Comparison of automated vs. manual extraction of 25-OH Vitamin D from plasma using ISOLUTE® SLE+

Introduction

Automated sample preparation using the Biotage® Extrahera™ was compared to an equivalent manual method utilizing a vacuum manifold. Analytes were extracted from pooled stripped plasma using a supported liquid extraction procedure. ISOLUTE® SLE+
400 µL sample volume plates, part number 820-0400-P01 were used for extraction.
Resulting extracts from both sample preparation methods were subsequently analysed by LC-MS/MS.

Procedure 

A pooled plasma sample was prepared in a sufficient quantity to run a full 96-well plate for each processing method. This pooled plasma sample was fortified with 25-OH Vitamin D2 and D3 at a concentration of 30 ng/mL respectively. 25-OH Vitamin D3-d6 was added as an internal standard at a concentration of 30 ng/mL.

From this pooled plasma sample 200 µL was transferred to all wells of two 96-well plates. All subsequent aspects of sample preparation were performed in duplicate on two separate plates utilizing either Extrahera or manual preparation using a calibrated air-displacement pipette. The pooled plasma sample was then pre-treated 1:1 (v/v) with Water:Propan-2-ol 1:1 (v/v) (200 µL).

After pre-wetting the pipette tips via aspirate/dispense cycling and to mix the samples, 300 µL of the pre-treated sample was loaded to each well of the ISOLUTE® SLE+ plates. Flows were initiated using a pulse of positive pressure (Extrahera) or vacuum (manual method).

After leaving for 5 minutes to allow the sample to completely absorb into the plates, elution was performed by the application of 2 x 750 µL of Heptane to the ISOLUTE® SLE+ plates. The extracts were collected in 2 mL 96-well collection plates under gravity elution, and as a final step to recover all available solvent from the media, by applying a pulse of positive pressure (Extrahera) or vacuum (manual method).

The extracts were evaporated to dryness in a TurboVap® 96 at 37 °C or a SPE Dry at 40 °C and reconstituted in 100 µL of 30:70 (v/v) water/methanol solution.
The plates were mixed on an orbital shaker for 10 minutes.

HPLC conditions

Instrument: Waters Aquity UHPLC
Cartridge: Restek Pinnacle DB Biphenyl, 50 mm x 2.1 mm 1.9 µm
Mobile Phase: 80:20 (v/v) 2mM ammonium formate with 0.1 % formic acid (aq.)/ Methanol with 0.1 % formic acid at 0.4 mL/min
Injection Volume:  15 µL

Mass spectrometry

Instrument: Waters Quattro Premier XE
MRM Conditions

Experimental precautions

The following precautions were performed to minimize differences between the manual and Extrahera extracted plates.

• Both plates were evaporated side by side on the same evaporation instrument (TurboVap® 96).
• During analysis on the LC-MS system samples were injected alternately from the two plates to reduce the effect of any sample stability issues.
• The same batch/bottles of samples, reagents and solvents were used for both methods.
  
  

Results

Average peak area data was calculated for all three compounds to compare any improvements in analyte recovery between Biotage® Extrahera^TM and manually processed samples.

Peak area ratio data was also generated for all samples by referencing the analyte vs. 25-OH Vitamin D3-d6. This provides standardized data to allow a comparison of the % RSD of the Extrahera vs. manual data sets.

Additional experiments were also completed using serum as the sample matrix. The average recovery comparison data is presented in Figure 1 below.

Figure 1. Average recovery (n=8) of 25-OH-Vitamin D2 and 25-OH-Vitamin D3 from serum

Calibration series samples prepared manually in Phosphate Buffered Saline with Bovine Serum Albumin (PBS-BSA) and those commercially available from Chromsystems were also processed and extracted using the procedure above with both Biotage® Extrahera^TM and manual sample processing methods. Calibration curves for both manual and Extrahera processed samples over concentration range 1–100 ng/mL for 25-OH Vitamin D2 and D3 in Phosphate Buffered Saline with Bovine Serum Albumin (PBS-BSA) are shown in Figures 2 to 5. Chromsystems calibration curves for both manual and Extrahera processed samples for 25-OH Vitamin D2 (15.8–61.6 ng/mL) and D3 (4.5–66.7 ng/mL) are shown in Figures 6 to 9.

 Figure 2. 25-OH Vitamin D2 in PBS-BSA – Biotage® ExtraheraTM 

 Figure 3. 25-OH Vitamin D2 in PBS-BSA – Manual 

 Figure 4. 25-OH Vitamin D3 in PBS-BSA – Biotage® ExtraheraTM 

 Figure 5. 25-OH Vitamin D3 in PBS-BSA - Manual 

 Figure 6. 25-OH Vitamin D2 Chromsystems – Biotage® ExtraheraTM 

 Figure 7. 25-OH Vitamin D2 Chromsystems – Manual 

 Figure 8. 25-OH Vitamin D3 Chromsystems – Biotage® ExtraheraTM 

 Figure 9. 25-OH Vitamin D3 Chromsystems - Manual 

Conclusion

A reduction to the % RSD was measured when using the Biotage® Extrahera^TM for 25-OH Vitamin D3, the precision of the D2 data was slightly better when processed manually.
For 25-OH Vitamin D3 the % RSD improved by 11.8 %, for 25-OH Vitamin D2 there was a slight drop in the % RSD of 3.6 % to 7.74 vs. 7.47, this is not considered to be statistically significant. The results suggest that methods performed on the Extrahera could give higher recoveries due to increases in the absolute average peak areas.

All analytes returned average peak areas that were higher when sample extraction was performed using the Biotage® Extrahera^TM, than the manual method with an average peak area increase of 5 %. The greatest improvement was measured with 25-OH Vitamin D2 where the average peak area was increased by 7 %. The serum samples extracted and analysed under the same conditions showed near identical recoveries between Extrahera and manual processing, 99 % versus 100 % for 25-OH Vitamin D3 respectively and 97 % for 25-OH Vitamin D2 for both methods. The calibration series data for samples prepared in PBS-BSA and the Chromsystems calibrators both returned good correlation coefficients with r2 greater than 0.99 for samples prepared using Extrahera and processed manually.

Literature number: PPS336