High-throughput screening methods for fatty acid (FA) determination are urgently needed due to their critical biochemical roles in human health while serving as biomarkers of habitual diet and chronic disease risk assessment. Herein, we introduce multisegment injection-nonaqueouscapillary electrophoresis-mass spectrometry (MSI-NACE-MS) as a multiplexed separation platform for analysis of more than 20 nonesterified FAs in human serum or plasma. Optimization of experimental conditions was required to overcome major technical hurdles in MSI-NACE-MS prior to a rigorous method validation and intermethod comparison with gas chromatography/mass spectrometry (GC/MS). Following a simple methyl-tert-butyl ether extraction, seven serum extracts were analyzed directly by MSI-NACE-MS within a single run (<4 min/sample) under negative ion mode detection that incorporates stringent measures for quality control, including batch correction adjustment. Overall, excellent technical variance (RSD = 10%) and good mutual agreement was demonstrated for 12 nonesterified FAs consistently measured in 50 serum samples analyzed independently by MSI-NACE-MS and GC/MS within the same laboratory (mean bias = 24%, n = 600). Also, total hydrolyzed plasma FAs using MSI-NACE-MS was compared to mean concentrations reported from a NIST standard reference material as an interlaboratory method validation (mean bias = 15%, n = 20). Accurate prediction of ion migration behavior in CE also supports structural elucidation of FAs in conjunction with high resolution MS. For the first time, we demonstrate that MSI-NACE-MS offers a rapid yet robust platform for direct quantification of circulating FAs using volume-restricted blood specimens that expands metabolome coverage to encompass anionic classes of lipids as required for large-scale epidemiological studies.

Capillary electrophoresis-mass spectrometry (CE-MS) represents a high efficiency microscale separation platform for untargeted profiling of polar/ionic metabolites that is ideal for volume-restricted biological specimens with minimal sample workup. Despite these advantages, the long-term stability of CE-MS remains a major obstacle hampering its widespread application in metabolomics notably for routine analysis of anionic metabolites under negative ion mode conditions. Herein, we report for the first time that commonly used ammonia containing buffers compatible with electrospray ionization (ESI)-MS can compromise the integrity of fused-silica capillaries via aminolysis of their outer polyimide coating. Unlike organic solvent swelling effects, this chemical process occurs under aqueous conditions that is dependent on ammonia concentration, buffer pH, and exposure time resulting in a higher incidence of capillary fractures and current errors during extended operation. Prevention of polyimide aminolysis is achieved by using weakly alkaline ammonia containing buffers (pH < 9) in order to preserve the tensile strength of the polyimide coated fused-silica capillary. Alternatively, less nucleophilic primary/secondary amines can be used as electrolytes without polyimide degradation, whereas chemically resistant polytetrafluoroethylene coating materials offer higher pH tolerance in ammonia. In this work, multisegment injection (MSI)-CE-MS was used as multiplexed separation platform for high throughput profiling of anionic metabolites when using optimized buffer conditions to prevent polyimide degradation. A diverse range of acidic metabolites in human urine were reliably measured by MSI-CE-MS via serial injection of seven urine samples within a single run, including organic acids, food-specific markers, microbial-derived compounds and over-the-counter drugs as their sulfate and glucuronide conjugates. This approach offers excellent throughput (<5 min/sample) and acceptable intermediate precision (average CV ≈ 16%) with high separation efficiency as reflected analysis of 30 anionic metabolites following 238 repeated sample injections of human urine over 3 days while using a single nonisotope internal standard for data normalization. Careful optimization and rigorous validation of CE-MS protocols are crucial for developing a rapid, low cost, and robust screening platform for metabolomics that is amenable to large-scale clinical and epidemiological studies.

New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection−nonaqueous capillary electrophoresis−mass spectrometry (MSI−NACE−MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high-from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI−NACE−MS greatly expands the metabolome coverage of conventional aqueous-based CE−MS protocols and is a promising platform for large-scale lipidomic studies.

A large body of evidence has linked unhealthy eating patterns with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake in nutritional epidemiology rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching single-spot urine and fasting plasma specimens were collected at baseline, and then following two weeks of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 plasma metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV < 30%) in the majority of participants (>75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following two weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high-resolution MS/MS, as well as co-elution after spiking with authentic standards if available. Overall, 3-methylhistidine and proline betaine concentrations increased in both plasma and urine samples after participants were assigned a Prudent diet (q < 0.05) with a corresponding decrease in the Western diet group. Similarly, creatinine-normalized urinary imidazole propionate, hydroxypipecolic acid, dihydroxybenzoic acid, and enterolactone glucuronide, as well as plasma ketoleucine and ketovaline increased with a Prudent diet (p < 0.05) after adjustments for age, sex, and BMI. In contrast, plasma myristic acid, linoelaidic acid, linoleic acid, α-linoleic acid, pentadecanoic acid, alanine, proline, carnitine, and deoxycarnitine, as well as urinary acesulfame K increased among participants following a Western diet. Most metabolites were also correlated (r > ± 0.30, p < 0.05) to changes in the average intake of specific nutrients from self-reported diet records reflecting good adherence to assigned food provisions. Our study revealed robust biomarkers sensitive to short-term changes in habitual diet, which is needed for accurate monitoring of healthy eating patterns in free-living populations, and evidence-based public health policies for chronic disease prevention.

New experiments for undergraduate students are needed to stimulate experiential learning in the laboratory while providing valuable training for future career development. Iodine deficiency remains a major public health concern that is monitored by measuring the median urinary iodide concentration of a population. In this context, we have introduced a kinetic spectrophotometric experiment based on the classic Sandell−Kolthoff reaction for second-year undergraduate students in analytical chemistry. This two-day laboratory experiment incorporates principles of quantitative chemical analysis, redox chemistry, reaction kinetics, optical spectroscopy, sample pretreatment, external calibration, method validation, statistical analysis, and quality assurance. Additionally, students gain real-world experience of implementation of a reliable analytical method used in global health initiatives to combat iodine deficiency, including participation in a round-robin study with the CDC. This colorimetric assay is widely used for continuous monitoring of mandatory iodized table salt programs to prevent the risk of iodine deficiency disorders, including developmental delays and intellectual impairment in children.