---
title: Comparison of automated vs manual extraction of NSAIDs from plasma using ISOLUTE® SLE+
description: A selection of non-steroidal anti-inflammatory drugs (NSAIDS) were extracted from pooled stripped plasma using a supported liquid extraction procedure.
---

[Literature ](https://www.biotage.com/literature)

# [Comparison of automated vs manual extraction of NSAIDs from plasma using ISOLUTE® SLE+](https://www.biotage.com/literature/application-note/comparison-automated-manual-extraction-nsaids-plasma-isolute-sle)

 Written by [Biotage](https://www.biotage.com/literature/author/biotage) | Dec 6, 2025 2:00:00 PM

## Introduction

Automated sample preparation using the Biotage® Extrahera™ was compared to an equivalent manual method utilizing a vacuum manifold. A selection of non-steroidal anti-inflammatory drugs (NSAIDS) 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 Sulindac, Ketoprofen and Indomethacin at a concentration of 25 ng/mL respectively.  
From this pooled plasma sample 250 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 pre-treated 1:1 (v/v) with 1 % formic acid (250 µL). This sample dilution results in approximate loading pH of 3.2.  
After pre-wetting the pipette tips via aspirate/dispense cycling and to mix the samples; 400 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 0.9 mL aliquots of methyl tertiary-butyl ether (MTBE) 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 and reconstituted in 200 µL of 60:40 (v/v) water/methanol solution. The plates were mixed on an orbital shaker for 10 minutes.

## HPLC conditions

Instrument: Waters Alliance 2795  
Cartridge: ACE Excel 2 C18-AR 50 x 2.1mm id cartridge  
Mobile Phase: 50:50 (v/v) 0.1 % (v/v) formic acid/acetonitrile at 0.25 mL/min  
Injection Volume:  25 µL

## Mass spectrometry

Instrument: Waters Quattro Ultima Pt

MRM Conditions

| ** Analyte** | ** Transition** | ** RT (min)** | ** Dwell (sec)** | ** **Cone (V) |  Col Energy (V) |
| --- | --- | --- | --- | --- | --- |
| Indomethacin | 358.1 to 139.0 | 2.5 | 0.1 | 35 | 18 |
| Sulindac | 357.1 to 233.1 | 0.9 | 0.1 | 35 | 26 |
| Ketoprofen | 255.1 to 209.1 | 1.2 | 0.1 | 35 | 13 |

 

 

 

 

 

 

 

## Experimental precautions

- 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™ and manually processed samples.  
Peak area ratio data was also generated for all samples by referencing the analyte vs. Ketoprofen. This provides standardized data to allow a comparison of the % RSD of the Extrahera vs. manual data sets. 

 

|   | Extrahera Sulindac Peak Area Ratio Summary | Manual Sulindac Peak Area Ratio Summary |
| --- | --- | --- |
| Average Sulindac Peak Area | 24622 | 22847 |
| Improvement (%) vs. Manual Method | 7.8 | - |
|   |   |   |
| Average IS Peak Area | 35199 | 32683 |
| Improvement (%) vs. Manual Method | 7.7 | - |
|   |   |   |
| Average Peak Area Ratio | 0.701 | 0.705 |
| % RSD of Extrahera™ Extraction | 4.01 | 6.43 |
| Improvement (%) vs. Manual Method | 37.6 | - |
|   |   |   |

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

|     | Extrahera Indomethacin Peak Area Ratio Summary | Manual Indomethacin Peak Area Ratio Summary |
| --- | --- | --- |
| Average Indomethacin Peak Area | 34395 | 27965 |
| Improvement (%) vs. Manual Method | 23.0 | - |
|   |   |   |
| Average IS Peak Area | 35199 | 32683 |
| Improvement (%) vs. Manual Method | 7.7 | - |
|   |   |   |
| Average Peak Area Ratio | 0.977 | 0.857 |
| % RSD of Extrahera™ Extraction | 5.46 | 8.56 |
| Improvement (%) vs. Manual Method | 36.2 | - |

 

 

 

 

 

 

 

 

 

 

 

 

 

## Conclusion

A significant reduction to the % RSD was measured when using the Biotage® Extrahera™ for both compounds compared to the manual processing approach.

| % RSD | Manual Procedure (n=96) | Extrahera (n=96) |
| --- | --- | --- |
| Sulindac | 6.43 | 4.01 |
| Indomethacin | 8.56 | 5.46 |

 

 

 

 

For Sulindac the % RSD improved by 37%, for Indomethacin there was also an improvement in the % RSD by 36 % to 5.46%. The results also suggest that methods performed on the Extrahera™ could give higher recoveries due to increases in the absolute average peak areas

 

| Average Peak Area | Manual Procedure (n=96) | Extrahera (n=96) |
| --- | --- | --- |
| Sulindac | 22847 | 24622 |
| Indomethacin | 27965 | 34395 |

 

 

 

 

 

Both compounds returned average peak areas that were higher when sample extraction was performed using Extrahera™ than the manual method with an average peak area increase of 15.4%. The greatest improvement was measured with Indomethacin where the average peak area was increased by 23%.

 

Literature number: PPS361

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