Acetonitrile and HPLC-grade water are purchased from EMD Millipore (Billerica, MA, USA). Formic acid was purchased from Amresco (Solon, OH, USA). Mitragynine with a concentration of 100 µg/mL and 7-hydroxy- mitragynine with a concentration of 100 µg/mL standards were purchased from Cerilliant (Round Rock, TX, USA). Internal standards mitragynine-D₃ with a concentration of 100 µg/mL and 7-hydroxymitragynine-D₃ with a concentration of 100 µg/mL were purchased from Cerilliant (Round Rock, TX, USA). Internal standards were used for quantification purposes as a mean for correct the loss of analytes of interest during sample preparation process or sample injection. The phenyl-hexyl HPLC column was purchased from Phenomenex (Torrance, CA, USA).

The assay was developed on a Shimadzu 20AD liquid chromatography (Columbia, MD, USA) coupled to an AB SciexQTrap 5500 quadrupole linear ion trap mass spectrometer (Framingham, MA, USA). A 2.6-µm 100 mm × 2.1 mm phenyl-hexyl analytical column was employed, and gradient elution with a 0.4-mL/min flow rate of water and acetonitrile as mobile phases was utilized. The LC-MS/MS conditions were optimized to achieve rapid and effective goals for the detection of kratom compounds.

Stock solutions were prepared weekly to keep the active component fresh. Methanol standards and urine standards were prepared for injection, respectively. The methanol standards were stored at −8˚C, and the urine standards were stored at 4˚C. For HPLC injection, 50 µL of working standards, 50 µL of 10 ng/mL internal standards and 150 µL of Mobile Phase A solution were mixed as the injection standards. For testing on urine samples, working standards were substituted with human urine.

Both blank and patient urine samples were stored at −20˚C until analysis. Urine samples were thawed and 1.0 mL aliquot was transferred to a 4 mL clear glass screw-top culture tube and spiked with 50 µL of 10 ng/mL internal standards. The urine samples were centrifuged at 15,000 rpm speed for 15 min and the supernatant was transferred to a clue tube. With addition of internal standards and diluent, the mixture was transferred to an HPLC vial for HPLC-MS/MS analysis.

Quantitation of mitragynine and 7-hydroxymitragynine were calibrated by internal standard technique. Deuterated internal standards purchased from Cerilliant (Round Rock, TX, USA) were added to the sample mixture as a calibration technique. The calibration curve was constructed by plotting the ratios of the peak area of mitragynine and mitragynine internal standard against the ratios of concentration of mitragynine and mitragynine internal standard. The 1/x regression model was employed to acquire the regression equation and coefficient (r).

Analytes were eluted with gradient mobile phases of water with 0.1% formic acid (Mobile Phase A) and acetonitrile (Mobile Phase B). Formic acid is a commonly-used additive for reversed-phase liquid chromatography, as it provides protons and promotes ionization for analytes. Acetonitrile is an organic solvent that provides advantages over methanol in terms of low back pressure, high sensitivity and less ghost peak for the gradient elution program.

The separation and identification of mitragynine and 7-hydroxymitragynine are demonstrated as Figure 2. The extracted chromatograms of both compounds are presented. As shown in the figure, 7-hydroxymitragynine eluted faster than mitragynine with the above mentioned conditions.

The operating conditions and parameters for the electrospray ionization source were optimized to obtain the best mass spectrometric performance for both mitragynine and 7-hydroxymitragynine. The mass spectrometry parameters are listed as Table 1.

With the assistance of direct infusion electrospray ionization mass spectrometry technique, mass spectra and information such as molecular weight and mass fragment were obtained. Possible cleavage and metabolites mechanisms are proposed as Figure 3 for mitragynine and Figure 4 for 7-hydroxymitragynine. As indicated by the mass spectrum parameters in method development, the transition 174.3 is the major fragment of protonated mitragynine compound under instrument conditions stated in Table 1 and Table 2.

As illustrated in Figure 3, protonated mitragynine with m/z 399.5 has fragment of m/z 174.3, 226.1 and 238.2. Figure 4 demonstrated the major fragment of protonated 7-hydroxymitragynine m/z 415.5 is m/z 190.2.

The analytical figures of merit were assessed and presented in Table 3. Linearity in terms of slope and R- squared value was determined. Sensitivity including LOD and LOQ, intraday precision and interday precision were calculated as well. Limit of detection down to 0.0123 ng/mL for mitragynine and 0.0691 ng/mL for 7-hydroxymitragynine were achieved. The LOD values show that the established method is very sensitive for trace amount analysis.