Cation-exchange Solid-phase Extraction Research Articles

Determination of Tetrodotoxin in Miso Soup byStrong Cation Exchange Solid Phase Extraction and LC-MS/MS

Determination of Tetrodotoxin in Miso Soup byStrong Cation Exchange Solid Phase Extraction and LC-MS/MS

Determination of three methylimidazole compounds in cosmetics by high performance liquid chromatography-tandem mass spectrometry

Methylimidazole compounds are byproducts formed during the caramel-coloring process and are used in various cosmetics. In addition metronidazole is an antibacterial and anti-inflammatory drug commonly used in modern medicine and is used in cosmetics to treat acne in the short-term. The illegal addition of metronidazole during cosmetics production can result in residual 2-methylimidazole (2-MEI), which, along with 4-methylimidazole (4-MEI), is a class 2B carcinogen. Therefore, establishing efficient, accurate, and sensitive analytical techniques for analyzing methylimidazole compounds in cosmetics is an urgent objective. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining 1-methylimidazole (1-MEI), 2-MEI, and 4-MEI in cosmetics was developed. Cosmetics samples were extracted via ultrasonication in acetonitrile and purified using a mixed cation-exchange (MCX) solid-phase extraction (SPE) column, with subsequent drying under a stream of nitrogen and redissolution in acetonitrile. The resulting solution was then filtered through a 0.22 μm organic filter membrane for further testing. The analytes were separated using an XBridge® shield RP18 chromatographic column (150 mm×4.6 mm, 3.5 μm) and isocratically eluted with 20 mmol/L ammonium formate solution (containing 0.1% formic acid)-acetonitrile (98∶2, v/v). The target compounds were ionized by electrospray ionization (ESI) source, analyzed in multi-reaction monitoring (MRM) mode, and quantified using the external standard method, with the peak area of the quantitative ion and the mass concentration of the compound taken as the longitudinal and transverse coordinates, respectively. Matrix-matching working curves were also constructed. 1-MEI exhibited good linear relationships in the range of 5-200 μg/L, with correlation coefficients (r2)≥0.9994, while 2-MEI and 4-MEI showed good linearities in the range of 2-100 μg/L with r2≥0.9984. The three methylimidazole compounds exhibited limits of detection (LODs) and quantification (LOQs) of 10-30 μg/kg and 25-100 μg/kg, respectively. Under three spiked levels (LOQ, 2LOQ, 10LOQ), the recoveries of three methylimidazole compounds were 80.9%-107.9%, with relative standard deviations (RSDs, n=6) of 1.2%-12.8%. The practicability of the method was examined using 48 cosmetic samples; 4-MEI was detected in nine samples at contents of 26-1000 μg/kg, while two samples contained 240 and 267 μg/kg of 2-MEI, respectively. 1-MEI was not detected in any of the 48 samples tested. The developed method is simple, fast, and highly sensitive, and provides methodological support for assessing risks and monitoring the three methylimidazole compounds in cosmetics through screening.

Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including β2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and β-glucuronidase/aryl sulfatase (40 μL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 μL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 μg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 μg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

Development of a Cation Exchange SPE-HILIC-MS/MS Method for the Determination of Ningnanmycin Residues in Tea and Chrysanthemum.

Ningnanmycin is a widely used antibiotic in agricultural production that effectively controls fungal and viral diseases in tea trees and chrysanthemums. The polarity characteristic of ningnanmycin has posed limitations on the development of robust detection methods, thereby hindering effective monitoring and control measures. By combining cation exchange solid phase extraction (SPE) with hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS), we have effectively tackled the issue pertaining to the separation and retention of ningnanmycin. The average recoveries of ningnanmycin in green tea, black tea, and chrysanthemum were 77.3-82.0%, 80.1-81.5%, and 74.0-80.0%, respectively. The intraday and interday relative standard deviations (RSDs) were below and equal to 7.7%. Good linearity was observed in the concentration range of 1-1000 μg/L (R2 > 0.998). The limits of detection (LODs) ranged from 1.1 μg/kg to 7.1 μg/kg, and the limits of quantification (LOQs) ranged from 3.6 μg/kg to 23.7 μg/kg for ningnanmycin. These results indicate the good accuracy, repeatability, reproducibility, and sensitivity of the method. It is suitable for detecting ningnanmycin in tea and chrysanthemum.

Pharmacokinetic study of osilodrostat and identification of mono-hydroxylated metabolite in equine plasma for the purpose of doping control.

Osilodrostat is an inhibitor of 11-beta-hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma. Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre- and post-administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS). [Correction added on 27 January 2023, after first online publication: In the preceding sentence, "C-HRMS" was changed to "LC/ESI-HRMS" in this version.] For quantification of osilodrostat, a strong cation exchange solid-phase extraction was employed, and the extracts were analyzed using LC/ESI-triple quadrupole tandem mass spectrometry (LC/ESI-QqQ-MS/MS)to establish its elimination profile. Such extracts were further analyzed using LC/ESI-HRMS to investigate the detectability of osilodrostat and its identified mono-hydroxylated metabolite over a 2-week sampling period. Mono-hydroxylated osilodrostat was identified based on the differential analysis and mass spectrometric interpretations, and it was found to be the most abundant metabolite in plasma. Elimination profile of osilodrostat in plasma was successfully established over the 24-h post-administration period. Both osilodrostat and its mono-hydroxylated metabolite were detected up to the last sampling point at 2weeks using HRMS, and osilodrostat could be confirmed up to 8-day post-administration with its reference material using HRMS as well. For doping control, screening of both the parent drug osilodrostat and its mono-hydroxylated metabolite in equine plasma would be recommended due to their extended detection windows of up to 2weeks. Given the availability of reference material for potential confirmation in forensic samples, osilodrostat is considered the most appropriate monitoring target.

A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine.

The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml-1, with the lowest LoD (1 pg ml-1) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.

Detection of 41 Drugs and Metabolites in Urine with Internal Hydrolysis Control by Liquid Chromatography-Tandem Mass Spectrometry.

Regular monitoring of pain management and substance use disorder patients through urine drug screening is important for assessing patient compliance with prescribed drugs and abstinence fromnon-prescribed drugs. Sample analysis is commonly performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as multiple drugs and metabolites can be monitored from one sample. However, challenges faced indeveloping an LC-MS/MS method for multiple analytes in urine include variability in matrix and concentration from sample to sample and variable chemistry of many pain management drugs and associated metabolites affecting accuracy and precision of results. We describe here an LC-MS/MS method for analysis of 41 drugs and metabolites commonly prescribed for pain management patients. The developed method uses enzymatic hydrolysis followed by cation exchange solid phase extraction. Resorufin-glucuronide is used as an internal hydrolysis control to monitor hydrolysis in each patient sample and minimize false negatives. Analysis was performed using an Agilent 6470 mass spectrometer in dynamic multiple reaction monitoring mode.

Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analyzing GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with an orbitrap. The present developed and validated method for some GHRHs (tesamorelin, CJC-1295, sermorelin (GRF 1–29), sermorelin (3–29)-NH2, somatorelin) is based on the triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE) with weak cation exchange and is able to detect concentrations as low as 0.2 ng/mL (LOD), a limit of quantification (LOQ) at 0.6 ng/mL, and linearity across the range of 0.1 ng/mL to 1.2 ng/mL. The present method developed by our doping control laboratory was validated according to WADA technical documents for selectivity, limit of detection (LOD), carryover, reliability of detection, stability and recovery. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories.

Determination of Nε-(1-Carboxymethyl)-L-Lysine in brewing soy sauce by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry

A method for the determination of Nε-(1-Carboxymethyl)-L-Lysine (CML) in brewing soy sauce was developed using ultrahigh performance liquid chromatography-tandem mass spectrometry. A mixed cation exchange solid phase extraction cartridge was used for purification, while gradient elution of a hydrophilic interaction chromatography column was performed with a 0.1% formic acid aqueous solution-acetonitrile. The adding standard recovery rate of this method ranged from 86.94 to 92.61%, and the relative standard deviation (RSD) was between 3.69 and 6.18%. The limit of detection (LOD) was 0.038 ng/mL, and the limit of quantification was 0.127 ng/mL. The RSD values of the repeatability test and the parallel test were 3.44 and 6.56%, respectively. Using this method, the CML contents determined in eight brewing soy sauce samples were 3.16 ± 0.18 – 15.10 ± 1.19 µg/mL. This method is simple, accurate, sensitive, and precise, and it is an effective method for monitoring the CML content in brewing soy sauce.

Determination of tetrodotoxin in bivalve mollusks by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry with internal standard calibration and its contamination in Zhejiang province, China

A method was established for determining tetrodotoxin (TTX) in bivalve mollusks by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry (HILIC-MS/MS) using kasugamycin as the internal standard for quantification. Samples were subjected to ultrasonic extraction with methanol–water (1:1, v/v) containing 0.5% acetic acid, protein precipitation with acetonitrile, clean-up using a cation exchange solid phase extraction cartridge, elution with acetonitrile:water (1:1, v/v) containing 0.3% hydrochloric acid, neutralization with ammonia before HILIC-MS/MS analysis. The average recovery of the samples spiked at 3 levels ranged in 84.6%-98.1% with the relative standard deviation less than 7.2%. Using this method, the contamination of TTX in 429 bivalve mollusk samples collected in the local markets during 2018 and 2020 was investigated. The detection rates were 12.0–18.8%, following the order of oyster > mussel > clam > scallop. High contaminated oysters and mussels with TTX were found in July to August. Moreover, TTX analogs found in bivalve mollusks included 4-epiTTX, 5,6,11-trideoxyTTX, 4,9-anhydroTTX, and 5-deoxyTTX/11-deoxyTTX.

Facile preparation of sulfonated covalent triazine frameworks for effectively detection of six carcinogenic aromatic amines in cigarette smoke

FDA reported a HPHCs (Harmful and Potentially Harmful Constituents) list for tobacco products and tobacco smoke in 2012, which contained six carcinogenic aromatic amines (AAs). In this work, a sulfonated covalent triazine frameworks (CTF-SO3H) material was synthesized and used as a solid phase extraction (SPE) absorbent for highly effective enrichment and determination of six carcinogenic AAs in cigarette mainstream smoke (MSS), coupled with HPLC-MS/MS technique. The CTF-SO3H material was prepared with a rapid and facile method, which was superior to the general methods. The characterization with Nitrogen adsorption and desorption isotherm experiment, High resolution transmission electron microscopy (HTEM), etc, indicated that the CTF-SO3H material possessed sulfonic acid groups and abundant microporous structure. And the CTF-SO3H material showed good enrichment effect for AAs due to the synergistic effect of π-π stacking and electrostatic interactions. Limits of detection (LODs) of 6 AAs with the developed method were in the range of 0.01–0.11 ng cig−1. Recoveries of 6 AAs were between 80.40% and 106.00%, while the intra-day and inter-day precisions were good (i.e. RSDintra ≤ 6.40%, RSDinter ≤ 9.90%). Commercial cigarette MSS samples were analyzed with the developed method, and a commercial cation exchange SPE column was chosen for comparison. Results obtained by two methods showed a good consistency. However, the CTF-SO3H material needed less dosage, even could be reused for 4 times. Thus, the developed method not only showed high enrichment efficiency, but also was economical.

Analytical Method Optimization of Tetrodotoxin and Its Contamination in Gastropods.

Tetrodotoxin (TTX) is an extremely potent marine biotoxin. An analytical method was developed for both trace contamination and extremely high levels of TTX in gastropods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with clean-up of cation exchange solid phase extraction (SPE) in this study. The limit of detection (LOD) in the sample matrix was 0.5 μg/kg. With the calibration of a screened internal standard (validamycin, IS), the linear range was 0.1-100 ng/mL (1.5-1500 μg/kg in sample matrix) with a correlation coefficient of r2 > 0.999. The average recoveries at three spiking levels (1.5 μg/kg, 44 μg/kg, and 1500 μg/kg) were 82.6-94.4% with relative standard deviations (RSDs) less than 8.4%. TTX levels in seven gastropods (741 samples) were studied. The contamination and analogues in Neverita didyma (N. didyma, 565 samples collected in Zhejiang province, China, from 2016 to 2022) were first reported. The detection rate of TTX in N. didyma was 34.2%. The average concentration was 23.1 μg/kg, and the maximum value was 2327 μg/kg. The time distribution study indicated that high contaminations of TTX occurred from May to August for N. didyma.

Applicable pharmacokinetic study: Development and validation of bioanalytical LC-MS/MS method for the simultaneous quantification of cytarabine and glasdegib used for the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is characteristic by high concentrations of immature bone marrow myeloid cells. Glasdegib (DaurismoTM, GSB) in combination with low dose cytarabin (DepocytTM, CTB) have recently been approved by the US Food and Drug Administration for the treatment of newly diagnosed AML in patients over 75 years of age or who have comorbidities that are prohibitive to intensive induction chemotherapy. However, the pharmacokinetic characteristics of the CTB and GSB is yet unknown. This study developed a selective, sensitive and fast analytical method using liquid chromatography-electrospray ionization (ESI) with tandem mass spectrometry (LC-MS/MS) to simultaneously quantify of CTB and GSB in rat plasma using duvelisib as internal standard (IS). The LC-MS/MS instrument was performed in the ESI interface operating at positive ionization and multiple reaction monitoring (MRM) mode. The CTB and GSB with the IS were extracted from rat plasma by using cation exchange solid-phase extraction cartridge before the analysis. The cartridge gave high recovery rates for both drugs without interference from plasma endogenous. Chromatographic separation was performed on reversed-phase polar-100 column (100 mm × 2.1 mm, 3 µm), with an isocratic mobile phase consisted of a mixture of water containing 0.1% formic acid and acetonitrile (85:15, v/v, pH 3.2) at a flow rate of 0.4 mL/min and the process of analysis was run for 3.5 min. The developed method was validated as per the FDA guidelines over a linear concentration range of 5–3000, for both CTB and GSB with high correlation coefficient (r2 ≥ 0.99). The lower limits of detection (LLOD) were 2.0 ng/mL for both drugs. The overall recoveries of CTB and GSB from rat plasma were in the range of 93.93–101.43%, and the mean relative standard deviation (RSD) of the results was 2.99%. The validated-method was successfully applied for the first time, to a pharmacokinetic study on concurrent oral administration of CTB and GSB in rats (12.0 mg/kg of CTB and 8.5 mg/kg of GSB). The maximum plasma concentration (Cmax) for CTB and GSB was 2312.23 ± 448.26 ng/mL and 1710.61 ± 166.04 ng/mL achieved at 2.83 ± 0.14 h and 2.39 ± 0.10 h, respectively. The AUC0-∞ for CTB and GSB was found to be 8592.49 ± 1714.12 and 4527.67 ± 390.01 ng/mL.h; respectively. The elimination half-life (t1/2kel) of CTB and GSB in rat plasma, were determined to be 2.00 ± 0.19 h and 1.58 ± 0.13 h, respectively. The newly developed approach stands out for its high extraction recovery and absence of matrix interference. Findings further demonstrated the high sensitivity of the developed method, which allowed for effective routine tests in pharmacokinetic investigations with LLOD of 2.0 ng/mL and a run time of 3.5 min.

Innovated single flush on-line solid-phase extraction in bead injection format for flow programming-based determination of creatinine in human urine

Reaction-based assays are commonly automated and miniaturized via flow analysis. However, aggressive reagents can affect or destroy even the chemically resistant manifold during long-term use. Using on-line solid-phase extraction (SPE) can eliminate this drawback and allow for high reproducibility and further advanced automation, as presented in this work. Determination of creatinine in human urine, an important clinical marker, by sequential injection analysis was achieved using bead injection on-line SPE with specific UV spectrophotometric detection, providing the necessary sensitivity and selectivity of the method for bioanalysis. The automated SPE column packing and disposal, calibration, and fast measurement highlighted the improvements in our approach. Variable sample volumes and a single working standard solution eliminated matrix effects, broadened the calibration range, and accelerated the quantification.Our method comprised an injection of 20 μL of 100 × times diluted urine with aqueous acetic acid solution pH 2.4, sorption of creatinine in a strong cation exchanger SPE column, washing out urine matrix with 50% aqueous acetonitrile, and elution of creatinine with 1% ammonium hydroxide. The SPE step was accelerated by a single flush of the column when the eluent/matrix wash/sample/standard zones sequence was created in the pump holding coil, and then the sequence of the zones was flushed into the column at once. The whole process was continually spectrophotometrically detected at 235 nm, subtracted from the signal at 270 nm. A single run duration was less than 3.5 min. Method relative standard deviation was <5.0% (n = 6). A calibration range was linear within the range of 0.02–0.30 μg creatinine (R > 0.999), covering 1.0–15.0 mmol/L creatinine in urine. The standard addition method used two different volumes of a single working standard solution for quantification. Results proved the effectiveness of our improvements in the flow manifold, bead injection, and automated quantification. The accuracy of our method was comparable to the routine enzymatic assay of real urine samples in a clinical laboratory.

Simultaneous determination of 10 first-generation histamine h1 receptor blockers in feeds by ultra-high-performance liquid chromatography triple quadrupole ion trap hybrid mass spectrometer.

A method for simultaneous determination of 10 first-generation histamine H1 receptor blockers in feeds by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry combined with solid phase extraction. Instrument conditions, extraction solvents, and purification methods have been optimized. Under the optimum conditions, these analytes were separated effectively at 6min. These feeds have been extracted by acid acetonitrile and purified by mixed cation exchange solid-phase extraction. The performance of this method meets the requirements of veterinary residue detection in feeds in China. It is appropriate for the confirmatory monitoring and quantitative analysis of 105 feed samples, five kinds of histamine H1 receptor blockers have been detected in 10 samples.

A New Broad-Spectrum Drug Screen for 127 Analytes by LC-MS/MS.

Broad-spectrum drug screening is offered by many clinical laboratories to support investigation of possible drug exposures. The traditional broad-spectrum drug screen employed at our laboratory utilizes several different analytical platforms, thus requiring relatively high volumes of sample and a cumbersome workflow. Here we describe the development and validation of a consolidated broad-spectrum drug screen assay designed to qualitatively detect 127 compounds in urine (Ur) and serum/plasma (S/P) samples. An LC-MS/MS method was developed using the Ultivo LC-MS/MS and designed to be qualitative with a 1-point calibration curve and 50% to 150% controls. Sample preparation included the addition of 122 internal standards (IS) followed by mixed-mode strong cation exchange solid-phase extraction and reverse-phase chromatographic separation on a biphenyl column. For the method described herein, ≥ 95% of analytes in urine and serum control samples had a CV of ≤20% for total imprecision. Accuracy testing included 46 external controls and demonstrated 99.9% accuracy. Method comparison studies to quantitative testing are discussed. The high level of coverage of the analytes with a stable isotope-labeled IS (SIL-IS) helped normalize for matrix effects when significant ion suppression (>25%) was present. Analyte stability in the matrix, the impact of potentially interfering compounds, and method ruggedness were demonstrated. Method limitations include limited detection of glucuronidated drugs and potential cross-contamination with samples at very high concentrations (>>100 × cutoff). The broad-spectrum drug screen method developed here qualitatively detected 127 drugs and select metabolites. This method could be used to support investigations of possible drug exposures in a clinical setting.

Quantification of dissolved metabolites in environmental samples through cation‐exchange solid‐phase extraction paired with liquid chromatography–mass spectrometry

AbstractSmall, biologically produced, organic molecules called metabolites play key roles in microbial systems where they directly mediate exchanges of nutrients, energy, and information. However, the study of dissolved polar metabolites in seawater and other environmental matrices has been hampered by analytical challenges including high inorganic ion concentrations, low analyte concentrations, and high chemical diversity. Here we show that a cation‐exchange solid‐phase extraction (CX‐SPE) sample preparation approach separates positively charged and zwitterionic metabolites from seawater and freshwater samples, allowing their analysis by liquid chromatography–mass spectrometry. We successfully extracted 69 known compounds from an in‐house compound collection and evaluated the performance of the method by establishing extraction efficiencies (EEs) and limits of detection (pM to low nM range) for these compounds. CX‐SPE extracted a range of compounds including amino acids and compatible solutes, resulted in very low matrix effects, and performed robustly across large variations in salinity and dissolved organic matter concentration. We compared CX‐SPE to an established SPE procedure (PPL‐SPE) and demonstrate that these two methods extract fundamentally different fractions of the dissolved metabolite pool with CX‐SPE extracting compounds that are on average smaller and more polar. We use CX‐SPE to analyze four environmental samples from distinct aquatic biomes, producing some of the first CX‐SPE dissolved metabolomes. Quantified compounds ranged in concentration from 0.0093 to 49 nM and were composed primarily of amino acids (0.15–16 nM) and compatible solutes such as trimethylamine N‐oxide (0.89–49 nM) and glycine betaine (2.8–5.2 nM).

A sensitive LC–MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow’s milk

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid–liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.Graphical abstract

Comparative amino acid composition and quality parameters of Moringa oleifera testa and cotyledon

Introduction: Moringa oleifera is a drought-resistant plant, widely used in the tropical region. The leaves and stems have been extensively utilised in foods and neutraceuticals preparation, with less attention to the seeds. In this study, amino acid (AA) compositions of M. oleifera testa and cotyledon were examined comparatively. Methods: Samples were separately defatted, hydrolyed, and neutralised. The AA solution was purified by cation-exchange solid-phase extraction, derivatised and analysed by gas chromatography. Results: Glutamic (acidic amino acid) and phenylalanine (essential amino acid, EAA) were the most concentrated in both samples. Total EAA (g/100g crude protein, cp) was higher in cotyledon (51.0) than testa (41.9). Predicted protein efficiency ratios (P-PERs) were higher in testa (0.605-1.530) than cotyledon 0.286-1.460). EAA index ranged between 0.951-1.13 (soybean comparison) and 83.0-96.9 (egg comparison) with corresponding biological value of 78.7-93.9. The following AA had scores &gt;1.0 in comparison to whole hen’s egg, testa: glycine (Gly), glutamic acid (Glu), phenylalanine (Phe), histidine (His), and cysteine (Cys); cotyledon (Gly), proline (Pro), Glu, Phe, His, arginine (Arg) and Cys. In comparison with requirements of pre-school children, six AA (6/9 or 66.7%) had scores &gt;1.0 in each sample. In provisional AA scoring pattern, isoleucine (Ile) (1.25) and Phe + tyrosine (Tyr) (1.68) had scores &gt;1.0 in testa while methionine (Met) + Cys, Phe+Tyr, and tryptophan (Trp) in cotyledon. However, tryptophan and lysine were the limiting AAs in testa and cotyledon, respectively. Conclusion: The study showed that both anatomical parts would complement each other in terms of amino acid supply.

Extraction, Isolation and Identification of Low Molecular Weight Peptides in Human Milk

Human milk contains numerous free low molecular weight peptides (LMWPs), which may play an important role in infant health and growth. The bioactivities of LMWPs are determined by their structures, especially the amino acid sequences. In the present study, 81 human milk samples were collected and purified by cation-exchange solid-phase extraction (SPE). Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for the separation and detection of free LMWPs in human milk. A total of 56 LMWPs were identified and quantified. These LMWPs were mainly derived from 3 regions of β-casein, which were the amino acid fragments of 16–40, 85–110, and 205–226. The predominant LMWPs were RETIESLSSSEESITEYK, RETIESLSSSEESITEYKQKVEKVK, ETIESLSSSEESITEYK, TQPLAPVHNPIS, and QPLAPVHNPISV with molecular weights of 2247.9573, 2860.2437, 2091.8591, 1372.7666, and 1271.7212, respectively. The results indicated that the technique based on SPE and UPLC-QTOF-MS might greatly facilitate the analysis of LMWPs in human milk.