Fatty Acids In Human Serum Research Articles

Determination of six volatile fatty acids in human serum, urine and faeces by low temperature derivatisation combined with HPLC–MS/MS

A stable isotope dilution-liquid chromatography tandem mass spectrometry method based on a low-temperature derivatization strategy with 3-nitrophenylhydrazine (3-NPH) was developed for the determination of six volatile fatty acids (VFAs) in serum, urine, and feces. Ice acetonitrile was used to precipitate proteins and extract the target analytes. The extract was derivatized with 3-NPH methanol solution at 4 °C. BEH C8 (1.7 μm, 2.1 × 100 mm) column was used for chromatographic separation, and acetonitrile–water (both containing 0.01 % formic acid) were used as the mobile phase with a gradient elution of 10 min. Electrospray ionization source (ESI) in negative ion multiple reaction monitoring (MRM) mode were used for analyte detection. The regression coefficients R2 of the calibration curves for the six VFAs were in the range of 0.9963–0.9994, and the LOQs were in the range of 0.02–0.5 μg mL−1, with the recoveries in the range of 85.3–104.3 %, and the intra- and inter-day precision in the range of 1.8–9.1 %. The method is simple, accurate and reliable, and has been applied in the sensitive determination of VFAs in complex biological samples.

Development and application of a sensitive liquid chromatography-tandem mass spectrometry method for the quantitative analysis of 11 free fatty acids in human serum using a derivatisation strategy

A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples.

Analysis of n-6 / n-3 ratio of polyunsaturated fatty acids in human serum by reactive thermal desorption-gas chromatography using a vertical microfurnace pyrolyzer

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) using a vertical microfurnace pyrolyzer and a flame ionization detector (FID) was applied to rapid analysis of chemical composition for polyunsaturated fatty acid (PUFA) components in human serum. Here, the authors used a reactive thermal desorption (RTD) method, where 2 μL of a serum sample was subjected to the THM reaction at 250 °C for 20 s, to detect PUFA components with high selectivity and sensitivity. The resulting chromatograms clearly showed a series of fatty acid methyl esters including PUFAs without any interference peaks derived from the matrix components of the serum samples. Furthermore, the observed chemical composition and n-6 / n-3 ratio for PUFA components were in good agreement with those obtained by a conventional analytical technique involving solvent extraction and transmethylation.

Analysis and Clinical Application of Polyunsaturated Fatty Acids in Human Serum Using One-step Extraction and Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Omega-3 and omega-6 fatty acids are reported to alleviate various inflammatory reactions such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, and inflammatory bowel disease. We have developed a new method for their quantitation in serum, utilizing ultra-high-performance liquid chromatography-electrospray ionizationtandem mass spectrometry (UHPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode, under negative ionization. MRM transitions were detected for five omega fatty acids, as follows: α-linolenic acid, m/z=277.20→277.20; eicosapentaenoic acid, m/z=301.10→257.30; docosahexaenoic acid, m/z=327.00→283.15; arachidonic acid, m/z=303.20→259.25; docosapentaenoic acid, m/z=329.10→285.25. After comparing several different solvents, we selected butanol:methanol (1:1, v/v) as the optimal extraction solvent. Chromatographic separations were carried out using an Acclaim RSLC 120 C18 column (150×2.1 mm, 2.2 μm) at 40oC. For the mobile phase, solvent A consisted of water containing 5% acetonitrile and 5% methanol, and solvent B consisted of methyl tert-butyl ether:acetonitrile:methanol (10:20:70, v/v/v) containing 2.5 mM ammonium acetate. The calibration curve showed excellent linearity within the range of 0.01-20 μg/mL (R2=0.9996-0.9999). The analytical method was validated and shown to have excellent sensitivity (LOD; 0.001-0.005 μg/mL, LOQ; 0.01-0.2 μg/mL), making it suitable for targeted analyses of omega fatty acids. Recovery ranged from 78.0 to 103.9% (RSD 0.5-5.6%). This new method uses relatively small amounts of serum and extraction solvent and is expected to be suitable for analysis of various clinical specimens, such as plasma, urine, and cerebrospinal fluid.

Sensitive and quantitative determination of short-chain fatty acids in human serum using liquid chromatography mass spectrometry

Short-chain fatty acids (SCFAs) are increasingly being monitored to elucidate the link between gut health and disease. These metabolites are routinely measured in faeces, but their determination in serum is more challenging due to their low concentrations. A method for the determination of eight SCFAs in serum is described here. High-resolution mass spectrometry and gas chromatography were used to identify the presence of isomeric interferences, which were then overcome through a combination of chromatographic separation and judicious choice of MS fragment ion. The SCFAs were derivatised to form 3-nitrophenylhydrazones before being separated on a reversed-phase column and then detected using liquid chromatography tandem mass spectrometry (LC-QQQ-MS). The LODs and LOQs of SCFAs using this method were in the range 1 to 7 ng mL−1 and 3 to 19 ng mL−1, respectively. The recovery of the SCFAs in serum ranged from 94 to 114% over the three concentration ranges tested.

A Fast and Accurate Way to Determine Short Chain Fatty Acids in Human Serum by GC–MS and Their Distribution in Children with Digestive Diseases

Short-Chain Fatty Acids (SCFAs) represent the largest group of metabolic nutrients produced by gut microbiota, and can be considered potential biomarkers for digestive tract diseases. A fast, cost-effective, and reproducible analytical method for the analysis of SCFAs is highly required. This work developed a one-step extraction of 14 SCFAs using a mixture of ethanol and n-hexane in human serum samples by GC–MS without derivatization. The recoveries of the 14 SCFAs ranged from 62.4% to 129.2% in human serum samples. The LODs and LOQs of the 14 SCFAs were 0.12–0.48 mg/L and 0.40–1.61 mg/L, respectively. The application of this method was carried out by comparing SCFAs in serum samples from children with health and digestive tract diseases, and the results demonstrated that SCFAs in the serum of children with digestive disorders were significantly higher than those of healthy children, implying that digestive tract diseases affect gut microbiota and their metabolisms.

In situ analysis of unsaturated fatty acids in human serum by negative-ion paper spray mass spectrometry

In situ identification and quantification of unsaturated fatty acid (FA) C=C positional isomers in human serum is herein performed by negative-ion paper spray (PS) mass spectrometry. Typically, by direct application of an alternating current (AC) voltage to the wet paper, the PS ionization could perform stably in the negative-ion mode without severe discharge. We suppose epoxidation reaction between unsaturated C=C bonds and reactive oxidative species might be initiated by a mild electrical discharge, which could be rapidly and controllably produced via a low amplitude AC voltage. Upon collision-induce dissociation (CID), the epoxide was fragmented to generate diagnostic ions indicating the C=C location. The intensity of the characteristic product ions could also be used for absolute quantification of the FA C=C positional isomers. The limits of detection (LODs) and limits of quantification (LOQs) were roughly in the range of 0.0178–0.0506 μM and 0.0218–0.3634 μM for standard FAs. Without the additional sample preparations or reactive chemical reagents, epoxidation of unsaturated FAs and ionization of the epoxide could be achieved in one-step by negative-ion mode PS, which enable a promising methodology for on-site clinical diagnosis.

Determination of total, free and esterified short-chain fatty acid in human serum by liquid chromatography-mass spectrometry.

Short-chain fatty acids are primarily absorbed through the portal vein during lipid digestion, which is utilized as the energy source, as well as prevent type 2 diabetes and some cancers. However, reports on the determination of these short-chain fatty acids in human serum are limited. Blood samples from human subjects ( n = 547, male/female = 246/301, age 58.85 ± 12.57) were collected. Saponification was applied to obtain total fatty acid. After derivatization by 2-nitrophenylhydrazine, fatty acid 4:0 and fatty acid 6:0 were measured by liquid chromatography-mass spectrometry. The developed method exhibited good linearity (R2 = 0.9996 for both). All the coefficients of variation of reproducibility and accuracy for fatty acid 4:0 and fatty acid 6:0 ranged 3.0%-6.1%, with the average recoveries of 87.8%-102.4% and 92.2%-98.2%, respectively. In all the samples, the concentration of fatty acid 4:0 (162.4 ± 76.4 μmol/L) was significantly higher than fatty acid 6:0 (2.0 ± 2.5 μmol/L, P < 0.001). Furthermore, the esterified form was predominant in both fatty acid 4:0 and fatty acid 6:0 (98.2% and 82.4% of total fatty acids, respectively). Besides, short-chain fatty acids showed no significant differences with regard to sex or age differences. This developed liquid chromatography-mass spectrometry method is convenient and reliable, which might be useful for monitoring the variations of short-chain fatty acids in blood.

Profiling of polyunsaturated fatty acids in human serum using off-line and on-line solid phase extraction-nano-liquid chromatography-quadrupole-time-of-flight mass spectrometry

Polyunsaturated fatty acids (PUFAs) play a pivotal role in the biological effects, and are the potential biomarkers for some diseases. However, the structural diversity and similarity, the low concentration, and the interference of high abundant endogenous components challenge the PUFAs profiling. Herein, a novel analytical approach, off-line and on-line solid phase extraction-nano-liquid chromatography-quadrupole-time-of-flight mass spectrometry (off-line and on-line SPE-nano-LC-Q-TOF-MS), was established to monitor the PUFAs. The combination of off-line and on-line SPE removed most of impurities, and the recoveries ranged from 80.1% to 93.0% and the matrix effects were from 85.1% to 92.8%. Using this method, 51 PUFAs could be separated well and quantified with the limits of quantification between 0.006 and 2.2 pg. Finally, this developed method was applied successfully to simultaneously qualify and quantify the potential biomarkers in the allergic patients. 21 PUFAs including LTB4, 5S-, 11S-, 15S-HETE and 15S-HEPE showed significant differences. Our study indicated that the established method has the potential to sensitively and accurately determine the PUFAs in biological samples.

Quantitative analysis of free fatty acids in human serum using biexciton auger recombination in cadmium telluride nanoparticles loaded on zeolite.

Nanometer-sized semiconductor particles (CdTe) were used as an inorganic matrix for the laser desorption/ionization mass spectrometry of fatty acids. The excitation power dependence of the peak intensity of stearic acid (Ste), [Ste - H](-), was observed, and it was proportional to the square of the excitation power. The peak intensity of [Ste - H](-) decreased by addition of a hole scavenger (KSCN). It was understood that the ionization of fatty acids were due to the biexciton Auger recombination and electron ejection from CdTe. CdTe were then loaded on zeolite surface. The peak intensity enhancement of the deprotonated ion of fatty acid were observed. This phenomenon was explained by measuring the carrier lifetime for Auger recombination in CdTe. In addition, reproducibility of fatty acid ions was highly improved reflecting homogeneous distribution of CdTe on zeolite surface. CdTe/HM20 was successfully applied to the quantitative analysis of Ste in human serum by isotope dilution using (13)C18-Ste. The concentration of Ste in human serum samples was estimated to be 76.62 mg/kg with the standard deviation (SD) of 2.37 mg/kg.

Determination of free fatty acids in human serum by HPLC with fluorescence detection.

It has been suggested that serum concentrations of polyunsaturated essential fatty acids correlate with the symptoms or severity of various diseases, including depression and Alzheimer-type dementia, and that determination of serum fatty acids might be important for disease diagnosis. Thus, we developed to analyze serum fatty acids in healthy individuals by using a high-performance liquid chromatography method with fluorescence detection, because free fatty acids have a carboxyl group that can be derivatized with a fluorescent reagent, 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. This approach could quantify five types of free fatty acids [α-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA) and oleic acid (OA)] in human serum. The detection limits of the method were in the range of 2.29-4.75 fmol (signal-to-noise ratio 3), and absolute concentrations of ALA, PLA, AA, LA and OA were 8.27 ± 1.04, 18.8 ± 2.95, 49.9 ± 4.03, 230 ± 18.1 and 201 ± 22.1 µM, respectively.

Identification and Quantitation of Fatty Acid Double Bond Positional Isomers: A Shotgun Lipidomics Approach Using Charge-Switch Derivatization

The specific locations of double bonds in mammalian lipids have profound effects on biological membrane structure, dynamics and lipid second messenger production. Herein, we describe a shotgun lipidomics approach that exploits charge-switch derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) and tandem mass spectrometry for identification and quantification of fatty acid double bond positional isomers. Through charge-switch derivatization of fatty acids followed by positive-ion mode ionization and fragmentation analysis, a marked increase in analytic sensitivity (low fmol/μL) and the identification of double bond positional isomers can be obtained. Specifically, the locations of proximal double bonds in AMPP-derivatized fatty acids are identified by diagnostic fragment ions resulting from the markedly reduced 1,4-hydrogen elimination from the proximal olefinic carbons. Additional fragmentation patterns resulting from allylic cleavages further substantiated the double bond position assignments. Moreover, quantification of fatty acid double bond positional isomers is achieved by the linear relationship of the normalized intensities of characteristic fragment ions vs the isomeric compositions of discrete fatty acid positional isomers. The application of this approach for the analysis of fatty acids in human serum demonstrated the existence of two double bond isomers of linolenic acid (i.e., Δ(6,9,12) 18:3, γ-linolenic acid (GLA), and Δ(9,12,15) 18:3, α-linolenic acid (ALA)). Remarkably, the isomeric ratio of GLA vs ALA esterified in neutral lipids was 3-fold higher than the ratio of their nonesterified moieties. Through this developed method, previously underestimated or unidentified alterations in fatty acid structural isomers can be determined facilitating the identification of novel biomarkers and maladaptive alterations in lipid metabolism during disease.

Rapid determination of C12–C26 non‐derivatized fatty acids in human serum by fast gas chromatography

The concentration of long-chain fatty acids (LCFAs) in human serum is closely related to human health. It is therefore important to develop a fast, low-cost, efficient method for their determination. In this study, by using fast temperature programming and micro-bore short capillary columns, a fast gas chromatography method was developed for the direct analysis of non-derivatized LCFAs including n-dodecanoic acid to n-hexacosanic acid (C12:0-C26:0, even numbers only), linoleic acid (C18:2), oleic acid (C18:1), and erucic acid (C22:1) within 4.0 min. Method optimization including extraction and separation conditions is considered, and the analysis of real serum samples is presented. The results show that ten LCFAs were well separated with sufficient resolution, and the detection limit was in the range of 2.8-9.6 microg/mL. The reproducibility (RSD) for both intra-day and inter-day determination was always less than 15%, and the recoveries for these LCFAs were from 63.1 to 97.0%.

Electrogenerated chemiluminescence derivatization reagent, 3-isobutyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ylamine, for carboxylic acid in high-performance liquid chromatography using tris(2,2'-bipyridine)ruthenium(II).

3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ylamine (IDHPIA) was found to be a selective and highly sensitive derivatization reagent for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(II). Free fatty acids and phenylbutylic acid were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid. Under the mild reaction conditions of room temperature for 45 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro-2H-pyridol1,2-a]pyrimidin-2-one, all the fatty acids tested were reacted with IDHPIA to produce highly sensitive derivatives. The chemiluminescence intensity was essentially the same for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limit (signal-to-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection was 0.5 and 0.6 fmol for myristic acid and phenylbutylic acid, respectively. IDHPIA was 100-fold more sensitive than previously developed reagents (Morita, H.; Konishi, M. AnaL Chem. 2002, 74, 1584-1589). The free fatty acids in human serum were successfully determined using the present method.

4-(5,6-Dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide as a fluorescent labelling reagent for determination of carboxylic acids in precolumn high-performance liquid chromatography

A fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide, was designed for the determination of carboxylic acids by precolumn HPLC. The reagent was reacted with carboxylic acids at 100°C for 15 min in the presence of an activating reagent (a mixture of triphenylphosphine, 2,2′-dipyridyldisulfide and pyridine) and produced highly fluorescent derivatives, which were separated on a reversed-phase column by fluorescence measurement at 317 nm (excitation) and 380 nm (emission). The detection limits were 4–12 fmol/injection. The reagent was used for HPLC assays of long chain fatty acids in human serum by isocratic elution.

Precolumn Fluorescence Derivatization of Carboxylic Acids Using 4-Aminomethyl-6,7-dimethoxycoumarin in a Two-Phase Medium

A new coumarin-based amine, 4-aminomethyl-6, 7-dimethoxycoumarin, was synthesized as a precolumn fluorescence derivatization reagent for carboxylic acids. The derivatization using the amine in a two-phase medium of water and ethyl acetate was examined. The two-phase derivatization method with the amine was found to be of value since it allows simple and rapid HPLC assays of carboxylic acids with a reasonable sensitivity and reparability. The detection limit was determined to be 20-50fmol per 10μl injection volume at a signal-to-noise ratio of 3 under partially optimized conditions. The method was also applied to the assay of fatty acids in human serum.

2-(5-Hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography and its application to the assay of fatty acids in human serum

A unique acid hydrazide, 2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole, that is characterized by the benzothiazole structure conjugated to an oxazoline moiety was synthesized, and its applicability as a precolumn derivation reagent for carboxylic acids in HPLC was examined in view of sensitivity and separability. The sensitivity of the hydrazide for carboxylic acids was determined using lauric acid to be 0.1 pmol (per 10-μ1 injection volume) at a signal-to-noise ratio of 3. The reagent allowed rapid assays of carboxylic acids within 20 min with satisfactory separability. The method was applied to the determination of fatty acids in human sera from healthy volunteers as well as from patients with diabetes or thyroid dysfunction.

2-(5-Hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole as a Precolumn Fluorescence Derivatization Reagent for Carboxylic Acids in High-Performance Liquid Chromatography and Its Application to the Assay of Fatty Acids in Human Serum.

A precolumn fluorescence derivatization reagent for carboxylic acids, 2-(5-hydrazinocarbonyl-2-furyl)-5, 6-dimethoxybenzothiazole, was developed and its applicability to fatty acids in biological samples was examined. This reagent was proved to be useful in the determination of fatty acids in high-performance liquid chromatography with the detection limit of approximately 50fmol per 10-μl injection volume. Fatty acids in human serum were assayed by the derivatization method using the reagent. The method was also applied to some prostaglandins.

Simple and highly sensitive determination of free fatty acids in human serum by high performance liquid chromatography with fluorescence detection.

A highly sensitive and simple reversed phase high performance liquid chromatographic (HPLC) method for the quantitative determination of free fatty acids in human serum is presented. The method is based on the direct derivatization of serum fatty acids with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37 degrees C. The resulting derivatives are separated within 75 min on a reversed phase column (YMC Pack C8) with a gradient elution of aqueous acetonitrile and detected fluorimetrically. The detection limits are 2.5-5 fmol in a 10 microL injection volume. The sensitivity permits precise determination of free fatty acids in 5 microL serum. The method is simple and is without the conventional liquid-liquid extraction steps of serum fatty acids.

Separation and quantitation of long-chain free fatty acids in human serum by high-performance liquid chromatography

A rapid, simple and highly sensitive reversed-phase high-performance liquid chromatographic method is described for the separation and quantitation of fatty acids in human serum using a very reactive fluorescent labeling reagent, 9-anthryldiazomethane. Quantitative esterification proceeds at room temperature without heat or catalysis. Baseline separation of nineteen select fatty acids from a standard mixture was achieved on two C 18-bonded silica columns connected in tandem using stepwise gradient elution of an acetonitrile-methanol-water mobile phase. The eluent was monitored by a fluorescence detector (maximum excitation wavelength, 365 nm; maximum emission wavelength, 412 nm). The procedure was applied to the analysis of both saturated and unsaturated long-chain free fatty acids (C 8 to C 22) extracted from human serum. Sera from fasting and non-fasting subjects were analyzed to show the applicability of this assay to biological samples. Detection limit and recovery of free fatty acids in serum were < 10 pmol/μl and > 92%, respectively.