Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells

Purinergic Signalling - Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson’s disease (PD) progression. However, the identification...     

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C₂₁-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time     

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.     

液相色谱-串联质谱法同时测定大鼠血浆中阿霉素和塞来昔布

目的:建立同时测定大鼠血浆中阿霉素和塞来昔布的液相色谱-串联质谱(LC/MS/MS)方法,研究这两种药物联合应用的药代动力学.方法:大鼠尾静脉注射阿霉素和塞来昔布,眼眶取血并抗凝,离心分离血浆,采用乙酸乙酯提取血浆中的阿霉素和塞来昔布,N2吹干乙酸乙酯,残留物用50μL甲醇溶解,取20μL用于LC/MS/MS分析.结果:用LC/MS/MS法检测大鼠血浆中阿霉素和塞来昔布的线性范围为1-800ng/mL,日内、日间精密度(RSD)均小于15%,检测血浆低、中、高三个浓度(8、50、500ng/mL)阿霉素的回收率分别为101.2%、95.1%和91.4%,检测血浆低、中、高三个浓度(8、50、500ng/mL)塞来昔布的回收率分别为105.6%、106.8%和93.7%.大鼠尾静脉注射5.8mg/kg阿霉素和3.8mg/kg塞来昔布的半衰期分别为2.3 h和3.6h,曲线下面积分别为670 ng·h·mL-1和1480ng·h·mL-1.结论:建立的方法灵敏、准确、快速,适甩于阿霉素和塞来昔布的药代动力学研究.     

Analysis of vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS

Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13′-hydroxychromanols (13′-OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13′-carboxychromanol (13′-COOH), appear to be more bioactive than tocopherols in anti-inflammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with efficacy of ≥ 89% and interday/intraday variation of 3–11% for major metabolites. To ensure accurate quantification, short-chain, long-chain, and sulfated carboxychromanols are included as external/internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with γ-tocopherol or δ-tocopherol. Although plasma levels of 13′-COOHs and 13′-OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo.     

Determination of endogenous tissue inflammation profiles by LC/MS/MS: COX- and LOX-derived bioactive lipids

Cyclooxygenase and lipoxygenase arachidonate products, including prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are known to modulate inflammation within tissues and can serve as important etiologic factors in carcinogenesis. Eicosanoid content in tissues is typically determined either as a single molecular species through antibody-based assays or by high-performance liquid chromatography after addition of an exogenous substrate such as arachidonic acid. Unfortunately, the methods currently in use are either time-consuming or complicated. Here we report a method for simultaneously identifying eicosanoids appearing as endogenous bioactive lipids in in vivo settings using LC/MS/MS. The analyses indicate marked differences in endogenous eicosanoid content between malignant tissue types suggesting a need for selective therapeutic approaches. As a demonstration of the utility of the method, we present data to show that the technique can be used to distinguish eicosapentaenoic acid-derived formation of PGE₃ from PGE₂ in murine prostate tissue. The method has also been applied to an examination of endogenous eicosanoid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer in hamsters demonstrating the inflammatory nature of this type of cancer with elevated levels of both PGE₂ and LTB₄. In addition, the concentration of the eicosanoid 13-hydroxyoctadecadienoic acid was 67.6% lower in DMBA treated specimens than in control specimens. Thus, our method provides a powerful tool for measuring modulation of eicosanoid metabolites in various preclinical and clinical tissues and may be useful in studies of the endogenous changes in eicosanoid metabolism at various stages of cancer development.     

Determination of endogenous tissue inflammation profiles by LC/MS/MS: COX- and LOX-derived bioactive lipids

Cyclooxygenase and lipoxygenase arachidonate products, including prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are known to modulate inflammation within tissues and can serve as important etiologic factors in carcinogenesis. Eicosanoid content in tissues is typically determined either as a single molecular species through antibody-based assays or by high-performance liquid chromatography after addition of an exogenous substrate such as arachidonic acid. Unfortunately, the methods currently in use are either time-consuming or complicated. Here we report a method for simultaneously identifying eicosanoids appearing as endogenous bioactive lipids in in vivo settings using LC/MS/MS. The analyses indicate marked differences in endogenous eicosanoid content between malignant tissue types suggesting a need for selective therapeutic approaches. As a demonstration of the utility of the method, we present data to show that the technique can be used to distinguish eicosapentaenoic acid-derived formation of PGE(3) from PGE(2) in murine prostate tissue. The method has also been applied to an examination of endogenous eicosanoid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer in hamsters demonstrating the inflammatory nature of this type of cancer with elevated levels of both PGE(2) and LTB(4). In addition, the concentration of the eicosanoid 13-hydroxyoctadecadienoic acid was 67.6% lower in DMBA treated specimens than in control specimens. Thus, our method provides a powerful tool for measuring modulation of eicosanoid metabolites in various preclinical and clinical tissues and may be useful in studies of the endogenous changes in eicosanoid metabolism at various stages of cancer development.     

An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.     

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS

An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.     

LC/MS determination of the intracellular concentration of two novel aryl phosphoramidate prodrugs of PMPA and their metabolites in dog PBMC

LC/MS assays were developed to determine the plasma and intracellular concentrations of two aryl phosphoramidate prodrugs of the nucleotide analog 9-[2-R-(phosphonomethoxy)propyl]adenine. LC/MS was used to demonstrate the presence of high concentrations of PMPA in peripheral blood mononucleocytes following oral administration of prodrugs in dogs. High concentrations of PMPA and active metabolite were detected in MT-2 cells incubated with prodrug using an ion-pairing LC/MS assay.     

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.     

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 μL) were subjected to deproteinization with acetonitrile (200 μL) and then, after centrifugation, 150 μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb^® 5 μm CNRP 4.6 × 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.     

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in <Emphasis Type="Italic">Butea monosperma</Emphasis> (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L^?1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L^?1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L^?1 2,4-d with 0.1 mg L^?1 Kn, and 0.5 mg L^?1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L^?1 thidiazuron with 0.5 mg L^?1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L^?1 AA with 3.0 mg L^?1 Kn and 0.5 mg L^?1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L^?1 Kn, 0.3 mg L^?1 indole-3-butyric acid, and 0.25 mg L^?1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.     

Development and validation of a LC/MS/MS method for simultaneous quantification of oxcarbazepine and its main metabolites in human serum

A fast, sensitive and specific LC/MS/MS method for the simultaneous analysis of oxcarbazepine (OXC), 10-hydroxycarbazepine (MHD) and trans-diol-carbazepine (DHD), in human serum, has been developed and validated. Serum drugs were extracted by C8 solid-phase cartridges (SPE) and separated in less than 3 min on a C18 reverse-phase column using an isocratic elution. A tandem mass spectrometer, as detector, was used for quantitative analysis in positive mode by a multiple reaction monitoring. Calibration curves, obtained on two ranges of concentration (0.78-50 mg/L for MHD and 0.078-5.0 mg/L for OXC and DHD), showed correlation coefficients (r) better than 0.997. Within day and between days quality controls imprecision, as CV%, ranged from 0.3 to 4.6% and from 1.9 to 5.8%, respectively. Cyheptamide (CYE) was used as internal standard. No detectable carry-over and no relevant cross-talk and matrix effect occurred. Samples from 24 treated patients were analysed and drug serum concentrations obtained by this method are in agreement with those of other methods and also are well correlated (r=0.88) in comparison to our routine HPLC-UV method. Based on the analytical results and short run time, the method is suitable to support routine analysis of therapeutic drugs monitoring from human serum of treated patients or for pharmacokinetic studies.     

Quantitative determination of lysophosphatidic acid by LC/ESI/MS/MS employing a reversed phase HPLC column

Lysophosphatidic acid (LPA) is a class of lipids that play multiple biological functions. Several reports show that they are potential biomarkers for diagnosing ovarian cancer. Therefore, it is necessary to accurately quantify their levels in biological samples. Here we report a high throughput LC/ESI/MS/MS (liquid chromatography electrospray tandem mass spectrometry) method employing a reversed phase C18 column to quantify LPA. In this method, a [(13)C(16)] labeled 16:0 LPA is used as the internal standard and the lipids are extracted out from biological samples using Bligh-Dyer method under highly acidic condition. The total run time is 8min. The detection limits of the assay reach fmol level and the CV% of the assay are within 10%. Using this method, we quantify the levels of six LPA species (16:0, 18:2, 18:1, 18:0, 20:4, and 22:6 LPA) in plasma samples. We find that some unknown compounds present in plasma can interfere with the quantification of LPA if they are not well separated from LPA. These unknown compounds are more hydrophobic than LPA and can be removed by thin-layer chromatography (TLC). We also find that the levels of LPA species in human plasma generally follow the order: 18:2 LPA>16:0 LPA, 20:4 LPA>18:1 LPA, 22:6 LPA, and 18:0 LPA.     

Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.     

Structure elucidation of ostreocin-A and ostreocin-E1, novel palytoxin analogs produced by the dinoflagellate Ostreopsis siamensis,using LC/Q-TOF MS

Palytoxin analogs are marine toxins with large complex polyol structures. A benthic dinoflagellate Ostreopsis siamensis produces more than ten palytoxins (ostreocins, OSTs). The limited sample availability of minor OSTs restricts the definition of their chemical structures. The present investigation characterizes structures of two minor OSTs, i.e., ostreocin-A (OSTA) and ostreocin-E1 (OSTE1), using ostreocin-D (OSTD) as a reference compound, by liquid chromatography/quadrupole-time-of-flight mass spectrometry. The molecular formulas of OSTA and OSTE1 were C₁₂₇H₂₁₉N₃O₅₄ and C₁₂₇H₂₁₇N₃O₅₂, respectively. Compared to OSTD, OSTA has an extra oxygen atom whereas OSTE1 lacks one oxygen atom and two hydrogen atoms. The MS/MS experiments (precursor ions: [M + H]⁺ and [M-H]^?) suggested a hydroxyl substitution at C82 in OSTA and alteration(s) between C53 and C100 in OSTE1. Further analysis of structural details in OSTE1 was performed through a pseudo-MS³ experiment (precursor ion: m/z 1432.748). Accordingly, the planar structures of OSTA and OSTE1 were assigned to 42,82-dihydroxy-3,26-didemethyl-19,44-dideoxypalytoxin and 42-hydroxy-3,26-didemethyl-19,44,73-trideoxypalytoxin-72-ene, respectively.

Abbreviations:CID: collision induced dissociation; HR-LC/MS/MS: high-resolution liquid chromatography/tandem mass spectrometry; LC/ESI/Q-TOF MS: liquid chromatography/quadrupole time-of-flight mass spectrometry equipped with an electrospray ionization source; NMR: nuclear magnetic resonance; OSTs: ostreocins; OSTA: ostreocin-A; OSTB: ostreocin-B; OSTD: ostreocin-D; OSTE1: ostreocin-E1; OVTX-a: ovatoxin-a; OVTXs: ovatoxins; PLTX: palytoxin     

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.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.