Methanol-water Gradient Research Articles

A Green Liquid Chromatography Method for Simultaneous Quantification of Caffeine and its Three Major Metabolites in Urine, Drinks, and Herbal Products

ABSTRACTA green reversed‐phase liquid chromatography method was developed to determine caffeine (CF), theobromine (TB), theophylline (TP), and paraxanthine (PX). The stationary phase was a silica‐based C18 monolithic column (100 × 3.0 mm2 i.d.), eluted with a gradient from 6% to 10% (v/v) of ethanol in water, at a flow rate of 0.75 mL/min (35°C) and UV detection at 272 nm. The compounds were separated in less than 6 min, with a total time of 10 min for column reconditioning. The effect of gradient composition, flow rate, injection volume, and column temperature was evaluated in the separation efficiency, especially regarding the critical pair PX/TP, for which the best resolution was 1.37. The linear range was between 0.5 and 100 µg/mL. The LODs were 0.065, 0.057, 0.040, and 0.044 mg/mL for TP, TB, PX, and CF, respectively. The method was applied to green tea and yerba mate infusions, soft drinks, energy drinks, and soluble coffee. Urine samples were analyzed before and after solid‐phase extraction. The accuracy was evaluated by the spiking/recovery strategy standing in the 79.6% to 116.4% range. The separation performance and the mobile phase consumption were compared to those of another method using a gradient of methanol in water.

Triazole Antifungal Quantification for Therapeutic Drug Monitoring in Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Posaconazole, Voriconazole, Itraconazole, and Hydroxyitraconazole.

Antifungal therapy with triazole drugs including posaconazole, voriconazole, itraconazole, and its active metabolite hydroxyitraconazole is routinely accompanied by therapeutic drug monitoring to ensure optimal dosing. The method presented here simultaneously quantitates these compounds in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specimen preparation includes protein precipitation with a methanol and acetonitrile mixture, centrifugation, and filtration. Analyte separation is achieved by reverse-phase chromatography using a dC18 column and a linear gradient of methanol in water. Analytes are detected by multiple reaction monitoring mass spectrometry and quantitated by comparison to a standard curve.

Simultaneous measurement of kynurenine metabolites and explorative metabolomics using liquid chromatography-mass spectrometry: A novel accurate method applied to serum and plasma samples from a large healthy cohort

Kynurenine metabolites are emerging as promising clinical biomarkers in several diseases, especially within psychiatry. Unfortunately, they are difficult to detect, particularly the challenging neurotoxic metabolite quinolinic acid (QUIN). The aim of this study was twofold: First, to develop a liquid chromatography-mass spectrometry method (LC-MS) for simultaneous targeted quantification of key kynurenine metabolites together with untargeted metabolomics, and second, to demonstrate the feasibility of the method by exploring serum/plasma and gender differences in 120 healthy young adults between 18 and 30 years of age. A range of analytical columns (C18 and biphenyl columns) and mobile phases (acidic and alkaline) were systematically evaluated. The optimized LC-MS method was based on a biphenyl column, a water-methanol gradient with 0.2% formic acid, and authentic isotope-labeled standards for each kynurenine metabolite. Precision and accuracy of targeted quantification of the key kynurenine metabolites tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and QUIN were excellent, far exceeding the acceptance criteria specified by international guidelines. Median inter- and intra-day precision were < 6% in serum and plasma; the median accuracy was 2.4% in serum and 8% in plasma. Serum concentrations were ≤ 10% different from the corresponding concentrations in plasma for all kynurenine metabolites in healthy young adults. Men had higher levels (8–18%) of TRP, KYN, and KYNA than women (p ≤ 0.009), while no differences were observed for 3-HK and QUIN (p > 0.70). Incurred sample reanalysis of 10% of the samples yielded a median difference < 5% from the initial measurement, demonstrating the robustness of the method. Besides the targeted quantification of key kynurenine metabolites, our method was found to be suitable for simultaneous untargeted metabolomics analyses of hundreds of metabolites. A range of compound classes could be detected including amino acids, nucleic acids, dipeptides, antioxidants, and acylcarnitines, making explorative studies highly feasible. For example, we identified an additional kynurenine metabolite, 2-Quinolinecarboxylic acid, which was 47% higher in males than females (adjusted p-value = 0.001). In conclusion, in this study, we present a reliable and robust LC-MS method for simultaneous targeted and untargeted metabolomics ready for both research and clinical use. We show that both serum and plasma can be used for kynurenine studies, and the reported gender differences are in accordance with the literature. Future studies should consider using biphenyl-based LC-MS columns to successfully detect QUIN.

Non-Steroidal Drug Interferences in a Quantitative Multisteroid LC-MS/MS Assay

Screening for possible interferences from steroidal compounds other than the target analytes (endogenous or exogenous) is well established in LC-MS/MS assay development for steroid quantification in a routine clinical setting. However, interferences from non-steroidal substances have, hitherto, not been explored. After screening more than 150 pharmaceuticals and their metabolites by analyzing commercial quality control samples from TDM analysis kits (Recipe, Chromsystems) with a multisteroid LC-MS/MS assay (protein precipitation followed by HybridSPE filtration, biphenyl column, methanol-water gradient with NH4F additive), we can report the finding of two newly discovered potential interferences from non-steroidal drugs. Antidepressant paroxetine (PX) was identified as an interference to 17-hydroxyprogesterone (17P), and α-hydroxytriazolam (α-OH-TZM)-a major metabolite of benzodiazepine triazolam (TZM)-was identified as an interference to aldosterone (ALDO). Despite different elemental and structural compositions and nominal masses, the M+1 isotopologues of PX and α-OH-TZM produced overlapping signals in ion traces monitored for the respective analytes (m/z 331 → 109/97 and 361→315/343, respectively). PX and TZM are frequently prescribed drugs, and their therapeutic ranges are far exceeding the reference ranges of 17P or ALDO (µmol vs nmol); therefore, these interferences should be considered clinically relevant. Striving for faster multi-analyte methods with high sample turnover, especially in the field of steroid quantification, can limit assay selectivity and specificity. Therefore, supported by the findings of this study, screening for potential interferences in multi-analyte LC-MS/MS method development should not cover only substances of the same class but also include a set of common drugs.

Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride

BackgroundMulti-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride. MethodsFollowing liquid–liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation. ResultsPost-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122–140 % for 3βOH-Δ5 steroids and 477–1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range −14 % to 11.9 % for 21/23, −21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from −29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. ConclusionsUHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of androgens, estrogens, glucocorticoids and progestagens in human serum.

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids and progestogens) in human serum. Samples (250 μl of matrix) were extracted with t-butylmethyl ether prior to LC-MS/MS analysis. The chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization source with acquisition in multiple reaction monitoring mode. The method was validated using surrogate matrices and human serum samples. The specificity of the method was confirmed for all of the considered steroids; linearity was also assessed (R2 > 0.99, lack-of-fit test) in the ranges of concentrations investigated. The lower limits of quantification were in the range 10-400 pg/ml depending on the target steroid. Accuracy was in the range 85-115% for all target steroids except for the lower limit of quantitation levels where it was 80-120%. The extraction recovery was always >65%. No significant matrix effects were observed. To test the reliability of the method, the analysis of serum samples collected from 10 healthy subjects (5M/5F) was performed. The present method can be used to identify the trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.

Development and Validation of a Method to Simultaneously Determine Multiple Sterols in Diversiform Food Substrates with UPLC-MS/MS

Sterols play an essential part in physiological activities of almost all creatures. However, there is a lack of a widely applicable routine method to investigate various sterols with both saturated and unsaturated forms in diversiform food materials. In this study, a new UPLC-MS/MS method with wider extensive applicability has been developed to fill this blank, which is considered time and cost saving, and can improve labor efficiency. The Agilent Eclipse Plus C18 column and methanol-water gradient elution system were employed to improve the separation at 40 °C and the flow rate was 0.5 mL/min. The method validation parameters include precision, recovery, linearity, and limit of detection and quantification (LOD and LOQ). The recoveries of sterols in all food matrices and the inter-day and intra-day precisions represented by RSDs were all within the recommended range of chemical analysis. The LODs and LOQs in different types of samples were in range of 0.0036–0.68 μg/mL and 0.01–2.0 μg/mL, respectively. All the results demonstrated that this method was sensitive and accurate enough. Additionally, the acid hydrolysis was found closely related to the degradation of unsaturated sterols in the pretreatment.

Isolation, Characterization, and Simultaneous Quantification of Main Chemical Constituents from Cassia Alata Linn. Leaves

The leaves of Cassia alata L. are used in traditional Vietnamese medicine to treat ringworm and fungal skin infection. This study aimed to isolate marker compounds from the crude methanol extract of C. alata leaves and develop a validated method to control these compounds using HPLC-DAD. The three isolated compounds were identified as rhein (1), aloe-emodin (2), and kaempferol (3) by NMR and HR-ESI-MS. A quick, precise, and accurate HPLC-DAD method in 20 min of analysis time to quantify isolated maker compounds of Cassia alata was developed for the first time. The analysis was performed on a Hypersil GOLD C18 column (150 x 2.1 mm, 3 µm) with 0.1% formic acid in water-methanol gradient elution. The linearity, specificity, sensitivity, precision, and accuracy parameters were validated. The accuracies of the method were 99.0 – 103.5% and linearity (r2 &gt; 0.9995) were achieved. The LODs and LOQs were less than 0.74 µg/mL and 2.25 µg/mL. The developed HPLC method could be applied for quality control testing of C. alata leaves. Keywords: Cassia alata L., kaempferol, rhein, aloe-emodin, HPLC-DAD.

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

Simultaneous quantification of ambrisentan, macitentan and sitaxentan in human plasma using UPLC-MS/MS.

Endothelin receptor antagonists (ERAs) such as, ambrisentan, macitentan and sitaxentan are primarily used for the treatment of pulmonary arterial hypertension. Considering the rise in endothelin in pre-eclampsia, ERAs may also be useful in its treatment. To evaluate the pharmacokinetics of ERAs, a rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated to determine the concentration of ambrisentan, macitentan and sitaxentan in human plasma. Plasma samples were treated with methanol to induce protein precipitation. A chromatographic separation was performed on a C18 column using a gradient of methanol-water containing 0.1% formic acid and 0.013% ammonium acetate and a flow rate of 0.5 ml/min. Multiple reaction monitoring was used for quantification. This method was validated in a linear range of 20.28-2028 μg/l for ambrisentan, 4.052-405.2 μg/l for macitentan and 205.4-10 270 μg/l for sitaxentan. The method was successfully validated according to US Food and Drug Administration guidelines to determine the concentrations of macitentan, ambrisentan and sitaxentan in human plasma. This method is now being used for study samples and clinical patient samples.

Liquid chromatography with tandem mass spectrometry method for trace level analysis of migrated secondary alkanesulfonate from poly(tetramethylene terephthalate) into food simulants.

Secondary alkanesulfonate has been widely used as an antistatic additive in polymers for producing anti-dust food packaging containers. Currently, no reported method exists for accurate quantification of secondary alkanesulfonate in ethanolic and acidic food simulants. A new liquid chromatography with tandem mass spectrometry method was developed to quantify the migrated amount of secondary alkanesulfonate at trace levels in food simulants from a poly(tetramethylene terephthalate) containing secondary alkanesulfonate. The poly(tetramethylene terephthalate) samples loaded with the antistatic additive were exposed to various food simulants. The collected extracts were directly analyzed by liquid chromatography with tandem mass spectrometry in electrospray ionization negative mode. As secondary alkanesulfonate is a mixture containing C14 to C17 chain lengths, it was separated on a Poroshell 120 EC-C8 column adopting methanol water gradient program. The migration of secondary alkanesulfonate ranged from 58 to 329 ppb; which is well within the allowed permissible regulatory limits and the adopted method was validated by conducting spiking studies and acceptable recoveries were obtained. The developed method was not only sensitive to detect lower levels of the migrated antistatic additive, but it also avoided more cumbersome sample preparation methodologies like sample enrichment and other derivatization approaches.

A precise, sensitive and stable LC-MSMS method for detection of picomolar levels of serum aldosterone

Background: Accurate quantification of aldosterone is essential in the diagnosis of primary aldosteronism. Liquid chromatography-tandem mass spectrometry (LC-MSMS) analysis is increasingly being used to improve analytical sensitivity and specificity, since this technology reduces most of the interferences observed with immunological methods.Methods: Serum samples with d7-aldosterone as internal standard were extracted with methyl tert-butyl ether, using liquid-liquid extraction (LLE). Chromatographic separation was performed on a C18 reverse phase column with a methanol-water gradient containing ammonium fluoride. Aldosterone detection was performed on an Agilent 6490 triple quadrupole using electro spray ionisation in positive mode.Results: Multiple reaction monitoring transitions were m/z 361.2–315.1 for aldosterone, and 368.5–323.3 for d7-aldosterone. Chromatographic retention time was 2.7 min. The method’s total CVs at aldosterone concentrations of 45.4 and 1080 pmol/L were 7.0% and 4.8%, respectively. The intra-assay CVs at concentrations of 60.0 and 637 pmol/L were 4.0% and 2.6%, respectively. The method’s LOQ and LOD were 10 and 5 pmol/L, respectively, demonstrating an excellent analytical sensitivity. The upper limit of quantification was set to 5000 pmol/L, corresponding to the highest calibrator concentration. The long-term stability of the method was evident from repeated measurements of external control pools from UKNEQAS over a period of about 3 years, showing CVs between 2.0 and 7.0%.Conclusions: We have described a precise, sensitive and stable LC-MSMS method for the measurement of serum aldosterone. In addition, due to the use of LLE and a short LC-column, the method is simple to perform, with a short chromatographic run time.

Analysis by LC–MS/MS of endogenous steroids from human serum, plasma, endometrium and endometriotic tissue

An LC–MS/MS method was developed and validated to analyze simultaneously estrogens (estradiol, E2; estrone, E1), androgens (testosterone, T; androstenedione, A4; dehydroepiandrosterone, DHEA), progestagens (17a-hydroxypregnenolone, 17OHP5; 17a-hydroxyprogesterone, 17OHP4; progesterone, P4), glucocorticoids (cortisol, F; cortisone E; corticosterone, B; 11-deoxycortisol, S; 21-hydroxyprogesterone, 21OHP4), and mineralocorticoids (aldosterone, A) from 150 μl of human serum, plasma, or endometrium and endometriotic tissue homogenates. Samples spiked with isotope-labeled steroids as internal standards were extracted with toluene prior to LC–MS/MS analysis. The chromatographic separation of underivatized steroids was achieved on a biphenyl column with 0.2 mM NH4F as the eluent additive and a water-methanol gradient to improve E2 and E1 ionization. Method validation was performed with human plasma samples, and analysis of certified E2, T, F, and P4 reference serums (BCR-576, ERM-DA346, ERM-DA192, ERM-DA347), as well as homogenates of endometrium and endometriotic tissue. A total of 27 steroids were included in the method development to ensure the specificity of the method. After validation, the method was found suitable for quantitative analysis of 11 steroids: E2 (6.7 pM-13 nM), E1 (1.3 pM-6.6 nM), T (3.3 pM-13 nM), A4 (13 pM-33 nM), 17OHP5 (32 pM-65 nM), 17OHP4 (33 pM-13 nM), F (33 pM-133 nM), E (13 pM-130 nM), B (33 pM-134 nM), S (13 pM-129 nM), and A (32 pM-32 nM). In addition, DHEA (333 pM-32 nM), P4 (13 pM-13 nM) and 21OHP4 (13 pM-13 nM) can be analyzed semiquantitatively.

Simultaneous Determination of Vinpocetine and its Major Active Metabolite Apovincaminic Acid in Rats by UPLC-MS/MS and its Application to the Brain Tissue Distribution Study.

A specific, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for simultaneous determination of vinpocetine (VP) and its active metabolite, apovincaminic acid (AVA) in rat brain regions, such as hypothalamus, striatum, cortex, cerebellum and hippocampus. Phenacetin was used as internal standard (IS). Brain tissue samples were precipitated protein by using 500 μL methanol. The separation was achieved on a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), using a methanol-water gradient elution at the flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via positive electrospray ionization source (ESI). The quantification was operated using the transitions of m/z 351 → m/z 280 for VP, m/z 323 → m/z 280 for AVA and m/z 180 → m/z 110 for IS, respectively. The calibration curve was linear in concentration range from 0.100 to 60.0 ng/mL for VP and 0.103 to 6.18 ng/mL for AVA. The intra-day and inter-day precision (relative standard deviation, RSD) values were within 11.8%, the accuracy (relative error, RE) was from -1.7% to 3.0% for VP and 2.7% to 9.5% for AVA at all the three concentration levels of quality-control (QC) samples. The improved UPLC-MS/MS method was specific, rapid and sensitive, which was further successfully applied to simultaneous determination of VP and AVA in different rat brain regions after intragastric administration of 4 mg/kg VP. It was indicated that VP could be eliminated quickly in brain, while the elimination of AVA was slow and it could be maintained for more than 12 h in brain. Moreover, it was found that the contents of VP and AVA were much higher in the hypothalamus, striatum and cortex than those in the cerebellum and hippocampus, which verified the distribution characteristics of VP and AVA in different brain regions from the point of quantitation in rats.

Abstract P2-09-11: Metabolite-guided long-term prediction of outcome in tamoxifen treated breast cancer patients

Abstract Background The genetic polymorphisms of CYP2D6 determine its enzymatic activity thereby the pharmacokinetic velocity by which tamoxifen (tam) is converted into active metabolites. Combined analyses of tam metabolites compared to CYP2D6 type and long-term survival could allow prediction of tam response and personalization of therapies. The clinical importance of defining such subgroups in the era of the new long-term (10-year) tam treatment paradigm is actualized. Material and Methods From May 1995 to December 1998, patients were included in an observational micro-metastasis study in the Oslo region and treated according to the national guidelines at the time (1). Serum samples were drawn at 3-year follow-up from 356 relapse-free patients, 106 of these were treated with tamoxifen. The median follow up time for breast cancer death was 16.8 years (3.5-19.4). Serum samples were processed using protein precipitation with acetonitrile. An Aquity UPLC system was used to chromatographically separate 10 tam metabolites using a BEH C18 Phenyl column (100 x 2.1 mm, 1.7 μm particle size) that was developed with a water-methanol gradient containing 0.1% formic acid. The LC system was coupled to a Xevo TQ-S tandem mass spectrometer equipped with an atmospheric pressure photoionization source. The method was validated with respect to linearity, imprecision, accuracy, and functional sensitivity according to FDA guidelines. Results The new LC-MS/MS method separated the active Z-isomers of 4OHtam and 4OHNDtam (endoxifen) from its inactive Z'-isomers and E-isomers. Imprecision (intra and inter-day CV %) was within 10 % for target concentrations for all metabolites and accuracies were in the range 95-106%. The method was validated with serum samples from 42 breast cancer patients using 20 mg of tamoxifen. The endoxifen concentrations ranged from 0 to 90 nM, with a median value of 25 nM. The previous observed endoxifen level of 10 nM in poor metabolizers (2) was used as cut-off for the grouping of patients. The nil endoxifen (NE) group (&amp;lt; 0.1nM, n=14) or low-endoxifene (LE) group (0.1-10 nM, n=8) were grouped together. Univariate survival analysis did not show a significant association between breast cancer specific survival and endoxifen levels. (p=0.15; logrank and p=0.18;Breslow). However, for the period beyond 10-years of follow-up the breast cancer survival differed between the high endoxifene (HE) group and the NE+LE groups. For patients surviving the first 10 years the breast cancer specific survival was 94.2% vs. 77.8% for the HE and NE+LE groups respectively (p=0.020, logrank and p=0.017, Breslow, HR=4.5, CI 95=1.1-17.9). In the multivariate analysis endoxifen ≤/&amp;gt; 10 nM remained the only factor in the final model. Discussion We developed a new accurate and precise LC-MS/MS method for the measurement of 10 tamoxifen metabolites. Importantly, the method separates active and inactive isomers of 4OHtam and 4OHNDtam/endoxifen. Despite the low number of patients, we observed a poorer long-term survival beyond 10 years in patients with nil or low serum concentration of endoxifen. A comprehensive analysis is presented addressing the relationship between genotyped based and metabolite based prediction of long-term outcome in tamoxifen treated breast cancer patients. Ref: 1. Wiedswang G, et al. Clin Ca Res; 2004 2. Mürdter TE, et al. Clin Pharmacol Ther; 2011. Citation Format: Helland T, Henne N, Bifulco E, Hustad SS, Kristensen VN, Lash T, Borgen E, Janssen EAM, Lien EA, Naume B, Mellgren G, Søiland H. Metabolite-guided long-term prediction of outcome in tamoxifen treated breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-11.

Determination of 5 nucleosides components in culture of Paecilomyces hepialid by HPLC

The concentration of 5 nucleosides, uracil, uridine, guanidine, adenine and adenosine in culture of Paecilomyces hepialid was determined by the developed method of HPLC. The HPLC method was performed on a Waters SunFire C18 (4.6 mm x 250 mm, 5 μm) column with methanol-water gradient elution as the mobile phase. The detection wavelength was 260 nm and the colunmn temperature was controlled at 30 °C. The linear range was 10.00-200.00 mg · L(-1) (r = 0.9994) for uracil, 10.10-202.00 mg · L(-1) (r = 0.9992) for uridine, 10.00-200.00 mg · L(-1) (r = 0.9991) for guanidine, 10.30-206.00 mg · L(-1) (r = 0.9992) for adenine and 10.45-209.00 mg · L(-1) (r = 0.9991) for adenosine, respectively. The RSD of precision was 0.032%, 0.035%, 0.039%, 0.049%, 0.00080%, respectively. The average recoveries of uracil, guanidine, adenine, and adenosine were 97.34%, 99.10%, 101.6%, 98.61% and 100.2% with RSD of 1.3%, 2.1%, 0.96%, 0.95%, and 1.3% respectively. The method showed high sensitivity, good selectivity, linearity and repeatability, which was suitable for the content analysis of 5 nucleosides components in P. hepialid and its extracts.

Development and validation of a method using supported liquid extraction for aldosterone determination in human plasma by LC-MS/MS

BackgroundAccurate quantitation of aldosterone is essential for screening, diagnosis and subtype classification in primary aldosteronism. A simple, sensitive method for aldosterone in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated. MethodsPlasma samples were diluted with water containing d7-aldosterone as internal standard. The samples were extracted with methyl-tert-butyl-ether (MTBE) on SLE cartridges. Separation was carried out on a Luna C18 (2) column using a methanol–water gradient. Detection was performed in the negative electrospray multiple reaction monitoring (MRM) quantitation. The use of water-based calibrators was evaluated against calibrators prepared in steroid-free serum. ResultsThe assay was linear up to 3265pmol/L with an LOQ of approximately 40pmol/L. Within-run and between-run precision for plasma aldosterone were less than 10% except at low level near LOQ but were still less than 14.7% (Westgard's desirable specification). The mean recovery of the analyte added to plasma was greater than 97.7% and matrix effects were less than 4%. Comparison with another LC-MS/MS method was performed on a more sensitive instrument (ABSciex TQ 5500) and gave the equation API 3000=0.957×TQ 5500+12.6, linear regression r2=0.974 (n=43). An estimation of the reference interval for adults was established on a group of healthy volunteers (n=53). Calibration with water-based calibrators was validated and can be used for measurement of aldosterone by LC-MS/MS. ConclusionsThis method is reliable, easy to perform on plasma specimens in a clinical environment and is attractive because of its simplicity.

Development of an analytical method by HPLC for quantification of galloylquinic compounds from Copaifera langsdorffii leaves

Copaifera langsdorffii Desf. (Fabaceae, Caesalpinioideae), popularly known as “copaiba”, is a large tree widely distributed in Brazil and used in folk medicine. Its leaves are composed mainly by phenolic compounds, such as galloylquinic acid derivatives (GQA) and flavonoids. The water soluble fraction of the crude hydroalcoholic extract was submitted to Sephadex LH-20 using a gradient of methanol-water (1:9 to 9:1) and purified by preparative RP-HPLC. Sixteen GQA were isolated and used as chromatographic standards. HPLC-PDA analyses were carried out on a Synergi Polar-RP (100 × 3.0 mm, 2.5 µm) column. The mobile phase was made up of formic acid-water (0.1:99.9, solvent A), and isopropanol-methanol-acetonitrile (0.5:4:6, solvent B). The elution gradient was A:B (90:10 to 85:15) in 8.0 minutes, followed by A:B (85:15 to 64:36) up to 30.0 minutes using a flow rate of 0.7 mL/min. The HPLC-PDA developed method was suitable to quantify nine GQA and two flavonoids, quercitrin and afzelin.

Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry

A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC–SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC–SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC–MS for analysis of nonpolar compounds.

An optimized method for corticosterone analysis in mouse plasma by ultra-performance liquid chromatography-full-scan high-resolution accurate mass spectrometry.

A simple method based on liquid chromatography-full-scan high-resolution accurate mass spectrometry (LC-HRMS) using a quadrupole time-of-flight (TOF) mass spectrometry was developed and optimized for corticosterone quantification in mouse plasma. Mouse plasma (100 μL) was extracted with methyl tert-butyl ether using prednisone as internal standard. Separation was performed on a short C18 column using a methanol-water gradient. Full-scan data were acquired in the TOF only mode, and extracted ion chromatograms were generated post-acquisition with the extract masses of the analytes. Enhanced sensitivity and reproducibility were acquired with optimized mass parameters. The calibration range was 8.24-412 ng/mL, and the limit of quantitation was 5.088 g/mL. Accuracy was between -5.9 and 8.6%. The precision of between-run (interday) and within-run (intraday) was within 5.6 and 6.9%, respectively. The LC-HRMS method was applied for plasma samples analysis from the stressed mice with and without ginseng treatment for the stress state estimation.