New LC-MS Method Development and Validation for Quantitative Estimation of 2-Acetoxy Ethyl Acetoxy Methyl Ether in Acyclovir

The objective of the proposed method is to develop and validate a specific, precise and accurate LC-MS method for the determination of 2-acetoxy ethyl acetoxy methyl ether (AEM) in Acyclovir. An isocratic separation was done by using YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column with a mobile phase composition of Buffer (0.1% Ammonia in water) : Methanol(25 :75v/v)at a flow rate of 0.4mL/min, diluent solution of 0.2% of 0.1N NaOH in Methanol were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) Acquisition mode and possitive polarity mode. The Retention time of AEM was found to be 5.344 minutes, LOD and LOQ were observed at 0.03 ppm and 0.11 ppm concentration respectively by signal to noise ratio method, linearity was found in the concentration range of 0.18-0.55 ppm with correlation coefficient of 0.996 and accuracy in the range of 97.7- 101% was obtained by performing percentage recovery studies. The % RSD was observed to be less than 10% for six replicates said to be precise. The proposed method was validated for accuracy, precision, sensitivity, and linearity successfully employed for quantitative determination of AEM in acyclovir.

Quantification of 15 Antibiotics Widely Used in the Critical Care Unit with a LC-MS/MS System: An Easy Method to Perform a Daily Therapeutic Drug Monitoring

Potential under- or overdose of antibiotics may occur in intensive care units due to high variability in plasma concentrations. The risk is either treatment failure or toxicity. Thus, therapeutic drug monitoring of antibiotics may guide dosing adjustment, maximising antibacterial efficacy and minimising toxicity. The aim of this study was to develop and validate a method for the analysis of 15 antibiotics including beta-lactams, linezolid, fluoroquinolones, daptomycin, and clindamycin to have a complete panel in the management of infections. We proposed to develop a fast, sensitive, and quantitative method for the analysis of 15 antibiotics using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-MS/MS) technology. this method required only 100 µL of plasma and consisted of a rapid liquid–liquid deproteinisation using methanol. Calibration curves ranged from 0.078 to 500 mg/L depending on the molecules, and were defined according to a therapeutic range. Inter- and intra-assay precisions values were less than 15%. This work described the development and the full validation of a precise, sensitive and accurate assay using UPLC-MS/MS technology. After validation, this new assay was successfully applied to routine therapeutic drug monitoring.

From the Autochthonous Grape Varieties of the Kastav Region (Croatia) to the Belica Wine

Research background. Coastal region of Croatia is rich in autochthonous grape varieties. Many of them have been almost abandoned such as the autochthonous varieties of Kastav (Croatia), used for the production of the Kastavska Belica wine. Therefore, the rationale of the presented study was to characterize autochthonous varieties 'Verdić', 'Mejsko belo', 'Jarbola', 'Divjaka' and 'Brajkovac' grape varieties. In addition, we performed a molecular characterization of the corresponding Belica wines. Experimental approach. Firstly, for five autochthons grape varieties, their genetic origin and ampelographic and economic characteristics were determined. Standard physico-chemical profiles and phenolic components by use of liquid chromatography coupled to triple quadrupole mass spectrometer (LC-QQQ) were determined for 12 corresponding wines from different producers. Fourier-transform infrared spectroscopy (FTIR) was used for determination of standard physico-chemical parameters Results and conclusions. Ampelographic analysis, which includes the data on producing characteristics and cluster and berry composition of varieties, revealed significant differences between analysed grape varieties. Results of physico-chemical analysis of the Belica wine showed that all wines met the requirements needed for the production of quality and top quality wines labelled with protected designation of origin (PDO) in Croatian coastal region. The LC-QQQ analysis confirmed the presence of different phenolic components in the Belica wines, where the most prominent phenols were flavonoids from the flavan-3-ol group. Overall, these results showed that autochthonous grapes from the Kastav region can be used for production of wines with added market value due to a growing demand for autochthonous products on the global market. Novelty and scientific contribution. The presented results represent scientific insight and a basis for further determination of the optimal cultivation technology aimed to take advantage of the best characteristics of each variety for production of a wine with desirable features.

Observations of Gas-Phase Alkylamines at a Coastal Site in the East Mediterranean Atmosphere

Atmospheric amines are ubiquitous compounds in the atmosphere, having both natural and anthropogenic origin. Recently, they have been identified as important contributors to new particle formation in the atmosphere, but observations of their atmospheric concentrations are scarce. In the present study we introduce the first systematic long-term observations of gas-phase amines measurements in the East Mediterranean atmosphere. Air samples were collected at the Finokalia monitoring station of the University of Crete during a 3.5-year period from January 2013 to July 2016, and analyzed after extraction using a high-performance liquid chromatography triple quadrupole mass spectrometer. The detected alkylamines were the sum of dimethylamine and ethylamine (DMA+EA), trimethylamine (TMA), diethylamine (DEA) and triethylamine (TEA). DMA+EA and TMA were the most abundant alkylamines, with concentrations spanning from the detection limit to 78.0 and 69.8 pptv, and average concentrations of 7.8 ± 12.1 and 7.5 ± 12.4 pptv, respectively. Amines showed pronounced seasonal variability with DMA+EA and TMA concentrations being higher in winter. Statistical analysis of the observations showed different sources for each of the studied amines, except for DMA+ΕA and DEA that appear to have common sources in the region. This analysis points to a marine source of TMA and animal husbandry in the area as a potential source of TEA. None of the alkylamines is correlated with other anthropogenic sources. Furthermore, no clear association was found between the seasonality of NPF events and alkylamines concentrations, while a clear correlation was detected between the seasonality of nucleation mode particle (dp < 25 nm) number concentrations and alkylamine concentrations, indicating that amines may contribute to nucleation mode particles’ production.

Ion-pairing chromatography and amine derivatization provide complementary approaches for the targeted UPLC-MS analysis of the polar metabolome

Liquid chromatography coupled to mass spectrometry is a key metabolomics technology. Reversed-phase liquid chromatog-raphy (RPLC) is very widely used as a separation step, possessing excellent characteristics with respect to reproducibility and reliability, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alter-native methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. We detected 132 metabolites for real biological samples (liver extracts) with good reproducibility (based on coefficients of variation in pooled biological quality control samples). Finally, we tested the combination of methods with real-world samples by analyzing a set of liver extracts from aged male and female mice that had been treated with the poly-phenol compound ampelopsin. Furthermore, we also compared the results of these LC-MS methods to 1H NMR spectrosco-py as an orthogonal method (also termed statistical heterospectroscopy (SHY)), and found a strong correlation between the results of these different analytical approaches. By these means, not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the di-rection of metabolite change was opposite for males and females.

Preparation of Anti-Aristolochic Acid I Monoclonal Antibody and Development of Chemiluminescent Immunoassay and Carbon Dot-Based Fluoroimmunoassay for Sensitive Detection of Aristolochic Acid I

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA.

Determination of Antibiotics, Pesticides, Herbicides, Fungicides and Hormones in Water Bodies in Italy in Occurrence with European Watch List Mechanism by Using an UHPLC-MS/MS System: Method Validation, Quantification and Evaluations

In recent years, the quality of aquatic ecosystems has received increasing attention from European institutions. The Commission Implementing Decision (EU) 2018/840 drafted a Watch List (WL) of compounds to be monitored in Europe. In this study, we report a method based on solid phase extraction with ultra-high-performance liquid chromatography, coupled with a triple-quadrupole mass spectrometer (UHPLC-MS/MS) to analyze the whole water sample. The method was developed and validated for the determination of 12 listed compounds. The employment of solid-phase extraction by a horizon system ensures the analysis of the entire body of samples and minimizes sample manipulation. Different ng L−1 detection limits (from 2 to 50 ng L−1), linearities (from 2 to 500 ng L−1), accuracy (from 70 to 130%) and levels of precision (RSD less 20% at LOQs levels) were assessed to be satisfactory for quantification and confirmation at the levels of interest. The developed method was applied for quantitative analysis for Watch List compounds (with the exception of hormones) in surface water samples from different Italian sites during monitoring activities by the Regional Environmental Protection Agencies in the years 2019 and 2020.

Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry

Background: A liquid chromatography tandem mass spectrometry method to quantify drugs in dried cervicovaginal secretions from flocked swabs was developed and validated using the antiretroviral efavirenz as an example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). A total of 98 samples (35 paired intensive CVS and DBS samples, 14 paired sparse CVS and DBS samples) from 19 participants were available for this analysis. Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC0-24h was 16370 ng*h/mL (5803-22088), Cmax was 1618 ng/mL (610-2438) at a Tmax of 8.0 h (8.0-12), and Cmin was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC0-24 ratio was 0.41 (0.20-0.59). Conclusions: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.

Determination of Cyanide in Blood for Forensic Toxicology Purposes—A Novel Nci Gc-Ms/Ms Technique

One of the recently evolving methods for cyanide determination in body fluids is GC-MS, following extractive alkylation with pentafluorobenzyl bromide or pentafluorobenzyl p-toluenesulfonate. The aim of this study was to improve previous GC methods by utilizing a triple quadrupole mass spectrometer, which could enhance selectivity and sensitivity allowing for the reliable confirmation of cyanide exposure in toxicological studies. Another purpose of this study was to facilitate a case investigation including a determination of cyanide in blood and to use the obtained data to confirm the ingestion of a substance, found together with a human corpse at the forensic scene. The blood samples were prepared following extractive alkylation with a phase transfer catalyst tetrabutylammonium sulfate and the PFB-Br derivatization agent. Optimal parameters for detection, including ionization type and multiple reaction monitoring (MRM) transitions had been investigated and then selected. The validation parameters for the above method were as follows—linear regression R2 = 0.9997 in the range of 0.1 µg/mL to 10 µg/mL; LOD = 24 ng/mL; LOQ = 80 ng/mL and an average recovery of extraction of 98%. Our study demonstrates the first attempt of cyanide determination in blood with gas chromatography-tandem mass spectrometry. The established method could be applied in forensic studies due to MS/MS confirmation of organic cyanide derivative and low matrix interferences owning to utilizing negative chemical ionization.