How to Prepare Samples for Prostaglandin Measurement

Why Pre-Analytics Makes or Breaks Prostaglandin Data

Prostaglandins form and decay quickly. What happens before the assay often decides the result. A short delay, warmth, or rough handling can change pathway readouts.

Small mistakes cause big bias. Platelets can activate after collection and inflate thromboxane readouts. Plastics can adsorb lipids and depress recovery. Heat and oxygen accelerate degradation and increase variability.

A good plan is simple: move fast, keep cold, and standardize each step. Decide anticoagulants and additives in advance. Use low-bind containers and single-use aliquots. Document what you did so results remain comparable across days and teams.

For an overview of prostaglandin biosynthesis, biological roles, and detection strategies, see our comprehensive guide to prostaglandin function and analysis.

Core Principles You Will Reuse Everywhere

• Move fast, keep cold. Pre-chill racks, tubes, and buffers. Minimize dwell time between steps without quoting rigid minutes.
• Handle gently. Avoid vigorous mixing, frothing, and repeated vortexing that can trigger platelet activation or oxidation.
• Block unwanted chemistry—only if validated. Use COX inhibitors, antioxidants, or mild acidification when your method panel supports them and the lab has validated their impact.
• Choose inert contact surfaces. Use low-bind plastics or verified glass. Match caps/liners to solvents to prevent leachables.
• Control light and oxygen. Use amber or foil-wrapped tubes when appropriate. Keep containers capped; limit headspace and air exposure.
• Aliquot once, measure once. Prepare single-use portions to avoid freeze–thaw cycles; fill to consistent volumes and seal tightly.
• Prevent carryover. Dedicate tips/tubes per sample; avoid long soaks of extracts in generic plastics.
• Standardize labeling and records. Log matrix, anticoagulant, any additives, storage state, and freeze–thaw count; note timing qualitatively (e.g., "immediate spin, kept on ice").
• Add a light QC layer at the bench. Plan for spike-recovery, a process blank, and a pooled QC so problems are caught before measurement.

Blood, Plasma, and Serum — From Draw to Freeze

Whole blood is the least forgiving matrix for prostaglandins. Platelets, enzymes, and lipids react to handling, so limit activation, heat, and time. Your goal is simple: limit activation, limit heat, limit time.

Collection. Choose an anticoagulant validated for your assay (EDTA or citrate are common). Invert gently to mix; avoid vigorous shaking. Keep tubes on a pre-chilled rack. Do not let samples sit uncapped or warm on the bench.

Plasma vs serum. Plasma helps minimize clot-induced formation of prostanoids and is often preferred for basal measurements. Serum reflects clot activation and can show higher TXB2 by design; use it only when your study intends to capture that biology. Always record which matrix you use.

Processing. Move from draw to separation promptly. Keep samples on ice during staging. Clarify the matrix with a standardized spin protocol and stick to it across the study. Avoid hemolysis; red-cell rupture complicates interpretation.

Optional inhibitors. If ex vivo synthesis is a concern, discuss validated COX inhibitors with your analytical team. Additives must be pre-approved for the target panel and matrix; unvalidated use can mask real biology.

Transfers. Use low-bind tips and tubes. For plasma, avoid scraping the buffy coat. Cap immediately after each transfer to limit air exposure and evaporation.

Aliquot and store. Prepare single-use aliquots. Minimize headspace. Freeze rapidly and store cold. Label clearly with matrix, anticoagulant, any additives, and freeze–thaw count.

Mini checklist

• Keep cold from draw to freeze.
• Standardize separation and avoid hemolysis.
• Prefer plasma for basal PGs; reserve serum for clot-related designs.
• Use low-bind plastics or verified glass for all contacts.
• Document matrix, anticoagulant, and any additives.

Tissue and Homogenate — Extraction Without Loss

Tissue work adds mechanical stress, oxidation, and adsorption risk. Build your workflow to control temperature, contact surfaces, and exposure from the first cut to the final vial.

Sampling. Excise tissue promptly and keep it cold. If appropriate, rinse or blot to remove excess blood that can confound prostanoid signals. Record anatomical region and handling notes for context.

Stabilize early. Chill tools, tubes, and solvents in advance. Work on cold blocks. Limit light and air exposure; amber or foil-wrapped containers help for light-sensitive analytes.

Homogenization. Use pre-chilled bead-mill tubes or rotor–stator probes. Keep strokes short to avoid heat. If sonication is required, do it in brief, cold bursts. Add validated antioxidants or mild acidification only when your analytical method supports them.

Extraction solvents. Choose a solvent system your lab has validated for recovery (e.g., cold methanol or methanol:water for protein precipitation, followed by an organic step if needed). Mix gently; avoid frothing. Clarify by a cold spin; transfer supernatant with low-bind tips.

Internal standards (if using LC-MS/MS). Where validated, add stable-isotope standards at the start of extraction to track losses and matrix effects through the full process.

Containers and contact time. Prefer low-bind plastics or verified glass, matched to your solvent. Minimize long soaks; cap immediately after transfers.

Storage. Split extracts into single-use aliquots. Seal tightly and keep cold. Avoid repeated freeze–thaw cycles.

Pilot first. Run a small spike–recovery in the exact tissue type to confirm extraction efficiency, oxidation control, and container compatibility before scaling.

Mini table — Tissue do's and don'ts

Cell Culture Supernatant and Lysate — Low-Abundance Reality

Cell systems often release prostaglandins at low concentrations and in bursty patterns. Your prep must protect faint signals while avoiding artefacts from media, serum, and plastics.

Design the model first. Define stimulation and vehicle controls up front. Decide whether you care about extracellular PGs (supernatant), intracellular pools (lysate), or both. Keep sampling consistent across wells and plates.

Mind the medium. Serum can carry arachidonic acid and enzymes that shift basal PGs. If background is a concern, consider charcoal-stripped serum or fatty-acid-free albumin as validated for your assay. Phenol red can bias colorimetric ELISA; use phenol-red-free media when practical.

Supernatants. Collect gently to avoid lifting cells. Chill immediately. Clarify with a brief, cold spin and transfer to low-bind tubes. For LC-MS/MS, consider protein precipitation (cold methanol) followed by a validated cleanup (e.g., SPE) to concentrate low-level PGs and reduce salts.

Lysates. Harvest on ice with a validated buffer. Limit vortex/sonication to short, cold bursts. Remove debris by cold spin. If your method supports it, add stable-isotope internal standards at harvest to track losses during prep.

Plastics and contact time. Use low-bind plates, tips, and tubes. Avoid long soaks of media or extracts in generic plastics. Cap promptly to limit air exposure.

Additives—only if validated. Antioxidants, mild acidification, or COX inhibitors can help arrest ex vivo changes, but only use them if your target panel and matrix have been explicitly validated for these additives.

Concentration and drying. If you must concentrate, use gentle evaporation (e.g., nitrogen with temperature control). Avoid high heat. Reconstitute in a solvent compatible with your method and matrix.

Mini table — Cell culture do's and don'ts

Containers, Additives, and Chain of Custody

Hardware and documentation shape data quality as much as technique. Choose contact materials that do not steal lipids, and keep a clean record of every hand-off.

Containers—minimize adsorption and leachables

• Use low-bind polypropylene for routine steps; validate borosilicate glass for long holds or organic extracts.
• Match cap liners to solvents (PTFE or foil-backed liners work well). Avoid rubbery stoppers that leach.
• Prefer amber vials or foil wraps for light-sensitive analytes.
• Cap immediately after transfers. Reduce headspace to limit oxygen exposure.
• Do not soak extracts in generic plastics; shorten contact time whenever possible.

Additives—helpful only when validated

• COX inhibitors, antioxidants, or mild acidification can limit ex vivo change.
• Use them only if the downstream method and matrix were explicitly validated.
• Record the name, lot, concentration, and addition point for each additive.

Chain of custody—make samples auditable

• Label with matrix, species, anticoagulant, additives, date, operator, and freeze–thaw count.
• Use barcodes where possible; avoid hand-written shorthand.
• Log each location change (bench → cold room → freezer → shipping).
• Note any deviations (e.g., "held on ice during centrifuge queue").

Mini table — Quick choices at a glance

Bench QC & Quick Fixes — Catch Issues Before Measurement

Light, well-placed QC catches problems before they reach the instrument. It protects scarce samples and prevents reruns.

Minimal QC set

• Spike–recovery. Add known prostaglandins to real matrix. Confirm recovery and linearity.
• Process blank. Run the full workflow without biological input. Check for contamination or carryover.
• Pooled QC. Combine small aliquots from study samples. Place at intervals to monitor drift and repeatability.
• Short stability check. Hold a test aliquot on ice and at room temperature. Verify that handling does not reshape the profile.
• Replicate consistency. Prepare technical duplicates from the same source. Track CV% to flag prep variance.

Bench QC dashboard showing spike recovery, clean blank, pooled QC drift, and quick fix references.

Common symptoms and fast fixes

Design a Panel That Fits Your Study

Match your question to the right measurement plan. Use ELISA for quick single-analyte screens. Choose LC-MS/MS when you need isomer resolution, multiplex efficiency, and pathway context.

Choosing between LC–MS/MS and ELISA depends on your study goals and analyte complexity. For method selection tips, see our comparison of prostaglandin measurement platforms.

Next steps

• Prostaglandins Analysis Service — targeted panels for accurate prostaglandin quantification
• Eicosanoid Analysis Service — broader pathway profiling across prostaglandins, thromboxanes, and leukotrienes
• Oxylipin Quantification & Pathway Profiling — COX/LOX/CYP context with ratios and panel-level insight
• Thromboxanes Analysis Service — focused TXA2/TXB2 readouts for platelet and vascular research
• Isoprostanes Analysis Service — oxidative-stress markers (e.g., 8-iso-PGF2α) complementing PG panels

References:

1. Brose, Stephen A., Brock T. Thuen, and Mikhail Y. Golovko. "LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes." Journal of Lipid Research 52(4) (2011): 850–859.
2. Cao, Hongmei, et al. "An improved LC–MS/MS method for the quantification of prostaglandins E₂ and D₂ production in biological fluids." Analytical Biochemistry 372(1) (2008): 41–51.
3. Thakare, R., Y. S. Chhonker, N. Gautam, et al. "Simultaneous LC–MS/MS analysis of eicosanoids and related metabolites in human serum, sputum and BALF." Biomedical Chromatography 32(3) (2018): e4102.
4. Montuschi, Paolo, Peter J. Barnes, and L. Jackson Roberts II. "Isoprostanes: Markers and mediators of oxidative stress." The FASEB Journal 18(15) (2004): 1791–1800.
5. Patrono, Carlo, et al. "Measurement of thromboxane biosynthesis in health and disease." Frontiers in Pharmacology 10 (2019): 1244.