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Evaluating total parenteral nutrition (TPN) products for quality assurance and quality control is crucial due to the chemical complexity of its components. With the advent of exploring different approaches for analysing TPN components using tandem mass spectrometry techniques, there is still a need for a robust and reproducible method for industrial routine analyses. This study allows simple, simultaneous determination of 22 fatty acids (FAs) commonly found in TPN components using gas chromatography-mass spectrometry (GC-MS). Five different transesterification techniques were applied for the FA standards and the sodium methoxide in methanol-dimethyl carbonate method was selected due to its good methylation efficiency. Fatty acid methyl esters (FAMEs) were separated in gas chromatography using an HP-5MS UI column with helium as the carrier gas. Mass spectrometry was used to fragment and quantify FAMEs using electron ionization (EI) and selected ion monitoring (SIM) mode. The analytical method was evaluated using the guidelines from the US Food and Drug Agency (FDA) and European Medicines Agency (EMA) in compliance with the International Council for Harmonization (ICH) document Q2(R2). Correlation coefficients (R-2) of the calibration curves for FAMEs were 0.99, except for C24:1 n-9 and C24:0, both R-2 = 0.98. The limits of detection (LOD) and quantification (LOQ) were found to be 1.69 mu g mL(-1) and 5.14 mu g mL(-1), respectively. The linear range was from 3.10-179.9 mu g mL(-1) for most FAMEs, except for C18:1 n-7 (3.96-224.9 mu g mL(-1)) and C18:1 n-9 (6.30-349.57 mu g mL(-1)). The intra-day and inter-day precision coefficients of variance (CV) of the method were less than 11.10% and 11.30%, respectively. Freeze-thaw cycles and ambient temperature measurements were performed for assessing sample stability. The validated method was applied to analyse major TPN components-fish and olive oils, and an unidentified lipid sample. The presented GC-MS method is simple and robust in the identification and quantification of 22 fatty acids simultaneously in the tested TPN components.

Free fatty acids (FFAs) are important energy sources and significant for energy transport in the body. They also play a crucial role in cellular oxidative stress responses, following cell membrane depolarization, making accurate quantification of FFAs essential. This study presents a novel supercritical fluid chromatography-mass spectrometry (SFC-MS) method using selected ion recording in negative electrospray ionization mode, enabling rapid quantification of 31 FFAs within 6 min without derivatization. FFAs are identified and quantified using an HSS C18 SB column and a secondary mobile phase consisting of methanol with formic acid by detecting their [M − H]⁻ ions. Calibration curves showed strong linearity (R² ≥ 0.9910), spanning 1000–12,000 ng/mL for short-chain FFAs and 50–1200 ng/mL for medium- and long-chain FFAs. The method achieves detection limits as low as 1 ng/µL for short-chain FFAs and 0.05 pg/µL for other FFAs per on-column injection. The method demonstrated high accuracy and precision, with bias and coefficients of variation maintained below 15% across five quality control levels. Freeze–thaw and autosampler stability studies confirmed the behavior of matrix-matched standards under optimal storage conditions. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using 13 deuterated FFAs as internal standards (IS) in comparison with heptadecanoic acid (C17:0). Significant variations in FFA quantification using two different IS approaches underscore the importance of selecting an appropriate IS. In summary, this study introduces a reliable and validated SFC-MS method for analyzing FFAs ranging from C4 to C26, requiring minimal sample preparation.

The increased presence of cholesterol oxidation products (COPs) over time may indicate pharmaceutical lipid emulsion (PLE) degradation, leading to decreased potency of total parenteral nutrition (TPN) products administered to clinical subjects. This study reports the analysis of cholesterol and 14 COPs potentially present in pharmaceutical-grade egg yolk powders using supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) within 15 minutes, without derivatization, in positive electrospray ionization (ESI⁺) mode. Identification and quantification of COPs were achieved using an HSS C18 SB column, with [M + H – H₂O]⁺ and [M + H – 2H₂O]⁺ as characteristic parent and daughter ions, respectively. Calibration curves demonstrated strong linearity (R² ≥ 0.9902), spanning 50–40000 ng/mL for cholesterol and most COPs per on-column injection. The method showed high accuracy and precision, with bias and coefficients of variation maintained below 20% across three quality control levels. Autosampler and freeze-thaw stability studies revealed that measurement variability for COPs is time- and temperature-dependent. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using three deuterated sterols and 19-hydroxycholesterol as internal standards (IS). Significant variations in the standard curves highlighted the importance of structural relevance in selecting an appropriate IS. To date, our method introduces a rapid SFC-MS/MS technique in ESI⁺ mode that quantifies cholesterol and COPs in their native form, reducing sample preparation and analysis time.

Changes in the lipid composition of pharmaceutical lipid emulsions (PLE) can serve as indicators of instability and potential long-term degradation of total parenteral nutrition (TPN) products. This study presents a targeted analysis of 30 key lipid species using ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), achieving a rapid analytical run time of 14 minutes. Lipids are identified and quantified using a CSH C18 column by monitoring specific precursor-to-product ion transitions in positive electrospray ionization mode. The method demonstrated excellent linearity (R² ≥ 0.9910), over a 5-1000 ng/mL linear range. Matrix-matched calibration curves revealed variable signal enhancement and suppression among targeted lipid species. Detection limits were as low as 0.01 pg/µL for certain lipid species, including SM (16:0, 18:1, 18:0), PG (34:0, 34:1), PC 38:2 and TAG (16:0, 18:1, 18:0). Accuracy and precision assessments showed biases and coefficients of variation below 20% across three quality control levels. Stability studies highlighted time- and temperature-dependent nature of matrix effects, even under optimal storage conditions. The validated method was applied to pharmaceutical-grade egg yolk powders, an important raw material for TPN products, using deuterated lipid internal standards for quantification. Key lipid species identified include PC (36:0, 34:0, 34:1, 34:0), TAG (16:0, 18:1, 18:0), and PE (34:2, 34:0, 36:1, 36:0), which greatly exceed LPC and LPE species, indicating that the tested samples are still in premium quality and found suitable for TPN formulations. This highlight the sensitivity and specificity of our method, as well as its potential application for pharmaceutical quality assurance and routine analysis.