Simultaneous determination of trimethylamine and trimethylamine N‐oxide in mouse plasma samples by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

A method was developed that applies hydrophilic interaction liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode to separate and accurately quantify trimethylamine and trimethylamine N‐oxide in a single chromatographic run. This was achieved by converting trimethylamine to ethyl betaine, which is less volatile and hence results in greatly improved quantitation. Ethyl betaine also gives a similar response to trimethylamine N‐oxide using positive‐ion electrospray ionization mass spectrometry. It is readily separated from trimethylamine N‐oxide by hydrophilic liquid chromatography in a 5 min run and with improved peak shape compared to underivatized trimethylamine. Validation of the method yielded a limit of detection (S/N ≥ 3) of 0.5 ng/mL for trimethylamine and 0.25 ng/mL for trimethylamine N‐oxide. Method accuracies of 91.4–105.3% with precisions of 0.4–5.5% were obtained for standard mixtures over the range of 2.5–500 ng/mL. Recoveries measured for the extraction of trimethylamine and trimethylamine N‐oxide spikes into mouse plasma were both >90%. The method, which simultaneously measures trimethylamine and trimethylamine N‐oxide, was successfully applied to mouse plasma samples and could be adapted for use with other biological fluids.     

Determination of gabapentin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry

A rapid, sensitive and selective method for the determination of gabapentin in human plasma was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS). The devised method involved protein precipitation with acetonitrile followed by separation on an Atlantis HILIC silica column using an acetonitrile/ammonium formate mobile phase (100 mM, pH 3.0) (85:15, v/v). Analytes were detected using an electrospray ionization mass spectrometer in the multiple-reaction monitoring mode. The standard curve was linear (r = 1.000) over the concentration range of 50.0-10000 ng/mL. The lower limit of quantification for gabapentin was 50.0 ng/mL (ca. 20 pg gabapentin) using a 10-microL plasma sample. The coefficients of variation and relative errors for intra- and inter-assay at four QC levels (i.e., 50.0, 125, 750, and 7500 ng/mL) were 4.7 to 9.4% and -4.1 to 1.6%, respectively. Absolute and relative matrix effects for gabapentin and metformin were practically absent. Gabapentin and metformin recoveries were 98.5% and 99.0%, respectively. This method was successfully applied to a bioequivalence study of gabapentin in humans.     

Quantification of doxazosin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry

Hydrophilic interaction LC with MS/MS (HILIC-MS/MS) was described as a rapid, sensitive, and selective method for the quantification of doxazosin in human plasma. Doxazosin and cisapride (internal standard) were extracted from human plasma with ethyl acetate at alkaline pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of ACN/ammonium formate (100 mM, pH 4.5) (93:7 v/v). The analytes were detected using an ESI MS/MS in the selective-reaction-monitoring mode. The standard curve was linear (r = 0.9994) over the concentration range of 0.2-50 ng/mL. The LOQ for doxazosin was 0.2 ng/mL using 100 microL plasma sample. The CV and relative error for intra- and interassay at four QC levels were 3.7-8.7% and 0.0-9.8%, respectively. The matrix effect for doxazosin and cisapride were practically absent. The recoveries of doxazosin and cisapride were 67.4 and 61.7%, respectively. This method was successfully applied to the pharmacokinetic study of doxazosin in humans.     

Hydrophilic interaction liquid chromatography/tandem mass spectrometry for the simultaneous determination of dasatinib,imatinib and nilotinib in mouse plasma

Hydrophilic interaction liquid chromatography (HILIC) interfaced with atmospheric pressure ionization (API) sources and a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of dasatinib, imatinib and nilotinib in mouse plasma samples. The retention profiles of all analytes on several silica stationary phases under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of all analytes in positive ion mode were investigated. The applicability of the proposed HILIC/MS/MS approach following a protein precipitation procedure for the quantitative determination of dasatinib, imatinib and nilotinib at low nano‐mole levels was examined with respect to assay specificity and linearity. The analytical results obtained by various HILIC/MS/MS approaches were found to be in good agreement with those obtained by reversed‐phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) methods in terms of assay sample throughputs, sensitivity and accuracy. Furthermore, the potential of matrix ionization suppression on the proposed HILIC/MS/MS systems was investigated using the post‐column infusion technique. Copyright © 2009 John Wiley & Sons, Ltd.     

Ultra‐high performance liquid chromatography tandem mass spectrometry for the detection of durum wheat contamination or adulteration

In this work, an ultra‐performance liquid chromatography electrospray ionization (UPLC‐ESI)‐MS/MS methodology based on multiple reaction monitoring (MRM) for the selective and sensitive detection and quantification of durum wheat adulteration has been developed and fully validated. The targeted analysis was performed by monitoring specific transitions at m/z 543.7 > 657.4 and m/z 543.7 > 299.2 of a species‐specific marker derived from a tryptic peptide of puroindoline a (Pin‐a), a cysteine‐rich protein selectively present only in common wheat. In addition, two transitions at m/z 500.4 > 725.4 and m/z 500.4 > 561.9 of a reference peptide belonging to purothionin A‐1, present in both species, were also monitored. The calibration curves obtained on binary mixtures with known percentages of common/durum wheat flours showed linearity (coefficient of regression, r ≥ 0.99) over concentrations that ranged between 80 and 1%. The limit of detection (LOD) and limit of quantification (LOQ) for the Pin‐a marker in wheat flours were 0.01 and 0.03%, respectively. The identified Pin‐a marker was also found to be highly diagnostic for the quantification of common wheat in raw materials (kernels) and processed products (pasta), thus offering new opportunities to assess food authenticity. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of gambogenic acid in dog plasma and its application to a pharmacokinetic study

A highly sensitive and rapid ultra‐high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of gambogenic acid in dog plasma. Gambogic acid was used as an internal standard (IS). After a simple liquid–liquid extraction by ethyl acetate, the analyte and internal standard were separated on an Acquity BEH C₁₈ (100 × 2.1 mm, 1.7 µm; Waters ) column at a flow rate of 0.2 mL/min, using 0.1% formic acid–methanol (10:90, v/v) as mobile phase. Electrospray ionization source was applied and operated in the positive ion mode. Multiple reaction monitoring mode with the transitions m/z 631.3 → 507.3 and m/z 629.1 → 573.2 was used to quantify gambogenic acid and the internal standard, respectively. The calibration curves were linear in the range of 5–1000 ng/mL, with a coefficient of determination (r) of 0.999 and good calculated accuracy and precision. The low limit of quantification was 5 ng/mL. The intra‐and inter‐day precisions (relative standard deviations) were <15%. The methodology recoveries were more than 66.63%. This validated method was successfully applied to a pharmacokinetic study after intravenous injection administration of gambogenic acid in dogs at a dose of 1 mg/kg. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography/tandem mass spectrometry in high‐throughput detection,quantification and confirmation of anabolic steroids in equine plasma

An ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast‐throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid‐liquid extraction using methyl tert‐butyl ether, separated on a 1.9 µm C₁₈ reversed‐phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screening results. Full product ion spectra of excellent quality for AAS, at 100 pg/0.5 mL in plasma, devoid of interfering spectra from impurities in plasma, were obtained. To our knowledge, this is the first report on the acquisition of full product ion spectra at such a low analyte concentration and plasma volume using a triple quadrupole instrument. In addition to product ion intensity ratios obtained from three SRM scans for identifying AAS in equine plasma, full product ion spectra were used as supporting evidence for confirmation. For quantification, deuterium‐labeled testosterone and stanozolol were used as internal standards (ISs). The limits of detection, quantification and confirmation were 6.25–12.5 pg/0.5 mL, 25 pg/0.5 mL and 50–100 pg/0.5 mL, respectively. There was no significant matrix effect on the analysis of all eight AAS. Intra‐day precision and accuracy were 2–15% and 91–107%, respectively. Inter‐day precision and accuracy were 1–21% and 94–110%, respectively. Total analysis time was 5 min. To date, the method has been successfully used in the analysis of >12 000 samples for AAS in plasma samples from racehorses competing in the State of Pennsylvania. The method is fast, selective, reproducible, and reliable. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of amprolium by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We present a fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core™ column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an “extract and shoot” strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 μg L⁻¹) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC–MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 μg kg⁻¹).     

Validation of a hydrophilic interaction ultra‐performance liquid chromatography–tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine‐¹³C, ¹⁵N₂ used as an internal standard, were extracted from human plasma treated with THU using a 96‐well Hybrid SPE‐Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2′‐2′‐difluoro‐2′‐deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra‐batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter‐batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC‐MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography tandem mass spectrometry for the rapid,simultaneous analysis of ziprasidone and its impurities

The separation and characterization of the unknown degradation product of second‐generation antipsychotic drug ziprasidone are essential for defining the genotoxic potential of the compound. The aim of this study was to develop a simple UHPLC method coupled with tandem mass spectrometry (MS/MS) for chemical characterization of an unknown degradant, and the separation and quantification of ziprasidone and its five main impurities (I–V) in the raw material and pharmaceuticals. Chromatographic conditions were optimized by experimental design. The MS/MS fragmentation conditions were optimized individually for each compound in order to obtain both specific fragments and high signal intensity. A rapid and sensitive UHPLC–MS/MS method was developed. All seven analytes were eluted within the 7 min run time. The best separation was obtained on the Acquity UPLC BEH C₁₈ (50 × 2.1 mm × 1.7 μm) column in gradient mode with ammonium‐formate buffer (10 mm ; pH 4.7) and acetonitrile as mobile phase, with the flow rate of 0.3 mL min^?1 and at the column temperature of 30°C. The new UHPLC–MS/MS method was fully validated and all validation parameters were confirmed. The fragmentation pathways and chemical characterization of an unknown degradant were proposed and it was confirmed that there are no structural alerts concerning genotoxicity.     

Ultra‐performance liquid chromatography–tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women

A highly sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid‐phase extraction, while E2 was first separated by liquid–liquid extraction with methyl tert‐butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC‐d5 and E2‐d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90–99%. The analytes were separated on UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (R_s) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra‐ and inter‐batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film‐coated tablets under fasting.     

A liquid chromatography/tandem mass spectrometry method for determination of aristolochic acid‐I in rat plasma

A sensitive, rapid and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the determination of aristolochic acid‐I (AA‐I) in rat plasma. Finasteride was used as the internal standard (IS). The analyte was separated on a Zorbax Eclipse XDB‐C₁₈ column by isocratic elution with methanol‐10 mM ammonium acetate (75:25, v/v, pH = 7.3) at a flow rate of 0.25 mL/min, and analyzed by mass spectrometry in positive multiple reaction monitoring mode. The precursor‐to‐product ion transitions of m/z 359.0 → 298.2 and m/z 373.1 → 305.2 were used to detect AA‐I and IS, respectively. Good linearity was achieved over a range of 0.4–600 ng/mL. Intra‐ and inter‐day precisions measured as relative standard deviation were less than 13.5%, and accuracy ranged from 94.2 to 97.5%. The developed method was successfully applied in the pharmacokinetic study of AA‐I in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of cyadox and its metabolites in plasma by high‐performance liquid chromatography tandem mass spectrometry

Cyadox is a novel antimicrobial growth‐promoter of the quinoxalines. For food safety and pharmacokinetic studies, a convenient, sensitive and reproducible LC‐ESI‐MS/MS method was developed for the simultaneous determination of cyadox and its major metabolites, quinoxaline‐2‐carboxylic acid, 1,4‐bisdesoxycyadox, cyadox‐1‐monoxide and cyadox‐4‐monoxide in chicken plasma. Plasma sample was subjected to a simple deproteinisation with acetonitrile. Analysis was performed on a C₁₈ column by detection with mass spectrometry in multiple reaction monitoring mode. A gradient elution program with 0.2% formic acid, methanol and acetonitrile was performed at a flow rate of 0.2 mL/min. The decision limits (CCαs) of five analytes in plasma ranged from 1.0 to 4.0 μg/L, and the detection capabilities (CCβs) were <10 μg/L. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of five analytes were between 87.4 and 93.9% in plasma at the spiked levels of 5 (10)–200 μg/L with the relative standard deviations <10% for each analyte. The developed method demonstrated a satisfactory applicability in real plasma samples.     

Quantification of total and free carnitine in human plasma by hydrophilic interaction liquid chromatography tandem mass spectrometry

Carnitine is an endogenous quaternary amine whose primary function is to shuttle long chain fatty acids to the mitochondrial matrix, where they subsequently undergo beta oxidation. Accurate quantification of total and free carnitine is essential for the accurate diagnosis of a number of inborn errors of metabolism, including disorders of fatty acid oxidation as well as various organic acidurias. Early methods for carnitine measurement were enzyme based. Recently, liquid chromatography tandem mass spectrometry has become the method of choice for carnitine measurement. Typically, carnitine is derivitized to from a butyl ester, thus improving its ionization and retention characteristics. A potential problem with this approach is that the acidic conditions used to carry out the reaction may hydrolyze other acyl esters, resulting in ex-vivo artifacts. Consequently, we developed a hydrophobic interaction chromatography (HILIC) tandem mass spectrometry method for the quantification of carnitine. The use of HILIC allows for the derivitization step to be circumvented, while still allowing for favorable chromatographic performance. The method was shown to be accurate, precise, and robust.     

Fast quantification of endogenous carbohydrates in plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Endogenous carbohydrates in biosamples are frequently highlighted as the most differential metabolites in many metabolomics studies. A simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, mass spectrometry conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, matrix effect, and stability of the method were verified. The results indicated that a methanol/acetonitrile (50:50, v/v) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at –20°C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, recovery, precision, matrix effect, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in terms of sensitivity, high throughput, and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research.     

Ultra‐performance liquid chromatography‐tandem mass spectrometry method for the determination of febuxostat in dog plasma and its application to a pharmacokinetic study

A rapid, sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the determination of febuxostat in dog plasma. Using paclitaxel as an internal standard (IS), a simple liquid–liquid extraction method with ethyl acetate was adopted for plasma sample pretreatment. Separation was carried out on an Acquity UPLC BEH C₁₈ column with a mobile phase consisting of acetonitrile and water (containing 0.2% formic acid). The assay was linear in the concentration ranged from 5 to 5000 ng/mL with a lower limit of quantification of 5 ng/mL for febuxostat. The single run analysis was as short as 2.0 min. Finally, the developed method was successfully applied to the pharmacokinetic study of febuxostat tablets following oral administration at a single dose of 40 mg in beagle dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d₇-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.     

High-performance liquid chromatography/tandem mass spectrometry method for the determination of arbidol in human plasma

An HPLC/MS/MS method for the determination of arbidol in human plasma was developed. Arbidol and internal standard (loratadine) were extracted from alkaline plasma with tert-butyl methyl ether and analyzed on a Zorbax SB C18 column (30 x 2.1 mm id, 3.5 microm particle size). The detection was by monitoring arbidol at m/z 479.1 --> 434.1 and the internal standard at m/z 383.2 --> 337.2. The method was validated according to U.S. Food and Drug Administration guidelines. The calibration curve was linear over the range of 0.5-500 ng/mL using a 100 microL sample volume. The intraday and interday precisions were less than 6.5%, and acceptable values were obtained for accuracy, recovery, and sensitivity. The developed method was selective, simple, sensitive, and easily applicable.     

A new hydrophilic interaction liquid chromatography tandem mass spectrometry method for the simultaneous determination of seven biogenic amines in cheese

An HILIC-PI APCI MS/MS method is developed for the determination of seven biogenic amines (cadaverine, histamine, putrescine, spermidine, spermine, tryptamine and tyramine) in cheese. Their presence and relative amounts give useful information about freshness, level of maturing, quality of storage and cheese typicization. The major drawback in the analysis is represented by the relevant matrix effect and the general unbalanced concentrations of the different amines in cheese. The method proposed represents an improvement with respect to an HPLC-MS/MS method already developed in this laboratory. The new method permits better sensitivities it makes use of a Waters Atlantis HILIC (150.0 mm x 2.1 mm i.d., 3 microm) stationary phase and of a mobile phase of acetonitrile and ammonium formate 50.0 mM in ultrapure water brought to pH 4.00 for formic acid, flowing under gradient conditions. The chromatographic system is interfaced with a 3200QTrap LC-MS/MS system (Applied Biosystem, Foster City, CA, USA) by a Turbo V interface equipped with Heated Nebuliser (APCI) and Turbo Ion Spray (TIS) probes. LOQ values lower than 10 microg L(-1) are obtained. The method is applied in the analysis of Castelmagno cheese.