(Peth) in Whole Blood Using Luna® Omega Polar C18 Column

TN-1222 APPLICATIONS Xianrong (Jenny) Wei Senior Scientist Jenny is a Senior Scientist in the Phenomenex PhenoLogix Simple and Robust LC-MS/MS Method of applications laboratory. Phosphatidylethanol (PEth) in Whole Blood Using Luna® Omega Polar C18 Column

Xianrong (Jenny) Wei, Daniel Spurgin, and Sean Orlowicz Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Introduction LC Conditions Phosphatidylethanols (PEth) are types of phospholipids that form Analytical Column: Luna Omega 1.6 µm Polar C18 in the presence of ethanol. Unlike other metabolites of ethanol Dimension: 30 x 2.1 mm such as fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG) and ethyl sulfate (EtS), the average half-life for PEth in circulation is Part No.: 00A-4748-AN around 4 days, which means that PEth may be detected weeks Recommended Guard: SecurityGuard™ ULTRA after ethanol has been consumed1,2. PEth is a phospholipid Part No: AJ0-9505 containing two fatty acids linked to a phosphoethanol. PEth Mobile Phase: A: Water / IPA / Acetonitrile with 5 mM Ammonium formate (30:10:60) 16:0/18:1 is the most abundant form and contains one palmitic B: Water / IPA / Acetonitrile with 5 mM Ammonium formate (1:79:20) acid and one oleic acid chain. It is one of nine PEth-homologues Gradient: Time (min) % B and is the focus of this study. In this study, we develop a LC- 0 10 0.3 10 MS/ MS research assay including a simple extraction procedure 0.31 40 with an internal standard using a reverse phase sub-2 μm Luna 2 100 Omega Polar C18 column for the separation. The assay has been 2.5 100 2.51 10 evaluated and meet ±15 % acceptance criteria. 3.5 10 Flow Rate: 0.45 mL/min Analyte Structures Figure 1. Temperature: 60 °C Phosphatidylethanol (PEth) 16:0/18:1 O O Injection Volume: 10 µL Instrument: Agilent® 1260 P O O O CH3 Detection MS/MS (SCIEX Triple Quad™ 4500), 4500, ESI, Neg Polarity (700 °C) O H O- + Na Mass Transitions O ID Q1 Mass (Da) Q3 Mass (Da) Dwell (msec) PEth 1 701.5 280.8 100 d5-PEth 16:0/18:1 trimethylammonium salt PEth 2* 701.5 254.9 100 O O PEth d5 1* 706.5 280.8 100

P D D PEth d5 2 706.5 254.8 100 O O O D *Quantitation mass O H O- D D H+ H C CH O 3 N 3 Results and Discussion CH3 To determine the appropriate LLOQ and linearity of the assay, we evaluated six individual lots of human blood to measure the average of endogenous level of PEth (Table 1). Lots from female Materials and/or under age 21 years old of human blood showed cleaner Analytical reference standard of Phosphatidylethanol 16:0/18:1 background of PEth. Figure 3 shows the endogenous levels of (PEth 16:0/18:1) was purchased from Avanti® Polar Lipids, Inc. two lots of blood. Red trace is from a 46 year old male and the (Alabaster, Alabama, USA); Internal standard of PEth 16:0/18:1 – blue trace is blank matrix from a 19 year old female. Lot 3 (blue) d5 was obtained from redhot diagnostics AB(Södertälje, Sweden) shows little to know endogenous levels and therefore can be used and human blood was purchased from BioreclamationIVT® for the calibration standard and quality control sample preparation (Chastertown, MD, USA) respectively. All other chemicals were to ensure the assay accuracy, precision and linearity. Figure 2, obtained from the Sigma-Aldrich® Company (St. Louis, MO). Water showed the blank extraction solvent response, no interference or purification via Sartorius Arium® Comfort II (Goettinger, Germany) analyte presented. Sample Extraction Procedure Phosphatidylethanol (PEth) is extracted from 100 μL of whole blood by addition of 400 μL of isopropyl alcohol (IPA) containing internal standard (10 μg/mL). After thorough mixing, the mixture is centrifuged at 13,500 rpm for 10 minutes, then 200 μL aliquot of the supernatant transferred to a vial.

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Results and Discussion (cont’d) Figure 3. Representative chromatograms of endogenous level of PEth in The assay was run on a Phenomenex Luna® Omega 1.6 µm blank human whole blood. Polar C18, 30 x 2.1 mm column with specified mobile phase and 5.2e5 5.0e5 Column Temp: Room temperature temperature which effectively ionizes the analytes while providing 4.5e5 Blue: Blank Matrix lot 3 sufficient selectivity, separation and sensitivity (Figure 4 and 5). Red: Blank Matrix lot 4 The assay carryover was also evaluated and there is no significate 4.0e5 carryover (Figure 6). Challenging matrices such as whole blood 3.5e5 sample can negatively impact column lifetime due to clogging, 3.0e5 PEth 16:0/18:1 Endogenous Level fouling, and creating robustness issues. For this reason an 2.5e5

evaluation test of column lifetime was performed with the results cps Intensity, 2.0e5 showing very good reproducibility after at least 500 injections (Figure 7). 1.5e5 1.0e5 The assay provides the dynamic range at 20–2000 ng/mL with the 5.0e4 linearity (R2) volume is greater than 0.99 (Figure 8). The accuracy 0.0 APP ID 24297 and precision were tested at three QCs levels at n=6, the low 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min QC accuracy presented at 101% with CV% at 11.97; mid QC at 108.2 % with CV% at 5.11 and High QC at 92.8 % with CV% 7.61, respectively (Table 1). The internal standard responses were Figure 4. Representative chromatogram of LLOQ at 20 ng/mL in human also performed. The assay showed the consistent response of the whole blood at column temp 60 °C. internal standard, which was within ±25 % of mean of standards 1.5e6 Column Temp: 60 ˚C and QCs in the run (Figure 9). 1.4e6 1.3e6 1.2e6 Table 1. Accuracy and Precision for 6 lots of blood 1.1e6 1.0e6 Analyte Low QC Mid QC High QC 9.0e5 & IS ame (60 ng/mL) (540 ng/mL) (1800 ng/mL) 8.0e5 7.0e5 Lot 1 62.2 560 1750 Intensity, cps Intensity, 6.0e5 Interference peaks Lot 2 62.9 558 1500 5.0e5 4.0e5 Lot 3 46.8 591 1520 3.0e5 PEth 16:0/18:1 Lot 4 60.8 560 1700 2.0e5 1.0e5 Lot 5 61.7 627 1770 0.0 APP ID 24298 Lot 6 68.5 611 1780 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min Mean 60.5 585 1670 S.D. 7.24 29.8 127.1 Figure 5. Representative chromatogram of ULOQ at 2000 ng/mL in human %CV 11.97 5.11 7.61 whole blood. %Theoretical 101 108.2 92.8 3.4e6 3.2e6 PEth 16:0/18:1 N 6 6 6 3.0e6 2.8e6 2.6e6 2.4e6 Figure 2. Representative chromatogram of blank extraction solvent (water/ 2.2e6 IPA 20:80) 2.0e6 1.8e6 290 1.6e6 280 1.4e6 Intensity, cps Intensity, 260 1.2e6 240 1.0e6 8.0e5 220 6.0e5 200 4.0e5 180 2.0e5

160 0.0 APP ID 24299 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min 140

Intensity, cps Intensity, 120 100 80 60 40 20

0 APP ID 24296 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min

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Figure 6. Representative chromatograms of carryover Figure 8. Representative calibration curve of PEth in human whole blood 3.4e6 Curve range: 3.2e6 STD 7 (2000 ng/mL) 55 3.0e6 20-2000 ng/mL 2.8e6 sample 19 50 2.6e6 45 2.4e6 40 2.2e6 2.0e6 35 1.8e6 30 1.6e6 25

Intensity, cps Intensity, 1.4e6 1.2e6 20 Analyte Area / IS Area 1.0e6 15 8.0e5 6.0e5 10 4.0e5 5 2.0e5 0 0.0 APP ID 24300 APP ID 24303 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min 200 400 600 800 1000 1200 1400 1600 1800 2000 Analyte Conc. / IS Conc. 1.3e6 1.2e6 1.1e6 Figure 9. Representative response of isotope internal standard of PEth 1.0e6 16:0/18:1-d5 9.0e5 2.2e5 8.0e5 2.0e5 1.9e5 7.0e5 1.8e5 1.7e5 6.0e5 1.6e5

Intensity, cps Intensity, 5.0e5 1.5e5 1.4e5 4.0e5 1.3e5 3.0e5 Matrix Blank 1.2e5 1.1e5 2.0e5 sample 20 1.0e5 9.0e4 1.0e5 8.0e4

IS Peak Area, counts Area, IS Peak 7.0e4 0.0 APP ID 24301 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 Figure 7. Representative chromatograms of reproducibility 1.0e4

0 APP ID 24304 9.9e5 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 9.5e5 STD 3: 100 ng/mL 9.0e5 Index 8.5e5 Blue: injection 14 8.0e5 Red: Injection 401 7.5e5 Conclusion 7.0e5 6.5e5 A simple and robust LC-MS/MS method is presented for the 6.0e5 quantitation of PEth 16:0/18:1 in human whole blood using a 5.5e5 ® 5.0e5 Phenomenex Luna Omega 1.6 µm Polar C18 UHPLC column. 4.5e5 The assay provides selectivity, separation of matrix interference

Intensity, cps Intensity, 4.0e5 3.5e5 peaks, and great reproducibility. This assay is accurate, time 3.0e5 PEth 16:0/18:1 2.5e5 saving, and automation friendly. 2.0e5 1.5e5 References 1.0e5 5.0e4 1. Steina Aradottir, Gulber Asanovska, Stefan Gjerss, Per Hasson and Christer

0.0 APP ID 24302 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 min Alling. Phosphatidylethanol (PEth) Concentrations in Blood Are Correlated To Reported Alcohol Intake in Alcohol- Dependent Patients. Alcohol & alcoholism Vol. 41, No. 4, pp.431-437, 2006 2. Guido Viel, Rafael Boscolo-Berto, Giovanni Cecchetto, Paolo Fais, Alessandro Nalesso and Santo Davide Ferrara. Phosphatidylethanol in Blood as a Marker of Chronic Alcohol Use: A Systematic Review and Meta- Analysis. Int J Mol Sci. 2012; 13(11): 14788-14812 3. Blomgren Anders, Hansson Therese, Isaksson Anders, Walther Lisa, Region Skane, Medical services. Fast and Robust LC-MS/MS Method for Determination of the alcohol Biomarker phosphatidylethanol (PEth) in whole blood using an Automated Extraction Procedure. Poster, 2013

If Phenomenex products in this technical note do not provide at least equivalent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 45 days for a FULL REFUND.

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Luna® Omega Polar C18 Ordering Information SecurityGuard™ 1.6 μm Minibore Columns (mm) ULTRA Cartridges‡ Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk Polar C18 00A-4748-AN 00B-4748-AN 00D-4748-AN 00F-4748-AN AJ0-9505

SecurityGuard 5 μm Minibore Columns (mm) Cartridges (mm) Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 4 x 2.0* 10/pk Polar C18 00A-4754-AN 00B-4754-AN 00D-4754-AN 00F-4754-AN AJ0-7600 for ID: 2.0 - 3.0 mm SecurityGuard 5 μm MidBore™ Columns (mm) Cartridges (mm) Phases 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0* 10/pk Polar C18 00B-4754-Y0 00D-4754-Y0 00F-4754-Y0 AJ0-7600 for ID: 2.0 - 3.0 mm SecurityGuard 5 μm Analytical Columns (mm) Cartridges (mm) Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 2.0* 10/pk Polar C18 00B-4754-E0 00D-4754-E0 00F-4754-E0 00G-4754-E0 AJ0-7601 for ID: 3.1-8.0 mm

‡ SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000 * SecurityGuard Analytical Cartridges require holder, Part No.: KJ0-4282

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions. Trademarks Luna and Strata are a registered trademark, and SecurityGuard trademarks of Phenomenex. Avanti is a registered trademark of Avanti Polar Lipids, Inc. BioreclamationIVT is a registered trademark of Bioreclamation-IVT Holdings, LLC. Sigma-Aldrich is a registered trademark of SIGMA-ALDRICH CO., LLC. Sartorius is a registered trademark of Sortorius AG. Agilent is a registered trademark of Agilent Technologies. Triple Quad is a trademark of AB SCIEX Pte. Ltd. SCIEX™ is being used under license. Disclaimer For RESEARCH USE ONLY. Not for use in diagnostic procedures. © 2017 Phenomenex, Inc. All rights reserved.

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