Precursor-product Ion Pairs Research Articles

Predicting Tandem Mass Spectra of Small Molecules Using Graph Embedding of Precursor-Product Ion Pair Graph.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics identification relies heavily on high-quality MS/MS data; MS/MS prediction is a good way to address this issue. However, the accuracy of the prediction, resolution, and correlation with chemical structures have not been well-solved. In this study, we have developed a MS/MS prediction method, PPGB-MS2, which transforms the MS/MS prediction into fragment intensity prediction, and the concept of precursor-product ion pair graph bags (PPGBs) was introduced to represent fragments, achieving uniform representation of precursor and product ion structures and MS/MS fragmentation information. The chemical structure information is kept before it is incorporated into machine learning models. Due to the PPGB representation, graph neural networks (GNNs) can be utilized to achieve MS/MS fragment intensity prediction. The system was trained and evaluated using [M+H]+ and [M-H]- data acquired by an Agilent QTOF 6530 in the NIST 20 tandem MS database. Results demonstrated that the average cosine similarity is 0.71 in the test set, which is higher than classical MS/MS prediction methods. PPGB-MS2 also achieves high-resolution MS/MS prediction due to its effective management of the correspondence between fragments and structures.

Determination of mitomycin C in rat plasma by liquid chromatography-tandem mass spectrometry and its application for determining pharmacokinetics in rat

To develop the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring mitomycin C in rat plasma, samples were processed using solid-phase extraction, with the internal standard being carbamazepine. A reversed phased C18 column was utilized for the LC-MS/MS study, and mobile phases consisting of 0.1 % formic acid in acetonitrile and water were injected into it at a rate of 0.3 mL/min. Multiple reaction monitoring in positive-ion mode with precursor-product ion pairs 335.3 → 242.3 (mitomycin C) and 237.1 → 194.1 (carbamazepine) was employed to quantify the compounds. The linear range in plasma was found to be 10–4000 ng/mL (r2 = 0.992). The inter-batch and intra-batch precision were <14.3 % (LLOQ: 14.7 %) and 13.4 % (LLOQ: 16.1 %), respectively. The recovery and the matrix effect of mitomycin C in plasma were 113 % and 111 %, respectively. Mitomycin C was stable under the conditions of this assay method. In the end, this approach proved effective in a pharmacokinetic investigation with the intravenous and oral administration of mitomycin C to rats.

Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high-resolution tandem mass spectrometry.

The manipulation of blood and blood components in sports is prohibited at all times, and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. But while most drug candidates based on stabilized hemoglobin (Hb) were found to be characterized by serious side effects, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be another candidate for transfusion medicine and cheating athletes, as it was found to be well tolerated in preclinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration, tryptic digestion, and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS). For the mass spectrometric identification of lugworm Hb, two precursor-product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T6 ), hbB1 (T3 and T6 ), and the linker chain (T16 ) were employed. The modified approach was comprehensively characterized and found to allow for the specific and sensitive detection of lugworm Hb down to concentrations of 10 μg/mL from 50 μL of serum/plasma. Therefore, it can serve as confirmation procedure for lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8h (dose: 600 mg/kg) suggest that the approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug candidate.

Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC–EI–QTOF) for comprehensive assessment of MS/MS fragments

Abstract Gas chromatography–tandem mass spectrometry with electron ionization (GC–EI–MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC–EI–MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor–product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof–of–concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. One sentence summary An analytical workflow that combines high-resolution mass spectrometry (MS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments, which provide positional information and therefore offering significant advantages over traditional MS to improve 13C-metabolic flux analysis.

Development and Validation of an LC-MS/MS Method for the Quantitative Determination of Contezolid in Human Plasma and Cerebrospinal Fluid

To develop and verify a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining contezolid in plasma and cerebrospinal fluid (CSF). Protein precipitation was performed on samples using linezolid as the internal standard. We used an Agilent EclipsePlus C18 column operating at 0.4 mL/min in conjunction with acetonitrile and water mobile phases for the LC-MS/MS analysis. Using the precursor-product ion pairs 409.15→269.14 (contezolid) and 338.14→195.1 (linezolid), multiple reaction monitoring was used to quantify the compounds. Plasma linearity range was 50.0 to 5000 ng/mL, and CSF was 20.0 to 1000 ng/mL (r2 = 0.999). The inter-batch and intra-batch precisions were ≤2.57% and ≤5.79%, respectively. Plasma recovered 92.94%, and CSF recovered 97.83%. Plasma, CSF, hemolytic plasma, and hyperlipidemic plasma all showed a coefficient of variation ≤ 7.44%. The stability and dilution integrity of this method were also acceptable. The study also demonstrated that artificial CSF can be used as a matrix for the preparation of standard curve samples. A simple and accurate method was developed and validated for the determination of contezolid concentrations in human plasma and CSF, which may be useful for monitoring the therapeutic effect of central nervous system medications.

A Sensitive HPLC-MS/MS Method for Determination of Obeticholic Acid in Human Plasma: Application to a Pharmacokinetic Study in Healthy Volunteers.

A rapid and sensitive LC-MS/MS method was developed and fully validated for the determination of obeticholic acid in human plasma. Glimepiride was used as internal standard. For this method, liquid-liquid extraction was performed to extract analyte from the plasma samples. Chromatographic separation was performed on a C18 (2.1 × 50mm, 2.7μm, Agilent) column with isocratic elution using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid at a flow rate of 0.4mL/min. The mass detection was performed in negative mode. The precursor-product ion pairs for MRM were m/z 465.3→419.3 for obeticholic acid and m/z 489.3→224.8 for the IS. The method exhibited great linearity over the concentration range of 0.150-100ng/mL for obeticholic acid. The sensitivity, linearity, accuracy, precision, recovery, matrix effect and stability of this method were all within the acceptable limits. The method was successfully validated and applied to the pharmacokinetic studies in healthy Chinese volunteers after a single oral dose administration of obeticholic acid tablets of 10mg, and the pharmacokinetic characteristics of obeticholic acid in human were reported for the first time.

Direct injection analysis of per and polyfluoroalkyl substances in surface and drinking water by sample filtration and liquid chromatography-tandem mass spectrometry

We developed and validated a method for direct determination of per- and polyfluoroalkylated substances (PFASs) in environmental water samples without prior sample concentration. Samples are centrifuged and supernatants passed through an Acrodisc Filter (GXF/GHP 0.2 um, 25 mm diameter). After addition of ammonium acetate, samples are analyzed by UPLC-MS/MS using an AB Sciex 6500 plus Q-Trap mass spectrometer operated in negative multiple reaction-monitoring (MRM) mode. The instrument system incorporates a delay column between the pumps and autosampler to mitigate interference from background PFAS. The method monitors eight short-/long-chain PFAS which are identified by monitoring specific precursor product ion pairs and by their retention times and quantified using isotope mass-labeled internal standard based calibration plots. Average spiked recoveries (n = 8) of target analytes ranged from 84 to 110% with 4–9% relative standard deviation (RSD). The mean spiked recoveries (n = 8) of four surrogates were 94–106% with 3–8% RSD. For continuous calibration verification (CCV), average spiked recoveries (n = 8) for target analytes ranged from 88 to 114% with 4–11% RSD and for surrogates ranged from 104–112% with 3–11% RSD. The recoveries (n = 6) of matrix spike (MX), matrix spike duplicate (MXD), and field reagent blank (FRB) met our acceptance criteria. The limit of detection for the target analytes was between 0.007 and 0.04 ng/mL. The method was used to measure PFAS in tap water and surface water.

Pharmacokinetics and Metabolism of Acetyl Triethyl Citrate, a Water-Soluble Plasticizer for Pharmaceutical Polymers in Rats.

Acetyl triethyl citrate (ATEC) is a water-soluble plasticizer used in pharmaceutical plasticized polymers. In this study, the pharmacokinetics and metabolism of ATEC were investigated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in rats. Plasma protein precipitation with methanol was used for sample preparation. For chromatographic separation, a C18 column was used. The mobile phases consisted of 0.1% formic acid and 90% acetonitrile, and gradient elution was used. The following precursor-product ion pairs were selected for reaction monitoring analysis: 319.1 m/z → 157 m/z for ATEC and 361.2 m/z → 185.1 m/z for tributyl citrate (internal standard) in positive ion mode. The LC–MS/MS method was fully validated and successfully applied to a pharmacokinetic study of ATEC in rats. The pharmacokinetic study showed that the volume of distribution and mean residence time of ATEC were higher after oral administration than after intravenous administration, pointing to extensive first-pass metabolism and distribution in tissue. In addition, the plasma concentration profile of the postulated metabolites of ATEC was investigated in plasma, urine, and feces. The resulting data indicated that ATEC was extensively metabolized and excreted mainly as metabolites rather than as the parent form. The developed analytical method and the data on the pharmacokinetics and metabolism of ATEC may be useful for understanding the safety and toxicity of ATEC.

Development and validation of an LC-MS/MS method for simultaneous quantification of voriconazole and its main metabolite voriconazole N-oxide in human plasma and its clinical application

ABSTRACTAn LC-MS/MS method was developed for simultaneous determination of voriconazole and its main metabolite voriconazole N-oxide in human plasma. Plasma pretreatment was simple one-step protein precipitation with acetonitrile, and separation was achieved on a Hypersil GOLD aQ column. A triple-quadrupole tandem mass spectrometer with electrospray ionization source was operated by multiple reaction monitoring (MRM) in positive ion mode, and the precursor–product ion pairs used for MRM were m/z 350.1→281.0, 365.8→224.1, and 531.3→489.2 for voriconazole, voriconazole N-oxide, and internal standard, respectively. The total analytical run time was 6 min. The proposed method was linear over the range 0.001–1 µg/mL for voriconazole and voriconazole N-oxide. The lower limit of quantification was 1 ng/mL for both of them. The specificity, accuracy, precision, recovery, matrix effect, stability as well as dilution integrity of this method were within acceptable limits during validation period. This method was successfully applied to therapeutic drug monitoring of voriconazole and to evaluate its metabolite profile.

Correction to: Simultaneous Quantification of 5-Aminoimidazole-4-Carboxamide-1-β-d-ribofuranoside and Its Active Metabolite 5-Aminoimidazole-4-Carboxamide-1-β-d-ribofuranotide in Mice Plasma by LC–MS/MS

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) and its active metabolite 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranotide (AICA ribotide) in mice plasma. Adenosine monophosphate was chosen as an internal standard, and plasma was deproteinized by adding 14% perchloric acid, and then, the resulting supernatant was diluted with water to decrease matrix effect. A 10 μL aliquot of the solution was injected into the LC–MS/MS system, and separation was achieved on a Gemini C6-Phenyl column with a mobile phase composed of 0.1% formic acid (v/v) and methanol. A triple quadrupole mass spectrometer with an electrospray turbo ion source operating in multiple reaction monitoring (MRM) positive ion mode was used as detector. The precursor–product ion pairs for MRM were m/z 259.2 → 110.1 and 259.2 → 242.1 for AICA riboside, 339.1 → 110.1 and 339.1 → 322.1 for AICA ribotide, and 348.3 → 136.0 and 348.3 → 119.1 for the internal standard, respectively. The total analytical run time was 10 min. The proposed method was linear over the range 0.01–10 μg/mL for AICA riboside and 0.01–5 μg/mL for AICA ribotide. The lower limit of quantification (LLOQ) was 10 ng/mL for both AICA riboside and AICA ribotide. The sensitivity, accuracy, and precision of this method were within acceptable limits during the validation period. The method was successfully applied to investigate the pharmacokinetics characteristics of AICA riboside and AICA ribotide in mice.

Lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG-4592) and its main metabolites into routine doping controls.

The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma- and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-for-purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1ng/mL and 1 to 5ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd.

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 Cmax and AUC0-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.

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

LC-MS/MS is useful for qualitative and quantitative analysis of 'doped' biological samples from athletes. LC-MS/MS-based assays at low-mass resolution allow fast and sensitive screening and quantification of targeted analytes that are based on preselected diagnostic precursor-product ion pairs. Whereas LC coupled with high-resolution/high-accuracy MS can be used for identification and quantification, both have advantages and challenges for routine analysis. Here, we review the literature regarding various quantification methods for measuring prohibited substances in athletes as they pertain to World Anti-Doping Agency regulations.

Development and validation of a UHPLC–MS/MS assay for colistin methanesulphonate (CMS) and colistin in human plasma and urine using weak-cation exchange solid-phase extraction

A rapid ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) assay method was developed for determination of CMS and formed colistin in human plasma and urine. After extraction on a 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the eluents were mixed and injected into the UHPLC–MS/MS system directly. A Phonomenex Kinetex XB-C18 analytical column was employed with a mobile phase consisting of solution “A” (acetonitrile:methanol, 1:1, v/v) and solution “B” (0.1% formic acid in water, v/v). The flow rate was 0.4mL/min with gradient elution over 3.5min. Ions were detected in ESI positive ion mode and the precursor-product ion pairs were m/z 390.7/101.3 for colistin A, m/z 386.0/101.2 for colistin B, and m/z 402.3/101.2 for polymyxin B1 (IS), respectively. The lower limit of quantification (LLOQ) was 0.0130 and 0.0251mg/L for colistin A and colistin B in both plasma and urine with accuracy (relative error, %)<±12.6% and precision (relative standard deviation, %)<±10.8%. Stability of CMS was demonstrated in biological samples before and during sample treatment, and in the extract. This new analytical method provides high-throughput treatment and optimized quantification of CMS and colistin, which offers a highly efficient tool for the analysis of a large number of clinical samples as well as routine therapeutic drug monitoring.

Development of an LC-MS/MS method for aromatase inhibitor screening

Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C18 column (3.0 × 50 mm, 2.7 μm) with a mobile phase consisting of 0.1% formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271 → 159 (estrone), and m/z 361 → 163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.

LC–ESI-MS/MS quantification of 4β-hydroxycholesterol and cholesterol in plasma samples of limited volume

A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500ng/ml and 50 to 2000μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor–product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis.The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC–ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.

Simultaneous determination of 9-dehydro-17-hydro-andrographolide and sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate in rat plasma by UHPLC-ESI-MS/MS after administration of xiyanping injection: application to a pharmacokinetic study

9-Dehydro-17-hydro-andrographolide (DHA) and sodium 9-dehydro-17-hydro-andrographolide-19-yl sulfate (DHAS) are active ingredients of xiyanping injection in clinical use. A simple, rapid and sensitive UHPLC-ESI-MS/MS method was developed for the determination of DHA and DHAS in rat plasma, and the pharmacokinetics of DHA and DHAS after intravenous administration of xiyanping injection was investigated. The plasma samples were treated with methanol to precipitate out protein, and the separation of DHA and DHAS was achieved on a Waters BEH C18 column with a mobile phase consisting of acetonitrile and 10 mmol/L ammonium acetate solution at a flow rate of 0.4 mL/min. DHA, DHAS and the internal standard (internal standard, IS) diethylstilbestrol were detected at negative ion mode. The precursor-product ion pairs used in multiple reaction monitoring mode were: m/z 349.1 → 286.9 (DHA), m/z 428.9 → 96.0 (DHAS) and m/z 267.1 → 236.9 (IS). Calibration curves offered satisfactory linearity within the test range, and all correlation coefficients were >0.995. The lower limit of detection of DHA and DHAS in plasma samples were determined to be 0.1 ng/mL. The lower limit of quantitation was 0.5 ng/mL for DHA and DHAS. All the recoveries of the quality control samples were in the range of 86.0-102.4%. The ratios of matrix effect were between 89.2 and 105.1%. The method was fully validated and successfully applied to the pharmacokinetic study of DHA and DHAS in rats. The study showed that both DHA and DHAS were distributed and eliminated rapidly in rats.

Conventional liquid chromatography/triple quadrupole mass spectrometry based metabolite identification and semi-quantitative estimation approach in the investigation of in vitro dabigatran etexilate metabolism

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometry based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor-product ion pairs were predicted to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects on tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using "metabolite standards" generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 converts DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 and M2 were further metabolized to DAB by CES2 and CES1, respectively. The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples.

Metabolite profiling of ginsenoside Re in rat urine and faeces after oral administration

Following oral administration of ginsenoside Re, the compound and its metabolites were identified and quantified in rat urine and faeces by liquid chromatography coupled with triple quadrupole mass spectrometry (LC–MS/MS). Ginsenoside Re (200mg/kg) was orally administered to rats by gastric intubation, and urine and faeces samples were then collected during the next 24h using metabolic cages. Samples were prepared by solid phase extraction and analysed by LC–MS/MS. The precursor-product ion pairs used for LC–MS/MS analysis were: m/z 945→475 for ginsenoside Re, 799→637 for ginsenoside Rg1, 783→475 for ginsenoside Rg2, 637→475 for ginsenosides Rh1 and F1, 475→391 for protopanaxatriol, and 779→641 for digoxin (internal standard). The major ginsenosides excreted in urine were ginsenosides Re and Rg1, and only minimal amounts of ginsenosides Rg2 and Rh1 were found. Greater amounts of ginsenoside metabolites were detected in the faeces samples; biotransformation to ginsenoside Rg1 was predominant but further deglycosylated metabolites including ginsenoside F1 and protopanaxatriol were additionally detected. The total recovery of ginsenosides over 24h was approximately 46%.

Determination of docetaxel in rat plasma and its application in the comparative pharmacokinetics of Taxotere and SID530, a novel docetaxel formulation with hydroxypropyl‐β‐cyclodextrin

In this study, a sensitive, simple and reliable method for the quantification of docetaxel in rat plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The plasma samples were prepared by protein precipitation, and paclitaxel was used as an internal standard (IS). Chromatographic separation was achieved using a Gemini C(18) column (2.0 × 150 mm, 5 µm) with a mobile phase consisting of 0.1% formic acid-acetonitrile (30:70, v/v). The precursor-product ion pairs used for multiple reaction monitoring were m/z 808.5 → 527.5 (docetaxel) and m/z 854.2 → 286.5 (IS, paclitaxel). A calibration curve for docetaxel was constructed over the range 1-1000 ng/mL. The developed method was specific, precise and accurate, and no matrix effect was observed. The validated method was applied in a comparative pharmacokinetic study in which two docetaxel formulations, SID530, a new parenteral formulation of docetaxel with hydroxypropyl-β-cyclodextrin (HP-β-CD), and Taxotere, were administered to rats at a dose of 5 mg/kg. For SID530 and Taxotere, the mean C(0) values were 1494 and 1818 ng/mL, respectively, and the AUC(last) values were 837 and 755 h ng/mL, respectively. These two formulations did not show any statistical differences with regard to the pharmacokinetic parameters, thus establishing that the SID530 and Taxotere products are pharmacokinetically comparable in male rats.