Ion-exchange Solid-phase Extraction Research Articles

Acute toxicity of petroleum asphalt seal coat leachates to Ceriodaphnia dubia is linked to polymer preservatives

Low-VOC waterborne asphalt-emulsion (AE) seal coat is considered more sustainable than solvent-based coal-tar emulsion seal coat because asphalt emulsions contain negligible amounts of carcinogenic PAHs and release fewer harmful volatile organic compounds. Yet, many low-VOC coatings leach water-soluble substances under outdoor conditions. To investigate the chemical composition of seal coat leachates, three AE formulations were cured under natural weathering conditions and exposed to simulated runoff over a 10-day field trial. Runoff was collected and concentrated using ion-exchange solid-phase extraction (SPE) and analyzed using gas chromatography/mass spectrometry (GC–MS). Leached compounds included hydrocarbons, esters, amines, siloxanes, plasticizers, biocides, polyethylene glycol (PEG) ethers, urethanes, and toluene diisocyanate (TDI). Glycol ethers comprised 29–97 % of the measured leachate mass. Two seal coat formulations contained isothiazolinone biocides, methylchloro- and methylisothiazolinone (CMIT/MIT; 0.5 mg/L in runoff), while a third seal coat formulation continuously leached TDI, a reactive polyurethane (PU) precursor (0.7 mg/L in runoff). Biocide-containing leachates showed acute toxicity to the freshwater water flea, Ceriodaphnia dubia after 48 h, while TDI-containing leachate showed no acute toxicity, suggesting that leachate toxicity was due to in-can polymer preservatives. As biocides are implicated in impaired reproductive signaling, these results support the use of alkaline pH to avoid biofouling and reinforce the goal of reducing and/or avoiding the use of biocides altogether, especially for environmentally friendly products.

An Optimized Safe Process from Bench to Pilot cGMP Production of API Eptifibatide Using a Multigram-Scale Microwave-Assisted Solid-Phase Peptide Synthesizer

A growing industrial interest toward the peptide drug market fueled the need for the development of effective and cGMP compliant manufacturing methods for these complex molecules. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only on the research scale but for multigram-scale production, as well. The possibility to use microwave-assisted technology on the multigram scale, recently introduced, prompted us to evaluate the possibility to conveniently set up a safe and fully cGMP-compliant pilot process to produce eptifibatide, a generic peptide active pharmaceutical ingredient. Accordingly, we developed an optimized process on the laboratory scale (1–5 mmol), which was subsequently successfully scaled up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot scale plant. The process consists of 5 steps: (1) automated microwave-assisted solid-phase synthesis of eptifibatide linear precursor; (2) cleavage from the resin with concomitant amino acid side-chains deprotection; (3) disulfide-bond formation in solution; (4) purification by flash column chromatography; (5) ion-exchange solid-phase extraction. Since the direct scale-up of a multigram-scale cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we initially performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result.

Quantitation of plasma metanephrines using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS): a candidate reference measurement procedure and its application to evaluating routine ID-LC/MS/MS methods.

Accurate measurement of plasma metanephrines (MNs) including metanephrine (MN) and normetanephrine (NMN) is crucial for the screening and diagnosis in pheochromocytomas and paragangliomas (PPGLs). Although the number of laboratories using liquid chromatography tandem mass spectrometry (LC-MS/MS) method to measure MNs has been increasing rapidly, those laboratory-developed assays showed incomparable results. There are no reference measurement procedures (RMPs) or reference materials (RMs) for MNs in Joint Committee for Traceability in Laboratory Medicine (JCTLM), which hindered the standardization of MNs measurement. We established a candidate RMP (cRMP) based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for plasma MNs measurement. Plasma samples were spiked with MN-D3 and NMN-D3 as internal standards; protein precipitation and ion-exchange solidphase extraction (SPE) were performed to extract samples, eventually analyzed by LC-MS/MS. The cRMP was applied to evaluate two routine ID-LC/MS/MS methods through split-sample comparisons. Fifty-three individual patient samples were determined by cRMP and two routine ID-LC/MS/MS methods; results were analyzed by ordinary linear regression and Bland-Altman plots. The cRMP exhibited desirable imprecision, with intra-run and total imprecision (coefficient variation, CV) for MN being 0.79-1.36% and 1.53-1.87% and for NMN being 1.10-1.34% and 1.15-1.64%. The analytical recoveries of MN and NMN ranged from 98.3 to 101.7% and from 98.5 to 101.9%, respectively. Significant calibrator biases and sample-specific deviations were observed in method comparison. An accurate, precise, and reliable cRMP for plasma MNs was developed, and RMs with value assigned following the cRMP would help minimize the calibration bias and improve the comparability of different measuring systems.

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.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis

BackgroundTherapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. ObjectiveThis study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. ResultsThe use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2–40 mg L−1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8–10.0%, intra-assay CV of 10.2–11.0%, and accuracy of 96.8–104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15–3.57 mg L−1 for GEN C1, 0.12–3.55 mg L−1 for GEN C1a, 0.20–5.77 mg L−1 for GEN C2, and 0.47–12.88 mg L−1 for the GEN complex, all within the linear range of the assay. ConclusionA sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.

TiO2 Decorated Low-Molecular Chitosan a Microsized Adsorbent for a 68Ge/68Ga Generator System.

We report column material for a 68Ge/68Ga generator with acid resistance and excellent adsorption and desorption capacity of 68Ge and 68Ga, respectively. Despite being a core element of the 68Ge/68Ga generator system, research on this has been insufficient. Therefore, we synthesized a low molecular chitosan-based TiO2 (LC-TiO2) adsorbent via a physical trapping method as a durable 68Ge/68Ga generator column material. The adsorption/desorption studies exhibited a higher separation factor of 68Ge/68Ga in the concentration range of HCl examined (0.01 M to 1.0 M). The prepared LC-TiO2 adsorbent showed acid resistance capabilities with >93% of 68Ga elution yield and 1.6 × 10−4% of 68Ge breakthrough. In particular, the labeling efficiency of DOTA and NOTA, by using the generator eluted 68Ga, was quite encouraging and confirmed to be 99.65 and 99.69%, respectively. Accordingly, the resulting behavior of LC-TiO2 towards 68Ge/68Ga adsorption/desorption capacity and stability with aqueous HCl exhibited a high potential for ion-exchange solid-phase extraction for the 68Ge/68Ga generator column material.

Development of a method for melamine determination in beer and beer-type beverages by GC-MS/MS

Based on the European regulation, the Czech Association of Breweries and Malthouses recommends 2.5 mg/kg as maximum permitted concentration of melamine for beer, barley, malt, hop, and their products. For this reason, the first analytical method for melamine determination in beer and beer-type beverages was developed. The sample preparation included ion-exchange solid-phase extraction and subsequent derivatization with BSTFA. Qualitative and quantitative analysis was performed by gas chromatography with tandem mass spectrometry. Validation parameters of the method, such as limits of detection and quantification were 0.1 mg/L and 0.4 mg/L, respectively. Extended uncertainty of the final method was evaluated to 0.1 mg/L. The method was applied on twenty samples of beer lagers, or other beer styles; all had the level of melamine under the limit of quantification (< 0.4 mg/L).

Development and Validation of a High-Performance Liquid Chromatography Method for the Determination of Histamine in Fish Samples Using Fluorescence Detection with Pre-column Derivatization

Histamine level determination in fish and fish products is important for human safety, fish quality and food industry. For this reason, a rapid, robust, and precise method is needed. To achieve this objective, an HPLC method with fluorescence detection was developed and validated. Histamine in fish samples was efficiently extracted with perchloric acid, and purified with ion-exchange solid-phase extraction cartridge. A pre-column derivatization was adopted with ortho-phthalaldehyde (OPA) in the presence of the reducing agent 2-mercaptoethanol, and the stability of the histamine-OPA derivatives was achieved with the acidification of the reaction medium. In terms of validation, besides the excellent linear correlations, satisfactory recoveries at all spiking levels ranging between 0 and 200 mg kg−1 were attained, with limit of detection calculated at 1.8 mg kg−1, whereas limit of quantification determined at 5 mg kg−1. The proposed method was successfully used in the analysis of reference materials and proficiency tests, and was found to be suitable, accurate, and rapid for detection and quantification of histamine in various fish samples.

Quantification of cytostatic platinum compounds in wastewater by inductively coupled plasma mass spectrometry after ion exchange extraction

Cytostatic platinum compounds (CPCs) are pharmaceutical compounds widely used in chemotherapy. However, these compounds have important side effects and can be toxic to the biota once they are excreted by patients and reach the aquatic medium, even at low concentrations.Most of the works have focused on the determination of CPCs in hospital wastewaters using inductively coupled plasma mass spectrometry (ICP–MS). However, the determination of CPCs in samples from wastewater treatment plants (WWTPs) is very limited, probably due to the difficulty of extracting such hydrophilic compounds from these complex aqueous matrices.This paper presents a new optimised and developed method for the extraction and preconcentration of CPCs in wastewater samples based on ion exchange solid phase extraction and their determination by ICP–MS.Under the optimal conditions, the procedure has good reproducibility and repeatability (with deviations lower than 15%), with a relative recovery between 47 and 90% and a low matrix effect (lower than 24%). We have obtained the lowest limit of quantification achieved up until now (0.74 ng L−1), thus allowing the determination of CPCs in new matrices. The described method was used for the determination of CPCs in wastewater from a WWTP and hospital wastewater of Gran Canaria Island (Spain). We have detected concentrations between 81.94 and 13,913 ng L−1 in hospital effluents and between 3.97 and 75.79 ng L−1 in wastewater treatment plants.

Identification of d-amino acids in tea leaves

During manufacturing processes and in the storage period of tea, amino acids may undergo enantiomeric isomerization, converting their l- to d-forms. To examine the hypothesis, a method was developed for the analysis of the enantiomers in tea leaves. After enriched by ion-exchange solid-phase extraction, the enantiomeric pairs were separated by a chiral high performance liquid chromatography (HPLC) and subsequently detected and identified by using a high resolution quadrupole time-of-flight mass spectrometry (QTOF MS). Only l-forms of amino acids were found in fresh tea leaves. A total of 11 d-amino acids were found in 19 tea samples, ranging from trace amount to 43 µg/g. The results indicated that the enantioisomerization of amino acids occurred in post-harvest tea leaves, and affected by process conditions and storage time.

Improved measurement of ethyl glucuronide concentrations in hair using UPLCMS/MS for the evaluation of chronic ethanol consumption

The presence of Ethyl glucuronide (EtG) in hair provides a strong indication of ethanol consumption and its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. However, due to the possibility of false negative results in cases of small ethanol intake or excessive hair washing, the combined measurement of ethyl palmitate (EtP) with EtG could be useful. In this study, a sensitive UHPLC-MS/MS procedure for the measurement of EtG in hair was developed and validated, using optimized sample preparation and chromatographic separation. Milled hair was extracted with water for 24 h at room temperature, followed by clean-up of the extract by ion-exchange solid phase extraction (SPE). Extraction was highly efficient, with yield of 96.93–101.06%. Chromatographic separation was performed with a Fluoro-Phenyl stationary phase. The assay was linear from 4 to 500pgmg−1, with accuracy in the range of 100.30–106.16%. Matrix effects (−0.87 to 5.89%) were adequately compensated by the use of deuterated EtG as internal standard. EtG was measured in hair samples of 46 volunteers, and results were compared with hair concentrations of ethyl palmitate (EtP) and the score in the AUDITC questionnaire. EtG hair concentrations were significantly correlated to the AUDIT-C classification (rs=0.365, p<0.05), but not to EtP hair levels. The diagnostic performance of EtG hair concentrations to identify excessive or moderate ethanol use was similar to the capability of AUDIT-C to identify severe and high health risk (Kappa, p=0.013). The developed assay is suitable for clinical use, providing a useful tool to evaluate chronic ethanol consumption.

Development and evaluation of a simultaneous and efficient quantification strategy for final prostanoid metabolites in urine

Prostanoids (PNs) play critical roles in various physiological and pathological processes. Therefore, it is important to understand the alternation of PN expression profiles. However, a simultaneous and efficient quantification system for final PN metabolites in urine has not yet been established. Here, we developed and evaluated a novel method to quantify all final PN metabolites. By purification using a reverse phase solid phase extraction (SPE) column, the matrix effects against the final PGD2, PGE2, and PGF2α metabolites were low, and their accuracies were nearly 100%. The matrix effects against the final PGI2 and TXA2 metabolites were high using reverse phase SPE column purification alone. By applying a tandem SPE method that combined reverse phase and ion exchange SPE columns, the matrix effects decreased so that the accuracy was nearly 100%. To validate the reliability of the method, each final metabolite was quantified from mouse urine to which the PNs (PGD2, PGE2, and PGI2) were intravenously administered. As a result, the amounts of PN metabolites were correlated with those of the PNs administered to the blood in a dose-dependent manner. To validate the method using human samples, the urinary metabolites of Crohn's disease (CD, a PN-related disease) patients and healthy individuals were quantified. All five metabolites were successfully quantified. Only final PGE2 metabolite levels were significantly higher in CD patients than those in healthy individuals, so that the urinary metabolite profiles of CD patients is determined. In conclusion, we developed a novel method to quantify all final PN metabolites simultaneously and efficiently and demonstrated the practicality of the method using human CD patient samples.

Dietary risk evaluation of acrylamide intake with bread in Poland, determined by two comparable cleanup procedures

ABSTRACTAcrylamide is regarded as a food chemical contaminant. The aims of this work included: (i) to develop sample cleanup procedures applicable for determination of AA in soft bread samples; (ii) to determine AA levels in soft bread available in retail trade in Poland and to compare them with currently standing benchmark levels; (iii) to determine dietary risk related to AA in soft bread. The procedure based on ion-exchange solid phase extraction was more suitable to obtain LOQs corresponding to AA concentrations in soft bread samples. AA levels found in bread samples were in 3.6–163 µg kg−1 range. AA levels varied greatly from sample to sample, which suggests that both food composition and manufacturing processes play a crucial role in AA generation. When considering reference point for neoplastic effects, Margin of Exposure calculated for AA taken with soft bread ranged between 543 and 3,035.

Isolation and characterization of hydrophilic dissolved organic matter in waters by ion exchange solid phase extraction followed by high resolution mass spectrometry

Dissolved organic matter (DOM) is a complex substance occurring in marine and freshwater environments. DOM has many functions that modify physical, chemical and biological processes in ecological systems. Solid-phase extraction (SPE) is widely used for the separation of DOM. However, a part of strongly hydrophilic compounds are lost using classical SPE separation. In this article, the strong hydrophilic components, which cannot be extracted by the hydrophobic reverse phase SPE, were further extracted in a natural organic matter sample by ion exchange SPE cartridges to obtain hydrophilic acid (HIA), hydrophilic neutral (HIN), and hydrophilic base (HIB) fractions. The extracts were characterized by three-dimensional excitation–emission matrix fluorescence spectroscopy and negative-ion electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The results revealed that the hydrophilic components were significantly different from the hydrophobic components in molecular composition. Hydrophilic components were identified as tryptophan-like and tyrosine-like compounds by three-dimensional fluorescence spectra. The HIA fraction contains mainly tannic acid-like compounds with high O/C ratio; the HIB fraction contains mainly amide-like compounds; and the HIN fraction contains mainly lignin and lipids.

Contamination status of bisphenol A and its analogues (bisphenol S, F and B) in foodstuffs and the implications for dietary exposure on adult residents in Zhejiang Province

An effective method has been developed for the simultaneous determination of four bisphenols (bisphenol A, S, F and B) in various foodstuffs. The contaminants were extracted by QuEChERS-based strategy and subjected to ion-exchange solid-phase extraction for further clean-up. The critical variables were screened by Plackett-Burman design and then optimized by central composite design. Under the optimized conditions, satisfactory accuracy (recoveries 76%–116%) and precision (RSDs < 12%) were achieved. The established method was then used to assess the contamination status of 379 real samples. Bisphenol A was demonstrated to be the predominant bisphenol with highest incidence (79.7%) and average concentration (14.3 μg/kg). The positive rates (mean concentration) of bisphenol S, F and B were 37.7% (1.6 μg/kg), 26.9% (1.4 μg/kg) and 0.0% (not detected), respectively. Finally, the daily dietary intakes of ∑4bisphenolsfor adult residents were estimated (55.9–76.1 ng/kg bw/day) according to the contamination concentrations and the daily recommended intakes.

Dispersive solid-phase extraction followed by triethylsilyl derivatization and gas chromatography mass spectrometry for perfluorocarboxylic acids determination in water samples

A new, simple derivatization method for the determination of perfluorocarboxylic acids (PFCAs, C3-C12) in water is presented. The method is based on an acid-catalyzed esterification of perfluorinated carboxylic acids with triethylsilanol. During the reaction, a triethylsilyl ester is formed which can be easily analyzed by gas chromatography-mass spectrometry (GC–MS). The classic ion exchange solid phase extraction used for PFCAs (DIN 38407-42) has been significantly modified and adopted for the newly developed derivatization method. The new method offers a linear range from 8 to 10,000 ng l−1 for PFOA, with a limit of quantification ranging from 4 to 48 ng l-1 depending on the actual PFCA. Due to sample preparation simplicity, the presented method can be applied to form PFCAs esters prior to GC–MS analysis of water samples. The method was successfully applied in the analysis of effluents from two water treatment plants in Baden-Württemberg, Germany. Additionally, to confirm the laboratory evaluation, the standard solutions and samples were also measured by an external laboratory using LC–MS2.

Feasibility of ion-exchange solid phase extraction inductively coupled plasma mass spectrometry for discrimination between inorganic As(III) and As(V) in phosphate-rich in vitro bioaccessible fractions of Ayurvedic formulations

Determination of potentially toxic elements in the μg/kg–mg/kg concentration range in some Ayurvedic formulations was performed after microwave-assisted acid digestion and inductively coupled plasma mass spectrometry. Arsenic and Hg concentration in the mineral formulations reached about 10% and 0.6% by weight, respectively. Arsenic bioaccessibility was not influenced by pepsin and pancreatin containing model suspensions of pH = 1.2 and 6.8 (with 0.05 M K2HPO4), respectively. The bioaccessible As fraction of the mineral formulations was about 10–20%, even if the estimated As intake from these products exceeded the 3.0 μg/BW kg/day benchmark dose limit with an increased incidence of 0.5% for lung cancer (BMDL05) caused by inorganic As by about 25 times. Ion-exchange solid phase extraction procedure was applied to discriminate between iAs species in either the initial bioaccessible fractions obtained by incubation in simulated duodenal juice or by spiking them with 75 and 150 μg/L arsenate ions [iAsV]. The complex sample/extractant matrix caused a 60–70% recovery for iAs(V) and a breakthrough of about 20%. Suppression of phosphate ions in the extractant improved iAs(V) recovery by about 10%. Arsenic breakthrough was not observable at the 75 μg/L iAs(V) spike level. Similar results were obtained on a homeopathic product confirming the interference of phosphate on As speciation in bioaccessible fractions of complementary and alternative medicines (CAMs). Conversion rate of iAs(III)/iAs(V) in Ayurvedic products with large As content could be estimated. This approach is more informative than extrapolation of results by X-ray based techniques on solid state of CAMs.

Analysis of Leafy Vegetable Nitrate Using a Modified Spectrometric Method.

A quick and cost-effective method to analyze leafy vegetable nitrate on spectrometry was developed and compared with a standard method using high performance liquid chromatography (HPLC). This method was designed to use ion-exchange solid phase extraction (SPE) cartridges in reducing interference from organic matrices to meet the criterion of an existing method for analyzing nitrate in wastewater. Nine vegetables (bok coy, cabbage, celery, Chinese cabbage, Chinese kale, lettuce, mustard green, pak choi, and water spinach) were selected for the method testing with three replicates being conducted for each vegetable. The nitrate contents ranged from 800 to 4,300 μg/g, with bok coy, celery, and pak choi being the highest. Data derived from spectrometry and HPLC were close to each other with most relative errors being within ±10% and were highly correlated with an R square value of 0.969. Stability testing and spike analysis resulted in a mean coefficient of variation lower than 6% and a mean recovery rate of 83.7%, suggesting reliability of the method. In addition, both the cost and time consumption for using this method were lower than the standard method using HPLC or ion chromatography, making this spectrometric method a good alternative for analysis of leafy vegetable nitrate.

Combining cationic and anionic mixed-mode sorbents in a single cartridge to extract basic and acidic pharmaceuticals simultaneously from environmental waters.

The aim of the present study is to broaden the applications of mixed-mode ion-exchange solid-phase extraction sorbents to extract both basic and acidic compounds simultaneously by combining the sorbents in a single cartridge and developing a simplified extraction procedure. Four different cartridges containing negative and positive charges in the same configuration were evaluated and compared to extract a group of basic, neutral, and acidic pharmaceuticals selected as model compounds. After a thorough optimization of the extraction conditions, the four different cartridges showed to be capable of retaining basic and acidic pharmaceuticals simultaneously through ionic interactions, allowing the introduction of a washing step with 15mL methanol to eliminate interferences retained by hydrophobic interactions. Using the best combined cartridge, a method was developed, validated, and further applied to environmental waters to demonstrate that the method is promising for the extraction of basic and acidic compounds from very complex samples.

A validated UPLC-MS/MS method coupled with protein precipitation and ion exchange solid phase extraction for the quantitation of porcine relaxin B29 in dog plasma and its application to a pharmacokinetic study.

Porcine relaxin is a 6 kDa peptide hormone of pregnancy with important physiological and pharmacological effects. It contains a number of analogs of which porcine relaxin B29 is one of the most important. To support the development of porcine relaxin B29 as a new drug, we established an UPLC-MS/MS method for its quantitation in dog plasma. Sample preparation by protein precipitation and ion exchange solid phase extraction was followed by UPLC on an XBridge™ BEH300 C18 column at 40°C in a run time of only 5.5min. Detection was performed on a Qtrap 6500 mass spectrometer using ESI in the positive ion mode with MRM of the transitions at m/z 831.7 [M+7H]7+→505.4 and m/z 1162.4 [M+5H]5+→226 for pRLX B29 and internal standard (recombinant human insulin), respectively. The method was linear over the concentration range 30-2000ng/mL with no matrix effects. Intra- and inter-day precisions were <15% with accuracies in the range 98.8-100.6%. The method was successfully applied to a pharmacokinetic study in beagle dogs after administration of a 0.15 mg/kg intravenous dose. Graphical abstract Sample preparation and detection procedure.