Determination of alprostadil in rat plasma by ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry after intravenous administration

A rapid, highly selective ultra performance liquid chromatography–electrospray ionisation–tandem mass spectrometry method (UPLC–ESI–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of alprostadil in rat plasma. After a simple sample preparation procedure involving a one-step liquid–liquid extraction, alprostadil and the internal standard, diphenhydramine, were chromatographed on an ACQUITY UPLC™ BEH C₁₈ column with gradient elution using a mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.25 mL min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r² = 0.99) over the concentration range 0.4–250.0 ng mL⁻¹, with a lower limit of quantification of 0.4 ng mL⁻¹ for alprostadil. The inter- and intra-day precision (%R.S.D.) was less than 8.5% and 2.4%, respectively, and the accuracy (RE%) was between 9.3% and 1.0% (n = 6). Alprostadil in rat plasma was stable when stored at room temperature for 0.5 h and at −20 °C for two weeks. The method was very rapid, simple and reliable, and was employed for the first time for the pharmacokinetic studies of alprostadil in rats after a single intravenous administration of 50 μg kg⁻¹.     

Determination of two COX-2 inhibitors in serum and synovial fluid of patients with inflammatory arthritis by ultra performance liquid chromatography–inductively coupled plasma mass spectroscopy and quadrupole time-of-flight mass spectrometry

The determination of two sulphur-containing drugs, the COX-2 inhibitors celecoxib and etoricoxib, in the serum and synovial fluid of inflammatory arthritis patients, is described using a sensitive ultra performance liquid chromatography–inductively coupled plasma mass spectroscopy (UPLC/ICPMS) method. Confirmation of the identity of the analytes in the samples was also performed by electrospray quadruple time-of-flight mass spectrometry in positive electrospray ionisation mode. The two COX-2 inhibitors were extracted from serum and synovial fluid following dilution with acetate buffer (pH 5) and liquid–liquid extraction (LLE) into ethyl acetate. Extracted samples were then analysed using UPLC/ICPMS with sulphur-specific detection. The limit of detection by UPLC/ICPMS was 0.45 ng/ml of sulphur in both serum and synovial fluid. The UPLC/ICPMS method was applied to the analysis of samples from patients receiving either 200 mg/day of celecoxib (2× 100 mg), 90 mg/day etoricoxib or placebo. The range of concentrations detected in the samples for the two drugs was from 0.3 to 3.3 μg/ml.     

Characterization of Heat-Labile toxin-subunit B from Escherichia coli by liquid chromatography–electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation.     

Plasma pharmacokinetics,tissue distribution and excretion study of 6-gingerol in rat by liquid chromatography–electrospray ionization time-of-flight mass spectrometry

A rapid resolution liquid chromatography coupled with electrospray ionization (ESI) time-of-flight mass spectrometry method was developed and validated for quantitative analysis of 6-gingerol in plasma and various tissues. Liquid–liquid extraction was employed as sample preparation technique. Biological samples were separated on an Agilent Zorbax StableBond-C₁₈ column (4.6 mm × 50 mm, 1.8 μm) and detected by TOF/MS with electrospray ionization (ESI) interface in positive ion mode. Calibration curves (1/x² weighted) offered satisfactory linearity (r² > 0.995) within the test range. The lower limit of quantification in different matrices was in a range of 10–100 ng/mL. Inter- and intra-day precision were in the range of 0.91–11.90% and 0.75–10.23%, respectively. Recoveries in plasma, urine and tissues ranged from 72.5% to 90.4%. Glucuronide of 6-gingerol, the major metabolite of 6-gingerol, was further determined after β-glucuronidase hydrolyzation. This developed method was successfully applied to pharmacokinetics, tissue distribution and excretion studies of 6-gingerol after oral or intraperitoneal administration in rats.     

Rapid identification of acetophenones in two Cynanchum species using liquid chromatography–electrospray ionization tandem mass spectrometry

Acetophenones in Cynanchum species, especially cynandione A and its derivatives, whose utilization and toxicity in herbal drugs and folk medicines has caused great interest in the chemical investigation, have extensive biological activities. In this paper, a facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MSⁿ) was developed for the analysis of cynandione A derivatives in the roots of the Cynanchum wilfordii and C. auriculatum. ESI-MS/MS and ESI-MSⁿ analysis of cynandiones A and B in negative ion mode were firstly performed employing two mass spectrometers each equipped with an ion-trap and a quadrupole time-of-flight (Q-TOF) mass analyzer. The results drawn from both instruments were similar to each other. Characteristic fragmentation pathways were proposed by comparing the spectra of two standards acquired in the experiments. The fragment ions at m/z 283 and 268 were obtained, and then were used as diagnostic ions to screen and identify cynandione A derivatives from the roots of above two species, together with an HPLC-MSⁿ method. Total of 28 cynandione A derivatives comprising 4 reported and 24 novel components were identified or tentatively identified. Furthermore, breakdown curves were constructed to distinguish two types of isomers among these compounds. To our knowledge, this is the first report on characterization of acetophenones by HPLC-ESI-MSⁿ, which allows a rapid and complete analysis of cynandione A derivatives in roots of Cynanchum species.     

Suspected-target and non-targeted screenings of phosphodiesterase 5 inhibitors in herbal remedies using liquid chromatography–quadrupole time-of-flight–mass spectrometry

The lucrative market of herbal remedies spurs rampant adulteration, particularly with pharmaceutical drugs and their unapproved analogues. A comprehensive screening strategy is, therefore, warranted to detect these adulterants and, accordingly, to safeguard public health. This study uses the data-dependent acquisition of liquid chromatography-quadrupole time-of-flight–mass spectrometry (LC–QTOF–MS) to screen phosphodiesterase 5 (PDE5) inhibitors in herbal remedies using suspected-target and non-targeted strategies. For the suspected-target screening, we used a library comprising 95 PDE5 inhibitors. For the non-targeted screening, we adopted top-down and bottom-up approaches to flag novel PDE5 inhibitor analogues based on common fragmentation patterns. LC–QTOF–MS was optimised and validated for capsule and tablet dosage forms using 23 target analytes, selected to represent different groups of PDE5 inhibitors. The method exhibited excellent specificity and linearity with limit of detection and limit of quantification of <40 and 80 ng/mL, respectively. The accuracy ranged from 79.0% to 124.7% with a precision of <14.9% relative standard deviation. The modified, quick, easy, cheap, effective, rugged, and safe extraction provided insignificant matrix effect within −9.1%–8.0% and satisfactory extraction recovery of 71.5%–105.8%. These strategies were used to screen 52 herbal remedy samples that claimed to enhance male sexual performance. The suspected-target screening resulted in 33 positive samples, revealing 10 target analytes and 2 suspected analytes. Systematic MS and tandem MS interrogations using the non-targeted screening returned insignificant signals, indicating the absence of potentially novel analogues. The target analytes were quantified from 0.03 to 121.31 mg per dose of each sample. The proposed strategies ensure that all PDE5 inhibitors are comprehensively screened, providing a useful tool to curb the widespread adulteration of herbal remedies.     

Rapid analysis of drugs of abuse and their metabolites in human urine using dilute and shoot liquid chromatography–tandem mass spectrometry

Liquid chromatography-tandem mass spectrometric method for analysis of 113 abuse drugs and their metabolites in human urine was developed and validated. A simple sample clean-up procedure using the “dilute and shoot” approach, followed by reversed phase separation, provided a fast and reliable method for routine analysis. Drugs were separated in a Capcell Pak MG-III C₁₈ column using a gradient elution of 1 mM ammonium formate with 0.1% formic acid in water and acetonitrile. The total time for analysis was 32 min. The multiple reaction monitoring mode using two transitions (e.g., quantifier and qualifier) was optimized for both identification and determination. The calibration curves for each analyte were linear over the concentration ranges of 1–100, 5–100, or 10–100 ng/mL using 400 μL of human urine sample with the coefficient of determination above 0.9921. The coefficient of variation and accuracy for the intra- and inter-assays of the tested drugs at three QC levels were 1.1–14.6 and 86.7–106.8%, respectively. The present method was successfully applied to the analysis of forensic urine samples obtained from 17 drug abusers. This method is useful for the rapid and accurate determination of multiple drug abuse with a small amount of urine in forensic and clinical toxicology.     

Rapid and global detection and characterization of aconitum alkaloids in Yin Chen Si Ni Tang,a traditional Chinese medical formula,by ultra performance liquid chromatography–high resolution mass spectrometry and automated data analysis

An improved method employing Metabolynx XS with mass defect filter (MDF), a post-acquisition data processing software, was developed and applied for global detection of aconitum alkaloids in Yin Chen Si Ni Tang, a traditional Chinese medical formula (TCMF). The full-scan LC–MS/MS data sets with extra mass were acquired using ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) with the MS^E mode in a single injection. To remove the interferences, Metabolynx XS was optimized to extract the ions of aconitum alkaloids located at the lower abundance. As a result, 62 ions were assigned rapidly to aconitum alkaloids and identified tentatively by comparing the accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literatures. Compared with the previous studies on Fuzi-containing TCMF, the report detected more aconitum alkaloids, and the analysis process was accelerated by automated data processing. It is concluded that the screening capability of Metabolynx XS with MDF, together with the utilization of MS^E in structural elucidation, can facilitate a rapid and comprehensive searching and effective structural characterization of aconitum alkaloids in TCMF.     

Simultaneous quantification of active components in the herbs and products of Si-Wu-Tang by high performance liquid chromatography–mass spectrometry

Si-Wu-Tang (SWT), comprising Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular Traditional Chinese Medicine (TCM) formulae for woman's health. Data mining from the available Chinese and English literatures indicated that the major bioactive components of SWT consist of paeoniflorin, paeonol, gallic acid, ferulic acid, Z-ligustilide, ligustrazine, butylphthalide, senkyunolide A and catalpol. Since content determination of the marker compounds is generally considered as an initial step for quality control of TCM product, a high performance liquid chromatography–mass spectrometric method employing both positive and negative electrospray ionization was developed for the simultaneous determination of the nine identified compounds in the raw herbs and products of SWT. The LOQ of the developed assay method for the tested components was 10 ng/ml for ligustrazine, 200 ng/ml for catalpol, and 100 ng/ml for the other seven compounds. The intra-day and inter-day variations of the current assay were within 17.5%. Paeoniflorin, ferulic acid, gallic acid, Z-ligustilide and senkyunolide A were found in all SWT products investigated. Variations in the contents of the studied compounds were observed among batches of raw herbs and SWT products. The currently developed method provides a sensitive and rapid quantification approach that can be useful in the quality control of raw herbs and products of SWT.     

Research on accurate quantitation technique using liquid chromatography coupled with triple quadrupole mass spectrometry and experiment optimization—take a novel diuretic as an example

Liquid chromatography coupled with the triple quadrupole mass spectrometry (LC-QQQ MS) technique is the most commonly used technique for quantitative analysis. It is widely used in pharmacology, targeted metabolomics, Chinese medicine quality control, and other research fields. The technique monitors only characteristic precursors and product ions through multiple reaction monitoring (MRM) mode and can detect targeted molecules in complex matrix with high specificity and sensitivity. In the present study, a diarylamide derivative diuretic was used as an example to introduce the method establishment and parameter optimization of this liquid chromatography-mass spectrometry technique. Diuretic and its internal standard could be completely separated within a 5-min gradient, and the quantitative linear range was 1–1000 ng/mL with R² = 0.9996 in practical samples. This study showed that the key to the method development for LC-QQQ MS was the selection of the LC mobile phase, the elution gradient, the declustering potential, and the collision energy of compounds in MS.     

A quantitative method for the simultaneous detection of SR9009 and SR9011 in equine plasma using liquid chromatography–electrospray ionization–tandem mass spectrometry

SR9009 and SR9011 are metabolic modulators pharmacologically targeting REV-ERB receptors as synthetic agonists. A liquid chromatography–tandem mass spectrometry method for the detection of SR9009 and SR9011 in equine plasma was developed and validated. Plasma samples were pretreated by protein precipitation with methanol and were loaded onto an ACQUITY ultra performance liquid chromatography high-strength silica C18 column (2.1 × 150 mm, 1.8 μm) for chromatographic separation. The mobile phase consisted of 5-mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in acetonitrile, and a gradient elution was used at a flow rate of 0.25 ml/min. For the mass spectrometry detection, the selected reaction monitoring mode was used with transitions of 438.2 → 124.9 for SR9009, 479.2 → 125.1 for SR9011, and 292.2 → 109.1 for the internal standard (testosterone-d3) in the positive ionization mode. The linearity, lower limit of quantification, intra- and inter-day precision, accuracy, matrix effect, recovery, and stability were evaluated. The method was found to be accurate and reproducible for the quantitation of SR9009 and SR9011. The developed method was successfully applied to plasma samples of thoroughbreds injected intramuscularly with SR9009.     

Comprehensive characterization of the in vitro and in vivo metabolites of geniposide in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer

1.?Geniposide (genipin 1-O-glucose), one of the major bioactive constituents isolated from Fructus Gardeniae, possesses many biological activities. In this study, an efficient strategy was developed using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer (UPLC–LTQ–Orbitrap) to profile the in vitro and in vivo metabolic patterns of geniposide in rat liver microsomes (RLMs), plasma, urine, and various tissues. And post-acquisition data-mining methods including extracted ion chromatogram (EIC), multiple mass defect filters (MMDF), fragment ion searching (FISh), and isotope pattern filtering (IPF) were adopted to characterize the known and unknown metabolites.

2.?A total of 33 metabolites were detected and interpreted according to accurate mass measurement, diagnostic fragment ions, relevant drug biotransformation knowledge, and bibliography data. Among them, 17 metabolites were detected in the plasma, 31 metabolites were identified in the urine, six metabolites could be found in rat heart, 12 in liver, three in spleen, six in lung, 12 in kidney, six in brain, and four in RLMs.

3.?A series of corresponding reactions such as hydrolysis, hydroxylation, taurine conjugation, hydrogenation, decarboxylation, demethylation, sulfate conjugation, cysteine S-conjugation, glucosylation, and their composite reactions were all discovered.

4.?The results could provide comprehensive insights and guidance for elucidation of side effect mechanism and safety monitoring as well as for rational formulation design in drug delivery system. The newly discovered geniposide metabolites could be targets for future metabolism studies on the important chemical constituents from herbal medicines.     

A novel approach to rapidly explore analytical markers for quality control of Radix Salviae Miltiorrhizae extract granules by robust principal component analysis with ultra-high performance liquid chromatography–ultraviolet–quadrupole time-of-flight mass spectrometry

In a well-controlled experiment, outliers discriminated by robust principal component analysis (RPCA) represent contents in samples which are of particular quality distinguishable from the rest of the others, therefore chemical constituents in a natural product causing discrimination between outliers and the majority of samples could be considered as analytical markers for quality control. Based on this strategy, a novel approach for rapidly exploring characteristic analytical markers was proposed for the quality control of extract granules of Radix Salviae Miltiorrhizae (EGRSM). In this study, large sizes of samples were analyzed via high-throughput ultra-high performance liquid chromatography–ultraviolet–quadrupole time-of-flight mass spectrometry (UHPLC–UV–Q-Tof MS). RPCA was first performed on the three groups of samples: RSM (the raw material), the in-house prepared aqueous extract of Radix Salviae Miltiorrhizae (AERSM) and commercial product of EGRSM, to determine the variation of specific constituents between raw material and the final products as well as the effect of manufacturing process on the overall quality. Then RPCA was performed on the commercial products of EGRSM to explore the applicability of identified characteristic markers for the quality control of EGRSM. Candidate markers were extracted by RPCA, and their molecular formulae were determined by high resolution electrospray ionization-mass spectrometric (ESI-MS) analysis. The suitability of identified markers was then evaluated by determining the relationship between quantities of the identified markers with their antioxidant activities biologically, and further confirmed in a variety of samples. In conclusion, the combination of RPCA with UHPLC–UV–Q-Tof MS is a reliable means to identify chemical markers for evaluating quality of herbal medicines.     

Separation and structural elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement by liquid chromatography–electrospray ionization mass spectrometry,infrared spectroscopy and nuclear magnetic resonance spectroscopy

MEGATON, a dietary supplement, was analyzed in order to detect PDE-5 inhibitors and their analogues. A new analogue of vardenafil could be detected by high-performance liquid chromatography (HPLC) analysis with a photodiode array detector (PDA). This compound was compared with sildenafil, tadalafil, and vardenafil as well as their structurally modified analogues such as hongdenafil and homosildenafil. The structure of this compound was elucidated by mass spectrometry (MS), infrared (IR) spectroscopy and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. When compared with vardenafil to verify the structural difference, this compound had an acetyl group instead of a sulfonyl group in the pyrazolopyrimidine portion without any substitution in the piperazine ring of the molecule. This compound was identified as 2-(2-ethoxy-5-(2-(4-ethylpiperazin-1-yl)acetyl)phenyl)-5-methyl-7-propyl-imidazo(5,1-f)-(1,2,4)triazin-4(3H)-one, which is also called acetylvardenafil.