Streamlined sample preparation of DoA panel in human nails using ISOLUTE® SLE+

Figure 1. Example structures by class.

Introduction

The testing of alternative matrices in forensic and/or clinical toxicology is gaining popularity, partly due to less invasive means of collection. Matrices such as hair or nail can provide a more rounded picture of abstinence or abuse and associated timeframes. This application note describes the sample pre-treatment and subsequent extraction of 49 drugs of abuse from human nails, prior to LC/MS analysis.

The method utalizes Biotage® Lysera for matrix micropulverization, prior to direct transfer to clean up using ISOLUTE® SLE+ supported liquid extraction products. Elimination of an evapo- ration step between the micropulverization and supported liquid extraction clean up stages provides a streamlined procedure for nail extraction.

Manual processing protocols were developed using the Biotage® PRESSURE+ 96 (plate format) or 48 (column format) Positive Pressure Manifolds. For automated processing, protocols were developed using Biotage® Extrahera^TM.

This application note contains procedures optimized for both individual column format and 96-well plate format for higher throughput applications. The methodology delivers clean extracts and analyte recoveries mostly greater than 80% with RSDs lower than 10% for all analytes and LLOQ from 1 pg/mg.

Both manual and automated procedures gave comparable results.

ISOLUTE® SLE+ Supported Liquid Extraction plates and cartridges offer an efficient alternative to traditional liquid- liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced preparation time.

Analytes

Amphetamine, Methamphetamine, 3,4-Methylenedioxyamphetamine (MDA), 3,4-Methylenedioxymethamphetamine (MDMA), 3,4-Methylenedioxy-N-ethylamphetamine (MDEA), Hydromorphone, Morphine, Benzoylecgonine (BZE), Oxymorphone, Dihydrocodeine, Oxycodone, Mephedrone, Norfentanyl, 7-amino-flunitrazepam, 7-amino-clonazepam, Hydrocodone, Codeine, 6-Monoacetylmorphine (6-MAM), Cocaine, Norketamine, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), Zaleplon, Zopiclone, Norbuprenorphine, Ketamine, Nitrazepam, Flunitrazepam, Clonazepam, α-OH-triazolam, Oxazepam, Estazolam, Temazepam, Zolpidem, Alprazolam, Methadone, Lorazepam, Bromazepam, α-OH-alprazolam, 2-OH-ethyl-flurazepam, Triazolam, Nordiazepam, Diazepam, Midazolam, Fentanyl, Flurazepam, Buprenorphine, Phencyclidine (PCP), Lysergic acid diethylamide (LSD)

Internal standards

Amphetamine-D₅, Morphine-D₃, Benzoylecgonine-D₃ (BZE-D3), 6-Monoacetylmorphine-D₃ (6-MAM-D3), Diazepam-D₅

Sample preparation procedure

Format

ISOLUTE® SLE+ 400 µL capacity cartridges. (p/n 820-0055-B) or ISOLUTE® SLE+ 400 µL capacity plates (p/n 820-0400-P01)

Matrix preparation

Weigh 10 mg of freshly clipped nails into 2 mL Biotage® Lysera tubes (p/n 19-620) containing 5 x 2.4 mm stainless steel beads (p/n 19-640).

Micropulverization procedure

Grind to a fine powder using Biotage® Lysera: 8 x 6.95 m/sec for 45 seconds with a 45s dwell.

Add 1 mL methanolic 0.1% (v/v) NH4OH to each nail sample after micropulverization. Also add 10 µL of a 100 pg/mL ISTD solution giving a 100 pg/mg spike.Mix.

Centrifuge tubes for 10 minutes at 13,300 rpm (Heraeus Pico 17 Microcentrifuge (Thermo Scientific) with 24 position, 2 mL rotor).

Post micropulverisation

Transfer an aliquot of supernatant directly to the appropriate ISOLUTE SLE+ product for clean up as described below.

Supported liquid extraction conditions

UHPLC conditions

Instrument

Shimadzu Nexera X2 UHPLC

Column

Restek Raptor™ Biphenyl 2.7 µm (100 x 2.1 mm) with a Restek EXP holder and Biphenyl guard column

Mobile phase

A: 2 mM Ammonium formate (aq) with 0.1% formic acid
B: 2 mM Ammonium formate in methanol with 0.1% formic acid

Flow rate

0.4 mL/min

Injection volume

5 µL

Column temperature

30 °C

Table 1. UHPLC gradient.

Mass spectrometry conditions

Instrument

Shimadzu 8060 Triple Quadrupole MS using ES interface

Nebulizing gas flow

3 L/min

Drying gas flow

3 L/min

Heating gas flow

17 L/min

Interface temperature

400 ^oC

DL temperature

250 ^oC

Heat block temperature

300 ^oC

CID gas flow

270 kPa

Table 2. MS conditions for target analytes in positive mode.

Results

This simple sample preparation method delivers clean extracts and analyte recoveries mostly greater than 80% with RSDs lower than 10% for all analytes (see fig 2), and LLOQs from 1pg/mL (see table 3) for all ISOLUTE® SLE+ formats used.

Figure 2. Representative analyte recoveries using the optimized ISOLUTE® SLE+ protocol for the 400 µL capacity cartridge format (p/n 820-0055-B) with manual or automated processing. Similar results were achieved using the 400 µL capacity plate formats.

Figure 3. Representative chromatography for application analytes spiked at 1 ng/mL.

Linearity was investigated for human nails spiked between 1–1000 pg/mg. Good linearity was observed for all analytes delivering r² values greater than 0.99. Table 3. details linearity performance and associated LOQ for each analyte, using the 400 µL capacity column format, p/n 820-0055-B. Similar results were obtained from both columns and plate formats, with either manual or automated processing.

Table 3. Analyte calibration curve r² and LOQ performance.

Figure 4. Calibration curves for Buprenorphine (a), 6-MAM (b), BZE (c) and Methamphetamine (d) extracted from human nails using the 400 µL capacity column format loading 400 µL of extract (manual processing). Similar results were achieved for the 400 µL capacity plate formats, and for automated processing procedures.

Chemicals and reagents

• Methanol (LC-MS grade), Ultra-Pure Methanol (Gradient MS), dichloromethane (99.8%), isopropanol (99.9%), MTBE (99%) and formic acid (98%) were purchased from Honeywell Research Chemicals (Bucharest, Romania).
• All analyte standards and deuterated internal standards, hydrochloric acid (37%) and ammonium formate (LC-MS grade) were purchased from Sigma- Aldrich Company Ltd. (Gillingham, UK).
• Ammonium hydroxide (28–30%) was purchased from Merck.
• Water used was 18.2 MOhm-cm, drawn daily from a Direct-Q5 water purifier.
• 0.1% NH4OH was prepared by adding 100 µL of ammonium hydroxide to 99.9 mL of methanol
• 50mM HCl in MeOH was prepared by adding 50 µL of hydrochloric acid to l to 12 mL of methanol.
• DCM: IPA (95:5, v/v) was prepared by adding 5 mL of isopropanol to 95 mL of DCM and mixing.
• Mobile phase A (2 mM ammonium formate (aq), 0.1 % formic acid) was prepared by adding 126 mg of ammonium formate to 500 mL of purified water, adding 1 mL of concentrated formic acid and making up to 1 L with purified water.
• Mobile phase B (2 mM ammonium formate (methanol), 0.1 % formic acid) was prepared by adding 126 mg of ammonium formate to 500 mL of HPLC grade methanol, adding 1 mL of concentrated formic acid and making up to 1 L with HPLC grade methanol.
• Internal standards( 100 pg/µL) were prepared from a 10 ng/µL stock solution by adding 10 µL of each of to 950 µL of MeOH. 10 µL of this solution was then added to each calibration.

Additional information

• All data shown in this application note was generated using freshly clipped nails provided by healthy human volunteers.
• Biotage®Lysera hints and tips.
  
  • A minimum of four tubes must be loaded in the tube carriage to ensure balance during processing.
  • Ensure vial caps are firmly tightened and Lysera locking mechanism is fully engaged.
  • To minimize sample transfer and manipulation steps, 2 mL Lysera tubes were placed directly into the centrifuge (Heraeus Pico 17 Microcentrifuge (Thermo Scientific) with 24 position, 2 mL rotor).

Ordering information

Appendix: Biotage®  Extrahera™  settings

The method described in this application note was automated on the Biotage® Extrahera™ using ISOLUTE® SLE+ 400 µL capacity cartridges and 96-well plates. This appendix contains the software settings required to configure Extrahera to run the column format method. As described in the main body of the application note, analyte recoveries, %RSDs, linearities and LOQs were comparable for both manually processed and automated methods, for both extraction formats.

Solvent Properties

Literature Number: AN916