Positive Ion Electrospray Ionization Research Articles

Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish

Rubiae Radix et Rhizoma (called “Qiancao”, QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called “Qiancaotan”, QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC–MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile–water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.

Pharmacokinetics and tissue distribution study of 15 ingredients of Polygonum chinense Linn extract in rats by UHPLC-MS/MS.

A rapid and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of 15 bioactive ingredients in rat plasma and tissues after oral administration of Polygonum chinense Linn extract (PCE). After addition of internal standards (ISs; rutin and danshensu), plasma and tissue samples were pre-treated by protein precipitation with acetonitrile-ethanol. The chromatographic separation was performed on an Agilent ZORBAX RRHD Eclipse Plus C18 column with gradient elution using a mobile phase composed of methanol and water (containing 0.2% acetic acid) at a flow rate of 0.3 mL min-1 . Mass spectrometric detection was carried out using a mass spectrometer in both positive and negative ion electrospray ionization modes by multiple reaction monitoring. The method provided excellent linearity, and the lower limit of quantification range 0.5-30 ng mL-1 for all analytes. The intra- and inter-day precision were less than 9.12% and the accuracy ranged from -4.02% to 6.32%, respectively. The mean extraction recovery and matrix effect of analytes and ISs ranged from 83.65% to 109.20%. The method was successfully applied to the pharmacokinetics and tissue distribution study of 15 ingredients of PCE in rats.

Simultaneous Determination of Three Coumarins in Rat Plasma by HPLC-MS/MS for Pharmacokinetic Studies Following Oral Administration of Chimonanthi Radix Extract.

Chimonanthi Radix (CR) is widely used in the treatment of influenza in China. Extensive studies revealed that the major bioactive constituents of CR were coumarins. However, pharmacokinetic study of coumarins in CR has not been fully studied. The purpose of this study was to establish a convenient and effective high-performance liquid chromatography-tandem mass spectrometry method that was used to simultaneously determine scopoletin, scopolin and isofraxidin in rat plasma after oral administration of CR extract using xanthotoxin as the internal standard. The chromatographic separation was carried out on a COSMOCORE C18 column (100×2mm, 2.6μm), using gradient elution with the mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Three coumarins and IS were quantified by positive ion electrospray ionization in multiple reaction monitoring mode. The method was fully validated in terms of specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analytes under various conditions. The results could provide further research foundation for anti-influenza mechanism of three coumarins in CR.

Electrospray ionization collision induced dissociation of thieno[3,2-d]pyrimidine derivatives

2,4-substituted thieno[3,2-d]pyrimidines, with different l-amino acid methyl ester substituent on 4-C and methyl or methylthio substituent on 2-C, showed a complex and analytically valuable fragmentation behaviour under positive ion electrospray ionization collision-induced dissociation ((+)-ESI-CID) mass spectrometry. Protonated 2,4-substituted thieno[3,2-d]pyrimidines firstly lose a neutral fragment C2H4O2 (MS2) and the ion [M + H - 60]+ thus formed, then dissociates by losing of extended series of neutral molecule fragmentation from amino acid methyl ester substituent, in competition with the expulsion of CS or CH3SH and other direct bond scissions. Moreover, losing of NH3, CHN and CH2NH from the precursor ions of [M + H - 60]+ were proposed to occur via rearrangements. The formation of diagnostic product ions and the series neutral eliminations provided information about the properties of substituents, assisting their characterization via MS/MS, high-resolution mass spectrometry, the hydrogen/deuterium exchange experiment and density functional theory (DFT) calculations. The proposed fragmentation patterns of these compounds give an insight into the understanding of gas-phase reactions in mass spectrometric studies of thieno[3,2-d]pyrimidine derivatives.

Validated Liquid Chromatography-Tandem Mass Spectroscopic Method for Simultaneous Determination of Certain Drugs Used in Cataract Surgery in Rabbit Aqueous Humor.

A sensitive, selective, accurate and precise ultra-high performance liquid chromatography-Tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery in aqueous humor. Cataract surgery requires a powerful mydriatic eye drops combination such as cyclopentolate hydrochloride and phenylephrine hydrochloride to dilate the pupil and facilitate eye lens replacement and also requires strong fluoroquinolone antibiotic such as lomefloxacin hydrochloride. The method was performed with positive ion electrospray ionization and the analytes were quantified and monitored on a triple quadrupole mass spectrometer using multiple reaction monitoring scanning mode. Liquid-liquid extraction was used for the purification and preconcentration of analytes from rabbit aqueous humor matrix. Chromatographic elution was performed using an Phenomenex Luna® C18 (150mm×2.1mm, 1.6μm) column and moxifloxacin hydrochloride as internal standard with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) at flow rate of 0.2mL/min. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. Full validation of the procedure was performed according to the US Food and Drug Administration guidance for industry: bioanalytical method validation and European Medicines Agency (EMA) guideline on bioanalytical method validation.

Composition and Structure of the Sulfur-Containing Compounds in the Extracts from the Chinese High-Organic-Sulfur Coals

The types and molecular structures of the sulfur-containing compounds in extracts of Chinese high-organic-sulfur coals with different ranks were studied using positive-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and methyl derivatization. The types and molecular structures of sulfur-containing compounds were described from three characteristics: the type of the heteroatom, the distribution of the carbon number, and the condensation degree. Nine types of sulfur-containing compounds were detected in the extracts, including S1, S2, O1S1, O1S2, O2S1, N1S1, N1S2, N2S1, and N1S1O1; these compounds are distributed differently in extracts from different high-sulfur coals according to their different ranks. As coal rank increases, sulfur-containing compounds, which have nitrogen atoms, gradually disappear, and sulfur-containing compounds in coal extracts are characterized by short side chains and high condensation.

Bioassay studies of Risperidone and its active metabolite in rat dried blood spots and dried plasma spots using LC‐ESI‐MS/MS: Comparison of their pharmacokinetic profiles

AbstractA highly sensitive and specific liquid chromatography‐elctrospray ionization tandem mass spectrometry method was developed and validated for rapid determination of risperidone and its active metabolite in rat dried blood spots and dried plasma spots. Chromatographic separation was achieved through ZIC®‐HILIC (250 × 4.6 mm, 5 µm) at 25°C using 10 mM ammonium acetate (pH: 3.0, adjusted with acetic acid) and acetonitrile (12:88, v/v) as a mobile phase. Tandem mass spectrometry detection was performed in positive ion electrospray ionization using multiple reaction monitoring mode to monitor precursor → product ion transitions m/z 411.2→191.1 for Risperidone, m/z 427.2→207.1 for its metabolite (Risperidone‐9‐OH), respectively. The lower limit of quantification of Risperidone and Risperidone‐9‐OH were 0.95 and 0.90 ng/mL in rat dried blood spots, respectively. The developed bioassay method was successfully applied to study the pharmacokinetic profile of Risperidone and Risperidone‐9‐OH using Iloperidone as an internal standard. As per pharmacokinetic studies, Cmax of Risperidone and Risperidone‐9‐OH in dried blood spots was 36.2 and 28.9 ng/mL; and in dried plasma spots was 38.2 and 30.2 ng/mL, respectively. Similarly, tmax of Risperidone and Risperidone‐9‐OH in dried blood spots was 1.4 and 14.7 h; and in dried plasma spots was 1.54 and 15.4 h, respectively.

Development of ultrahigh-performance liquid chromatography/mass spectrometry and ultrahigh-performance supercritical fluid chromatography/mass spectrometry assays to determine the concentration of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions.

RationaleFast and easily transferable chromatography/mass spectrometry assays were required to detect and quantify the amount of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions.MethodsBitrex™ solutions were analysed using reversed‐phase ultrahigh‐performance liquid chromatography coupled with positive ion electrospray ionisation mass spectrometry (UHPLC/ESI‐MS). Separation was achieved using a mobile phase gradient with an Acquity BEH C18‐packed column. Sodium saccharin solutions were analysed using ultrahigh‐performance supercritical fluid chromatography coupled with negative ion electrospray ionisation (UHPSFC/ESI‐MS). Separation was achieved using isocratic elution with an Acquity UPC2 Torus Diol packed column and a methanol (25 mM ammonium acetate) co‐solvent.ResultsThe calibration curves obtained using the ratio of the active compound to an internal standard generated linear regression values (R2) >0.99. Samples analysed prior to and after an autoclave sterilisation process and bottling gave repeatable measurements within 10% of the expected concentration.ConclusionsThe two assays afford a fast robust and quantitative analytical method for the detection of the active components used to test the efficacy of the homemade facemask testing solutions.

Analysis of aristolochic acid in asarum (Xixin) and its preparations by liquid chromatography/tandem mass spectrometry

A method for analysis of aristolochic acids (AAs: AA-I, AA-II) in Asarum (Xixin, named in Chinese) and its preparations by liquid chromatography coupled with tandem mass spectrometry has been developed. The chromatographic analysis was carried out using a C18 column (2.1x150 mm) and a mixture of 0.1% formic acid (containing 0.1% ammonium acetate) and acetonitrile (65:35, v/v) as the mobile phase at a flow rate of 0.3 mL/min. Piromidic acid was used as an internal standard. The mass detection was measured as the positive ion electrospray ionization mode. The precursor and daughter ions for quantitation were m/z 359 and 298 for AA-I, m/z 329 and 268 for AA-II. The instrument detection limit of AA-I and AA-II was 2 and 2.8 ng/mL, respectively. The result showed that Asarum contained AA-I while AA-II was not detected. AA-I distribution in different parts of Asarum was examined in detail and AA-I content in both methanol and water extracts was also compared in this study. In our twenty samples of Asarum, the average amounts of AA-I in 75% methanol extracts of roots, rhizomes, petioles and leaves were 1.23 ± 0.64, 1.72 ± 1.38, 7.81 ± 4.04 and 27.13 ± 25.31 ppm, respectively. The average amounts of AA-I in water extracts of roots, rhizomes, petioles and leaves were 0.33 ± 0.32, 0.45 ± 0.50, 3.98 ± 3.09 and 6.32 ± 9.75 ppm, respectively. The results indicated that the amounts of AA-I in roots were less than those in petioles and leaves, and the amounts of AA-I in water extracts were about 25 to 50 percent of those in 75% methanol extracts. For Asarum preparations, the amounts of AA-I in each six of Xi-xin-extract- powder and Ma-Huang-Fu-Zi-Xi-Xin-Tang ranged between 0.35-2.07 ppm and 0.21-1.00 ppm, respectively. Results of this study could be provided for the regulatory authority as references.

A simplified and reliable LC-tandem mass spectrometry method for determination of ulipristal acetate in human plasma and its application to a pharmacokinetic study in healthy Chinese volunteers.

In this study, a simplified, sensitive and reliable LC-tandem mass spectrometry method was established and validated for the quantification of ulipristal acetate (UPA) in human plasma and for the investigation of pharmacokinetic profile of UPA following a single oral administration of ella (UPA 30-mg tablet) in healthy Chinese volunteers. Plasma samples were analyzed after being processed by protein precipitation with methanol. Chromatographic separation was performed on a Kinetex EVO C18 column (2.1 × 50 mm, 2.6 μm) using gradient elution with a mobile phase composed of methanol and water containing 2 mm ammonium acetate and 0.3% formic acid at a flow rate of 0.3 mL/min. The chromatographic running time was 4.0 min per sample. The MS detection was performed via an LC system with the positive ion electrospray ionization interface in multiple reaction monitoring mode using the transition of m/z 476.2 → 134.1 for UPA and m/z 479.3 → 416.2 for UPA-d3 [internal standard (IS)], respectively. UPA and IS were monitored without severe interference from the biological matrices. The method was linear over the wide concentration range of 0.300-300 ng/mL. The intra- and inter-day precision and accuracy were well within the limits required for bioanalytical assays. The method was first used to describe the pharmacokinetic characteristic of UPA after a single oral administration of ella in healthy Chinese volunteers. Based on a between-study comparison, there were statistically significant differences (p < .05) between Chinese and Caucasian volunteers for the systemic exposure of UPA, suggesting that race seems to significantly impact the systemic exposure of UPA.

Direct sulfur-containing compounds analysis in petroleum via (+) ESI FT-ICR MS using HBF4 as ionization promoter

Molecular-level characterization of sulfur-containing compounds has attracted particular attention due to its relative high abundance and negative impact on the refining process. In this paper, a novel analytical method for selective ionization of sulfidic and/or isolated thiophenic compounds (thiophene rings that are not condensed with aromatic systems) and condensed thiophenic compounds (thiophene rings that are condensed with aromatic systems, DBE ≥ 6, DBE = number of rings plus double bonds to carbon) in petroleum samples was developed by using fluoroboric acid (HBF4) as ionization promoter. Positive-ion electrospray ionization [(+) ESI] Fourier transform ion cyclotron resonance mass spectrometry was followed to characterize the molecular composition. Sulfidic and/or isolated thiophenic compounds were mainly ionized as protonated molecular ions ([M+H]+), while condensed thiophenic compounds (DBE ≥ 6) were mainly ionized as molecular radical cations (M+). The method was validated by various sulfur-containing model compounds and then applied to vacuum gas oil, crude oil and heavy oil samples. (+) ESI coupled with HBF4 additive is a simple method for selective ionization of sulfides and thiophenes in high-sulfur-content petroleum samples and has the potential for the characterization of sulfides and thiophenes in other complex mixtures.

Characterization of heteroatom class species in asphaltenes from medium/low temperature coal tar

ABSTRACT The classes, structures, and distributions of heteroatom compounds in asphaltenes from medium/low temperature coal tar (LCT) were characterized by using positive-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The NX, S1OX, S2OX, SX, S1N1OX, OX, N1OX, N2OX, N3OX, S1NX, and S2NX class species were identified in LCT asphaltenes. The O1 class species were the most abundant species of the total heteroatom class species, of which structures were likely furan derivatives or ketone oxides. There were mainly N1 class species of the basic nitrides, and the structures were likely pyridine derivatives with DBE of 16 − 25. The S1 class species were predominant of the sulfur-containing compounds, of which structures were likely thiophene derivatives or thioether derivatives with low degree of condensation. Nitrogen oxides of LCT asphaltenes mainly belong to N1O1 class species composed of 4 − 8 aromatic rings with a keto group and a pyridine ring. The sulfur oxides were mainly S1O1 class species consisted of 5 − 8 aromatic rings with a thiophene sulfoxide ring. The oxygen and sulfur heteroatoms in LCT were mainly concentrated in asphaltenes, which were mainly high-condensation compounds. The N1 class species in LCT asphaltenes have a higher degree of condensation, which occupied only smaller proportion in LCT.

Development of UPLC–MS/MS method for studying the pharmacokinetic interactions of pexidartinib with antifungal drugs in rats

Pexidartinib was approved in the USA for targeted therapy of adult patients with symptomatic tenosynovial giant cell tumour (TGCT) by the FDA. The purpose of our study was to develop and establish a quick assay based on ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) for the measurement of pexidartinib concentrations in plasma and to survey whether antifungal drugs (isavuconazole, posaconazole, fluconazole and itraconazole) could change the pharmacokinetic parameters of pexidartinib in rats. After the quick protein crash with acetonitrile, the chromatographic separation of pexidartinib and upadacitinib (used as the internal standard in this study, IS) were conducted on an Acquity BEH C18 (2.1 × 50 mm, 1.7 μm) column, and the detection of the analyte was also accomplished with a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electro-spray ionization (ESI) interface. The assay showed good linearity in the range of 1−7000 ng/mL. The accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery, matrix effect, stability, and carry-over were also met the requirements. The application of the validated UPLC–MS/MS bioanalytical method was further successfully involved in the drug-drug interactions study from rats. It was found that fluconazole and itraconazole significantly increased the concentration of pexidartinib and had the inhibitory effect on the metabolism of pexidartinib, while not isavuconazole and posaconazole. Thus, more attention should be paid to the concurrent use of pexidartinib with fluconazole or itraconazole to reduce the risk of unexpected clinical outcomes.

Inhibitory effects of voriconazole, itraconazole and fluconazole on the pharmacokinetic profiles of ivosidenib in rats by UHPLC-MS/MS

Ivosidenib, as an oral mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor, was awarded approval in the USA for the targeted therapy of relapsed or refractory acute myeloid leukemia (AML) in adult patients, who also had a susceptible enzyme to mIDH1. The aim of our present study was to develop and validate an accurate and fast assay based on the ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique for the quantification of ivosidenib in plasma and to investigate the possible effects of different CYP3A4 inhibitors (voriconazole, itraconazole and fluconazole) on ivosidenib metabolism in rats. After the fast protein crash with acetonitrile, chromatographic separation of ivosidenib and erlotinib (used as the internal standard in this experiment, IS) was accomplished using an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column, and detection of the analyte was also performed using a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electrospray ionization (ESI) interface. The assay showed enough linearity over a 0.5−6000 ng/mL calibration range. The application of the validated bioanalytical method based on the UHPLC-MS/MS technique was further successfully exhibited in an animal study of the drug-drug interaction between ivosidenib (50 mg/kg) and voriconazole (20 mg/kg)/itraconazole (20 mg/kg)/fluconazole (20 mg/kg) in rats. Voriconazole, itraconazole and fluconazole increased the exposure of ivosidenib in plasma by different degrees and also had a potential inhibitory effect on the metabolism of ivosidenib. Thus, a dose reduction or interruption of ivosidenib may be important to guide the practice of clinical medicine.

Impurity identification and quantification for arginine vasopressin by liquid chromatography/high-resolution mass spectrometry.

For pharmaceutical quality control, impurities may have unexpected pharmacological or toxicological effects on quality, safety, and efficacy of drugs. Arginine vasopressin (AVP) is an important cyclic peptide drug that is mainly used for the treatment of diabetes insipidus and esophageal varices bleeding. With the advancement made in analytical techniques, liquid chromatography/high-resolution mass spectrometry (LC/HRMS) has emerged as a critical technique for the identification and quantification of structurally related peptide impurities in AVP. An LC/HRMS/MS-based method using a quadrupole ion trap-Orbitrap mass spectrometer operated in the positive ion electrospray ionization mode was developed for the determination and quantification of structurally related peptide impurities in AVP. Under optimized experimental conditions, three deamidation products, ([Glu4 ]AVP, [Asp5 ]AVP, and AVP acid), two amino acid deletion impurities (des-Pro7 -AVP and des-Gly9 -AVP), one amino acid insertion impurity (endo-Gly10a -AVP), one end chain reaction product (N-acetyl-AVP), and one AVP isomer were detected. Subsequent quantification using an external standard method estimated the total mass fraction of all structurally related peptide impurities in the AVP study material to be 30.3 mg/g with an expanded uncertainty of 3.0 mg/g (k = 2). This study complements the AVP impurity profile and improves the separation and discovery of other potential impurities in vasopressin analogues.

Molecular composition of vanadyl porphyrins in the gilsonite

A gilsonite from Qingchuan (Sichuan province, China) was separated into several fractions and characterized by various techniques. The vanadyl porphyrins were determined by inductively coupled plasma optical emission spectrometer, ultraviolet-visible spectrophotometry, high-temperature gas chromatography-atomic emission detector, and positive-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The results show that the gilsonite contains a large amount of metal elements, especially the vanadium content is as high as 3888 μg/g. A large number of vanadyl porphyrins are detected, in which the deoxophylloerythroetio porphyrins are the most abundant, indicating high molecular condensation but low thermal maturity. The huge reserves and high metal content imply important utilization value for this natural source.

SAP fractions from light, medium and heavy oils: Correlation between chemical profile and stationary phases

The chemical composition of oil samples is fundamentally important for planning the recovery, transportation and refining processes of the oil industry. In this work, we examined how stationary phase affects the recovery of non-retained substances and the separation of saturated, aromatic and polar fractions from different density oils by column chromatography. We evaluated four stationary phases: silica gel, alumina, strong anion exchange (SAX) and C-18 silica gel, the latter being the only nonpolar one. The experiment was carried out in quintuplicate and the fractions obtained were analyzed by gas chromatography coupled with mass spectrometry and positive-ion mode electrospray ionization FT-ICR mass spectrometry (ESI(+)-FT-ICR MS). The influence of the stationary phase on the separation of the various classes of substances found in petroleum was observed by chemometrics. For saturated compounds, light and medium oils presented n-paraffins, iso-paraffins, naphthenes, terpanes and steranes, while heavy oil presented intense biodegradation, with a higher concentration of terpanes and steranes. When heavy oil was fractionated with C-18 silica gel, the presence of n-paraffins could also be observed. Alumina generated a saturated fraction, that was less contaminated with aromatics, and it showed a separation capacity similar to that of silica gel and SAX. For aromatics, all stationary phases generated fractions contaminated with saturated compounds, and polar fractions showed a greater presence of basic heteroaromatics compounds containing predominantly one nitrogen. A more reliable SAP separation of crude oil fractions depends on their density.

Determination of nitenpyram dissipation and residue in kiwifruit by LC-MS/MS

ABSTRACT To investigate the presence of nitenpyram in kiwifruit, a field experiment was conducted in six kiwifruit producing regions in China. A modified quick, easy, cheap, effective, rugged and safe method (QuEChERS) method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of nitenpyram in kiwifruit. Quantitative detection was performed by LC-MS/MS in multiple reaction monitoring (MRM) mode under positive-ion electrospray ionisation. The proposed method is linear and accurate in the range of 0.005 to 4.5 μg/mL. The recovery of nitenpyram was satisfactory (71.96–96.67%) with reasonable RSDs (9.83 %) for 0.01–4.5 mg/kg spiked levels. The limit of quantification (LOQ) was 0.01 mg/kg for nitenpyram. Nitenpyram degraded rapidly in kiwifruit with a half-life <4 days. Residues of nitenpyram were <0.01 − 0.54 mg/kg in kiwifruit 7, 14 and 21 days after spraying at six sites.

Development and Validation of UPLC-MS/MS Method for Determination of Enasidenib in Rat Plasma and Its Pharmacokinetic Application.

In our research, a straightforward UPLC-MS/MS method, with diazepam as the internal standard (IS), was proposed and acknowledged to determine the concentrations of enasidenib in rat plasma. When preparing the sample, we used acetonitrile for protein precipitation. The gradient elution method was used, and the mobile phase was acetonitrile and 0.1% formic acid. Diazepam was used as the IS. We used the Acquity UPLC BEH C18 column to separate enasidenib and IS. Under the positive ion electrospray ionization (ESI) source conditions, the mass transfer pairs of enasidenib were monitored by multiple reaction monitoring (MRM) to be m/z 474.2 ⟶ 456.1 and m/z 474.2 ⟶ 267.0, and the IS mass transfer pairs were m/z 285.0 ⟶ 154.0. Enasidenib had good linearity (r2 = 0.9985) in the concentration range of 1.0–1000 ng/mL. Besides, the values of intraday and interday precision were 2.25–8.40% and 3.94–5.46%, respectively, and the range of the accuracy values varied from −1.44 to 2.34%. Matrix effect, extraction recovery, and stability were compliant with FDA approval guidelines in terms of bioanalytical method validation. We had established a new method that had been applied to the pharmacokinetic study of enasidenib in rats.

A rapid and convenient derivatization method for quantitation of short‐chain fatty acids in human feces by ultra‐performance liquid chromatography/tandem mass spectrometry

Short-chain fatty acids (SCFAs) are associated with intestinal microbiota and diseases in humans. SCFAs have a low response in mass spectrometry, and in order to increase sensitivity, reduce sample consumption, shorten analysis time, and simplify sample preparation steps, a derivatization method was developed. We converted seven SCFAs into amide derivatives with 4-aminomethylquinoline. The reaction occurred for 20 min at room temperature. The analytes were separated on a reversed-phase C18 column and quantitated in the positive ion electrospray ionization mode using multiple reaction monitoring. Acetic acid-d4 was used as the stable-isotope-labeled surrogate analyte for acetic acid in the working solutions, while the other stable-isotope-labeled standards were used as internal standards (ISs). Method validation showed that the intra-day and inter-day precision of quantitation for the seven SCFAs over the whole concentration range was ≤3.8% (n = 6). The quantitation accuracy ranged from 85.5% to 104.3% (n = 6). Most important, the collected feces were vortexed immediately with ethanol. This study provides a new derivatization method for a precise, accurate, and rapid quantitation of SCFAs in human feces using ultra-performance liquid chromatography/tandem mass spectrometry. This method successfully determined the concentration of SCFAs in human feces and could assist in the exploration of intestinal microbiota and diseases.