Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

Identification of hydroxy fatty acids by liquid chromatography-atmospheric pressure chemical ionization mass spectroscopy in Euglena gracilis

Hydroxy fatty acids from Euglena gracilis were identified by reverse-phase high performance liquid chromatography coupled to a mass spectrometer run in atmospheric pressure chemical ionization positive ion mode. These metabolites were converted to methyl esters to improve stability and chromatographic properties. A detection limit of 20 pg/microl per injection was determined for 5-HETE methyl ester based on the signal to noise ratio of the m/z 317 ion which corresponds to the loss of a hydroxyl group (M-17) and the major fragment in all HETE methyl esters studied. This is the first report for these metabolites in E. gracilis.     

Pharmacokinetics of ergosterol in rats using rapid resolution liquid chromatography-atmospheric pressure chemical ionization multi-stage tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC-MS(n)) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS(n). The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7-2000, 6-2000 and 8-7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Metabolic fingerprinting of rat urine by LC/MS Part 1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.     

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.     

Fast and reliable determination of voriconazole in human plasma by LC-APCI-MS/MS

A fast and reliable liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method was developed and validated for the quantification of voriconazole in human plasma. The proposed method was validated in a linear range of 50-10,000 ng/ml, and the total run time was 1.5 min. This method was successfully used to support routine therapeutic drug monitoring of voriconazole.     

Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.     

Development and validation of bioanalytical methods for Imidafenacin (KRP-197/ONO-8025) and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

Imidafenacin (KRP-197/ONO-8025, IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a new antimuscarinic agent currently under application for the indication of treatment of overactive bladder in Japan. We developed and validated the sensitive and selective bioanalytical methods for the extremely low levels of IM and its metabolite, M-2 (Method 1), M-4 (Method 2) and M-9 (Method 3) in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In each method, plasma sample was extracted by solid phase extraction, separated on a semi-micro high performance liquid chromatography column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization or ionspray interface. Selected reaction monitoring mode was used for quantification. Each method was found to have acceptable accuracy, precision, stability, selectivity and linearity over the concentration range of 10-500 pg/mL for IM and M-2, 10-1000 pg/mL for M-4 and 50-5000 pg/mL for M-9. Using these analytical methods, concentration profiles of IM and its metabolites in human plasma were successfully determined even in the low pg/mL levels after oral administration of IM at the therapeutic dosage of 0.1 mg.     

A Mass Spectrometric Study for Comparative Analysis and Evaluation of Metabolite Recovery from Plasma by Various Solvent Systems

A solvent system that extracts a maximum number of metabolites belonging to diverse chemical classes from complex biofluids, such as plasma, may offer useful inputs to understand the metabolic and physiological state of an individual. The present study compared seven solvent systems for extraction of metabolites from plasma. The extracts were analyzed by mass spectrometry (MS) and MS/MS (MS2) using a quadrupole time-of-flight liquid chromatography/MS system in positive and negative modes of ionization. Metabolites with molecular mass below 400 were identified using Human Metabolome Database MS2 and MS search interfaces. The acetone/isopropanol (2:1) system yielded promising results in positive ionization mode, as the maximum number of MS and MS2 features was detected in the extract. It was found to be superior in extraction of various classes of metabolites, especially organic acids, nucleosides and nucleoside derivatives, and heterocyclic molecules. Glycerophosphocholines in the mass range of 400–700 were found to be efficiently extracted by the methanol/chloroform/water (8:1:1) system. In negative mode as well, the maximum number of MS2 features was detected in methanol/chloroform/water and acetone/isopropanol extracts. The fingerprints of molecular features obtained in the negative and positive modes differed from each other to a significant extent.     

Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry

Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.     

Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization-tandem mass spectrometry

The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 degrees C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI-tandem mass spectrometry, where CA-GSH was detected on the product ion mass chromatograms monitored with stable and abundant dehydrated positive-ion [M+HH(2)O](+) at m/z 680.3 and fragmented negative-ion [GSHH](-) at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro.     

Metabolite Profiling in Withania somnifera Roots Hydroalcoholic Extract Using LC/MS,GC/MS and NMR Spectroscopy

Ashwagandha (Withania somnifera) is a very well‐known herbal medicine and it was well studied for its active metabolites throughout the World. Although, nearly 40 withanolides were isolated from W. somnifera root extract, still there is remaining unidentified metabolites due to very low abundance and geographical variation. Advanced separation technology with online identification by mass and nuclear magnetic resonance (NMR) are nowadays used to find out the new compounds in the crude herbal extract. This article described the metabolite profiling of ashwagandha root hydroalcoholic extract using ultra‐performance liquid chromatography coupled with a positive ion electrospray ionization tandem mass spectrometry through gas chromatography mass spectrometry (GC/MS) and NMR spectroscopy. A total of 43 possible withanolides was identified and proposed their structures based on the mass of molecular and fragment ions. GC/MS and NMR analysis indicated the presence of several known withanolides including withaferin A, withanolide D, withanoside IV or VI, withanolide sulfoxide, etc. To the best of our knowledge, dihydrowithanolide D at m/z 473 (t_R 7.86 min) and ixocarpalactone A at m/z 505 (t_R 8.43 min) were first time identified in the ashwagandha root hydroalcoholic extract. The current study that described the identification of withanolides with summarized literature review might be helpful for designing the experiment to identify of the new chemical constituents in Withania species.     

Isolation by high pressure liquid chromatography and characterization of benzo(a)pyrene-4'5-epoxide as a metobolite of benzoapyrene.

A high-pressure liquid chromatography (HPLC) system is described that separates at least nine benzo(a)pyrene metabolites including an epoxide. The epoxide metabolite has been isolated and characterized as benzo(a)pyrene-4,5-epoxide by comparison of its HPLC retention times, ultraviolet and mass spectral analysis with synthetic benzo(a)pyrene-4,5-epoxide and its conversion by liver microsomes to benzo(a)pyrene-4,5-dihydrodiol.     

Identification of incurred sulfonamide residues in eggs: methods for confirmation by liquid chromatography-tandem mass spectrometry and quantitation by liquid chromatography with ultraviolet detection

Two complementary methods for identifying and measuring sulfonamide residues in eggs were developed for use in surveying eggs for potential drug residues. The first method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) to confirm the presence of sulfonamide residues in eggs. During its validation the limit of confirmation was estimated to be 5-10 ng/g (ppb) depending on the drug. Also, a method for measuring residue level by liquid chromatography with ultraviolet detection (LC-UV) was validated using the same extraction procedure as the confirmatory method. The determinative method was validated over the 50-200 ppb range. Samples were prepared by homogenizing whole egg, extracting with acetonitrile, and cleaning up with a C(18) solid-phase extraction cartridge. For confirmation, analytes were separated by gradient LC on a C(18) column, ionized by electrospray ionization (ESI), and detected by MS-MS with an ion trap mass spectrometer. For determination, analytes were separated by a different gradient LC procedure and detected by UV at 287 nm. Fifteen drugs were dosed individually in laying hens, and residues of parent drug and/or metabolites were found in eggs for all the drugs. Validation was based on repetitive analyses of control samples, control samples fortified at 100 ppb sulfonamides, and samples of blended incurred eggs.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Identification of felodipine metabolites in rat urine

After intragastric administration of 100 mumol kg-1 [14C]felodipine to rats eight urinary metabolites were isolated. Batch extraction at pH 2.2 and semipreparative reversed-phase liquid chromatography were used for trace enrichment of the metabolites. Trimethylsilylation followed by transesterification with diazomethane blocked the carboxylic acid and alcohol groups selectively before gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. Deuterated derivatives of the metabolites and chemical ionization measurements added complementary structural information. All metabolites reported in this study were formed from oxidized felodipine by ester hydrolysis. Hydroxylation of the pyridine methyl group represented an important metabolic pathway and metabolites oxidized to the corresponding carboxylic acids were detected as well. Lactone formation from hydroxy acid metabolites in urine as a possible analytical artefact is discussed.     

Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.     

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 2-ethyl-5-methyl-3,3-diphenylpyraline and methadol in meconium by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

This paper details a validated liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method for the quantification of methadone, and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) and methadol in human meconium. Limits of detection (LOD) were determined to be 1.0 ng/g for methadone, EDDP and EMDP and 2.5 ng/g for methadol. The limits of quantitation (LOQ) for methadone, EDDP, EMDP were 5 and 25 ng/g for methadol. Linearity ranged from 5.0 to 500 ng/g. Following solid-phase extraction, no matrix effect was observed. This method proved to be suitable for the quantification of methadone, EDDP and EMDP and the semi-quantitation of methadol in meconium. Literature review revealed no other published LC-APCI-MS/MS method for the detection of methadone and its three main metabolites in meconium specimens.     

Investigation of the in vitro biotransformation of R-(+)-thalidomide by HPLC, nano-HPLC, CEC and HPLC–APCI-MS

High-performance liquid chromatography (HPLC), nano-HPLC, capillary electrochromatography (CEC) and on-line HPLC–atmospheric pressure chemical ionization mass spectrometry (APCI-MS) techniques were used for the identification and detailed characterization of two new metabolites of the former sedative drug thalidomide (TD). The advantages of nano-HPLC and CEC are higher peak efficiency and a drastic decrease in the analysis time, which, together with lower sample dilution during the analyses, allowed to obtain a detection sensitivity that was comparable to HPLC with common-sized columns. Both, nano-HPLC and CEC could be realized in the commercially available capillary electrophoresis system HP^3D. On-line HPLC–APCI-MS coupling is a very useful technique for the rapid identification of metabolites without any need for reference compounds.