High-sensitivity Liquid Chromatography-tandem Mass Spectrometry Research Articles

Low Progesterone and Low Estradiol Levels Associate With Abdominal Aortic Aneurysms in Men.

ContextMale sex is a major risk factor for abdominal aortic aneurysms (AAA) but few studies have addressed associations between sex hormone levels and AAA.ObjectiveWe aimed to describe the associations between serum sex steroids and early, screening-detected AAA in men.MethodsWe validated a high-sensitivity liquid chromatography–tandem mass spectrometry assay for comprehensive serum sex hormone profiling. This assay was then employed in a case-control study including 147 men with AAA (infrarenal aorta ≥ 30 mm) and 251 AAA-free controls recruited at the general population-based ultrasound screening for AAA in 65-year-old Swedish men.Outcomes includedassociations between dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, androstenedione, estrone, testosterone, dihydrotestosterone, and estradiol and AAA presence.ResultsDehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, testosterone, and estradiol, but not the other hormones, were lower in men with AAA. In models with adjustments for known AAA risk factors and comorbidity, only progesterone (odds ratio per SD decrease 1.62 [95% CI, 1.18-2.22]) and estradiol (1.40 [95% CI, 1.04-1.87]) remained inversely associated with the presence of AAA. Progesterone and estradiol contributed with independent additive information for prediction of AAA presence; compared with men with high (above median) levels, men with low (below median) levels of both hormones had a 4-fold increased odds ratio for AAA (4.06 [95% CI, 2.25-7.31]).ConclusionMeasured by a high-performance sex steroid assay, progesterone and estradiol are inversely associated with AAA in men, independent of known risk factors. Future studies should explore whether progesterone and estradiol, which are important reproductive hormones in women, are protective in human AAA.

A fast and high-sensitivity liquid chromatography-tandem mass spectrometry method combined with in vivo microdialysis for quantification of meropenem in rabbits with sepsis under the simultaneous infusion of total parenteral nutrition: Application to a pharmacokinetic study.

A fast and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method assisted by microdialysis was established for the determination of meropenem in total parenteral nutrition (TPN) infused plasma. A 10-times dilution was arranged for sample preparation to overcome the severe matrix effect caused by the high salt content in dialysate and complex composition of TPN. This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (R2 > 0.998), recovery, matrix effect and stability. In the optimized conditions, the calibration curve range was set from 2 to 2000 ng/ml. This validated method was applied to pharmacokinetics study of meropenem in rabbits with sepsis (induced by cecal ligation and punctures) under simultaneous infusion of TPN to simulate the clinical practice. The results demonstrated that the LC-MS/MS method assisted by microdialysis can be used successfully for the determination of meropenem in TPN-infused plasma. Moreover, the area under the curve and the maximum concentrations in the plasma of meropenem in control rabbits were significantly smaller (P < 0.05), while clearance and distribution volumes were significantly greater (P < 0.05) than in those with sepsis. It could be speculated that drug monitoring in patients with sepsis may be necessary.

Alpha-Methylfentanyl and Beta-Hydroxyfentanyl LC-MS-MS Quantification in Rat Plasma after Long-Term Ethanol Exposure.

Fentanyl and its analogues are highly abused drugs that dominate the illicit drug trade. alpha-Methylfentanyl (A-F) and beta-hydroxyfentanyl (B-F) are two fentanyl analogues that require the development of rapid detection technologies. The current study established and validated a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure A-F and B-F concentrations in rat plasma following intravenous drug administration (20 μg/kg). Because fentanyl is primarily metabolized by the liver, we evaluated the concentrations of A-F and B-F in vivo in rats, in a control group and a group with liver damage induced by 55 days of oral ethanol gavage (6.5 g/kg, 22.5% v/v). Liquid-liquid extraction and LC-MS-MS operating in the positive ion multiple reaction monitoring mode were used. A C18 column was used, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile. The limit of detection was 3 pg/mL (S/N > 5) for A-F and B-F. The calibration curves were linear within the concentration range of 0.01-5 ng/mL (R2 = 0.9991) and 0.005-20 ng/mL (R2 = 0.9999) for A-F and B-F, respectively. Extraction recoveries were 91.3%-97.6% with RSD ≤ 11.2% and 90.5%-94.3% with RSD ≤ 10.5% for A-F and B-F, respectively. Plasma matrix effects were 80.61%-84.58% for A-F and 80.67%-81.33% for B-F with RSD ≤ 13.9%. The validated assay indicated no significant differences in pharmacokinetic parameters (AUC0-t, Cmax and T1/2) derived from the assessment of A-F and B-F plasma concentrations between control and ethanol-exposed rats. This assay, for which the LOD was 3 pg/mL for A-F and B-F may help the forensic science field to determine fentanyl analogue-related causes of death and identify illicit drug tampering.

Innovating the Concept and Practice of Two-Dimensional Gel Electrophoresis in the Analysis of Proteomes at the Proteoform Level.

Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given “protein” is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr. Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.

Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma.

Background Ropivacaine is a widely used local anaesthetic drug, highly bound to plasma proteins with a free plasma fraction of about 5%. Therefore, the monitoring of free drug concentration is most relevant to perform pharmacokinetic studies and to understand the drug pharmacokinetic/pharmacodynamic (PK/PD) relationship. Methods A high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using reverse-phase LC and electrospray ionisation mass spectrometry with multiple reaction monitoring (MRM) is described for the quantitation of both free and total ropivacaine in human plasma. Ropivacaine-d7 was used as an internal standard (IS). Results The method was validated in the range 0.5-3000 ng/mL, with five levels of QC samples and according to the European Medicine Agency and Food and Drug Administration guidelines. The performance of the method was excellent with a precision in the range 6.2%-14.7%, an accuracy between 93.6% and 113.7% and a coefficient of variation (CV) of the IS-normalised matrix factor below 15%. This suitability of the method for the quantification of free and total ropivacaine in clinical samples was demonstrated with the analysis of samples from patients undergoing knee arthroplasty and receiving a local ropivacaine infiltration. Conclusions A method was developed and validated for the quantification of free and total ropivacaine in human plasma and was shown suitable for the analysis of clinical samples.

Azo coupling-based derivatization method for high-sensitivity liquid chromatography–tandem mass spectrometry analysis of tetrahydrocannabinol and other aromatic compounds

An azo coupling-based derivatization method is reported for high-sensitivity liquid chromatography–tandem mass spectrometry (LC–MS/MS) quantitation of tetrahydrocannabinol (THC) and other aromatic compounds, i.e. phenols and amines. Through the azo coupling of a diazonium to an analyte, it produces a derivatized analyte which has enhanced ionization efficiency and results in high-response fragments in tandem mass spectrometry. The derivatization method was applied to six typical aromatic compounds using three different diazonium salts as derivatization reagents, demonstrating its applicability to a variety of analytes and reagents. The derivatization reaction can be directly carried out in neat samples, and after derivatization the samples can be immediately sent to the LC–MS/MS instrument for analysis. These advantages facilitate a one-step sample preparation procedure that can be completed in less than one hour, allowing for a “derivatize & shoot” lab workflow. The derivatization method was applied to establish an LC–MS/MS assay for the quantitation of THC in human breath samples. The derivatization conditions were studied in this application, including the effects of acidity, organic solvent, and diazonium concentration in the reaction. The THC derivatization assay was validated and achieved a limit of quantitation (LOQ) of 0.50 pg/ml using either of the two regio-isomers of the azo-derivative of THC (THC-DRV). To prove that the derivatization method has compatibility with complex-matrix samples, a THC derivatization assay for serum samples was established, in which the azo coupling reaction was directly carried out in crude protein-precipitated supernatants. An LOQ of 5.0 pg/ml was achieved. In addition, excellent correlation between THC derivatization and non-derivatization assays was found in the analysis of whole blood samples.

Free D-amino acids produced by commensal bacteria in the colonic lumen

D-amino acids (D-AAs) have various biological activities, such as activation of N-methyl-D-aspartic acid (NMDA) receptor as a co-agonist by D-Ser. Since several free D-AAs are released in the broth monocultured with bacterium and D-AAs are probably utilized for bacterial communication, we presume that intestinal microbiota releases several kinds of free D-AAs, which may be involved in the hosts’ health. However, presently, only four free D-AAs have been found in the ceacal lumen, but not in the colonic lumen. Here, we showed, by simultaneous analysis of chiral AAs using high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS), that 12 free D-AAs (D-Ala, D-Arg, D-Asp, D-Gln, D-Glu, D-allo-Ile, D-Leu, D-Lys, D-Met, D-Phe, D-Ser, and D-Trp) are produced by intestinal microbiota and identified bacterial groups belonging to Firmicutes as the relevant bacterial candidates.

Defects in 15-HETE Production and Control of Epithelial Permeability by Human Enteric Glial Cells From Patients With Crohn’s Disease

Enteric glial cells (EGCs) produce soluble mediators that regulate homeostasis and permeability of the intestinal epithelial barrier (IEB). We investigated the profile of polyunsaturated fatty acid (PUFA) metabolites produced by EGCs from rats and from patients with Crohn's disease (CD), compared with controls, along with the ability of one of these metabolites, 15-hydroxyeicosatetraenoic acid (15-HETE), to regulate the permeability of the IEB. We isolated EGCs from male Sprague-Dawley rats, intestinal resections of 6 patients with CD, and uninflamed healthy areas of intestinal tissue from 6 patients who underwent surgery for colorectal cancer (controls). EGC-conditioned media was analyzed by high-sensitivity liquid-chromatography tandem mass spectrometry to determine PUFA signatures. We used immunostaining to identify 15-HETE-producing enzymes in EGCs and tissues. The effects of human EGCs and 15-HETE on permeability and transepithelial electrical resistance of the IEB were measured using Caco-2 cells; effects on signal transduction proteins were measured with immunoblots. Levels of proteins were reduced in Caco-2 cells using short-hairpin RNAs or proteins were inhibited pharmacologically. Rats were given intraperitoneal injections of 15-HETE or an inhibitor of 15-lipoxygenase (the enzyme that produces 15-HETE); colons were collected and permeability was measured. EGCs expressed 15-lipoxygenase-2 and produced high levels of 15-HETE, which increased IEB resistance and reduced IEB permeability. 15-HETE production was reduced in EGCs from patients with CD compared with controls. EGCs from patients with CD were unable to reduce the permeability of the IEB; the addition of 15-HETE restored permeability to levels of control tissues. Inhibiting 15-HETE production in rats increased the permeability of the IEB in colon tissues. We found that 15-HETE regulates IEB permeability by inhibiting an adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1. Enteric glial cells from patients with CD have reduced production of 15-HETE, which controls IEB permeability by inhibiting adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1.

High-sensitivity liquid chromatography–tandem mass spectrometry method for the simultaneous determination of sodium picosulfate and its three major metabolites in human plasma

Sodium picosulfate (PICO-Na) is a member of the polyphenolic group of stimulant laxatives. Its major metabolites in humans are its active aglycone BHPM (bis-(p-hydroxyphenyl)-pyridyl-2-methane), the monoglucuronide (M1) and the monosulfate (M2) of BHPM. A sensitive, specific and rapid liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of picosulfate (PICO) and its three major metabolites in human plasma to investigate the pharmacokinetics of PICO and its major metabolites. Following protein precipitation with acetonitrile, chromatographic separation was achieved on a Luna 5u C18(2) column using gradient elution starting with 10% of 10mM ammonium acetate followed by increasing percentages of acetonitrile to eliminate interferences due to in-source conversion of the conjugated metabolites. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive mode, using the transitions of m/z 438.1→m/z 278.1 for PICO, m/z 278.1→m/z 184.2 for BHPM, m/z 454.1→m/z 184.2 for M1, and m/z 358.1→m/z 184.2 summed with m/z 358.1→m/z 278.1 for M2. Deuterium labeled compounds of the analytes were used as the internal standard, two of which, M1-d12 and M2-d12, were synthesized in-house. The method was validated in concentration ranges of 0.150–40.0ng/mL for PICO and M2, 0.600–160ng/mL for BHPM, and 0.045–12.0ng/mL for M1 with acceptable accuracy and precision. The method was successfully applied to characterize the pharmacokinetic profiles of PICO and its metabolites in healthy volunteers after a single oral administration of 5mg PICO-Na.

High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma

A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100mm×2.1mm, 3.5μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500ng/mL for caffeine and paraxanthine, 0.1–40ng/mL for losartan and EXP3174, 0.05–20ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8ng/mL for dextromethorphan and dextrorphan, 0.01–1.0ng/mL for midazolam, and 0.04–4ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.

High-Sensitivity Tandem Mass Spectrometry Assay for Serum Estrone and Estradiol

High-sensitivity measurement of serum estrogens is important in adult and pediatric endocrinology and oncology. We developed a high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of estrone (E1) and estradiol (E2). Aliquots of 200 muL of serum were spiked with internal standard, extracted, derivatized with dansyl chloride, and analyzed by LC-MS/MS using 2-dimensional chromatographic separation. Total imprecision for the method was less than 11%; the limit of quantitation was 1 pg/mL. Reference intervals were established with samples from more than 900 healthy postmenopausal women, men, girls, and boys. Concentrations of estrogens in children reached adult levels by Tanner stage 3. In men and postmenopausal women, the median concentrations of total estrogens (E1 + E2) were 39 and 22 pg/mL, and the median E2/E1 ratios were 0.98 and 0.55, respectively. The method requires a small sample volume and has adequate sensitivity and specificity for analyzing estrogens in samples from postmenopausal women, men, and children.