Dansyl Hydrazine Research Articles

Profiling organic pollutants in environmental water by dansylation-based non-targeted liquid chromatography-high resolution mass spectrometry analysis

Monitoring organic pollutants in rainwater is important to understand relations between air pollutants and water safety. Non-targeted liquid chromatography–mass spectrometry (LC–MS) is a powerful tool but because of big differences of pollutants in polarity, ionization efficiency, and low concentrations, it is challenging to detect all pollutants in a single analysis. Chemical derivatization is a widely adapted strategy to fractionate complex samples with enhanced sensitivity and selectivity. Herein, we propose the usage of dansylation as a chemical derivatization method to improve both LC retention and MS ionization of organic pollutants containing amine, hydroxyl, and carboxyl for non-targeted LC–MS analysis. We first evaluated the labeling coverage of dansylation to organic pollutants in water matrix. Using dansyl chloride (DnsCl) and dansyl hydrazine (DnsHz) to label 100 amine- and hydroxyl-containing compounds and 100 carboxyl compounds, respectively, we found DnsCl and DnsHz had over 60% labeling coverage for 8 categories of compounds. Then dansylation was applied to label the rainwater, source water, disinfected rainwater, and drinking water samples. To facilitate the annotation of dansylated compounds, we also established a web-based tool termed DansylFinder. Using DansylFinder, 3889, 5813, 6077, and 4050 tentative annotations were found in rainwater, source water, disinfected rainwater, and drinking water samples by dansylation-based non-targeted LC-HRMS analysis. Four hundred fifty four were persistently detected in all water samples, suggesting significant organic overlaps of the four water samples. In addition, two degradation pathways reported in drinking water disinfection process were also detected in disinfected rainwater, suggesting rainwater is a potential path for air pollutants to infiltrate drinking water system.

A Julolidine Aldehyde Dansyl Hydrazine Schiff Base as Fluorescence Chemosensor for Zn2+ ions Recognition and its Application.

The Schiff base fluorescent probe (Dz-Jul), containing julolidine aldehyde and dansyl hydrazine, was derived using a simple condensation. This chemosensor showed high selectivity towards Zn2+ and quick response (170s) in DMSO/H2O solutions (8/2, v/v, pH 7.2 buffer). A fluorometric titration determined that Dz-Jul-Zn2+ has a binding ratio of 1:1, and the association constant (Ka) is 1.03 × 105M-1. The Dz-Jul detection limit of Zn2+ ions was 15nM, much lower than the WHO standard (76.0nM). DFT, ESI mass, and FTIR spectral demonstrated a plausible complexation mode between Dz-Jul and Zn2+ ions. In actual water samples, Zn2+ has been detected with good detection performance using Dz-Jul. Additionally, Dz-Jul-coated test strips allowed for rapid and qualitative monitoring of Zn2+ ions in a visible manner.

A versatile internal filtration effect-based ratiometric probe for fluorescence and naked eye sensing of 2,2′,4,4′,6,6′-hexanitrostilbene

2,2′,4,4′,6,6′-hexanitrostilbene (HNS) detection is an essential issue in homeland security screening and environment monitoring. Herein, Mn doped ZnS quantum dots (QDs), ethanolamine (EA) and dansyl hydrazine (DNSH) were proposed as ratiometric probes (DNQDs) for fluorescent (FL), visual FL (VFL), and naked-eye-based visual (NEBV) detection of HNS on surfaces and in water. The amino-HNS complex was formed due to the reaction between HNS and EA after adding HNS into DNQDs. The complex could quench the FL emission of the QDs and DNSH with different rates because of the internal filtration effect. HNS sensing was acquired by comparing the ratio change between their FL intensities. Meanwhile, DNQDs could be used for NEBV sensing of HNS by recognizing the orange-red colored complex. DNQDs demonstrated a good sensitivity: the lowest detectable concentrations/amounts of FL, VFL, and NEBV methods for HNS in water were 0.025, 2.5, and 5 mg/L, and those of VFL and NEBV methods for HNS on surfaces were 0.64 and 1.59 μg/mm2, respectively. Finally, DNQDs were successfully utilized for HNS detection on palm and travel bag surfaces as well as in environmental water. DNQDs-based approaches might be a promising potential for HNS analysis owing to the high sensitivity and versatility.

Profiling of carbonyl compounds in fresh cabbage with chemometric analysis for the development of freshness assessment method

Abstract The aim of this study was to screen the distribution of carbonyl compounds (CCs) in fresh cabbage as a sample model of multi-layered leafy vegetables for discovering the universal freshness marker of fresh produces. The distribution of CCs was observed in the three outer leaf layers of the cabbage. The profile of CCs in each leaf will guide the selection of which leaf is the appropriate part to be used to further discover a freshness marker of cabbage during storage treatment at the postharvest stage. The carbonyl compounds in each leaf were extracted using a mixture of chloroform and methanol (2:1). The extracted CCs from samples were derivatized with dansyl hydrazine. A high-performance liquid chromatography-mass spectrometer with multiplexed multiple reactions monitoring (MRM) was used for the comprehensive detection of carbonyl compounds. More than 400 peaks were observed in the MRM chromatogram from all leaves. The distribution of m/z values that represent CCs were analyzed employing the principle component analysis-discriminant analysis by relating it to the leaf position. The distribution of CC was different for each leaf where the leaves of the second and third layers were similar and significantly different from the leaves of the first layer. The accumulation of trans-2-hexenal was dominant in the first layer; therefore, the utilization of the first layer is not suitable as the part of the sample to discover the freshness marker of multi-layered leafy vegetables.

Ratiometric fluorescence sensing of hypochlorite ion by dansyl hydrazine - Spectroscopic and TD-DFT studies

Probe dansyl hydrazine (DH) exhibits highly selective and sensitive fluorescence detection properties towards hypochlorite (OCl−) ions in DMSO and aq. DMSO media. The probe comprises a fluorophore N,N-dimethylnaphthalene unit and an electron-withdrawing –SO2NHNH2 moiety attached to it. Titration of OCl− ions with DH caused C-S bond cleavage reaction that enhanced the fluorescence of DH with a blue shift. 1H NMR and HRMS spectral techniques were used to confirm the OCl−ion-triggered C-S bond cleavage mechanism. Based on DH's significant fluorescence enhancement response to OCl− ions, the detection limit was estimated as low as 18 µM. Moreover, computational density functional theory (DFT) and time-dependant density functional theory (TD-DFT) calculations provided more insight into the probe's absorption and emission spectral properties before and after the addition of OCl− ions. Additionally, the probe DH would be a potential tool for detecting OCl− ion concentration in bleaching powder.

Online coupling of matrix solid-phase dispersion to direct analysis in real time mass spectrometry for high-throughput analysis of regulated chemicals in consumer products

The current work is the first study on online coupling of matrix solid-phase dispersion (MSPD) to direct analysis in real time mass spectrometry (DART-MS) bridging with solid-phase analytical derivatization (SPAD) based on a graphene oxide nanosheets (GONs)-coated cotton swab. Proof-of-concept demonstrations were explored for high-throughput analysis of a diversity of regulated chemicals in consumer products such as textiles, toys, and cosmetics. On-demand sorbent combinations were blended with samples, packed into MSPD columns, and mounted on a homemade 3D-printed rack module for automated sample feeding. To achieve good synergy between MSPD and DART-MS, a cotton swab with a conical tip deposited with GONs was attached to the bottom of the MSPD column. The swabs serve as a solid-phase microextraction probe for convenient enrichment of the eluted analytes from MSPD, thermal desorption of the enriched analytes by DART, and sensitive detection by a hybrid quadrupole-Orbitrap mass spectrometer. Furthermore, the utility of an on-swab SPAD strategy was demonstrated for the detection of formaldehyde by use of the derivatizing reagent of dansyl hydrazine, contributing to improved ionization efficiency without compromising the overall coherence of the analytical workflow. The MSPD-DART-MS methodology was systematically optimized and validated, obtaining acceptable recovery (71.7–110.3%), repeatability (11.8–19.3%), and sensitivity (limits of detection and quantitation in the ranges of 6.2–19.5 and 23.7–75.9 μg/kg) for 32 target analytes. The developed protocol streamlined sample extraction, clean-up, desorption, ionization, and detection, highlighting the appealing potential for high-throughput analysis of samples with complex matrices.

Rapid determination of aldehydes in food by high-throughput reactive paper spray ionization mass spectrometry

Due to endogenous existence and hazardous properties, the quantification of fatty and aromatic aldehydes in food matrices is indispensable to food safety. Current analytical methodologies for aldehydes in food suffer from several drawbacks (typically complicated pretreatment, long analysis time and low analytical throughput, low sensitivity, etc.). In this work, a highly sensitive, high-throughput and sample pretreatment-free method for aldehyde analysis was developed to address the above problems, where in situ extraction and derivatization of aldehydes with hydrazines to hydrazones were integrated into reactive paper spray ionization mass spectrometry (reactive PSI-MS) considering weakly ionizable properties of aldehydes. Four aldehydes, formaldehyde, acetaldehyde, propionaldehyde and benzaldehyde were reacted with dansyl hydrazine in 1.5 % v/v HAc acidified methanol on the qualitative fast-speed filter paper for 5 min, and then a voltage (+4 kV) was applied to initiate paper spray for mass spectrometry detection with internal calibration by dansyl amide. Once the completion of MS analysis, the glass plate was moved to introduce the second triangular tip towards the MS inlet (5 mm apart) for next trial, achieving high-throughput reactive PSI-MS analysis (60 h−1). The four aldehydes showed good linearity in the concentration range from 2 to 150 μM with low detection limits of 0.03–0.15 μM. The method was applied to direct quantification of four aldehydes in 15 kinds of foods such as vegetables, fruits and meat products. The proposed method has the advantages of simple operation, high efficiency and high sensitivity, making it attractive to routine analysis of aldehydes in food, environmental and biological samples.

Stable isotope labeling method with sensitive identification and accurate quantitation function for aldehydes in fried foods

Deuterated dansylhydrazine (d6-DH) was prepared by one step simple reaction, and applied to the stable isotope labeling (SIL) of aldehydes with the aid of commercially available dansylhydrazine (DH). Common multiple reaction monitoring (MRM) parameters was obtained by a few standard samples and then applied to other aldehydes. Hence levels of unknown aldehydes in real samples was identified using the sensitive MRM mode but not the traditional scan mode. The SIL strategy also enhanced the quantitation accuracy because samples and standards were d6-DH and DH labeled respectively, and then mass spectrum (MS) analyzed simultaneously. Analytes and standards were analyzed in identical MS conditions, and thus matrix effect which caused by different ionization efficiency between samples and standards was greatly minimized. The proposed method was applied to the SIL analysis of 21 aldehydes with limits of detection (LODs) ranged from 8.0 − 14.2 ng/kg. The recoveries were in the range of 85.3–96.4%, and the matrix effect was in the range of 90.1–102.6%.

Integrated LC-MS metabolomics with dual derivatization for quantification of FFAs in fecal samples of hepatocellular carcinoma patients

FFAs display pleiotropic functions in human diseases. Short-chain FAs (SCFAs), medium-chain FAs, and long-chain FAs are derived from different origins, and precise quantification of these FFAs is critical for revealing their roles in biological processes. However, accessing stable isotope-labeled internal standards is difficult, and different chain lengths of FFAs challenge the chromatographic coverage. Here, we developed a metabolomics strategy to analyze FFAs based on isotope-free LC-MS-multiple reaction monitoring integrated with dual derivatization. Samples and dual derivatization internal standards were synthesized using 2-dimethylaminoethylamine or dansyl hydrazine as a “light” label and N,N-diethyl ethylene diamine or N,N-diethyldansulfonyl hydrazide as a “heavy” label under mild and efficient reaction conditions. General multiple reaction monitoring parameters were designed to analyze these FFAs. The limit of detection of SCFAs varied from 0.5 to 3 nM. Furthermore, we show that this approach exhibits good linearity (R2 = 0.99374–0.99929), there is no serious substrate interference, and no quench steps are required, confirming the feasibility and reliability of the method. Using this method, we successfully quantified 15 types of SCFAs in fecal samples from hepatocellular carcinoma patients and healthy individuals; among these, propionate, butyrate, isobutyrate, and 2-methylbutyrate were significantly decreased in the hepatocellular carcinoma group compared with the healthy control group. These results indicate that the integrated LC-MS metabolomics with isotope-free and dual derivatization is an efficient approach for quantifying FFAs, which may be useful for identifying lipid biomarkers of cancer.

LC-MS Quantification of Malondialdehyde-Dansylhydrazine Derivatives in Urine and Serum Samples.

We developed a robust analytical method for quantification of malondialdehyde (MDA) in urine and serum samples using dansylhydrazine (DH) as a derivatizing reagent. The derivatization procedure was partially carried out using an autosampler injection program to minimize errors associated with the low-volume addition of reagents and was optimized to yield a stable hydrazone derivative of MDA and its labeled d2-MDA analogue. The target MDA-DH derivatives were separated on an Agilent Zorbax Eclipse Plus Phenyl-Hexyl (3.0×100mm, 3.5μm) column. The mass-to-charge ratios of the target derivatives [(M+H)+ of 302 and 304 for MDA-DH and d2-MDA-DH, respectively] were analyzed in single ion monitoring mode using a single quadrupole mass spectrometer operated under positive electrospray ionization. The method limits of quantification were 5.63nM (or 0.405ng/mL) for urine analysis and 5.68nM (or 0.409ng/mL) for serum analysis. The quantification range for urine analysis was 5.63-500nM (0.405-36.0ng/mL) while the quantification range for serum analysis was 5.68-341nM (0.409-24.6ng/mL). The method showed good relative recoveries (98-103%), good accuracies (92-98%), and acceptable precisions (relative standard deviations 1.8-7.3% for inter-day precision; 1.8-6.1% for intra-day precision) as observed from the repeat analysis of quality control samples prepared at different concentrations. The method was used to measure MDA in individual urine samples (n=287) and de-identified archived serum samples (n=22) to assess the overall performance of the method. The results demonstrated that our method is capable of measuring urinary and serum levels of MDA, allowing its future application in epidemiologic investigations.

Controlled microwave derivatization reaction for reproducible trace analysis of budesonide in human plasma

Microwave reactors with on-line controlled reaction conditions have been recently innovated to improve reaction kinetics and reproducibility. Herein, a modern microwave reactor was dedicated to develop a new, sensitive and reproducible approach for trace analysis of budesonide (BUD) in human plasma. The method was based on fast microwave reaction of BUD with dansyl hydrazine (DNS-HZ) reagent under controlled conditions coupled with high-performance liquid chromatography (HPLC)-fluorescence detection. The microwave irradiation and dansylation conditions were optimized for the best sensitivity and selectivity. The controlled microwave derivatization reaction (CMDR) decreased the reaction time, amplified the reaction yield and enhanced product purities by reducing the unwanted side reactions. The chromatographic separation was attained by isocratic elution on reversed phase column via a mobile phase consisted of methanol and phosphate buffer (10 mM, pH 7.0) at ratio 80:20 (v/v). The fluorescence detector was set at 500 nm after excitation at 330 nm. Betamethasone dipropionate (BDP) was used as an internal standard. CMDR-HPLC method validation was performed in agreement with bioanalytical method validation guidelines by the US food and drug administration (US-FDA). The obtained linearity range was 0.2–100 ng mL−1 with correlation coefficient 0.9991 and the lower limit of quantitation (LLOQ) in human plasma was 0.21 ng mL−1. The developed CMDR -HPLC method was applied successfully for assessment of plasma levels of BUD in allergic rhinitis patients after intranasal administration of the micronized BUD.

Identification of a freshness marker metabolite in stored soybean sprouts by comprehensive mass-spectrometric analysis of carbonyl compounds

The objective of this study was to identify metabolites that quantitatively indicate degrees of freshness of soybean sprouts. Self-cultivated soybean sprouts were stored at 5 °C, 10 °C or 20 °C, and respiratory CO2 production rates were monitored using gas chromatography during storage. Carbonyl compounds (CCs) were analyzed comprehensively using mass-spectroscopic metabolomics analyses. CCs were derivatized using dansyl hydrazine (DH) and were then analyzed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-MS/MS) with multiplexed multiple reaction monitoring (MRM). In the MRM chromatogram, 171 to 358 peaks were observed from stored soybean sprouts. Principle component analysis and discriminant analysis (PCA-DA) selected the CC-DH derivative ion with a m/z 512 at a retention time of 9.34 min as the most significant metabolite. Searching online metabolomics databases and matching fragment patterns of product ion mass spectra of an authentic standard revealed abscisic acid is a freshness marker of soybean sprouts.

Determination of formaldehyde and acetaldehyde in packaging paper by dansylhydrazine derivatization-high performance liquid chromatography-fluorescence detection

A high performance liquid chromatography with fluorescence detection (HPLC-FLD) method was developed for the simultaneous determination of formaldehyde and acetaldehyde in packaging paper by dansylhydrazine (DNSH) derivatization. The samples were extracted by derivatization reagent for 30 min, and derived for 24 h. After purifying treatment with a PSA/C18 cartridge, a Diamonsil® C18 column (150 mm×4.6 mm, 5 μ m) was used as stationary phase for separation, the mixtures of acetic acid aqueous solution (pH 2.55)-acetonitrile were used as mobile phases by gradient elution, and the excitation and emission wavelengths were 330 nm and 484 nm, respectively. The results showed that the recoveries of formaldehyde and acetaldehyde spiked in the samples were 81.64%-106.78%, and the relative standard deviations (RSDs) were 2.02%-5.53% (n=5). The limits of detection of formaldehyde and acetaldehyde were 19.2 μ g/kg and 20.7 μ g/kg, respectively. The limits of quantification of formaldehyde and acetaldehyde were 63.9 μ g/kg and 69.1 μ g/kg, respectively. The method is simple, sensitive and reproducible. It provides a basic approach for the determination of trace formaldehyde and acetaldehyde.

Comprehensive analysis of the lipophilic reactive carbonyls present in biological specimens by LC/ESI-MS/MS.

A new analytical method has been developed for profiling lipophilic reactive carbonyls (RCs) such as aldehydes and ketones in biological samples using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with selected reaction monitoring (SRM). The method consists of several phases, including (1) extraction of lipophilic RCs with a chloroform/methanol mixture; (2) derivatization of the extracted RCs with dansyl hydrazine (DH); and (3) SRM detection of the characteristic product ion of the 5-dimethylaminonaphthalene-1-sulfonyl moiety (m/z 236.1). The analytical results were expressed as RC maps, which allowed for the occurrence and levels of different lipophilic RCs to be visualized. We also developed a highly reproducible and accurate method to extract, purify and derivatize RCs in small volumes of biological specimens. This method was applied to the detection of free RCs in mice plasma samples, and resulted in the detection of more than 400 RCs in samples obtained from C57BL/6J mice. Thirty-four of these RCs were identified by comparison with authentic RCs. This method could be used to investigate the levels of RCs in biological and environmental samples, as well as studying the role of lipid peroxidation in oxidative stress related-disorders and discovering new biomarkers for the early diagnosis of these diseases.

Improved conditions for periodate/Schiff's base‐based fluorescent staining of glycoproteins with dansylhydrazine in SDS‐PAGE

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8ng of glycoproteins can be selectively detected within 2h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.

Selective recognition of sulfate anions in a 95% ethanol solvent with a simple neutral salicylaldehyde dansyl hydrazine Schiff base tuned by Brønsted–Lowry acid–base reaction

A new Schiff base compound, 5-(dimethylamino)-N′-(2-hydroxybenzylidene)naphthalene-1-sulfonohydrazide (R), has been synthesized, characterized, and employed as a selective fluorescence receptor for the recognition of sulfate anions. UV–vis absorption, fluorescence emission, 1H NMR spectra and DFT calculation studies on the system have been carried out to determine the nature of the interactions between R and anions. The results reveal that the deprotonation of the phenol without the need of a strong base leads to the formation of a hydrogen-bonding complex with a SO2NH group, which is responsible for the spectra changes. The deprotonation process for the selectivity recognition of sulfate can be tuned by the Brønsted–Lowry acid–base reaction in nonaqueous solutions, revealing that suitable phenolic hydroxyl acidity is the key factor for anion recognition selectivity.

Simultaneous Quantitation of Alpha-ketoglutaric Acid and 5-Hydroxymethylfurfural in Plasma by HPLC with UV and Fluorescence Detection

Alpha-ketoglutaric acid (KG) and hydroxymethylfurfural (HMF) are currently being investigated in clinical trials as an approach in targeted cancer therapy. Hence, a method for the simultaneous determination of KG and HMF in plasma has been developed. Due to the strongly discriminative chemical properties of KG and HMF, SPE purification is performed using an ion-exchange cartridge to separate KG, and a hydrophobic polymeric cartridge to separate HMF. The cartridges are connected together for several steps, thus resulting in a quicker approach for the purification of plasma samples. The derivatization step is based on the reaction of the carbonyl groups of KG and HMF with dansylhydrazine (DNSH) catalyzed by trifluoroacetic acid. The formed derivatives could be separated by reversed-phase LC on a C8-column, and analyzed by UV and fluorescence detection in a single run using a gradient program. The obtained results show good reproducibility, specificity, and detection limits down to the low picomole range.

Selective Fluorescent Detection of Aspartic Acid and Glutamic Acid Employing Dansyl Hydrazine Dextran Conjugate

Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1 × 10(-4) M to 25 × 10(-3) M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.

Metabolism of Cyclophosphamide by CYP 2B6 and Associated Polymorphisms

Cyclophosphamide (CPA) is one of the most widely used drugs in cancer treatment, yet, there is considerable interpatient variability with regards to efficacy and toxicity with CPA treatment. This interpatient variability is not well understood. CPA is an oxazaphosphorine prodrug, requiring bioactivation by a cytochrome P450. Cytochrome P450 2B6 (CYP2B6) is the major P450 involved in CPA activation. CYP2B6 is known to be highly polymorphic, and these polymorphisms have been demonstrated to affect the metabolism of other known CYP2B6 substrates such as bupropion and efavirenz. We hypothesized that CYP2B6 polymorphisms have varying ability to metabolize CPA, resulting in varying amounts of activated CPA. This is a potential cause for the efficacy and toxicity discrepancies with regard to CPA treatment. We examined two different assays for the measurement of a cyclophosphamide metabolite, using 3‐aminophenol and dansyl hydrazine to derivitze a fluorescent product. We also evaluated the metabolism of cyclophosphamide by the wild type CYP2B6 and its polymorphisms CYP2B6*4, CYP2B6*5, CYP2B6*6, CYP2B6*7, CYP2B6*8, and CYP2B6*9. We found that there were no significant differences in between metabolism of CYP2B6 polymorphisms and wild type.

Occurrence of cytotoxic 9-oxononanoyl secosterol aldehydes in human low-density lipoprotein

The reaction products of three major cholesteryl esters, cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE), and cholesteryl linoleate (C18:2-CE), present in human low-density lipoprotein (LDL) treated with ozone were isolated and characterized. In vitro ozonization of C16:0-CE was found to form the palmitoyl ester of secosterol-A (3β-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product secosterol-B (3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde). On the other hand, when C18:1-CE and C18:2-CE were oxidized by ozone, the aldehyde 9-oxononanoyl cholesterol (9-ONC) was formed as a primary product, which was then further oxidized to form 9-oxononanoyl secosterol-A (9-ON-secoA) and 9-oxononanoyl secosterol-B (9-ON-secoB). The compounds 9-ON-secoA and -B, but not 9-ONC, were found to exhibit strong cytotoxicity against human leukemia HL-60 cells. An LC–ESI–MS/MS method was developed for the detection of these cholesteryl ester ozonolysis products by derivatizing them with dansyl hydrazine. Using this method, we found for the first time that low concentrations of 9-ON-secoA and -B, but not palmitoyl secosterols, were present in human LDL. These novel oxidized cholesterol esters, 9-ON-secoA and -B, probably play important roles in the pathogenesis of several inflammatory disorders such as cancer, diabetes, atherosclerosis, and neurodegenerative diseases.