Automated extraction of DoA panel from urine using ISOLUTE® HCX with the Biotage® Extrahera™ HV-5000

Figure 1. Selected analyte structures representing the analyte classes extracted.

Introduction

This application note describes the automated extraction of multiple drugs of abuse from human urine using ISOLUTE® HCX mixed-mode solid phase extraction cartridges prior to LC-MS/MS analysis. The extraction methodology is suitable for raw or hydrolyzed urine samples. Hydrolysis protocols are provided.

The Biotage® Extrahera® HV-5000 automated sample preparation system allows rapid and efficient cartridge format extractions to a high level of accuracy and precision.
The simple sample preparation procedure, based on a mixed- mode (non-polar/strong cation exchange) extraction mechanism, delivers clean extracts and analyte recoveries mostly greater than 80% with RSDs lower than 5% for most analytes. Linearity of greater than 0.99 is achieved for most analytes from 0.25–250 pg/mL without the use of internal standards.

Sample preparation procedure

Format:

ISOLUTE® HCX 130 mg/3 mL, p/n 902-0013-B

Automated processing:

Automated sample processing was performed using the Biotage® Extrahera™ HV-5000 system. Detailed processing conditions are included in the appendix.

Sample pre-treatment:

Non-hydrolyzed urine

Dilute 1.5 mL urine with an equal volume of phosphate buffer (100 mM, pH 6).

Hydrolyzed urine

Spike sample with internal standard and/or controls as necessary. Dilute urine (1.5 mL) with ammonium acetate buffer (100 mM, pH 5, 1.425 mL) and β-glucuronidase (0.075 mL). Mix and incubate at 60 °C for 2 hours.
After pre-treatment, transfer to 16 x 75 mm tubes for Extrahera HV-5000 processing.

Condition:

Add methanol (2 mL) to each cartridge and push through under positive pressure.

Equilibration:

Add phosphate buffer (pH 6, 0.1 M, 2 mL) to each cartridge and push through under positive pressure.

Sample loading:

Load 2 mL of the pre-treated urine samples onto each SPE cartridge using positive pressure.

Wash 1:

Elute interferences with acetic acid (1 M, 2mL) using positive pressure.

Wash 2:

Elute interferences with methanol (1 mL) using positive pressure.

Elution:

Elute analytes with DCM:MeOH:NH4OH (78:20:2, v/v, 2 x 3 mL)*. Collect the elution solvent into 12 x 75 mm glass tubes containing 100 µL of 50 mM HCl in methanol.

*Note: this volume is used ensure high recoveries of the large, diverse analyte panel. For reduced panels, a smaller volume may be appropriate.

Post elution and reconstitution:

Dry the extract in a stream of air or nitrogen using a TurboVap® LV at 40 °C, 1.5 L/min.
Reconstitute evaporated samples with H2O:MeOH (90/10, v/v) containing 0.1% formic acid (30 µL). Vortex mix, transfer to a 96 well collection plate and cap.

LC conditions

Instrument:

Shimadzu Nexera UHPLC

Cartridge:

Restek Raptor™ Biphenyl 2.7 µm (100 x 2.1 mm)

Mobile phase:

A: 2 mM ammonium formate (aq) containing 0.1% formic acid
B: 2 mM ammonium formate (MeOH) containing 0.1% formic acid

Flow rate:

0.4 mL/min

Injection volume:

10 µL

Cartridge temperature:

30 °C

MS 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 Temp:

400 °C

DL Temp:

250 °C

Heat Block Temp:

300 °C

CID Gas Flow:

270 kPa

Results

Recovery and Reproducibility

High (mostly > 80%) and reproducible (RSD mostly < 5%) recoveries were achieved using the method described in this application note using the Biotage® Extrahera™ HV-5000.

Figure 2. Extraction recoveries and precision for target analytes in non-hydrolysed urine.

Figure 3. Extraction recoveries and precision for target analytes in hydrolysed urine.

Figure 4. Representative chromatography with analytes spiked at 2 ng/mL

Linearity

Calibration curve performance was investigated for analytes spiked into urine (before hydrolysis) at concentrations of 0.025-25 ng/mL. Good linearity was observed for all analytes typically delivering r2 values greater than 0.99 without the use of an internal standard. Tables 4 and 5 detail linearity performance and associated LLOQ for each analyte extracted from non-hydrolyzed and hydrolyzed urine matrix respectively. In some instances saturation was observed at the top of the calibration line resulting in the calibration range being reduced. This can be corrected by increasing the reconstitution volume above 0.5 mL or reducing the injection volume below 10 µL. Alternatively method sensitivity may be improved by reducing the reconstitution volume and increasing the injection volume.

Improved linearity is likely to be obtained by using structurally similar internal standards.
Reduced LLOQ may be achieved, as this is dependant on the make and model of the LC-MS/MS system being used. In this case, using the Shimadzu LCMS-8060, and the conditions shown, the LLOQ was estimated by extrapolating analyte only calibration lines down to a level where the signal to noise was estimated to be 10:1. No calibration lines covered a concentration range below the LLOQ

Chemicals and reagents

•  Methanol (LC-MS grade), propan-2-ol (HPLC grade) and dichloromethane (DCM, HPLC grade) were purchased from Rathburn Chemicals (Walkerburn, Scotland, UK).
•  All analyte standards, ammonium acetate, ammonium formate, acetic acid, formic acid and ammonium hydroxide (27–30%) were purchased from Sigma- Aldrich Company Ltd. (Gillingham, UK).
• Beta glucuronidase was purchased from Sigma Aldrich (β glucuronidase from Helix Pomata, G7017-10 mL)
•  Water used for all solutions was 18.2 MOhm-cm, drawn daily from a Direct-Q5 water purifier.
•  Phosphate buffer 0.1 M pH 6 was prepared by diluting 1.15 g K2HPO4 and 11.81g KH2PO4 in 1 litre of water.
• Wash 1 solvent (1M Acetic acid) was made by adding 28.74 mL of glacial acetic acid to 400 mL of water and then making up to 500 mL with water
• Elution solvent (DCM : IPA : ammonium hydroxide (78:20:2, v/v)) was made up by measuring out 78 mL of DCM and 20 mL of IPA and adding both to a bottle with 2 mL concentrated ammonium hydroxide solution.
• Reconstitution solvent was made by measuring out 90 mL of purified water and 10 mL of MeOH and adding them to the same bottle with 100 µL formic acid.
•  Mobile phase A (2 mM ammonium formate (aq), 0.1% formic acid) was prepared by adding 0.126 mg of ammonium formate to 1 L purified water with 1 mL formic acid.
• Mobile phase B (2 mM ammonium formate (aq), 0.1% formic acid) was prepared by adding 0.126 mg of ammonium formate to 1 L ultra-pure MeOH with 1 mL formic acid.
  
  

Additional information

• All data shown in this application note was generated using human urine donated by anonymous healthy volunteers.
•  The Biotage® Extrahera^TM method detailed in this application note was used for the analysis of drugs of abuse in both hydrolyzed and non-hydrolyzed urine. Because the sample pre-treatment conditions used were different for each matrix, this step was not performed on the Extrahera.
•  In the method detailed here 3 mL of pre-treated urine is prepared with a 2 mL aliquot subsequently extracted. When processing samples using the Extrahera system, it is recommended that a pre-treated sample volume larger than 2 mL is prepared so that 2 mL can be accurately aspirated from the sample tube. With an optimized sample aspiration depth sample volumes as low as 2.5 mL can be extracted.
• The Extrahera method detailed here allows the batch extraction of 24 samples cartridges. For a batch size of 48 samples, use a 48 Position Configuration kit for the Biotage® Extrahera^TM HV-5000, and tables SPE cartridges.
• If a 48 position configuration kit is used then this should be in conjunction with 96 well pressure head seals.

Reduction in running time

Using a Biotage® ExtraheraTMHV-5000 with 5 mL capacity tips resulted in a shorter run time than when performed on an Biotage® Extrahera^TM Classic (which uses 1 mL capacity tips). The method described here took 46 minutes and 18 seconds (batch size 24) to run on a Extrahera HV 5000 compared to 58 minutes 52 seconds using the Extrahera Classic. The sample throughput can be improved further if desired by setting the addition of aqueous solvents (phosphate buffer equilibration and acetic acid wash) to serial dispensing.

Ordering information

for batch size of 24 samples

for batch size of 48 samples

Appendix

Biotage® Extrahera^TM HV-5000 settings

The method described in this application note was automated on the Biotage® Extrahera™ using ISOLUTE® HCX 130 mg/ 3 mL SPE cartridges. This appendix contains the software settings required to configure Extrahera to run this method. Analyte recoveries, % RSDs, linearities and LOQs were comparable for both manually processed and automated methods.

Method name: DoA Urine HCX HV5000
Sample plate/rack: 16 x 100 mm Test Tube, C40708
Extraction Media: ISOLUTE® HCX 130 mg, 902-0013-B

Literature number: AN982