Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography.
This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.
Bispo
Marcia S
MS
Centro Federal de Educação Tecnologica da Bahia-CEFET, Universidade Federal da Bahia-UFBA, Brazil.
Veloso
Márcia Cristina C
MC
Pinheiro
Heloísa Lúcia C
HL
De Oliveira
Rodolfo F S
RF
Reis
José Oscar N
JO
De Andrade
Jailson B
JB
D001628:Beverages / Q000032:analysis; D002110:Caffeine / Q000032:analysis* / Q000652:urine; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013805:Theobromine / Q000032:analysis* / Q000652:urine; D013806:Theophylline / Q000032:analysis* / Q000652:urine
D002110:Caffeine; D013806:Theophylline; D013805:Theobromine