5. AOACSPDSMethods-2018AwardsV3

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standards were obtained from ChromaDex. The certified reference standards were certified to 100 μg/mL and used to determine the concentration and purity of the working reference standards. Stock solutions were prepared by weighing 10 mg of each standard into separate 10 mL volumetric flasks. Flasks were diluted to volume with extraction solvent. A working stock solution of 7-OH mitragynine was prepared at 100 μg/mL prior to the preparation of calibration standards with extraction solvent as the diluent. ( a ) Mitragynine (C 23 H 30 N 2 O 4 ) .—FW of 398.495 g/mol, CAS 4098-40-2, 85.5% purity, acquired from ChromaDex (Product No. 00013890), stored at 4°C and desiccated. ( b ) 7-OH mitragynine (C 23 H 30 N 2 O 5 ) .—FWof 414.49 g/mol, CAS 174418-82-7, 84.6% purity, acquired from ChromaDex (Product No. 0008624), stored at –20°C and desiccated. ( c ) Mitragynine (C 23 H 30 N 2 O 4 ) .—FW of 398.495 g/mol, CAS 4098-40-2, 100.0 ± 0.5 μg/mL, acquired from Cerilliant Corp. (Product No. M-152), stored at –80°C. ( d ) 7-OH mitragynine (C 23 H 30 N 2 O 5 ) .—FW of 414.49 g/mol, CAS 174418-82-7, 100.0 ± 0.5 μg/mL, acquired from Cerilliant Corp. (Product No. H-099), stored at –80°C. F. Apparatus ( a ) Analytical balance .—Mettler Toledo AE 260, analytical range (±0.1 mg; VWR International) or equivalent. ( b ) Centrifuge .—Eppendorf 5804 table-top centrifuge (VWR International) or equivalent. ( c ) Wrist action shaker .—Burrell Scientific Model 75 DD or equivalent. ( d ) Vortex mixer .—Thermolyne Maxi Mix 1 (Thermo Scientific, Asheville, NC) or equivalent. ( e ) Micropipets .—Eppendorf Reference Series 100, 200 and 1000 μL or equivalent. ( f ) HPLC system .—Agilent 1200 Series Liquid Chromatograph (Agilent, Mississauga, ON, Canada), equipped with binary pump and degasser, temperature-controlled column compartment, autosampler, flow cell, 10 mm, 1.0 μL, 60 bar, diode array detection, ChemStation software, or equivalent HPLC system capable of operating up to 600 bar. ( g ) Syringes .—3 mL Luer-lok fitted with a PTFE filter, 0.22 μm pore size, or equivalent. ( h ) Polypropylene centrifuge tubes .—50 mL. G. HPLC Conditions ( a ) Analytical column .—Kinetex 5 μm EVO C18 100 Å (Phenomenex, Torrance, CA, USA), 150 × 4.6 mm, 5 μm particle size. ( b ) Column temperature .—25°C. ( c ) Detector conditions .—Monitor at 226 nm (4 nm bandwidth), no reference. ( d ) Flow rate .—1.5 mL/min. ( e ) Run time .—18 min, plus 4 min column re-equilibration time. ( f ) Injection volume .—5 μL. ( g ) Gradient conditions .— See Table 2017.14 . H. Calculations Mitragynine from solid samples was quantified in mg/g using the following equation:

AOAC Official Method 2017.14 Mitragynine in Mitragyna speciosa Raw Materials and Finished Products Liquid Chromatography with UV Detection First Action 2017

A. Principle The HPLCmethod is suitable for detection and quantitation of the two alkaloids of interest—mitragynine and 7-OH mitragynine—in several matrixes, including raw materials, dry extracts, capsules, and liquid beverages. B. Test Materials All test materials were stored at room temperature. ( a ) M. speciosa raw material .—Red vein powder spiked with 0.05% 7-OH mitragynine (KT-RM001). ( b ) M. speciosa raw material .—White vein powder (KT- RM002). ( c ) M. speciosa raw material .—Maeng Da powder (KT-RM003). ( d ) M. speciosa powdered bulk extract .—Obtained from a commercial supplier, spiked with 0.25% 7-OH mitragynine (KT- BE001). ( e ) M. speciosa liquid beverage .—Obtained from a commercial supplier (KT-LB001). ( f ) M. speciosa liquid beverage .—Obtained from a commercial supplier (KT-LB002). ( g ) M. speciosa finished product, powder .—Obtained from a commercial supplier (KT-FP001). ( h ) M. speciosa finished product, capsule .—Obtained from a commercial supplier (KT-FC001). C. Reagents and Supplies All reagents were purchased from VWR International (Mississauga, ON, Canada) unless otherwise specified. ( a ) Methanol (CH 3 OH) .—Formula weight (FW) of 32.04 g/mol, CAS 67-56-1, HPLC grade or equivalent. ( b )  Water (H 2 O) .—FW of 18.01 g/mol, CAS 7732-18-5, submicron filtered, HPLC grade or equivalent. ( c ) Acetic acid (C 2 H 4 O 2 ) .—FW of 60.05 g/mol, CAS 64-19-7, glacial, ACS grade or equivalent. ( d ) Formic acid (CH 2 O 2 ) .—FW of 46.03 g/mol, CAS 64-18-6, 3 ) .—FW of 79.065 g/mol, CAS 1066-33-7, eluent additive (Sigma-Aldrich, Oakville, ON, Canada) for LC-MS or equivalent. ( f ) Acetonitrile (C 2 H 3 N) .—FW of 41.05 g/mol, CAS 75-05-8, HPLC grade or equivalent. ( g ) Ammonium hydroxide (NH 4 OH) .—FW of 35.04 g/mol, CAS 1336-21-6, HPLC grade or equivalent. D. Solutions ( a )  Reference standard and liquid sample diluent .—100% methanol. ( b ) Extraction solvent for dry test materials. —0.5 M acetic acid in 70% methanol. ( c ) Mobile phase A .—5.0 mM ammonium bicarbonate buffer, pH 9.50. ( d ) Mobile phase B .—Acetonitrile. E. Reference Standards Certified reference standards for both alkaloids were obtained from Cerilliant Corp. (Round Rock, TX, USA). Working reference HPLC grade purity or equivalent. ( e ) Ammonium bicarbonate (NH 4 HCO

where P 0

= peak area of the target analyte in the sample

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