Pre-column Derivatization Method Research Articles

Liquid-liquid addition reaction of ethylene oxide with hydrazine hydrate in microreactors: Kinetics and process optimization

The addition reaction of ethylene oxide (EO) and hydrazine hydrate (N2H4·H2O) to β-hydroxyethyl hydrazine (HEH) is an important process for the synthesis of hydrazine-derived monopropellant in the aerospace industry. Due to the complexity of this reaction mechanism, it is difficult to reduce the formation of byproducts and efficiently obtain pure HEH. Hence it is necessary to investigate process behavior and establish kinetic network for further optimization. Here a continuous microflow system was developed to systematically investigate the synthesis of HEH under high pressure in the liquid–liquid form. A simple and effective pre-column derivatization method was applied to characterize and quantify the product. Subsequently, a thorough optimization of parameters, including N2H4·H2O concentration, molar ratio (N2H4·H2O/EO), temperature and reaction time, was conducted. Additionally, response surface methodology was employed to optimize the reaction conditions, illuminating the intricate interactions among the specified variables and achieving a high HEH yield of 96.97 %. Finally, a kinetic model was established, and the activation energies for the main reaction and side reaction were measured to be 38.31 kJ·mol−1 and 44.94 kJ·mol−1, respectively. This kinetic model can enable the accurate determination of kinetic parameters and provide guidance for the process intensification of this EO addition reaction.

Highly sensitive analysis of fatty aldehydes in vegetable oils using a novel coumarin-based fluorescent probe by HPLC for quality control

A novel fluorescent probe, 3-(4-((hydroxylamino)methyl)-1H-pyrazol-1-yl)-7-methoxyl coumarin (HAPMC), has been designed and synthesized. The compound exhibits exceptional fluorescence properties due to a coumarin-pyrazolyl fluorophore. Moreover, it incorporates an N-hydroxylamine functional group capable of reacting with aldehydes to yield nitrone derivatives under mild conditions. This unique combination renders it an ideal derivatization reagent for aldehyde analysis. A standard method was established for the quantitative analysis of eight fatty aldehydes by utilizing high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and a pre-column derivatization process, which has low detection limits (0.3–1.2 nM), satisfactory reproducibility (intraday and interday relative standard deviations in the range of 1.29 %-2.92 % and 1.73 %-3.45 %), and an extensive linear range (25–5000 nM) with excellent linearity (R2 ≥ 0.9989). The spiked recoveries of the fatty aldehydes range from 89.6 % to 106.0 %. The greenness and sustainability of our approach are reinforced by evaluations conducted with the AGREE and RGB 12 algorithms. The established method was successfully applied to the qualitative and quantitative detection of fatty aldehydes in nine real vegetable oil samples, including peanut, canola, rice, soybean, olive, rapeseed, corn, sunflower, and blended oils. The fatty aldehydes are known to have adverse effects on human health due to their carcinogenic and mutagenic properties. The findings revealed that hexanal and nonanal were the most prevalent saturated fatty aldehydes, with a notable increase in the total aldehyde concentration observed as the temperature and duration of heating increased. Compared to alternative analytical techniques, this pre-column derivatization method utilizing HAPMC displayed simplicity, reliability, and sensitivity. The results demonstrate the potential for broad application of this approach in various important research domains, including food quality control, environmental monitoring, and medical research.

Determination of genotoxic impurities of aromatic aldehydes in pharmaceutical preparations by high performance liquid chromatography after derivatization with N-Cyclohexyl-4-hydrazino-1,8-naphthalenediimide

The detection of aromatic aldehydes, considered potential genotoxic impurities, holds significant importance during drug development and production. Current analytical methods necessitate complex pre-treatment processes and exhibit insufficient specificity and sensitivity. This study presents the utilization of naphthalenediimide as a pre-column derivatisation reagent to detect aromatic aldehyde impurities in pharmaceuticals via high-performance liquid chromatography (HPLC). We screened a series of derivatisation reagents through density functional theory (DFT) and investigated the phenomenon of photoinduced electron transfer (PET) for both the derivatisation reagents and the resulting products. Optimal experimental conditions for derivatisation were achieved at 40 °C for 60 min. This approach has been successfully applied to detect residual aromatic aldehyde genotoxic impurities in various pharmaceutical preparations, including 4-Nitrobenzaldehyde, 2-Nitrobenzaldehyde, 1,4-Benzodioxane-6-aldehyde, and 5-Hydroxymethylfurfural. The pre-column derivatisation method significantly enhanced detection sensitivity and reduced the limit of detection (LOD), which ranged from 0.002 to 0.008 μg/ml for the analytes, with relative standard deviations < 3 %. The correlation coefficient (R2) >0.998 demonstrated high quality. In chloramphenicol eye drops, the concentration of 4-Nitrobenzaldehyde was measured to be 8.6 µg/mL below the specified concentration, with recoveries ranging from 90.0 % to 119.2 %. In comparison to existing methods, our work simplifies the pretreatment process, enhances the sensitivity and specificity of the analysis, and offers comprehensive insights into impurity detection in pharmaceutical preparations.

Development of a Triphenylmethyl Alkylation Pre-Column Derivatization Method for HPLC Quantitative Analysis of Chemically Unstable Halogenated Compounds.

The primary limitations of the quantitative analysis of thermally labile halogenated compounds by traditional gas chromatography (GC) are the inadequacy of identifying the insufficiently volatile impurity (often with a high boiling point) and the difficulty in obtaining a standard substance with a reliable standardized assay. Taking the 4-(Chloromethyl)-5-methyl-1,3-dioxol-2-one (DMDO-Cl, 1) as an example, we reported a triphenylmethanamino-derivatization method to overcome the challenges of the assay determination of such species. During the quantification of 1, the presence of GC-undetectable polymeric impurity 10 poses a critical challenge in assessing the material quality. Moreover, the standard substance of 1 is not available on the market due to its inherent instability during storage and handling, further complicating the quantitative analysis. In this work, a precolumn HPLC-UV derivatization method based on triphenylmethanamino-alkylation was developed to quantitatively analyze 1. The resulting derivative 2 exhibits excellent crystallinity and superior physical and chemical stability and possesses effective chromophores for UV detection. The conversion from analyte 1 to derivative 2 demonstrates desirable reactivity and purity, facilitating quantitative analysis using the external standard method. The chemical derivatization-chromatographic detection method was optimized and validated, demonstrating its high specificity, good linearity, precision, accuracy, and stability. This method offers a valuable alternative to the general quantitative NMR (qNMR) detection technique, which exhibits reduced specificity in the presence of increased levels of impurities in compound 1.

Determination of the stability of sodium cyclamate during deep-frying using HPLC.

The oil used to fry food is often used multiple times to reduce costs. However, when foods containing sweeteners are processed in this way, the sweeteners may produce substances harmful to the body as a result of repeated frying at high temperatures. This article investigated the stability of sodium cyclamate during deep-frying by HPLC using a pre-column derivatization method. The results showed that cyclohexylamine was a decomposition product of a standard sample of sodium cyclamate when deep-fried at 200°C for 25 min. A pre-column derivatization/HPLC method was established to determine cyclohexylamine, a decomposition product of sodium cyclamate, under these conditions. Dansyl chloride was used as the derivatization reagent, the derivatization temperature was 60°C, the derivatization time was 20 min, the pH of sodium bicarbonate buffer solution was 11, and the concentration of dansyl chloride was 2.0 mg/mL. Detection was carried out by using an Agilent 1260 high-performance liquid chromatograph coupled with an ultraviolet detector. The ultraviolet detection wavelength was 254 nm, and the mobile phase was acetonitrile-1.0 g/L potassium dihydrogen phosphate solution at a flow rate of 1.0 mL/min. Gradient elution was adopted, the peak of the cyclohexylamine derivative appeared at a retention time of 17.75 min, and the peak area response value was the largest. The methodological validation analysis showed that the detection limit of cyclohexylamine was 0.5 mg/kg, the quantification limit was 2.0 mg/kg, and the spiked recoveries were in the range of 99.37-110.16%. The relative standard deviations (RSDs) were in the range of 0.17-1.26%. Four samples were tested and analyzed by the established method, and cyclohexylamine was not detected.

Polysaccharide of Asparagus cochinchinensis (Lour.) Merr regulates macrophage immune response and epigenetic memory through TLR4-JNK/p38/ERK signaling pathway and histone modification

BackgroundInnate immune memory of macrophages is closely linked to histone modifications. While various studies have demonstrated that the polysaccharide of Asparagus cochinchinensis (Lour.) Merr (ACMP), extracted through alcohol-alkali extraction, enhances macrophages' non-specific immune function; no literature currently addresses whether ACMP's regulatory effect is related to innate immune memory and histone modification. PurposeThis study aims to investigate if ACMP induces innate immune memory emergence in macrophages via pattern recognition receptor (PRR). Study designAfter co-incubating different doses of ACMP with RAW264.7 cells and BMDM cells, we observed changes in signaling pathways related to PRR and assessed the presence of innate immune memory phenomenon in the cells. MethodsWe observed the morphological characteristics of the ACMP using a scanning electron microscope, infrared spectrum, and HPLC pre-column derivatization method. We used q-PCR, Western blot, RNA-seq, and CUT&Tag-seq methods to examine ACMP's regulation of macrophage immune response and innate immune memory and explored its specific mechanism. ResultsACMP, primarily composed of Man, GlcN, Rha, Fuc, GalA, Xyl, Glc, Gal, Ara, and, exhibited a molar ratio of each monosaccharide (1.41: 0.35: 0.49: 0.18: 1.00: 97.12: 0.36: 3.58: 1.14). ACMP regulated immunological function in macrophages through the TLR4-MAPK-JNK/p38/ERK pathway. ACMP induced elevated levels of chromosomal H3K4me1, enhancing TNF-α, IL-1β, and other genes' responsiveness, allowing macrophages to develop innate immune memory to ACMP stimulation. ConclusionThis study first time demonstrates that ACMP regulates immunological function through the TLR4-MAPK-JNK/ERK/p38 signaling pathway, distinct from prior reports. ACMP induces innate immune memory in macrophages in response to its immune stimulation by promoting increased H3K4me1 on chromosomes. This mechanism may be crucial in how plant polysaccharides regulate macrophages and the body's immune function.

Determination of lincomycin residues of animal derived food by pre-column derivatization with HPLC -UVD

The excessive accumulation of antibiotic residue in animal-derived foods pose a threat to our heath. Therefore, it is necessary to establish a rapid, simple, effective, safe and highly sensitive lincomycin (LIN) residues detection method by high-performance liquid chromatography equipped with ultraviolet. Here, due to the weak UV absorption of LIN, the p-Toluenesulfonyl isocyanate (PTSI, has a strong UV absorption) was binded to the hydroxyl group of LINs by using pre-column derivatization method, improving the UV absorption of LIN. This 9-min-run analytical method detects and quantifies LIN residues with acceptable validation performance parameters in terms of sensitivity, selectivity, linearity, the limit of quantification, accuracy, and precision.Our result showed that the limits of detection and quantification for LIN in samples were 25–40 μg/kg and 40–60 μg/kg, respectively, under the optimized conditions. Furthermore, the LIN recoveries ranged from 72.4 % to 96.7 %; the determination coefficients were 0.9912–0.9996, and the relative standard deviations were 5.6 %-12.7 %. Therefore, the HPLC-UVD method for detecting LIN residues in animal-derived food products has been established.

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method.

Homotaurine (HOM) is considered a promising drug for the treatment of Alzheimer's and other neurodegenerative diseases. In the present work, a new high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (λex. = 340 nm and λem. = 455 nm) method was developed and validated for the study of substance permeability in the central nervous system (CNS). Analysis was performed on a RP-C18 column with a binary gradient elution system consisting of methanol-potassium phosphate buffer solution (pH = 7.0, 0.02 M) as mobile phase. Samples of homotaurine and histidine (internal standard) were initially derivatized with ortho-phthalaldehyde (OPA) (0.01 M), N-acetylcysteine (0.01 M) and borate buffer (pH = 10.5; 0.05 M). To ensure the stability and efficiency of the reaction, the presence of different nucleophilic reagents, namely (a) 2-mercaptoethanol (2-ME), (b) N-acetylcysteine (NAC), (c) tiopronin (Thiola), (d) 3-mercaptopropionic acid (3-MPA) and (e) captopril, was investigated. The method was validated (R2 = 0.9999, intra-day repeatability %RSD < 3.22%, inter-day precision %RSD = 1.83%, limits of detection 5.75 ng/mL and limits of quantification 17.43 ng/mL, recovery of five different concentrations 99.75-101.58%) and successfully applied to investigate the in vitro permeability of homotaurine using Franz diffusion cells. The apparent permeability (Papp) of HOM was compared with that of memantine, which is considered a potential therapeutic drug for various CNSs. Our study demonstrates that homotaurine exhibits superior permeability through the simulated blood-brain barrier compared to memantine, offering promising insights for enhanced drug delivery strategies targeting neurological conditions.

Well-oriented immobilized immunoaffinity magnetic beads for detection of fumonisins in grains and feeds via pre-column automatic derivatization of high-performance liquid chromatography.

In this study, based on the high-throughput automatic sample pretreatment with immunoaffinity magnetic beads with oriented immobilized antibodies, grain and feed fumonisin (FB) content was detected using pre-column automatic derivatization of high-performance liquid chromatography (HPLC). The FB capacity of well-oriented antibody immunoaffinity magnetic beads was 1.5-1.8 times that of magnetic beads with randomly fixed antibody. This pre-column automatic derivatization method using an autosampler can reduce error from manual injection and improve detection efficiency. The spiked recoveries for three different concentrations in maize, husked rice, and pig feed under optimized conditions were 84.6-104.0% (RSD<9.3%). Our novel method was also applied to the analysis of FBs in 63 maize samples collected from the main maize-production regions in China. The results showed that as latitude increased, the contamination level of FBs tended to decrease. High temperature and high humidity are also more favorable for FB growth.

Chiral Amino Acid Profiling in Serum Reveals Potential Biomarkers for Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease, and increasing evidence has linked dysregulation of amino acids to AD pathogenesis. However, the existing studies often ignore the chirality of amino acids, and some results are inconsistent and controversial. The changes of amino acid profiles in AD from the perspective of enantiomers remain elusive. The purpose of this study is to investigate whether the levels of amino acids, especially D-amino acids, are deregulated in the peripheral serum of AD patients, with the ultimate goal of discovering novel biomarkers for AD. The chiral amino acid profiles were determined by HPLC-MS/MS with a pre-column derivatization method. Experimental data obtained from 37 AD patients and 34 healthy controls (HC) were statistically analyzed. Among the 35 amino acids detected, D-proline, D/total-proline ratio, D-aspartate, and D/total-aspartate ratio were decreased, while D-phenylalanine was elevated in AD compared to HC. Significant age-dependent increases in D-proline, D/total-proline ratio, and D-phenylalanine were observed in HC, but not in AD. Receiver operator characteristic analyses of the combination of D-proline, D-aspartate, D-phenylalanine, and age for discriminating AD from HC provided satisfactory area under the curve (0.87), specificity (97.0%), and sensitivity (83.8%). Furthermore, the D-aspartate level was significantly decreased with the progression of AD, as assessed by the Clinical Dementia Rating Scale and Mini-Mental State Examination. The panels of D-proline, D-phenylalanine, and D-aspartate in peripheral serum may serve as novel biomarker candidates for AD. The latter parameter is further associated with the severity of AD.

One-step pre-column derivatization method for HPLC-UV determination of organic acids in fruit juices

A simple and effective one-step pre-column derivatization method for reversed-phase HPLC-UV determination of the major organic acids (citric, malic, tartaric, and succinic acids) in fruit juices has been developed. The method is based on the coupling reaction of organic acids with 1-naphthylamine in the presence of N,N′-diisopropylcarbodiimide. The resulting organic acid amide derivatives were successfully separated on an AQ-C18 column and detected by a UV detector at 222 nm. Isocratic elution was performed using an acetonitrile-methanol-tetrahydrofuran-water (37:4:1:58; v/v) mixture as a mobile phase at a flow rate of 2.0 mL/min. Validation studies showed good recoveries ranging from 88.97 to 92.52% with RSD % between 2.95 and 4.56%. The detection limits obtained were 0.18, 0.48, 0.14, and 0.69 µg/mL for tartaric acid, malic acid, succinic acid, and citric acid, respectively. The method was successfully applied to three different commercial fruit juice and nectar samples with little or no clean-up procedures.

Age-associated polyamines in peripheral blood cells and plasma in 20 to 70 years of age subjects

Dietary polyamines have been associated with slowing ageing processes and various pathologies, raising the importance of establishing reference values at different ages throughout life. This study aimed to analyse age-dependent variations in polyamine content using peripheral blood cells and plasma in a healthy and homogeneous population. Peripheral blood of 193 volunteers of both sexes (20–70 years), selected by convenience, was processed to separate cells and plasma. A pre-column derivatization method was used to determine the amines by HPLC (nmol or pmol/mg protein or nmol/ml) to analyse their association with the age (continuous or ordinal in decades) of the subjects. Putrescine and spermine weakly declined significantly in mononuclear cells with age. In erythrocytes and plasma, putrescine showed an evident decrease in the 60–70-year-old group compared to the rest. The ratios between polyamines, mainly in erythrocytes, decreased in the 60–70 years age group and increased the ratio of putrescine in mononuclear cells/erythrocytes. The ratio of putrescine in mononuclear cells/erythrocytes was higher in the 60–70-year-old age group than in the rest. In a sample of subjects (20–29 vs. 60–70 years), whole blood polyamines were not significantly different when differences existed in erythrocytes. Polyamine homeostasis in blood cells and plasma changed with age. Putrescine declined in mononuclear cells and decreased in erythrocytes and plasma in the decade of the 60 s. Further studies should establish an age-dependent phenotype and whether polyamines’ supplementation could restore the decreased values and be associated with long-term overall biological benefits.

A novel reagent pre-column derivatization method for UHPLC-MS/MS to determine four α-dicarbonyl compounds in beer

A novel reagent pre-column derivatization method for UHPLC-MS/MS to determine four α-dicarbonyl compounds in beer

A highly sensitive pre-column derivatization method for fluorescence detection of tobramycin using 2-naphthalenesulfonyl chloride as a derivatizing agent

A new, simple, rapid and highly sensitive high performance liquid chromatographic method was developed for the determination of tobramycin (TOB) in ophthalmic solutions. The developed method was based on derivatizing this non-chromophoric drug with 2-naphthalenesulfonyl chloride (NSCl), at 80 ºC for 40 min, and measuring the fluorescent product. Elution from the column was isocratic, using a mobile phase consisting of acetonitrile –1% phosphoric acid in water, (90:10, v/v), with a flow rate of 2 mL/min, at 40 ºC. Determination was developed using fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 380 nm. The chromatographic separation was achieved on Knauer-Eurospher 100–5 C18 silica column (300 mm × 4 mm i.d, 5 μm). Different approaches were used to optimize the conditions of the method and to reach the maximum yield. The linear regression data for the calibration plot showed acceptable correlation coefficient (r2 = 0.9998) at the concentration ranges of 2.5–1000 ng/mL for TOB. The method was successfully validated according to the guidelines of the International Council on Harmonization, and was found suitable for pharmaceutical applications. The method was new, simple and precise for analysis of TOB and applicable for routine analysis in quality control laboratories.

In-Needle Pre-Column Derivatization for Amino Acid Quantification (iPDAQ) Using HPLC.

Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.

Precolumn Derivatization High-Performance Liquid Chromatography for Determination of Perfluorocarboxylic Acids in Catalytic Degradation Solutions.

Perfluoroalkyl carboxylic acids (PFCAs), a series of ubiquitous contaminants in the global environment, attracted much attention due to their potential for high bioaccumulation and toxicity to various organisms. There are a lot of measurement requests in currently increasing degradation studies of PFCAs, which usually rely on expensive liquid chromatography-mass spectrometry (LC-MS). The degradation solutions containing high-concentration PFCAs can easily cause the pipeline pollution of the LC/MS instrument, which is usually used for trace analysis of environmental samples. In this study, a simple and reliable precolumn derivatization LC method coupled with an ultraviolet detector (UV) was developed for the determination of the main PFCAs (C4-9) of environmental concern. These PFCAs in degradation solutions were crosslinked to UV-responsive 3, 4-diphenylamine (DCA) by a carbodiimidization method, followed by a simple solid-phase extraction (SPE) cleanup, and quantitatively measured using a conventional LC-UV instrument. Compared to previously reported precolumn derivatization methods, this new derivatization approach has the advantages such as mild reaction conditions, easy operation, enhanced stability of derivatives, and low cost. The instrumental limits of detection (ILDs) for the targeted PFCAs in organic and aqueous mediums were 0.2–0.5 and 0.6–1.5 mg/L, respectively. The method has been successfully applied to the determination of PFCAs in catalytic degradation solutions and recommended for use in other assays involving relatively high-concentration PFCAs.

Development and Validation of an RP-HPLC-FLD Method for the Determination of Biogenic Amines after Pre-column Derivatization with a New Pyrazoline Based Reagent, 3- Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline

Application of 3-Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline (NFCP) as a novel fluorigenic label for the derivatization of biogenic amines (BAs) and separation by liquid chromatography (LC) was investigated. Optimum conditions for the pre-column derivatization and chromatographic separation were investigated. The resulting derivatives are fluorescent at λex/λem: 380/460 nm. For the sake of evaluating the efficacy of the proposed analysis of BAs of different nature, the formation of nine NFCP-BAs derivatives was tested individually. Simultaneous derivatization and separation of four BAs (histamine, tyramine, tryptamine, and phenylethylamine) were achieved under the isocratic elution mode. Furthermore, the derivatives were identified by the characteristic product ion obtained during LC-MS/MS analysis. The viability of the method was established by measuring levels of histamine in fresh and spoiled fish samples. Histamine concentrations were found to be 0.4 µgg-1 and 4.1 µgg-1 in fresh and spoiled tuna fish samples respectively. As a simple, reliable, and sensitive pre-column derivatization method, this work provides for future research to develop a novel HPLC method for the analysis of BAs from food samples such as fish and various other matrices by utilizing the new pyrazoline compound.

Accurate determination of amino acids by quadruple isotope dilution-reverse phase liquid Chromatography-Tandem mass spectrometry after derivatization with 2-Naphthoyl chloride

The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 – 0.266 µmol kg−1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled (15N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision.

MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal carcinoma cells

Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological and pathological significances, a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A one-step extraction of cells with 85% methanol followed by a simple derivatization reaction within 5 min at room temperature contributed to shortened analysis time. The derivatives of 22 nucleoside mono-, di- and triphosphates were retained on the typical C18 column and eluted by ammonium acetate and acetonitrile in 9 min. Under these optimal conditions, good linearity was achieved in the tested calibration ranges. The lower limit of quantitation (LLOQ) was determined to be 0.1–0.4 μM for the tested RNs and 0.001–0.1 μM for dRNs. In addition, the precision (CV) was <15% and the RSD of stability was lower than 10.4%. Furthermore, this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT 116 exposed to 10-hydroxycamptothecin. In conclusion, our method has proven to be simple, rapid, sensitive, and reliable. It may be used for specific expanded studies on intracellular pharmacology in vitro.

High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography

Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis, anti-oxidative, anti-tumor, and immunity boosting activities. The variation in the structure, composition, and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities. Therefore, extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control. Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region. At present, the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection, gas chromatography-ultraviolet detection, and mass spectrometry. Pressurized capillary electrochromatography is a high-efficiency microseparation technology. In this study, two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method, and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria. The interaction effects of four factors, the liquid-solid ratio, ultrasonic extraction time, ultrasonic extraction temperature, and ultrasonic power, on the extraction rate of the polysaccharides were analyzed. By combining the optimal conditions predicted by the software and the actual equipment conditions, the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature, 90℃; liquid-solid ratio of Pueraria thomsonii Benth, 20 mL/g; liquid-solid ratio of Pueraria lobata Ohwi, 40 mL/g; ultrasonic extraction time, 30 min; ultrasonic power, 180 W. Through data fitting, the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established. A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established. The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method. The separation conditions for these monosaccharides were explored, and the buffer concentration, buffer pH, applied voltage, type of chromatographic column, and mobile phase ratio were optimized. Finally, the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples. The results of response surface analysis showed that among the four experimental factors, ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria, followed by the liquid-solid ratio; the influence of the ultrasonic extraction time and ultrasonic power was relatively weak. The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7 μm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution (18:82, v/v, pH 4.1) as the mobile phase, by detection at 250 nm under an applied voltage of-20 kV. The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method. The results show that the proposed method has a good linear relationship and good repeatability. The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose, mannose, rhamnose, and fucose in the molar ratio 1.00:0.16:0.14:0.07. The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00:0.70. This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides, and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.