22 Buyers’ Guide 2021 Results and Discussion
Chromatography A chromatogram of all the compounds from the 150 ng/mL calibration standard is shown in Figure 2. The peak assignments for the chromatogram can be found in Table 1.
The LC method starts with a 100% water solution to ensure that salts and the most polar components that may still be present in the injected solution are eluted at the beginning of the run before any of the opioids are eluted from the column. This prevents those compounds from interfering with ionisation of the opioid compounds.
The first opioid peak to elute is morphine at a time of 3.60 minutes and the last peak is methadone at 7.71 minutes. All peaks were resolved apart from hydrocodone and its metabolite norhydrocodone (compounds 4,5). Due to the differing precursor ions, this means there is no interferences with the signals.
Figure 2. Chromatogram of 150 ng/mL calibration standard of opioids and metabolites. Peak assignments can be found in Table 1.
The 12 analysed opioids and metabolites are listed in Table 1. There is variety of polarities across the range of compounds - the most hydrophilic compound at a LogP value of 0.70 for noroxycodone and the most hydrophobic at a LogP value of 5.20
Table 1. Properties and MS parameters for the compounds analysed - a The Metabolomics Innovation Centre [11].
Predicted value from Drugbank [9] b
for EDDP. All the compounds analysed are weak bases with pKas ranging from 8.2 - 10.1 which means the SCX (Strong Cation Exchange) resin is the best suited SPE resin to capture and produce clean samples to inject onto an LC-MS system.
Predicted value from Pubchem [10], c Predicted value from
Number Compound Opioid Class R.T (min) Formula Molecular Mass LogP pKa] MRM Transitions Coll. Energy (V) 1
Morphine 2 3 4 5 6 7 8 9 10 11 12 Natural Oxymorphone Semi-synthetic Noroxycodone
Semi-synthetic metabolite
Hydrocodone Semi-synthetic
Norhydroco- done
O-desmethyl-cis- tramadol
Norfentanyl Cis-tramadol Meperidine Fentanyl EDDP Methadone
Semi-synthetic metabolite
Synthetic metabolite
Synthetic metabolite
Synthetic Synthetic Synthetic
Synthetic metabolite
Synthetic 3.60 C17H19 3.90 C17H19 5.23 C17H19 5.32 C18H21 5.35 C17H19 5.50 C15H23 6.11 C14H20 6.23 C16H25 6.49 C15H21 6.89 C22H28 NO3 NO4 NO4 NO3 NO3 NO2 N2 285.14 301.13 301.13 299.15 285.14 249.17 O 232.16 NO2 NO2 N2 7.15 C20H23 7.71 C21H27 263.19 247.16 O 336.22 N 277.18 NO 309.21 0.87a 0.83a 0.70b 2.20b 1.70b 2.30 b 1.60b 1.34a 2.72a 4.05a 5.20b 3.93a 8.2a 8.2a 9.5a 8.6a 10.1a 9.0a 10.0c 9.4a 8.6a 9.0a 9.6c 9.2a
286.13>165.1 286.13>201.1
302.17>227.1 302.17>284.2
302.1>227.1 302.1>284.1
300.1>171.1 300.1>199.1
286.1>171.1 286.1>199.2
250.1>56.1 250.1>58.1
233.1>84.1 233.1>177.1
264.1>56.1 264.1>58.1
248.1>174.2 248.1>220.2
337.1>105.1 337.1>188.2
278.1>234.1 278.1>249.2
310.1>105.1 310.1>265.2
40.92 25.55
27.83 19.39
28.32 15.76
38.69 29.42
36.71 27.50
41.00 16.67
17.22 15.11
46.00 16.09
19.41 20.88
37.03 21.99
30.31 22.88
26.90 14.74
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