Inter-day RSD Research Articles

Evaluation of different extraction procedures for the quantification of seven cannabinoids in cannabis-based edibles by the use of LC-MS

Although still illegal in many countries, food products containing cannabis or marijuana extracts have become very popular in recent years. In the present study, an LC-MS method was developed for the quantitative analysis of seven cannabinoids in various solid and liquid cannabis-based goods. The proposed analytical approach demonstrated satisfactory performance characteristics in terms of linearity (R2≥0.995), accuracy (recovery: 70.0–110 %), precision (intraday RSD: 0.950–6.03 %, interday RSD: 1.02–6.94 %), sensitivity (LOD≤2.19 ng/mL, LOQ≤6.59 ng/mL) and carry-over effect (average carryover signals ≤3.90 %). Solid-phase extraction (SPE), and ultrasound-assisted extraction (UAE) were utilized for the extraction of the analytes from liquid cannabis edibles (beer and energy drink), while Soxhlet and ultrasound-assisted extraction (UAE) were used for solid products (chocolates, hemp seeds, hemp tea). Infusion and decoction processes were followed for cannabis hemp tea and roasted coffee, respectively. UAE provided higher extraction efficiencies for cannabis-based edibles in solid form, while infused-cannabis beverages were extracted more efficiently using the SPE procedure. Cannabidiol (CBD) and cannabigerol (CBG) were the most detectable cannabinoids in all examined samples. Significantly high levels of cannabinoids were detected in cannabis tea extract prepared by the UAE procedure (total cannabinoids: 5440 μg/g). According to the suppliers, all examined samples were supposed to be free of Δ9-tetrahydrocannabinol (Δ9-THC). However, five products were found to contain considerable amounts of this compound (0.600–180 μg/g). Only in the case of cannabis beer, cannabis roasted coffee, and cannabis energy drink, Δ9-THC was not detected.

Two-Dimensional Liquid Chromatography Method for the Determination of Gelsemium Alkaloids in Honey.

Toxic Chinese medicine residues in honey pose a serious threat to consumer health. Gelsemium is one of the nine ancient poisons, making the whole plant virulent. The residue of Gelsemium alkaloid in honey causes poisoning from time to time. Therefore, it is very important to establish a method for the detection of Gelsemium alkaloids in honey. In this study, a method of solid phase extraction (SPE) with two-dimensional liquid chromatography (2D-LC) was developed for the first time for the simultaneous determination of Gelsemium alkaloids in honey, including gelsemine, koumine and humantenmine. First, the honey samples were purified by a PRS cation exchange column and extracted with 5% ammoniated methanol. Then, we verified the methodological indicators, which were in line with the Codex Guideline requirements. The verification results are as follows: matrix-matched calibrations indicated that the correlation coefficients were higher than 0.998. The recovery was in the range of 81%–94.2% with an intraday precision (RSD) of ≤5.0% and interday RSD of ≤3.8%. The limit of detection for the three alkaloids was 2 ng/g. The limits of quantification for gelsemine and koumine were 5 ng/g, and humantenmine was 20 ng/g. This method can be applied to the monitoring of Gelsemium alkaloids in honey.

Application of nanostructured supramolecular solvent based on C12mimBr ionic liquid surfactant to direct extraction of some chlorophenols in soil and rice samples

In the proposed research, ionic liquid surfactant (C12-mimBr)-based nanostructured supramolecular solvent (IL-SUPRAS) was applied for direct extraction and preconcentration of three chlorophenols (CPs) in soil and rice samples before using gas chromatography-flame ionization detection (GC-FID) analysis. Mainly for extraction of analyte from a solid sample, an initial extraction step is performed using harmful organic solvents. Then, after evaporation of those solvents, the secondary extraction method is applied. The purpose of this research is direct extraction of some analytes from solid samples without performing the initial extraction step and using an adverse organic solvent. At first, IL-SUPRAS aggregation was prepared in a water solution. After that, an appropriate amount of it was introduced to the Eppendorf vial containing the solid sample. In the next step, to effectively extract the analytes from samples to the SUPRAS phase, the Eppendorf vial was vortexed and centrifuged, respectively. Finally, to prevent the entrance of the SUPRAS phase into the GC instrument, a back extraction step of the analytes was performed. The influence of effective parameters (i.e., pH of sample solution, vortex time, centrifuge time, and volume of THF) on the extraction procedure was investigated. Under optimum experimental conditions, the enrichment factors were obtained in the reasonable range of 103.2–134.6. The calibration curves also were linear in the range of 1.0–2000 µg L-1 with coefficients of determination (R2) more than 0.9962 for all three analytes. Limits of detection (S/N = 3) of 0.3–0.7 µg L-1, thelimits of quantification (LOQ) of 0.99–2.1 µg L-1, the relative standard deviations (RSDs %) composed of inter-day RSD (7.6–8.7) and intra-day RSD (6.3–7.6),and relative recoveries in the range of 77.4–93.6 % were obtained. The proposed method (IL-SUPRAS-DME) was successfully applied, for direct and fast extraction of CPs analytes in soil and rice samples.

Determination of Local Anesthetic Drugs in Human Plasma Using Magnetic Solid-Phase Extraction Coupled with High-Performance Liquid Chromatography.

In this work, magnetic tetraethylenepentamine (TEPA)-modified carboxyl–carbon nanotubes were synthesized, characterized, and used as adsorbents to conduct magnetic solid-phase extraction (MSPE) for the preconcentration of seven local anesthetic drugs (procaine, lidocaine, mepivacaine, oxybuprocaine, bupivacaine, tetracaine, and cinchocaine) from human plasma. The separation and determination of analytes were performed on high-performance liquid chromatography with UV detection. Several factors affected the extraction efficiency, such as the amount of adsorbents used, extraction time, sample pH, and optimization of elution conditions. Under optimal conditions, satisfactory linear relationships were obtained in the range of 0.02–5.00 mg/L, with the limits of detection (LOD) ranging from 0.003 mg/L to 0.008 mg/L. The recoveries of analytes for spiked human plasma were in the range of 82.0–108%. Moreover, the precision with intra-day and inter-day RSD values were obtained in the range of 1.5–7.7% and 1.5–8.3%. The results indicated that this method could determine the concentration of seven local anesthetic drugs in human plasma with high precision and repeatability and provide support for the clinical monitoring of the concentration of local anesthetic drugs in human plasma.

Screening of prolyl hydroxylase 2 inhibitors based on quantitative strategy of peptides

Prolyl hydroxylase 2 (PHD2) is a key oxygen receptor regulating oxygen homeostasis in human body, and it is one of the important targets for drug research and development of hypoxia related diseases. In PHD2 enzymatic reaction, the structure of substrate (HIF-1α556–574) and product (hydroxylated HIF-1α) peptide only differ from one oxygen atom (MW>2000), which makes it a great challenge to separate them accurately and efficiently. In this work, the direct separation and detection of HIF-1α and hydroxylated HIF-1α has been firstly reported based on micellar electrokinetic chromatography (MEKC). Under optimized conditions, the intraday RSD of peak area and apparent electrophoretic mobility of hydroxylated HIF-1α were 1.87% and 0.81% respectively, and the interday RSD were 2.01% and 1.03% respectively. The LOD and LOQ of the MEKC method were 10 µM and 50 µM respectively, and the recoveries was 98.42–105.38%. Subsequently, the feasibility and accuracy of MEKC method to screen PHD2 inhibitors were confirmed by using roxadustat, and the IC50 (10.36 µM) and inhibitor type (competitive) were consistent with literature. Finally, the method was used to screen the PHD2 inhibitory activity of five traditional Chinese medicines (TCMs). The present work not only overcomes the difficulties of direct quantitative detection of hydroxylated HIF-1α, but also provides technical support for exploring and discovering new drug leads for hypoxia-related diseases from complex matrix such as TCMs.

Amylodextrin hydrogel as a green sorbent for pipette-tip micro-solid phase extraction followed by ion mobility spectrometry for analysis of triazole fungicides in environmental water samples

Triazole compounds have been globally used for controlling fungal diseases such as rusts, powdery mildew, and many leaf-spotting fungi. These compounds also have adverse effects on the environment and human health. In the present work, a hydrogel-based sorbent was synthesized by a combination of hydrophilic acrylic polymer and biodegradable amylodextrin. The sorbent was characterized by Fourier-transform infrared (FT-IR) spectroscopy, Brunauer-Emmett-Teller (BET) surface area, elemental CHN analysis, field emission scanning electron microscopy (FE-SEM), and zeta potential. The synthesized hydrogel was used as a green sorbent in the pipette-tip micro-solid phase extraction procedure for extraction and quantification of triazole fungicides (penconazole, hexaconazole, and tebuconazole) in environmental water samples. The corona discharge-ion mobility spectrometry (CD-IMS) was used as a rapid and sensitive detection technique. The effects of important variables on the extraction efficiency were optimized by response surface methodology (RSM). The proposed method provided good linearity (r2 ≥ 0.995) over a concentration range of 0.5–1000 ng/mL, acceptable intra-day RSD (≤ 5.7%, n = 5) and inter-day RSD (≤ 8.7%, n = 5), low limits of detection (0.10–0.20 ng/mL), good preconcentration factors (16.9–17.9 times), and high extraction recoveries (93.1–98.3%) under optimum extraction conditions. The extraction conditions were as follows: the amount of sorbent: 6.0 mg, pH of sample solution: 7.5, the flow rate of sample solution: 2.0 mL/min, adsorption time: 16.5 min, volume of desorption solvent: 1.1 mL, the flow rate of desorption solvent: 0.5 mL/min, and desorption time: 11.7 min. Finally, the proposed method was successfully applied for the quantification of triazoles in river and agricultural water samples.

A Simple and Reliable Liquid Chromatographic Method for Simultaneous Determination of Five Benzodiazepine Drugs in Human Plasma

Benzodiazepines (BZDs) are one of the most important drugs that have been used in the treatment of neuropsychological disorders. Indeed, BZDs are abused by drug addicts regardless of their therapeutic uses. Therefore, it was important in forensic and clinical toxicology to reach an easy and reliable method for the screening and quantification of BZDs in the human plasma matrix. In the current work, five BZDs, namely bromazepam, clonazepam, lorazepam, nordiazepam and diazepam were simultaneously separated and detected by a simple and reliable RPLC method in a human plasma matrix. Isocratic mobile elution consisting of 20 mmol L−1 phosphate buffer (pH 7.0) and methanol (50:50, v/v) on a Symmetry C18 column was employed. The flow rate, wavelength and column temperature were fixed at 1.0 mL min−1, 214 nm and 40 °C, respectively. The proposed method was validated, giving a linearity within the concentration ranges 5–500 ng mL−1 for bromazepam and diazepam, 3–500 ng mL−1 for clonazepam and lorazepam and 1–500 ng mL−1 for nordiazepam with a determination coefficient (R2) more than 0.9992. The LOD values for the selected BZDs ranged from 0.54 to 2.32 and from 1.78 to 7.65 ng mL−1 for standard methanolic and plasma matrices, respectively. Precision, accuracy, selectivity, stability, and robustness were some of the terms considered in validating the current RPLC method. Based on these results, a simple and reliable RPLC method was successfully applied to quantify BZDs in human plasma matrix appearing with recoveries ranging from 96.5 to 107.5% and interday RSD less than 4%. The current developed method was useful for rapidly screening the most commonly used BZDs in the market within their therapeutic concentration ranges.

Validated HPTLC Method Development for Simultaneous Estimation of Spironolactone and Hydrochlorothiazide in Formulation

This study describes a new TLC method for simultaneous quantification of Spironolactone and Hydrochlorothiazide in bulk and tablet dosage forms that is simple, precise and accurate. The drugs were separated using aluminium precoated plate with silica gel 60 F-254 as the stationary phase using Toluene: Ethyl acetate: Methanol 6: 4: 1 (v/v/v) as the mobile phase. At 231 nm, the separated zones were densitometrically evaluated. The two drugs were satisfactorily resolved with RF values of 0.78 ± 0.02 and 0.33 ± 0.02 for Spironolactone and Hydrochlorothiazide. The accuracy and reliability of the method were assessed by evaluation of linearity (120-270 ng/spot for Spironolactone and 100-350 ng/spot for Hydrochlorothiazide), precision (intra-day RSD 0.98-1.80 % and inter-day RSD 0.76-1.92 % for Spironolactone, and intra-day RSD 0.84-1.85 % and inter-day RSD 0.31-1.68 % for Hydrochlorothiazide), accuracy (100.78 ± 0.49 % for Spironolactone and 98.54 ± 1.80 % for Hydrochlorothiazide, by ICH guidelines. These methods have been applied to formulation and no interference of excipients is seen which proves that the method is specific.

Liquid chromatography coupled to tandem mass spectrometry for comprehensive quantification of crustacean tropomyosin and arginine kinase in food matrix

Crustacean has been recognized as one of the most critical food allergens whose impact could develop or persist into adulthood. It's urgently needed to establish accurate and reliable methods to quantify tropomyosin (TM) and arginine kinase (AK), the primary allergenic proteins in crustaceans. Herein, we developed an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method based on stable isotope-labeled internal standard (SIIS) to quantify TM and AK simultaneously. The amino acid sequences of TM and AK from the mostly consumed crustaceans in the world were analyzed, and the signature peptides of the two allergenic proteins were selected given the specificity, stability, and mass spectrometry responsiveness. Under the optimal detection conditions, signature peptide FLAEEADR and VSSTLSSLEGELK were able to quantify TM and AK in a complex food matrix with high accuracy (recoveries: 94.11%–102.16%) and precision (intra- and inter-day RSD<10%). At the same time, a signature peptide of TM, ALSNAEGEVAALNR, was found to be absent in crab. The proposed method exhibited a broad linear range (0–1000 ng/mL) of the absolute quantification (AQUA) peptides with R2 > 0.99 and took approximately 11 h per analysis. Moreover, it was successfully applied for quantifying TM and AK in commercial food samples, indicating the great applicable potential. The proposed method can reveal the allergenic risk of crustacean-related food and thus help allergic individuals avoid such food intake.

Profiling of polar ionogenic metabolites in Polish wines by capillary electrophoresis-mass spectrometry.

The composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties), and winemaking practices (including technology and storage). Metabolomics using NMR spectroscopy, GC-MS, and/or LC-MS has shown to be a useful approach for assessing the origin, authenticity, and quality of various wines. Nonetheless, the use of additional analytical techniques with complementary separation mechanisms may aid in the deeper understanding of wine's metabolic processes. In this study, we demonstrate that CE-MS is a very suitable approach for the efficient profiling of polar ionogenic metabolites in wines. Without using any sample preparation or derivatization, wine was analyzed using a 10-min CE-MS workflow with interday RSD values for 31 polar and charged metabolites below 3.8% and 23% for migration times and peak areas, respectively. The utility of this workflow for the global profiling of polar ionogenic metabolites in wine was evaluated by analyzing different cool-climate Polish wine samples.

A versatile method for multiclass determination of β-lactam drugs in urine by solid-phase extraction followed by HILIC-UV

A method for multiclass determination of β-lactam antibiotics in human urine by solid-phase extraction (SPE) prior to quantification by hydrophilic interaction liquid chromatography (HILIC) was developed. Four cephalosporins, one carbapenem and four penicillins were selected among the most prescribed β-lactam drugs. A HILIC column – silica-linked zwitterionic sulfobetaine groups – proved to be valuable for analysis of these three classes of highly hydrophilic compounds, with suitable capacity factors, peak shape and resolution gained by proper selection of the chromatographic conditions. Sample extraction has been performed on Strata™-X. The quick SPE procedure entirely developed in urine successfully combined sample clean-up, avoiding protein precipitation before extraction, with recovery higher than 66% (interday RSD < 15%, n = 5), and method quantification limits in urine in the range 1–4 µg mL−1. Selectivity has been proven on blank urine samples, also in presence of several non-target pharmaceuticals, and the method was applied to the analysis of samples from four patients under β-lactams therapy.

Fabric Phase Sorptive Extraction for the Determination of Anthracyclines in Sewage

Anthracyclines are a group of antineoplastic compounds used to treat acute leukemia and other cancers, and they are excreted after consumption by patients. These chemicals are often found in sewage at very low concentration levels. For this reason, the development of sensitive analytical methodologies capable of determining them at low concentrations is of prime importance. A simple, fast and sensitive analytical method using fabric phase sorptive extraction (FPSE) followed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FD) has been developed and validated for the extraction of anthracyclines from sewage samples. FPSE is a green, cheap, simple, selective and rapid sample preparation technique. The different parameters that affect the performance of the FPSE process, including extraction time, eluting solvent, elution time and pH, were optimized. The developed method showed satisfactory reproducibility, with intraday and interday RSD values lower than 15% for all the compounds and limits of detection between 0.1–0.15 µg·L−1. The unique combination of sample preparation by this micro-extraction technique with fluorescence detector have resulted in the satisfactory extraction of highly polar anthracyclines, without any noticeable matrix effect, a very common shortcoming of exhaustive sample preparation technique such as solid phase extraction (SPE) and mass spectrometry.

Detection of clenbuterol residues in beef sausages and its enantiomeric analysis using UHPLC-MS/MS: A risk of unintentional doping in sport field.

Clenbuterol (Clb) can be present in Mexico often but not all over the world in animal tissues and organs, therefore, potentially is derived from animal sources as well. The aims of this study were to develop and validate a method for detecting traces of clenbuterol in beef sausages. A calibration curve showed linearity in the range of 20–500 pg ml−1. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 5 and 10 pg g−1, respectively. The analyte recovery was from 95.70% to 100.40% with an intraday relative standard deviation (RSD%) of 0.99%–2.10% and an interday RSD% of 0.54%–2.34%, R 2 = 0.9998. The methodology developed was applied successfully in 15 samples of beef sausage, and 73.3% of the samples tested contained racemic clenbuterol in concentrations between 30 and 471 pg g−1. The UHPLC–MS/MS method developed combines high sensitivity with good selectivity and short chromatographic run time. Additionally, the enantiomeric analysis of clenbuterol performed in beef sausages showed a 59% for R‐(−)‐Clb and 41% for S‐(+)‐Clb. The presence of clenbuterol in beef sausages could represent a risk of unintentional doping in sport field, because the clenbuterol is a banned substance included in the World Anti‐Doping Agency's (WADA) list of prohibited substances.

Phytochemical investigation on Vitex negundo leaves and their anti-inflammatory and analgesic activities

The phytochemical investigation on Vitex negundo leaves has led to the isolation of one new iridoid glucoside (8α-hydroxy-4-carboxyl-5βH-9βH-iridoid-1α-O-(6′-O-(6,7-dihydrofoliamenthonyl)-β-ᴅ-glucopyranoside, 3), together with three known compounds, namely agnuside (1), 6′-O-E-caffeoylmussaenosidic acid (2), and 3,5-dicaffeoylquinic acid (4). The HPLC analytical study was also performed to quantify the content of agnuside (1) in dried leaves. The results indicated the very high content of 1 (3.04 ± 0.02%). The method was also validated by various parameters, including linearity (R2= 0.9999), precision (intra-day RSD ≤ 2.50%, inter-day RSD= 0.76%), and accuracy (recovery rates 96.58-101.86%). The animal testing data showed that the extract did not reduce pain at the doses of 9.6 and 28.8 g /kg (leaf weight/body weight) in the hot plates and pain measuring models but showed the pain reduction in the acetic acid-induced pain model. The extract at the dose of 5.6 g/kg (leaf weight/body weight) also had effects on the acute inflammation in the carrageenin-induced edema model. The extract at the dose 9.6 and 28.8 g/kg (leaf weight/body weight) also showed significant chronic anti-inflammation, comparable to methylprednisolone at the dose 10 mg/kg on the mouse peritoneal.

Simultaneous determination of febuxostat and montelukast in human plasma using fabric phase sorptive extraction and high performance liquid chromatography-fluorimetric detection

In the present work, a new sensitive and selective high-performance liquid chromatography-fluorimetric detection (HPLC-FLD) method was developed and validated to quantify febuxostat (FBX) and montelukast (MON) in human plasma. The developed procedure was successfully applied to a study aimed at evaluating the pharmacokinetic profiles of febuxostat and montelukast in human plasma. A sol-gel poly (caprolactone)-block-poly(dimethylsiloxane)-block-poly(caprolactone) (sol-gel PCAP-PDMS-PCAP) extraction sorbent coated fabric phase sorptive extraction membrane was used in the extraction process. The entire chromatographic analysis was performed with isocratic elution of the composition of the mobile phase (acetonitrile:water, 60:40, v:v, 0.032% glacial acetic acid) on the C18 column. The flow rate is varied during the analysis, particularly from 0.5 mL min−1 at the start and linearly increased to 1.5 mL min−1 in 7 min. The detection and quantification of the analytes was carried out by means of a fluorimetric detector at 320 nm and 350 nm as absorption wavelengths and at 380 and 400 nm as emission wavelengths for FBX and MON, respectively. The calibration curves demonstrated linearity in the range 0.3–10 ng mL−1 and 5–100 ng mL−1 for FBX and MON, respectively, while the LOD and LOQ values were 0.1 and 0.3 ng mL−1 for FBX and 1.5 and 5 ng mL−1 for MON. Intraday and interday RSD% values were found lower than 5.79%. As reported, the method was applied to real plasma samples obtained from a volunteer who was co-administered both the drugs. Pharmacokinetic data reveal that the concentration of both the drugs reaches the plateau approximately at the same time, but exhibits an elimination phase at different rates. This study demonstrated the usefulness of the new method and its applicability in therapeutic drug monitoring (TDM).

Development of An HPLC Method for the Determination of Mesalazine in Human Plasma by Fluorimetric Derivatization and Application to A Prototype Pharmacokinetic Study.

In this study, a new, fast and sensitive HPLC method with fluorometric detection was developed for the determination of mesalazine in human plasma and applied to a pharmacokinetic study. Mesalazine was precolumn derivatized with NBD-Cl and the fluorescent derivative was separated on a C18 (150 × 4.6mm × 2.6μm) analytical column at 30 ºC using a mobile phase composed of acetonitrile-0.1% o-phosphoric acid in water (70:30, v/v) by isocratic elution with flow rate of 1.0mLmin-1. The method was based on the measurement of the derivative using fluorescence detection (λex = 280nm, λem = 325nm). The retention time of mesalazine is 3.08 ± 0.06min. Nortriptiline was used as internal standard. This currently developed method was validated according to ICH criteria by evaluating the specificity, linearity, precision, accuracy and robustness. The method was determined to be linear in a concentration range of 0.25-1.5μgmL-1 with the correlation coefficient of 0.9997. LOD and LOQ were found to be 0.075 and 0.25μgmL-1, respectively. Intraday and interday RSD values were less than 5.92%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC0-t, AUC0-∞, Cmax, tmax, t1/2, were calculated according to the assays. The presented method can certainly be used for bioequivalence and bioavailability investigations and routine analysis of the drug in plasma.

A novel electronic tongue using electropolymerized molecularly imprinted polymers for the simultaneous determination of active pharmaceutical ingredients

The combination of chemometrics and electrochemical sensors modified with molecularly imprinted polymers (MIPs) towards the development of MIP-based electronic tongues (ETs) was explored herein. To demonstrate the potential of such an approach, the simultaneous determination of paracetamol, ascorbic acid and uric acid mixtures in pharmaceutical samples was evaluated. To this aim, MIP-based sensors for the different compounds were prepared by in situ electropolymerization of pyrrole in the presence of p-toluenesulfonate anion (pTS−), which acted as functional doping ion of the polypyrrole (PPy) MIP backbone. Morphological characterization of the MIPs was done by scanning electron microscopy (SEM), while functionalization of the electrodes was monitored electrochemically. Under the optimized measuring conditions, the developed sensors showed a good performance, with good linearity at the μM level (R2 > 0.992, limits of detection between 1 and 24 μM) as well as good repeatability (intra- and inter-day RSD values between 3 and 6% over 30 consecutive measurements). Finally, the quantification of the individual substances in different pharmaceutical samples was achieved by an artificial neural networks (ANNs) model, showing satisfactory agreement between expected and obtained values (R2 > 0.987).

Tyrosol and Hydroxytyrosol Determination in Extra Virgin Olive Oil with Direct Liquid Electron Ionization-Tandem Mass Spectrometry

Extra virgin olive oil (EVOO) is one of the main ingredients of the Mediterranean diet. It is claimed as a functional food for its unique content of health-promoting compounds. Tyrosol (Tyr), Hydroxytyrosol (Htyr), and their phenolic derivatives present in EVOO show beneficial properties, and their identification and quantification, both in their free form and after the hydrolysis of more complex precursors, are important to certify its quality. An alternative method for quantifying free and total Tyr and Htyr in EVOO is presented using an LC–MS interface based on electron ionization (EI), called liquid electron ionization (LEI). This method requires neither sample preparation nor chromatography; the sample is diluted and injected. The selectivity and sensitivity were assessed in multiple reaction monitoring mode (MRM), obtaining confirmation and quantification in actual samples ranging from 5 to 11 mg/Kg for the free forms and from 32 to 80 mg/Kg for their total amount after hydrolysis. Two MS/MS transitions were acquired for both compounds using the Q/q ratios as confirmatory parameters. Standard addition calibration curves demonstrated optimal linearity and negligible matrix effects, allowing a correct quantification even without expensive and difficult to find labeled internal standards. After several weeks of operation, the system’s repeatability was excellent, with an intraday RSD (%) spanning from five to nine and an interday RSD (%) spanning from 9 to 11.

Determination of clenbuterol at trace levels in raw gelatin powder and jellies using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry

Clenbuterol is present in animal tissues and organs and, therefore, potentially present in gelatin derived from animal sources. The objective of this study was to develop a method for identify an quantify traces of clenbuterol in gelatin and jellies. The clenbuterol calibration curve showed linearity in the range of 20–1000 pg mL−1. The detection and quantification limits were 5 pg g−1 and 10 pg g−1, respectively. The recovery of the analyte ranged from 93.4 to 98.7% with an intra-day RSD% (n = 4) of 1.25%-3.25%, and an inter-day RSD% (n = 12) of 0.5%-2.25%, with good linearity (R2 = 0.99). The method developed and validated was successfully applied in 54 gelatin samples, 57.4% of which showed clenbuterol. This UHPLC-MS/MS method combines high sensitivity with good selectivity and short chromatographic run time.

Pharmacokinetic comparison between Tanreqing Capsules Substitute and Tanreqing Capsules in rats by LC-MS/MS

Due to the limited resource of bear bile powder, the major raw material of Tanreqing Capsules(TRQ), cultured bear bile powder is used as a replacement to develop the Tanreqing Capsules Substitute(TRQS). An LC-MS/MS method was established in this study for simultaneous quantitation of 8 compounds from TRQS in rat plasma: tauroursodeoxycholic acid(TUDCA), taurocheno-deoxycholic acid(TCDCA), ursodeoxycholic acid(UDCA), chenodeoxycholic acid(CDCA), ferulic acid, wogonoside, baicalin, and forsythoside A. Thereby, the pharmacokinetic behaviors of TRQ and TRQS were evaluated. Concentration of endogenous compounds TUDCA, TCDCA, UDCA, and CDCA was determined with the stable isotope surrogate analytes: D4-TUDCA, D4-TCDCA, D4-UDCA, and D4-CDCA. Plasma samples were extracted by acetonitrile-induced protein precipitation. The LC conditions are as follows: Waters BEH C_(18) column(2.1 mm×100 mm, 1.7 μm), mobile phase of 10 mmol·L~(-1) ammonium formate aqueous solution(containing 0.01% formic acid) and acetonitrile-methanol mixture(1∶5). MS conditions are as below: multiple reaction monitoring(MRM), ESI~(+/-). Concentration of UDCA, CDCA, TUDCA, and TCDCA was corrected with a response factor, which is the ratio between the responses recorded for the surrogate and the authentic analyte at the equal concentration. Each of the plasma components showed good linearity(r &gt; 0.995 1). Accuracy and precision met the criteria(inter-day RSD&lt;7.0%, RE 89.98%-112.0%; intra-day RSD&lt;12%, RE 90.41%-111.2%). The recovery was 64.83%-119.9% and matrix effect was 87.15%-113.8%. The validated method was applied for pharmacokinetic study of TRQS and TRQ(po, 0.94 g·kg~(-1)). There was no significant difference in C_(max) and AUC_(0-24 h) of baicalin, UDCA, TUDCA, and TCDCA between the two groups, indicating similar pharmacokinetic behaviors between TRQS and TRQ in rats.