Targeted Analysis of Veterinary Drugs in Food Samples by Developing a High-Resolution Tandem Mass Spectral Library.

Veterinary drug residues present in foods can pose severe health threats to the population. The present study aims to develop a high-resolution mass spectral library of 158 veterinary drugs of 16 different classes for their rapid identification in food samples through liquid chromatography-high-resolution electrospray ionization-tandem mass spectrometry (LC-HR-ESI-MS/MS). Standard drugs were pooled according to their log P values and exact masses before analysis. Spectra were collected at system automated collision energy, i.e., of 25-60 eV and four predetermined collision energies (10, 20, 30, and 40 eV) for each compound using a schedule precursor list of [M + H]+, [M + Na]+, and [M + NH4]+ ions. The utility of the developed database was checked by analyzing food samples. A total of 17 veterinary drugs based on the reference standard retention times (RTs), HR-MS spectra, and MS/MS spectra were identified in the analyzed samples. Moreover, five veterinary drugs were selected for quantitative analysis, including doxycycline hyclate, lincomycin, sulfasalazine, moxifloxacin, and diphenoxylate, using liquid chromatography-ion trap mass-spectrometry (LC-IT-MS). Concentrations of the drug were obtained to vary from 0.0805 to 0.9731 mg/kg in food samples and were found to be exceeded in most of the cases as per the maximum residue levels described by Food and Agriculture Organization (FAO)/World Health Organization (WHO). The MS data were submitted to the MetaboLights online database (MTBLS2914). This study will help in the high-throughput screening of multiclass veterinary drugs in foodstuffs.

A rapid colorimetric method for the detection of carminic acid in samples based on visible color change.

Carminic Acid (CA), an insect-derived red color, is widely used as a colorant and additive in food and non-food items. The detection of CA is of great concern since it is unacceptable for vegetarians and vegans consumers. Therefore, it is important for food authorities to have a rapid detection method for CA. We describe here a simple and rapid method for the qualitative detection of CA, using Pb2+ for complex formation. As a result, the sample solution shows a visible change from pink to purple (bathochromic shift) which could also be analyzed through a spectrophotometer at λmax = 605 nm. The structure of the CA-Pb2+ complex was also studied through advanced spectroscopic techniques. Moreover, the presence of iron results in the formation of a stable CA-Fe2+ complex without any significant color change, as Fe2+ has a stronger binding affinity with CA. Thus, sodium fluoride (NaF) was used to prevent CA-Fe2+ complex formation. Therefore, two methods were developed based on the absence (method I) and presence (method II) of NaF. The LOD and LOQ for the method I was 0.0025 and 0.0076 mg mL-1, and for method II, values were 0.0136 and 0.0415 mg mL-1, respectively. The methods were also validated by intra and inter-day analyses. A total of 45 commercials, including food and non-food samples, were screened for the detection of CA. The developed methods are applicable for the effective and rapid surveillance of CA in various samples without the use of high-tech instruments.

Sensitive Detection of Pharmaceutical Drugs and Metabolites in Serum Using Data-Independent Acquisition Mass Spectrometry and Open-Access Data Acquisition Tools.

Data-independent acquisition (DIA) based strategies have been explored in recent years for improving quantitative analysis of metabolites. However, the data analysis is challenging for DIA methods as the resulting spectra are highly multiplexed. Thus, the DIA mode requires advanced software analysis to facilitate the data deconvolution process. We proposed a pipeline for quantitative profiling of pharmaceutical drugs and serum metabolites in DIA mode after comparing the results obtained from full-scan, Data-dependent acquisition (DDA) and DIA modes. using open-access software. Pharmaceutical drugs (10) were pooled in healthy human serum and analysed by LC-ESI-QTOF-MS. MS1 full-scan and Data-dependent (MS2) results were used for identification using MS-DIAL software while deconvolution of MS1/MS2 spectra in DIA mode was achieved by using Skyline software. The results of acquisition methods for quantitative analysis validated the remarkable analytical performance of the constructed workflow, proving it to be a sensitive and reproducible pipeline for biological complex fluids.

Phytochemical analysis and biological activities of "Cherchoomoro" (Nepeta adenophyta Hedge).

ETHNOPHARMACOLOGICAL RELEVANCE: Nepeta adenophyta Hedge (Lamiaceae) is an endemic therapeutic herb from Astore, Gilgit (Pakistan). This plant species has been reported among the local communities, especially for treating abdominal pain, kidney pain, menstrual pain, headache, and controlling bleeding disorders. Therefore, the scientific basis is provided for the relief of pain as it is used in various pain management among the natives, especially as ethnogynecological herbal remedy. AIM OF THE STUDY: The present study investigates the analgesic and anti-inflammatory effects of the ethanolic extract of N. adenophyta in animal models. Furthermore, the extract was also studied to determine their valuable phytoconstituents. MATERIAL AND METHODS: The biological effects were determined via tail-flick, hot plate, and acetic-acid-induced abdominal writhing methods. At the same time, anti-inflammatory activity was assesed via oxidative burst and antioxidant DPPH assay. Gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS) techniques were employed to understand the phytochemicals present in the crude ethanolic extract of Nepeta adenophyta. RESULTS: In the current study, Nepeta adenophyta extract exhibited potent analgesic and anti-inflammatory effects on different pain models and indicated that the analgesic effect of N. adenophyta extract is mediated both in central and peripheral ways. Dose-dependent and significant (P < 0.05) increases were shown in pain threshold, at 45 min post-treatment, with 20 and 40 mg/kg of the extract in the tail-flick model. The effects of the extract were similar to aspirin but lower to those by morphine (2.5 mg/kg) in the same tests. The extract (20-40 mg/kg) showed dose-dependent inhibition of writhing with a significant (P < 0.001) increase protection against thermal stimuli in hot plate test as compared to control and similar to aspirin and morphine. Further, the anti-inflammatory activity of the crude in oxidative burst and DPPH assays showed significant inhibitory activity. The chemical profile analysis showed major phytochemicals, including long chain derivatives of alkane and alcohol, phenolics, naphthalene, naphthopyran, androsten phenanthrenone, nepetalactones, flavonoids etc. CONCLUSIONS: Nepeta adenophyta Hedge is suggested as a natural alternative for mild pain relief. Our findings endorse the folklore use of N. adenophyta in different pain managements which can be attributed to the presence of polyphenolic compounds, naphthalene derivatives, flavanoids and nepetalactones etc.

High-Throughput Detection of an Alkaloidal Plant Metabolome in Plant Extracts Using LC-ESI-QTOF-MS.

Plant alkaloids represent a diverse group of nitrogen-containing natural products. These compounds are considered valuable in drug discovery and development. High-throughput identification of such plant secondary metabolites in complex plant extracts is essential for drug discovery, lead optimization, and understanding the biological pathway. The present study aims to rapidly identify different classes of alkaloids in plant extracts through the liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach using 161 isolated and purified alkaloids. These are biologically important unique alkaloids belonging to different sub-classes such as isoquinoline, quinoline, indole, tropane, pyridine, piperidine, quinolizidine, aporphine, steroidal, and terpenoid. The majority of these are not available commercially and are known to manifest valuable biological activities. Four pools of a maximum of 50 phytostandards each were prepared, based on their log P value to minimize co-elution for rapid and cost-effective analyses. MS/MS spectra were acquired in the positive ionization mode by using their [M + H]+ and/or [M + Na]+ with both the average collisional energy (25.5-62 eV) and individual collisional energies (10, 20, 30, and 40 eV). Accurate mass, high-resolution mass spectrometry (HR-MS) data, MS/MS data, and retention times were curated for each compound. The developed LC-MS/MS method was successfully used to interrogate and fast dereplicate alkaloids in 13 medicinal plant extracts and a herbal formulation. A total of 56 alkaloids were identified based on the reference standard retention times (RTs), HR-MS spectra, and/or MS/MS spectra. The MS data have been submitted to the MetaboLights online database (MTBLS2914). The mass spectrometric and chromatographic data will be useful for the discovery of new congeners and the study of biological pathways of alkaloids in the plant kingdom.