Hydrophilic Interaction Liquid Chromatography-tandem Mass Research Articles

Rapid and sensitive HILIC–MS/MS analysis of carnitine and acetylcarnitine in biological fluids

Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.

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.

Simultaneous Determination of Trimethoprim and Diaveridine in Tissues of Chicken, Porcine, and Fish by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

A sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of trimethoprim and diaveridine in edible tissue samples (chicken and porcine muscle; liver, kidney, and fat tissues as well as fish muscle) was developed and validated in this study. Trimethoprim, diaveridine, and the internal standard trimethoprim-D3 were extracted from samples using acetonitrile. The Oasis MCX Solid-Phase Extraction Cartridge was selected for cleaning up the extracts. Chromatographic separation was performed on a hydrophilic interaction liquid chromatography column with gradient elution. The analytes were determined using triple–quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. The relative recoveries from spiked samples ranged from 82.5 to 117.2 %, with the relative standard deviations generally being below 15.4 %. Limits of detection and quantification for analytes were within 0.3–1 and 1–2 μg kg−1, respectively. The proposed method was successfully applied to detect trimethoprim and diaveridine in real samples.

Erratum to: Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry

Erratum to: Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry

Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatographic-tandem mass spectrometric (HILIC-MS-MS) method for investigation of the in vivo metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen, in rabbit blood has been developed and validated. This method achieved excellent repeatability and accuracy. Recovery ranged from 76.9 to 116.3 % and precision (as RSD) between 0.53 and 6.52 %. Linearity was good for all compounds (R(2)>0.9990) and the limit of detection (LOD) ranged from 0.016 to 0.082 ng mL(-1). Pharmacokinetic analysis indicated that NNK was rapidly eliminated in vivo in rabbit blood and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite. The hydroxy acid, keto acid, and NNAL-N-oxide were also important metabolites in rabbit blood. It is probable that α-methylene hydroxylation was the major pathway of α-hydroxylation of NNK and NNAL in the rabbit.

Rapid quantitative analysis of carnitine and acylcarnitines by ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry

l-Carnitine and its acyl esters (acylcarnitines) play an important role in the metabolism of fatty acids. However, most of the present methods for the quantitative analysis of acylcarnitines have restrictions both in sample preparation and in chromatographic separation. Herein we present a validated method for determination of carnitine and eleven acylcarnitines in human serum and rat tissue biopsies by using ultra-high performance–hydrophilic interaction liquid chromatography–tandem mass spectrometry (UHP–HILIC–MS/MS). The procedure uses minimal sample preparation including only addition of organic solvent, labeled internal standard, incubation and centrifugation. The separation is performed without derivatization or addition of ion-pairing reagent within 7min on a hydrophilic interaction liquid chromatographic column with mass spectrometric detection. The method is linear in response over the concentration range from 20 to 600ng/ml for carnitine and acetylcarnitine and 5–200ng/ml for the other acylcarnitines, with correlation coefficients higher than 0.994. Recoveries were higher than 88% for most of the compounds. Limits of detection were 5ng/ml for carnitine and acetylcarnitine and approximately 0.5ng/ml for other acylcarnitines. The method was applied to the analysis of serum and tissue samples.

RRLC-MS/MS method for the quantitation of atractyloside in Fructus Xanthii (Xanthium sibiricum)

Fructus Xanthii (Xanthium sibiricum) is used as a traditional Chinese medicine (TCM) but contains the toxin atractyloside. Historically the bur's spines are thought to be a reservoir of the toxins and removal of these spines theoretically reduced toxicity. However, this has not been proven and, furthermore, the Chinese Pharmacopoeia does not provide a quantitative method to evaluate the potential toxicity of this herb. Thus, the objective of this study was to provide a quantitative method using hydrophilic interaction liquid chromatography-tandem mass spectrometry to determine atractyloside content and concentration of Fructus Xanthii (Xanthium sibiricum) for the first time. Additionally, this method was validated and applied to evaluate the distribution and concentration of the atractyloside of this TCM in different plant parts including the spines, shell, seed-coat and seed kernel. The results of this analytical methodology applied to 27 samples of Fructus Xanthii (Xanthium sibiricum) demonstrated that this process improved sensitivity and accuracy and reduced the time commitment to evaluate this herb and others which potentially contain atractyloside. Furthermore, this study demonstrated that atractyloside is mainly stored in the seed kernel and not the spines as the historical thought.

Determination of nitenpyram and its metabolites in agricultural products by using hydrophilic interaction liquid chromatography-tandem mass spectrometry

Determination of nitenpyram and its metabolites in agricultural products by using hydrophilic interaction liquid chromatography-tandem mass spectrometry

Determination of eight artificial sweeteners in wastewater by hydrophilic interaction liquid chromatography-tandem mass spectrometry

The development of a method for the simultaneous determination of eight artificial sweeteners, namely aspartame (ASP), alitame (ALI), neotame (NEO), acesulfame (ACS), saccharin (SAC), cyclamate (CYC), sucralose (SCL) and neohesperidin dihydrochalcone (NHDC), under hydrophilic interaction liquid chromatography (HILIC) conditions is presented herein. The ionization of all the compounds was achieved by negative electrospray. Two silica-based columns, a fully porous (XBridge HILIC) and a fused-core one (Kinetex HILIC), were compared. For HILIC optimization, the effect of the following parameters was studied: the mobile phase, column temperature, pH and ionic strength of the ammonium formate buffer. The optimized mobile phase was 5 mM ammonium formate buffer pH 3.5–methanol–acetonitrile (15/10/75 v/v), with a flow rate of 100 μL min−1 at 50 °C. The asymmetry factor of the peaks was lower on the Kinetex HILIC column than that on the XBridge HILIC. The predominant retention mechanism of all the analytes on silica columns seems to be partition to the aqueous layer. The method was validated in tap water and wastewater samples using Kinetex HILIC. Three deuterated internal standards were used, one for each category of sweeteners (dipeptides, sulfamates and sugars). The instrumental limits of detection ranged from 0.24 (SAC) to 4.4 (SCL) μg L−1. The recoveries in influent samples ranged from 39% (CYC) to 97% (NEO), while recoveries were better for the other two matrices. Inter-day precision (%RSD, n = 6) ranged from 4.2% (SAC) to 20% (ALI) in influent samples. The method was applied to influents and effluents from the wastewater treatment plant of Athens for seven consecutive days and high levels of sucralose and sulfamates were found.

Quantification of intact carboplatin in human plasma ultrafitrates using hydrophilic interaction liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Carboplatin is a platinum agent that is used for treatment of non-small-cell lung cancer and ovarian cancer. A sensitive and selective analytical method for the quantification of carboplatin in human plasma ultrafiltrates using liquid chromatography-tandem mass spectrometry was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin-d4 as an internal standard and were further diluted with acetonitrile. Chromatographic separation was performed on a Accucore HILIC (50mm×2.1mm i.d., 2.6μm) column using mobile phase (acetonitrile-water-acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. Detection was performed on electrospray ionization triple quadrupole tandem mass spectrometer using low-energy collision induced dissociation (CID-MS/MS) analysis operating in the selected reaction monitoring (SRM) scan mode. The lower limit of quantification for carboplatin was 0.025μg/mL. This method covered a linearity range of 0.025-50μg/mL. The intra-day precision and inter-day precision (R.S.D.) ranged from 1.5 to 4.3%, and the accuracy (R.E.) was within ±2.9%. The present method was applied to a clinical pharmacokinetic study of carboplatin in a cancer patient.

Direct and simultaneous quantitation of 5‐aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry

Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Validation of an LC–MS/MS method for the quantification of choline-related compounds and phospholipids in foods and tissues

A hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC LC–MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenized phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d3 and Cho-d3. Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6% to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using 31P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database.

A full validated hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the quantification of oxaliplatin in human plasma ultrafiltrates

Oxaliplatin is a platinum agent that is used for treatment of colorectal cancer. A sensitive and selective hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the quantification of oxaliplatin was developed. Human plasma ultrafiltrates were precipitated by acetonitrile containing carboplatin as an internal standard and further diluted with acetonitrile. Chromatographic separation of oxaliplatin and the internal standard was achieved with a column modified with phosphorylcholine and an isocratic mobile phase (acetonitrile/water/acetic acid=90:10:0.1, v/v/v) at the flow rate of 0.2mL/min. The lower limit of quantification for oxaliplatin was 25ng/mL. The linearity range of the method was from 25 to 5000ng/mL. The intra-day precision and inter-day precision (RSD) ranged from 0.8 to 6.1%, and the accuracy (RE) was within ±4.5%. The extraction recoveries from human plasma ultrafiltrates were 83.6–91.6%, and ion suppression caused by matrix components was 86.7–88.5% at three different levels, respectively. This method was applied to a clinical pharmacokinetic study of oxaliplatin in a cancer patient. The maximum concentration of colorectal cancer patient administered oxaliplatin was 1650ng/mL.

A HILIC–MS/MS method for the simultaneous determination of seven organic acids in rat urine as biomarkers of exposure to realgar

Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R2>0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.

Quantification of atractyloside in Fructus Xanthii using HILIC-ESI-MS/MS

Fructus Xanthii is thought to be toxic in traditional Chinese medicine. The bur is usually taken orally to treat certain diseases, as accordingly results in poisoning incidents happen occasionally in China. Atractyloside, a highly hydrophilic compound, is the main lethal component in Fructus Xanthii. Unfortunately, so far the quantitative method of this toxic compound is unavailable in Chinese Pharmacopoeia. In this study, a simple and rapid method using hydrophilic interaction liquid chromatography-tandem mass spectrometry has been developed to quantify the toxic component in Fructus Xanthii. The method was further validated from linearity, sensitivity, accuracy, and precision and extraction efficiency. As a result, the limits of detection and quantification were 0.0625 and 0.25ng/mL, respectively, and the accuracy ranged from 99.37 to 103.97% with relative standard deviation less than 4.86%. The established method was then used to determine the toxin contained in 28 batches of commercial herbal drugs, as showed that the method was satisfying for determination of atractyloside contained in these samples over a large content range of 0.19 to 80.75mg/g, which, together with time-saving, sensitive and accurate characteristics, makes this method very attractive for quantitative analysis of the toxic component in the crude drug and related Chinese medicine preparations.

Direct and simultaneous determination of 5‐aminolaevulinic acid and porphobilinogen in urine by hydrophilic interaction liquid chromatography–electrospray ionisation/tandem mass spectrometry

Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatisation steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive electrospray ionisation. Urine samples (N = 46) from active and latent mutation-confirmed acute hepatic porphyria patients and normal subjects (N = 45) were analysed and the results compared with those of a commercially available spectrophotometric method. The validated calibration range was 3-3000 µmol/L for ALA and 2-2000 µmol/L for PBG. For both analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was between 88 and 109%. The lower limit of quantitation was 0.1 μmol/L for both analytes. The calculated LC-MS/MS and spectrophotometric results from patient samples compared well [Pearson correlation (r²) of 0.99 and 0.95, for ALA and PBG, respectively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Analysis of ibotenic acid and muscimol in Amanita mushrooms by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We have established the most modern method for analysis of ibotenic acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an ibotenic acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for ibotenic acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic acid aqueous solution (A) and 0.5 % formic acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for ibotenic acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both ibotenic acid and muscimol with correlation coefficients not <0.99. Intraday and interday accuracy and precision were also generally satisfactory. Using the above new method, the concentrations of ibotenic acid and muscimol were actually measured for a mushroom (most probably Amanita ibotengutake) obtained from a poisoning case; they were 210 and 107 μg/g, respectively. The novel points of our method are no requirement for derivatization before LC–MS–MS analysis, the use of anion exchange solid-phase extraction, the use of hydrophilic interaction column for LC separation, and the use of acivicin as IS.

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the quantification of lipid-related extracellular metabolites in Saccharomyces cerevisiae

A highly sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150mm×4.6mm, 5μm) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r2≥0.995) over the range of 0.5–150nM, with the lower limit of quantitation validated at 0.5nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between −8.42 to 8.22% and −9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae.

Optimization of a solid phase extraction and hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the determination of metformin in dietary supplements and herbal medicines

Metformin is one of the most common adulterants found in anti-diabetic dietary supplements and herbal medicines. An analytical method based on hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry was developed and validated for the determination of metformin in dietary supplements and herbal medicines. Sample preparation involved solid phase extraction of the analyte using Plexa PCX cartridges, and cleanup by a primary–secondary amine (PSA) adsorbent for minimization of the matrix effect. Chromatographic separation was performed on a HILIC column using a mixture of 0.025mol/L ammonium formate (pH 3) and acetonitrile (18:82, v/v) as the mobile phase at flow rate of 0.25mL/min. The method was validated in four matrices (capsules, honeyed pills, tablets and oral solutions). The method had a good linear range (5–500ng/mL), and the overall precision and accuracy ranged from 2.2% to 9.9% and 4.6% to 11.3%, respectively.