Katherine Yahvah (Presenter)
Kashi Clinical Laboratories
Bio: Katherine Yahvah, PhD, is currently the associate director of the toxicology division of Kashi Clinical Laboratories in Portland, OR. Her team focuses on the development and implementation of HPLC-MS/MS methods for quantitation of licit and illicit drugs in a clinical setting.
Authorship: Katherine Yahvah, Zahra Kashi, Andrea DeBarber
Kashi Clinical Laboratories, Portland, OR
| Short Abstract We describe development and validation of a 6.5 minute LC-ESI-MS/MS method for quantification of mitragynine in human urine for monitoring of kratom use. Methadone-d3 is used as internal standard. A between-laboratory comparison of concentrations was performed with mitragynine concentrations across the analytical measurement demonstrating acceptable correlation. Our laboratory has routinely measured mitragynine in urine samples for > 1 year with an overall CV for QC samples of 9.3% and accuracy of 102.3%. The methodology we describe allows for measurement of mitragynine within routinely used sample analysis techniques for pain medication and illicit drugs, enabling easy implementation of mitragynine testing. |
Long Abstract
Kratom is derived from the leaves of the Mitragyna speciose tree native to Southeast Asia and has been traditionally used for medicinal purposes in the region. The primary active constituent of kratom is the alkaloid mitragynine, which acts as a μ-opioid receptor agonist. Kratom is considered a controlled substance in Australia and many Southeast Asian countries. The US Drug Enforcement Agency has designated kratom a drug of concern but has not yet moved to classify kratom as a scheduled substance. Kratom can easily be purchased in the US. We describe development and validation of a 6.5 min LC-ESI-MS/MS method for quantitative analysis of mitragynine in human urine for clinical toxicity monitoring of kratom use.
Method calibrators and QCs were generated using dilutions of authentic standard in methanol spiked into surine. For calibrators, QCs or samples 2.5 ng methadone-d3 internal standard was added to 50 µl urine. After hydrolysis of glucuronide conjugates using beta glucuronidase incubation for two hours with acetate buffer (pH 4.0), samples were diluted with 560 ul of 1:9 methanol:water solution. Prior to analysis any precipitate was removed by centrifuging at 22,000x g for 10 min. LC-ESI-MS/MS experiments were performed and the method validated using a QTRAP® 5500 mass spectrometer (SCIEX, Framingham MA), equipped with a TurboIonSpray® ESI source. The ionization interface was operated in the positive mode using the following settings: TEM 600°C, IS 2.5 kV; CUR, GS2 and GS1 nitrogen gas flow rates, 35, 60 and 50 psi respectively. For mitragynine the multiple reaction monitoring (MRM) transitions monitored for quantification were as follows (all at EP 10 V): Mitragynine m/z 399.4→174.0 (CE 33eV, DP 90eV and CXP 12eV) and m/z 399.4→226.2 (CE 23eV, DP 90eV and CXP 12eV), for methadone-d3 internal standard m/z 313.2→102.1 (CE 72eV, DP 56eV and CXP 6eV). The QTRAP® 5500 was coupled to a Shimadzu UPLC system (Columbia, MD) composed of a SIL-20ACXR auto-sampler and two LC-20ADXR LC pumps. Analytes were resolved using a 50x4.6 mm (i.d.), 2.6 mm Kinetex phenyl-hexyl reversed-phase HPLC column with guard (Phenomenex; Torrance, CA). The gradient mobile phase was delivered at a flow rate of 0.7 ml/min. The mobile phase consisted of two solvents: A, 10 mM ammonium formate in water and B, methanol at 0.08% formic acid. Solvent B was increased from 5-40% over 2.2 min then from 40-95% over 2.5 min. The column was washed at 95% B for 1 min, decreased to 5% B over 0.1 min and re-equilibrated at 5% B for 1 min. The column temperature was kept at 35°C using a Shimadzu CTO-20AC column oven. The sample injection volume was 5 µl. To calculate analyte concentrations, calibration curves were routinely generated by performing a weighted (1/x) least-squares linear regression for peak area ratios (analyte/internal standard) plotted against specified calibrant concentration in urine (ng/ml).
The LOD for mitragynine under these conditions (based on a S/N ratio of 3:1) was 0.25 ng/ml. Surine calibration curves for both mitragynine transitions with methadone-d3 internal standard demonstrated acceptable linearity with correlation coefficients >r2=0.998 across the range 4-100 ng/ml (with n=6 samples for each calibrant precision for all calibrants <10% RSD, accuracy within ±10%). The selectivity of the method was assessed with accuracy determined to be within ±20% for QC samples fortified with commonly used over the counter and prescribed medications and their metabolites (caffeine, cotinine, acetaminophen, ibuprofen, baclofen, diphenylhydramine and clonidine). Between injection carry-over for a concentration of up to 1,000 ng/ml was assessed and determined to be <20% of the LLOQ. The matrix effect from 10 different lots of urine was assessed and determined to be within ±25%. A between-laboratory comparison of calculated concentrations was performed with mitragynine concentrations o across the analytical measurement demonstrating acceptable correlation.
Our laboratory has routinely measured mitragynine in urine samples over a 12 month time period with an overall CV for QC samples of 9.3% and an accuracy of 102.3% (over 233 batches). Using a 10 ng/ml cut-off concentration 13 positives have been identified out of 9635 samples analyzed from patients in an addiction rehabilitation setting to provide a positive rate for this population of 0.13%. The methodology we describe allows for measurement of mitragynine within routinely used sample analysis methodology for pain medication and illicit drugs, enabling easy implementation of mitragynine testing.
References & Acknowledgements:
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