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.     

Simultaneous quantification of the major bile acids in Artificial Calculus bovis by high‐performance liquid chromatography with precolumn derivatization and its application in quality control

An accurate and sensitive high‐performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2‐bromine‐4’‐nitroacetophenone and 18‐crown ether‐6 were used for derivatization. The chromatographic separation was performed on an Agilent SB‐C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r² ≥ 0.9980), recovery (94.24–98.91%), limits of detection (0.25–0.31 ng) and limits of quantification (0.83–1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.     

Determination of Fatty Acids in Saliva of Smokers and Nonsmokers by HPLC with Fluorescence Detection Using a Hydrazine-Based Difluoro-boraindacene Reagent

1,3,5,7-Tetramethyl-8-daminozide-difluoroboradiaza-s-indacence (TMBODIPY-H), a pre-column fluorescent derivatization reagent, was applied to the analysis of fatty acid by high-performance liquid chromatography with fluorescence detection. Using this reagent, 12 fatty acids from propionic acid (C3) to stearic acid (C18) can be derivatized at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide within 2 h. The baseline separation of these derivatives can be achieved within 46 min by gradient elution. The detection limits of these fatty acids are in the range of 0.1–1 nM and the linear ranges of most of them are 0.5–100 nM. This was the first application of hydrazine-based difluoro-boraindacene reagent for the analysis of fatty acids, and the proposed method has been successfully used for the determination of saliva samples of smokers and nonsmokers with recoveries of 88–110 %.     

Determination of low-molecular-weight carboxylic acids in aqueous samples by gas chromatography and nitrogen-selective detection of 2-nitrophenylhydrazides

A gas chromatographic method was developed for the determination of low-molecular-weight carboxylic acids in aqueous samples based on a derivatization procedure compatible with aqueous solutions. The technique uses nitrogen-selective detection with a thermionic-specific detector after derivatization of carboxylic acids as 2- nitrophenylhydrazides. The hydrazides were extracted with ethyl acetate prior to injection into the gas chromatograph. The derivatives appear to be stable in ethyl acetate at 0–5° C for long periods and, therefore, can be stored for analysis at a later date. The detection limits of different short-chain acids are in the range 0.8–1.4 pmol per injected sample. The relative standard deviation is less than 10% at the 1 μM level. Examples of the use of the method are given for the determination of carboxylic acids in anoxic marine sediment pore waters, coastal sea water and Black Sea water samples.     

Design and synthesis of a hydrophilic fluorescent derivatization reagent for carboxylic acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), and its application to capillary electrophoresis with laser-induced fluorescence detection

A hydrophilic fluorescent derivatization reagent for fatty acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH(2)), was designed and synthesized. NBD-PZ-NH(2) possesses not only a fluorophore and a reacting group but also a positive charge group and, thus, was hydrophilic and suitable for application to capillary electrophoresis. NBD-PZ-NH(2) reacted with fatty acids in the presence of triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS) at room temperature within 10 min. The derivatives were strongly fluoresced and were positively charged at pH below 3. The derivatives of C4-C20 fatty acids were separated within 10 min in 50% acetonitrile in water containing 30 mM ammonium acetate and 1.0 M acetic acid by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The detection limits attained were 6.5 nM (signal-to-noise ratio of 3). It is proposed that NBD-PZ-NH(2) is a prominent derivatization reagent for fatty acids which is suitable for CE-LIF application.     

Determination of Fatty Acids in Three Nitraria Species by Precolumn Fluorescence Labeling for High-Performance Liquid Chromatography and Atmospheric Pressure Chemical Ionization–Mass Spectrometry

A novel fluorescence method using 2-(7-methyl-1H-pyrazolo-[3,4-b] quinoline-1-yl) ethyl-4-methyl benzenesulfonate as the labeling reagent was established for high-performance liquid chromatography determination of fatty acids. The conditions were optimized, including the identity of the organic solvent, identity, and amount of catalyst, amount of derivatization reagent, derivatization temperature, and derivatization time. The results indicated that quantitative yields of derivative were obtained with a five-fold molar excess of reagent at 90°C for 30 min using 25 mg of potassium carbonate as the catalyst. Atmospheric pressure chemical ionization mass spectrometry results indicated that collision-induced dissociation of protonated fatty acid derivatives produced fragments at m/z 228.2, m/z 210.2, and m/z 183.8. The method was validated in terms of linearity, limits of detection, limits of quantification, precision, and accuracy, and the results showed that the method exhibited excellent sensitivity, selectivity, and reproducibility. Limits of detection and limits of quantification were in the ranges of 0.52–2.34 ng mL^?1 and 1.23–6.63 ng mL^?1, respectively. This method was successfully applied to the determination of fatty acids in sarcocarps, seeds, and leaves of Nitraria tangutorum Bobr., Nitraria sibirica Pall., and Nitraria roborowskii Kom. The results indicated that the main components were oleic, linoleic, linolenic, and hexadecanoic acids. However, the composition of fatty acids in the tissues varied considerably.     

A Novel Labeling Reagent of 2-(12-Benzo[b]acridin-5-(12H)-yl)-acetohydrazide for Determination of Saturated and Unsaturated Fatty Acids in Traditional Chinese Herbs by HPLC-APCI-MS

A new fluorescence labeling reagent 2-(12-benzo[b]acridin-5(12H)-yl)-acetohydrazide (BAAH) has been designed for fatty acids labeling. Eleven fatty acids containing seven saturated and four unsaturated fatty acids were used to evaluate the analytical potential of this reagent. The labeling reaction of BAAH with fatty acids was completed at 85?°C for 60?min using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) as the condensing agent. Separation of the derivatized fatty acids was carried out on a reversed-phase Thermo Hypersil Gold C18 column (4.6?mm?×?250?mm, 5?μm) in combination with a gradient elution with a good baseline resolution. The fluorescence excitation and emission wavelengths were set at λ_ex 280 and λ_em 510?nm, respectively. The identification was carried out by the online APCI-MS in positive-ion detection mode. Linear correlation coefficients for all fatty acid derivatives were of >0.9994. Detection limits, at a signal-to-noise ratio of 3:1, were 3.89–12.5?nmol?L^?1 for the labeled fatty acids. The developed method was successfully applied to the accurate determination of fatty acids in five traditional Chinese herbs with satisfactory results.     

Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68 degrees C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at lambda(em) = 460 nm with lambda(ex) = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10-450 and 35-1400 fmol, respectively.     

A pre-column derivatization high-performance liquid chromatography method for simultaneous determination of short-chain and medium-chain fatty acids in a fecal sample

Short-chain and medium-chain fatty acids have plentiful biological functions, which play a crucial role in the diagnosis and therapy of many diseases. Herein, a new method for simultaneous quantifying 17 short-chain and medium-chain fatty acids with high-performance liquid chromatography coupled with an ultraviolet detector was developed and the pre-column derivatization by indole-3-acetic acid hydrazide was performed to improve the separation and detection. The conditions of the derivatization reaction were systematically investigated. Subsequently, the method was validated and the results showed a satisfactory linearity (linear regression coefficients > 0.9969), the limit of detection (4.0×10⁻³–1.9×10⁻² μmol/L), precision (0.9%–7.3% for intra-day and 2.0%–9.8% for inter-day), recovery (90.0%–109.1% with relative standard deviation <7.7%) and stability (0.1%–3.3% for standard solution and 0.2%–3.9% for fecal sample). Finally, the established method was successfully applied to quantify short-chain and medium-chain fatty acids in the feces of healthy control and diabetic rats. Eleven kinds of short-chain and medium-chain fatty acids were detected and six of them showed a significant difference between the control group and the model group.     

6-Oxy-(acetyl ethylenediamine) fluorescein, a novel fluorescent derivatization reagent for carboxylic acids and its application in HPLC

A novel fluorescent derivatization reagent for carboxylic acids, 6-oxy-(acetyl ethylenediamine) fluorescein (AEF), was well designed, synthesized, and applied to HPLC. The derivatization reaction with 12 fatty acids, including n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), and linoleic acid (C18:2), was completed at 55 degrees C within 40 min. The derivatives of fatty acids were separated on a C18 RP column and detected by fluorescence detection. The LODs attained were 0.4-1.2 nM (S/N of 3). It has been demonstrated that AEF is a prominent derivatization reagent for carboxylic acids which is suitable for HPLC.     

Identification of lipid binders in old oil paintings by separation of 4-bromomethyl-7-methoxycoumarin derivatives of fatty acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for identification of drying oils from binding media or protective film used in pictorial works of art prior to conservation or restoration is proposed. Fluorescence derivatization of fatty acids released by hydrolysis of structural drying oils is studied. The derivatization reagent was 4-(bromomethyl)-7-methoxycoumarin with 18-crown-6 as catalyst. Mobile phase was programmed from methanol-water (90:10 v/v) to methanol-water (100:0 v/v) in 25 min. The excitation and emission wavelengths were 325 and 395 nm, respectively. Under these chromatographic conditions, coumarin derivatives of myristic, palmitic, oleic and stearic acids were satisfactorily resolved. The method shows good sensitivity, with a detection limit of 6.0 x 10(-8) mmol, and good linearity between 1.0 x 10(-7) and 1.8 x 10(-4) mmol of each analyte. Peak area ratios among fatty acids derivatives, especially the stearic acid/palmitic acid peak area ratio, are useful to identify the drying oils. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

Optimization of a SPME/GC/MS Method for the Simultaneous Determination of Pharmaceuticals and Personal Care Products in Waters

A headspace solid phase microextraction (HS-SPME) method coupled with gas chromatography and MS detection (GC/MS) was optimized for the simultaneous determination of 21 target Pharmaceuticals and Personal Care Products (PPCPs) in water samples. The analytes included fragrances, UV-filters, antiseptics, estrogens, anti-inflammatory drugs, and pesticides. An on-fiber SPME derivatization, using silyl reagents, was performed for the analysis of more polar acidic compounds. An experimental design approach was applied to systematically investigate and optimize the operative parameters affecting the extraction recovery, namely: extraction temperature and time, derivatization time, desorption temperature and time. The optimum operating conditions were: extraction time of 125?min at a temperature of 40?°C; derivatization time of 30.5?min; desorption time of 2?min at a temperature of 300?°C. Under these conditions, good reproducibility was assessed as RDS% values ≤10% for underivatized PPCPs and ≤20% for derivatized compounds. The method detection limits (LOD) were between 0.7 and 9.0?ng?L^?1, with the highest values in the range 2.5–9.0?ng?L^?1 for the derivatized analytes. Method accuracy was evaluated on spiked tap water samples: recoveries varied from 85 to 103% and from 75 to 110% for non-derivatized and derivatized compounds, respectively.     

Simultaneous Derivatization and Dispersive Liquid–Liquid Microextraction for Fatty Acid GC Determination in Water

Simultaneous derivatization and dispersive liquid–liquid microextraction technique for gas chromatographic determination of fatty acids in water samples is presented. One hundred microlitre of ethanol:pyridine (4:1) were added to 4 mL aqueous sample. Then a solution containing 0.960 mL of acetone (disperser solvent), 10 μL of carbon tetrachloride (extraction solvent) and 30 μL of ethyl chloroformate (derivatization reagent) were rapidly injected into the aqueous sample. After centrifugation, 1 μL sedimented phase with the analytes was analyzed by gas chromatography. The effects of extraction solvent type, derivatization, extraction, and disperser solvents volume, extraction time were investigated. The calibration graphs were linear up to 10 mg L^?1 for azelaic acid (R ² = 0.998) and up to 1 mg L^?1 for palmitic and stearic acids (R ² = 0.997). The detection limits were 14.5, 0.67 and 1.06 μg L^?1 for azelaic, palmitic, and stearic acids, respectively. Repeatabilities of the results were acceptable with relative standard deviations (RSD) up to 13%. A possibility to apply the proposed method for fatty acids determination in tap, lake, sea, and river water was demonstrated.     

柱前衍生化-超高效液相色谱法快速测定酱油中的18种氨基酸

建立了一种6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,超高效液相色谱(UPLC)对酱油中18种氨基酸进行快速分离检测的方法。采用BEH C18色谱柱分离,在260 nm波长下检测,以乙酸铵-乙酸-乙腈-水和乙腈-乙酸为流动相,将流动相梯度和流速梯度相结合,在12 min内实现了18种氨基酸衍生物的分离。方法的线性回归系数(r2)均大于0.999,检出限为0.032～0.12 mg/L,日间相对标准偏差(RSD)为0.72%～4.05%,在酱油中18种氨基酸的加标回收率为90.2%～103.7%。该方法前处理过程简单,分离时间短,是检测酱油中氨基酸的有效手段,可用于酱油的质量评定。     

Simultaneous quantification of fatty acids in serum with dual derivatization by liquid chromatography-tandem mass spectrometry

Fatty acids have multitudinous biological functions and play a crucial role in many biological processes, but due to poor ionization efficiency and lack of appropriate internal standards, the comprehensive quantification of fatty acids by liquid chromatography-tandem mass spectrometry is still challenging. In this study, a new, accurate, and reliable method for quantifying 30 fatty acids in serum using dual derivatization was proposed. Indole-3-acetic acid hydrazide derivants of fatty acids were used as the internal standard and indole-3-carboxylic acid hydrazide derivants of them were used to quantify. The derivatization conditions were systematically optimized and the method validation results showed good linearity with R² > 0.9942, low detection limit (0.03–0.6 nM), precision (1.6%–9.8% for intra-day and 4.6%–14.1% for inter-day), recovery (88.2%–107.2% with relative standard deviation < 10.5%), matrix effect (88.3%–105.2% with the relative standard deviation < 9.9%) and stability (3.4%–13.8% for fatty acids derivants in 24 h at 4°C and 4.2%–13.8% for three freeze-thaw cycles). Finally, this method was successfully applied to quantify fatty acids in serum samples of Alzheimer's patients. In contrast to the healthy control group, nine fatty acids showed a significant increase in the Alzheimer's disease group.     

A fluorimetric liquid chromatography for highly sensitive analysis of very long chain fatty acids as naphthoxyethyl derivatives

Summary A simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with 2-(2-naphtoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were analyzed by HPLC with fluorimetric detection (λex=235 nm; λem=366 nm). The linear range for the determination of docosanoic, tetracosanoic and hexacosanoic acids was 0.028–1.4 μM with a detection limit of about 5.6 nM (S/N=3) (56 fmol per 10 μL injection). Application of the method to the analysis the non-esterified (free) very long chain fatty acids spiked in plasma proved feasible.     

Qualitative/quantitative strategy for the determination of glufosinate and metabolites in plants

A simple method for the simultaneous determination of glufosinate and its metabolites in plants based on liquid chromatography–ultraviolet (LC–UV) absorption detection after derivatization with fluorenylmethoxycarbonyl chloride (FMOC-Cl) of some analytes to facilitate separation is reported here. Nonavailable standard metabolites were identified by LC–TOF/mass spectrometry (MS), which also confirmed all target analytes. Ultrasound-assisted extraction was used for sample preparation (power of 70 W and duty cycle of 0.7 s/s for 10 min) with subsequent evaporation of the extractant, reconstitution and filtration as the cleanup/concentration step prior to derivatization, and chromatographic separation and detection at 270 nm for underivatized analytes and 340 nm for those that were derivatized. The chromatographic analysis was completed in 40 min using a Luna® column (C18 phase). The analytical characteristics of the method were linear dynamic range of the calibration curves within 0.047–700 μg/mL with a regression coefficient (rc) of 0.999 for glufosinate, 0.077–700 μg/mL with a rc of 0.998 for N-acetyl-glufosinate, and 0.116–600 μg/mL with a rc of 0.998 for 3-(methylphosphinico)propanoic acid. The precision for the determination of glufosinate (studied at two levels, 0.1 and 5 μg/mL) was 2.7 and 6.0 % for repeatability and 4.7 and 7.2 % for within-laboratory reproducibility, respectively. Identification and confirmatory analysis of the presence of glufosinate and metabolites in the extracts from treated plants was carried out by LC–TOF/MS in high-resolution mode for the precursor ion. The method was validated by analyzing wheat (Triticum aestivum) samples (resistant and susceptible biotypes) treated with 300 g of glufosinate/ha following conventional agronomical practices.     

Development of a New Pre-Derivatization LC Method for Analysis of Branched-Chain and Aromatic Amino Acids in Rat Plasma and to Monitor their Dynamic Variation as a Result of Acute Hepatic Injury

9,10-Anthraquinone-2-sulfonyl chloride, a new derivatization reagent with strong UV absorbance, has been used to develop a reversed-phase liquid chromatographic method for analysis of branched-chain and aromatic amino acids in rat plasma. Because the reagent has a sulfonyl chloride group it is highly reactive with primary and secondary amino groups and reaction is complete within 5 min at room temperature in phosphate–borate buffer (pH 8.41)–acetonitrile. The resulting adducts were separated on a C₁₈ column at 30 °C and detected at 254 nm. Good linearity was achieved over the concentration range 10–1,000 μmol L^?1 for each amino acid. Intra-day precision (relative standard deviation; RSD, %) and accuracy (bias, %) for all quality-control concentrations, were ≤8.9% and 16.0%, respectively. Inter-day precision and accuracy were ≤9.5% and 11.7%, respectively. The method was used for analysis of branched-chain and aromatic amino acids in the plasma of rats with hepatic injury induced by d-galactosamine.     

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

Atpresentseparationsandquantitativedeterminationsofaminoacidsbymeansofnewfluorescencereagentsforpre-columnorpost-columnderivatizationinRP-HPLCarestillanactivefiled,developmentshavingbeensummarizedbySnyder'.MostaminoacidsdonotshowUVabsorptionin220-254urn,henceinordertoincreasedetectionsensitivityandimproveselectivity,generallyderivatizationreagentsareemployed.Phenylisothiocyanate(PITC)',OPAand3,5-dinitrobenzoylchloride3arewellknownderivatizationreagefltsforthedeterminationofaminocompounds…     

HPLC of amino acids and oligopeptides by pre-column fluorescence derivatization with N-hydroxysuccinimidyl-α-(9-phenanthrene)-acetate

Summary A sensitive LC method for the detection of amino acids and oligopeptides with pre-column fluorescence derivatization has been developed. Glycine, glycylglycine, triglycine, glutathione, glutamic acid, and cysteine were separated on a reversed-phase C₁₈ column with methanol-water-triethylamine eluent, derivatization and chromatographic conditions were optimized. The six derivatives were eluted in 20 min with good reproducibility. The relative standard derviations (n=6) at an analytical concentration of 2×10⁻⁶ M are <5%. Detection limits (signal-to-noise ratio=3) for the six derivatives are 23–68 fmol.