--- title: Comparison of automated vs. manual extraction of 25-OH Vitamin D from plasma using ISOLUTE® SLE+ description: Automated sample preparation using the Biotage® Extrahera™ was compared to an equivalent manual method utilizing a vacuum manifold. --- [Literature ](https://www.biotage.com/literature) # [Comparison of automated vs. manual extraction of 25-OH Vitamin D from plasma using ISOLUTE® SLE+](https://www.biotage.com/literature/application-note/comparison-automated-manual-extraction-vitamin-d-plasma-isolute-sle) Written by [Biotage](https://www.biotage.com/literature/author/biotage) | Dec 6, 2025 6:00:00 PM ## 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 | Analyte | Transition | RT (min) | Dwell (sec) | Cone (V) | Col Energy (V) | | --- | --- | --- | --- | --- | --- | | 25-OH Vitamin D2 | 395.5 to 119.2 | 1.7 | 0.1 | 30 | 26 | | 25-OH Vitamin D3 | 383.5 to 107.2 | 1.6 | 0.1 | 30 | 25 | | 25-OH Vitamin D3-d6 | 389.6 to 263.5 | 1.5 | 0.1 | 30 | 16 |   ## 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® ExtraheraTM 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.   |   | Extrahera Peak Area Ratio Summary | Manual Peak Area Ratio Summary | | --- | --- | --- | | Average 25-OH D2 Peak Area | 1697 | 1577 | | Improvement (%) vs. Manual Method | 7.6 | - | |   |   |   | | Average 25-OH D3-d6 Peak Area | 1441 | 1384 | | Improvement (%) vs. Manual Method | 4.1 | - | |   |   |   | | Average Peak Area Ratio | 1.1771 | 1.1421 | | % RSD of Extrahera Extraction | 7.7 | 7.5 | | Improvement (%) vs. Manual Method | -3.6 | - |                           |   | Extrahera Peak Area Ratio Summary | Manual Peak Area Ratio Summary | | --- | --- | --- | | Average 25-OH D3 Peak Area | 4758 | 4528 | | Improvement (%) vs. Manual Method | 5.1 | - | |   |   |   | | Average 25-OH D3-d6 Peak Area | 1441 | 1384 | | Improvement (%) vs. Manual Method | 4.1 | - | |   |   |   | | Average Peak Area Ratio | 3.3051 | 3.2813 | | % RSD of Extrahera Extraction | 5.9 | 6.7 | | Improvement (%) vs. Manual Method | 11.8 | - |   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® ExtraheraTM 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® ExtraheraTM 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. | % RSD | Manual Procedure (n=93) | Extrahera (n=92) | | --- | --- | --- | | 25-OH Vitamin D2 |  7.47 | 7.74 | | 25-OH Vitamin D3 | 6.70 | 5.91 |   | Average Peak Area | Manual Procedure (n=93) | Extrahera (n=962) | | --- | --- | --- | | 25-OH Vitamin D2  | 1577 | 1697 | | 25-OH Vitamin D3  |  4728 | 4758 | | 25-OH VitaminD3-d6  | 1384 | 1441 |   All analytes returned average peak areas that were higher when sample extraction was performed using the Biotage® ExtraheraTM, 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 [View full post](https://www.biotage.com/literature/application-note/comparison-automated-manual-extraction-vitamin-d-plasma-isolute-sle) ```json { "@context" : "http://schema.org", "@type" : "BlogPosting", "author" : { "@type" : "Person", "name" : "Biotage" }, "dateModified" : "2025-12-06T18:00:00.736Z", "datePublished" : "2025-12-06T18:00:00Z", "headline" : "Comparison of automated vs. manual extraction of 25-OH Vitamin D from plasma using ISOLUTE® SLE+", "image" : { "@type" : "ImageObject", "height" : 961, "url" : "https://www.biotage.com/hubfs/Imported_Blog_Media/biotage-pps366-figure1-Average-recovery-25-OH-VitaminD2-and-25-OH-Vitamin.jpg", "width" : 1587 }, "mainEntityOfPage" : "https://www.biotage.com/literature/application-note/comparison-automated-manual-extraction-vitamin-d-plasma-isolute-sle", "publisher" : { "@type" : "Organization", "logo" : { "@type" : "ImageObject", "height" : 60, "url" : "/hs/hsstatic/content_shared_assets/static-1.4092/img/default-amp-logo.png", "width" : 60 }, "name" : "Literature" } } ```