Isotope Internal Standards Research Articles

Quantitation of phenylalanine and tyrosine from dried Blood/Plasma spots with impregnated stable isotope internal standards (SIIS) by FIA-SRM

BackgroundDried Blood Spot (DBS) analysis has been used for identification and quantification of diseases and disorders in large populations. Simply collecting blood or plasma samples on cotton paper, followed with an organic solvent extraction, many small molecules can be detected and quantified. In a typical procedure of DBS analysis in newborn screening, stable isotope internal standards (SIIS) are added to extraction solvent as a reference. However, this way of employing SIIS does not reflect extraction efficiency, or protein binding issues, nor does it reflect potential degradation that could occur. In addition, punched-out discs from larger DBS are known to have imprecision typically ≥ 15%. MethodsWe developed and tested an approach, internal quantitative DBS (iqDBS), which delivers an exact volume of whole blood or plasma to a paper disc that is impregnated with a dried concentration of SIIS for quantitation. Amino acids were derivatized to make butyl esters and measured using Flow Injection Analysis with Selected Reaction Monitoring (FIA-SRM). ResultsWe demonstrated with phenylalanine and tyrosine improved sensitivity and accuracy by applying iqDBS. ConclusionsWe established a new method for quantitative analysis of small molecules from dried blood spots that incorporates stable isotope internal standard at the time of blood collection.

Parts per billion of nitrite in microcrystalline cellulose by ion chromatography mass spectrometry with isotope labeled internal standard

Regulatory authorities like the U.S. Food and Drug Administration (FDA) have set strict specification levels for N-nitrosamines in finished drug products. Nitrite is a potential precursor for the formation of probable carcinogenic N-nitrosamines when secondary or tertiary amines are also present in the active pharmaceutical ingredient (API) synthesis or drug formulation process. An accurate and sensitive determination of nitrite will be useful when a drug product manufacturer chooses to investigate the reaction kinetics between nitrite and amines or to select appropriate excipients for its drug formulation. Pharmaceutical excipient manufacturers may also need an accurate nitrite measurement to investigate the nitrite content in their excipients. This study details the development and validation of an ion chromatography mass spectrometry (IC-MS) method for trace nitrite determination in microcrystalline cellulose materials, one of the important pharmaceutical excipients used in many drug formulations. MS operated under selected ion monitoring mode was used to solve the commonly encountered interference issue with conductivity detection, and nitrite isotope internal standard was employed to address the ion suppression issue with MS detection. The installation of an after-column “jumper” to flush water with an auxiliary pump through the MS when it is not used for data collection avoided sensitivity loss due to trace salt accumulation in the ion source. Validation of the optimized method was satisfactory, with linearity of nitrite in the concentration range of 0.02–7.50 ppm (µg/g) having a regression coefficient of > 0.999, precision of RSD < 9.5% at 0.03 ppm and RSD < 3.4% at 0.4 ppm and recovery of 92.0–103.0%. The limit of detection and limit of quantitation were 0.005 and 0.016 ppm, respectively.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography–mass spectrometry

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC–MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC–HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at −20 °C and were used to develop an isotope-dilution LC–MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.Graphical

Confirmation ofcannabinoids inforensic toxicology casework by isomer-selective UPLC-MS-MS analysis inurine.

Confirmation of cannabinoid use by forensic toxicology testing in urine has been traditionally focused on ∆9-tetrahydrocannabinol (∆9-THC) with analysis of its major metabolite, 11-nor-9-carboxy-∆9-THC (∆9-cTHC), in free and conjugated forms. Legalization of hemp, however, has led to the widespread production and sale of cannabidiol (CBD) derivatives with psycho-activity, including ∆8-THC and ∆10-THC isomers. The increasing availability and growing use of isomer derivatives necessitate an expanded scope of cannabinoid confirmation test protocols. We report a quantitative, isomer-selective method of cannabinoid confirmation by liquid chromatography-tandem mass spectrometry determination of parent drug isomers (∆8-THC, ∆9-THC, ∆10-THC and CBD) as well as isomeric metabolites (∆8-cTHC and ∆9-cTHC). An efficient C18 phase chromatography on 1.6-µm solid core particles was used with a step gradient for near isocratic separation of both early-eluting THC metabolite isomers and later-eluting CBD and THC isomers. A rapid method of hydrolysis, dilution and analysis was employed for the quantitative co-determination of free and conjugated analytes, using stable isotope internal standardization. Method validation is reported, along with interference assessment from a prior confirmation method. Casework experience with the isomer-selective method revealed a 14% prevalence of ∆8-cTHC positive cases with a pattern of concomitant ∆8-THC and ∆9-THC use. A comparison of ∆8-cTHC and ∆9-cTHC phase two metabolism is also reported.

Chemical QuantArray: A Quantitative Tool for Mass Spectrometry Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a powerful analytical technique that provides spatially preserved detection and quantification of analytes in tissue specimens. However, clinical translation still requires improved throughput, precision, and accuracy. To accomplish this, we created "Chemical QuantArray", a gelatin tissue microarray (TMA) mold filled with serial dilutions of isotopically labeled endogenous metabolite standards. The mold is then cryo-sectioned onto a tissue homogenate to produce calibration curves. To improve precision and accuracy, we automatically remove pixels outside of each TMA well and investigated several intensity normalizations, including the utilization of a second stable isotope internal standard (IS). Chemical QuantArray enables the quantification of several endogenous metabolites over a wide dynamic range and significantly improve over current approaches. The technique reduces the space needed on the MALDI slides for calibration standards by approximately 80%. Furthermore, removal of empty pixels and normalization to an internal standard or matrix peak provided precision (<20% RSD) and accuracy (<20% DEV). Finally, we demonstrate the applicability of Chemical QuantArray by quantifying multiple purine metabolites in 14 clinical tumor specimens using a single MALDI slide. Chemical QuantArray improves the analytical characteristics and practical feasibility of MALDI-MSI metabolite quantification in clinical and translational applications.

Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100–5.00 μg/mL for rhamnose, 0.0500–25.0 μg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00–500 μg/mL for galactose, 10.0–5000 μg/mL for mannose, and 50.0–25,000 μg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.

Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100µg/kg for chlorpromazine, 44 ± 4.4µg/kg for ciprofloxacin, 3.3 ± 1.4µg/kg for clenbuterol, 9.5 ± 0.8µg/kg for dexamethasone, 57 ± 4.8µg/kg for enrofloxacin, 3.0 ± 0.4µg/kg for meloxicam, 12.4 ± 1.2µg/kg for ractopamine, and 2290 ± 120µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Determination of 15 halogenated polycyclic aromatic hydrocarbons in aquatic products by stable isotope dilution coupled with gas chromatography-triple quadrupole mass spectrometry

Halogenated polycyclic aromatic hydrocarbons (H-PAHs), including chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) and brominated polycyclic aromatic hydrocarbons (Br-PAHs), are compounds in which one or more hydrogen atoms replaced by chlorine or bromine atoms. These compounds are not only difficult to degrade but also highly fat soluble and toxic. They are a new type of high-risk organic pollutants with structures similar to those of dioxins, and their toxicity is even higher than that of the parent polycyclic aromatic hydrocarbons (PAHs). The bioaccumulation of H-PAHs can be predicted by their octanol-water partition coefficient (Kow); in general, higher bioaccumulation capacity and Kow values indicate greater fat solubility. Therefore, animal-derived foods with higher fat contents, such as animal meat, milk, aquatic products, and their processed forms, are more likely to be contaminated with higher contents of H-PAHs than those with lower fat contents. In this work, a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method coupled with stable isotope dilution was established to determine 15 H-PAHs in aquatic products. The instrument and pretreatment methods were systematically optimized. The GC-MS/MS used in this method can effectively eliminate matrix interferences and features high sensitivity and low analytical cost; thus, it has good application prospects. The samples were added with an isotope internal standard before extraction to calibrate the loss of the tested substance during the pretreatment process, extracted by accelerated solvent extraction, purified using gel permeation chromatography and PRiME HLB columns, and then analyzed by GC-MS/MS. The use of two DB-5MS chromatographic columns (30 m×0.25 mm×0.25 μm) and microplate fluidics technology to connect chromatographic columns 1 and 2 in series led to better separation effects, good peak shapes, and high target compound responses. The 15 H-PAHs demonstrated good linearities in the range of 1-50 μg/L, with correlation coefficients (r) greater than or equal to 0.993. The relative standard deviation (RSD) values of the relative response factor (RRF) of the H-PAHs were less than 9%, the method detection limit (MDL) was 0.009-0.072 μg/kg, and the method quantification limit (MQL) was 0.031-0.240 μg/kg. Three spiked levels of 0.25, 1.0, 2.5 μg/kg were added to the blank samples to determine the recovery and precision. The recoveries for these spiked levels were 74.6%-116.8%, 77.8%-123.2%, and 71.9%-124.8%, respectively, and the corresponding RSDs were 0.6%-8.2%, 0.6%-9.0%, and 0.4%-10.6%, respectively. The total actual content of H-PAHs in aquatic product samples was 0.60-3.54 μg/kg. Among the H-PAHs investigated, 9-chlorophenanthrene (9-ClPhe) showed the greatest detection rate (100%) and highest content (1.15 μg/kg), indicating that H-PAHs widely exist in aquatic products. Thus, further assessment of the dietary exposure risk of these compounds is necessary. The developed method simplifies the pretreatment step, and has the advantages of simplicity, rapid analysis, high recoveries, and good stability. It is suitable for the qualitative and quantitative analysis of H-PAHs in actual aquatic product samples and provides reliable technical support for the residue status and risk assessment of H-PAHs in aquatic products.

Development of a UPLC-MS/MS method for simultaneous therapeutic drug monitoring of anti-hepatocellular carcinoma drugs and analgesics in human plasma.

In hepatocellular carcinoma treatment, sorafenib, oxaliplatin, 5-fluorouracil, capecitabine, lenvatinib, and donafenib are first-line drugs; regorafenib, apatinib, and cabozantinib are second-line drugs; and oxycodone, morphine, and fentanyl are commonly used analgesics. However, the high degree of inter- and intra-individual variability in the efficacy and toxicity of these drugs remains an urgent issue. Therapeutic drug monitoring (TDM) is the most reliable technical means for evaluating drug safety and efficacy. Therefore, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous TDM of three chemotherapy drugs (5-fluorouracil, oxaliplatin, and capecitabin), six targeted drugs (sorafenib, donafenib, apatinib, cabozantinib, regorafenib, and lenvatinib), and three analgesics (morphine, fentanyl, and oxycodone). We extracted 12 analytes and isotope internal standards (ISs) from plasma samples by magnetic solid phase extraction (mSPE) and separated them using a ZORBAX Eclipse Plus C18 column with water containing 0.1% formic acid and methanol containing 0.1% formic acid as the mobile phase. The analytical performance of our method in terms of sensitivity, linearity, specificity, carryover, precision, limit of quantification, matrix effect, accuracy, dilution integrity, extraction recovery, stability, and crosstalk of all the analytes under different conditions met all the criteria stipulated by the guidelines of the Chinese Pharmacopoeia and U.S. Food and Drug Administration. The response function was estimated at 10.0-10 000.0ng/mL for sorafenib, donafenib, apatinib, cabozantinib, regorafenib, and lenvatinib, and 20.0-20 000.0ng/mL for 5-fluorouracil, oxaliplatin, capecitabin, morphine, fentanyl, and oxycodone, with a correlation of > 0.9956 for all compounds. The precision and accuracy of all analytes were < 7.21% and 5.62%, respectively. Our study provides empirical support for a simple, reliable, specific, and suitable technique for clinical TDM and pharmacokinetics.

Simultaneous determination of 24 antiepileptic drugs and their active metabolites in human plasma by UHPLC-MS/MS

Antiepileptic drugs (AEDs) have narrow therapeutic ranges with large individual variability. Routine therapeutic drug monitoring of AEDs was useful for dose optimization, but the common immunoassays could not meet the detection requirements of AEDs, especially for new generation AEDs. The aim of this study was to validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously quantification of 24 AEDs and their active metabolites in human plasma and comparison with a chemiluminescent immunoassay (Simens ADVIA Centaur). The method validation was performed according to FDA and EMEA guidelines. A one-step protein precipitation by acetonitrile followed a five-fold dilution was performed for sample pretreatment. A 5.2 min gradient separation by methanol and 10 mM ammonium acetate was used for separation at 0.6 mL/min under 45 °C. Both positive and negative electrospray ionization were used. Isotopic internal standard was used for all analytes. The inter-day (36 days) accuracy and precision of quality control samples were − 1.07–13.69% and < 6.70% for all analytes. The stability was acceptable for all analytes under routine storing conditions. A total of 436 valproic acid, 118 carbamazepine, and 65 phenobarbital samples were determined twice by each of the UHPLC-MS/MS and immunoassay. Evaluated by Bland-Altman plot, the mean overestimation of the immunoassay compared to UHPLC-MS/MS was 16.5% for valproic acid, 5.6% for carbamazepine, and 40.3% for phenobarbital.

Determination of sixteen antibiotics and four β-agonists in human urine samples using ultra-performance liquid chromatography-tandem mass spectrometry based on high-throughput automatic solid-phase extraction

An analytical method combining high-throughput automatic solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine 16 antibiotics (macrolides, tetracyclines, quinolones, and sulfonamides) and 4 β-agonists (terbutaline, salbutamol, ractopamine, and clenbuterol) in human urine samples. After thawing at room temperature, 1 mL of urine was sampled and the internal standard was added, followed by the addition of 200 μL ammonium acetate buffer and 20 μL β-glucuronidase, and the mixture was incubated at 37 ℃ overnight. Automatic solid-phase extraction was used to extract the target compounds from the urine samples, and the recoveries were compared using different solid-phase extraction 96-well plates (PRiME MCX, Sep-Pak C18, PRiME HLB), types and volumes of rinse solutions and eluents. Satisfactory recoveries of the 20 target compounds were obtained using the Oasis PRiME HLB 96-well plate, with 1.5 mL 10% (v/v) methanol aqueous solution and 2.0 mL methanol as the rinse solution and eluent, respectively. The eluent was concentrated under nitrogen gas at 45 ℃, and the recoveries of the target compounds were compared under different conditions (completely or almost dry, drying to 1 mL, and adding water as a protective agent), and the recovery rate was optimal when water was added as a protective agent. In this study, two types of analytical columns (ACQUITY BEH C18 and ACQUITY HSS T3) and different gradient elution procedures and mobile phases were compared. The optimal chromatographic effect was realized using an HSS T3 column (100 mm×3.0 mm, 1.8 μm) and 0.1% (v/v) formic acid aqueous solution-0.1% (v/v) formic acid in acetonitrile as the mobile phase in gradient elution at a flow rate of 0.3 mL/min. Comparing the peaks observed using different proportions of methanol aqueous solution and the initial mobile phase as the injection solvent revealed that 30% (v/v) methanol aqueous solution was the optimal solution in terms of peak shape and signal-to-noise ratio. MS was conducted using positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, and the MS parameters were optimized, including the curtain (CUR) and collision gases (CAD). The standard curve obtained using this method exhibited a good linearity (correlation coefficient>0.997), and the respective limits of detection and quantification were 0.02-0.12 ng/mL and 0.06-0.41 ng/mL. At spiked levels of 0.25, 2.5, and 12.5 ng/mL, the recoveries were in the range of 81.7%-120.0% (except that of tetracycline), the intra- and inter-day RSDs (n=6) were 1.1%-11.0% and 1.2%-13.0%, respectively. Azithromycin, trimethoprim, terbutaline, salbutamol, ractopamine, and clenbuterol displayed moderate matrix effects, but all targets exhibited weak matrix effects after correction using the isotope internal standard. To evaluate the accuracy of this method, BCR-503 (containing salbutamol and clenbuterol) and internal quality control samples were used and the concentrations of salbutamol and clenbuterol were within the reference ranges. Additionally, the mean concentrations of the 20 target compounds of two different internal quality control samples after 7 measurements were in the ranges of 0.44-0.59 ng/mL (0.5 ng/mL) and 1.72-2.16 ng/mL (2.0 ng/mL), respectively, which were satisfactory. In this study, the analytical method employed automatic sample pretreatment with a 96-well solid-phase extraction plate, and the detection efficiency was considerably improved. This method displays the advantages of simple operation, ideal recovery, a high sensitivity and weak matrix effect, which satisfies the requirements for the simultaneous determination of 16 antibiotics and 4 β-agonists in human urine samples. This study provides a crucial method for use in monitoring antibiotics and β-agonists in human urine and studying their exposure characteristics and health risks.

Simultaneous determination of UDCA and its major metabolites in human plasma with surrogate matrix by a rapid and specific LC-MS/MS method

A rapid, convenient, and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of ursodeoxycholic acid (UDCA), and its major metabolites, glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. Methanol was chosen as surrogate matrix for preparation the calibrators to establish calibration curves. Isotope internal standard was used for each analyte. After plasma samples were deproteinized with methanol, the post-treatment samples were analyzed on a ZORBAX SB-C18 column (2.1 × 50 mm, 1.8 μm) with 2 mM ammonium acetate and acetonitrile as mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing negative ESI interface using API5500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 391.4 → m/z 391.4, m/z 448.3 → m/z 73.9, m/z 498.4 → m/z 80.1, m/z 395.3 → m/z 395.3, m/z 453.3 → m/z 74.0, and m/z 503.2 → m/z 79.9 for UDCA, GUDCA, TUDCA, UDCA-d4, GUDCA-d5, and TUDCA-d5, respectively. The calibration curve ranges were 5.00–2500 ng/mL for UDCA and GUDCA and 0.500–250 ng/mL for TUDCA. The intra- and inter-day precision was within 7.00% in terms of relative standard deviation (RSD%) and the accuracy within 11.75% in terms of relative error. The selectivity, sensitivity, extraction recovery, matrix effect, dilution reliability, and stability were within the acceptable range. The method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after oral administration of 250 mg UDCA.

A simple and rapid LC-MS/MS method for the simultaneous determination of 15 bile acids in human serum and its application to patients with decompensated cirrhosis.

Liver cirrhosis is currently the twelfth leading cause of death globally and the sixth leading cause of death in China. Its treatment is expensive. Changes in the composition of the serum bile acid pool are sensitive indicators of the severity of liver cirrhosis. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and used to simultaneously determine 15 bile acids in human serum in patients with decompensated cirrhosis. Sample preparation involved spiking with isotope internal standards (ISs) followed by protein precipitation and the analytical run time was 5 min. The LC-MS/MS method was fully validated according to CLSI C62A and the Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories. The method achieved an acceptable coefficient of variation for precision (0.83-14.80%) and accuracy (89.39-107.62%). Finally, as proof of applicability, the method was applied to patients with decompensated cirrhosis in routine clinical sample analysis. The degree of variation of different bile acids was clearly shown. These results indicated that abnormal metabolic pathways might play important roles in decompensated cirrhosis.

Unwanted Ingredients—Highly Specific and Sensitive Method for the Extraction and Quantification of PFAS in Everyday Foods

Perfluorinated alkyl substances (PFAS) are anthropogenic substances and can only come from polluted sources. There are many classes of PFAS which can be transformed to perfluoalkyl carboxylic acids and perfluoroalkyl sulfonic acids in the environment. Once they are in this form, they are environmentally mobile and extremely stable with half-lives of decades. These compounds are not innocuous and are implicated in causing many different diseases. In this publication, a method for the extraction and quantitation of perflurocarboxylic acids and perfluorosulfonic acids, using high-resolution liquid chromatography mass spectrometry (HRMS), is evaluated. A QuEChERS extraction method was performed on tomatoes, strawberries and milk purchased from different supermarkets on different days. Recoveries from milk and tomatoes were between 80 and 120%. Matrix effects were significant for strawberries and tomatoes and stable isotope internal standards were required to compensate. PFBA was found in all 3 food types. The total PFAS was 0.68 ng g−1 in milk, 0.20 ng g−1 in strawberries and 0.36 ng g−1 in tomatoes. This study demonstrates the importance of using a robust analytical method to investigate the PFAS content of complex food matrices and in particular the contribution of short-chain PFAS to overall dietary consumption.

Effective Extraction of Bisphenol Compounds from Milk with Stable Zr(IV)-Based Metal-Organic Framework Particles.

Bisphenol compounds (BPs) have recently been the subject of growing interest due to their wide use in industrial and consumer products. Besides their adverse effects on human endocrine system, effective extraction of BPs and their elimination from complex sample matrix are still significant challenges in food analysis. Herein, a novel Zr(IV)-based metal-organic framework (MOF), named BUT-16, has been synthesized and utilized for the extraction and enrichment of BPs in milk samples. Bisphenol A (BPA), one of the highest production volume BPs, is used as a model molecule. The uptake capacity for BPA can reach up to 48 mg/g, and the adsorption rate is rapid (∼10 min), because of the larger surface area and cooperation of multiple functionalities of BUT-16. Employing BUT-16 in solid-phase extraction, coupled with ultra-performance liquid chromatography-tandem mass spectrometry detection, we generated a rapid, facile, and robust method for the enrichment and detection of trace BPA and its 12 substitutes in milk samples. After optimization, the limits of detection and quantification for BPs can be achieved as low as 0.05 and 0.2 ng/mL, respectively. Without the correction of the isotopic internal standard, the average recoveries of BPs at the different spiked concentrations varied from 63.8 to 120.6%, with a satisfactory precision (RSD ≤ 8.2%). Furthermore, the proposed method was successfully applied to the detection of BPs in real milk samples, and the results were in accordance with those of methods reported previously.

Capillary-in-Capillary Electrospray Ionization (CC-ESI) Source Enabling Convenient Sampling and Quantitative Analysis for Point-of-Care Testing.

With outstanding analytical performances, mass spectrometry (MS) has shown great potential for clinical applications. To facilitate the sampling process and quantitative analysis, a capillary-in-capillary electrospray ionization (CC-ESI) source was developed in this study. Utilizing two nested capillaries as a sampler and an ESI emitter, the source enabled spontaneous liquid sampling based on the capillary phenomenon and electrospray ionization mass spectrometry (ESI-MS) analysis. Apart from the cheap price, high portability, and disposability, the CC-ESI had merits of quantitation capability as well as adequate sensitivity. By coupling CC-ESI to a miniature mass spectrometer (mini-MS), a limit of detection (LOD) of 1 ng/mL was achieved for standard imatinib at collision-induced dissociation (CID) tandem MS mode, and a LOQ of 1 ng/mL was obtained for atenolol and imatinib (with isotopic internal standard) at multiple ion reaction monitoring (MRM) modes. As two demonstrations for analysis of practical samples, rapid analysis of abused drugs on surface and quantitative analysis of therapeutic drugs in whole blood were also performed with a CC-ESI mini-MS.

Robust quantitation of gangliosides and sulfatides in human brain using UHPLC-MRM-MS: Method development and application in Alzheimer's disease

Gangliosides (GAs) and sulfatides (STs) are acidic glycosphingolipids that are particularly abundant in the nervous system and are closely related to aging and neurodegenerative disorders. To explore their roles in brain diseases, in-depth molecular profiling, including structural variations of sphingoid backbone, fatty acyl group, and sugar chain of GAs and STs was performed. A total of 210 GAs and 38 STs were characterized in the inferior frontal gyrus (IFG) of human brain, with 90 GAs discovered in brain tissues for the first time. Influential MS parameters for detecting GAs and STs in multiple reaction monitoring (MRM) mode were systematically examined and optimized to minimize in-source fragmentation, resulting in remarkable signal intensity enhancement for GAs and STs, especially for polysialylated species. To eliminate analytical variations, isotopic interference-free internal standards were prepared by simple and fast reduction reaction. The final established method facilitated the simultaneous quantitation of 184 GAs and 30 STs from 25 subtypes, which represents the highest number of GAs quantitated among all quantitation methods recorded in literature so far. The method was further validated and applied to reveal the aberrant change of GAs and STs in the IFG of 12 Alzheimer's disease (AD) patients. Four GAs exhibited high classification capacity for AD (AUC ≥0.80) and were thereby considered the most promising signatures for AD. These findings suggested the close correlation between GAs and the pathogenesis of AD, highlighting the achievements of our robust method for investigating the roles of GAs and STs in various physiological states and diseases.

Determination of 25-hydroxyvitamin D in serum by pre-column derivatization-stable isotope labeling-ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry

To establish a rapid and accurate method for the determination of 25-hydroxyvitamin D_3(25(OH)D_3) and 25-hydroxyvitamin D_2(25(OH)D_2)in serum by pre-column derivatization with stable isotope labeling and ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry, which could be used to diagnose vitamin deficiency and to assess the nutritional status of vitamin D in the population. The serum samples with isotopic internal standard were extracted by mixed extraction solvent(ethyl acetate∶hexane = 2∶1, V/V), centrifuged and dried by the nitrogen blowing, and derivatized with 4-phenyl-1, 2, 4-triazoline-3, 5-dione(PTAD). The reaction products were separated on a BEH C_(18) column(2.1 mm×50 mm, 1.7 μm), and eluted with a 0.1% formic acid/water solution-acetonitrile gradient. The mass spectrometry was performed in positive electrospray ionization(ESI~+) and parallel reaction monitoring(PRM) for the detection of the targets, and quantified by isotope internal standard. The limits of detection both for 25(OH)D_3 and 25(OH)D_2 were 0.01 ng/mL and the limits of quantification were 0.03 ng/mL. The concentration series of 25(OH)D_2 ranged from 0.5 to 40.0 ng/mL and the concentration series of 25(OH)D_3 ranged from 2.5 to 200.0 ng/mL. The recoveries(n=5) were 95.7%-101.3% for 25(OH)D_3, and 98.7%-108.6% for 25(OH)D_2, with the relative standard deviations(RSDs) of 0.5%-4.9% and 2.2%-3.4%, respectively. The accuracy of the determination of 25(OH)D_3 and 25(OH)D_2 in Level 2 serum of the 25(OH)D standard reference material NIST SRM 972a was 104.8% and 94.9%, respectively. The M(P25, P75)serum levels of 25(OH)D_3 and 25(OH)D_2 for 116 pregnant women at the first trimester from Beijing were 25.7(20.8, 32.6) ng/mL and 0.8(0.4, 1.1) ng/mL, respectively. This method is highly sensitive, qualitatively accurate and suitable for evaluation and monitoring the nutritional status of vitamin D in the population.

Normalization Approach by a Reference Material to Improve LC-MS-Based Metabolomic Data Comparability of Multibatch Samples.

Large cohorts of samples from multiple batches are usually required for global metabolomic studies to characterize the metabolic state of human disease. As such, it is critical to eliminate systematic variation and truly reveal the biologically associated alterations. In this study, we proposed a reference material-based approach (Ref-M) for data correction by liquid chromatography-mass spectrometry and represented by an analysis of multibatch human serum samples. The reference material was generated by mixing serum from healthy donors and distributed to each extraction batch of subject samples. Pooled quality control samples and isotopic internal standards were then applied in each acquisition batch for data quality control. Finally, each metabolite in subject samples was normalized by its counterpart in the reference serum. We demonstrated that Ref-M significantly enhanced the numbers of efficient features and effectively eliminated the batch variation of 522 serum samples of healthy individuals, benign pulmonary nodules, and lung cancer patients. Twenty differential metabolites were identified to distinguish lung cancer from healthy controls in the training set. The discriminant model was validated in an independent data set with an area under the receiver operating characteristics (ROC) curve (AUC) of 0.853. Another 40 serum samples further tested with Ref-M were achieved an AUC of 0.843 by the established model. Our results showed that the reference material-based approach presents the potential to improve the data comparability and precision for biomarker discovery in large-scale metabolomic studies.

A sensitive and accurate GC–MS method for analyzing microbial metabolites short chain fatty acids and their hydroxylated derivatives in newborn fecal samples

Short chain fatty acids (SCFAs), crucial intestinal bacterial metabolites, have been widely accepted as potential diagnostic markers in neonatal medicine. Nevertheless, it is still a great challenge to accurately quantify SCFAs in newborn fecal samples due to the huge variation of water content, limited commercial isotope-labeled internal standards and poor sensitivity. In this study, Na2CO3 solution (50 μg/mL) was applied to convert the free SCFAs to SCFA sodium salts, which could prevent the loss of violate SCFAs during lyophilization process. Furthermore, N-methylbenzylamine-d0/d3 was applied as the chemical derivatization regent to enhance the sensitivity and accuracy. Based on this method, the SCFA contents in meconium and neonatal fecal samples were analyzed to illustrate the change of SCFAs during the gut microbiome development. Chemical derivatization based on N-methylbenzylamine-d0/d3 could not only significantly promote the sensitivity (323–1280 folds compared to free SCFAs) by promoting the ionization efficiency, but also provide one-to-one isotope internal standards. Moreover, 7 SCFAs, including acetic acid (2), n-butyric acid (4), isobutyric acid (5), 2-hydroxybutyric acid (11), 2-hydroxy-3-methylbutyric acid (13), 3-hydroxybutyric acid (14), 2-hydroxy-2-methylbutyric acid (17) were found to be significantly increased in neonatal fecal samples compared to the meconium fecal samples. All these results proved that this method could be applied for SCFA analysis in newborn fecal samples with perfect accuracy and sensitivity.