Review: A Quantitative, Selective and Fast Ultra-High Performance Liquid Chromatography Mass Spectrometry Method for the Simultaneous Analysis of 33 Basic Drugs in Hair (Amphetamines, Cocaine, Opiates, Opioids and Metabolites)

Emily C. Lennert





drug, analysis, ultra-high performance liquid chromatography, tandem mass spectrometry, UPLC-MS/MS, cocaine, amphetamine, opiates, opioids, metabolites, fentanyl, norfentanyl, MDMA, oxycodone, codeine, ephedrine, morphine

Article Reviewed

Fernández, M. M. R.; Di Fazio, V.; Wille, S. M.R.; Kummer, N.; Samyn, N . A quantitative, selective and fast ultra-high performance liquid chromatography mass spectrometry method for the simultaneous analysis of 33 basic drugs in hair (amphetamines, cocaine, opiates, opioids and metabolites). Journal of Chromatography B. 2014, 965, 7-18.


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.


Testing of hair samples for the presence of drugs has become a common practice in the forensic field. Hair analysis can be used to determine chronic drug use as well as to establish exposure to drugs over time by segmental analysis, in which segments of hair may be analyzed to establish a timeline of drug exposure based on average hair growth. Due to the wide variety of drugs and metabolites that may be present in a sample, an extraction method that allows for the analysis of several drugs is desirable. This study presented a method for the analysis of 33 drugs and metabolites in hair, including amphetamines, cocaine, opiates/opioids, and metabolites.

Drugs and metabolites analyzed included amphetamine, methamphetamine, MDMA, ephedrine, morphine, codeine, oxycodone, methadone, cocaine, benzoylecgonine, fentanyl, norfentanyl, and more. The full list of drugs and metabolites can be found in section 2.1 within the study. Blank hair samples were obtained for method development and validation. Samples were divided into two groups: group A and group B. Group A contained all drugs/metabolites except for fentanyl, hydromorphone, hydrocodone, oxycodone, norfentanyl, 6-monoacethylmorphine (6-MAM), buprenorphine, norbuprenorphine, and normeperidine, which were group B compounds. A mixed working solution containing 4 g/mL each of group A compounds and 0.08 g/mL of group B compounds was prepared. An internal standard working solution, containing a mixture of deuterated forms of the 33 drugs/metabolites, was also prepared at 0.2 g/mL. Calibration solutions were prepared to create calibration curves allowing for quantification of the 33 compounds. A quality control (QC) low solution was prepared at 0.075 ng/mL for group A and 0.015 ng/mL for group B. A QC high solution was prepared at 3 ng/mL for group A and 0.6 ng/mL for group B.

Hair samples, both blank and those spiked with target compounds, were prepared as follows. Samples were decontaminated in dichloromethane and water, then dried and cut into 1-2 cm segments. 20 mg of hair sample was placed in a Precellys tube, and the hair was powdered using a ball mill Precellys 24. 50 L of internal standard solution was added, followed by incubation with 1 mL methanol at 45C for 4 hours with sonication. Samples were then centrifuged and the supernatant, i.e. top layer of liquid, transferred into a clean tube. The sample was then evaporated down to 100 L and then 2.5 mL of sodium acetate buffer was added. Samples were then extracted by solid phase extraction (SPE), the details of which can be found in section 2.3.1 in the study. Following extraction, sample extracts were analyzed by ultra-high performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS).

Extraction efficiency was evaluated by spiking 500 mg of hair with 1000 L of the drug mixture working solutions. 20 mg of sample was then processed as described above, with alteration to the incubation time. Incubation was performed for either 2 or 4 hours under sonication or 24 hours under mechanical shaking. Hair samples were then extracted and analyzed by UPLC-MS/MS. Higher recovery with sonication was observed at 4 hours for several drugs, and 4 hours was selected as the incubation time.

The method was then validated for selectivity, imprecision, bias, stability of processed samples over 72 hours, matrix effects, and recovery. The authors reported that no interferences were observed from the analysis of blank hair samples, which ensured the selectivity of the method towards drugs only. Imprecision was evaluated using QC standards at high and low concentrations. Inter- and intra-assay imprecision were considered satisfactory. Inter-assay imprecision refers to imprecision observed between separate runs; whereas, intra-assay imprecision refers to the imprecision observed between replicates of the same sample. Furthermore, when stability of the samples, i.e. whether the target drugs degraded over time, was checked over a 72 hour period, no instability was reported. When assessing matrix effects, i.e. the effect caused by all components except for the target analyte in a sample, significant matrix effects were observed for several drug/metabolite compounds. However, these effects were diminished when the internal standard was considered. Extraction recovery of the target drugs ranged from 37 – 107%, with the exception of one sample, which fell below 37%. The results of the extraction recovery and matrix effect experiments are summarized in Table 3 within the study. The authors stated that extraction recovery was not a problem as long as the instrument exhibited sufficient sensitivity and reproducibility. Therefore, the results were acceptable, particularly considering that the method is a quantitative method for 33 drugs and metabolites. The method was then tested on several real world samples, and was successfully applied to authentic casework samples.

Scientific Highlights

  • The presented method allowed for the simultaneous analysis of 33 drugs and metabolites from hair samples.
  • The method was determined to be selective and display acceptable precision and accuracy.
  • Samples were stable over 72 hour periods, and acceptable extraction recoveries were observed.


Hair is a common form of forensic evidence; a quantitative, selective method that allows for the detection of multiple drugs in a sample is therefore desirable.

Potential Conclusions

The presented method may be useful for the quantitative analysis of the 33 drugs and metabolites examined in this study.


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