James Byrd, Marta Kozak 28 Apr 2011 LC-MS analysis of six opiates in urine with Quantum Ultra triple quadrupole mass spectrometer James Byrd, Marta Kozak 28 Apr 2011

Purpose Develop LC-MS method to analyze six opiates in urine Method specification: Analysis time: max 6 min LOQ of 40 ng/mL (20 ng/mL even better) to support cut off value of 50 ng/mL Sample preparation: glucuronide enzymatic hydrolysis followed by urine dilution

Conclusion 6 minutes method to analyzed six opiates in urine was developed Method meets laboratory standards for linearity, accuracy and precision Linearity: 10-500 ng/mL Precision: QC1 (16.7 ng/mL): <15.7% QC2 (167 ng/ml); <5.2 Accuracy: QC1 (16.7 ng/ml): 97.7-132% QC2 (167ng/ml): 91.0-108% Method was tested for matrix effects-no significant matrix effects were observed

List of analytes and internal standards Analayte Internal Standard Morphine Morphine-D3 Hydromorphone Hydromorphone-D6 Oxymorphone Oxymorphone-D3 Codeine Codeine-d3 Hydrocodone Hydrocodone-D3 Oxycodone Oxycodone-D3 Two pairs of analytes - Morphine-Hydromorphone and Codeine-Hydrocodone - are isobaric (have the same SRM transitions) and these compounds have to be chromatographically separated.

Method validation and acceptance criteria Linearity range Calibration standards acceptance criteria: 20% accuracy for the lowest calibrator and 15% accuracy for higher calibrators Precision %RSD equal to 20% or better for the low QC sample and equal to 15% or better for high QC sample analyzed in five replicates Spike recovery %Recovery within 30% for the low spiked concentration (10ng/mL) and within 20% for the high concentration (125 ng/mL)

Instrumentation Quantum Ultra triple quadruple mass spectrometer with HESI source TLX-1

Calibration standards Calibration standards spiking solutions were prepared in MeOH by diluting 1 mg/mL solutions purchased from Cerillant Calibration standards were prepared in urine by mixing 10 uL of standard spiking solution with 990 uL of blank urine

QC samples and spiked urine samples Two QC samples with concentrations of 16.7 ng/mL and 167 ng/mL were prepared in urine Six urines from different subjects were spiked with mix of analytes to final concentrations of 10 ng/mL and 125 ng/mL

Sample Processing Method Aliquot 0.2 mL urine into microcentrifuge tube Add 10 µL IS (4000 ng/mL of each deuterated analyte in MeOH) Add 0.1 mL ß-glucuronidase (Sigma G-8132: dissolve at 10,000 U/mL in 1M acetate buffer adjusted to pH 5 w/glacial acetic acid,) Incubate at 60°C for 60 min Cool samples to room temperature Add 200 µL MeOH Vortex Centrifuge at 12 000 rpm for 5 minutes Mix 0.05 mL supernatant + 0.95 mL water in HPLC vial Inject 20 - 50 µL into LC-MS system

LC method Analytical Column: 50 x 4.3 mm, 1.9 um Hypersil GOLD aQ (part number 25302-054630) Column temperature: 25 deg C Mobile Phase A: 10 mM NH4FA, 0.1%FA in DI water B: 10 mM NH4FA, 0.1%FA in MeOH C: ACN/IPA/Acetone=20/20/10/v/v/v Gradient: total time = 5.67 min

Mass Spec source parameters HESI source Spray Voltage: 3500 mV Heater temp: 350 deg C Capillary temp: 370 deg C Sheet gas: 45 units Aux gas: 25 units Probe position: C/D

Mass Spec acquisition method Timed SRM transitions-two SRM transitions for each analyte Cycle time 0.4 sec Resolution: 0.7 FWHM Collision gas pressure: 1.4 mTorr Divert valve in 3 positions: 1.5-4.5 minutes to mass spectrometer

Mass Spec acquisition method: List of SRM transitions and acquisition time windows

Mass Spec acquisition method-software screen capture

Mass spec acquisition method-divert valve positions

Processing method Each analyte and internal standard were identified with ion ratio. Ion ratio accuracy window was set to 30%(relative) for Morphine and Oxymorphone and to 20% (relative) for rest of analytes and internal standards 30%(relative) ion ratio accuracy windows for Morphine and Oxymorphone was needed to allow LOQ of 5 ng/mL for Morphine and 10 ng/mL for Oxymorphone. If LOQ of 5-10 ng/mL is not required then ion ratio accuracy window can be set to 20% (relative)

Processing method: Morphine-D3 and Morphine

Results

Results: 10 ng/mL calibration standard in urine Morphine Morphine-D3 Hydromorphone Hydromorphone-D6 Oxymorphone Oxymorphone-D3 Codeine Codeine-D3 Hydrocodone Hydrocodone-D3 Oxycodone-D3 Oxycodone

Results Summary – Calibration curves: injection volume =20 uL Morphine R2=0.9870 Hydromorphone R2=0.9874 Oxymorphone R2=0.9868 Cal Std % Diff 10 ng/mL -5.99 25 ng/mL 11.3 50 ng/mL 8.34 150 ng/mL 6.00 250 ng/mL -10.9 500 ng/mL -8.76 Cal Std % Diff 10 ng/mL -4.73 25 ng/mL 11.1 50 ng/mL 3.62 150 ng/mL 1.71 250 ng/mL -16.4 500 ng/mL 4.64 Cal Std % Diff 10 ng/mL -1.05 25 ng/mL -4.44 50 ng/mL 15.8 150 ng/mL 3.36 250 ng/mL -13.8 500 ng/mL 0.19 Codeine R2=0.9967 Hydrocodone R2=0.9961 Oxycodone R2=0.9923 Cal Std % Diff 10 ng/mL 2.24 25 ng/mL -7.13 50 ng/mL 1.37 150 ng/mL 7.04 250 ng/mL -2.84 500 ng/mL -0.68 Cal Std % Diff 10 ng/mL -0.97 25 ng/mL -0.24 50 ng/mL 7.53 150 ng/mL -3.72 250 ng/mL -7.00 500 ng/mL 4.40 Cal Std % Diff 10 ng/mL -1.65 25 ng/mL -1.12 50 ng/mL 11.6 150 ng/mL 3.83 250 ng/mL -10.8 500 ng/mL -1.88

Internal standard signal precision in sequence of 19 samples %RSD n=29 Morphine-D3 10.4 Hydromorphone-D6 10.5 Oxymorphone Codeine 6.9 Hydrocodone 8.7 Oxycodone 5.3

Results Summary – QC1 samples: injection volume =20 uL Spiked concentration = 16.7 ng/mL Morphine %RSD=11.9 Hydromorphone %RSD=11.8 Oxymorphone %RSD=7.85 Sample # Conc (ng/mL) % Diff QC1-1 15.5 -7.27 QC1-2 19.7 17.7 QC1-3 16.4 -1.56 QC1-4 20.7 23.8* QC1-5 18.3 9.72 Sample # Conc (ng/mL) % Diff QC1-1 18.3 9.78 QC1-2 21.6 29.2* QC1-3 16.2 -3.0 QC1-4 20.0 19.7 QC1-5 17.0 1.78 Sample # Conc (ng/mL) % Diff QC1-1 17.5 4.91 QC1-2 20.3 21.4* QC1-3 17.3 3.60 QC1-4 20.4 21.9* QC1-5 19.6 17.4 Codeine %RSD=11.4 Hydrocodone %RSD=15.7 Oxycodone %RSD=11.5 Sample # Conc (ng/mL) % Diff QC1-1 17.8 6.78 QC1-2 20.0 20.1* QC1-3 15.9 -4.81 QC1-4 19.6 17.4 QC1-5 15.7 -6.14 Sample # Conc (ng/mL) % Diff QC1-1 17.5 4.89 QC1-2 22.1 32.4* QC1-3 15.4 -7.91 QC1-4 19.2 15.0 QC1-5 15.5 -7.33 Sample # Conc (ng/mL) % Diff QC1-1 15.8 -5.66 QC1-2 19.4 15.9 QC1-3 15.3 -8.25 QC1-4 17.8 6.79 QC1-5 14.8 -11.0 *) results which did not meet accuracy criteria of 20% if calculated against intended concentration of 16.7 ng/mL All samples (except one) meet accuracy criteria if calculated against mean value

Results Summary – QC2 samples: injection volume =20 uL Spiked concentration = 167 ng/mL Morphine %RSD=5.06 Hydromorphone %RSD=5.16 Oxymorphone %RSD=4.06 Sample # Conc (ng/mL) % Diff QC2-1 162 -3.13 QC2-2 165 -0.97 QC2-3 175 4.60 QC2-4 180 8.08 QC2-5 181 8.36 Sample # Conc (ng/mL) % Diff QC2-1 154 -7.86 QC2-2 158 -5.42 QC2-3 168 0.34 QC2-4 174 4.09 QC2-5 170 2.12 Sample # Conc (ng/mL) % Diff QC2-1 180 7.83 QC2-2 167 0.02 QC2-3 185 11.0 QC2-4 178 6.74 QC2-5 184 10.2 Codeine %RSD=1.75 Hydrocodone %RSD=4.10 Oxycodone %RSD=3.57 Sample # Conc (ng/mL) % Diff QC2-1 171 2.32 QC2-2 170 1.65 QC2-3 174 4.18 QC2-4 168 0.92 QC2-5 176 5.29 Sample # Conc (ng/mL) % Diff QC2-1 154 -7.48 QC2-2 -9.02 QC2-3 159 -4.66 QC2-4 166 -0.73 QC2-5 168 -0.74 Sample # Conc (ng/mL) % Diff QC2-1 166 -0.75 QC2-2 155 -7.23 QC2-3 154 -7.52 QC2-4 160 -4.26 QC2-5 -0.31

Results Summary – Spiked urine Level 1: injection volume =20 uL Spiked concentration = 10 ng/mL Morphine %RSD=13.7 Recovery 79.5-123% Hydromorphone %RSD= 19.7 Recovery = 73.5-146% Oxymorphone %RSD=7.0 Recovery=102-120% Cal Std Conc (ng/mL) % Diff Urine#1 12.9 29.4 Urine#2 10.5 5.35 Urine#3 8.07 -19.3 Urine#4 7.35 Urine#5 24.3 Urine#6 46.4 Cal Std Conc (ng/mL) % Diff Urine#1 8.76 -12.3 Urine#2 11.2 11.9 Urine#3 9.20 -7.95 Urine#4 11.1 10.6 Urine#5 12.0 20.5 Urine#6 12.3 23.5 Cal Std Conc (ng/mL) % Diff Urine#1 10.2 2.18 Urine#2 11.9 18.8 Urine#3 11.0 9.72 Urine#4 10.3 2.64 Urine#5 12.0 19.5 Urine#6 10.7 6.89 Codeine %RSD=14.0 Recovery=76.5-114% Hydrocodone %RSD=9.7 Recovery 98.9-127% Oxycodone %RSD=13.2 Recovery=89-124% Cal Std Conc (ng/mL) % Diff Urine#1 10.4 3.88 Urine#2 7.65 -23.5 Urine#3 8.69 -13.1 Urine#4 8.98 -10.2 Urine#5 9.66 -3.39 Urine#6 11.4 14.2 Cal Std Conc (ng/mL) % Diff Urine#1 12.7 26.7 Urine#2 11.5 15.0 Urine#3 9.89 -1.07 Urine#4 11.1 Urine#5 10.3 3.30 Urine#6 12.4 23.7 Cal Std Conc (ng/mL) % Diff Urine#1 10.2 2.56 Urine#2 8.90 -11.0 Urine#3 9.44 -5.57 Urine#4 9.62 -3.76 Urine#5 11.7 17.0 Urine#6 12.4 23.9

Results Summary – Spiked urine Level 1: injection volume =20 uL Spiked concentration = 10 ng/mL Morphine %RSD=13.7 Recovery =79.5-123% Hydromorphone %RSD= 19.7 Recovery = 73.5-146% Oxymorphone %RSD=7.0 Recovery=102-120% Cal Std Conc (ng/mL) % Diff Urine#1 12.9 29.4 Urine#2 10.5 5.35 Urine#3 8.07 -19.3 Urine#4 7.35 Urine#5 12.4 24.3 Urine#6 14.6 46.4 Cal Std Conc (ng/mL) % Diff Urine#1 8.76 -12.3 Urine#2 11.2 11.9 Urine#3 9.20 -7.95 Urine#4 11.1 10.6 Urine#5 12.0 20.5 Urine#6 12.3 23.5 Cal Std Conc (ng/mL) % Diff Urine#1 10.2 2.18 Urine#2 11.9 18.8 Urine#3 11.0 9.72 Urine#4 10.3 2.64 Urine#5 12.0 19.5 Urine#6 10.7 6.89 Codeine %RSD=14.0 Recovery=76.5-114% Hydrocodone %RSD=9.7 Recovery =98.9-127% Oxycodone %RSD=13.2 Recovery=89.0-124% Cal Std Conc (ng/mL) % Diff Urine#1 10.4 3.88 Urine#2 7.65 -23.5 Urine#3 8.69 -13.1 Urine#4 8.98 -10.2 Urine#5 9.66 -3.39 Urine#6 11.4 Cal Std Conc (ng/mL) % Diff Urine#1 12.7 26.7 Urine#2 11.5 15.0 Urine#3 9.89 -1.07 Urine#4 11.1 Urine#5 10.3 3.30 Urine#6 12.4 23.7 Cal Std Conc (ng/mL) % Diff Urine#1 10.2 2.56 Urine#2 8.90 -11.0 Urine#3 9.44 -5.57 Urine#4 9.62 -3.76 Urine#5 11.7 17.0 Urine#6 12.4 23.9

Results Summary – Spiked urine Level 2: injection volume =20 uL Spiked concentration = 125 ng/mL Morphine %RSD=3.7 Recovery =95.0-105% Hydromorphone %RSD= 8.4 Recovery = 73.5-95.4% Oxymorphone %RSD=7.57 Recovery=96.9-120% Cal Std Conc (ng/mL) % Diff Urine#1 120 -4.16 Urine#2 131 5.20 Urine#3 125 0.322 Urine#4 119 -4.94 Urine#5 124 -0.978 Urine#6 -0.300 Cal Std Conc (ng/mL) % Diff Urine#1 110 -11.9 Urine#2 119 -4.55 Urine#3 94.1 -24.7 Urine#4 115 -7.97 Urine#5 118 -5.28 Urine#6 116 -6.61 Cal Std Conc (ng/mL) % Diff Urine#1 128 2.13 Urine#2 140 11.8 Urine#3 142 13.2 Urine#4 121 -3.08 Urine#5 135 8.68 Urine#6 150 20.0 Codeine %RSD=6.8 Recovery=85.4-102% Hydrocodone %RSD=11.1 Recovery 77.6-104% Oxycodone %RSD=6.0 Recovery=85.6-101% Cal Std Conc (ng/mL) % Diff Urine#1 111 -11.2 Urine#2 125 -0.041 Urine#3 127 2.03 Urine#4 107 -14.6 Urine#5 119 -4.58 Urine#6 120 -4.04 Cal Std Conc (ng/mL) % Diff Urine#1 97.0 -22.4 Urine#2 122 -2.61 Urine#3 109 -12.4 Urine#4 130 3.90 Urine#5 106 -14.8 Urine#6 102 -18.1 Cal Std Conc (ng/mL) % Diff Urine#1 112 -10.2 Urine#2 123 -1.88 Urine#3 126 0.954 Urine#4 107 -14.4 Urine#5 119 -4.64 Urine#6 115 -7.34