Poster Presentation Australian Society for Medical Research Annual Scientific Meeting 2016

Development and Validation of a Rapid High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-UV/MS) assay for determining caffeine and dimethylxanthines in pre-workout supplements (#139)

Andy Koh 1 , Anna Lohning 1
  1. Bond University, Robina, QLD, Australia

Dietary pre-workout supplements usually contain caffeine and other botanical extracts to promote weight-loss and to improve athletic performance [1]. The increase in popularity of these products [2] and reports of adverse effects [3] drives the need to investigate the amount of caffeine and its dimethylxanthine derivatives present. A rapid, sensitive and simple HPLC-UV/MS method was developed for the simultaneous quantitation and detection of caffeine, theobromine, paraxanthine, and theophylline in domestic and overseas pre-workout supplements. Five supplements were prepared and extracted using solid phase extraction. The chromatography involved a gradient elution (A: water and B: acetonitrile, adjusted to pH 4.5 with 0.1% formic acid) on a CSH phenyl-hexyl column, detected with a UV/Vis-detector (272nm) coupled with mass confirmation on a single quadrupole (QDa) mass detector in positive electrospray ionisation (ESI) scan mode. Calibrations were linear with determination coefficients of r2> 0.999, except for theobromine (r2=0.991). The supplement with the highest concentration of caffeine (210 mg/serve) did not state the presence of caffeine on its label. Furthermore, a supplement stating to contain 100mg/serve of caffeine was found to contain 188mg/serve (Figure 1). The intermediate precisions (RSD, %) were less than 10.5%, with overall recoveries (RSD, %) between 81.5-89.5%. This method offers a reliable and fast analysis that is also applicable to biological fluids analysis as it boasts the separation of theophylline and paraxanthine with good resolution.


Amount of Caffeine Quantified  (mg//mL)

Caffeine listed on the label (mg)

Actual amount of caffeine/ (mg)


20.10 ± 1.81

Not listed



15.74 ± 1.53

Proprietary Blend



25.30 ± 1.21

Proprietary Blend



9.89 ± 0.39




24.00 ± 0.16



Table 1: Amount of caffeine reported compared to respective label contents after back-calcuations. Equivalent caffeine content in 1 cup of coffee= 100mg

  1. 1. Eudy, A.E., et al., Efficacy and safety of ingredients found in preworkout supplements. Am J Health Syst Pharm, 2013. 70(7): p. 577-88.
  2. 2. Gurley, B.J., S.C. Steelman, and S.L. Thomas, Multi-ingredient, caffeine-containing dietary supplements: history, safety, and efficacy. Clin Ther, 2015. 37(2): p. 275-301.
  3. 3. Di Lorenzo, C., et al., Adverse effects of plant food supplements and botanical preparations: a systematic review with critical evaluation of causality. Br J Clin Pharmacol, 2015. 79(4): p. 578-92.