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Drug of Abuse Analysis Using Agilent Chem Elut S Supported Liquid

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Drug of Abuse Analysis Using Agilent Chem Elut S Supported Liquid Application Note Forensics Drug of Abuse Analysis in Human Urine Using Agilent Chem Elut S Supported Liquid Extraction by LC/MS/MS Author Abstract Derick Lucas Agilent Technologies, Inc. Agilent Chem Elut S is a new sample preparation product that uses synthetic sorbent for supported liquid extraction (SLE). This Application Note demonstrates the use of Chem Elut S 96-well plates (400 mg) for the quantitative analysis of 24 drugs of abuse (DOAs) in human urine by LC/MS/MS. Urine samples were enzyme- digested with β-glucuronidase then loaded into Chem Elut S 400 mg 96-well plates. Aqueous sample was absorbed onto the media and equilibrated for five minutes, then eluted with methyl tert-butyl ether (MTBE) to give a clean extract containing target analytes. The method was verified for accuracy and precision, and delivered excellent recovery (100 ±20%) and precision (%RSD <20%) for all but one compound. Significant advantages are demonstrated against traditional liquid-liquid extraction (LLE) and diatomaceous earth SLE methods. Results indicate that Chem Elut S, based on synthetic SLE sorbent, provides fast, simple, and consistent sample preparation for this application. Introduction eluting them into a collection tube for Experimental post-treatment and analysis. Compared In forensic toxicology, fast and reliable to traditional LLE, the interaction surface All reagents and solvents were HPLC DOAs quantitation in biological between aqueous and organic phases or analytical grade. Methanol (MeOH) specimens is of great interest in forensic using SLE is significantly increased, and acetonitrile (ACN) was from testing.1–3 This is primarily due to the which improves analyte partition from Honeywell (Muskegon, MI, USA), and increasing number of DOAs and samples the aqueous to organic phase and methyl tert-butyl ether (MTBE) was from submitted for analysis. The DOAs urine obsoletes the mixing step. This provides VWR-BDH Chemicals (Radnor, PA, USA). test is one of the most common forensic significant time and labor savings over A concentrated solution of hydrochloric tests for screening and quantitation of conventional LLE with simplified and acid (HCl, 38%) was obtained from DOAs in the human body. Urine matrix improved reproducibility. VWR. A combined DOA standard stock is not as complex as other biological Additionally, DE is a natural material, solution was from Agilent Technologies matrices, such as plasma and serum. consisting of irregular fossilized (part number 5190-0470), and However, it contains a large amount microorganisms, and it is difficult to internal standard (IS) stock solutions of various salts, which can interfere control the sorbent batch-to-batch (1 mg/mL) were from Cerilliant with the test’s reliability and instrument particle consistency. The sorbent (Round Rock, TX, USA). β-Glucuronidase usability. Therefore, appropriate variability complicates product enzyme was purchased as a solution sample preparation is necessary for manufacturing and quality control, (100,000 units/mL) from Sigma-Aldrich matrix cleanup and DOAs extraction. and leads to product performance (St. Louis, MO, USA). Human urine (Mass Additionally, DOA analytes are often inconsistency. DE can give lower and Spect Gold) was purchased from Golden glycosylated and require enzymatic inconsistent water-holding capacity West Biologicals, Inc. (Temecula, CA, digestion with β-glucuronidase prior to when compared to synthetic media. The USA). Reagent solutions were prepared sample preparation to release the free Chem Elut S sorbent greatly improves fresh for every batch to avoid variability. drugs for the following extraction and water-holding capacity, batch-to- 4 Standards and solutions analysis. batch consistency, and performance Combined standard and IS intermediate Common techniques for preparing consistency. The 96-well plate design spiking solutions were prepared at samples for DOA analysis include LLE, offers large headspace for samples and 20 µg/mL in MeOH with the dilution of solid phase extraction (SPE), and SLE. eluent, a square upper frit that holds appropriate volume of individual stock LLE can be particularly labor-intensive, sample until pressure/vacuum is applied, solutions. All spiking solutions are stored requiring extensive sample mixing, a full skirt for hardware compatibility, at –20 °C until use. manual phase separation, and and fast, consistent elution. occasionally centrifugation to break This study uses Chem Elut S 2 mL Equipment and supplies up emulsions. Furthermore, LLE is 96-well plates (400 µL) to quantitatively • Agilent Chem Elut S 400 µL difficult and cumbersome to automate, determine 24 DOAs in human urine. (p/n 5610-2004) in contrast to SLE. In traditional SLE, Experimental comparisons were also • Agilent positive pressure manifold, an aqueous sample is loaded onto the evaluated between the Chem Elut S, LLE, PPM-96 (p/n 5191-4116) sorbent bed containing diatomaceous and DE-based SLE based on recovery, • Agilent square 96-well 2 mL earth (DE), where the aqueous sample reproducibility, and ease-of-use. is coated as a thin film on the material. collection plate (p/n 5133009) A water-immiscible solvent is then • Agilent square 96-well sealing caps passed through the SLE bed, extracting (p/n 5133005) target analytes from the sample and • SPE 96 evaporator • Multitube Vortexer (VWR, PA, USA) • Eppendorf pipettes and repeater • ViaFlo 96 liquid handler (Integra, Hudson, NH, USA) 2 Instrument conditions LC Parameters • Agilent 1290 Infinity quaternary Analytical Column Agilent Poroshell 120 EC-C8, 2.7 µm, 2.1 × 100 mm (695775-906T) Agilent Poroshell 120 EC-C18, 2.1 × 5 mm, 2.7 µm, guard column (821725-911) pump (G4204A) Column Temperature 50 °C • Agilent 1290 Infinity autosampler Injection Volume 2 µL (G4226A) Mobile Phase A 5 mM ammonium formate + 0.1% formic acid in water • Agilent 1290 Infinity thermostatted Mobile Phase B 0.1% formic acid in ACN column compartment (G1316C) Flow Rate 0.5 mL/min • Agilent 1290 Infinity thermostat Hold at 5% B to 0.5 minutes, Gradient Ramp to 50% B at 5 minutes, (G1330B) Ramp to 95% B at 6 minutes, Hold at 95% B to 7 minutes Table 1 shows the analyte acquisition Post Time 2 minutes parameters, and Figure 1 shows the LC/MS/MS chromatogram of 1 ng/mL MS/MS configuration and parameters DOA in urine. See Table 1 for peak identification following the elution order. Agilent 6490 Triple Quadrupole LC/MS with iFunnel (G6490A) MS/MS Mode Dynamic MRM Ion mode Positive Drying Gas Temperature 250 °C Drying Gas Flow 5 L/min Nebulizer Pressure 45 psi Sheath Gas Temperature 325 °C Sheath Gas Flow 11 L/min Capillary Voltage 3,000 V EMV 200 V Nozzle Voltage 1,500 V iFunnel Parameters High-pressure RF: 90(Pos), 90(Neg) Low-pressure RF: 70(Pos), 60(Neg) 3 Table 1. Target drugs, retention time, and MRM parameters. Product Ion (m/z) Internal Retention Precursor Quantifier Qualifier Analyte Standard Time (min) Ion (m/z) Ion CE (V) Ion CE (V) Codeine IS 1 2.35 300.2 128.1 60 165.1 40 Oxycodone IS 1 2.68 316.2 241.1 28 256.1 24 Amphetamine IS 1 2.64 136.1 93.0 13 124.1 5 Amphetamine-d3 (IS 1) 2.64 141.1 91.1 20 119.1 10 MDA IS 1 2.72 180.1 163.1 4 105.1 24 Hydrocodone IS 1 2.84 300.2 128.1 60 199.1 28 Methamphetamine IS 1 2.87 150.1 91.1 20 119.1 8 MDMA IS 1 2.91 194.1 163.1 8 105.1 24 Strychnine IS 1 3.10 335.2 184.1 40 156.1 40 Phentermine IS 1 3.10 150.1 133.1 8 133.1 8 MDEA IS 1 3.19 208.1 163.1 8 105.1 48 Heroin IS 2 3.81 370.2 328.2 20 165.1 24 Cocaine IS 2 3.94 304.2 182.1 16 82.0 40 Cocaine-d3 (IS 2) 3.94 307.2 185.1 30 82.0 48 Meperidine IS 2 4.03 248.2 220.1 20 174.1 48 Trazodone IS 2 4.35 372.2 176.1 24 148.1 16 PCP IS 2 4.51 244.2 159.1 8 159.1 8 Nitrazepam IS 2 5.20 282.1 180.1 40 236.1 24 Oxazepam IS 2 5.27 287.1 241.1 20 104.1 40 Verapamil IS 2 5.44 455.3 165.1 28 150.1 48 Lorazepam IS 3 5.41 321.0 229.1 32 275.0 20 Alprazolam IS 3 5.51 309.1 205.1 48 281.1 40 Methadone IS 3 5.54 310.2 265.2 12 105.0 28 Temazepam IS 3 5.73 301.1 177.0 44 255.1 16 Proadifen IS 3 6.24 354.2 167.1 40 209.1 20 Diazepam IS 3 6.18 285.1 193.1 32 154.1 24 Diazepam-d3 (IS 3) 6.18 290.1 198.1 32 154.1 24 ×104 2.2 2.0 1.8 1.6 1.4 1.2 Counts 1.0 0.8 0.6 0.4 0.2 0 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 Acquisition time (min) Figure 1. LC/MS/MS MRM chromatogram overlay of 24 drugs at 1 ng/mL in urine after Agilent Chem Elut S treatment. 4 Calibration standards and QC sample preparation The DOA standard spiking solution (1 µg/mL) was used to prepare Aqueous 1: Load 3: Add solvent 4: Elute calibration curve standards in human sample Gravity, urine.
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