Analysis of chlormequat and mepiquat by hydrophilic interaction chromatography coupled to tandem mass spectrometry in food samples

In this work a LC–MS/MS method for the determination of two quaternary ammonium growth regulators (chlormequat and mepiquat) in food is reported. The separation was based on hydrophilic interaction liquid chromatography (HILIC) without the use of ion-pair reagents. A gradient elution of acetonitrile and formic acid/ammonium formate buffer from 60 to 40% acetonitrile was enough to achieve a resolution >1.5 in less than 4.0 min. The HILIC system was coupled to a triple quadrupole mass spectrometer equipped with a heated electrospray probe (H-ESI) providing sub-pg LODs in SRM mode. A straightforward sample treatment (SPE C18 clean-up) was enough to provide MLODs at low ppb levels when analysing a range of food samples that covered different kinds of matrices such as fresh fruit, vegetables, fruit juices, baby food, bread, coffee and beer. Chlormequat was found in seven samples (0.8–126 ng/g) but mepiquat was only detected in bread and coffee samples (0.9–166 ng/g).     

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug–drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C₁₈ column and quantitated with a 16 min run in a triple‐quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC‐MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid determination of glyphosate, glufosinate, bialaphos, and their major metabolites in serum by liquid chromatography-tandem mass spectrometry using hydrophilic interaction chromatography

We developed a simple and rapid method for the simultaneous determination of phosphorus-containing amino acid herbicides (glyphosate, glufosinate, bialaphos) and their major metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA), in human serum. Serum samples were filtrated through an ultrafiltration membrane to remove proteins. The filtrate was then washed with chloroform, and injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Chromatographic separation was achieved on a hydrophilic interaction chromatography (HILIC) column. Determination of the target herbicides and metabolites was successfully carried out without derivatization or solid phase extraction (SPE) cartridge clean-up. The recoveries of these compounds, added to human serum at 0.2μg/mL, ranged from 94% to 108%, and the relative standard deviations (RSDs) were within 5.9%. The limits of detection (LODs) were 0.01μg/mL for MPPA, 0.02μg/mL for AMPA, 0.03μg/mL for both glyphosate and glufosinate, and 0.07μg/mL for bialaphos, respectively.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry for pharmacokinetic study

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC‐MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 μm) was used for hydrophilic‐based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor–product ion pairs for multiple‐reaction monitoring were m /z 409.1 → 391.0 for eurycomanone and m /z 449.1 → 303.0 for IS. The linear range was 2–120 ng/mL. The intra‐ and inter‐day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The C _max and AUC_0–t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.     

Simultaneous determination of N‐ethylpentylone,dopamine, 5‐hydroxytryptamine and their metabolites in rat brain microdialysis by liquid chromatography tandem mass spectrometry

N‐Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography–tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4‐dihydroxyphenylacetic acid, 3‐methoxytyramine and 5‐hydroxyindole‐3‐acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C₁₈ column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4–113.5%) and precision (relative standard deviation <15%) at three quality control levels. The limits of detection were 0.2 ng/mL for NEP and 0.2–50 nm for the others and good linearity was obtained. This study pioneered a method to integrate exogenous drugs and endogenous neurotransmitters as the drugs act on the same determination system, which means that this innovation can provide support for further study of the addictive effects of NEP or other synthetic cathinones on extracellular levels of dopamine and 5‐hydroxytryptamine.     

Comparison of hydrophilic interaction and reversed-phase liquid chromatography coupled with tandem mass spectrometric detection for the determination of three pharmaceuticals in human plasma

In the present study, hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) combined with tandem mass spectrometric detection (MS/MS) were evaluated and compared for the determination of donepezil, cetirizine and loratadine in human plasma, in terms of sensitivity and sample preparation procedure. A retention study for the above compounds of various polarities was performed, using both C(18) and silica columns, with several aqueous-organic mobile phase ratios, in order to investigate their retention mechanism profile under HILIC and RPLC. Both chromatographic conditions were compared for chromatographic analysis of plasma samples processed with a liquid-liquid extraction (LLE) method for donepezil determination, resulting in significantly higher sensitivity under HILIC. Furthermore, HILIC and RPLC were compared for direct injection, and novel methods including LLE, solid-phase extraction and protein precipitation protocols were developed. Direct injection technique significantly reduced sample preparation time, increasing at the same time method sensitivity. The current study contributes to broadening the range of analyzable compounds by HILIC-MS/MS to molecules of medium polarity.     

Simultaneous determination of sesquiterpene lactones isoalantolactone and alantolactone isomers in rat plasma by liquid chromatography with tandem mass spectrometry: Application to a pharmacokinetic study

A selective, sensitive, and accurate LC–MS/MS method for the simultaneous determination of isoalantolactone and alantolactone in rat plasma has been developed using psoralen as the internal standard. LC–MS/MS analysis was carried out on a Triple Quadrupole mass spectrometer using positive ion ESI and the selected reaction monitoring mode. The assays were linear in the range of 7.5–750 ng/mL for isoalantolactone and 5.5–550 ng/mL for alantolactone. The average recoveries in plasma samples both were better than 85%. The intra‐ and inter‐day precision and accuracy values were found to be within the assay variability criteria limits according to the US FDA guidelines. The method was successfully applied to pharmacokinetic studies of the two structural isomers after an intravenous injection of Inula helenium formulation to rats.     

Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: the determination of choline containing compounds in foods

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method using multiple scan modes was developed to separate and quantify 11 compounds and lipid classes including acetylcholine (AcCho), betaine (Bet), choline (Cho), glycerophosphocholine (GPC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphocholine (PCho) and sphingomyelin (SM). This includes all of the major choline-containing compounds found in foods. The method offers advantages over other LC methods since HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The LC-MS/MS method allows quantification of all choline-containing compounds in a single run. Tests of method suitability indicated linear ranges of approximately 0.25-25 μg/ml for PI and PE, 0.5-50 μg/ml for PC, 0.05-5 μg/ml for SM and LPC, 0.5-25 μg/ml for LPE, 0.02-5 μg/ml for Cho, and 0.08-8 μg/ml for Bet, respectively. Accuracies of 83-105% with precisions of 1.6-13.2% RSD were achieved for standards over a wide range of concentrations, demonstrating that this method will be suitable for food analysis. 8 polar lipid classes were found in a lipid extract of egg yolk and different species of the same class were differentiated based on their molecular weights and fragment ion information. PC and PE were found to be the most abundant lipid classes consisting of 71% and 18% of the total phospholipids in egg yolk.     

Practical liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of amitriptyline,nortriptyline and their hydroxy metabolites in human serum

Amitriptyline (AMI) has been in use for decades in treating depression and more recently for the management of neuropathic pain. A highly sensitive and specific LC–tandem mass spectrometry method was developed for simultaneous determination of AMI, its active metabolite nortriptyline (NOR) and their hydroxy‐metabolites in human serum, using deuterated AMI and NOR as internal standards. The isobaric E‐10‐hydroxyamitriptyline (E‐OH AMI), Z‐10‐hydroxyamitriptyline (Z‐OH AMI), E‐10‐hydroxynortriptyline (E‐OH NOR) and Z‐10‐hydroxynortriptyline (Z‐OH NOR), together with their parent compounds, were separated on an ACE C₁₈ column using a simple protein precipitation method, followed by dilution and analysis using positive electrospray ionisation with multiple reaction monitoring. The total run time was 6 min with elution of E‐OH AMI, E‐OH NOR, Z‐OH AMI, Z‐OH NOR, AMI (+ deuterated AMI) and NOR (+ deuterated NOR) at 1.21, 1.28, 1.66, 1.71, 2.50 and 2.59 min, respectively. The method was validated in human serum with a lower limit of quantitation of 0.5 ng/mL for all analytes. A linear response function was established for the range of concentrations 0.5–400 ng/mL (r² > .999). The practical assay was applied on samples from patients on AMI, genotyped for CYP2C19 and CYP2D6, to understand the influence of metaboliser status and concomitant medication on therapeutic drug monitoring.     

Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC^® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5 μm with an average pore diameter of 200 Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5 picomole (20 mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of two novel highly lipophilic anticancer drug candidates in rat plasma and tissues

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) method was developed and validated for determination of two highly lipophilic anticancer drug candidates, LG1980 and GH501, in rat plasma and tissues (liver, kidney and femur bones). LG1980 and GH501 were extracted from rat plasma and tissue homogenates using liquid–liquid extraction. The method provided a linear range of 1.0–200.0 ng/mL for GH501 in plasma and LG1980 in plasma and liver. For both analytes in other tissue homogenates the linear range was 2.0–400.0 ng/mL. The method was validated with precision within 15% relative standard deviation, accuracy within 15% relative error and a consistent recovery. This method has been successfully applied in two preclinical studies for LG1980 and GH501 to determine their concentrations in rat plasma, liver, kidney and bone over 24 h after intravenous injection of compounds.     

Simultaneous determination of evobrutinib and its metabolite evobrutinib‐diol in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography hyphenated with electrospray ionization tandem mass spectrometric method (LC–ESI–MS/MS) was developed and validated for simultaneous determination of evobrutinib and evobrutinib‐diol in dog plasma. The plasma sample was processed using acetonitrile and chromatographic separation was carried out on a Waters Acquity BEH C₁₈ column (50 × 2.1 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile, with an optimized gradient elution at a flow rate of 0.4 mL/min. Detection was accomplished in selective reaction monitoring mode via electrospray ionization interface operated in positive ion mode. The precursor‐to‐product transitions for quantification were m/z 430.2 → 98.1 for evobrutinib, m/z 464.2 → 98.1 for evobrutinib‐diol and m/z 441.2 → 138.1 for ibrutinib (internal standard). The developed assay was linear over the tested concentration ranges with correlation coefficient >0.995. The LLOQ was 0.1 ng/mL for both analytes. The inter‐ and intra‐day precisions were <9.65% and the accuracy ranged from ?3.94 to 6.37%. The extraction recovery was >85.41% and no significant matrix effect was observed. The developed assay was successfully applied to the pharmacokinetic study of evobrutinib and evobrutinib‐diol in dogs after oral administration of evobrutinib at a single dose of 5 mg/kg.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.     

Enantioselective determination of ketamine in dog plasma by chiral liquid chromatography–tandem mass spectrometry

Ketamine is an N‐methyl‐d ‐aspartate receptor antagonist that is usually used clinically as a racemic mixture. Its two enantiomers exhibit different pharmacological activities. To determine whether the enantiomers have different pharmacokinetic profiles, a chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of ketamine enantiomers in dog plasma. The enantiomers of ketamine were extracted from 50 μL of plasma by methyl tert‐butyl ether. Adequate chromatographic retention and baseline resolution of the enantiomers were achieved within a runtime of 5 min on a chiral column coated with polysaccharide derivatives, using a gradient mobile phase of acetonitrile and 10 mm ammonium bicarbonate aqueous solution. Ketamine enantiomers were detected by mass spectrometry with multiple reaction monitoring mode using the transitions of m/z 238.3 → 125.9 for the analytes and m/z 237.1 → 194.1 for carbamazepine (internal standard). The method was linear over the concentration range from 0.5 to 500 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was 0.5 ng/mL. The intra‐ and inter‐day precision was <7.3% and 8.5% for R‐ and S‐ketamine, respectively. The accuracy was 92.9–110.4% for R‐ketamine and 99.8–102.4% for S‐ketamine. The method was successfully applied to characterize the stereoselective pharmacokinetic profiles of ketamine in beagle dogs.     

Study on bioequivalence of beraprost in healthy volunteers by liquid chromatography with tandem mass spectrometry

Beraprost sodium is an oral prostacyclin analog that was first approved in 1992 (Japan) for the treatment of peripheral vascular disorders. It is administered orally as a tablet available in strength 20 μg. In this paper, we described a liquid chromatography tandem mass spectrometry method that was developed for the quantification of beraprost in human plasma with high sensitivity at picogram per milliliter concentration. The method had been validated in terms of selectivity, sensitivity, accuracy and precision, matrix effect, linearity, recovery and carry‐over according to the Guideline on Bioanalytical Validation from the European Medicines Agency. The standard calibration curve for beraprost was 9.5–1419 pg/mL. This method has been applied successfully to a bioequivalence study with 60 μg of beraprost (three tablets) in 29 healthy volunteers. The results showed that the two formulations of beraprost are bioequivalent.     

Rapid and simultaneous determination of hexapeptides (Ac-EEMQRR-amide and H2N-EEMQRR-amide) in anti-wrinkle cosmetics by hydrophilic interaction liquid chromatography-solid phase extraction preparation and hydrophilic interaction liquid chromatography with tandem mass spectrometry

A rapid method for the simultaneous determination of Ac-EEMQRR-amide and H(2)N-EEMQRR-amide in cosmetic products was developed and evaluated. This analytical procedure involved extracting samples with 0.1:0.1:85:15 (v:v) trifluoroacetic acid (TFA):formic acid:acetonitrile (ACN):water and determination by hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Samples showing serious ion suppression were further cleaned up using HILIC-SPE prior to HILIC-MS/MS analysis. Stable isotopically labeled peptides, corresponding to the above two peptides, were used as internal standards to correct for loss of recovery and matrix effects. Electrospray ionization (ESI) in the positive mode was used. The linear range was 2.0-1000 ng/mL for Ac-EEMQRR-amide and 25.0-2500 ng/mL for H(2)N-EEMQRR-amide. Thirteen commercial products were analyzed for the two peptides using this method. The amounts of Ac-EEMQRR-amide in the samples ranged from none detected to 42.3 μg/g. H(2)N-EEMQRR-amide was not detected in any of the samples. The recoveries for Ac-EEMQRR-amide and H(2)N-EEMQRR-amide ranged from 85% to 110% and 84% to 119%, respectively, at the spiking level of 30 μg/g.     

Pharmacokinetics and bioavailability study of ginsenoside Rk1 in rat by liquid chromatography/electrospray ionization tandem mass spectrometry

Ginsenoside Rk1 (Rk1) exhibited various potent biological activities. However, its pharmacokinetic profile in vivo remains unclear. In the present study, a simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for determination of Rk1 in rat plasma and applied in a pharmacokinetic study. The sample was precipitated with acetonitrile and separated on a Zorbax Eclipse XDB C18 column (50 × 2.1 mm, 1.8 μm). The mobile phase was composed of 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Rk1 and internal standard (ginsenoside Rg3) were quantitatively monitored with precursor‐to‐product ion transitions of m/z 765.4 → 441.5 and m/z 783.5 → 621.4, respectively. The assay was linear over the concentration range of 5–1000 ng/mL (r > 0.99) with the LLOQ of 5 ng/mL. Other parameters including intra‐ and inter‐day precision and accuracy, extraction recovery and matrix effect were within the acceptable limits. The analyte was stable under the tested storage conditions. The validated method has been successfully applied to a pharmacokinetic study of Rk1 in rat plasma after intravenous (5 mg/kg) and oral (25 mg/kg, 50 mg/kg) administration. After oral administration, Rk1 could be detected in blood at 30 min and reached the highest concentration at 4.29~4.57 h. Our results demonstrated that Rk1 showed low clearance, moderate half‐life (3.09–3.40 h) and low bioavailability (2.87–4.23%). The study will provide information for the further application of Rk1.     

Quantification and pharmacokinetic study of entrectinib in rat plasma using ultra‐performance liquid chromatography tandem mass spectrometry

To characterize the preclinical plasma pharmacokinetics of entrectinib, a reproducible and precise assay is necessary. In this study, we developed and validated a simple ultra‐performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the measurement of entrectinib using carbamazepine as the internal standard in rat plasma. Sample preparation was a simple protein precipitation with acetonitrile, then entrectinib was eluted on an Acquity UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 μm) using a gradient elution with a mobile phase composed of acetonitrile (A) and 0.1% formic acid in water (B). Detection was achieved using multiple‐reaction monitoring in positive ion electrospray ionization mode. The method showed good linearity over the concentration range of 1–250 ng/mL (r² > 0.9951). The intra‐ and inter‐day precision was determined with the values of 6.3–12.9 and 2.6–6.9%, respectively, and accuracy values of 0.5–11.6%. Matrix effect, extraction recovery, and stability data all met the acceptance criteria of US Food and Drug Administration guidelines for bioanalytical method validation. The method was successfully applied to a pharmacokinetic study. In this study, we developed the complete validated method for the quantification of entrectinib in rat plasma.     

Metabolic profiles and pharmacokinetics of goitrin in rats through liquid chromatography combined with electrospray ionization–tandem mass spectrometry

Several chemical and biological studies have revealed R,S‐goitrin as the main bioactive constituent of Isatis indigotica Fort., responsible for antiviral antiendotoxin activity; however, few pharmacokinetic studies have been conducted. To comprehend the kinetics of R,S‐goitrin and promote its curative application, a rapid and sensitive UHPLC–MS/MS method was developed. The selected reaction monitoring transitions were m/z 130.0 → 70.0 for R,S‐goitrin and m/z 181.1 → 124.0 for the internal standard in a positive‐ion mode. The established UHPLC–MS/MS method achieved good linearity for R,S‐goitrin at 10–2000 ng/mL. The intra‐ and interday accuracy levels were within ±9.7%, whereas the intraday and interday precision levels were <11.3%. The extraction recovery, stability and matrix effect were within acceptable limits. The validated method was successfully applied for the pharmacokinetic analysis of R,S‐goitrin in rats after oral administration. Moreover, a total of six metabolites were structurally identified through UHPLC–Q/TOF–MS. The proposed metabolic pathways of R,S‐goitrin in rats involve demethylation, acetylation, glutathionylation and oxygenation.