Review: Analysis of Isomeric Opioids in Urine Using LC-TIMS-TOF MS

Emily C. Lennert





opioids, drugs of abuse, liquid chromatography, trapped ion mobility spectrometry, time of flight mass spectrometry, LC-TIMS-TOF MS, isomers

Article Reviewed

Adams, K. J.; Ramirez, C. E.; Smith, N. F.; Muñoz- Muñoz, A. C.; Andrade, L.; Fernandez-Lima, F. Analysis of isomeric opioids in urine using LC-TIMS-TOF MS. Talanta. 2018, 183, 177-183.


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.


Commonly, preliminary drug testing targeting drugs of abuse in urine will take place using immunoassays, followed by confirmatory testing with liquid chromatography – tandem mass spectrometry (LC-MS/MS). Immunoassays allow for the detection of broad drug classes, without specific drug identification. However, with LC-MS/MS, positive drug identification may be made based on retention time and mass spectra of both the intact and fragmented molecule. Ion mobility spectrometry, which separates molecules based on size, shape, and charge, adds a third dimension to the analysis. Trapped ion mobility spectrometry (TIMS) is a form of ion mobility spectrometry that was employed in this study to aid in the separation of opioid isomers. Isomers are compounds with the same molecular formula, and therefore the same mass, but different arrangement of atoms within the molecule. In the class of opioids, several isomers exist, such as 6-acetylmorphine and naloxone or codeine and hydrocodone. The purpose of this study was to use LC-TIMS-time-of-flight (TOF) MS to provide a comprehensive, high throughput analysis of opioids in urine. Opioids were identified based on retention time from LC, collisional cross section (CCS) from TIMS, and accurate mass from MS.

Eight opioids as well as their deuterated analogues were purchased for this study (Table 1). A deuterated analogue is a form of a compound where one or more hydrogen atoms have been replaced with the heavier deuterium atom. Deuterium is an isotope of hydrogen. Deuterated analogues were used as internal standards for the study. An internal standard is a constant, known concentration of a compound, which may then be used to quantify other components of a mixture. Opioid free human urine was also purchased to serve as the sample matrix.

Table 1. Opioids and deuterated opioid analogues. Three sets of isomers were analyzed, and are distinguished by color.

Calibration curves were prepared in urine and water using seven calibration points, ranging from 0.1 – 500 ng/mL with a constant 50 ng/mL deuterated internal standard mix. Spiked samples were diluted with a 10% methanol in water mix to reach a volume of 300 L, with no further sample preparation prior to analysis, i.e. the “dilute-and-shoot” method. Limits of detection were determined based on the calibration curves, and limits of quantification were reported as five time the limit of detection. Additionally, matrix effect experiments were conducted using ten urine samples containing 50 ng/mL of internal standard and spiked at low and high concentrations of opioid mixture, 75 ng/mL and 400 ng/mL, respectively. These urine samples were compared to samples prepared in water to determine a matrix factor. Theoretical calculations were then made for the collisional cross section (CCS) of each molecule, i.e. the information derived from TIMS. First, structures were proposed for each molecule being studied, and then theoretical CCS were calculated using MOBCAL software.

Ion mobility profiles of isomeric sets of opioids showed a single peak for each opioid’s protonated molecule [M + H]+. Small differences in CCS were observed within opioid isomer sets; for example, codeine and hydrocodone showed CCS values of 168.2 and 167.8 Å2, respectively. Theoretical and experimental CCS values showed good agreement, as shown in Table 1 within the study. Agreement was also noted with previously reported drift tube ion mobility spectrometry (DTIMS) CCS values. Differences in the compound structure, e.g. size and shape, lead to differences in the CCS values between isomers. For example, for 6-AM and naloxone, significant differences exist in the orientation of nitrogen group and methyl group on the oxygen atom, leading to different CCS values. Similarly, hydrocodone and codeine differ by the presence of a carbonyl group on a six membered ring, which produces a minor change in size, resulting in little difference in CCS value. Morphine, hydromorphone, norcodeine, and norhydrocodone all differ in structure at the nitrogen, where norcodeine and norhydrocodone have a secondary amine while morphine and hydromorphone have a tertiary amine. Due to similar amine groups, hydromorphone and morphine cannot be separated based on CCS; however, morphine and norcodeine can be baseline separated based on CCS, as well as hydromorphone and norhydrocodone. While the above ion mobility profiles were obtained at fast scan rates, the authors note that at slow scan rates, 6-AM and naloxone, hydromorphone and norhydrocodone, and morphine and norcodeine were able to be baseline separated. However, codeine and hydrocodone, morphine and hydromorphone, and norcodeine and norhydrocodone were unable to be baseline separated by TIMS.

Matrix effects were studied through comparison of the separation of opioid standards in solutions prepared in urine and in water. The authors concluded that potential interferences from molecules in the urine may lead to higher limits of detection in the urine samples compared to the water samples. The authors noted that LC allows for the separation of potential matrix interferants from the opioid target analytes. Additionally, the addition of LC allowed for the separation of opioid isomers that were not possible by TIMS-MS only. Limits of detection and quantification were studied and compared to traditional LC-TOF MS; results are summarized in Table 2 within the study. Generally, limits of detection and quantification were found to be in good agreement between the two techniques, with higher limits observed for urine samples compared to water samples for both techniques, consistent with the previously observed matrix effects. Reproducibility of the three identification parameters, CCS< retention time, and m/z, was evaluated across several calibration points and in water and urine. All parameters were found to be minimally affected by matrix or by concentration. Within-day reproducibility was found to be high, with a low relative standard deviation observed between replicates. Reproducibility results indicated that the LC-TIMS-TOF MS technique was highly reproducible.

Scientific Highlights

  • LC-TIMS-TOF MS was successfully applied for the first time for the identification of opioid isomers.
  • Good agreement was observed between theoretical, previously reported DTIMS, and experimental TIMS CCS values. CCS values varied due to differences in size and shape of compounds.
  • Higher limits of detection and quantification were observed in urine samples compared to water samples, suggesting some matrix effect were present.
  • LC-TIMS-TOF MS was shown to be a highly reproducible technique.


Opioids exist in the form of isomeric compounds and may be difficult to distinguish without the proper techniques.

Potential Conclusions

LC-TIMS-TOF MS may provide a reliable method of differentiating opioid isomers in human urine.