All the confirmation procedures for amphetamines and related compounds should include preventative measures to avoid loss of these volatile compounds during the evaporation step of extraction or during analysis. Procedures to reduce/ eliminate loss of amphetamines during evaporation include lowering the temperature for evaporation, performing incomplete evaporation, or adding methanolic hydrochloric acid prior to evaporation to produce more stable hydrochloride salts [24 - 26]. Derivatization using reagents, such as heptafluorobutyric anhydride (HFBA), pentafluoropropionic anhydride (PFPA), trifluoroacetic anhydride (TFAA), 4-carboxyhexafluorobutyryl chloride (4-CB), N-methyl-N-t-butyldimethylsilyl trifluoroacetamide (MTBSTFA), N-trifluoroacetyl-1-prolyl chloride (TPC), R(-)-α-methoxy-α-trifluoromethylphenylacetyl chloride (R-MTPAC), chlorodifluoroacetic anhydride (CIF2AA), 2,2,2-trichloroethyl chloroformate, and propylchloroformate decreases the volatility of amphetamines in addition to improving chromatography and quantitation, and forming higher molecular weight fragments yielding different mass ions and ion ratios than potentially interfering compounds [18]. Use of deuterated internal standard (IS) solution contaminated with nondeuterated analyte can lead to inaccurate concentrations and potential false results. IS solutions should be monitored for the presence of the nondeuterated analyte of interest. Error in addition of IS solution is another potential source of inaccurate results. A recommended quality assurance measure to prevent this type of error is to monitor the relative IS abundance of every sample and establish acceptable limits (such as a requirement to be .50% and ,200% of the calibrator IS area) [8].

Although amphetamine and methamphetamine are primarily excreted unchanged in urine, some metabolites of designer amphetamines are excreted as conjugated metabolites [3,24,27,28]. For this reason and because most multianalyte procedures include a hydrolysis step using acid, base, or glucuronidase the efficiency of the hydrolysis should be verified for the initial and periodic evaluations of method parameters.

Reference: [Chapter 16 - Critical Issues When Testing for Amphetamine-Type Stimulants: Pitfalls of Immunoassay Screening and Mass Spectrometric Confirmation for Amphetamines, Methamphetamines, and Designer Amphetamines, p206-213]

Immunoassays detect substances above a set threshold using antibodies[1] [2]. While a useful tool, immunoassays have poor specificity that may lead to false-positive results [1-3].

Reference:

1. Standridge JB, Adams SM, Zotos AP. Urine drug screening: a valuable office procedure. Am Fam Physician. 2010;81(5):635-640.
2. Moeller KE, Lee KC, Kissack JC. Urine drug screening: practical guide for clinicians. Mayo Clin Proc. 2008;83(1):66-76.
3. Saitman A, Park HD, Fitzgerald RL. False-positive interferences of common urine drug screen immunoassays: a review. J Anal Toxicol. 2014;38(7):387-396.

Reference: Urine Drug Screening: Minimizing False-Positives and False-Negatives to Optimize Patient Care

Structure similarity to amphetamine or methanphetamine creates potential problems due to cross-reactivity of the antibodies in immunoassays.

Reference: [Chapter 16 - Critical Issues When Testing for Amphetamine-Type Stimulants: Pitfalls of Immunoassay Screening and Mass Spectrometric Confirmation for Amphetamines, Methamphetamines, and Designer Amphetamines, p206-213]