Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Mass spectrometry quantitation of immunosuppressive drugs in clinical specimens using online solid-phase extraction and accurate-mass full scan-single ion monitoring.

Introduction: Therapeutic drug monitoring (TDM) of immunosuppressants is essential for optimal care of transplant patients. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) are the most commonly used methods for TDM. However, immunoassays can suffer from interference from heterophile antibodies and structurally similar drugs and metabolites. Additionally, nominal-mass LC-MS assays can be difficult to optimize and are limited in the number of detectable compounds. Objectives: The aim of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using online solid-phase extraction (SPE) and accurate-mass full scan-single ion monitoring (FS-SIM) data acquisition mode. Methods: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spectrometer. TurboFlow online SPE was used for sample clean up. The accurate-mass MS was set to positive electrospray ionization mode with FS-SIM for quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A. MS2 fragmentation pattern was used for compound confirmation. Results: The method was validated in terms of precision, analytical bias, limit of quantitation, linearity, carryover, sample stability, and interference. Quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated well with results from an independent reference laboratory (r = 0.926-0.984). Conclusions: Accurate-mass FS-SIM can be successfully utilized for immunosuppressant TDM with good correlation with results generated by standard methods. TurboFlow online SPE allows for a simple "protein crash and shoot" sample preparation protocol. Compared to traditional MRM, analyte quantitation by FS-SIM facilitates a streamlined assay optimization process.

Semi-automated serum steroid profiling with tandem mass spectrometry.

Objectives: Highly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels. Methods: Sample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications. Results: Validation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples. Conclusions: It was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry.

Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients. Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase. Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione. Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

Targeted metabolic profiling of urinary steroids with a focus on analytical accuracy and sample stability.

Introduction: Preoperative diagnostic workup of adrenal tumors is based on imaging and hormone analyses, but charged with uncertainties. Steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 24-h urine has shown potential to discriminate benign and malignant adrenal tumors. Our aim was to develop and validate a specific and accurate LC-MS/MS method for the quantification of deconjugated urinary marker steroids, to evaluate their pre-analytical stability and to apply the method to clinical samples of patients with adrenal tumors. Methods: A method for the quantification of 11 deconjugated steroids (5-pregnenetriol, dehydroepiandrosterone, cortisone, cortisol, α-cortolone, tetrahydro-11-deoxycortisol, etiocholanolone, pregnenolone, pregnanetriol, pregnanediol, and 5-pregnenediol) in human urine was developed and validated based on international guidelines. Steroids were enzymatically deconjugated and extracted by solid phase extraction before LC-MS/MS quantification in positive electrospray ionization mode. Results: Excellent linearity with R2 > 0.99 and intra- and inter-day precisions of < 10.1 % were found. Relative matrix effects were between 96.4 % and 101.6 % and relative recovery was between 98.2 % and 115.0 %. Sufficient pre-freeze stability for all steroids in urine was found at 20-25 °C for seven days and at 4-6 °C for up to 28 days. Samples were stable during long-term storage at -20 °C and -80 °C for 6 months. Conclusions: A sensitive and robust LC-MS/MS method for the quantification of 11 urinary steroids was developed and validated according to international guidelines. Pre-analytical matrix stability was evaluated and the suitability of the method for the analysis of clinical samples and prospective validation studies was shown.

Co-pigmentation of strawberry anthocyanins with phenolic compounds from rooibos.

Anthocyanin-rich strawberry model solutions were co-pigmented with rooibos phenolics to enhance color and heat stability. The addition of green and fermented rooibos extracts at pigment-to-co-pigment molar ratios of 1:10, 1:50, and 1:100 pelargonidin-3-glucoside equivalents: orientin equivalents induced hyper- and bathochromic shifts at room temperature and during thermal processing at 80 °C for an hour. Co-pigmentation effects on hyperchromic shift were up to 96%, and bathochromic shift reached 19 nm when adding flavonoid-rich fractions of green rooibos phenolics. Following the co-pigmentation tests with rooibos extracts, selected pure phenolic co-pigments were tested for their monomeric contribution to the observed co-pigmentation effects. Orientin was identified as a potent co-pigment for pelargonidin-3-glucoside, showing stronger co-pigmentation effects than that of its aglycon luteolin. Additionally, orientin had the most pronounced bathochromic shift in heat-treated solutions. Rooibos extracts, particularly flavonoid-rich fractions composed of luteolin, apigenin, and quercetin glycosides, are suggested as color enhancers and stabilizers for strawberry products.

Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry.

Background: Although measurement of 25(OH)D3 is a routine analytical method to determine plasma vitamin D status, 1α,25(OH)2D3 is the biologically active form. Hence, simultaneous measurement of 25(OH)D3 and 1α,25(OH)2D3 could provide better insight into vitamin D status and pharmacokinetics. However, 1α,25(OH)2D3 has a low plasma concentration, making its quantification challenging for most analytical techniques. Here, we demonstrate use of liquid chromatography tandem mass spectrometry (LC-MSMS) for the development of a simple and rapid method for the simultaneous quantification of 25(OH)D3 and 1α,25(OH)2D3. Methods: Samples were purified from 250 µL human plasma. Chromatography was performed on an analytical column, under gradient conditions using a mobile phase consisting of methanol-lithium acetate. The mass detector was operated in positive multiple reaction monitoring mode. The established method was validated according to the guidance issued by ICH and FDA. Furthermore, a clinical study was performed using this method to detect the plasma concentrations of 1α,25(OH)2D3 after oral administration of calcitriol. Results and conclusion: The method was acceptably linear over the concentration ranges of 20-1200 pg/mL for 1α,25(OH)2D3 and 1-60 ng/mL for 25(OH)D3, respectively, with correlation coefficients of r2 > 0.993. Both the inter-assay and intra-assay precision was < 15%, and the analytical recoveries were within 100% ± 10%, with no significant matrix effect or carryover. Thereby, we, provide a facile method for the simultaneous detection of vitamin D metabolites in plasma.

Biotechnological formation of dairy flavor inducing δ-lactones from vegetable oil.

Agroscope Culture Collection was screened to identify bacterial strains effective in production of dairy flavor inducing lactones using grapeseed oil as a substrate. Lentilactobacillus parafarraginis FAM-1079, Lactococcus lactis subsp. lactis FAM-17918, and L. lactis subsp. lactis biovar diacetylactis FAM-22003 showed the most efficient formation of targeted δ-lactones. The application of sublethal heat stress significantly increased target lactone production. The most profound improvement was for L. lactis subsp. lactis biovar diacetylactis where δ-octadecalactone generation was improved by factor of 9. The pre-fermentation step as well as growth phase in which bacteria are harvested did not have a significant impact on lactones yield. The lactone production process from vegetable oil developed in this study offers a new way of developing a natural flavor ingredient for incorporation into plant-based products.

Detection of seven Alternaria toxins in edible and medicinal herbs using ultra-high performance liquid chromatography-tandem mass spectrometry.

Alternaria mycotoxins are ubiquitous mycotoxins that contaminate food and animal feed. Here, an UPLC-MS/MS was developed and used for the detection of seven Alternaria mycotoxins in 19 different edible and medicinal herbs. Extensive optimization resulted in a simple and convenient sample preparation procedure with satisfactory extraction and a lower matrix effect. LOQs ranged from 0.01 to 2.0 ng/mL. Recoveries varied between 71.44% and 112.65%, with RSD less than 12%. The method was successfully applied for use in the mycotoxin analysis of 260 samples. A high percentage (28.46%) of samples were contaminated by 1-5 mycotoxins. Alternariol mono methylether was the predominant mycotoxin with high percentage of positive samples (37.5%), followed by alternariol (22.5%), alternariol (17.5%), tentoxin (10.83%), altertoxin Ⅰ (7.5%), and altenusin (4.17%). Collectively, the natural incidence data obtained from this study will help with better, validated risk assessments and efforts towards more comprehensive, future regulation.

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease.

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Endocannabinoids as potential biomarkers: It's all about pre-analytics.

INTRODUCTION: Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are central lipid mediators of the endocannabinoid system. They are highly relevant due to their involvement in a wide variety of inflammatory, metabolic or malign diseases. Further elucidation of their modes of action and use as biomarkers in an easily accessible matrix, like blood, is restricted by their susceptibility to deviations during blood sampling and physiological co-dependences, which results in high variability of reported concentrations in low ng/mL ranges. OBJECTIVES: The objective of this review is the identification of critical parameters during the pre-analytical phase and proposal of minimum requirements for reliable determination of endocannabinoids (ECs) in blood samples. METHODS: Reported physiological processes influencing the EC concentrations were put into context with published pre-analytical research and stability data from bioanalytical method validation. RESULTS: The cause for variability in EC concentrations is versatile. In part, they are caused by inter-individual factors like sex, metabolic status and/or diurnal changes. Nevertheless, enzymatic activity in freshly drawn blood samples is the main reason for changing concentrations of AEA and 2-AG, besides additional non-enzymatic isomerization of the latter. CONCLUSION: Blood samples for EC analyses require immediate processing at low temperatures (>0 °C) to maintain sample integrity. Standardization of the respective blood tube or anti-coagulant, sampling time point, applied centrifugal force and complete processing time can further decrease variability caused by sample handling. Nevertheless, extensive characterization of study participants is needed to reduce distortion of clinical data caused by co-variables and facilitate research on the endocannabinoid system.

Green extraction of polyphenols from citrus peel by-products and their antifungal activity against Aspergillus flavus.

Aspergillus flavus is a pathogenic fungus associated with food safety issues worldwide. This study investigated the antifungal activity of citrus peel extracts prepared using food-grade solvents (hot water or ethanol). Mandarin (Citrus reticulata) peel ethanol extracts inhibited the mycelial growth of A. flavus (39.60%) more effectively than those of orange (32.31%) and lemon (13.51%) after 7 days of incubation. The growth of A. flavus could be completely inhibited by mandarin extracts at 300-400 mg mL-1, depending on the extraction solvent. Solid-phase extraction (SPE) separated the polyphenol-rich fractions, which showed up to 40% higher antifungal activity than crude extracts. Twelve polyphenols (2 phenolic acids and 10 flavonoids) were identified by HPLC-DAD, narirutin and hesperidin were the most abundant. In conclusion, citrus peels are promising bioresources of antifungal agents with potential applications in food and other industries.

Poisoning by organophosphorus nerve agents and pesticides: An overview of the principle strategies and current progress of mass spectrometry-based procedures for verification.

Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis.

A high sensitivity LC-MS/MS method for measurement of 3-methoxytyramine in plasma and associations between 3-methoxytyramine, metanephrines, and dopamine.

INTRODUCTION: Diagnosis of pheochromocytoma and paraganglioma (PPGL) is aided by the measurement of metanephrine (MN) and normetanephrine (NMN). Research suggests that 3-methoxytyramine (3MT), a dopamine (DA) metabolite, may serve as a biomarker of metastasis in patients with paraganglioma. Considering the very low endogenous plasma 3MT concentrations (<0.1 nM), highly sensitive and specific methods for 3MT are needed. METHODS: We developed a simple method for measurement of 3MT. Sample preparation was performed using solid phase micro-extraction with the eluates injected directly onto the LC-MS/MS. Data acquisition was performed in multiple reaction monitoring mode with an instrumental analysis time of 3 min per sample. We evaluated the method's performance and analyzed samples from healthy individuals and pathological specimens. RESULTS: The limit of quantitation and upper limit of linearity were 0.03 nM and 20 nM, respectively. The intra-/inter-day imprecision for pooled plasma samples at concentrations of 0.04 nM, 0.2 nM, and 2 nM was 10.7%/18.3%, 4.5%/8.9%, and 3.1%/0.9%, respectively. Among samples with MN, NMN, or both MN and NMN above the reference intervals (RIs), 0%, 16% and 46%, respectively, showed 3MT greater than the proposed upper RI value of 0.1 nM; 12% of samples with DA above the RI had 3MT above 0.1 nM. CONCLUSIONS: The developed method allowed accurate quantitation of 3MT in patient samples and would provide valuable information to clinicians diagnosing or monitoring patients with PPGL. High 3MT concentrations in patient samples with MN and NMN within the respective RIs may alert clinicians of the possibility of a DA-producing tumor.

Application of d-SPE before SPE and HPLC-FLD to Analyze Bisphenols in Human Breast Milk Samples.

In this study, we propose a simple, cost-effective, and sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the simultaneous determination of seven bisphenols (bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB), BADGE (bisphenol A diglycidyl ether), BADGE∙2H2O, BADGE∙H2O, BADGE∙2HCl) in human breast milk samples. The dispersive solid phase extraction (d-SPE) coupled with solid phase extraction (SPE) procedure performed well for the majority of the analytes with recoveries in the range 57-88% and relative standard deviations (RSD%) of less than 9.4%. During the d-SPE stage, no significant matrix effect was observed thanks to the application of different pairs of salts such as zirconium-dioxide-based sorbents (Z-Sep or Z-Sep +) and primary secondary amine (PSA) or QuEChERS Enhanced Matrix Removal-Lipid (EMR-Lipid) and PSA. The method limits of quantification (mLOQs) for all investigated analytes were set at satisfactory low values in the range 171.89-235.11 ng mL-1. Analyte concentrations were determined as the average value from human breast milk matrix samples. The results show that the d-SPE/SPE procedure, especially with the application of EMR-Lipid and PSA, could be used for further bisphenol analyses in human breast milk samples.

From Vietnamese plants to a biflavonoid that relieves inflammation by triggering the lipid mediator class switch to resolution.

Chronic inflammation results from excessive pro-inflammatory signaling and the failure to resolve the inflammatory reaction. Lipid mediators orchestrate both the initiation and resolution of inflammation. Switching from pro-inflammatory to pro-resolving lipid mediator biosynthesis is considered as efficient strategy to relieve chronic inflammation, though drug candidates exhibiting such features are unknown. Starting from a library of Vietnamese medical plant extracts, we identified isomers of the biflavanoid 8-methylsocotrin-4'-ol from Dracaena cambodiana, which limit inflammation by targeting 5-lipoxygenase and switching the lipid mediator profile from leukotrienes to specialized pro-resolving mediators (SPM). Elucidation of the absolute configurations of 8-methylsocotrin-4'-ol revealed the 2S,γS-isomer being most active, and molecular docking studies suggest that the compound binds to an allosteric site between the 5-lipoxygenase subdomains. We identified additional subordinate targets within lipid mediator biosynthesis, including microsomal prostaglandin E2 synthase-1. Leukotriene production is efficiently suppressed in activated human neutrophils, macrophages, and blood, while the induction of SPM biosynthesis is restricted to M2 macrophages. The shift from leukotrienes to SPM was also evident in mouse peritonitis in vivo and accompanied by a substantial decrease in immune cell infiltration. In summary, we disclose a promising drug candidate that combines potent 5-lipoxygenase inhibition with the favorable reprogramming of lipid mediator profiles.

Use of a deactivated PTV injector liner and GCMS/MS for the quantitative determination of multiple pesticide residues in fruit and vegetables.

The quantitative determination of multiple pesticide residues in food is an iterative process given the frequent changes in monitoring specifications set by regulatory authorities, introduction of new pesticide active ingredients, variety of commodities encountered and advances in the capability of analytical instrumentation and software platforms. The method described here:•replaces our previous methodology [1] that was based on an ethyl acetate extraction [2], two different sample extract clean-up regimes depending on the commodity; either Gel Permeation Chromatography (GPC) or Solid Phase Extraction (SPE) and GC/MSMS analysis using cool on-column injection and permits higher throughput using the same QuEChERS extraction method used for LCMS/MS analysis [3]•uses PTV injection incorporating a deactivated (baffled) injection liner required to improve performance for 'difficult to analyse' pesticides e.g. captan, dichlofluanid, folpet, tolylfluanid.•has been validated for the quantitative determination of 113 different pesticides and their metabolites in a range of fruit and vegetables of high water content and high acid and high water content i.e. cabbage, lemon, pepper, plum and spinach and complies with requirements of European Commission guidance document on Analytical Quality Control and Method Validation Procedures for Pesticides Residues Analysis in food and feed - SANTE/12682/2019 [4].

Data on the optimisation of a solid phase extraction method for fractionating estrogen metabolites from small urine volumes.

Certain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis.