Detection of phenobarbital adulteration in dietary supplements: simultaneous analysis of 16 sedative-hypnotics and sleep-inducers by ultra-high-performance liquid chromatography with UV detection (UPLC-UV) and quadruple Orbitrap mass spectrometry (Q-Orbitrap-MS)

  • Lee, Ji Hyun ;
  • Choi, Ji Yeon ;
  • Park, Hanna ;
  • Min, Ah Young ;
  • Kim, Nam Sook ;
  • Park, Seong Soo ;
  • Park, Sung-Kwan ;
  • Kang, Ho-il
  • Received : 2018.09.09
  • Accepted : 2019.01.15
  • Published : 2019.02.25


The safety of food is occasionally questionable, as there have been some reports of products contaminated with illegal adulterants. In this study, the presence of 16 sedative-hypnotics and sleep inducers in dietary supplements was determined by ultra-high-performance liquid chromatography with UV detection (UPLC-UV) and quadruple Orbitrap mass spectrometry (Q-Orbitrap-MS). The UPLC method was validated, providing a linearity (R2) of more than 0.999, and LODs and LOQs that ranged from 0.2 to 0.5 and 0.6 to $1.5{\mu}g\;mL^{-1}$, respectively. The repeatabilities were 0.2-8.4 % (intra-day) and 0.3-4.5 % (inter-day), and the accuracies were 89.0-117.0 % (intra-day) and 87.8-111.9 % (inter-day). The mean recoveries of the spiked samples ranged from 98.7 to 107.3 %. The relative standard deviation (%RSD) of the stability was less than 2.4 %. Using the developed method, one sedative-hypnotic compound, phenobarbital, was detected in one of the nineteen samples tested. In addition, the major characteristic fragment ions of each target compound were confirmed using Q-Orbitrap-MS for higher accuracy. Monitoring the presence of these 16 sedative-hypnotics and sleep inducers in dietary supplements should be pursued in the interest of human health, and the results of this study confirmed that the developed method has value for this application.


Sedative-Hypnotics;Adulterants;UPLC-UV;Q-Orbitrap-MS;Method Validation

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Fig. 1. LC chromatogram of the 16 sleep inducers analysed in this study and the individual PDA spectra of each compound.

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Fig. 2. XIC of a phenobarbital standard (a) and 500-fold diluted positive sample (b).

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Fig. 3. Mass spectra of a phenobarbital standard (a) and a sample that contains phenobarbital (b).

Table 1. Recovery of each compound under different extraction conditions

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Table 2. Summary of the calibration curves, limits of detection (LOD), and limits of quantification (LOQ) for 16 sleep inducers by UPLC

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Table 3. Intra-and inter-day variation in the three concentrations of the 16 sleep inducers using UPLC

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Table 3. Continued

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Table 4. The recovery efficiency of the 16 sleep inducers from dietary supplement samples using UPLC

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Table 5. Stability of the 16 sleep inducer compounds over 48 h analysed with UPLC.

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Table 5. Continued

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Supported by : Ministry of Food and Drug Safety