Review: Identification and Quantification of 35 Psychotropic Drugs and Metabolites in Hair by LC-MS/MS: Application in Forensic Toxicology

Emily C. Lennert


Category: Chemistry

Keywords: psychotropic, drug, drug facilitated crime, DFC, drug facilitated sexual assault, DFSA, benzodiazepines, anesthetics, liquid chromatography, mass spectrometry, tandem mass spectrometry, triple quadrupole, LC-MS, LC-MS/MS, hair

Article to be reviewed:

  1. Maublanc, J.; Dulaurent, S.; Morichon, J.; Lachâtre, G.; Gaulier,J. Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology. International Journal of Legal Medicine. 2015, 129, 259-268.

Disclaimer: The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


In drug facilitated crimes (DFC), particularly in drug facilitated sexual assaults (DFSA), it is common for days or even weeks to pass between the time of suspected drug intake and sample collection. In situations like this, hair may be the only way to test for drugs. In DFC, a number of drugs could be used to facilitate the crime, including illicit drugs like LSD and ketamine, and prescription drugs, such as lorazepam (Ativan®) and alprazolam (Xanax®). The authors of this study intended to develop and validate a liquid chromatography – tandem mass spectrometry (LC-MS/MS) method capable of detecting 35 drugs or metabolites that have been identified in DFC cases. A full list of the drugs investigated can be found in the table below. The drugs investigated included: alprazolam, buprenorphine, clonazepam, diazepam, flunitrazepam (Rohypnol®, commonly known as “roofies”), ketamine, lorazepam, LSD, tramadol, methadone, and others. Validation methods and parameters were based on international guidelines, cited within the paper.

35 psychotropic drugs

7-aminoclonazepam diphenhydramine LSD tetrazepam
7-aminoflunitrazepam doxylamine methadone tramadol
alimemazine flunitrazepam midazolam triazolam
alprazolam haloperidol niaprazine zolpidem
bromazepam hydroxyzine nordazepam zopiclone
buprenorphine ketamine oxazepam
clobazam levomepromazine prazepam
clonazepam loprazolam propoxyphene
cyamemazine lorazepam scopolamine
diazepam lormetazepam temazepam

Hair samples were prepared by decontamination in a series of baths: 2 water baths followed by 2 dichloromethane baths, with hair dried between baths. Hair samples were then cut into small pieces, less than 1mm, and weighed to create 50 mg samples. A stock solution containing the 35 target drugs and metabolites was prepared; working solutions were prepared immediately before use. Working solutions were prepared by diluting the stock solution to concentrations of 1, 10, 100, and 1,000 μg/L. An internal standard (IS) solution containing 5 compounds was prepared at a concentration of 0.1 mg/L. Calibration standards were prepared by adding the volume of working solution required to obtain the desired concentration, which ranged from 0.5 to 500 pg/mg, to a sample of 50 mg hair and 25 μL of IS solution. Samples were then incubated by adding 3 mL of phosphate buffer, pH 5, and allowing the sample to sit for 16 hours a room temperature. Following incubation, samples were centrifuged, or spun rapidly to separate layers within the sample, and the supernatant, i.e. top layer, was collected and transferred to a new vial. Then, 1.0 mL of sodium hydroxide (NaOH), 0.25 concentration, and 5 mL of a 70:30 dichloromethane/ether solution were added to each vial, and samples were centrifuged again. The organic layer (top layer) was retained and allowed to evaporate to dryness. The dry residue was then reconstituted in 70 μL of a 90:10 formate buffer/acetonitrile solution and subsequently analyzed by LC-MS/MS.

Samples were analyzed via LC-MS/MS. Liquid chromatography separates samples in liquid prior to analysis by MS. The sample is injected and enters a mobile phase; the mobile phase is a solution under high pressure that forces the sample through the stationary phase, i.e. column. The column is a tube-like component that works like a filter to separate the analytes within a sample based on the molecule’s weight and size. After separation, the analytes elute from, or exit, the column and go to the mass analyzer, where the molecular weight (i.e. molecular mass) can be determined from the resulting mass spectrum. Separation within the column leads to analytes eluting at different times, called retention times. Retention times are characteristic of compounds, and can help to identify an analyte, and can help to identify which compound is present. The identification is further determined by the mass spectra obtained from the mass analyzer. A mobile phase of 2 mM, pH 3 ammonium formate and acetonitrile/2 mM, pH 3 ammonium formate (90:10) was used in this study. The LC was coupled to a triple quadropole MS, with an electrospray ion source. Electrospray ion sources operate by forcing the liquid sample through a needle, creating a fine aerosol. An electrical charge is applied at the tip of the needle, which ionizes the sample as it is sprayed. The charged droplets evaporate, decreasing in size as they head toward and enter the mass analyzer. The triple quadropole MS system utilizes three quadropole mass spectrometers. The first quadrupole can be used to select and filter one analyte out of a mixture. The second quadrupole is used to induce fragmentation prior to reaching the third quadrupole. The third and last quadrupole is used to identify the molecular weight of all of the fragments so that the analyst identify the chemical structure of the initial analyte.

All 35 drugs and metabolites were detected and quantified by LC-MS/MS in this study. After analysis, the limit of detection (LOD), limit of quantification (LOQ), intra- and inter-day precision, relative standard deviation (RSD), and percent deviation were calculated for each analyte. Calibration curves were created for each analyte to allow for quantification, and the coefficient of determination, also known as r-squared value, was greater than 0.99 for each, indicating a very good calibration curve for each analyte. The closer to 1 the value is, the better the calibration curve is.

  • LOD is the lowest concentration of an analyte that can be differentiated from the background, i.e. detected, with confidence. LOQ is the lowest concentration that can be measured with certainty, i.e. quantified. The LOD and LOQ for each analyte was very low, between 0.5 and 5 pg/mg, with the exception of lorazepam at 10 pg/mg for both LOD and LOQ. This means that the method is effective at detecting low concentrations of the target compounds.
  • Intra-day precision is the measure of precision between replicates analyzed on the same day, and inter-day precision is the measure of precision between replicates analyzed on different days. The precision was found to be acceptable for all analytes, with variation below 25% at the LOQ and below 20% for all other concentrations.
  • RSD is used to estimate precision; in this study, RSD was determined for all replicates, inter- and intra-day replicates, together. Percent deviation is used to estimate the accuracy of measurements. Precision differs from accuracy: precision is the closeness of measurements to one another, and accuracy is the closeness of a measurement to a known or standard value. For example, if a measurement is made repeatedly and determined to be 1.2 each time, the measurement is precise, but if the true value is 3, the measurement is not accurate. RSD and percent deviation were found to be acceptable for all analytes, with variation below 25% at the LOQ and below 20% for all other concentrations.

Additionally, a variety of hair colors and textures were examined to determine if variations in the matrix, i.e. hair, had any effect on the results; no relationship or interference was found between the quantification of drugs and the hair type. After the method was validated, it was tested on 32 real world samples. Positive results were obtained in 22 cases. In many cases, more that one target drug was identified in the sample. The identification of multiple drugs within a sample highlights the utility of this method for the simultaneous detection and quantification of 35 analytes.

Scientific Highlights:

  • A LC-MS/MS method was developed and validated for the analysis of 35 drugs commonly reported in drug facilitated crimes.
  • Hair type, i.e. color and texture, did not affect the recovery and quantification of drugs from the hair samples.
  • The described LC-MS/MS method was found to be precise and accurate during validation studies.
  • Analysis of real world samples resulted in identification of multiple drugs in several samples.

Relevance: Oftentimes, in cases of drug facilitated crimes and sexual assaults, a case is not reported or samples are not collected for days or weeks after the crime occurs. In these cases, drug traces in hair may be the only viable evidence that drugs were involved. The ability to identify a variety of drugs associated with these crimes is vital to successful analysis of the evidence.

Potential Conclusions:

  • LC-MS/MS is a verified, precise and accurate method for the analysis of common drugs used in DFC and DFSA.
  • LC-MS/MS is capable of detecting multiple drugs within a single sample.
  • The given LC-MS/MS method is tested and viable for real world application.