Evaluating Novel Markers for Specimen Validity Testing

Arch Pathol Lab Med. 2020 Feb;144(2):168-171. doi: 10.5858/arpa.2019-0197-OA. Epub 2019 Nov 22.


Context.—: Synthetic urine products are commercially marketed for the purpose of specimen substitution for urine drug screens. These products are widely popular because they yield negative drug screen results, meet criteria for specimen validity testing, and are easily accessible and affordable. Current specimen validity criteria are ineffective for detecting these synthetic products, and new markers of specimen validity are required.

Objective.—: To develop and evaluate a multicomponent liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for urine specimen validity testing.

Design.—: A quantitative LC-MS/MS assay was developed for caffeine, cotinine, theobromine, and urobilin in urine. The assay was applied to known synthetic urine products (n = 10) as well as human specimens received for pre-employment testing (n = 500), for-cause workplace testing (n = 100), and medical pain management monitoring (n = 200). Specimens devoid of all 4 validity markers were subjected to follow-up testing that involved microscopic urinalysis and comprehensive gas chromatography mass spectrometry for drugs, pharmaceuticals, hormones, and lipids.

Results.—: Of the experimental groups, 10 of 10 synthetic urine products (100%), 12 of 500 pre-employment specimens (2.4%), and 4 of 200 pain management specimens (2.0%) failed the experimental LC-MS/MS assay. Follow-up testing indicated that each of the failed specimens was nonphysiologic in nature.

Conclusions.—: Simultaneous application of the 4 experimental validity markers appeared to be a robust method for detecting nonphysiologic specimens. New markers of specimen validity must be developed in order to identify commercially available synthetic urine products.

MeSH terms

  • Biomarkers / urine*
  • Caffeine / urine
  • Chromatography, Liquid
  • Cotinine / urine
  • Humans
  • Substance Abuse Detection / methods*
  • Tandem Mass Spectrometry
  • Theobromine / urine
  • Urinalysis / methods*
  • Urobilin / urine


  • Biomarkers
  • Urobilin
  • Caffeine
  • Cotinine
  • Theobromine