Dansyl Chloride Research Articles

Visual and Rapid Detection of Nerve Agent Mimics in Gas and Solution Phase by a Simple Fluorescent Probe.

Chemical nerve agents are highly toxic organophosphorus compounds that are easy to obtain and can be utilized by terrorists to threaten homeland security and human safety. Those organophosphorus nerve agents contain nucleophilic ability that can react with acetylcholinesterase leading to muscular paralysis and human death. Therefore, there is great importance to explore a reliable and simple method to detect chemical nerve agents. Herein, the o-phenylenediamine-linked dansyl chloride as a colorimetric and fluorescent probe has been prepared to detect specific chemical nerve agent stimulants in the solution and vapor phase. The o-phenylenediamine unit serves as a detection site that can react with diethyl chlorophosphate (DCP) in a rapid response within 2 min. A satisfied relationship line was obtained between fluorescent intensity and the concentration of DCP in the range of 0-90 μM. In the optimized conditions, we conducted the fluorescent titration to measure the limits of detection (0.082 μM) with the fluorescent enhancement up to 18-fold. Fluorescence titration and NMR studies were also conducted to explore the detection mechanism, indicating that the formation of phosphate ester causes the intensity of fluorescent change during the PET process. Finally, probe 1 coated with the paper test is utilized to detect DCP vapor and solution by the naked eye. We expect that this probe may give some admiration to design the small molecule organic probe and applied in the selectivity detection of chemical nerve agents.

Fluorescence digital image-based method to measure biogenic amines in Buffalo Mozzarella and other cheeses produced in Brazil

A fluorescence digital image-based (FDIB) method was applied for the determination of total biogenic amines in Mozzarella, Buffalo Mozzarella, Prato and Coalho cheese. For the first time in the literature a FDIB has been applied to quantify biogenic amines (BA) in cheese samples. No analytical methods have been reported before for the analysis of BA in Buffalo Mozzarella. Therefore, two BAs, i.e., Histamine and Tyramine were selected as models to evaluate the behavior of BAs during the derivatization reaction with dansyl chloride (DNS-Cl). The total biogenic amines ranged from 16.98 ± 0.98 mg L−1 to 72.48 ± 1.78 mg L−1 for cheese samples. Similar results with a confidence level of 95% were obtained using high performance liquid chromatography with spectrophotometric detection at 254 nm. The FDIB method developed reduces analytical steps such as extraction of excess of derivatizing agent, purification, removal of the used solvent, pre-concentration of analytes, filtration, and other analytical procedures that are used in HPLC, colorimetry, spectrophotometry, spectrofluorometry and other analytical methods. Thus, the FDIB is faster, low cost and generates less waste, being a more green method compared to the mentioned methods.

Determination of biogenic amines in chicken, beef, and mutton by dansyl chloride microwave derivatization in Malaysia

In this study, an improved dansyl-chloride derivatization technique using a microwave synthesizer was used for the qualitative and quantitative analyses of biogenic amine in the fresh meat samples. The derivatization technique was optimized in terms of temperature, reaction time, and spinning speed. The derivatization method together with a validated reversed-phase HPLC-DAD method was used for the determination of biogenic amines in chicken, beef, and mutton sold in the wet market. The results of the analyses showed that tryptamine, putrescine, and histamine were generally detected in all the three types of meat. Higher levels of histamine were found in chicken and beef. However, low levels of histamine were observed in mutton. Tyramine was either detected low or moderate in all the three types of meat. The biogenic amines of the fresh meat sold in the wet market is generally higher than the reported values. The mechanisms of biogenic amines-dansyl-chloride formation were investigated and proposed. PRACTICAL APPLICATION: The biogenic amine derivatization method was improved. The improved derivatization method can be potentially used for various food products beside meats for routine biogenic amine analyses due to its fast analysis time and simplicity. High levels of biogenic amines were generally found in the meat sold in the wet markets. However, proper handling of the raw meat can reduce the risk of infection.

Utility of dansyl chloride for the establishment of a sensitive spectrofluorimetric approach for estimating midodrine hydrochloride: application to content uniformity testing.

A new, straightforward spectrofluorimetric approach with high sensitivity was established for determining midodrine hydrochloride based on derivatizing this drug through its reaction with 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride). The highly fluorescent product was extracted with methylene chloride, and then its emission was measured at 532 nm after excitation at 339 nm. The reaction was conducted in aqueous medium containing 0.1 M borate buffer (pH 8.2). The results showed that the proposed method is sensitive with high linearity in the range from 0.1 to 3 μg mL-1. The lower limits of detection and quantitation were 29 and 88 ng mL-1, respectively. Furthermore, the proposed approach was analytically assessed by applying the ICH guidelines. The suggested approach was effectively utilized for the estimation of the medicine in its marketable tablet formulations with excellent recovery and without any interfering effect from excipients. Moreover, the presented approach was utilized to test the content uniformity of commercial tablets following the USP guidelines.

A robust multi-residue method for the monitoring of 25 endocrine disruptors at ultra-trace levels in surface waters by SPE-LC-MS/MS.

Estrogenic endocrine disruptors are one of the biggest ecotoxicological threats in water that pose a significant ecological burden and health-risk for humans due to their high biological activity and proven additive effects. Therefore, we have developed and validated the most comprehensive and ultra-sensitive analytical method published to date, for reliable quantification of 25 high-risk endocrine disruptors at their ecologically relevant concentrations: naturally excreted hormones (estradiol, estrone, estriol, testosterone, corticosterone, and progesterone), synthetic hormones used for contraception and menopausal symptoms (ethinylestradiol, drospirenone, chlormadinone acetate, norgestrel, gestodene, tibolone, norethindrone, dienogest, and cyproterone) and bisphenols (BPS, BPA, BPF, BPE, BPAF, BPB, BPC, and BPZ). It is based on a solid-phase extraction of water samples, followed by a robust dansyl chloride derivatization with detection by liquid chromatography-tandem mass spectrometry with a single sample preparation and two analytical methods using the same analytical column and mobile phases. The achieved limits of quantitation are in the sub-ng L-1 range, and detection limits as low as 0.02 ng L-1, meeting the newest proposal for environmental quality standards (EQS) by the EU water framework directive for estradiol and ethinylestradiol. The method was extensively validated and applied to seven representative Slovenian water samples, where we detected 21 out of 25 analytes; 13 were quantified in at least one sample. Estrone and progesterone were quantified in all samples, reaching levels up to 50 ng L-1; ethinylestradiol was higher than the current EQS (0.035 ng L-1) in three samples, and estradiol was above its EQS (0.4 ng L-1) in one sample, proving the method's applicability and the necessity for monitoring these pollutants.

Validation of a methodology for determination of polyamines in urine by High Performance Liquid Chromatography (HPLC)

The levels of polyamines (PAs) in urine are an important biomarker of physiological and pathological phenomena for humans.Due to its importance, the validation of a PAs determination methodology was validated for (Putrescine (Pu), Cadaverine(Cdv), Spermidine (Spd), Spermine (Spm), Epinephrine (Epf) and Histamine (Hst) in urine by Reversed-phase HighPerformance Liquid Chromatography (RP-HPLC) with pre- column derivatization using dansyl chloride and UV detection at254 nm. To this aim, selectivity, working range and linearity, detection /quantification limit were determined. The precision (asrepeatability and precision under intermediate conditions), accuracy (measured as recovery) and robustness were established.The methodology was adapted from the Chilean Standard for the detection of histamine and other biogenic amines in fish andshellfish ME-711.O4-070, 2014, so it was necessary to implement adjustments for its application in urine. The results showedthat amino acids (AAs) did not cause interference in the analysis of PAs, and allow determination of concentrations in thelinear range between 4 and 28 μg/mL. However, good recovery (accuracy) was not obtained in the experiment, possiblybecause the standards used were not acetylated (42.6% Pu and 69% Cdv). The present methodology allows determining thelevels of Pu, Cdv, Spd, Spm Hst and Efd in urine samples from humans and other mammalian species.

Validation of HPLC Method for Analysis of Gamma-Aminobutyric and Glutamic Acids in Plant Foods and Medicinal Plants.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system of mammals and plays an important role in the suppression of neurons' excitability. GABA is formed from the decarboxylation of glutamic acid (Glu), and both GABA and Glu could be considered as important biologically active food components. In the current study, we validated a HPLC method for concomitant detection of GABA and Glu in plant samples after derivatization with dansyl chloride. The validated method had high precision and a high recovery rate and was successfully used for GABA and Glu quantification in 55 plant foods (fruits, vegetables, legumes, cereals, pseudocereals, and nuts) and 19 medicinal plants. Vegetables were the most important dietary source of these amino acids, with the highest quantity of GABA found in potatoes-44.86 mg/100 g fresh weight (FW) and yellow cherry tomatoes-36.82 mg/100 g FW. The highest amount of Glu (53.58 mg/100 g FW) was found in red cherry tomatoes. Analyzed fruits were relatively poor in GABA and Glu, and European gooseberry was the richest fruit with 13.18 mg/100 g FW GABA and 10.95 mg/100 g FW Glu. Cereals, pseudocereals, nuts, and legumes contain much higher amounts of Glu than GABA. The obtained results enrich the available information on the content of gamma-aminobutyric and glutamic acids in plant foods and could be used for the development of GABA-enriched functional foods.

Pyridinium and ammonium stable isotope labeling agents and their performance in the analysis of alkylamines in food and food packaging materials

Two pairs of stable isotope labeling (SIL) agents, (4-carboxyphenyl)trimethylammonium iodide (d0-CPTA) and its deuterated counterpart d3-CPTA, 1-methyl-nicotinamide iodide (d0-MNA) and its deuterated counterpart d3-MNA, were designed and synthesized. Their mass spectrometry (MS) sensitivity enhancement effect was studied and compared with commercial dansyl chloride to provide inspiration for labeling agent design. CPTA with quaternary ammonium group showed much higher MS sensitivity enhancement effect and was applied to the SIL analysis of alkylamines in food and food packaging materials. The matrix effect was minimized due to the SIL strategy and the permanent charge of the CPTA. The limits of detection (LODs) were in the range of 2.9–5.1 ng/L, and the limits of quantitation (LOQs) were in the range of 9.6–16.8 ng/L. The recoveries ranged from 91.2 % to 97.1 % with relative standard deviations of less than 6.6 %, and the matrix effect ranged from −1.8 % to −4.9 %.

Bioactive Amines in Wines. The Assessment of Quality Descriptors by Flow Injection Analysis with Tandem Mass Spectrometry.

Biogenic amines (BAs) occur in a wide variety of foodstuffs, mainly from the decomposition of proteins by the action of microorganisms. They are involved in several cellular functions but may become toxic when ingested in high amounts through the diet. In the case of oenological products, BAs are already present in low concentrations in must, and their levels rise dramatically during the fermentation processes. This paper proposes a rapid method for the determination of BAs in wines and related samples based on precolumn derivatization with dansyl chloride and further detection by flow injection analysis with tandem mass spectrometry. Some remarkable analytes such as putrescine, ethanolamine, histamine, and tyramine have been quantified in the samples. Concentrations obtained have shown interesting patterns, pointing out the role of BAs as quality descriptors. Furthermore, it has been found that the BA content also depends on the vinification practices, with malolactic fermentation being a significant step in the formation of BAs. From the point of view of health, concentrations found in the samples are, in general, below 10 mg L-1, so the consumption of these products does not represent any special concern. In conclusion, the proposed method results in a suitable approach for a fast screening of this family of bioactive compounds in wines to evaluate quality and health issues.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17β-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Development of a High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) Method for Determination of Biogenic Amines in Ripened Cheeses.

Biogenic amines (BAs) are organic, basic nitrogenous compounds formed during the decarboxylation of amino acids. A method for the determination of eight biogenic amines (tryptamine, 2-phenyletylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine) in ripened cheeses was developed and validated. Cheese samples with the addition of internal standards were extracted with 0.2 M perchloric acid and pre-column derivatized with dansyl chloride at 60 °C for 15 min, purified with toluene and dried under a stream of nitrogen. The samples were analyzed using high performance liquid chromatography with diode array detector (HPLC-DAD). The method was validated with the BAs at three concentration levels: 50, 100, and 200 mg/kg, respectively. The obtained values of correlation coefficient (R2) ranged at 0.9997−0.9998 for all of compounds. The limits of detection (LOD) and quantification (LOQ) were in ranges 1.53−1.88 and 5.13−6.28 mg/kg, respectively. The recovery for all of biogenic amines ranged from 70 to 120% and the precision (RSDr) value were <20%. The validated method was applied to analysis of 35 real ripened cheese samples purchased in Poland.

Sample multiplexing for increasing throughput for quantification of estrogens in serum by LC-MS/MS.

Estrogens are involved in many physiological processes in vivo. The accurate and rapid quantification of estrogens is required for the diagnosis and prognosis of estrogen-related diseases. To achieve high-volume assays, we developed and validated a sample-multiplexing liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of serum estrogens including estrone (E1), estradiol (E2), and estriol (E3). A total of 100 μL serum samples were extracted using ethyl acetate. After derivatization with either dansyl chloride or pyridine-3-sulfonyl chloride, derivatized samples were combined. Then we performed the second liquid-liquid extraction using hexane to purify the mixture. Finally, the reconstitution solutions were injected into LC-MS/MS. In addition, the proposed LC-MS/MS method was validated according to FDA and CLSI guidelines. Within a single run (7min), this sample-multiplexing LC-MS/MS method could simultaneously analyze E1, E2, and E3 in 2 serum samples. Meanwhile, the method demonstrated satisfactory analytical characteristics including accuracy (87.7-110.3%), linearity (2-1000pg/mL, R2 > 0.99), precision (intra-assay CV, 1.7-8.7%; inter-assay CV, 1.9-9.4%), and negligible interference and carry-over effect as well as acceptable matrix effect. In conclusion, this sample-multiplexing LC-MS/MS method has achieved a doubled-throughput assay for simultaneous quantification of E1, E2, and E3 without compromising analytical characteristics.

Development and Validation of an HPLC-FLD Method for the Determination of NDMA and NDEA Nitrosamines in Lisinopril Using Pre-Column Denitrosation and Derivatization Procedure

In order to meet the analytical requirements of the European Medicines Agency (EMA), a new HPLC-FLD method was successfully developed using dansyl chloride for the derivatization and determination of the genotoxic impurities N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) in Lisinopril API and its final product. Samples’ pretreatment includes liquid–liquid microextraction, denitrosation, and derivatization steps. To optimize the process, the parameters contributing to high sensitivity and yielding reliable results were thoroughly studied and optimized using one-factor-at-a-time and experimental design approaches. The analytes were pre-column derivatized with Dansyl-Cl and analyzed by HPLC-fluorescence (λem/λem = 340/530) using a C18 column and a mixture of phosphate buffer (pH = 2.8; 20 mM)/acetonitrile 55:45 v/v as the mobile phase. The six-level concentration calibration was shown to be linear, with R equal to 0.9995 for both analytes. The limit of detection (LOD) was satisfactory and equal to 4.7 and 0.04 ng/mL for NDMA and NDEA, respectively. Precision was less than 13.4% in all cases, and the average recoveries were equal to 109.2 and 98.1% for NDMA and NDEA, respectively. The proposed procedure is relatively easy, rapid, and suitable for the determination of the two nitrosamines in routine analysis tests.

Simultaneous Determination of Amino Acids and Biogenic Amines by Liquid Chromatography Coupled to Mass Spectrometry for Assessing Wine Quality

Biogenic amines (BAs) and free amino acids (AAs) are low-molecular nitrogenous compounds occurring in a wide range of foodstuffs, found in increased amount in different fermented foods, seafood, and wines. This study deals with the development of an analytical method based on liquid chromatography with tandem mass spectrometry with precolumn derivatization with dansyl chloride for the determination of BAs and AAs in musts, wines, and sparkling wines. The resulting compositional profiles have been exploited as potential descriptors of quality and other oenological issues using chemometric methods including principal component analysis (PCA) and partial analysis of least squares-discriminants (PLS-DA). Proline is the most abundant compound, and other remarkable species are lysine, ethanolamine, tyramine, histamine, and putrescine. Fermented samples (wines and sparkling wines) are much richer in both BAs and free AAs than the initial musts. Significant differences have also been noticed in the quality, as the best products display, in general, lower levels. The dissimilarities in the content of the analytes between the two grape varieties studied (pinot noir and xarel·lo) and those dealing with quality aspects have made it possible to establish a tree to classify the samples based on these two features with excellent classification rates.

An HPLC-based assay for improved measurement of glutamate decarboxylase inhibition/activation

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals.

A dansyl-based fluorescent probe for turn-off and turn-on detection of Hg2+ in a full water system

A dansyl-based fluorescent probe, 2-((5-dimethylamino)naphthalene-1- sulfonamido)acetic acid (DNSG), was synthesized from dansyl chloride and glycine. In HEPES buffer (20 mM, pH 6.7), DNSG could detect Hg2+ by fluorescence quenching. The maximal fluorescence intensity showed a good linear relationship with the Hg2+ concentration (0 ~ 80 μM). The detection limit was 0.33 μM. With the help of silk fibroin (0.076 g/L), DNSG could detect Hg2+ by fluorescence enhancement and blueshift. The linear concentration range for Hg2+ was 0 ~ 160 μM with a detection limit of 0.81 μM. DNSG could detect Hg2+ quickly and reversibly with excellent anti-interference ability and further could assay environmental water sample efficiently. The sensing mechanism was examined by Job’s plot, mass spectrum and 1H NMR analysis.

Fluorogenic gemcitabine based light up sensor for serum albumin detection in complex biological matrices

Herein we report fluorogenic derivative of gemcitabine (GEM-DNS), synthesized from gemcitabine hydrochloride and dansyl chloride in a single step. Owing to its large stoke shift of ∼200 nm and intriguing photophysical properties, the said dye has been utilized to estimate albumin concentration in complex bio-media such as human urine and blood serum. High sensitivity and selectivity towards albumin make the aforementioned dye a powerful diagnostic tool to detect ailments such as liver cirrhosis, diabetes, hypertension etc.

Determination of trace concentrations of simple phenols in ambient PM samples.

Phenols are hazardous, but yet ubiquitous in the environment, including in atmospheric aerosols due to combustion emissions. There, phenols are subjected to secondary transformations, producing even more toxic nitrophenolic air pollutants. However, primary simple phenols, i.e. those containing only hydroxyl, methyl and methoxy substituents are not easy to detect. Trace concentrations, semi-volatile character and poorly ionizable functional groups prevent us from their determination by the most common analytical techniques, such as gas and liquid chromatography with mass spectrometric detection (GC/LC-MS). Here, we present a new derivatization method for MS/MS detection with positive ion electrospray ionization (+ESI-MS/MS) of simple phenols in atmospheric particulate matter (PM) extracts. The method is sensitive, selective, and robust, and requires no sample concentration step, which is critical due to the volatile character of the target analytes. After derivatization with dansyl chloride, phenol, catechol, cresols and guaiacol were detected in urban PM samples from Ljubljana, Slovenia. This method finally enables to study the abundance of primary phenols in atmospheric PM from different sources, which will improve understanding of secondary aerosol (trans)formation pathways and allow for more targeted mitigation strategies in respect to airborne phenolic pollutants.

Single-Stranded DNA Recognition over Fluorescent Gold-Aryl Nanoparticles

Fluorescence labeling of gold-aryl nanoparticles, AuNPs-COOH, was achieved by the covalent derivatization with dansyl chloride (DNS-Cl) reagent (5-naphthalene-1-sulfonyl chloride) for potential ssDNA recognition. The fluorescent gold nanoparticles of AuNPs-C6H4-4-COO-dansyl (AuNPs-DNS) of spherical shape and a size of 19.3 ± 8.3 nm were synthesized in a carbonate-bicarbonate buffer (pH = 10.6) at 37 °C. The fluorescence emission at 475 nm was acquired using fluorescence spectroscopy and investigated using time-resolved photoluminescence. The conjugation of ssDNA to AuNPs-DNS using the freeze-thaw and salt-aging methods was confirmed by fluorescence emission quenching, gel electrophoresis separation, and lifetime decrease. Conjugated ssDNA to AuNPs-DNS using the freeze-thaw method was more efficient than the salt-aging method. The purity of ssDNA upon conjugation was measured with optical density, and the obtained A260/A280 ratio was in the range of 1.7–2.0. This research can be applied to other nucleotide recognition and theranostics.

Determination of estrogens in human serum using a novel chemical derivatization-assisted liquid chromatography-electrospray ionization-tandem mass spectrometry method.

Assessing estrogen concentrations in biological systems can provide valuable information on physiological processes, which is crucial for the early diagnosis of many diseases. Because estrogens are present in the human body in low concentrations and in a wide dynamic range, analytical methods with high sensitivity and specificity are required for their determination in complex biological matrices. To discover an appropriate derivatization reagent for estrogen mass spectrometry (MS) analysis, we compared five sulfonyl chloride derivatization reagents, namely 3-methyl-8-quinolinesulfonyl chloride (MQSCl) and 8-quinolinesulfonyl chloride (QSCl), 1-methyl-1H-pyrazole-4-sulfonyl chloride, 1,2-methyl-imidazole-5-sulfonyl chloride, and dansyl chloride. By selecting the derivatization reagent with the best performance, we developed and validated a novel chemical derivatization-assisted-liquid chromatography-electrospray ionization-tandem mass spectrometry (CD-LC-ESI-MS/MS) method to simultaneously determine the concentrations of estrone, estradiol, and estriol (E1, E2, and E3) in human serum. It was found that among the five investigated reagents, MQSCl-derivatized estrogens presented the highest sensitivity using LC-ESI-MS/MS. Based on this discovery, MQSCl was chosen to derivatize the analyzed estrogens to assist LC-ESI-MS/MS analysis. The limit of quantification of E1, E2, and E3 was measured as 2.7, 4.6, and 5.1pg/mL, respectively. Inter- and intra-day precision, expressed as the coefficient of variation, was shown to be lower than 13.2% for all concentrations. The mean recovery was 72.4% overall, with good reproducibility at low, medium, and high concentrations in the calibration range. The developed method was successfully applied to the quantitative determination of estrogens in clinical human serum from pediatric and adult women, demonstrating the suitability of estrogen analysis in the biological matrix at low concentration (pg/mL).