Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation. Graphical abstract

Quantitation and Validation of 34 Fentanyl Analogs from Liver Tissue Using a QuEChERS Extraction and LC–MS-MS Analysis

Since 2013, drug overdose deaths involving synthetic opioids (including fentanyl and fentanyl analogs) have increased from 3,105 to 31,335 in 2018. Postmortem toxicological analysis in fentanyl-related overdose deaths is complicated by the high potency of the drug, often resulting in low analyte concentrations and associations with toxicity, multidrug use, novelty of emerging fentanyl analogs and postmortem redistribution. Objectives for this study include the development of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and subsequent liquid chromatography–mass spectrometry/mass spectrometry analysis, validation of the method following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 requirements and application to authentic liver specimens for 34 analytes including fentanyl, metabolites and fentanyl analogs. The bias for all 34 fentanyl analogs did not exceed ±10% for any of the low, medium or high concentrations and the %CV did not exceed 20%. No interferences were identified. All 34 analytes were within the criteria for acceptable percent ionization suppression or enhancement with the low concentration ranging from −10.2% to 23.7% and the high concentration ranging from −7.1% to 11.0%. Liver specimens from 22 authentic postmortem cases were extracted and analyzed with all samples being positive for at least one target analyte from the 34 compounds. Of the 22 samples, 17 contained fentanyl and metabolites plus at least one fentanyl analog. The highest concentration for a fentanyl analog was 541.4 μg/kg of para-fluoroisobutyryl fentanyl (FIBF). The concentrations for fentanyl (n = 20) ranged between 3.6 and 164.9 μg/kg with a mean of 54.7 μg/kg. The fentanyl analog that was most encountered was methoxyacetyl fentanyl (n = 11) with a range of 0.2–4.6 μg/kg and a mean of 1.3 μg/kg. The QuEChERS extraction was fully validated using the ASB Standard 036 requirements for fentanyl, metabolites and fentanyl analogs in liver tissue.

Quantitation of Fentanyl and Metabolites from Liver Tissue Using a Validated QuEChERS Extraction and LC–MS-MS Analysis

According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were −10.0% and 1.4% for fentanyl, −2.1% and −0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from −8.5% to −19.9% for the low concentrations, −3.6% to −14.7% for the medium concentrations and 1.5% to −16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5–100 and 4-ANPP had a linear range of 0.4–80 μg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3 μg/kg with a mean of 149.2 μg/kg (n = 10). The range of norfentanyl concentrations were 1.9 to 50.0 μg/kg with a mean of 14.1 μg/kg (n = 10). The range of 4-ANPP concentrations were 3.2 to 23.7 μg/kg with a mean of 7.5 μg/kg (n = 7).

A pneumatically assisted nanospray desorption electrospray ionization source for increased solvent versatility and enhanced metabolite detection from tissue

Pneumatically assisted nano-DESI offers improved sensitivity for metabolite species by 1–3 orders of magnitude through more complete desolvation and reduced ionization suppression.

An On-Line Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Perfluoroalkyl Acids in Drinking and Surface Waters

An UHPLC-MS/MS multiresidue method based on an on-line solid phase extraction (SPE) procedure was developed for the simultaneous determination of 9 perfluorinated carboxylates (from 4 to 12 carbon atoms) and 3 perfluorinated sulphonates (from 4 to 8 carbon atoms). This work proposes using an on-line solid phase extraction before chromatographic separation and analysis to replace traditional methods of off-line SPE before direct injection to LC-MS/MS. Manual sample preparation was reduced to sample centrifugation and acidification, thus eliminating several procedural errors and significantly reducing time-consuming and costs. Ionization suppression between target perfluorinated analytes and their coeluting SIL-IS were detected for homologues with a number of carbon atoms less than 9, but the quantitation was not affected. Total matrix effect corrected by SIL-IS, inclusive of extraction efficacy, and of ionization efficiency, ranged between −34 and +39%. The percentage of recoveries, between 76 and 134%, calculated in different matrices (tap water and rivers impacted by different pollutions) was generally satisfactory. LODs and LOQs of this on-line SPE method, which also incorporate recovery losses, ranged from 0.2 to 5.0 ng/L and from 1 to 20 ng/L, respectively. Validated on-line SPE-LC/MS/MS method has been applied in a wide survey for the determination of perfluoroalkyl acids in Italian surface and ground waters.