Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry

Analysis of nucleosides and nucleotides is desirable in many biological studies, but the task is analytically challenging due to the high polarity of the analytes. In this study, resolution of mixtures containing nucleosides and their mono-, di- and triphosphates was achieved using a porous graphitic carbon (PGC) stationary phase, Hypercarb, under conditions suitable for liquid chromatography/mass spectrometry (LC/MS). Different organic mobile phases and modifiers were evaluated and the separation of 16 nucleosides and nucleotides was optimized using gradient elution with a water/acetonitrile mobile phase containing ammonium acetate and diethylamine as modifiers. The ammonium acetate concentration proved to be critical for retention and diethylamine was found to improve the peak shapes of di- and triphosphates for mass spectrometric detection. A variety of silica-based columns designed for polar compound separation were also tested using optimized LC conditions and compared with results obtained with the Hypercarb column. Only the Hypercarb column provided separations suitable for accurate quantitation of mixed nucleosides and their phosphates.     

Phase I and II metabolites of speciogynine, a diastereomer of the main Kratom alkaloid mitragynine, identified in rat and human urine by liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry

Mitragyna speciosa (Kratom in Thai), a Thai medical plant, is misused as herbal drug of abuse. Besides the most abundant alkaloids mitragynine (MG) and paynantheine (PAY), several other alkaloids were isolated from Kratom leaves, among them the third abundant alkaloid is speciogynine (SG), a diastereomer of MG. The aim of this present study was to identify the phase I and II metabolites of SG in rat urine after the administration of a rather high dose of the pure alkaloid and then to confirm these findings using human urine samples after Kratom use. The applied liquid chromatography coupled to low- and high-resolution mass spectrometry (LC-HRMS-MS) provided detailed information on the structure in the MS(n) mode particularly with high resolution. For the analysis of the human samples, the LC separation had to be improved markedly allowing the separation of SG and its metabolites from its diastereomer MG and its metabolites. In analogy to MG, besides SG, nine phase I and eight phase II metabolites could be identified in rat urine, but only three phase I and five phase II metabolites in human urine. These differences may be caused by the lower SG dose applied by the user of Kratom preparations. SG and its metabolites could be differentiated in the human samples from the diastereomeric MG and its metabolites comparing the different retention times determined after application of the single alkaloids to rats. In addition, some differences in MS(2) and/or MS(3) spectra of the corresponding diastereomers were observed.     

Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.     

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC–MS/MS with metal-free column

The metabolome presence of nucleobases, nucleosides, nucleotides and related phosphorylated metabolites has been examined for Alzheimer's disease (AD). Although reversed-phase liquid chromatography tandem mass spectrometry (LC–MS/MS) has been used for the determination of these analytes, they were limited in chromatographic signal intensity and reproducibility owing to significant peak tailing caused by complexing with metallic cations and phosphate groups. In this work, we applied LC–MS/MS analysis with a metal-free column for comprehensive quantification of 40 analytes regarding to purine and pyrimidine metabolism in postmortem cerebrospinal fluid (pCSF) from AD patients. For the analytical column, an InertSustain AQ-C₁₈ metal-free PEEK column was used. MS detection was by electrospray positive ionization. The metal-free column allowed for sharp peak detection of highly polar metabolites within a running time of 17 min. In validation, the limits of detection (LOD), the limit of quantitation (LOQ) and recovery value using a pooled pCSF sample are 1–500 nM, 0.5–250 nM and a range of 53.1–144.0% (RSD ranged from 0.4 to 19.6%). The developed LC–MS/MS method utilizing a metal-free column provides an accurate quantification of some metabolites regarding purine and pyrimidine metabolism in pCSF samples obtained from AD patients.     

The need for chromatographic and mass resolution in liquid chromatography/tandem mass spectrometric methods used for quantitation of lactones and corresponding hydroxy acids in biological samples

Because of the potential in-source conversion between a lactone and the corresponding hydroxy acid, it has been recognized that a liquid chromatography/tandem mass spectrometric (LC/MS/MS) method developed for quantitation of a lactone drug in the presence of its hydroxy acid metabolite (or vice versa) must incorporate chromatographic separation between the two compounds, unless in-source conversion between the two compounds has been eliminated by the appropriate selection of the LC/MS/MS parameters. We now report that chromatographic separation between a lactone and its hydroxy acid will be required under certain LC/MS/MS conditions used even in the absence of in-source conversion. This is due to the fact that the 18-mass-unit difference between a lactone and its hydroxy acid is, by coincidence, different by only one mass unit from the 17-mass-unit difference between the [M + H](+) and [M + NH(4)](+) ions of the lactone or the hydroxy acid. Thus, the [M + H](+) ion of a hydroxy acid is higher than the [M + NH(4)](+) ion of its lactone by only one mass unit. Therefore, in a method developed for quantitation of a hydroxy acid drug utilizing a selected-ion-monitoring (SRM) scheme that incorporates its [M + H](+) ion as the precursor ion, the quantitation would be inaccurate due to the interference by the contribution of the A + 1 isotope response from the [M + NH(4)](+) ion of the lactone metabolite present in the sample, unless there is a chromatographic separation between the two compounds. This is true even if Q1 is operated under a unit-mass resolution. The implication of this type of interference, arising from the presence of both the [M + H](+) and [M + NH(4)](+) ions of a drug and its metabolite, to the selection of LC and MS conditions (including mass resolution) will be discussed using the data obtained with a model lactone drug and its hydroxy acid metabolite.     

A strategy for a post-method-validation use of incurred biological samples for establishing the acceptability of a liquid chromatography/tandem mass-spectrometric method for quantitation of drugs in biological samples

Validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) methods are now widely used for quantitation of drugs in post-dose (incurred) biological samples for the assessment of pharmacokinetic parameters, bioavailability and bioequivalence. In accordance with the practice currently accepted within the pharmaceutical industry and the regulatory bodies, validation of a bioanalytical LC/MS/MS method is performed using standards and quality control (QC) samples prepared by spiking the drug (the analyte) into the appropriate blank biological matrix (e.g. human plasma). The method is then declared to be adequately validated for analyzing incurred biological samples. However, unlike QC samples, incurred samples may contain an epimer or another type of isomer of the drug, such as a Z or E isomer. Such a metabolite will obviously interfere with the selected reaction monitoring (SRM) transition used for the quantitation of the drug. The incurred sample may also contain a non-isomeric metabolite having a molecular mass different from that of the drug (such an acylglucuronide metabolite) that can still contribute to (and hence interfere with) the SRM transition used for the quantitation of the drug. The potential for the SRM interference increases with the use of LC/MS/MS bioanalytical methods with very short run times (e.g. 0.5 min). In addition, a metabolite can potentially undergo degradation or conversion to revert back to the drug during the multiple steps of sample preparation that precede the introduction of the processed sample into the LC/MS/MS system. In this paper, we recommend a set of procedures to undertake with incurred samples, as soon as such samples are available, in order to establish the validity of an LC/MS/MS method for analyzing real-life samples. First, it is recommended that the stability of incurred samples be investigated 'as is' and after sample preparation. Second, it is recommended that potential SRM interference be investigated by analyzing the incurred samples using the same LC/MS/MS method but with the additional incorporation of the SRM transitions attributable to putative metabolites (multi-SRM method). The metabolites monitored will depend on the expected metabolic products of the drug, which are predictable based on the functional groups present in the chemical structure of the drug. Third, it is recommended that potential SRM interference be further investigated by analyzing the incurred samples using the multi-SRM LC/MS/MS method following the modification of chromatographic conditions to enhance chromatographic separation of the drug from any putative metabolites. We will demonstrate the application of the proposed strategy by using a carboxylic acid containing drug candidate and its acylglucuronide as a putative metabolite. Plasma samples from the first-in-man (FIM) study of the drug candidate were used as the incurred samples.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the clinical reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C8 column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria

Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid and malonyl-CoA/3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate/fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one (13)C-labeled I.S., the addition of global (13)C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global (13)C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in Methylobacterium extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of M. extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies.     

Analysis of iron-phytosiderophore complexes in soil related samples: LC-ESI-MS/MS versus CE-MS

Phytosiderophores (PS) form stable complexes with various transition metals. These ligands are exuded by the roots of graminacous plants as a mechanism for mobilizing and acquiring soil iron. To investigate iron mobilization and transport, a novel LC method in combination with ESI-MS/MS for the determination of three Fe(III)-complexes with mugineic acid (MA), 2'-epi-MA and 2'-deoxymugineic acid (DMA) has been developed. Liquid chromatographic separation was realized using a silica-based mixed-mode reversed phase/weak-anion exchange type stationary phase and a 50 mM ammonium acetate buffer, pH 6.5. Baseline separation of the two complex diastereomers Fe(III)-MA and Fe(III)-epi-MA could be achieved. ESI-MS/MS detection allowed for simultaneous quantification of the complexes and the free ligands. Limits of detection were determined to be 0.001 and 0.05 μM for DMA and Fe(III)-DMA, respectively. The analytical figures of merit of the novel method were evaluated and compared with a CE-ESI-MS method that we had published earlier. The LC-ESI-MS/MS method has been successfully applied to real samples derived from preliminary extraction experiments.     

Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.     

High-performance liquid chromatographic determination of loxoprofen and its diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6,7-methylenedioxycoumarin

A simple and sensitive high-performance liquid chromatographic procedure to determine loxoprofen and its diastereomeric alcohol metabolites in biological specimens is described. The analysis involves liquid-liquid extraction with benzene, pre-column derivatization with a highly fluorogenic reagent, 4-bromomethyl-6,7-methylenedioxycoumarin (BrMDC) and subsequent separation on a reversed-phase column. Loxoprofen, its pharmacologically active metabolite, trans-alcohol, and less active cis-alcohol were completely separated within 20 min with a mobile phase of 55% of aqueous acetonitrile containing acetic acid. Any endogenous substances do not interfere in the analysis of either plasma or urine samples. The quantitation limit was 0.01 micrograms/ml for human plasma and 0.05 micrograms/ml for urine. The method was applied to a pharmacokinetic study in healthy human subjects who had received 60 mg of loxoprofen sodium.     

Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.     

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.     

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.     

A Nano-Chip-LC/MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ × 150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS ⁿ identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh⁵C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh⁵C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh⁵C was readily identified by its MH⁺ m/z 260 and MSⁿ fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic beta-substituted alpha-amino acids on a teicoplanin chiral stationary phase

High-performance liquid chromatographic (HPLC) separation of stereomeric cyclic beta-substituted or-quaternary alpha-amino acids was performed on a chiral stationary phase based on the glycopeptide antibiotic teicoplanin. The investigated amino acids are the 1-amino-2-methylcyclohexanecarboxylic acids, the 1-amino-2-hydroxycyclohexanecarboxylic acids, Ala, Cha, Phe and Tle. The effects of the mobile phase composition (type and content of organic modifier, pH) and of the temperature on the enantio- and diastereoselectivity were studied and the conditions were optimised to resolve the four stereomers of one amino acid in a single chromatographic run. The influence of the modifier concentration and the pH of the mobile phase reveal two enantiomeric and diastereomeric discrimination mechanisms based on different interactions with the stationary phase. For optimal separation of diastereomers the column has to be conditioned with an acidic eluent.     

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism.     

Pharmacokinetics and excretion study of bergenin and its phase II metabolite in rats by liquid chromatography tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of bergenin and its phase II metabolite in rat plasma, bile and urine has been developed. Biological samples were pretreated with protein precipitation extraction procedure and enzymatic hydrolysis method was used for converting glucuronide metabolite to its free form bergenin. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reaction monitoring. Negative electrospray ionization was employed as the ionization source. Sulfamethoxazole was used as the internal standard. The separation was performed on a reverse‐phase C₁₈ (250 × 4.6 mm, 5 μm) column with gradient elution consisting of methanol and 0.5% aqueous formic acid. The concentrations of bergenin in all biological samples were in accordance with the requirements of validation of the method. After oral administration of 12 mg/kg of the prototype drug, bergenin and its glucuronide metabolite were determined in plasma, bile and urine. Bergenin in bile was completely excreted in 24 h, and the main excreted amount of bergenin was 97.67% in the first 12 h. The drug recovery in bile within 24 h was 8.97%. In urine, the main excreted amount of bergenin was 95.69% in the first 24 h, and the drug recovery within 24 h was <22.34%. Total recovery of bergenin and its glucuronide metabolite was about 52.51% (20.31% in bile within 24 h, 32.20% in urine within 48 h). The validated method was successfully applied to pharmacokinetic and excretion studies of bergenin.