High-performance liquid chromatographic determination of free fatty acids and esterified fatty acids in biological materials as their 2-nitrophenylhydrazides

doi:10.1016/S0003-2670(01)01582-3

Analytica Chimica Acta

Volume 465, Issues 1–2, 16 August 2002, Pages 237-255

https://doi.org/10.1016/S0003-2670(01)01582-3 Get rights and content

Abstract

High-performance liquid chromatography (HPLC) in conjunction with direct derivatization is described for the determinations of both free fatty acids (FFAs) and esterified fatty acids (EFAs) in biological materials. The method is based on the reaction of these acids with acidic 2-nitrophenylhydrazine hydrochloride (2-NPH·HCl) with and without saponification of the samples, and there are no sample work-up steps. The derivatized FAs were extracted into n-hexane and separated isocratically with short retention times. These acids were consisted saturated and mono- and polyunsaturated FAs including cis–trans isomers and double-bond positional isomers. The analytical results showed good recovery and reproducibility using an internal standard. The method is simple, rapid and reliable and has several advantages with regard to resolution, analysis time, selectivity and sensitivity over previous methods. Thus, the present method can serve as a useful tool for routine determinations of FFAs and EFAs in various fields.

Introduction

The development of an analytical method for the routine simultaneous identification and quantification of a variety of fatty acids (FAs) is desirable for use in various fields. The determination of FAs has been developed mainly by gas chromatography (GC) of methyl esters [1], [2], [3], [4], [5], a technique introduced in 1952 by James and Martin [6]. As an alternative to GC, liquid chromatography (LC) has better sensitivity and selectivity. However, most FAs show no useful absorption in the visible and ultraviolet (UV) regions or no fluorescence for detection in high-performance liquid chromatography (HPLC). Therefore, several HPLC methods have been developed for the analysis of saturated and unsaturated FAs, employing pre-column derivatization techniques to increase the sensitivity and selectivity of detection [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48].

In most of these methods, one drawback is that lengthy and cumbersome clean-up procedures, such as liquid–liquid extraction or extrelut disposable column extraction, are needed for the quantitative isolation of the FAs from the biological materials prior to the derivatization. Another drawback is that, in spite of a fairly long analysis time (40–70 min), simultaneous measurements of biologically important polyunsaturated FAs, such as α-linolenic acid (n=3), γ-linolenic acid (n=6), eicosapentaenoic acid (n=3) (EPA), arachidonic acid (n=6) (AA) and docosahexaenoic acid (n=3) (DHA) could not been achieved. Therefore, the establishment of a simpler and more rapid method with complete separation capability is needed for the screening of large numbers of samples.

We have developed the utility of the reagent 2-nitrophenylhydrazine hydrochloride (2-NPH·HCl) for the derivatization of various carboxylic acids including FAs, and their separation and quantitation by HPLC in various fields [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65]. I have already reviewed the HPLC determination of mono-, poly- and hydroxycarboxylic acids in foods and beverages as their 2-nitrophenylhydrazides [66]. The present review demonstrates the direct derivatization of free fatty acids (FFAs) in serum and esterified fatty acids (EFAs) in platelet phospholipids with 2-NPH·HCl without any pre-treatment and/or extraction steps, and the determinations of the acid hydrazides using a reversed-phase HPLC method with simple isocratic elution systems.

Section snippets

Reagent solutions

Phospholipid standards, l-α-phosphatidylcholine (PC), l-α-phosphatidylethanolamine (PE), l-α-phosphatidyl-l-serine (PS), l-α-phosphatidylinositol (PI), l-α-phosphatidyl-dl-glycerol (PG), l-α-lysophosphatidylcholine, sphingomyelin, cardiolipin and triglycerides (TG) were purchased from Sigma (St. Louis, MO, USA). All FA solutions in ethanol were obtained from Yamamura Chemical Laboratories (Kyoto, Japan). 2-NPH·HCl (Tokyo Kasei Kogyo, Tokyo, Japan) solutions (20 mM) were prepared by dissolving

Instrumentation

Chromatographic analyses were carried out using a Shimadzu LC-6A liquid chromatograph (Shimadzu Seisakusho, Kyoto, Japan) equipped with an on-line degasser ERC-3310 (Erma, Tokyo, Japan) and a Shimadzu SPD-6AV variable-wavelength UV–VIS detector. The detector signals were recorded on a Rikadenki multi-pen recorder (Tokyo, Japan). The column temperature was kept constant at 30–50 °C using a Shimadzu GTO-6A column oven. All columns were packed at Yamamura Chemical Labs.

HPLC conditions

The separation of 15 FA

Calculation

Calibration curves were constructed by derivatizing increasing amounts of FAs in the presence of margaric acid as the internal standard and analyzing as described above. The calibration test was replicated five times. From the chromatograms obtained, the relationships between the peak-height ratios of the acid hydrazides to that of the internal standard and the concentrations of the acids were calculated by the least-squares method. Previous works [51], [52], [58], [59], [62] demonstrated that

Determination of FFAs in human serum

FFAs in blood, derived mainly from triacylglycerols in adipose tissues by the action of hormone-sensitive lipase or from lipoprotein fractions by the action of lipoprotein lipase, play a central role in energy metabolism. The monitoring of individual FA levels in serum or plasma is known to be useful in the management of patients with several diseases, such as diabetes mellitus [78], thyremphraxis and hepatic dysfunction [79].

To confirm the clinical utility, serum samples from 16 fasting normal

Conclusions

The FFAs in serum can be reacted directly with 2-NPH·HCl without hydrolytic or oxidative degradation. This method also allows the direct derivatization of EFAs after saponification of platelet phospholipids, and there are no necessary of cumbersome and rigorous sample work-up steps. The HPLC analysis described here permit the isocratic separation of saturated and mono- and polyunsaturated FAs including cis–trans isomers and double-bond positional isomers in samples with good accuracy,

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ReviewHigh-performance liquid chromatographic determination of free fatty acids and esterified fatty acids in biological materials as their 2-nitrophenylhydrazides

Abstract

Introduction

Section snippets

Reagent solutions

Instrumentation

HPLC conditions

Calculation

Determination of FFAs in human serum

Conclusions

J. Chromatogr.

Chem. Phys. Lipids

Chem. Phys. Lipids

J. Chromatogr.

Anal. Chim. Acta

J. Chromalogr.

J. Chromatogr.

J. Chromatogr.

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J. Chromatogr.

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Anal. Biochem.

Clin. Chim. Acta

J. Chromatogr.

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J. Chromatogr.

J. Chromalogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr. B

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Clin. Chim. Acta

J. Chromatogr., Biomed. Appl.

J. Chromatogr., Biomed. Appl.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr., Biomed. Appl.

J. Chromatogr. A

J. Chromatogr. B, Biomed. Appl.

J. Chromatogr. B, Biomed. Appl.

J. Chromatogr. A

J. Chromatogr.

Review
High-performance liquid chromatographic determination of free fatty acids and esterified fatty acids in biological materials as their 2-nitrophenylhydrazides