Interaction Liquid Chromatography Tandem Mass Research Articles

Hydrophilic interaction liquid chromatography–tandem mass spectrometry methylphosponic and alkyl methylphosphonic acids determination in environmental samples after pre-column derivatization with p-bromophenacyl bromide

Once exposed to the environment organophosphate nerve agents readily degrade by rapid hydrolysis to the corresponding alkyl methylphosphonic acids which do not exist in nature. These alkyl methylphosphonic acids are finally slowly hydrolyzed to methylphosphonic acid. Methylphosphonic acid is the most stable hydrolysis product of organophosphate nerve agents, persisting in environment for a long time. A highly sensitive method of methylphosphonic acid and alkyl methylphosphonic acids detection in dust and ground mixed samples has been developed and validated. The fact that alkyl methylphosphonic acids unlike methylphosphonic acid did not react with p-bromophenacyl bromide under chosen conditions was discovered. This allowed simultaneous chromatographic separation and mass spectrometric detection of derivatized methylphosphonic acid and underivatized alkyl methylphosphonic acids using HILIC–MS/MS method. Very simple sample pretreatment with high recoveries for each analyte was developed. Methylphosphonic acid pre-column derivate and alkyl methylphosphonic acids were detected using tandem mass spectrometry with electrospray ionization after hydrophilic interaction liquid chromatography separation. The developed approach allows achieving ultra-low detection limits: 200pgmL−1 for methylphosphonic acid, 70pgmL−1 for ethyl methylphosphonic acid, 8pgmL−1 for i-propyl methylphosphonic acid, 8pgmL−1 for i-butyl methylphosphonic acid, 5pgmL−1 for pinacolyl methylphosphonic acid in the extracts of dust and ground mixed samples. This approach was successfully applied to the dust and ground mixed samples from decommissioned plant for the production of chemical weapons.

Development of a hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the determination of kinsenoside, an antihyperlipidemic candidate, in rat plasma and its application to pharmacokinetic studies

Kinsenoside is a major bioactive constituent isolated from Anoectochilus formosanus and is investigated as an antihyperlipidemic candidate. In this study, a rapid, sensitive, and reliable bioanalytical method was developed for the determination of kinsenoside in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The plasma sample was pretreated with 1% acetic acid, followed by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC silica column (2.1mm×100mm, 3μm). The mobile phases consisted of 0.1% acetic acid in distilled water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). A gradient program was used at a flow rate of 0.2mL/min. For mass spectrometric detection, the multiple reaction monitoring mode was used; the MRM transitions were m/z 265.2→m/z 102.9 for kinsenoside and m/z 163.3→m/z 132.1 for the internal standard (IS) nicotine in the positive ionization mode. A calibration curve was constructed in the range of 2–500ng/mL. The intra- and interday precision and accuracy were within 5%. The HILIC–MS/MS method was specific, accurate, and reproducible and was successfully applied in a pharmacokinetic study of kinsenoside in rats.

Development, validation, and comparison of four methods to simultaneously quantify l-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry.

To understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality.

Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

Short peptides in food protein hydrolysates are of significant interest as they may be highly bioactive whilst also being bioavailable. A dipeptidyl peptidase IV (DPP-IV) inhibitory whey protein hydrolysate (WPH) was fractionated using nanofiltration (NF) with a 200Da MWCO membrane. The DPP-IV half maximal inhibitory concentration of the NF permeate (IC50=0.66±0.08mgprotein equivalentmL−1) was significantly more potent (P>0.05) than that of the starting WPH (IC50=0.94±0.24mgprotein equivalentmL−1) and associated retentate (IC50=0.82±0.13mgprotein equivalentmL−1). This confirmed the contribution of short peptides within the NF permeate to the overall DPP-IV inhibitory activity. An hydrophilic interaction liquid chromatography (HILIC-) and reverse-phase (RP-) liquid chromatography tandem mass spectrometry (LC–MS/MS) strategy, based on two retention time models, allowed detection of eight free amino acids and eight di- to tetrapeptides in the NF permeate. The potential sequences of the peptides within the NF permeate were then ranked on the basis of their highest probability of occurrence. A confirmatory study with synthetic peptides showed that valine–alanine (VA), valine–leucine (VL), tryptophan–leucine (WL) and tryptophan–isoleucine (WI) displayed DPP-IV IC50 values <170μM. The NF and LC–MS strategies employed herein represent a new approach for the targeted identification of short peptides within bioactive food protein hydrolysates.

Analysis of paralytic shellfish toxins, potential chemical threat agents, in food using hydrophilic interaction liquid chromatography–mass spectrometry

A novel method for determining paralytic shellfish toxin (PST) profiles in food was developed using a combination of silica and strong cation exchange (SCX) solid phase extraction (SPE) coupled to hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). Besides the risk for natural contamination of seafood and drinking water, PSTs also pose potent threats through intentional contamination of food, due to their high toxicity and the wide distributions of toxin-producing algae. The new preparation method aim to maintain the samples’ original toxin profiles by avoiding conditions known to induce interconversion or degradation of the PSTs. The method was evaluated for PST extraction from water, milk, orange juice, apple purée, baby food, and blue mussels (Mytilus edulis). The extracts were found to produce reproducible retention times in HILIC–MS/MS analysis. When an authentic toxic mussel sample was analyzed using the novel method, saxitoxin and gonyautoxin-3 were identified, in agreement with data acquired using the Lawrence pre-column oxidation high-performance liquid chromatography–fluorescence detection (HPLC–FLD) method. Overall recoveries of the PSTs from tested foods by the novel method ranged from 36% to 111%.

Simultaneous determination of residues of dipyrone metabolites in goat tissues by hydrophilic interaction liquid chromatography tandem mass spectrometry

A reliable LC–MS/MS method with high sensitivity was developed and validated for the determination of dipyrone (DIP) metabolites in goat muscle, fat, liver, and kidney samples. Analytes were extracted using acetonitrile mixed with ammonia solution. After dehydration and evaporation to dryness, extracts were purified using an Oasis MAX cartridge. Chromatographic separation was performed on a hydrophilic interaction liquid chromatography column. The analytes were then detected using triple-quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration plots were constructed using matrix-matched standards and showed good linearity. Limits of quantification for 4-methylaminoantipyrine (MAA), 4-formylaminoantipyrine (FAA), and 4-acetylaminoantipyrone (AAA) ranged from 0.4μgkg−1 to 6μgkg−1, while those for 4-aminoantipyrone (AA) ranged from 10μgkg−1 to 125μgkg−1 in all tissues. The developed method was successfully applied in the determination of DIP metabolite residues in actual goat tissues.

Computational analysis and ratiometric comparison approaches aimed to assist column selection in hydrophilic interaction liquid chromatography–tandem mass spectrometry targeted metabolomics

In the present work two different approaches, a semi-quantitative and a Derringer function approach, were developed to assist column selection for method development in targeted metabolomics. These approaches were applied in the performance assessment of three HILIC columns with different chemistries (an amide, a diol and a zwitterionic phase). This was the first step for the development of a HILIC UPLC–MS/MS method that should be capable to analyze a large number of polar metabolites. Two gradient elution profiles and two mobile phase pH values were tested for the analysis of multi-analyte mixtures. Acquired chromatographic data were firstly treated by a ratiometric, “semi-quantitative” approach which quantifies various overall analysis parameters (e.g. the percent of detected compounds, retentivity and resolved critical pairs). These parameters were used to assess chromatographic performance in a rather conventional/traditional and cumbersome/labor-intensive way. Secondly, a comprehensive and automated comparison of the three columns was performed by monitoring several well-known chromatographic parameters (peak width, resolution, tailing factor, etc.) using a lab-built programming script which calculates overall desirability utilizing Derringer functions. Derringer functions exhibit the advantage that column performance is ultimately expressed in an objective single and quantitative value which can be easily interpreted. In summary, results show that each column exhibits unique strengths in metabolic profiling of polar compounds. The applied methodology proved useful for the selection of the most effective chromatographic system during method development for LC–MS/MS targeted metabolomics, while it could further assist in the selection of chromatographic conditions for the development of multi-analyte methods.

Toxin profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese coast, as determined by liquid chromatography tandem mass spectrometry.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1–4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry Methylphosphonic Acid Determination in Water Samples after Derivatization with p-Bromophenacyl Bromide

Methylphosphonic acid is the most stable hydrolysis product of organophosphate nerve agents, which persist in environmental samples for a long time. A highly sensitive method of methylphosphonic acid detection in environmental waters has been developed and validated. Derivatization process and stability of p-bromophenacyl bromide methylphosphonic acid derivate were studied. Derivate was stable for 2 days and water content no greater than 7 % did not influence the reaction yield. The analyte concentration step in a rotary evaporator was employed prior to analysis. Detection of the pre-column methylphosphonic acid derivatization product was carried out using tandem mass spectrometry with electrospray ionization after hydrophilic interaction liquid chromatography separation. Evaporation recovery of 90 % was obtained. The intraday and interday repeatability were 6 and 25 %, respectively. Matrix effect influence was not observed. The hydrophilic interaction chromatography method results in excellent peak shape at adequate retention time. Developed approach successfully combines relatively fast derivatization and sample preparation with hydrophilic interaction chromatography and tandem mass spectrometry which allows to achieve the lowest limit of detection (0.1 ng mL−1) for methylphosphonic acid in spiked environmental waters in comparison with other HPLC techniques.

Heterogeneity of peptide adducts with carbonylated lipid peroxidation products.

Highly reactive lipid peroxidation-derived carbonyls (oxoLPP) modify protein nucleophiles via Michael addition or Schiff base formation. Once formed, Michael adducts can be further stabilized via cyclic hemiacetals with or without loss of water. Depending on the mechanism of their formation, peptide-oxoLPP can carry aldehyde or keto groups and thus be a part of the total protein carbonylation level. If a carbonyl function is lost during consecutive reactions, the oxoLPP-peptide adducts will not be detected using the common carbonyl labeling protocols. Because of the differences in adduct stabilities, it is possible to address the heterogeneity of peptide/protein-oxoLPP adducts by careful evaluation of tandem mass spectra of modified peptides. Here, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of lysine, cysteine and histidine containing model peptides co-incubated with oxidized 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine to characterize the collision-induced dissociation behavior of peptide-carbonyl adducts. Numerous modifications were detected based on the analysis of tandem mass spectra, including Schiff bases on lysine (two), Michael adducts on lysine (six), cysteine (eleven) and histidine (two), as well as 4-hydroxy-2-aldehydes derived dehydrated cyclic hemiacetals on cysteine (five) and histidine (one). Additionally, cysteine and histidine side chains were modified by lipid-bound aldehydes as Michael adducts and dehydrated hemiacetals. The tandem mass spectra revealed collision-induced dissociation characteristics specific for each class of oxoLPP-peptide adducts.

An integrated metabolomics workflow for the quantification of sulfur pathway intermediates employing thiol protection with N-ethyl maleimide and hydrophilic interaction liquid chromatography tandem mass spectrometry.

The sulfur metabolic pathway is involved in basic modes of cellular metabolism, including methylation, cell division, respiratory oscillations and stress responses. Hence, the implicated high reactivity of the sulfur pathway intermediates entails challenges for their quantitative analysis. In particular the unwanted oxidation of the thiol group-containing metabolites glutathione, cysteine, homocysteine, γ-glutamyl cysteine and cysteinyl glycine must be prevented in order to obtain accurate snapshots of this important part of cellular metabolism. Suitable analytical methodologies are therefore needed to support studies of drug metabolism and metabolic engineering. In this work, a novel sample preparation strategy targeting thiolic metabolites was established by implementing thiol group protection with N-ethyl maleimide using a cold methanol metabolite extraction procedure. It was shown that N-ethyl maleimide derivatization is compatible with typical metabolite extraction procedures and also allowed for the stabilization of the instable thiolic metabolites in a fully (13)C-labeled yeast cell extract. The stable isotope labeled metabolite analogs could be used for internal standardization to achieve metabolite quantification with high precision. Furthermore, a dedicated hydrophilic interaction liquid chromatography tandem mass spectrometry method for the separation of sulfur metabolic pathway intermediates using a sub-2 μm particle size stationary phase was developed. Coupled with tandem mass spectrometry, the presented methodology proved to be robust, and sensitive (absolute detection limits in the low femtomole range), and allowed for the quantification of cysteine, cysteinyl glycine, cystathionine, cystine, glutamic acid, glutamyl cysteine, reduced glutathione, glutathione disulfide, homocysteine, methionine, S-adenosyl homocysteine and serine in a human ovarian carcinoma cell model.

Application of Carboxymethyl Chitosan on Adsorbent Detoxification of Paralytic Shellfish Poisoning Toxins from &lt;i&gt;Chlamys nobilis&lt;/i&gt;

This study investigates the effectiveness of adding carboxymethyl chitosan during steam cooking as an adsorbent for detoxifying paralytic shellfish poisoning toxins (PSP toxins) in the scallop Chlamys nobilis. Toxin analysis using a mouse bioassay test and hydrophilic interaction liquid chromatography–tandem mass spectrometry method (LC–MS) showed that most of the PSP toxins (&gt;80%) were contained in the visceral compartment of the raw scallops. Overall, 82.2Mu/100g of PSP toxins were released from scallop tissues during steaming. The toxicity of the soup steamed with 0.1%, 0.3%, 0.6%, 1.0% and 1.5% carboxymethyl chitosan decreased the toxin content by 35.6%, 44%, 48.4%, 53.3% and 56.9% (p&lt;0.005), respectively. The relative PSP toxin content in the raw adductor muscle was comparable to those after steaming or steaming with carboxymethyl chitosan (p &gt; 0.05).The PSP toxin concentrations in the adductor muscles, gills + mantle and visceral compartments steamed with 0.1%, 0.3%, 0.6%, 1% and 1.5% carboxymethyl chitosan were not significantly different from those in the corresponding raw samples. The LC–MS analysis showed that the adsorbance of carboxymethyl chitosan for each of the PSP toxins was positive correlation with concentration, although the uptake efficiency of each toxin was different. The reduction in toxin content of all analyzed toxins reached 45.6%, 30.2%, 44.5%, 55.9% and 37.6% under the corresponding carboxymethyl chitosan concentrationsis.

Evaluation of different hydrophilic stationary phases for the simultaneous determination of iminosugars and other low molecular weight carbohydrates in vegetable extracts by liquid chromatography tandem mass spectrometry

Iminosugars are considered potential drug candidates for the treatment of several diseases, mainly as a result of their α-glycosidase inhibition properties. A method by hydrophilic interaction liquid chromatography tandem mass spectrometry has been optimized for the first time for the simultaneous determination of complex mixtures of bioactive iminosugars and other low molecular weight carbohydrates (LMWC) in vegetable extracts. Three hydrophilic stationary phases (sulfoalkylbetaine zwitterionic, polyhydroxyethyl aspartamide and ethylene bridge hybrid (BEH) with trifunctionally bonded amide) were compared under both basic and acidic conditions. The best sensitivity (limits of detection between 0.025 and 0.28ngmL−1) and overall chromatographic performance in terms of resolution, peak width and analysis time were obtained with the BEH amide column using 0.1% ammonium hydroxide as a mobile phase additive. The optimized method was applied to the analysis of extracts of hyacinth bulbs, buckwheat seeds and mulberry leaves. Iminosugar and other LMWC structures were tentatively assigned by their high resolution daughter ions mass spectra. Several iminosugars such as glycosyl-fagomine in mulberry extract were also described for the first time. Among the extracts analysed, mulberry showed the widest diversity of iminosugars, whereas the highest content of them was found in hyacinth bulb (2.5mgg−1) followed by mulberry (1.95 mgg−1).

An evaluation of sucrose as a possible contaminant in e-liquids for electronic cigarettes by hydrophilic interaction liquid chromatography–tandem mass spectrometry

The influence of sucrose combustion products on smoking and nicotine addiction is still controversial because the presence of the sucrose may be treated as a source of aldehydes and organic acids. In e-liquids used as refills for electronic cigarettes, which are made primarily of poly(propylene glycol), glycerine and ethanol, sucrose may be present at trace levels, and its impact on mainstream smoke formation, and hence on human health and smoking/nicotine addiction is unknown. An analytical method was developed where high-performance liquid chromatography in hydrophilic interaction liquid chromatography mode and tandem mass spectrometry were used for fast and simple determination of sucrose and other saccharides in e-liquids for electronic cigarettes. Minimal effort was required in the sample preparation step, and satisfactory results were obtained, and the sample matrix had an insignificant impact. The chromatographic separation was done using an Ascentis Express OH5 column (150 mm × 2.1 mm, 2.7 μm). The coefficients of variation for within-day precision for three concentrations were 2.4 %, 1.6 % and 2.3 %, and the between-day coefficients of variation for a single concentration were 2.1 %, 2.5 % and 1.7 % measured on the next 3 days. The detection limit was 0.73 μg/g, and the sucrose content in e-liquids ranged from 0.76 to 72.93 μg/g among 37 samples. Moreover, with the method presented it is possible to determine the presence of other saccharides such as fructose, glucose, maltose and lactose. However, only sucrose was found in all samples of e-liquids. The proposed method is rapid, simple and reliable in terms of high-performance liquid chromatography coupled with tandem mass spectrometry.

PEP Search in MyCompoundID: Detection and Identification of Dipeptides and Tripeptides Using Dimethyl Labeling and Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry

Small peptides, such as dipeptides and tripeptides, are naturally present in many biological samples (e.g., human biofluids and cell extracts). They have attracted great attention in many research fields because of their important biological functions as well as potential roles as disease biomarkers. Tandem mass spectrometry (MS/MS) can be used to profile these small peptides. However, the type and number of fragment ions generated in MS/MS are often limited for unambiguous identification. Herein we report a novel database-search strategy based on the use of MS/MS spectra of both unlabeled and dimethyl labeled peptides to identify and confirm amino acid sequences of di/tripeptides that are separated using hydrophilic interaction (HILIC) liquid chromatography (LC). To facilitate the di/tripeptide identification, a database consisting of all the predicted MS/MS spectra from 400 dipeptides and 8000 tripeptides was created, and a search tool, PEP Search, was developed and housed at the MyCompoundID website ( www.mycompoundid.org/PEP). To evaluate the identification specificity of this method, we used acid hydrolysis to degrade a standard protein, cytochrome c, to produce many di/tripeptides with known sequences for LC/MS/MS. The resultant MS/MS spectra were searched against the database to generate a list of matches which were compared to the known sequences. We correctly identified the di/tripeptides in the protein hydrolysate. We then applied this method to detect and identify di/tripeptides naturally present in human urine samples with high confidence. We envisage the use of this method as a complementary tool to various LC/MS techniques currently available for small molecule or metabolome profiling with an added benefit of covering all di/tripeptide chemical space.

Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometric Determination of Creatinine in Human Urine

The ability to measure creatinine with good accuracy is essential as its urinary concentration is used to quantify other analytes in urinalysis. In this study, an assay for the determination of creatinine in human urine by hydrophilic interaction liquid chromatography and electrospray ionization tandem mass spectrometry was developed and validated. Hydrophilic interaction chromatography provided retention of creatinine well away from the early eluting impurities that can reduce sensitivity by suppression of the mass spectrometry signal. Ion suppression effects were further eliminated by using a co-eluting, stable-isotope labeled creatinine as the internal standard. The resultant limit of detection was 1.5 picomoles, requiring a sample size of less than 10 µL of urine. The use of tandem mass spectrometry with a stable isotope labeled internal standard provided high specificity, accuracy, and precision for this assay resulting a recovery of 93.9% and a repeatability standard deviation of 3.78%.

Synthesis of molecularly imprinted polymer sorbents and application for the determination of aminoglycosides antibiotics in honey

Aminoglycoside antibiotic (AA)-selective molecularly imprinted polymer (MIP) sorbent was synthesized by polymerization of methacrylic acid and ethylene glycol dimethacrylate in the presence of streptomycin as template molecule. The MIP sorbent was in detail characterized and its performance was evaluated by selecting four model AAs including streptomycin, gentamicin, spectinomycin and dihydrostreptomycin. Relative to non-imprinted polymer (NIP), the MIPs exhibited much higher recognizable capacity and specificity to the AAs and negligible adsorption for non-AAs as well. In combination of hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS), the MIP sorbent-based solid phase extraction (SPE) was able to effectively determine the residue of four model AAs in honey samples with recovery ranging from 90% to 110%. The limit of detection (and the limit of quantitation) of streptomycin, gentamicin, spectinomycin and dihydrostreptomycin was 4.5 (15.0), 2.4 (8.0), 6.0 (20.0) and 1.8 (6.0)μg/kg, respectively. Relative standard deviations of intraday and inter-day assay under three spiked levels were 4.4–12.0% and 6.8–14.6%, respectively.

Simultaneous determination of β-hydroxybutyrate and β-hydroxy-β-methylbutyrate in human whole blood using hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry

ObjectivesFor the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) was developed, which does not require chemical modification of the analytes. Design and methodsSamples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis. ResultsSeparation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10–20μM BHB and 1μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10–1000μM BHB and 1–20μM HMB. The lower limits of quantification were estimated to be 3μM for BHB and 0.4μM for HMB. ConclusionsA simple and highly sensitive and selective HILIC–MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.

Determination of ethyl glucuronide in human hair by hydrophilic interaction liquid chromatography–tandem mass spectrometry

Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been utilized as a marker for alcohol intake. This study presents development, validation and application of a new hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method for the analysis of EtG in human hair samples. The linearity was assessed in the range of 5–2000 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.05 pg/mg and 0.18 pg/mg in hair, respectively. Differently from the extraction procedures in the literature, a fast and simple liquid–liquid method was used and highest recoveries and cleanest extracts were obtained.The method was successfully applied to 30 human hair samples which were taken from those who state they consume alcohol. EtG concentrations in the hair samples of alcohol users participated in this study, ranged between 1.34 and 82.73 pg/mg. From the concentration of EtG in hair strands 20 of the 30 subjects can be considered regular moderate drinkers.

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma

A new, sensitive and fast method for the simultaneous determination of pyrazinamide, isoniazid, streptomycin, ethambutol, and rifampicin in human plasma was developed and validated. The method required only 100 μL of plasma and one step for sample preparation by protein precipitation. The drugs were separated by using a hydrophilic interaction liquid chromatography (HILIC) column. The mobile phase was methanol and water (0.1% formic acid and 5 mM ammonium acetate, pH 3.0 ± 0.1) in a ratio of 65:35 (v/v), which was eluted at an isocratic flow rate of 0.5 mL/min. Tandem mass spectrometry was performed with a triple-quadrupole tandem mass spectrometer. By use of the HILIC column, the detection was free of ion-pair reagents in the mobile phase, with no significant matrix effects. The total run time was less than 2 min for each sample. The method was validated by evaluating its selectivity, sensitivity, linearity, accuracy, and precision according to US Food and Drug Administration guidelines. The lower limit of quantification was 4.0 ng/mL for pyrazinamide, isoniazid, and rifampicin, 0.5 ng/mL for ethambutol, and 10.0 ng/mL for streptomycin. The intraday precision and interday precision were less than 9%, with the accuracy ranging between -9.3 and 7.3%. The method was successfully applied to therapeutic drug monitoring of 33 patients with tuberculosis after administration of standard antituberculosis drugs. The method has been proved to meet the high-throughput requirements in therapeutic drug monitoring.