Mixed-mode Solid-phase Extraction Research Articles

Use of Commercial Mixed-Mode Stationary Phases and Sorbents in the High-Performance Liquid Chromatography Analysis and Solid-Phase Extraction of Ionized and Hydrophilic Bioactive Compounds.

Mixed-mode high-performance liquid chromatography (HPLC) is increasingly used for the analysis of ionic and highly hydrophilic drugs, which are difficult to separate by conventional single-mode HPLC. In the former case, chromatographic separation is achieved using one of the several commercially available mixed-mode stationary phases, typically combinations of reversed and ion-exchange phases. Moreover, mixed-mode stationary phases can be used as solid-phase extraction (SPE) sorbents. This review focuses on the recent applications of mixed-mode stationary phases in the chromatographic analysis of bioactive compounds, such as drugs, herbicides, and pesticides. Specifically, we briefly summarize HPLC methods utilizing mixed-mode stationary phases and SPE pretreatment procedures utilizing mixed-mode sorbents developed in the last decade, thus providing a reference work for overcoming the difficulties in analyzing ionized or hydrophilic drugs by conventional reversed-phase chromatography.

Sample Preparation Techniques for Growth-Promoting Agents in Various Mammalian Specimen Preceding MS-Analytics.

The misuse of growth-promoting drugs such as beta-2 agonists and steroids is a known problem in farming and sports competitions. Prior to the analysis of biological samples via liquid chromatography (LC)-mass spectrometry (MS) or gas chromatography (GC)-MS, sufficient sample preparation is required to reliably identify or determine the residues of drugs. In practice, broad screening methods are often used to save time and analyze as many compounds as possible. This review was conceptualized to analyze the literature from 2018 until October 2023 for sample preparation procedures applied to animal specimens before LC- or GC-MS analysis. The animals were either used in farming or sports. In the present review, solid phase extraction (SPE) was observed as the dominant sample clean-up technique for beta-2 agonists and steroids, followed by protein precipitation. For the extraction of beta-2 agonists, mixed-mode cation exchanger-based SPE phases were preferably applied, while for the steroids, various types of SPE materials were reported. Furthermore, dispersive SPE-based QuEChERs were utilized. Combinatory use of SPE and liquid-liquid extraction (LLE) was observed to cover further drug classes in addition to beta-2 agonists in broader screening methods.

Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and β-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of β-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of β-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.

A selective dual quenching sensor (EY/BG@CDs) for simultaneous monitoring of gentamicin and ketorolac levels in plasma: a highly efficient platform that caters to the needs of therapeutic drug monitoring.

This research work introduces a novel sensor that utilizes two fluorophores to enable simultaneous monitoring of gentamicin sulphate (GNT) and ketorolac tromethamine (KET). The innovative sensor is composed of carbon dots (CDs) derived from black grapes (BG) and eosin Y (EY) dye. The interaction between the studied drugs and EY/BG@CDs sensor components allows for their simultaneous detection where GNT quenches the fluorescence of EY at 535 nm without affecting the fluorescence of CDs, while KET quenches the fluorescence of BG@CDs at 385 nm without impacting EY fluorescence. The BG@CDs probe was successfully characterized using various techniques such as absorption spectrophotometry, spectrofluorimetry, TEM imaging, infrared spectroscopic analysis, and XRD analysis. The suggested methodology was observed to be highly sensitive for the simultaneous determination of GNT and KET in their spiked rabbit plasma samples, with wide linear ranges and low limit of detection (LOD) values. The studied drugs were extracted using a highly selective extraction method involving protein precipitation followed by mixed mode solid phase extraction using an Oasis WCX cartridge. The simultaneous determination of GNT and KET is essential due to the potential interactions between the studied drugs. Therefore, this analysis can be used to evaluate the necessity of dose monitoring and the potential adverse effects of co-administration of these drugs.

Simple and fast determination of tetrodotoxin in human plasma based on hydrophilic-interaction/ion-exchange mixed-mode solid phase extraction combined with liquid chromatography-tandem mass spectroscopy

In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the measurement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant matrix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic interaction and ion exchange. 100μL plasma sample was precipitated rapidly with acetonitrile containing 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed with 95% acetonitrile, TTX was eluted with 200μL of 50% acetonitrile containing 1% trichloroacetic acid. 2μL of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide column was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and timesaving (<30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using m/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transition for TTX) and 179.2→63.0 (transition for IS), respectively. The method was linear in the range of 0.1–20 ng/mL for TTX with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra- and inter-assay accuracies were in the range of 98.5%-99.8% (relative standard deviations, RSDs ≤ 5.92%) and 98.8–99.5% (RSDs ≤ 6.23%), respectively. Biases of spiking analysis were ranged from -7.00% to 7.43% for healthy human plasma samples (RSDs ≤ 8.83%) and from -5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high bilirubin plasma samples (RSDs ≤ 6.40%), which proved the good anti-interference property of the method. The results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.

Development and Validation of a Uplc-Qtof-Ms Method for Blood Analysis of Isomeric Amphetamine-Related Drugs

The identification of isomeric drugs is gaining increasing importance in forensics and doping control. Isomers vary in terms of safety, effectiveness, and regulation, particularly for amphetamine-related drugs (ARDs). This study developed and validated a pseudo-isocratic UPLC-qTOF-MS analytical method for the identification of isomeric Amphetamine-related drugs (ARDs) in blood following mixed-mode solid-phase extraction (MMSPE). The procedure requires 250 μL of blood to achieve a limit of quantification (LOQ) and detection (LOD) of 20 ng/mL for all analytes. In aged animal blood samples, extraction recoveries of 63–90% and matrix effects of 9–21% were observed. Precision and accuracy for all analytes were within 20% and 89–118%, respectively. The analytical method was developed and validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard. It has acceptable accuracy and precision for use in doping control and forensic toxicology.

Determination of three oryzanols in rice by mixed-mode solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry

建立了利用高效液相色谱-串联质谱结合混合型阴离子交换固相萃取柱测定稻米中3种谷维素(环木菠萝烯醇阿魏酸酯(CA-FA)、24-亚甲基环木菠萝烯醇阿魏酸酯(24MCA-FA)和菜油甾醇阿魏酸酯(Camp-FA))含量的分析方法。实验通过优化3种谷维素的多反应监测(MRM)质谱参数,比较了在不同流动相中的分离度以及响应强度,同时考察了不同提取条件、净化条件对3种谷维素提取率和净化效果的影响,再结合外标法定量,实现了对稻米中3种谷维素的定量分析。实验结果表明,采用5 mmol/L乙酸铵水溶液为流动相A,甲醇∶乙腈=1∶1(v/v)为流动相B,梯度洗脱,3种谷维素在Agilent Eclipse XDB-C8色谱柱(150 mm×2.1 mm, 3.5 μm)上基本分离且响应强度和峰形最佳;提取条件经正交实验优化后可得:料液比为1∶20(g/mL)、提取溶剂为甲醇、浸泡时间为12 h、超声温度为40 ℃以及超声时间为20 min时,3种谷维素提取率最高;对混合型阴离子交换固相萃取柱的上样溶剂以及洗脱溶剂优化后,样品基质效应为1.6%~10.8%。在各优化条件下,3种谷维素在各自的线性范围内线性良好,相关系数(r2)均≥0.9983,检出限(LOD)为0.5~1.0 μg/L,定量限(LOQ)为2.0~3.5 μg/L。在稻米样品本底浓度2、5和10倍的加标水平下3种谷维素的平均回收率为86.1%~110.6%,相对标准偏差(RSD)为0.9%~3.2%。该方法可快速准确测定稻米中3种谷维素的含量,为后续稻米中谷维素类化合物测定及鉴定奠定基础。

Probing for peptidic drugs (2-10kDa) in doping control blood samples.

Bioactive peptides with a molecular mass between 2 and 10kDa represent an important class of substances banned in elite sports, which has been recognized with an increasing number and variety of substances by anti-doping organizations. Also, the annually renewed list of prohibited substances of the World Anti-Doping Agency (WADA) explicitly mentions more and more of these peptides, and efficient testing procedures are required. Even under simplified sample preparation conditions, liquid chromatography coupled to high-resolution mass spectrometry (with resolution properties>100,000 full width at half maximum) offers suitable conditions for this task and can therefore be used as an initial testing procedure. In contrast to urine, blood analysis essentially relies on the detection of intact peptide hormones, and the expected concentrations are commonly higher in blood samples than in urine. This facilitates the analysis, and a generic sample preparation by means of mixed-mode solid-phase extraction could be realized in this study. Co-extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin and porcine insulin), growth hormone releasing hormones (sermorelin, CJC-1295 and tesamorelin), insulin-like growth factors (long-R3-IGF-I, R3-IGF-I and Des1-3-IGF-I) and mechano growth factors (human MGF and MGF-Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls. The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogues.

What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine

Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5–52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference—with knockout samples measuring below the assay’s limit of detection—while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction. These findings imply that previous conclusions based on urinary oxytocin measurements—especially those involving unextracted samples—may need to be reassessed.

Supercritical fluid chromatography time-of-flight mass spectrometry enantiomeric determination of basic drugs in sewage samples

Many pharmacologically active compounds are chiral species, and their therapeutic or toxicological effects might differ between isomers. Herein, we develop a fast and sensitive chiral analysis methodology for the determination of eight pharmaceuticals, considered as emerging environmental pollutants and belonging to two different chemical classes, in wastewater and sludge samples. Compounds were separated using supercritical fluid chromatography (SFC) combined with time-of-flight mass spectrometry (TOF-MS) detection. The stationary phase, the modifier and the additive combined with supercritical carbon dioxide (CO2), in the SFC mobile phase, played a major effect in the enantiomeric resolution of selected compounds. Moreover, the composition of the mobile phase affected their ionization efficiency in the electrospray ionization source. Methanol (MeOH), containing a 0.1% of ammonia, was used as CO2 modifier for the separation of compounds in a polysaccharide-type column. Total analysis time was 15.5 min, achieving resolution factors between 1.03 and 2.49 for the eight pairs of enantiomers. In combination with mixed-mode solid-phase extraction and matrix solid-phase dispersion protocols, compounds were determined in wastewater and sludge samples, with limits of quantification in the range of 0.010–0.020 µg L−1 and 3.7–11.1 ng g−1, for aqueous and solid samples, respectively. The amine-type drugs (tramadol, propranolol and venlafaxine) were mostly found in wastewater samples, whilst azolic antimycotics were mainly quantified in sludge. The first group of compounds showed enantiomeric fractions significantly different to those existing in the commercial counterpart pharmaceuticals.

Determination of trace organic contaminants by a novel mixed-mode online solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry

In this study, a novel mixed-mode online solid-phase extraction (SPE) method was developed to recover miscellaneous trace organic contaminants (TrOCs) from environmental water samples. Six kinds of sorbents, including C18 substances, hypercross-linked polymers (2), cation-exchange resins, anion-exchange resins, and graphitized nonporous carbons, were packed into a single online SPE cartridge. Furthermore, a fully automated analytic method was established by coupling this mixed-mode online SPE with liquid chromatography tandem mass spectrometry (online SPE-LC-MS/MS). Sixty-nine TrOCs with diverse properties were selected to examine the performance of this mixed-mode SPE cartridge in comparison with solo-mode online SPE cartridges. The method quantification limit (MQL) and the relative recovery coefficient of TrOCs in diverse water matrices, including groundwater, surface water and sewage effluent were evaluated. The MQL of most TrOCs was lower than 10 ng L−1. The relative recovery coefficients for most TrOCs in the groundwater (50/69) and surface water (38/69) matrix fit in the satisfactory range. Moreover, mixed-mode online SPE coupled with LC-high-resolution MS was applied for a suspect screening of TrOCs in sewage effluents. A series of highly polar TrOCs that had scarcely been reported by previous studies were identified by this practical and easily accessible method. Finally, this novel mixed-mode online SPE with LC-MS/MS method was applied to quantify the TrOCs in the environmental water samples.

Solid-Phase Extraction Approaches for Improving Oligosaccharide and Small Peptide Identification with Liquid Chromatography-High-Resolution Mass Spectrometry: A Case Study on Proteolyzed Almond Extract.

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

Bio-analytical liquid chromatographic-based method with a mixed mode online solid phase extraction for drug monitoring of fluconazole in human serum

A simple, cost-effective and sensitive liquid chromatography-based bio-analytical method has been developed and validated for therapeutic drug monitoring of fluconazole (FLUC) in human serum. Integration of online mixed-mode solid-phase extraction (SPE) into the analytical system was the key for direct injection of untreated serum samples. A short protein-coated (PC) µBondapak CN silica column (PC-µB-CN-column) as a SPE tool and phosphate buffer saline (PBS) (pH 7.4) as an eluent were applied in the extraction step. PC-µB-CN-column operates in two different chromatographic modes. Using PBS, proteins were extracted from serum samples by size-exclusion liquid chromatography, while FLUC trapping was reversed-phase liquid chromatography dependent. FLUC was then eluted from the PC-µB-CN-column onto the quantification position using a mixture of acetonitrile-distilled deionized water (20:80, v/v) as an eluent and ODS analytical column. FLUC was separated at ambient temperature (22 ± 1 °C) and detected at 260 nm. The method was linear over the range of 200–10000 ng/mL. FLUC recovery in untreated serum samples ranged from 97.8 to 98.8% and showed good accuracy and precision. The reliability of the developed method was evaluated by studying the pharmacokinetic profile of FLUC in humans after an oral administration of a single 150 mg tablet.

Sensitive determination of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and complementary cannabinoids in hair using alkaline digestion and mixed-mode solid phase extraction followed by liquid-chromatography-tandem mass spectrometry

Hair drug testing can be used for the evaluation of cannabis use with a large detection window, and is required for professional driving license granting in Brazil. A positive hair result for cannabis use requires quantification of the metabolite THC-COOH above the cutoff value of 0.2 ng/g. The achievement of such lower limit of quantification is challenging, particularly with the use of liquid chromatography coupled to triple quadrupole mass spectrometers (LC-MS/MS). In this study, a very sensitive LCMS/ MS assay for the simultaneous quantification of THC-COOH along with THC, CBD, and CBN was developed and validated. Sample preparation was based on hair hydrolysis, followed by selective ion-exchange solid-phase extraction. The extraction yield was 101.5–101.6% for THC-COOH, 92.3–97.4% for THC, 89.7–95.2% for CBN, and 104.9–121.1% for CBD. Internal standard corrected matrix effects were − 2.7 to − 1,1 for THCCOOH and − 11.5 to − 0.1% for the other analytes. The lower limit of quantification was 01 ng/g for THC-COOH and 25 ng/g for THC, CBD, and CBN. The assay fulfilled validation guidelines acceptance criteria. The measurement uncertainties were determined and the assay was ISO17025 accredited, being currently used in routine testing.

Protein Targets of Acetaminophen Covalent Binding in Rat and Mouse Liver Studied by LC-MS/MS.

Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS.

The increasing importance to determine bioactive peptide hormones such as insulin, its synthetic analogs, and C-peptide in urine samples represents an analytical challenge. The physiological concentrations of insulin in urine are commonly found at sub-ng/mL levels and thus represent a complex analytical task. C-peptide concentrations, on the other hand, tend to be in the moderate ng/mL range and are hence much easier to determine. Insulin and C-peptide are important in the diagnostics and management of metabolic disorders such as diabetes mellitus and are also particularly relevant target analytes in professional sports and forensics. All insulins are classified on the World Anti-Doping Agency’s (WADA) list of prohibited substances and methods in sports with a minimum required performance level (MRPL) of 50 pg/mL. Until now, methods combining immunoextraction and subsequent mass spectrometric detection have mostly been used for this purpose. With the method developed here, sample preparation has been simplified considerably and does not require an antibody-based sample purification. This was achieved by a sophisticated mixed-mode solid-phase extraction and subsequent separation with liquid chromatography coupled to high-resolution mass spectrometry. Included target insulins were human, lispro, glulisine, aspart, glargine metabolite, degludec, and additionally, human C-peptide. The method was validated for the synthetic insulin analogs considering WADA requirements including specificity, limit of detection (10–25 pg/mL), limit of identification, recovery (25–100%), robustness, carry over (<2%), and matrix effects. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely, [[2H10] LeuB6, B11, B15, B17]-insulin and [[13C6] Leu26, 30] C-peptide. Finally, the method was applied to samples from patients with diabetes mellitus treated with synthetic insulins.

Development and optimization of modified nucleosides and deoxynucleosides simultaneous extraction with the use of Design of Experiments approach

Modified nucleosides and deoxynucleosides are investigated as potential markers of pathological disorders. Due to different molecular properties, their concurrent extraction from biological matrices is challenging. The aim of the present work was the development of sample preparation procedure allowing for simultaneous extraction of 12 selected modified nucleosides and deoxynucleosides with the use of solid phase extraction (SPE) technique. Commercially available mixed-mode SPE cartridges based on phenylboronic acid and cation-exchange sorbents were utilized. In order to decrease the number of experiments and, consequently the volume of applied solvents, the developed method was optimized with the use of Design of Experiments approach. Optimization of SPE method was carried out by employing Fractional Factorial Design with selected input variables such as composition of conditioning, washing and elution solvents. Next, Full Factorial Design with conditioning, extraction and elution duration time as factors was employed. Evaluated responses in these two experimental sets were peak areas of selected analytes. Levels of assessed factors were determined. Developed and optimized SPE procedure allowed for simultaneous extraction of modified nucleosides and deoxynucleosides with mean recovery of 76.0%. Procedure was applied on the exemplary urine samples with the use of high performance liquid chromatography hyphenated with triple quadrupole mass spectrometry (HPLC-QqQ/MS).

Quantification of cannabinoids in human hair using a modified derivatization procedure and liquid chromatography-tandem mass spectrometry.

The aim of this work was to develop and validate a liquid chromatography-tandem mass spectrometry method for detecting of the main cannabinoids, cannabinol (CBN) and tetrahydrocannabinol (THC) and the primary metabolite 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in hair samples. Extraction of the cannabinoids was carried out by a polymeric strong anion mixed-mode solid-phase extraction cartridge and then employing methanolic HCl followed by 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS) as a derivatization procedure of carboxyl and phenolic groups, respectively, offering enhanced sensitivity for the detection of THC-COOH in hair matrices. Formation of a methyl ester increased its lipophilicity and removed the negative charge on the carboxyl group. Calibration curves were prepared over the range of 0.02-4pg/mg of hair for THC and CBN and 0.2-12pg/mg of hair for THC-COOH. The extraction recovery was between 81% and 105% for all compounds. The limit of detection (LOD) and limit of quantification (LOQ) were 2 and 20pg/mg, respectively, for both CBN and THC and 0.1 and 0.2pg/mg, respectively, for THC-COOH, which met the society of hair testing recommendation. Intra-assay and interassay precision were always lower than 4% and 11%, respectively for these cannabinoids, whereas intra-assay and interassay bias were between +14% and -18% and +15% and -12%, respectively. Twenty-seven hair specimens from cannabis users were investigated. The concentrations of CBN, THC and THC-COOH gave ranges of (0.022-2.562ng/mg), (0.049-0.431ng/mg) and (0.222-4.867pg/mg), respectively. This new method of derivatization improves the LOD to ensure detection of the metabolite.

Analysis of 17 fentanyls in plasma and blood by UPLC-MS/MS with interpretation of findings in surgical and postmortem casework

The opioid crisis is linked to an increased misuse of fentanyl as well as fentanyl analogs that originate from the illicit drug market. Much of our current understanding of fentanyl and fentanyl analog use in our communities comes from postmortem toxicology findings. In the clinical settings of addiction medicine and pain management, where the opioid abuse potential is high, the use of fentanyl, as well as specific fentanyl analogs, may be underestimated due to limited plasma testing and limited availability of assays with suitable analytical sensitivity and selectivity to detect misuse of fentanyls. We report plasma and blood assays for 17 fentanyls (these include fentanyl, fentanyl analogs, fentanyl metabolites and synthetic precursors) in clinical, and medical examiner, casework. A mixed-mode solid phase extraction of diluted plasma or precipitated blood was optimized for maximum recovery of the fentanyls with minimized matrix effects. Analysis was performed using a Waters ACQUITY UPLC I-Class interfaced with a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer. Method parameters were optimized and validated for precision, accuracy, carryover, linearity and matrix effects. Application studies were performed in postmortem blood obtained in 44 fentanyl-related fatalities and in serial plasma samples from 18 surgical patients receiving intravenous fentanyl therapy while undergoing parathyroidectomy. Fentanyls found in postmortem cases included fentanyl, norfentanyl, despropionyl-fentanyl (4-ANPP), beta-hydroxy fentanyl (β-OH fentanyl), acetyl fentanyl, acetyl norfentanyl, methoxyacetyl fentanyl, furanyl fentanyl, cyclopropyl fentanyl, and para-fluorobutyryl fentanyl, with fentanyl, norfentanyl, 4-ANPP and β-OH fentanyl predominating in frequency. Fentanyl concentrations ranged from 0.2 to 56 ng/mL and fentanyl was nearly always found with 4-ANPP, norfentanyl and β-OH fentanyl. Concentrations of other fentalogs ranged from <1 to 84 ng/mL (extrapolated). In the surgical cases, fentanyl was detected and quantified along with norfentanyl and β-OH fentanyl, but without detection of 4-ANPP in any of the samples. The association and relative concentrations of β-OH fentanyl, fentanyl and norfentanyl in the postmortem and clinical studies indicated a metabolic, rather than an illicit, source of β-OH fentanyl.