Converting a liquid-liquid extraction method for vitamin D to supported liquid extraction in 96-well plate format

Introduction

The objective of this study was to determine feasibility of transferring a validated liquid-liquid extraction method to a supported liquid extraction method using Biotage ISOLUTE® SLE+ 96-well plates (400µL sample capacity). The laboratory supporting this study processes ~550 samples per month supporting vitamin D analysis in plasma and serum (mixed samples / not separated). During this evaluation, it was determined that sample processing time was reduced by ~50% for a de-identified patient set (n=30, split and processed by both methods). Towards high throughput analysis platforms, ISOLUTE SLE+ was demonstrated as a viable solution in the sample preparation workflow for vitamin D applications.

Figure 1: Vitamin D structures

Experimental overview

The strategy for this development workflow began with the liquid-liquid extraction procedure and the direct transfer to supported liquid extraction was evaluated. To optimize the method for analyte recovery, pH adjustment was evaluated in the sample pre-treatment. Elution volumes were also optimized for analyte recovery. Once optimized, the proof-of-concept was determined on a real patient sample set. The samples were split and process by each method for verification.

Analytical conditions

Reagents (all obtained from Fisher Scientific (LC-MS grade))

Internal standard d6-25-hydroxyvitamin D2, d3-25-hydroxyvitamin D3. 200 ng/mL in MeOH
Sample pre-treatment mix (SLE) 5M NaOH/H2O/IPA (50:50:100, v/v/v)
Aqueous mobile phase 2 mM ammonium acetate, 0.1% formic acid in H2O
Methanol mobile phase 2 mM ammonium acetate, 0.1% formic acid in MeOH

Equipment

Instrument

Waters Quattro Micro triple quadrupole tandem mass spectrometer

Electrospray source (+)

Agilent 1100 or Waters 2795 Alliance HT HPLC

Column

Phenomenex Kinetex 2.6 µm PFP 100A

100 mm x 3 mm

Pre-column

Phenomenex Security Guard ULTRA cartridges

UHPLC PFP for 3.0mm ID columns

Security Guard ULTRA holder

UPLC conditions

*The analytical window runs to 5.5 min and then the cartridge equilibrates for additional time. The total time is 10 min.

Mass spectrometry conditions

Sample preparation methodology

 
Method 1: Gold standard referee method – validated liquid-liquid extraction method

1. Set up labelled 2 mL Eppendorf centrifuge tubes for each blank, standard, control, and patient sample.
2. Vortex patient specimens, controls, and standards for 10 seconds. Centrifuge the tube if the volume is close to needed volume (200 μL).
3. Pipette 200 μL of sample into the 2 mL Eppendorf tube.
4. Add 50 μL NaOH (5 M/L). Mix on Mix-Mate at 2000 RPM for 1 minute. Tubes are OK uncapped for this step.
5. Pipette 200 μL of internal standard into each tube. MixMate for 3 minutes.
6. Extract by adding 1.5 mL of hexane to each tube. Cap and vortex for 10 seconds. MixMate for 4 minutes.
7. Centrifuge in micro-centrifuge at 13,000 rpm for 5 min.
8. Cool in -20 °C freezer for 20 min. Use tube racks previously pre-cooled in the freezer.
9. Using a transfer pipette, carefully transfer as much of the upper solvent layer as possible without removing any of the bottom layer to a 2 mL labelled LCMS autosampler vial.
10. Dry down extracts under nitrogen in Reactitherm Evaporator with the bath temperature set up at 35 °C. Before the evaporation, clean nozzles with paper towel wetted by 20% methanol or isopropanol wipe.
11. Reconstitute with 80 µL 70% MeOH 30%H2O. Vortex well.
12. Transfer all into a glass insert. Return the insert to the original vial and cap .
13. Centrifuge for 10 min at 2,500 rpm.
14. Inject 20 μL into LCMS.

Total time for processing 30 samples >4 hours

Method 2: Candidate supported liquid extraction procedure using ISOLUTE® SLE+ plates

1. Label 0.6 mL microfuge tubes.
2. Add 200 µL patient sample (plasma or serum) to each tube.
3. Add 30 µL IS to each tube.
4. Add 200 µL sample pre-treatment mix to each tube.
5. Cap, mix on MixMate for 5 seconds.
6. Transfer entire sample to ISOLUTE SLE+ 400µL plate.
7. Wait 5 minutes.
8. Add 750 µL hexane to wells.
9. Wait 5 minutes. If solvent isn’t completely eluted, pulse with pressure using a Biotage® PRESSURE+ 96. Repeat step 8 and 9 with a further aliquot of hexane.
10.  Dry under nitrogen, 45 °C.
11. Reconstitute with 80 µL 70% MeOH 30%H2O.
12. Mix on MixMate 5 seconds.
13. Transfer to vial inserts.
14. Inject 20 μL into LCMS.

Total time for processing 30 samples <2 hours

Results

Example chromatograms for samples prepared using a) original liquid-liquid extraction method and b) supported liquid extraction method are shown in Figures 2 and 3 below:

Figure 2. Example chromatogram for vitamin D metabolites extracted using original liquid-liquid extraction method.

Figure 3. Example chromatogram for vitamin D metabolites extracted using supported liquid extraction method on ISOLUTE® SLE+ plate.

Levels of Vitamin D metabolites from split samples extracted using each technique are shown in Table 3, and correlation plots for each metabolite are shown in Figures 4 and 5.

Figure 4. Correlation plot: Split patient sample study, determination of vitamin D3 in plasma and serum

Figure 5. Correlation plot: Split patient sample study, determination of vitamin D2 in plasma and serum.

Conclusions

Transfer of methodology from a traditional liquid-liquid extraction method to a supported liquid extraction method was simple and straightforward, with minimal optimization required. Good correlation between measured levels of vitamin D metabolites in split samples extracted using the original validated liquid-liquid extraction method and the optimized supported liquid extraction method using ISOLUTE SLE+ plates was achieved.

Time for processing a batch of 30 patient samples (serum and plasma) was approximately halved using the supported liquid extraction approach, from > 4 hours to < 2 hours. Due to the cleaner nature of the extraction, centrifugation steps were not required in the supported liquid extraction method.

This study suggests that supported liquid extraction using ISOLUTE®SLE+ 96-well extraction plates is a viable alternative to liquid-liquid extraction in a busy clinical laboratory.

Ordering information 

Literature Number: PPS367