Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring.

A novel test using dried blood spots for the chromatographic assay of methadone

A novel test has been developed for the analysis of methadone in dried blood spot specimens from patients undergoing methadone maintenance treatment. An isocratic reversed-phase high-performance liquid chromatography method with coulometric detection has been optimized for the determination of methadone. The clean-up of dried blood spots was performed by means of an original microextraction by packed sorbent procedure after microwave-assisted extraction of the drug with a suitable solvent. Extraction yields were satisfactory, always being higher than 90.0 %. The calibration curve was linear over the 4–500 ng mL^-1 concentration range. The method had satisfactory sensitivity (limit of quantitation of 4 ng mL^-1), precision (relative standard deviation less than 5.8 %), selectivity and accuracy (recovery greater than 87.0 %). It was successfully applied to dried blood spot samples collected from heroin-addicted patients undergoing methadone maintenance therapy at dosages between 40 and 240 mg day^-1. The statistical analysis (Bland–Altman plot) showed that the results were in good agreement with those found from the analysis of plasma samples obtained from the same patients. Thus, the method has proved to be suitable for the monitoring of methadone by means of dried blood spots.

LC/ESI-MS/MS method for quantification of 28 synthetic cannabinoids in neat oral fluid and its application to preliminary studies on their detection windows

Serum and urine samples are commonly used for the analysis of synthetic cannabinoids in biofluids; however, their utilization as analytical matrices for drug abstinence control features some substantial drawbacks. While for blood collection invasive sampling is inevitable, the urinary analysis of synthetic cannabinoids is limited by the lack of available reference standards of the respective major metabolites. Moreover, the long detectability of synthetic cannabinoids in both matrices hampers the identification of a recent synthetic cannabinoid use. This article describes the development, validation and application of an LC/ESI-MS/MS method for the quantification of 28 synthetic cannabinoids in neat oral fluid (OF) samples. OF samples were prepared by protein precipitation using ice-cold acetonitrile. Chromatographic separation was achieved by gradient elution on a Luna Phenyl Hexyl column (50?×?2 mm, 5 μm), while detection was carried out on a QTrap 4000 instrument in positive ionization mode. The limits of detection ranged from 0.02 to 0.40 ng/mL, whereas the lower limits of quantification ranged from 0.2 to 4.0 ng/mL. The method was applied to authentic samples collected during two preliminary studies in order to obtain insights into the general detectability and detection windows of synthetic cannabinoids in this matrix. The results indicate that synthetic cannabinoids are transferred from the blood stream into OF and vice versa only at a very low rate. Therefore, positive OF samples are due to contamination of the oral cavity during smoking. As these drug-contaminations could be detected up to approximately 2 days, neat oral fluid appears to be well suited for detection of a recent synthetic cannabinoid use.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS, potassium (K⁺) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K⁺ concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18–0.47). A reference subset (n?=?50) was used to generate an algorithm based on K⁺ concentrations in DBS. Application of the developed algorithm on an independent test set (n?=?50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from ?29.1 to 21.1 %. The mean difference, as obtained by Bland-Altman comparison, was ?6.6 % (95 % confidence interval (CI), ?9.7 to ?3.4 %). After application of the algorithm, differences between corrected and whole blood concentrations lay between ?19.9 and 13.9 % with a mean difference of ?2.1 % (95 % CI, ?4.5 to 0.3 %). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17–0.47), yielding similar results. In conclusion, a K⁺-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine. Graphical Abstract

Percentage differences between uncorrected DBS and whole blood paraxanthine concentrations (upper panel) and between corrected and whole blood paraxanthine concentrations (lower panel) (n = 103)     

A New Splitting Method for Both Analytical and Preparative LC/MS

This paper presents a novel splitting method for liquid chromatography/mass spectrometry (LC/MS) application, which allows fast MS detection of LC-separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min.

Sensitive analytical methods for 22 relevant insecticides of 3 chemical families in honey by GC-MS/MS and LC-MS/MS

Several methods for analyzing pesticides in honey have been developed. However, they do not always reach the sufficiently low limits of quantification (LOQ) needed to quantify pesticides toxic to honey bees at low doses. To properly evaluate the toxicity of pesticides, LOQ have to reach at least 1 ng/g. In this context, we developed extraction and analytical methods for the simultaneous detection of 22 relevant insecticides belonging to three chemical families (neonicotinoids, pyrethroids, and pyrazoles) in honey. The insecticides were extracted with the QuEChERS method that consists in an extraction and a purification with mixtures of salts adapted to the matrix and the substances to be extracted. Analyses were performed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) for the pyrazoles and the pyrethroids and by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the neonicotinoids and ethiprole. Calibration curves were built from various honey types fortified at different concentrations. Linear responses were obtained between 0.2 and 5 ng/g. Limits of detection (LOD) ranged between 0.07 and 0.2 ng/g, and LOQ ranged between 0.2 and 0.5 ng/g. The mean extraction yields ranged between 63 % and 139 % with RSD <25 %. A complete validation of the methods also examined recovery rates and specificity. These methods were applied to 90 honey samples collected during a 2009–2010 field study in two apiaries placed in different anthropic contexts.

Structural characterization of electrochemically and in vitro biologically generated oxidation products of atorvastatin using UHPLC/MS/MS

Ultrahigh-performance liquid chromatography coupled with high-mass-accuracy tandem mass spectrometry (UHPLC–MS–MS) has been used for elucidation of the structures of oxidation products of atorvastatin (AT), one of the most popular commercially available drugs. The purpose of the study was identification of AT metabolites in rat hepatocytes and comparison with electrochemically generated oxidation products. AT was incubated with rat hepatocytes for 24 h. Electrochemical oxidation of AT was performed by use of a three-electrode off-line system with a glassy carbon working electrode. Three supporting electrolytes (0.1 mol L^?1 H₂SO₄, 0.1 mol L^?1 HCl, and 0.1 mol L^?1 NaCl) were tested, and dependence on pH was also investigated. AT undergoes oxidation by a single irreversible process at approximately +1.0 V vs. Ag/AgCl electrode. The results obtained revealed a simple and relatively fast way of determining the type of oxidation and its position, on the basis of characteristic neutral losses (NLs) and fragment ions. Unfortunately, different products were obtained by electrochemical oxidation and biotransformation of AT. High-mass-accuracy measurement combined with different UHPLC–MS–MS scans, for example reconstructed ion-current chromatograms, constant neutral loss chromatograms, or exact mass filtering, enable rapid identification of drug-related compounds. β-Oxidation, aromatic hydroxylation of the phenylaminocarbonyl group, sulfation, AT lactone and glycol formation were observed in rat biotransformation samples. In contrast, a variety of oxidation reactions on the conjugated skeleton of isopropyl substituent of AT were identified as products of electrolysis.

Implementation of a Gaussian Beam Laser and Aspheric Optics for High Spatial Resolution MALDI Imaging MS

We have investigated the use of a Gaussian beam laser for MALDI Imaging Mass Spectrometry to provide a precisely defined laser spot of 5 μm diameter on target using a commercial MALDI TOF instrument originally designed to produce a 20 μm diameter laser beam spot at its smallest setting. A Gaussian beam laser was installed in the instrument in combination with an aspheric focusing lens. This ion source produced sharp ion images at 5 μm spatial resolution with signals of high intensity as shown for images from thin tissue sections of mouse brain.

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C₁₈ column (3.0?×?50 mm, 2.7 μm) with a mobile phase consisting of 0.1 % formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271?→?159 (estrone), and m/z 361?→?163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Determination of benzyl isothiocyanate metabolites in human plasma and urine by LC-ESI-MS/MS after ingestion of nasturtium (Tropaeolum majus L.)

A liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine the concentration of benzyl isothiocyanate (BITC) metabolites in human plasma and urine. In this study, the following BITC metabolites have been considered: BITC–glutathione, BITC–cysteinylglycine, BITC–cysteine, and BITC–N-acetyl-l-cysteine. The assay development included: (1) synthesis of BITC conjugates acting as reference substances; (2) sample preparation based on protein precipitation and solid-phase extraction; (3) development of a quantitative LC-MS/MS method working in the multiple-reaction monitoring mode; (4) validation of the assay; (5) investigation of the stability and the reactivity of BITC conjugates in vitro; (6) application of the method to samples from a human intervention study. The lower limits of quantification were in the range of 21–183 nM depending on analyte and matrix, whereas the average recovery rates from spiked plasma and urine were approximately 85 and 75 %, respectively. BITC conjugates were found to be not stable in alkaline buffered solutions. After consumption of nasturtium, containing 1,000 μM glucotropaeolin, the primary source of BITC, quantifiable levels of BITC–NAC, BITC–Cys, and BITC–CysGly were found in human urine samples. Maximum levels in urine were determined 4 h after the ingestion of nasturtium. With regard to the human plasma samples, all metabolites were determined including individual distributions. The work presented provides a validated LC-MS/MS method for the determination of BITC metabolites and its successful application for the analysis of samples collected in a human intervention study.

Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots

The segregation in dried droplet MALDI sample spots was analyzed with regard to the matrix-to-sample ratio using optical microscopy, MALDI imaging mass spectrometry (MALDI MSI) and IR imaging spectroscopy. In this context, different polymer/matrix/solvent systems usually applied in the analysis of synthetic polymers were investigated. The use of typical matrix concentrations (10 mg mL^?1) in almost every case resulted in ring patterns, whereas higher concentrated matrix solutions always led to homogeneous sample spot layers. The data revealed that segregation is predominantly caused by matrix transport in the drying droplet, whereas polymer segregation seems to be only secondary.

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5–100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1–1,000 nM for all MTXPGn (R ²?>?0.99). The coefficient of variation ranged from 1–4% for intraday precision and 6–15% for interday precision. Samples were stable for at least 1 month at ?80 °C. Recovery ranged from 98–100%, and the relative matrix-effect varied from 95–99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

LC-MS/MS method for the simultaneous quantification of artesunate and its metabolites dihydroartemisinin and dihydroartemisinin glucuronide in human plasma

Artesunate (AS), a hemisuccinate derivative of artemisinin, is readily soluble in water and can easily be used in formulations for parenteral treatment of severe malaria. AS is rapidly hydrolyzed to the active metabolite dihydroartemisinin (DHA) and primarily eliminated by biliary excretion after glucuronidation. To investigate systematically the AS metabolism and pharmacokinetics, a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of AS and its metabolites DHA and DHA glucuronide (DHAG) in human plasma samples was developed. Compared to previous methods, our method includes for the first time the quantification of the glucuronide metabolite using a newly synthesized stable isotope-labeled analogue as internal standard. Sample preparation was performed with only 50 μL plasma by high-throughput solid-phase extraction in the 96-well plate format. Separation of the analytes was achieved on a Poroshell 120 EC-C₁₈ column (50*2.1 mm, 2.7 μm, Agilent Technologies, Waldbronn, Germany). The method was validated according to FDA guidelines. Calibration curves were linear over the entire range from 1 to 2,500 nM (0.4–961.1 ng/mL), 165 to 16,500 nM (46.9–4,691.8 ng/mL), and 4 to 10,000 nM (1.8–4,604.7 ng/mL) for AS, DHA, and DHAG, respectively. Intra- and interbatch accuracy, determined as a deviation between nominal and measured values, ranged from ?5.7 to 3.5 % and from 2.7 to 5.8 %, respectively. The assay variability ranged from 1.5 to 10.9 % for intra- and interbatch approaches. All analytes showed extraction recoveries above 85 %. The method was successfully applied to plasma samples from patients under AS treatment.

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product

Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine eight-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers [5-methyltetrahydrofolate (5-methylTHF), folic acid (FA), plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF] and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 μL instead of 275 μL) by using 96-well SPE plates with 50 mg instead of 100 mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, two replicates/day) for three serum quality control pools was 2.8–3.6 % for 5-methylTHF (19.5–51.1 nmol/L), 6.6–8.7 % for FA (0.72–11.4 nmol/L), and ≤11.4 % for the minor folate forms (<1–5 nmol/L). The mean (±SE) recoveries of folates spiked into serum (3 days, four levels, two replicates/level) were: 5-methylTHF, 99.4?±?3.6 %; FA, 100?±?1.8 %; minor folates, 91.7–108 %. SPE extraction efficiencies were ≥85 %, except for THF (78 %). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method [n?=?150, r?=?0.99 for 5-methylTHF, FA, and total folate (tFOL, sum of folate forms)] and produced slightly higher tFOL (5.6 %) and 5-methylTHF (7.3 %) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h), resulting in improved SPE efficiency and recovery compared to the eight-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ～2 h, and all relevant folate forms can be accurately measured using a small serum volume.

False Results Caused by Solvent Impurity in Tetrahydrofuran for MALDI TOF MS Analysis of Amines

Tetrahydrofuran (THF) is one of the most frequently used solvents in the MALDI TOF MS analysis of synthetic compounds. However, it should be used with caution because a trace amount of 4-hydroxybutanal (HBA) might be generated and accumulated in THF during storage. Since only a tiny amount of analytes is required in MALDI MS measurements, a trace amount of HBA might have a significant effect on the MS results. It was found that HBA will quickly react with primary and secondary amino compounds, leading to false results about the sample composition with an extra series of ions with additional mass of 70 Da in between. The formation of HBA can be inhibited by butylated hydroxytoluene (BHT) antioxidant. Therefore, when THF is required as the solvent for sample preparation, it is strongly recommended to use a BHT-stabilized one, at least for the analysis of compounds with amino groups.