injection volume (μL): 0.5	SIT β-sitosteryl glucoside (BSSG)	[139]
GC	FID	Saponification: ethanolic KOH (1 M/hot plate/80–90 min) Unsaponifiable extraction: toluene Derivatization: TMCS Addition internal standard solution (5 alfa-cholestane)	Column: (25 m × 0.32 mm, 17 μm/5% phenyl-methylsilicone or methyl silicone gum stationary phase) Temperatures (°C): Column 190→255, injector: 250, detector: 300 Gas (mL/min): helium 2/15/3; makeup helium 20/hydrogen 35/air 380 Injection volume (μL): 1	Campesterol Stigmasterol SIT	[119]
GC	FID/MS	Addition internal standard solution (cholestanol), chloroform Saponification: methanol KOH (0.3M/80 °C/60 min) Unsaponifiable extraction: hexane (twice) Derivatization: MTSA, TMCS, and pyridine	Column: (60 m × 0.25 mm, 0.25 um/5% phenyl methyl siloxane stationary phase) Temperatures (°C): column 80→325, detector 230. Gas (mL/min): helium (1) Injection volume (μL): 1	campesterol stigmasterol SIT total sterols	[120]
LC	APCI–MS	Solvent extraction: hexane Saponification with base (ethanolic KOH)/acid hydrolysis Solvent extraction of unsaponifiable material: toluene	1. Column: phenyl (150 mm × 3.9 mm, 3,5 μm) Mobile phase: 58% acetonitrile, 42% water Flow rate (mL/min): 1.1 (isocratic) 2. Column: ACE C18 (150 mm × 3.0 mm, 3 μm) Mobile phase: 90% methanol, 10% water Flow rate (mL/min): 0.80 (isocratic) Injection volume (μL): 5 for qualitative, 10 for quantitative measurement	campesterol, cycloartenol, lupenone, lupeol, SIT, and stigmasterol (Standard Reference Materials containing saw palmetto)	[39]
HPLC	ESI-MS	Saponification: ethanolic KOH (70 °C/30 min) Unsaponifiable extraction: hexane Derivatization: dansyl chloride (40 °C/30 min)	Column: C18 (250 × 3.0 mm, 5 μm) Mobile phase: 95% methanol: 5% water Flow rate (mL/min): 0.5 (isocratic)	campesterol stigmasterol β-sitosterol	[141]
HPLC	UV	Ultrasound-Assisted Emulsification Microextraction (USAEME): Solubilization: methanol Addition: calcium chloride 2mol/L Fat extraction: hexane (heating for 10 min at 50 °C/ultrasonic time 25 min/centrifugation 2600 rpm, 15 min)	Column: RP-18 (125-4 mm, 5 μm) Mobile phase: hexane, propan-1-ol (99.5:0.5; v/v) Flow rate (mL/min): 0.8−1 (isocratic) Injection volume (μL): 50 UV detection: 212 nm	β-sitosterol	[142]
UHPLC	UV/CAD	Addition internal standard solution (cholesterol) Saponification: ethanolic KOH (80 °C/30 min) Acid hydrolysis: HCl Solvent extraction: hexane (3 min)	Column: (100 × 2.1 mm, 1,7 μm) Phenyl-hexyl Mobile phase: acetonitrile: water Flow rate (mL/min): 0.9 (gradient) Temperature: 60 °C Inj. vol. (μL): 2 Run time (min): 8.5	ergosterol, brassicasterol, campesterol, campestanol, fucosterol, stigmasterol, stigmastanol, SIT esterified form	[122]

GC: gas chromatography; FID: flame ionization detection; BSTFA: bis-(trimethylsilyl) trifluoroacetamide; LLE: Liquid-liquid extraction; MS: mass spectrometry; TMCS: trimethylchlorosilane; MTSFA: N- methyl-N-(trimethylsilyl) fluoroacetamide; LC-liquid chromatography; APCI: atmospheric pressure chemical ionization; ESI: ionization by electrospray; UV: ultraviolet; HPLC: high liquid chromatography; UHPLC: ultra-high liquid chromatography; CAD: Charged aerosol detector.