HPLC-MS Method Research Articles

An Antiviral Drug-Peramivir: Degradation and Identification of Impurities and the Endorsement of an HPLC-MS Method.

Peramivir is a neuraminidase inhibitor that serves as a transition state analogue for influenza neuraminidase, inhibiting the formation of new viruses in infected cells, and has been approved for intravenous administration. To validate an HPLC method used to identify the degraded products of the antiviral drug peramivir. Herein, we report the identification of compounds formed after the degradation of peramivir through acid, alkali, peroxide, thermal, and photolytic degradation. At the level of toxicology, a technique was devised for the isolation and measurement of peramivir. A sensitive and reliable LC-tandem mass spectrometry technique for the quantitative measurement of Peramivir and its impurities was developed and verified in order to comply with the recommendations made by the International Council for Harmonisation (ICH). The proposed protocol was in the 50-750 µg/mL range. Relative Standard Deviation values of less than 2.0% indicated good recovery in the range of 98.36-102.57%. Within the studied range, the calibration curves demonstrated good linearity and, in addition, the fitting of correlation coefficient was more than 0.999 for every impurity. Quantitative analysis of contaminants revealed the high efficiency at a low level. Given its ability to separate degradation products, quantitative analysis is used to detect and quantify known and unknown impurities and degradants in the peramivir drug substance during routine analysis and stability studies. No significant degradation was found in peroxide and photolytic degradation studies. An HPLC method was developed and put to the test in order to analyze the behavior of the impurities of peramivir as they degraded when subjected to the stress conditions suggested by the ICH. Peramivir was found to be stable under peroxide and photolysis conditions but not stable or degradable when exposed to the acid, base, and thermal stress conditions. The method developed was extremely precise, linear, accurate, robust, and rugged. As a result, this technology has the potential to be used in the medication production process for regular impurity analysis as well as for the stability analysis of peramivir.

Photon localization-assisted visible light photocatalysis of photonic crystal CdS-N/ZnO heterojunction for efficient photodegradation tetracycline hydrochloride

Herein, ZnO based materials are modified to narrow the photonic band gap, improved utilization rate of visible light and recombination efficiency of photoexcited carriers for the photocatalytic degradation of tetracycline hydrochloride (TCH). For this purpose, a photonic crystal (PC) heterojunction was constructed comprising of nitrogen (N) doped cadmium sulfide (CdS) coupled with ZnO (CdS-N/ZnO) via sacrificial template and multilayer in-situ deposition methods. The constructed PC junction acted as photocatalyst for the effective degradation of TCH under visible light by synergistically promoting the separation efficiency of electron-hole pairs, narrowing the band gap and broadening the light absorption spectrum of ZnO. Moreover, a slow light effect was achieved by adjusting the band gap of the PC overlapped with the wavelength of incident light, which finally enhanced the light harvesting performance of the CdS-N/ZnO photocatalyst. The optimized PC CdS-N/ZnO composites exhibited efficient TCH removal rate of 97.0 % in 90 min and kb as 4.00–15.17 times that of other reported catalysts. Moreover, PC CdS-N/ZnO catalyst achieved 45.2 % mineralization rates reached in 120 min with high stability and reusability for six cycles. HPLC-MS method was used for monitoring the intermediates of TCH degradation, and also analyzing the possible pathways of products. In addition, the toxicity of TCH and its intermediates were also investigated, which showed that the toxicity has effectively been alleviated. The photocatalytic mechanism endowed that ·O2– and h+ are the major active free radicals in the photocatalytic degradation process, and the electron transport path is type Ⅱ heterostructure. This work will thus envisage a novel vision of designing ZnO photocatalysts for the antibiotic degradation in the practical application of wastewater treatment.

Extraction and Quantification of Azelaic Acid from Different Wheat Samples (Triticum durum Desf.) and Evaluation of Their Antimicrobial and Antioxidant Activities

Azelaic Acid (AzA) is a 9-carbon atom dicarboxylic acid, with numerous pharmacological uses in dermatology. Its effectiveness in papulopustular rosacea and acne vulgaris, among other dermatological disorders such as keratinization and hyper-pigmentation, is thought to be related to its anti-inflammatory and antimicrobial properties. It is a by-product of Pityrosporum fungal mycelia metabolism but also it is found in different cereals such as barley, wheat, and rye. Diverse topical formulations of AzA exist in commerce, and it is mainly produced via chemical synthesis. In this study we describe the extraction of AzA from whole grains and whole-grain flour (Triticum durum Desf.) through green methods. Seventeen different extracts were prepared and analyzed for their AzA content by HPLC-MS methods and then screened for their antioxidant activity using spectrophotometric assays (ABTS, DPPH, and Folin-Ciocalteu). Minimum-inhibitory-concentration (MIC) assays against several bacterial and fungal pathogens were performed, to validate their antimicrobial activity. The obtained results indicate that whole grain extracts provide a wider spectrum of activity than the flour matrix; in particular, the Naviglio® extract showed higher AzA content, while the hydroalcoholic ultrasound-assisted extract provided better antimicrobial and antioxidant activity. The data analysis was performed using principal component analysis (PCA), as an unsupervised-pattern-recognition technique, to extract useful analytical and biological information.

Portable colorimetric enzymatic disposable biosensor for histamine and simultaneous histamine/tyramine determination using a smartphone

Tyramine oxidase (TAO), peroxidase (HRP), and Amplex Red (AR) have been immobilized on cellulose to obtain disposable biosensors for the determination of histamine. During the enzymatic reaction, AR is oxidized and a pink spot is obtained. Using a smartphone and measuring the G (green) color coordinate, histamine can be determined in the presence of other biogenic amines (putrescine and cadaverine) in concentrations ranging from 2·10−5 M to 5·10−4 M with a 7.5·10−6 M limit of detection (LoD). Despite tyramine interference, experimental conditions are provided which allow rapid and simple histamine and simultaneous histamine/tyramine (semi)quantitative determination in mixtures. Finally, tyramine and histamine were determined in a tuna extract with good results (compared to the reference HPLC–MS method). The methodology can also be applied in solution allowing histamine (and simultaneous histamine/tyramine) determination with a lower LoD (1.8·10−7 M) and a similar selectivity.Graphical abstract

Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction – Parallel Ion Parking

β-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of β-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/β ratio can act as a biomarker to identify β-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/β ratio as a thalassemia biomarker.

Cytokinin localization and dynamics in rye plants under chilling and kernel priming with zeatin

In the early stages of plant development, a sharp chilling can pose a significant threat to cold-resistant crops, such as winter cereals, in particular, rye. Phytohormones from the cytokinin family play a crucial role in regulating plant response to low-temperature stress. Pre-sowing seed priming with cytokinin solutions is a promising approach to improving the stress tolerance of cereals. The aim of this study was to investigate the impact of chilling on the dynamics and localization of cytokinins in plants of rye cv. Boguslavka in the early stages of development, to determine the possibility of mitigating the adverse effects of cold stress through kernel priming with a zeatin solution, and to assess the effect of this substance on the balance of endogenous cytokinins. Kernels were soaked in either water or a zeatin solution (10—6 М), and plants were grown under controlled conditions of the vegetation chamber. Chilling was induced by exposing 7-day-old plants to the refrigerator for 2 h or twice for 6 h over two days. Endogenous cytokinins were analyzed using the HPLC-MS method. We found significant changes in the cytokinin pool in plants exposed to 2 °C for 2 h while no morphological alterations were observed. The content of trans-zeatin and trans-zeatin riboside increased considerable in the shoots of the plants grown from the kernels soaked in water, whereas in the roots, the level of these cytokinins dropped sharply. Prolonged chilling led to retarded shoot growth and elongated roots. Simultaneously, the level of trans-zeatin, isopentenyladenosine and isopentenyladenine in the shoots decreased to minimum values, while the amount of trans-zeatin riboside increased almost twofold. Transformations of the cytokinin pool in the roots followed a similar pattern. Overall, under the cold stress at the alarm stage, cytokinin balance changes were aimed at maintaining growth processes, while during the acclimation stage, they contributed to growth inhibition. Pre-sowing priming of rye kernels with a zeatin solution allowed plants to maintain normal growth and morphology under of prolonged chilling. This treatment significantly affected the homeostasis of endogenous cytokinins, resulting in a more moderate rearrangement of the cytokinin pool in stressed plants than in the control. We assume that changes in the balance of cytokinins due to seed priming allow plants to improve cold tolerance. Seed treatment with cytokinins could be a promising approach to increasing the adaptive properties of plants.

Aptamer-locker probe coupling with truncated aptamer for high-efficiency fluorescence polarization detection of zearalenone

Zearalenone (ZEN) is a kind of mycotoxin with estrogen-like effect. The presence of ZEN in cereals is potentially harmful to the health of humans. Herein, for the first time, the binding mechanism of ZEN aptamer with 19 bases (Z19) were systematically studied by molecular dynamics, circular dichroism and isothermal titration calorimetry. The binding driving forces, key bases of Z19 to ZEN, the conformational changes and thermodynamic information were originally found. On this basis, by employing the inherent rapid nature of fluorescence polarization and target recycling induced by Exo III, a novel fluorescence polarization aptasensor was constructed. The realized “add and measure” detection mode can be completed within one hour. Under the optimal conditions, the detection platform showed excellent analytical performance with a linear range of 0.01–100 ng/mL and a limit of detection as low as 0.004 ng/mL. Moreover, the practicability and reliability of the constructed strategy were confirmed with standard HPLC-MS method in corn and wheat samples. The established aptasensor with enhanced sensitivity broaden the application scope of fluorescence polarization technique, especially in detection targets of low molecular weight.

Quantitative analysis of lignans from the fruits of wild chervil (Anthriscus sylvestris (L.) Hoffm.)

Anthriscus sylvestris (L.) Hoffm., known as wild chervil, is a perennial widespread Apiaceae species. Being rich in lignans, it was used in traditional medicine to treat headaches, as analgesic, antipyretic, diuretic, antitussive, antihypertensive etc. Quantitative studies of lignans in Anthriscus sylvestris are limited to a few dominant lignans in roots and herbs. Hereby, the HPLC-MS method was used to quantify 14 lignans in fruit extract of A. sylvestris. A much higher content of lignans (970.2 mg/g) was found, compared to the previously analyzed root and herb extracts (4.3-66 mg/g) from the same location. The three most abundant lignans were found to be deoxypodophyllotoxin (867 mg/g), yatein (61.0 mg/g) and dimethylmatairesinol (27.7 mg/g). A significant amount of deoxypicropodophyllotoxin was also detected, but not quantified due to lack of reference standard. Due to previously observed spontaneous interconversion of cis/trans isomers, nemerosin and isochaihulactone, as well kaerophyllin and isokaerophyllin, were quantified together. Based on the obtained results, the fruits of A. sylvestris could be used as an industrial raw material for obtaining deoxypodophyllotoxin. This is also supported by a significantly simpler lignan profile of the fruit extract.

AZƏRBAYCAN FLORASINDAN DATURA STRAMONİUM L. NÖVÜNÜN KİMYƏVİ TƏRKİBİNİN TƏDQİQİ

By using GC-MS and HPLC-MS methods, alkaloids have been investigated in the Datura stramonium (thorn apple) from the flora of Azerbaijan. Totally 40 nortropane, tropane and pyrrolidine derivatives have been identified in the plant samples. Atropines have been quantified in the aerial parts of plant by using HPLC-DAD method in comparison with analytical standard of substance. Atropine was 4.353 mg/g in the dried plant sample. Spermidine and tyramine derived natural polyamines have been detected in the aerial parts of plant by performing HPLC-MS method. Sterols have been tested in the seed oil of plant in comparison with analytical standards of cholesterol, kaempesterol, stigmasterol and β-sitosterol by using GC-FID method. ICP-MS have been performed for qualitative and quantitative determination of 6 macroelements and 13 microelements in the roots, stems, leaves, flowers, fruits and seeds of plant. Along with heavy metals, several elements, which have macro- and micronutrient importance, have been determined in the samples.

Lipid biomarkerek HPLC-HRMS vizsgálata történeti embertani minták tbc diagnosztikája során

Tuberculosis (TB) is not only an infectious disease but one of the top 10 causes of death, spreading mainly with aerosol transmission and accompanying the history of humankind for several millennia. TB is caused in humans and animals by members of the Mycobacterium tuberculosis complex. For better understanding of the disease and the evolutional background of its causative agent, involvement of palaeopathological investigations is surpassingly important. Traditionally, palaeopathology is using a broad variety of markers, which are observable by macroscopic investigations. These markers are mainly related to different extra-pulmonary forms of TB. However, these manifestations develop only in a few cases. Moreover, many markers are not pathognomonic of TB. Therefore, to avoid underestimation of TB prevalence in paleopathological studies, the diagnosis on archaeological material requires a multidisciplinary approach. For better estimation of TB incidence in past populations, an array of specific biomarkers can be brought into play to confirm initial, macromorphology-based skeletal diagnoses, namely aDNA and lipid biomarkers. The three lipid biomarker groups, which are mainly involved in such investigations are the mycolic acids (MAs), the mycocerosic acids (MCs) and the C27 mycolipenic acid (ML). These unique lipids can be located in the lipid-rich mycobacterial cell wall. The application of lipid-based TB diagnostic approach has been proved to be robust and reliable through many examples. A variety of analytical methods have been employed for mycobacterial lipid biomarker profiling. Fluorescence HPLC is well-developed for the analysis of mycolic acids and phthiocerols. Furthermore, one isolated case of direct mass spectrometric detection of M. tuberculosis free mycolic acids has been also introduced. Our aim was to establish a lipid-biomarker-based HPLC-MS method for TB diagnosis in historical human samples, as this instrumentation is available in many laboratories, has the potential of a quick and sensitive and at the same time an affordable measurement protocol. We successfully developed and optimised a method, which is capable to separate and detect MAs and MCs. Moreover, we optimised the sample pre-treatment process. We started to build an MA and MC lipid profile library, which can serve as a comparison to diagnose TB. For the verification of new methods and the estimation of reliability of new markers in macroscopic analysis, palaeopathologists usually use well-documented collections of skeletons and mummies from the pre-antibiotic era. For this purpose, we chose to test our method on the Vác Mummy Collection. In the case of four out of six mummified individuals, MC profiles of characteristic M. tuberculosis MCs were recorded. The HPLC-ESI-MS method, developed for the detection of MCs, opens a new avenue for the detection of ancient mycobacterial disease, encompassing both tuberculosis, leprosy and joint cases.

Development and Validation of Eltrombopag Determination in Human Plasma Blood by HPLC-MS Method

Introduction. "Eltrombopag" is a thrombopoietin receptor agonist (TPO-RA) that is approved for the treatment of immune thrombocytopenic purpura (ITP). According to the literature, very few analytical methods for determining eltrombopag in biological samples have been reported. To study the pharmacokinetics of new formulations of eltrombopag, a sensitive and specific method is required that allows one to accurately determine the concentration of eltrombopag in human blood plasma. Normally, HPLC methods should provide time, accuracy, and sensitivity as a result, it is necessary to develop fast or ultra-fast methods such as LC-MS/MS without any loss in sensitivity or separation efficiency.Aim. We aimed to develop and validate a method for the quantitative determination of eltrombopag levels in human plasma by using HPLC with mass spectrometric detection for performing the analytical part of pharmacokinetic studies.Materials and methods. Eltrombopag levels were determined in human plasma by HPLC with mass spectrometric detection. The samples were prepared using protein deposition.Results and discussion. The method was validated for selectivity, matrix effect, calibration curve, accuracy, precision, the limit of quantification, carry-over effect, and sample stability.Conclusion. The method for the determination of eltrombopag levels in human plasma has been developed and validated by HPLC-MS. The analytical range of eltrombopag levels in human plasma was 10–6750 ng/ml. This method could be used to determine eltrombopag levels in plasma for PK and BE studies.

Activity assay based on the immobilized enzyme kallikrein and mass spectrometry

Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.

Validation of high performance liquid chromatography coupled to mass spectrometry to determine albendazole sulfoxide in human plasma

Context: Albendazole is used in the treatment of cysticercosis and hydatid disease, and its prolonged administration can generate adverse effects attributed to the active metabolite, albendazole sulfoxide (ABZSO), whose measurement requires validated methods that help monitor patients under treatment. Aims: To validate a high-performance liquid chromatography method with mass detection (HPLC-MS) for the determination of ABZSO in human plasma. Methods: HPLC-MS was performed on C18 columns (100 × 4.6 mm × 3.5 µm) and mobile phase composed of methanol, water and ammonia solution (25%) (800:200:0.1 v /v/v) at a flow rate of 0.5 mL/minute. The calibration curve was built with an albendazole standard, and detection in a triple quadrupole system. Results: The method was linear (1-1500 ng/mL, R2 = 0.999) with a detection limit of 1 ng/mL. ABZSO concentrations at 1, 5, 50, 100, 200, 500, 1000, and 1500 ng/mL had relative standard deviations of 10.4, 8.4, 7.8, 2.9, 7.4, 3.2, 3.4 and 1.3%. It was highly precise with a mean coefficient of variation of 1.411%, and accurate with a bias of -14.7; 2.8 and -0.8% in ABZSO concentrations of 5, 500 and 1000 ng/mL. The average recovery was 89.2%, and the stability of ABZSO after refrigeration had an average coefficient of variation of 2.3%. Conclusions: The HPLC-MS method is valid and reliable to determine ABZSO, and could be used in pharmacokinetic, bioequivalence, and other studies that evaluate the biological monitoring of patients receiving oral treatment with albendazole.

Quality by Design Approach for a Multicomponent Quantification Using HPLC-PDA and HPLC-MS: Application to Dosage Form and Biological Body Fluids

A multicomponent pharmaceutical that contains loratadine, paracetamol, and pseudoephedrine was quantified using HPLC-PDA. The three analytes were well-separated and quantified in the dosage form on a C-18 column using a gradient mobile phase. A quality by design strategy was followed to achieve the challenging separation. Screening and optimization steps were carried out to investigate the effect of many factors on the studied responses with a minimum number of runs. The ANOVA of the factorial model showed that % acetonitrile (factor A), flow rate (factor B), and pH (factor C) were significant. The detection of the analytes’ peaks was carried out using a PDA detector at 248nm for loratadine and paracetamol, and 214 nm for pseudoephedrine. The second method was SPE-HPLC-MS, where the three analytes and desloratadine, the active metabolite of loratadine, were quantified in spiked plasma and urine, using betamethasone valerate as an internal standard. The recovery of the analytes from body fluids was above 96%, and the LOQ was below 0.5 ng/mL. The validation of the developed HPLC-PDA method was achieved as per ICH guidelines, whereas the HPLC-MS method was validated according to FDA guidelines for bioanalytical method validation. The results were compared with the reported method, and no significant differences were found.

Development of a Novel HPLC-MS Method to Separate Polar and Non-Polar Compounds in Biodiesel/Petrodiesel Mixtures

Due to a trend to higher sustainability, biodiesel is often mixed into petrodiesel. The analysis of these blends on a molecular level is not trivial, since huge differences in concentrations and polarity of the analytes require a large dynamic range of the analytical method, as well as the ability to investigate molecules of widely different polarities. A combination of high-performance liquid chromatography (HPLC) with high resolution mass spectrometry (HRMS) was identified as a promising method and a normal-phase (NP)-HPLC using amino-functionalized silica gel-based stationary phase delivered the best results with very fast (under 4 min) measurements, with distinct separation of the compounds and clean mass spectra of singular compounds. This method can also be easily modified to elute all FAMEs (fatty acid methyl esters) in one singular peak, thus making the separation even faster (under 3 min).

Quantitation of the ataxia-telangiectasia-mutated and Rad3-related inhibitor elimusertib (BAY-1895344) in human plasma using LC-MS/MS.

Ataxia-telangiectasia-mutated and Rad3-related (ATR) is master regulator of the DNA-damage response that, through multiple mechanisms, can promote cancer cell survival in response to replication stress from sources, including chemotherapy and radiation. Elimusertib (BAY-1895344) is an orally available small-molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. To support these studies and define elimusertib pharmacokinetics, we developed a HPLC-MS method for its quantitation. A 50-μL volume of plasma was subjected to acetonitrile protein precipitation and then chromatographic separation using a Phenomenex Polar-RP column (2 × 50 mm, 4μm) and a gradient mobile phase consisting of 0.1% formic acid in acetonitrile and water during a 7-min run time. Mass spectrometric detection was achieved using a SCIEX 4000 triple-stage mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, the assay was linear from 30 to 5000 ng/mL and proved to be both accurate (93.5-108.2%) and precise (<6.3% coefficient of variation) fulfilling criteria from the Food and Drug Administration guidance on bioanalytical method validation. This LC-MS/MS assay will support several ongoing clinical studies by defining elimusertib pharmacokinetics.

Building comprehensive glucosinolate profiles for brassica varieties

Brassica plants play an important role in common agricultural practices, such as livestock feed or biofumigation, due to the bioactivity of the natural degradation products of glucosinolate metabolites. Therefore, the ability to survey comprehensive glucosinolate profiles for individual brassicas is essential for informing proper species selection for the intended application. Current methods for glucosinolate identification and quantification involve complex or unconventional procedures, and proper reference materials are not readily available. Therefore, researchers with limited resources that require glucosinolate profiles are at an extreme disadvantage. In this work, a simple and accurate HPLC-MS method was developed and validated to build preliminary glucosinolate profiles for three agriculturally relevant forage brassica varieties [turnip (B. rapa L.), canola (B. napus L.), and rapeseed (B. napus L.)]. The average glucosinolate content across three herbage collection dates for canola, rapeseed and turnip were 2.9 ± 0.9 mg g−1, 6.4 ± 1.3 mg g−1, and 14 ± 3.4 mg g−1, respectively. GLS concentrations are reported in milligrams of glucosinolate, calculated as sinigrin equivalents, per gram of dry plant material. This semi-quantitative approach for reporting total GLS content in brassicas is accurate within 15%. Several minor individual glucosinolates were identified that have not been previously reported in canola, rapeseed and turnip species, including glucotropaeolin and 4-hydroxyglucobrassicin (canola), glucoraphanin and glucoberteroin (rapeseed), and glucosinalbin and glucobarbarin (turnip). This non-targeted screen of several forage brassica varieties demonstrates the inherent variation in both the individual glucosinolate content and the total glucosinolate profile among brassicas, and highlights the importance of such glucosinolate characterization in agricultural practices. Additionally, the method developed in this study can be used as a tool for researchers with limited resources to build accurate glucosinolate profiles of brassica plants.

Quantitative Analysis and Differential Evaluation of Radix Bupleuri Cultivated in Different Regions Based on HPLC-MS and GC-MS Combined with Multivariate Statistical Analysis.

The quality of Radix Bupleuri is greatly affected by its growing environment. In this study, Radix Bupleuri samples that were harvested from seven different regions across northwest China were examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) to reveal significant differences in quality contributed by the cultivation region. An HPLC-MS method was firstly established and used in the multiple reaction monitoring mode for the quantitative analysis of five saikosaponins in Radix Bupleuri so as to evaluate the difference in the absolute content of saikosaponins attributable to the cultivation region. The effect on the components of Radix Bupleuri was further investigated based on the profiles of the representative saponins and volatile compounds, which were extracted from the Radix Bupleuri samples and analyzed by HPLC-MS and GC-MS. Multivariate statistical analysis was employed to differentiate the Radix Bupleuri samples cultivated in different regions and to discover the differential compositions. The developed quantitative method was validated to be accurate, stable, sensitive, and repeatable for the determination of five saikosaponins. Further statistical tests revealed that the collected Radix Bupleuri samples were distinctly different from each other in terms of both saponins and volatile compounds, based on the provinces where they were grown. In addition, twenty-eight saponins and fifty-eight volatile compounds were identified as the differentially accumulated compositions that contributed to the discrimination of the Radix Bupleuri samples. The Radix Bupleuri samples grown in Shouyang county showed the highest content of saikosaponins. All of the results indicated that the cultivation region significantly affected the accumulation and diversity of the main chemical components of Radix Bupleuri. The findings of this research provide insights into the effect of the cultivation region on the quality of Radix Bupleuri and the differentiation of Radix Bupleuri cultivated in different regions based on the use of HPLC-MS and GC-MS combined with multivariate statistical analysis.

Production and Selection of Antibody-Antigen Pairs for the Development of Immunoenzyme Assay and Lateral Flow Immunoassay Methods for Carbofuran and Its Analogues.

To produce a sensitive monoclonal antibody (mAb) for the simultaneous detection of carbofuran, benfuracarb, carbosulfan and 3-hydroxy-carbofuran, 2,3-dihydro-2,2-dimethyl-7-benzofuranmethanamine (DDB) was conjugated to bovine serum albumin (BSA) to prepare the immunogen DDB-BSA and mice were immunized. Coating antigens were prepared by conjugating DDB and 5-methoxy-2,3-dihydrobenzofuran-3-acetic acid (MDA) to BSA and ovalbumin (OVA), respectively. Furthermore, the effect of different antibody–antigen pairs on the sensitivity of ELISA and LFIA methods for the detection of carbofuran was investigated. After the immunization, a high-affinity mAb 13C8 was obtained. The ability of the coating antigen to compete with carbofuran for binding antibodies was found to be significantly different between ELISA and LFIA methods. With the antibody–antigen pair 13C8-MDA-OVA, the IC50 values of the ELISA and QD-LFIA methods for carbofuran were 0.18 ng/mL and 0.67 ng/mL, respectively. The cross-reactivity (CR) values of the two methods for benfuracarb, carbosulfan and 3-hydroxy-carbofuran ranged from 72.0% to 83.7%, while, for other carbamate pesticides, the CR values were less than 1%. The spiked recoveries of carbofuran in vegetables by the QD-LFIA method were 83–111%, with a coefficient of variation below 10%, and the test results of the actual samples were consistent with the HPLC-MS method. Overall, this study provides key materials for the development of immunoassays for carbofuran and its analogues, and the antibody–antigen pair selection strategy established in this study provides useful insights for the development of sensitive immunoassays for other compounds.

Development of a Sensitive and Rapid HPLC-MS Method for Dihydrocodeine and Dihydromorphine: Application to Bioequivalence Studies.

A ultraperformance liquid chromatography-tandem mass spectrometry method has been developed to determine dihydrocodeine (DHC) and dihydromorphine (DHM) in human plasma using dihydrocodeine-d6 and desomorphine as internal standards (IS). Acetonitrile-water-ammonium format was used as the mobile phase, in gradient elution on a C18 column. The concentration of DHC and DHM was determined in the positive ionization mode of mass spectrometry. The total chromatogram run time was 3.2min, and the linear ranges of DHC and DHM were 1.000-400.0ng/mL and 0.050-20.00ng/mL, respectively. The method was fully validated concerning precision, accuracy, selectivity, linearity, recovery, stability and matrix effect. The method had been successfully applied to the bioequivalence test. In addition, we found that a high-fat diet impacts the Tmax and t1/2 of DHC.