Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.     

Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

Detection of banned nitrofuran metabolites in animal plasma samples using UHPLC-MS/MS

The use of nitrofurans as veterinary drugs in food-producing animals has been banned in the EU since the 1990s. Monitoring programs in the EU are based on the detection of protein-bound metabolites after slaughter. An UHPLC-MS/MS method was developed and validated for pre slaughter determination of four nitrofuran metabolites (AHD, AOZ, SEM, AMOZ) in animal plasma (bovine, ovine, equine and porcine). This method is proposed as an alternative method for on-farm surveillance. Plasma samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with organic solvent. Extracts were concentrated and then analysed by UHPLC-MS/MS. The method was validated according to Commission Decision 2002/657/EC. Inter-species recovery for AHD, AOZ, SEM and AMOZ was 72, 74, 57 and 71%, respectively. Decision limits (CCα) were calculated from within laboratory reproducibility experiments to be 0.070, 0.059, 0.071 and 0.054 μg kg(-1), respectively. In addition, the assay was applied to incurred plasma samples taken from pigs treated with furazolidone.     

Pharmacokinetics of kakkalide and its main metabolites in rat plasma determined by HPLC-DAD and LC-MSn

This study was undertaken to assess the plasma pharmacokinetic profile of kakkalide (KA), the major isoflavone found in extracts from the dried flower of Pueraria lobata. The main metabolites were identified using HPLC-DAD or LC/MS/MS method, and a HPLC-UV method for simultaneous quantification of the metabolites as well as the parent compound in plasma was developed. Rat plasma contained three glucuronide metabolites, irisolidone-7-O-glucuronide (Ir-7G), tectorigenin-7-O-glucuronide (Te-7G) and 6-OH biochanin A-glucuronide (6-OH BiA-G), as well as KA and trace amount of irisolidone (Ir) after oral administration of 200 mg/kg KA. The pharmacokinetics of KA and three glucuronide conjugates in rat plasma was determined for the first time using a simple, selective and accurate HPLC method. The AUC(0-t) values of the glucuronide metabolites are significantly greater than that of KA. They were detectable in rat plasma at different time points, indicating that glucuronidation during KA metabolism in vivo may occur in different sites, first in intestine and then in liver. Moreover, enterohepatic recirculation may result in the slow elimination of these glucuronide metabolites.     

Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-beta-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-beta-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid-liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C(18) column (5 microm, 4.6mm i.d. x 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 x 10(-4) to 70.4 x 10(-4) micromol/mL (0.050-2.00 microg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 microg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.     

Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro C18 column eluted with a mobile phase consisting of hexafluoro-2-propanol and triethylamine in methanol at 50 degrees C. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC-MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.     

Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB.     

Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma

Glycerophosphocholines (GPCho's) are known to cause liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) matrix ionization effects during the analysis of biological samples (i.e. blood, plasma). We have developed a convenient new method, which we refer to as "in-source multiple reaction monitoring" (IS-MRM), for detecting GPCho's during LC-MS/MS method development. The approach uses high energy in-source collisionally induced dissociation (CID) to yield trimethylammonium-ethyl phosphate ions (m/z 184), which are formed from mono- and disubstituted GPCho's. The resulting ion is selected by the first quadrupole (Q1), passed through the collision cell (Q2) in the presence of collision gas at low energy to minimize fragmentation, and m/z 184 selected by the third quadrupole. This approach can be combined with standard multiple reaction monitoring (MRM) transitions with little compromise in sensitivity during method development and sample analysis. Hence, this approach was used to probe ionization matrix effects in plasma samples. The resulting information was employed to develop LC-MS/MS analyses for drugs and their metabolites with cycle times less than 5 min.     

A sensitive and selective HPLC/ESI-MS/MS assay for the simultaneous quantification of 16-dehydropregnenolone and its major metabolites in rabbit plasma

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M(1)), 5-pregnene-3beta-ol-20-one (M(2)), 5-pregnene-3beta, 20-diol (M(3)), 5-pregnene-3beta-ol-16, 17-epoxi-20-one (M(4)) and 5,16-pregnadien-3beta, 11-diol-20-one (M(5)) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS-MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M(3) and M(5) where linearity was over the 3.13-400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.     

Determination of risperidone and enantiomers of 9-hydroxyrisperidone in plasma by LC-MS/MS

A robust and validated liquid-liquid extraction LC-MS/MS method was developed for population pharmacokinetic analysis and therapeutic drug monitoring of risperidone and the enantiomers of its major active metabolite (+)-and (-)9-hydroxyrisperidone in pediatric patients. The method was rapid, sensitive and used a low sample amount (200 microL), which is very desirable for the pediatric population. The assay was validated from 0.2 to 50 ng/mL in plasma for all analytes. LLOQ for all analytes was 0.2 ng/mL. The extracts were analyzed by normal phase LC-MS/MS. The sample run time was 8 min. Intra- and interday precision for all analytes was < or =6%; method accuracy was between 89 and 99%. Additional experiments were performed to analyze matrix effects and identify a proper internal standard for each analyte. The validated method was used to study risperidone and its enantiomer metabolites in plasma as part of a population pharmacokinetic study in pediatric patients with pervasive developmental disorder (PDD).     

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.     

Quantification of steroid glycosides from Hoodia gordonii in porcine plasma using high performance liquid chromatography-mass spectrometry

An HPLC-ESI-MS/MS method using collision induced dissociation - multiple reaction monitoring was developed for the quantification of eight Hoodia gordonii steroid glycosides and their metabolites in porcine plasma samples. The method was validated for the three most important glycosides and was successfully applied also for the related glycosides and metabolites. The limits of quantification were 0.04 ng ml(-1) for the two main steroid glycosides and 0.1 ng ml(-1) for the detiglated metabolites. These limits are sufficiently low to allow monitoring the concentration-time profiles in plasma after feeding H. gordonii. The standard deviations of the intra-day measurements were better than 20% for concentrations below 5 ng ml(-1) and better than 10% for concentrations above 5 ng ml(-1). The method was successfully applied to plasma samples collected from a porcine pharmacokinetics study.     

Determination of xanomeline (LY246708 tartrate), an investigational agent for the treatment of Alzheimer's disease, in rat and monkey plasma by capillary gas chromatography with nitrogen-phosphorus detection

A GC method is described for the determination of xanomeline (LY246708 tartrate) and selected metabolites in rat and monkey plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane. The organic extract was evaporated to dryness and the residue was reconstituted in hexane. The analytes were separated from metabolites and endogenous substances using a DB1701 capillary column. The analytes were detected using nitrogen-phosphorus detection (NPD). The limit of quantitation was determined to be 8 ng/ml, and the response was linear from 8 to 800 ng/ml. The method has been successfully applied to rat and monkey samples pursuant to the development of xanomeline as an agent for the symptomatic treatment of Alzheimer's disease.     

A GC-MS/MS method for the quantitative analysis of low levels of the tyrosine metabolites maleylacetone, succinylacetone, and the tyrosine metabolism inhibitor dichloroacetate in biological fluids and tissues

We developed a sensitive method to quantitate the tyrosine metabolites maleylacetone (MA) and succinylacetone (SA) and the tyrosine metabolism inhibitor dichloroacetate (DCA) in biological specimens. Accumulation of these metabolites may be responsible for the toxicity observed when exposed to DCA. Detection limits of previous methods are 200 ng/mL (1.2 pmol/microL) (MA) and 2.6 microg/mL (16.5 pmol/microL) (SA) but the metabolites are likely present in lower levels in biological specimens. To increase sensitivity, analytes were extracted from liver, urine, plasma and cultured nerve cells before and after dosing with DCA, derivatized to their pentafluorobenzyl esters, and analyzed via GC-MS/MS.     

Routine determination of [18F]-L-6-fluorodopa and its metabolites in blood plasma is essential for accurate positron emission tomography studies.

A batch-contact alumina-extraction method has been used to separate [18F]-L-6-fluorodopa (FD) from its principal metabolite, 3-O-methyl-[18F]-6-fluorodopa (3-OMe-FD), in arterial blood plasma samples collected from subjects pretreated with carbidopa during positron emission tomography (PET) scans. The time course of the metabolite-corrected blood plasma activity is then used as an input function for kinetic analysis of striatal FD uptake. Results obtained from using the batch-contact alumina-extraction method were compared with those from high performance liquid chromatography, and also with those from a chromatographic alumina cartridge technique developed in this laboratory. In 60 human subjects including normal healthy volunteers and patients diagnosed as having a movement disorder, arterial blood plasma samples were collected after FD injection during a two-hour PET scan and analyzed by the batch-contact alumina-extraction method. The activity ratio (metabolites/FD) increased linearly with time for all subjects. However, there was a wide variation in the slope of the plot of the activity ratio (metabolites/FD) versus time among the subjects. No significant linear or curved relationship was observed between the slope and the age of the subject. Separation of FD from its metabolites is therefore necessary for each PET-FD study conducted.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.     

Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S-(+)-(5-2H3)hexobarbital and R-(-)-(5-2H3)hexobarbital were synthesized for clinical studies along with (+/-)-(1,5-2H6)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid-phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M-pentafluorobenzyl anion [M-PFB]- except for 1,5-dimethylbarbituric acid, where M-. was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S-(+)-2(H0)hexobarbital and R-(-)-(2H3)hexobarbital. Marked stereoselective disposition was observed, with the R-(-)-enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid—liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate—acetonitrile—methanol—nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C₁₈ reversed-phase column. The standard curves were linear in the range 50–5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2–14%. In the range 50–5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.