A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 μL. A sample throughput of 7 h-1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL-1, with LOD values ranging from 0.4 to 1.5 ng mL-1. Accuracy values in the range of 88.2-106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.
Breast milk analysis provides useful information about acute newborn exposure to harmful substances, such as psychoactive drugs abused by a nursing mother. Since breast milk represents a complex matrix with large amounts of interfering compounds, a comprehensive sample pre-treatment is necessary. This work focuses on determination of amphetamines and synthetic cathinones in human breast milk by microextraction techniques (liquid-phase microextraction and electromembrane extraction), and their comparison to more conventional treatment methods (protein precipitation, liquid-liquid extraction, and salting-out assisted liquid-liquid extraction). The aim of this work was to optimize and validate all the extraction procedures and thoroughly assess their advantages and disadvantages with special regard to their routine clinical use. The applicability of the extractions was further verified by the analysis of six real samples collected from breastfeeding mothers suspected of amphetamine abuse. The membrane microextraction techniques turned out to be the most advantageous as they required low amounts of organic solvents but still provided efficient sample clean-up, excellent quantification limit (0.5 ng mL-1), and good recovery (81-91% and 40-89% for electromembrane extraction and liquid-phase microextraction, respectively). The traditional liquid-liquid extraction as well as the salting-out assisted liquid-liquid extraction showed comparable recoveries (41-85% and 63-88%, respectively), but higher quantification limits (2.5 ng mL-1 and 5 ng mL-1, respectively). Moreover, these methods required multiple operating steps and were time consuming. Protein precipitation was fast and simple, but it demonstrated poor sample clean-up, low recovery (56-58%) and high quantification limit (5 ng mL-1). Based on the overall results, microextraction methods can be considered promising candidates, even for routine laboratory use.
Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6-9.6%), linearity (r2, 0.9991-0.9999), enrichment (EF, 29-97), sensitivity (LOD, 0.2-3.4 μg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.
Liquid-liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid-liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1-200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of - 1.67-5.67 %, while the inter-day accuracy was in the range of 0.20-6.90 %. The intra-day precision for IBT was in the range of 3.20-4.37 % and 3.13-4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of - 11.2-3.71 % and the inter-day accuracy in the range of - 5.76-0.92 %. The intra-day precision for PCI was in the range of 3.49-7.64 % and 3.63-8.61 % for the measurement between days. In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63-3.78 ng/mL, PCI-45227 concentration range: 1.84-14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34-89.0 ng/mL, PCI-45227 concentration range: 5.64-124 ng/mL).
- MeSH
- 3D tisk MeSH
- adenin analogy a deriváty MeSH
- extrakce kapalina-kapalina metody MeSH
- koronární angioplastika * MeSH
- lidé MeSH
- piperidiny MeSH
- pyrazoly MeSH
- rozpouštědla MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
INTRODUCTION: Organic molecules that interact with the cannabinoid receptors are called cannabinoids, which can be endogenous, natural or synthetic compounds. They possess similar pharmacological properties as produced by the plant, Cannabis sativa L. Before cannabinoids can be analysed, they need to be extracted from the matrices. OBJECTIVE: To review literature on the methods and protocols for the extraction of naturally occurring cannabinoids. METHODOLOGY: An extensive literature search was performed incorporating several databases, notably, Web of Knowledge, PubMed and Google Scholar, and other relevant published materials. The keywords used in the search, in various combinations, with cannabinoids and extraction being present in all combinations, were Cannabis, hemp, cannabinoids, Cannabis sativa, marijuana, and extraction. RESULTS: In addition to classical maceration with organic solvents, e.g. ethanol, pressurised solvent extraction, solvent heat reflux, Soxhlet extraction, supercritical fluid extraction, ultrasound-assisted extraction and microwave-assisted extraction, are routinely used nowadays for the extraction of cannabinoids from plant materials and cannabis consumer products. For the extraction of cannabinoids from biological samples, e.g. human blood, and also from food and beverages, and wastewater, solid-phase extraction and its variants, as well as liquid-liquid extraction are commonly used. Parameters for extraction can be optimised by response surface methodology or other mathematical modelling tools. There are at least six US patents on extraction of cannabinoids available to date. CONCLUSIONS: Irrespective of the extraction method, extraction temperature, extraction time and extraction pressure play a vital role in overall yield of extraction. Solvent polarity can also be an important factor in some extraction methods.
Novel method for the determination of a tumor marker homovanillic acid (HVA) in human urine was developed. Combination of hollow fiber - based liquid-phase microextraction (HF-LPME) and differential pulse voltammetry (DPV) at a cathodically pre-treated boron doped diamond electrode (BDDE) was applied for these purposes. Optimum conditions were: butyl benzoate as supported liquid membrane (SLM) formed on polypropylene HF, 0.1 mol L-1 HCl as donor phase, 0.1 mol L-1 sodium phosphate buffer of pH 6 as acceptor phase, and 30 min extraction time. HF-LPME-DPV concentration dependence was linear in the range from 1.2 to 100 μmol L-1. Limits of quantification (LOQ) and detection (LOD) were 1.2 and 0.4 μmol L-1, respectively. The applicability of the developed method was verified by analysis of human urine. Standard addition method was used, found HVA concentration was 13.5 ± 1.3 μmol L-1, RSD = 9.3% (n=5).
- MeSH
- kyselina homovanilová MeSH
- lidé MeSH
- mikroextrakce kapalné fáze * MeSH
- nádorové biomarkery * MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 μg/L with linear working ranges of 50-5000 μg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 μg/L. Average recovery values were 102.7 ± 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.
In the present work, a disposable microextraction device with a polyamide 6 nano-fibrous supported liquid membrane (SLM) is employed for the pretreatment of minute volumes of biological fluids. The device is placed in a sample vial for an at-line coupling to a commercial capillary electrophoresis instrument with UV-Vis detection (CE-UV) and injections are performed fully automatically from the free acceptor solution above the SLM with no contact between the capillary and the membrane. Up to 4-fold enrichment of model basic (nortriptyline, haloperidol, loperamide, and papaverine) and acidic (ibuprofen, naproxen, ketoprofen, and diclofenac) drugs is achieved by optimizing the ratio of the donor to the acceptor solution volumes (16 to 4 μL, respectively). The actual setup enables SLM extractions from less than a drop of sample and is suitable for pretreatment of scarce human body fluids. Two unique methods are reported for efficient clean-up and enrichment of the basic and acidic drugs from capillary blood (formed as dried blood spot), serum, and urine samples, which enable their determination at therapeutic and/or toxic levels. The hyphenation of the SLM extraction with CE-UV analysis provides good repeatability (RSD, 2.4-14.9%), linearity (r2, 0.988-1.000), sensitivity (LOD, 0.017-0.22 mg L-1), and extraction recovery (ER, 20-106%) at short extraction times (10 min) and with minimum consumption of samples and reagents. Graphical abstract.
- MeSH
- elektroforéza kapilární metody MeSH
- koncentrace vodíkových iontů * MeSH
- léčivé přípravky metabolismus MeSH
- lidé MeSH
- membrány umělé * MeSH
- mikroextrakce kapalné fáze metody MeSH
- nanovlákna * MeSH
- reprodukovatelnost výsledků MeSH
- tělesné tekutiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10-1000 ng/mL), with adequate accuracy (90.1-114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR.
- MeSH
- elektroforéza kapilární metody MeSH
- elektrolyty MeSH
- extrakce kapalina-kapalina MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- pyrazoly krev chemie izolace a purifikace farmakokinetika MeSH
- pyrimidiny krev chemie izolace a purifikace farmakokinetika MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Essential oils obtained via the hydrodistillation of two Asian herbs (Houttuynia cordata and Persicaria odorata) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Additionally, both the liquid and vapor phase of essential oil were tested on antimicrobial activity using the broth microdilution volatilization method. Antimicrobial activity was tested on Gram-negative and Gram-positive bacteria-Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes, Klebsiella pneumoniae, Seratia marcescense and Bacillus subtilis. Hydrodistillation produced a yield of 0.34% (Houttuynia cordata) and 0.40% (Persicaria odorata). 41 compounds were identified in both essential oils. Essential oils contained monoterpenes and their oxidized forms, sesquiterpenes and their oxidized forms, oxidized diterpenes, derivates of phenylpropene and other groups, such as, for example, aldehydes, alcohols or fatty acids. Both essential oils were antimicrobial active in both vapor and liquid phases at least in case of one bacterium. They expressed various antimicrobial activity in the range of 128-1024 μg∙mL-1, 512-1024 μg∙mL-1 in broth and 1024 μg∙mL-1, 512-1024 μg∙mL-1 in agar, respectively. Research showed new interesting information about P. odorata and H. cordata essential oils and demonstrated that both essential oils could be possibly used in the field of natural medicine or natural food preservation.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- bakteriální infekce farmakoterapie mikrobiologie MeSH
- extrakce kapalina-kapalina MeSH
- grampozitivní bakterie účinky léků MeSH
- Houttuynia chemie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- oleje prchavé chemie farmakologie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- Polygonaceae chemie MeSH
- rostlinné extrakty chemie farmakologie MeSH
- Staphylococcus aureus účinky léků patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
