High Sensitivity Analysis of Opioids in Oral Fluid Using Ionkey/MS Gregory T

High Sensitivity Analysis of Opioids in Oral Fluid Using ionKey/MS Gregory T. Roman, Robert Lee, James P. Murphy, and Michelle Wood

GOAL The ionKey/MS System facilitates high sensitivity To separate and quantify opioids at very low and robust analysis of opioids in oral fluid. levels in oral fluid with superior sensitivity compared to 2.1 mm I.D. chromatography.

BACKGROUND Over the past three decades oral fluid has emerged as a highly valuable biological specimen and is commonly used in numerous settings, including therapeutic drug monitoring, and workplace and roadside drug testing. Oral fluid analysis has many advantages compared with other matrices such as blood or urine. These include collection convenience and reduced sample collection overheads. Whilst oral fluid itself is a relatively clean Figure 1. The ionKey/MS System with the ACQUITY UPLC® M-Class System and the Xevo® TQ-S matrix, comprising mostly of water and a Mass Spectrometer. small percentage of proteins, the popularity of the specimen has led to the development of Flow rate Composition Composition Time (min) Curve a large variety of collection devices, aiming (µL/min) A (%) B (%) to further simplify and standardize collection. 0 3 5 95 Initial Typically these devices will include additives 6 3 45 55 6 and preservatives to improve the stability of 8 3 85 15 6 the collected sample. One of the key analytical 11 3 5 95 6 challenges with oral fluid analysis is the Mobile phase A: Water, 0.1% formic acid limited amount of sample available for testing Mobile phase B: Acetonitrile, 0.1%formic acid compared with blood or urine, requiring Trapping conditions very sensitive instrumentation to reach the 1 min 15 99.50% 0.50% 6 low levels of detection and quantification. Table 1. LC/MS conditions. The ionKey/MS™ System offers the capability of improving sensitivity in sample-limited situations, making it ideally suited for this application. Performing analysis of oral fluid samples taken 12 1: Oxymorphone-3b-D-glucuronide 100 directly from a collection device provides a 2: Morphine-3b-D glucuronide 3: Morphine-6b-D-glucuronide streamlined method for reducing workflow and 4: Hydromorphone-3b-D-glucuronide 5: Morphine 5 increasing throughput. The use of a trapping 6: Codeine-6b-D-glucuronide 7: Dihydrocodeine column prior to analytical separation allows the 10

8: Oxymorphone % removal of additives (e.g., surfactants) that can 9: Hydrocodone 10: Oxycodone 13 cause suppression in LC-MS and lead to reduced 11: 6-Acetylmorphone 1 12: O-desmethyl Tramadol 11 sensitivity. Trapping can also provide improved 13: Codeine 2 14: Norbuprenorphine-glucuronide 3 7 4 8 peak shape for hydrophilic small molecules, 15: Norfentanyl 6 9 0 Time 16: Tramadol 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 and crucially enables enhanced loading for 17: Normeperedine 100 25 ionKey/MS. Furthermore, the trapping column 18: Meperidine 19: buprenorphine-glucuronide adds a layer of protection similar to that of a guard 20: Norbuprenorphine 21: Fentanyl 23 column, for precious downstream consumables, 22: Buprenorphine 23: EDDP+ making the analytical method more robust. 24: Propoxyphene 25: Methadone %

12 17 THE SOLUTION 21 4 10 Here we demonstrate the separation and high 3 6 2 7 13 sensitivity detection of opioids in oral fluid with 1 8 11 14 9 1819 15 20 22 24 the ionKey/MS System comprised of an ACQUITY 0 Time 4.00 5.00 6.00 7.00 8.00 UPLC M-Class System in combination with a Xevo TQ-S Mass Spectrometer. Preliminary trapping was Figure 2. MRM chromatograms for 25 opioids and metabolites. achieved using an ACQUITY UPLC M-Class HSS T3, 100Å, 1.8 μm, 300 μm x 50 mm Trap Column (P/N 186008029) in combination with a 5 μL loop (Figure 1 and Table 1). Analytical separation was

Morphine oral fluid matrix calibration curve Morphine oral fluid matrix MRM 100 pg/mL Compound name: Morphine Correlation coefficient: r = 0.995468, r^2 = 0.990956 092914_Trapping_ Morphine_Calibration Curve_11 F7:MRM of 2 channels,ES+ Calibration curve: 50780.8 * x + 1832.39 Morphine 286.1 > 165.10, 286.1>201.1 100 Response type: External Std, Area 3.49 1.927e+005 Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 5761.06 78.18*

450000 LOQ: 13 pg/mL S/N: 78.2 400000 R2 = 0.99 Peak-peak 350000 ignoring zeros

300000 % no extra processing on se 250000 Re sp 200000

150000

Morphine 100000 3.49 3.30 5761.06 78.18* 50000 4.47 4.53 5.19

-0 Conc 0 min -0.0 1.02.0 3.04.0 5.06.0 7.08.0 9.010.0 2.50 3.00 3.50 4.00 4.50 5.00

Figure 3. Calibration curve and LOQ of morphine spiked into oral fluid. performed using an iKey™ HSS T3, 100Å, 1.8 µm, ACQUITY UPLC 2.1 mm x 10 cm Column 150 µm x 100 mm (P/N 186007261) Separation

Device. Trapping-enabled quantitative analysis of 092914_2pt1 Acquity_ Morphine_Calibration Curve_3 7: MRM of 2 Channels ES+ S/N:PtP=8.58 100 TIC (Morphine) 5.96e4 25 early eluting opioids and metabolites in oral 1.16 1.17 fluid is illustrated in Figure 2. 1.15 100 pg/mL % 1.15 1.17 Figure 3 shows the calibration curve and LOQ of 0.79 0.78 1.15 0.80 0.91 1.11 0.85 0.89 0.97 1.01 1.24 1.24 1.40 morphine spiked into oral fluid. The LOQ (based on 1.04 1.20 1.31 1.39 1.40 1.50 1.71 0 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 a signal to noise ratio of 10:1) was calculated to be 0929142t1A it MhiClibtiC 4 7MRM f2Ch lES 13 pg/mL. The peak width of morphine, at 10% peak height, was 3.0 seconds and is shown in the insert ionKey/MS in Figure 2. Figure 4 compares morphine detected 092914_Trapping_ Morphine_Calibration Curve_7 7: MRM of 2 Channels ES+ S/N:PtP=87.67 100 TIC (Morphine) using an ionKey/MS System to an ACQUITY UPLC 4.22e5 2.1 mm I.D. Column. The ionKey/MS System shows ~10X greater S/N 100 pg/mL an improvement of 9X over the analogous 2.1 mm % column format. The trapping column and separation

0 device used in these studies were specifically chosen 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 092914 TiMhiClibtiC 8 7MRM f2Ch lES for their increased retentivity for hydrophilic opioids and metabolites. These comparisons were performed Figure 4. Comparison of morphine detection using an ionKey/MS System to an with equivalent injection volumes of 5 μL. ACQUITY UPLC 2.1 mm I.D. Column.

SUMMARY Specifically, morphine-3b glucuronide was demonstrated to be baseline separated from morphine, with a sensitivity improvement of 11X. Whilst this The utilization of the ionKey/MS System enabled particular glucuronide may not be relevant for oral fluid, the increased sensitivity a 9X improvement in sensitivity for morphine presents a clear advantage for analysis in other biological specimens. compared to an ACQUITY UPLC 2.1 mm I.D. Column when injecting the same volume. In addition The assay described here is robust and reproducible using oral fluid matrix to morphine, many of the glucuronides were injections of over 500 injections. Retention time reproducibilities were also identified as having improved sensitivity. <1% RSD, while peak area reproducibility of morphine was 11.1% RSD.

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