Unique biomedical application of fluorescence derivatization based on palladium-catalyzed coupling reactions for HPLC analysis of pharmaceuticals and biomolecules.

Palladium-catalyzed coupling reactions are versatile and powerful tools for the construction of carbon-carbon bonds in organic synthesis. Although these reactions have favorable features that proceed selectively in mild reaction conditions using aqueous organic solvents, no attention has been given to their application in the field of biomedical analysis. Therefore, we focused on these reactions and evaluated the scope and limitations of their analytical performance. In this review, we describe the pros and cons and future trends of fluorescence derivatization of pharmaceuticals and biomolecules based on palladium-catalyzed coupling reactions such as Suzuki-Miyaura coupling, Mizoroki-Heck coupling, and Sonogashira coupling reactions for HPLC analysis.

Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application.

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin-avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0-100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%).

A Turn-On Quinazolinone-Based Fluorescence Probe for Selective Detection of Carbon Monoxide.

Carbon monoxide (CO) is a toxic, hazardous gas that has a colorless and odorless nature. On the other hand, CO possesses some physiological roles as a signaling molecule that regulates neurotransmitters in addition to its hazardous effects. Because of the dual nature of CO, there is a need to develop a sensitive, selective, and rapid method for its detection. Herein, we designed and synthesized a turn-on fluorescence probe, 2-(2'-nitrophenyl)-4(3H)-quinazolinone (NPQ), for the detection of CO. NPQ provided a turn-on fluorescence response to CO and the fluorescence intensity at 500 nm was increased with increasing the concentration of CO. This fluorescence enhancement could be attributed to the conversion of the nitro group of NPQ to an amino group by the reducing ability of CO. The fluorescence assay for CO using NPQ as a reagent was confirmed to have a good linear relationship in the range of 1.0 to 50 µM with an excellent correlation coefficient (r) of 0.997 and good sensitivity down to a limit of detection at 0.73 µM (20 ppb) defined as mean blank+3SD. Finally, we successfully applied NPQ to the preparation of a test paper that can detect CO generated from charcoal combustion.

Determination of Anthraquinone-Tagged Amines Using High-Performance Liquid Chromatography with Online UV Irradiation and Luminol Chemiluminescence Detection.

Quinones are frequently used as derivatization reagents in HPLC analysis to improve detection sensitivity. In the present study, a simple, sensitive, and selective chemiluminescence (CL) derivatization strategy for biogenic amines, prior to their HPLC-CL analysis, was developed. The novel CL derivatization strategy was established based on using anthraquinone-2-carbonyl chloride as derivatizing agent for amines and then using the unique property of the quinones' moiety to generate reactive oxygen species (ROS) in response to UV irradiation. Typical amines such as tryptamine and phenethylamine were derivatized with anthraquinone-2-carbonyl chloride and then injected into an HPLC system equipped with an online photoreactor. The anthraquinone-tagged amines are separated and then UV-irradiated when they pass through a photoreactor to generate ROS from the quinone moiety of the derivative. Tryptamine and phenethylamine can be determined by measuring the chemiluminescence intensity produced by the reaction of the generated ROS with luminol. The chemiluminescence disappears when the photoreactor is turned off, suggesting that ROS are no longer generated from the quinone moiety in the absence of UV irradiation. This result indicates that the generation of ROS could be controlled by turning the photoreactor on and off. Under the optimized conditions, the limits of detection for tryptamine and phenethylamine were 124 and 84 nM, respectively. The developed method is successfully applied to determine the concentrations of tryptamine and phenethylamine in wine samples.

Development of a Selective Assay of Tyrosine and Its Producing and Metabolizing Enzymes Utilizing Pulse-UV Irradiation-Induced Chemiluminescence.

A new pulse UV irradiation-induced chemiluminescence (CL) determination method was developed for l-tyrosine using the luminol derivative L-012. The proposed method depends on the formation of reactive oxygen species (ROS) upon pulse UV irradiation of l-tyrosine; then, these ROS react with L-012 producing strong CL. The proposed method showed excellent sensitivity and ultraselectivity toward l-tyrosine. The mechanism of the developed CL method was studied using ROS scavengers, HPLC, and mass spectrometry. The method was linear for l-tyrosine in the range of 0.03-50 µM. Minor changes in the l-tyrosine structure, including hydroxylation, dehydroxylation, phosphorylation, or decarboxylation, were found to lead to a strong decrease in CL. Using the excellent selectivity of the proposed method for l-tyrosine, we have developed a CL assay for measuring alkaline phosphatase activity in the range of 0.02-15 U/L with the limit of detection (LOD) of 4 mU/L using the nonchemiluminescent O-phospho-l-tyrosine as a substrate. Furthermore, the CL reaction was applied for tyrosinase activity assay as this enzyme can convert l-tyrosine to the nonchemiluminescent l-dopa. The decrease in CL is correlated with the tyrosinase activity in the range of 0.025-0.75 U/mL with an LOD of 1.5 mU/mL. Moreover, the tyrosinase activity assay was successfully applied for the determination of IC50 of the tyrosinase inhibitors kojic acid and benzoic acid. Therefore, our novel pulse UV irradiation CL method for the determination of l-tyrosine was not only suitable for the determination of this vital amino acid but also extended to the successful determination of its producing and metabolizing enzymes and their inhibitors.

HPLC Fluorescence Method for Eugenols in Basil Products Derivatized with DIBI.

Eugenols (Eugs) such as eugenol (Eug), methyleugenol (MeEug), and linalool (Lin) in basil product are the main bioactive components of basil products and have a terminal double-bond. A sensitive HPLC-fluorescence method for Eugs derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) was developed. Good separation of DIB-Eugs was achieved within 20 min on an Atlantis T3 column (50 × 2.1 mm i.d., 3 µm) with a mobile phase of methanol-water. The calibration curves obtained with Eug standards showed good linearities in the range of 0.1-50 µM (r ≥ 0.999). The limits of detection at a signal-to-noise ratio (S/N) = 3 for Eug, MeEug, and Lin were 1.0, 6.0, and 4.8 nM, respectively. The limits of quantitation (S/N = 10) of the Eugs were lower than 19.9 nM. The accuracies for the Eugs were within 96.8-104.6%. The intra- and inter-day precisions as relative standard deviations for the Eugs were less than 1.2 and 9.6% (n = 3). The recoveries of Eug, MeEug, and Lin were 99.0 ± 0.1, 98.0 ± 0.2, and 96.0 ± 0.4% (n = 3), respectively. The DIB-Eugs were confirmed to be stable for 2 h (>90%) at room temperature and 24 h (>95%) at 4 °C. These parameters of the proposed method were useful for the simultaneous determination of Eugs in basil products. Therefore, the developed method may be a powerful tool for the quality evaluation of dried commercially available basil products.

Determination Method for Pyrroloquinoline Quinone in Food Products by HPLC-UV Detection Using a Redox-Based Colorimetric Reaction.

We have developed an HPLC-UV method for the determination of pyrroloquinoline quinone (PQQ), which utilizes a redox-based colorimetric reaction. In the proposed colorimetric reaction, the redox reaction between PQQ and dithiothreitol generates superoxide anion radicals that can convert 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazan dye. After PQQ separation on an octadecyl silica column, it was mixed online with dithiothreitol and INT, and the formed formazan dye was monitored by absorbance at 490 nm. The detection limit (S/N = 3) of the proposed method was 7.6 nM (152 fmol/injection). The proposed method could selectively detect PQQ in food products without any clean-up procedures.

A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction.

Ubiquinone (UQ) is considered one of the important biologically active molecules in the human body. Ubiquinone determination in human plasma is important for the investigation of its bioavailability, and also its plasma level is considered an indicator of many illnesses. We have previously developed sensitive and selective chemiluminescence (CL) method for the determination of UQ in human plasma based on its redox cycle with dithiothreitol (DTT) and luminol. However, this method requires an additional pump to deliver DTT as a post-column reagent and has the problems of high DTT consumption and broadening of the UQ peak due to online mixing with DTT. Herein, an HPLC (high-performance liquid chromatography) system equipped with two types of online reduction systems (electrolytic flow cell or platinum catalyst-packed reduction column) that play the role of DTT was constructed to reduce reagent consumption and simplify the system. The newly proposed two methods were carefully optimized and validated, and the analytical performance for UQ determination was compared with that of the conventional DTT method. Among the tested systems, the electrolytic reduction system showed ten times higher sensitivity than the DTT method, with a limit of detection of 3.1 nM. In addition, it showed a better chromatographic performance and the best peak shape with a number of theoretical plates exceeding 6500. Consequently, it was applied to the determination of UQ in healthy human plasma, and it showed good recovery (≥97.9%) and reliable precision (≤6.8%) without any interference from plasma components.

A Smart Advanced Chemiluminescence-Sensing Platform for Determination and Imaging of the Tissue Distribution of Natural Antioxidants.

Antioxidants have gained marked attention owing to their ability to prevent the oxidation of biological components and to protect the body from reactive oxygen species, thereby maintaining human health. Thus, antioxidant-rich dietary supplements and natural foods can be effective against oxidative stress and can even act as chemopreventive agents. Therefore, a simple and rapid assay for evaluation of antioxidant capacity and assessment of their distribution profile in natural sources is vital. Herein, we report a rapid, innovative chemiluminescence (CL) platform for evaluation and visualization of antioxidant capacity. We found that intense and long-lasting CL was formed upon the redox reaction of quinones, e.g., menadione, with antioxidants, e.g., l-ascorbic acid, in the presence of luminol. The produced CL intensities were proportional to the antioxidants' concentrations with a detection limit of 0.18 µM for the model antioxidant, l-ascorbic acid. As the formed CL was long-lasting, it could be easily captured and detected with a charge-coupled device (CCD) camera. To evaluate the quantification ability of the CCD camera, we developed a smart and fast microplate-based assay based on photographing the generated CL with a cooled CCD camera. The photographed CL intensities were linearly proportional with the antioxidant concentrations, and then the method was applied for photographing multiple food sample extracts. Ultimately, we utilized our method for the distribution profiling of antioxidant capacity in food cut sections. Samples were dipped in luminol and then in quinone, followed by CCD camera photography, without the need for any pulverization/extraction procedure, giving precise antioxidant distribution information.

Current trends in isotope-coded derivatization liquid chromatographic-mass spectrometric analyses with special emphasis on their biomedical application.

Currently, LC-MS has various applications in different areas such as metabolomics, pharmacokinetics, and pathological studies. Yet, matrix effects resulting from co-existing constituents remain a major problem for LC-MS [or LC-tandem mass spectrometry (LC-MS/MS)]. Moreover, technical problems and instrumental drifts may lead to ion abundance variance. Thus, an internal standard (IS) is required to guarantee the accuracy and precision of the method. Because of their limited number, isotope-coded derivatization (ICD) has been recently introduced to overcome this problem. For ICD, a stable heavy isotope-coded moiety is used for labeling the standard or the control sample and the formed products can act as ISs. A light form of the reagent is used for labeling the sample. Then, both are mixed and analyzed by LC-MS(/MS). This strategy permits the identification of different unknown analytes including potential metabolites and disease biomarkers. All these attributes lead to persistent growth in the applications of ICD LC-MS(/MS) in various biomedical branches. In this article we review the ICD methods published in the last eight years for biomedical applications as well as briefly summarize other applications for environmental and food analyses as some of their used ICD reagents were further applied for analyzing biological specimens or have the potential for that.

Novel Isotope-Coded Derivatization Method for Aldehydes Using 14N/15N-Ammonium Acetate and 9,10-Phenanthrenequinone.

Isotope-coded derivatization (ICD) is used as a promising alternative approach to isotope internal standards in order to overcome matrix effects caused by coexisting substances that often occur while analyzing metabolites by LC-MS/MS. ICD introduces two different mass tags to every analyte via the use of heavy and light forms of the derivatization reagents. Herein, we report the first ICD approach for aldehydes that uses commercially available reagents avoiding the need for expensive and tedious multisteps synthetic procedures. The method is based on the reaction of the safe and stable derivatizing agent, 9,10-phenanthrenequinone, and the cheap and commercially available ICD reagent, 14N/15N-ammonium acetate, with aldehydes followed by LC/ESI+-MS/MS. Multiple reaction monitoring is done at the transitions m/ z [M + H]+ → m/ z [Product ion A] and m/ z [M + 2 + H]+ → m/ z [Product ion A + 2] for 14N- and 15N-labeled analytes, respectively. Among lipid peroxidation products, 4-hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexenal (HHE) are considered the most toxic produced aldehydes as they contain additional two reactive functional groups, the unsaturated bond and the hydroxyl group, besides the aldehyde one. Thus, they were chosen as representative analytes in this study. The developed method was able to detect HHE and HNE in human serum with very high sensitivity down to LOQ of 0.2 and 0.05 nM, respectively, employing an expedient salting out liquid-liquid extraction method. The developed method was able to differentiate between the levels of HHE and HNE in serum samples of healthy subjects and diabetic, rheumatic, and cardiac disorder patients.

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High-Performance Liquid Chromatography with Fluorescence Detection after pre-Column Derivatisation.

INTRODUCTION: Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen. OBJECTIVE: To develop a sensitive and selective analytical method for tanshinones by high-performance liquid chromatography (HPLC) with fluorescence detection after pre-column derivatisation. METHODOLOGY: The proposed method depends on derivatisation reaction of tanshinones with 4-carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative. RESULTS: The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen. CONCLUSION: The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o-quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.

Immune complexome analysis reveals the specific and frequent presence of immune complex antigens in lung cancer patients: A pilot study.

Cancer immunotherapies such as antibodies targeting T cell checkpoints, or adaptive tumor-infiltrating lymphocyte (TIL) transfer, have been developed to boost the endogenous immune response against human malignancies. However, activation of T cells by such antibodies can lead to the risk of autoimmune diseases. Also, the selection of tumor-reactive T cells for TIL relies on information regarding mutated antigens in tumors and does not reflect other factors involved in protein antigenicity. It is therefore essential to engineer therapeutic interventions by which T cell reactivity against tumor cells is selectively enhanced (i.e., "focused cancer immunotherapy") based on tumor antigens that are specifically expressed in the tumor of a certain cancer and in many patients with this cancer. Immune complexes (ICs) are the direct and stable products of immunological recognition by humoral immunity. Here, we searched for tumor-specific IC antigens in each of five cancers (lung (n = 28), colon (n = 20), bladder (n = 20), renal cell (n = 15) and malignant lymphoma (n = 9)), by using immune complexome analysis that comprehensively identifies and profiles the constituent antigens in ICs. This analysis indicated that gelsolin and inter-alpha-trypsin inhibitor heavy chains were specifically and frequently detected (at a frequency higher than 80%), and that phosphoproteins (VENTX, VCIP135) were also specifically present in the ICs of lung cancer patients. Immune complexome analysis successfully identified several tumor-specific IC antigens with high detection frequency in lung cancer patients. These specific antigens are required to validate the clinical benefit by further analysis using a large number of patients.

Spatial correlativity of atmospheric particulate components simultaneously collected in Japan.

The simultaneous sampling of total suspended particles was performed at 14 sites in Japan during July 2008-June 2009. The spatial correlativity of each particulate composition toward Osaka was obtained for nine selected sites to overview the chemical composition and geographical distribution of particulate components across a wide range of areas nationwide. The spatial correlatives of atmospheric particulate components were extended to an even wider range of areas up to 950 km distance (meso-alpha scale region, >200 km) for a far-reaching distance analysis unique in the literature. Overall, the spatial correlations of ionic species and both organic and elemental carbons were significant, suggesting their shared advections, including their long-range transport from East Asia. Although sulfate ions are widely dispersed across Japan, such is not necessarily correlated with organic and elemental carbon, possibly indicating that the sulfate emission source, including long-range transport, differs from that of carbonaceous particulates. By contrast, the characteristics of spatial correlatives of metallic constituents vary; for example, particulate Pb and Cd show a significantly wide range of spatial correlatives to Osaka, while Mn-though limited to cities neighboring Osaka-shows significant spatial correlations. Other metallic constituents showed no significant spatial correlatives, indicating the effects of local pollutants. Moreover, the extent of the spatial dispersion of the particulate components and the relationships among chemical components were analyzed via factor analysis to highlight the effects of long-range inflow and local original emissions. In this treatment, 13 particulate components among the 19 measured were implicated in long-range transport.

Immune complexome analysis of antigens in circulating immune complexes isolated from patients with IgG4-related dacryoadenitis and/or sialadenitis.

OBJECTIVES: IgG4-related disease (IgG4-RD) is characterized by various serological abnormalities. Some patients with IgG4-RD present with hypergammaglobulinemia, hypocomplementemia, and autoantibodies recognizing rheumatoid factors and nuclear factors. However, whether IgG4-RD is an autoimmune disease remains unclear. METHODS: Here, we used immune complexome analysis to comprehensively identify constituent antigens of circulating immune complexes isolated from 10 patients with IgG4-related dacryoadenitis and/or sialadenitis (IgG4-RDS) which is one condition associated with IgG4-RD. RESULTS: We detected each of 125 distinct antigens in independent samples from two or more patients with IgG4-RDS. Of them, 17 antigens were found to be specific to patients with IgG4-RDS by comparing 125 antigens with all the antigens found in other connective tissue diseases (antineutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus) or healthy donors. The number of disease-specific antigens associated with IgG4-RDS was comparable to those autoimmune diseases. CONCLUSIONS: Studies of the 17 IgG4-RDS-specific antigens might lead to a better understanding of the pathogenesis of IgG4-RD, but further study of the prevalence of these CIC-associated antigens that involves a selective and sensitive assay will be needed to determine their potential as diagnostic or pathogenic biomarkers.

Quinones as novel chemiluminescent probes for the sensitive and selective determination of biothiols in biological fluids.

Altered plasma aminothiol concentrations are thought to be a valuable risk indicator and are interestingly utilized for routine clinical diagnosis and for the monitoring of various metabolic disorders and human diseases, and accordingly there is a need for an accurate and reliable assay capable of simultaneously determining aminothiols including glutathione (GSH), N-acetylcysteine (NAC), homocysteine (Hcys), and cysteine (Cys) in human plasma. Herein, a highly sensitive, selective, and very fast HPLC-chemiluminescence (HPLC-CL) coupled method is reported, exploiting for the first time the strong nucleophilicity and high reactivity of aminothiols toward quinones for a CL assay. The unique redox-cycling capability of quinone and/or Michael addition adducts, thioether-quinone conjugates, was utilized to establish a novel analytical method based on the reaction of adducts with dithiothreitol (DTT) to liberate reactive oxygen species (ROS), which are detected by using a luminol-CL assay. Specimen preparation involved the derivatization of aminothiols with menadione (MQ) for 5 minutes at room temperature. A unique green chemistry synthesis of thioether-quinones in HEPES buffer (pH 8.5) was introduced by using our reaction methodology without needing any hazardous organic solvent or catalyst. The aminothiol-MQ adducts were separated using solid-phase extraction followed by isocratic elution on an ODS column. Linearity was observed in the range of 2.5-500, 5-500, 10-1500, and 20-2000 nM with detection limits (S/N of 3) of 3.8, 4.2, 8, and 16 (fmol per injection) for GSH, NAC, Hcys, and Cys, respectively. The method was successfully applied for the selective determination of aminothiols in human plasma from healthy people and patients with rheumatic arthritis and diabetes mellitus. The obtained results postulated the usefulness of our method for investigating the relationship between aminothiol metabolism and related human disorders.

Poly(L-lactic acid)-modified silica stationary phase for reversed-phase and hydrophilic interaction liquid chromatography.

Poly(L-lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(L-lactic acid)-modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(L-lactic acid) chain. The poly(L-lactic acid)-silica column was characterized in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(L-lactic acid)-silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited "U-shaped" curves, which was an indicator of reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention behavior. In addition, carbonyl groups included into the poly(L-lactic acid) backbone work as an electron-accepting group toward a polycyclic aromatic hydrocarbon and provide π-π interactions.

Determination of acrolein in serum by high-performance liquid chromatography with fluorescence detection after pre-column fluorogenic derivatization using 1,2-diamino-4,5-dimethoxybenzene.

Acrolein is a major unsaturated aldehyde that is generated during the lipid peroxidation process. The measurement of acrolein in biological samples should be useful to estimate the degree of lipid peroxidation and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liquid chromatography with fluorescence detection method was developed for the determination of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent derivative of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein precipitation treatment.

Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography-fluorescence detection.

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2'-furil to form fluorescent difurylimidazole derivatives that were separated on a C18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355nm, respectively. The method showed good linearity over the concentration ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions.