UPLC-MS Method Development for Phospholipid Quantification: Advancing Parenteral Nutrition Analysis

Dive into the cutting-edge world of pharmaceutical analysis with Mark Dennis Retrato, a PhD candidate at Uppsala University. In this exclusive interview, Mark shares his groundbreaking research on developing a novel UPLC-MS method for analyzing phospholipids in total parenteral nutrition (TPN) products.

Key highlights:

• Innovative UPLC-MS method for phospholipid quantification
• Importance of nitrogen evaporation in sample preparation
• Unexpected findings in phosphatidylcholine species
• Implications for quality control in pharmaceutical manufacturing
• Potential impact on patient care and TPN product stability

Learn how this research is pushing the boundaries of analytical chemistry and contributing to improved healthcare outcomes. Whether you're a scientist, healthcare professional, or simply curious about advancements in medical technology, this interview offers valuable insights into the future of parenteral nutrition analysis.

Video Transcription

How do you ensure that lipid-based nutrition products for critically ill patients remain stable and safe from production to administration?

For Mark, an industrial PhD student at Örebro University in Sweden, this question is at the heart of his research. Working in close collaboration with a pharmaceutical company, he is developing and validating a robust UPLC–MS method to quantify key phospholipids in raw materials used for total parenteral nutrition (TPN).

In this interview, Mark explains why he chose UPLC–MS, how he designed the method to be technically transferable, and what his findings mean for quality control and patient care.

From the Philippines to Sweden: an industrial PhD journey

Originally from the Philippines, Mark moved to Sweden to pursue his PhD at Örebro University. His project is firmly rooted in real-world needs: he is officially employed as an industrial PhD student, developing methods that must work not only in an academic setting, but also in routine pharmaceutical laboratories.

“Even if I’m doing an academic work, the main goal is to actually do an academic work that is reproducible outside,” Mark explains. “We wanted a method that is simple, doable, and suitable for method transfer.”

Why UPLC–MS for phospholipid quantification?

The focus of Mark’s project is a phospholipid-rich pharmaceutical raw ingredient used in TPN formulations. While he has experience with GC–MS and supercritical fluid chromatography (SFC), this study required a different approach.

Several criteria guided the choice of technique:

• Minimal sample preparation – only a few simple steps such as pre-concentration and evaporation
• Compatibility with large, non-volatile molecules like phospholipids
• Suitability for routine QC workflows

GC×GC was considered but would have required extensive derivatization to render the lipids volatile. SFC remained an option, but ultimately the team chose UPLC coupled to tandem mass spectrometry.

“We leaned towards UPLC–MS because it was the best approach for our type of analysis and our sample,” says Mark. “It allowed us to keep sample preparation straightforward while still getting robust, targeted quantification.”

Building the method: from direct infusion to matrix-matched calibration

Method development began on the MS side, with direct infusion experiments to check ionization, interferences, and transitions for the target phospholipids. Once this foundation was in place, the team moved on to chromatographic optimization:

• Screening different columns and mobile phases
• Adjusting gradients for robust separation
• Designing a polar initial mobile phase to efficiently elute phospholipids, followed by stronger conditions for more hydrophobic species

Because the analyte is a pharmaceutical raw material, the method also had to meet regulatory expectations. Mark and his colleagues aligned their validation strategy with the ICH Q2(R2) guideline, which underpins FDA and EMA requirements for analytical methods.

Key validation elements included:

• System suitability tests before each run
• Matrix-matched calibration curves, using a matrix closely resembling the actual product
• Structurally related internal standards, grouped by analyte type
• Quality control samples across the working range

This regulatory-aware design ensures that the method is not only scientifically sound, but also ready for use in regulated environments.

Surprising complexity: four peaks where two were expected

One of the most striking findings emerged when the team looked more closely at phosphatidylcholines (PCs) in the egg-yolk-derived material.

“Sometimes we only expect two or three PC species,” Mark notes. “But for one nominal composition, we actually observed four distinct peaks.”

These four peaks correspond to different combinations of fatty acid chains that share the same condensed formula. The result highlights the hidden structural complexity of lipid mixtures that might otherwise appear simple on paper.

Another important observation was the role of lysophosphatidylcholines (LPCs). Increases in LPC/PC ratios were associated with changes in product stability, making LPCs a potential marker for degradation processes.

From raw material to finished product: implications for QC

The method’s design and performance make it highly relevant for quality control of TPN products. Its main advantages are:

• Targeted quantification of key phospholipids, not just profiling
• Technical transferability to QC labs
• Flexibility across the manufacturing chain

The same method can be used at different stages:

• Raw material assessment (incoming control)
• In-process monitoring during compounding
• Final product testing of finished goods

With adjustments in sample preparation and pre-concentration, the method can also be adapted to clinical samples, such as plasma, where lipid levels are lower.

Sample preparation, nitrogen evaporation, and time pressure

Sample preparation presented its own challenges, particularly in terms of solubility, stability, and analyte losses. The team optimized solvent systems and extraction protocols, and fine-tuned the use of internal standards to avoid signal masking by the highly concentrated raw material.

A critical final step is nitrogen evaporation, used to concentrate samples before injection:

• Starting material: ca. 1 mg of phospholipid raw material
• Extraction volume: up to ~2 mL
• Pre-evaporation volume: ~1 mL
• Post-evaporation reconstitution: ~200 µL

Evaporation speed must be carefully controlled. Too slow, and the long preparation time can increase the formation of degradation products such as LPCs; too fast, and the risk of losses and instability grows.

A personal connection to patient care

Beyond the technical and regulatory aspects, this project has a strong personal meaning for Mark.

“The ending of this work is to actually assess the stability of pharmaceutical products that are consumed by critically ill patients who cannot ingest food,” he says. “With my small contribution, I feel like I am part of something valuable for patients.”

By delivering a robust, transferable UPLC–MS method for phospholipid analysis, his work aims to support more reliable quality control of TPN formulations – and ultimately, more consistent care for some of the most vulnerable patients.

This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.

Concentrating on Chromatography Podcast

Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!

You can find Concentrating on Chromatography Podcast in podcast apps:

• Spotify
• Apple podcast
• podscan

and on YouTube channel