Stable Isotope Dilution Liquid Chromatography-tandem Research Articles

Quantification of midostaurin in plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry: Application to a cohort of patients with acute myeloid leukemia.

Midostaurin is an oral multitargeted tyrosine kinase inhibitor for the treatment of acute myeloid leukemia (AML). Therapeutic drug monitoring of midostaurin may support its safe use when suspecting toxicity or combined with strong CYP3A4 inhibitors. A stable isotope dilution liquid chromatography-tandem mass spectrometry method was developed and validated for the determination and quantification of midostaurin in human plasma and serum. Midostaurin serum concentrations were analyzed in 12 patients with FMS-like tyrosine kinase 3 (FLT3)-mutated AML during induction chemotherapy with cytarabine, daunorubicin, and midostaurin. Posaconazole was used as prophylaxis of invasive fungal infections. Linear quantification of midostaurin was demonstrated across a concentration range of 0.01-8.00 mg/L. Inter- and intraday imprecisions of the proposed method were well within ±10%. Venous blood samples were taken in nine and three patients in the first and second cycle of induction chemotherapy. Median (range) midostaurin serum concentration was 7.9 mg/L (1.5-26.1 mg/L) as determined in 37 independent serum specimens. In a real-life cohort of AML patients, interindividual variability in midostaurin serum concentrations was high, highlighting issues concerning optimal drug dosing in AML patients. A personalized dosage approach may maximize the safety of midostaurin. Prospective studies and standardization of analytical methods to support such an approach are needed.

The associations between TMAO-related metabolites and blood lipids and the potential impact of rosuvastatin therapy

BackgroundTrimethylamine N-oxide (TMAO)-related metabolites are associated with the pathogenesis of atherosclerotic cardiovascular disease (ASCVD) and are known to disrupt lipid metabolism. The aims of this study were to evaluate the associations between TMAO-related metabolites and blood lipids and determine how lowering the lipid profile via rosuvastatin therapy influences TMAO-related metabolites.MethodsA total of 112 patients with suspected ASCVD were enrolled in this study. The levels of plasma TMAO-related metabolites, including TMAO, choline, carnitine, betaine, and γ-butyrobetaine (GBB), were analyzed by stable isotope dilution liquid chromatography-tandem mass spectrometry (LC/MS/MS) before and after rosuvastatin therapy in all patients. Statistical methods were used to detect the associations between TMAO-related metabolites and blood lipids and determine how rosuvastatin therapy alters the levels of these metabolites.ResultsA significant positive correlation was found between TMAO and triglycerides (TG) (r = 0.303, P < 0.05). Furthermore, significant negative correlations were found between TMAO and high-density lipoprotein cholesterol (HDL-c) and between betaine and low-density lipoprotein cholesterol (LDL-c) (r = − 0.405 and − 0.308, respectively, both P < 0.01). Compared to baseline, significantly lower TMAO levels and higher carnitine, betaine and GBB levels were observed after rosuvastatin therapy, while the lipids decreased significantly (P < 0.05). The significant correlation between TMAO and TG or between betaine and LDL-c disappeared after rosuvastatin therapy (r = 0.050 and − 0.172, respectively, both P > 0.05). However, a significantly positive association between carnitine and TC and a negative association between carnitine and LDL-c or between betaine and TG were found after adjustment for sex, age, body mass index (BMI) and lipids (P < 0.05).ConclusionsThis study suggests that TMAO-related metabolites are significantly associated with blood lipids, although some of them are changed postrosuvastatin therapy. Lower TMAO and higher TMAO precursors were observed after rosuvastatin therapy compared to baseline. This study indicates that elevated TMAO precursors after rosuvastatin therapy and their potential impact on ASCVD should be considered in the clinic.

Plasma desmosine, a biomarker of elastin degradation, predicts outcomes in coronary artery disease

Abstract Background Recent evidence from animal studies suggests that elastin degradation accelerates atherosclerosis and increases risk of plaque rupture and subsequent myocardial infarction and stroke. Desmosine is an elastin-specific degradation product. We analysed the prognostic value of plasma desmosine (pDES) in a cohort of patients with coronary artery disease (CAD). Methods Patients with CAD (n=400) undergoing elective coronary angiography were prospectively recruited over 3 years and had bloods drawn for analysis of pDES using a validated stable isotope dilution liquid chromatography-tandem mass spectrometry method. Patients were followed up for 12 months for major adverse cardiovascular events (MACE: composite of death, myocardial infarction, target vessel repeat revascularisation, target lesion revascularisation, and heart failure hospital admission). The upper limit of normal for pDES is 0.35 ng/mL. The predictive value of pDES for MACE was analysed with Cox-proportional hazards ratio (HR) model and Kaplan-Meier survival analysis. Results During follow-up, there were 36 MACE events. Median pDES level across the entire cohort was 0.3 ng/mL (IQR 0.23–0.41 ng/mL). Patients with a pDES level &amp;gt;0.35 ng/mL were more likely to be male and older with a mean age of 69.7±10.3 years, have a history of prior stroke or transient ischaemic attack (TIA) and COPD. In univariable analysis, a pDES level of &amp;gt;0.35 ng/mL was associated with an increased risk of MACE (HR 4.76, 95% CI: 2.34–9.68, p&amp;lt;0.001). In multivariable analysis, pDES &amp;gt;0.35 ng/mL was associated with risk of MACE after adjustment for age, sex, COPD status and previous stroke and TIA (HR 3.97; 95% CI: 1.82–8.67, p=0.001) (Figure). Conclusion Increased elastin degradation as measured by elevated pDES levels, predicts outcomes in patients with CAD independent of traditional cardiovascular risk factors and may play a role as a future biomarker in these patients. Kaplan-Meier survival analysis Funding Acknowledgement Type of funding source: None

Luhong Granules Prevent Ventricular Remodelling after Myocardial Infarction by Reducing the Metabolites TMAO and LPS of the Intestinal Flora.

Background Ventricular remodelling is a common pathological change at all stages of heart disease. Luhong granules are widely used in patients with chronic ventricular remodelling after myocardial infarction and can alleviate chest tightness, shortness of breath, and other symptoms. However, its effect on ventricular remodelling remains to be studied. Purpose In this study, we investigated the effects of these granules on myocardial fibrosis in a rat model of myocardial infarction in vivo. Methods Male Wistar rats were randomly divided into four groups: the sham operation group, the acute myocardial infarction (AMI) group, the Luhong granule group, and the vancomycin group, with a sample size (n) of 10 rats in each group. The AMI model was established in all rats by ligation of the left anterior descending (LAD) coronary artery (the sham operation group did not undergo ligation). Luhong granules (0.5 ml·kg−1·d−1), vancomycin (0.075 g·ml−1·d−1), and 0.9% saline (5 ml·kg−1·d−1 for the sham operation and AMI groups) were administered orally for 6 weeks. Echocardiography was used to check cardiac structure and function. Myocardial and small intestinal tissue morphology was observed by haematoxylin and eosin (H&E) staining, and heart samples were stained with Masson's trichrome to analyse myocardial fibrosis. 16S rDNA sequencing was performed to detect changes in the gut flora. The level of trimethylamine N-oxide (TMAO) in plasma samples was quantified by stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS). Results H&E and Masson's trichrome staining of cardiac tissues showed that Luhong granules could partially reverse ventricular remodelling and improve intestinal barrier function (P < 0.05). Echocardiographic analysis showed that, compared with the AMI group, the left ventricular ejection fraction (LVEF) in the Luhong granule group was increased (P < 0.05). Stool sequencing and microbiological analysis showed changes in Bacteroidales, Alistipes, Phascolarctobacterium, etc., which can produce TMAO. We found that Luhong granules can reduce Bacteroidales, Alistipes, and Phascolarctobacterium at the genus level. The levels of TMAO and lipopolysaccharides (LPS) in plasma samples were reduced in the Luhong granule group (P < 0.05). Conclusions Our results indicate that Luhong granules reduce TMAO and LPS levels in circulating blood by improving intestinal flora and intestinal barrier function to delay ventricular remodelling after myocardial infarction.

Plasma concentration of trimethylamine-N-oxide and risk of gestational diabetes mellitus

The microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) has been reported as a novel and independent risk factor for the development of cardiovascular and metabolic diseases, but the association with gestational diabetes mellitus (GDM) remains unclear. The aim of this study was to investigate the association between plasma TMAO concentration and GDM in a 2-phase study. A 2-phase design was used in the current study. An initial phase included 866 participants (433 GDM cases and 433 matched controls) with fasting blood samples collected at the time of GDM screening (24-32 wk of gestation). An independent-phase study, with 276 GDM cases and 552 matched controls who provided fasting blood samples before 20 wk of gestation and who had GDM screened during 24-32 wk of gestation, was nested within a prospective cohort study. These 2 studies were both conducted in Wuhan, China, and the incidence of GDM in the cohort study was 10.8%. Plasma TMAO concentrations were determined by stable isotope dilution liquid chromatography-tandem mass spectrometry. GDM was diagnosed according to the American Diabetes Association criteria by using an oral-glucose-tolerance test. In the initial case-control study, the adjusted OR of GDM comparing the highest TMAO quartile with the lowest quartile was 1.94 (95% CI: 1.28, 2.93). Each SD increment of ln-transformed plasma TMAO was associated with 22% (95% CI: 5%, 41%) higher odds of GDM. In the nested case-control study, women in the highest quartile also had increased odds of GDM (adjusted OR: 2.06; 95% CI: 1.28, 3.31) compared with women in the lowest quartile, and the adjusted OR for GDM per SD increment of ln-transformed plasma TMAO was 1.26 (95% CI: 1.08, 1.47). Consistent findings from this 2-phase study indicate a positive association between plasma TMAO concentrations and GDM. Future studies are warranted to elucidate the underlying mechanisms. This trial was registered at www.clinicaltrials.gov as NCT03415295.

Reliable LC-MS/MS assay for the estimation of rilpivirine in human plasma: application to a bioequivalence study and incurred sample reanalysis.

A simple, precise, and rapid stable isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of rilpivirine, a non-nucleoside reverse transcriptase inhibitor in human plasma. Rilpivirine and its deuterated analogue, rilpivirine-d6, used as an internal standard (IS) were quantitatively extracted by liquid-liquid extraction with methyl-tert-butyl ether and diethyl ether solvent mixture from 50 μL plasma. The chromatography was achieved on Gemini C18 (150 × 4.6 mm, 5 µm) analytical column in a run time of 2.2 min. The precursor → product ion transitions for rilpivirine (m/z 367.1 → 128.0) and IS (m/z 373.2 → 134.2) were monitored on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL. The mean extraction recovery for rilpivirine (94.9%) and IS (99.9%) from spiked plasma samples was consistent and reproducible. The IS-normalized matrix factors for rilpivirine ranged from 0.98 to 1.02 across three quality controls. Bench top, freeze-thaw, wet extract, and long-term stability of rilpivirine was examined in spiked plasma samples. The application of the method was demonstrated by a bioequivalence study with 25 mg rilpivirine tablet formulation in 40 healthy subjects. The assay reproducibility was shown by reanalysis of 200 study samples and the % change in the concentration of repeat values from the original values was within ±15%.

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

Analysis of Organic Acids in Fruit Juices by Liquid Chromatography−Mass Spectrometry: An Enhanced Tool for Authenticity Testing

Organic acid analysis plays a fundamental role in the testing of authenticity of fruit juices. Analytical methods used routinely for organic acids suffer from poor reproducibility, often give false positives/negatives for tartaric acid, and do not offer the possibility of analyte confirmation. There are conflicting reports in the literature on the presence/absence of tartaric acid in pomegranate juice, a potential indicator of adulteration with grape juice. In this work, a method based on stable isotope dilution liquid chromatography-tandem mass spectrometry is described for citric, malic, quinic, and tartaric acid in fruit juices. Validation data including precision and recovery in six types of juice are presented. Tartaric and quinic acids were confirmed in pomegranate juice at concentrations of 1-5 and ∼1 mg/L, respectively. These concentrations are much lower than those resulting from adulteration with grape juice and apple juice, respectively, at the 5% level. A separate method for isocitric acid in orange juice based on the single standard addition method is also described.

Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography–tandem mass spectrometry

An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography-tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, 13C3-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-microm 2.1 x 150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 microg kg(-1)) and was found to vary between 0.6-2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 microg kg(-1).

Quantitative Measurement of Porphobilinogen in Urine by Stable-Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

Determination of 17-Hydroxyprogesterone in Plasma by Stable Isotope Dilution/Benchtop Liquid Chromatography-Tandem Mass Spectrometry

An assay based on stable isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS-MS) was developed for the quantification of 17-hydroxyprogesterone, the most important indicator of 21-hydroxylase deficiency in human plasma. Plasma was extracted using ethyl acetate and Extrelut^® columns. LC was performed on a reversed-phase C18 column using a water/methanol gradient. A benchtop triple quadrupole mass spectrometer, operating in selected reaction monitoring mode, served as mass detector. The analytical run time was 9 min per sample. The sensitivity was high: 0.06 pmol of 17-hydroxyprogesterone yielded a signal-to-noise ratio of 13. Precision (CV) and accuracy (relative error) derived from the analyses of unspiked and spiked validation samples were 7.4–12.0% and 6.4%, respectively. When analyzing the same samples – median (range), in nanomoles per liter – from neonates and adults independently by ID/LC-MS-MS as well as by ID/gas chromatography (GC)-MS, corresponding results were obtained: neonates (n = 10), ID/LC-MS-MS 3.99 (0.48–16.05), ID/GC-MS 5.39 (1.57–13.02); adults (n = 10), ID/LC-MS-MS 2.66 (1.39–6.15), ID/GC-MS 2.54 (0.51–5.12). The technique permitted reliable detection of classical and nonclassical forms of 21- hydroxylase deficiency. The much simpler sample preparation, the faster analytical run time and the operational ease possible with ID/LC-MS-MS permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity. We expect that benchtop tandem mass spectrometry will open new avenues in clinical steroid analysis.