Unlocking the power of Zc3h12c: Orchestrating Macrophage activation and elevating the innate immune response.

The activation of macrophages, essential for the innate defense against invading pathogens, revolves around Toll-like receptors (TLRs). Nevertheless, a comprehensive understanding of the molecular mechanisms governing TLR signaling in the course of macrophage activation remains to be fully clarified. Although Zc3h12c was originally identified as being enriched in organs associated with macrophages, its precise function remains elusive. In this study, we observed a significant induction of Zc3h12c in macrophages following stimulation with TLR agonists and pathogens. Overexpression of Zc3h12c significantly mitigated the release of TNF-α and IL-6 triggered by lipopolysaccharide (LPS), whereas depletion of Zc3h12c increased the production of the cytokines mentioned above. Notably, the expression of IFN-ß was not influenced by Zc3h12c. Luciferase reporter assays revealed that Zc3h12c could suppress the TNF-α promoter activity. Moreover, Zc3h12c exerted a notable inhibitory effect on JNK, ERK, p38, and NF-κB signaling induced by LPS. In summary, the findings of our study suggest that Zc3h12c functions as a robust suppressor of innate immunity, potentially playing a role in the pathogenesis of infectious diseases.

Comparison of the characterization of allergenic protein 3 (Pla a3) released from Platanus pollen grains collected in Shanghai during the spring of 2019 and 2020.

Due to the COVID-19 pandemic in early 2020, large-scale industrial production has been stagnant and reduced, the urban air quality has been greatly improved. It provided an excellent opportunity to explore the effects of air pollutants on the sensitization of pollen allergen proteins in the environment. Platanus pollen grains sampled in the spring of 2019 and 2020 were used for detailed characterization and analysis. Scanning electron microscopy, Fourier transform infrared, X-ray spectroscopy (XPS), trypan blue staining, and western blot analysis were employed to characterize Platanus pollen protein released from pollen grains. Our data showed that the viability of the pollen grains in 2019 was lower compared that in 2020, and the pollen grains collected in 2019 had a higher absorption peak of protein functional groups. The XPS spectra assay result demonstrated that the binding energy of the high-resolution components had not variation on the surface of pollen grains, but relative content of nitrogen and peptide chain in the pollen grains sampled in 2019 were higher than in 2020. These results suggested that more protein in the pollen grains was released onto the surface of pollen grains. In addition, western blot assay showed that the expression of Pla a3 protein in pollen grains sampled in 2019 was significantly higher than that in 2020, revealing that air pollutants could enhance the expression of Pla a3 proteins in Platanus pollen. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10453-021-09731-6.

Measurement of serum 17-hydroxyprogesterone using isotope dilution liquid chromatography-tandem mass spectrometry candidate reference method and evaluation of the performance for three routine methods.

OBJECTIVES: Accurate measurements of serum 17-hydroxyprogesterone (17OHP) are essential for diagnosis and treatment monitoring for congenital adrenal hyperplasia patients. The performance of serum 17OHP routine methods remains highly variable that calls for a candidate reference measurement procedure (cRMP) to improve the standardization of serum 17OHP measurements. METHODS: Serum samples spiked with internal standards were extracted with a combination of solid-phase extraction and liquid-liquid extraction. The 17OHP was quantified by the isotope dilution coupled with liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) with electrospray ionization in positive ion mode. Nine structural analogs of 17OHP were evaluated for interferences. The precision and analytical recovery were assessed. Twenty native and 40 spiked serum for performance evaluation were measured by the cRMP and two clinical LC/MS routine methods. RESULTS: No apparent interferences were found with the 17OHP measurement. The within-run, between-run, and total precision for our method were 0.4-0.8%, 0.6-2.0%, and 1.0-2.1% for four pooled serum (2.46-102.72 nmol/L), respectively. The recoveries of added 17OHP were 100.0-100.2%. For the performance of two LC/MS routine methods, they showed relative deviation ranges of -22.1 to 1.1% and -6.7 to 12.8%, respectively. CONCLUSIONS: We developed and validated a reliable serum 17OHP method using ID-LC/MS/MS. The desirable accuracy and precision of this method enable it to serve as a promising cRMP to improve the standardization for serum 17OHP routine measurements.

Lithium chloride confers protection against viral myocarditis via suppression of coxsackievirus B3 virus replication.

Viral myocarditis (VMC) is a type of inflammation affecting myocardial cells caused by viral infection and has been an important cause of dilated cardiomyopathy (DCM) worldwide. Type B3 coxsackievirus (CVB3), a non-enveloped positive-strand RNA virus of the Enterovirus genus, is one of most common agent of viral myocarditis. Till now, effective treatments for VMC are lacking due to lack of drugs or vaccine. Lithium chloride (LiCl) is applied in the clinical management of manic depressive disorders. Accumulating evidence have demonstrated that LiCl, also as an effective antiviral drug, exhibited antiviral effects for specific viruses. However, there are few reports of evaluating LiCl's antiviral effect in mice model. Here, we investigated the inhibitory influence of LiCl on the CVB3 replication in vitro and in vivo and the development of CVB3-induced VMC. We found that LiCl significantly suppressed CVB3 replication in HeLa via inhibiting virus-induced cell apoptosis. Moreover, LiCl treatment in vivo obviously inhibited virus replication within the myocardium and alleviated CVB3-induced acute myocarditis. Collectively, our data demonstrated that LiCl inhibited CVB3 replication and negatively regulated virus-triggered inflammatory responses. Our finding further expands the antiviral targets of LiCl and provides an alternative agent for viral myocarditis.

Application of a micro plasma collection card for the detection of homocysteine by liquid chromatography with tandem mass spectrometry.

Generally, it is recommended that blood samples for homocysteine detection should be centrifuged immediately to separate plasma in order to avoid continuous synthesis by blood cells. The use of a micro plasma collection card may improve sample stability and result accuracy by offering automatic and instant plasma separation. We compare a micro plasma collection method with routine wet plasma to explore applications of the dried plasma spots for homocysteine determination by using liquid chromatography with tandem mass spectrometry. The method was validated for both dried plasma spots and wet plasma. The assay was linear from 0.5-45 µmol/L with good precisions and accuracies. The extraction recovery and matrix effect for dried plasma spots were >97% and 0.98 after internal standard normalization, respectively. It was reproducible for retaining homocysteine in dried plasma spots and kept stable for 30 days. The plasma conversion factor was 7.77 ± 0.7% by calculating the ratio of homocysteine concentration between dried plasma spots and wet plasma (n = 165). Neither hematocrit nor homocysteine concentration affected the plasma conversion factor as long as the hematocrit was within the normal range. The results support the clinical usefulness of the dried plasma spots as a convenient and stable biological matrix for testing homocysteine.

Determination of the novel antiarrhythmic drug sulcardine sulfate in human plasma by liquid chromatography tandem mass spectrometry and its application in a clinical pharmacokinetic study.

Sulcardine sulfate (Sul), a novel antiarrhythmic agent, is currently in phase I and phase II clinical trials. To elucidate its clinical pharmacokinetic characteristics, a rapid and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of Sul in human plasma. Plasma samples were precipitated by acetonitrile and isotope-labeled sulcardine was added as internal standard. The analysis was carried out on a Capcell Pak C18 MG III column (100 × 2.0 mm, 5 µm) with 0.1% formic acid in acetonitrile solution and water (17:83, v/v) as mobile phase. The linear range was 5.0-1000 ng/mL for Sul, with a lower limit of quantification of 5.0 ng/mL. The intra- and inter-batch CVs were within ±11.0% and the accuracies were 4.9-107.3%. Our method, for the first time, allows the rapid (only 3.0 min) and accurate quantification of Sul in human plasma. The method has been successfully applied in the pharmacokinetic study of Sul in a clinical trial following oral administration of Sul to healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

Hair analysis, a reliable and non-invasive method to evaluate the contamination by clenbuterol.

The illegal use of clenbuterol has been an increasingly serious issue in today's livestock products industry. It becomes an important project to develop a reliable approach to detect its content in food animals. A simple and sensitive LC-MS/MS method was developed to detect clenbuterol residue in hair, with the low limit of quantitation (LLOQ) about 0.5ng/g. Hogs fed with 340µg/day of clenbuterol for 2 weeks were found a high clenbuterol residue in their hair approximately at 1-2 months after withdrawal. There remained 3.31ng/g clenbuterol in hog hair approximately 5 months after the last administration, focused on the tip of the hair (mainly in hogs with dark hair). An extensive contamination was observed in twenty investigated market hogs whose dark hair obviously had a higher clenbuterol residue than the light ones (p=0.017, t test). Volunteers (60.3 percent) from Xuhui district (Shanghai) were found to have a detectable amount of clenbuterol in their hair (>0.5ng/g). In conclusion, hair residue detection is a reliable method to evaluate the clenbuterol contamination in animals and humans. Meat supply in the Xuhui district might have serious potential safety risks which should be further investigated and discussed to determine the safety range of clenbuterol residue.

Contribution of carboxylesterase in hamster to the intestinal first-pass loss and low bioavailability of ethyl piperate, an effective lipid-lowering drug candidate.

Ethyl piperate is an effective lipid-lowering drug candidate synthesized from piperine. However, its pharmacokinetic characteristics and oral absorption process remain unclear. A liquid chromatography-tandem mass spectrometry method was applied to determine the oral bioavailability of ethyl piperate. Simulated gastrointestinal pH conditions and intestinal washings were prepared to investigate their contributions to the loss of ethyl piperate. Hydrolysis by carboxylesterase (CES) was evaluated in vitro using microsomes and S9 fractions. In situ intestinal single-pass perfusion experiments were performed to estimate the role of CES in ethyl piperate absorption. The bioavailability of ethyl piperate was extremely low (0.47%) in hamster independent of gastrointestinal environmental effects. Ethyl piperate was a typical substrate of CES with kinetic parameters K(m) and V(max) of 7.56 ± 1.491 µM and 0.16 ± 0.008 nmol · min(-1) · mg protein(-1), respectively. CES was responsible for 85.8% of the intestinal hydrolysis of ethyl piperate. Specific inhibition of CES with bis-p-nitrophenyl phosphate (BNPP), decreased degradation clearance to 36% of control with no significant change in absorption clearance. This contrasted with the results of Caco-2 monolayer experiments, which showed a dramatic increase in the apparent permeability coefficient after BNPP treatment. mRNA levels for the CES isozyme, CES2A3, were similar among the three regions of hamster intestine and 60% less than those in liver; CES1B1 mRNA levels were even lower in the intestine and showed a proximal-to-distal decrease. In conclusion, CES markedly contributes to intestinal first-pass hydrolysis of ethyl piperate that is sufficient, but not necessary, to cause the observed extremely low bioavailability.

Liquid chromatography tandem mass spectrometry method for determination of N-acetylcysteine in human plasma using an isotope-labeled internal standard.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine total N-acetylcysteine in human plasma. Mass spectrometric detection was achieved in positive electrospray ionization and multiple reaction monitoring mode. The mass transition pairs of N-acetylcysteine and the isotope-labeled internal standard d3-N-acetylcysteine were 164 → 122 and 167 → 123, respectively. The method was linear over the range of 10-5000 ng/mL in human plasma. The adoption of trichloroacetic acid significantly enhanced the extraction recovery. The blank matrix was screened to minimize the influence of endogenous N-acetylcysteine. After being fully validated, the method was successfully applied to the pharmacokinetic and bioequivalent study of N-acetylcysteine after oral administration of 600 mg tablets to 24 healthy Chinese volunteers.

Characterization of allergenicity of Platanus pollen allergen a 3 (Pla a 3) after exposure to NO2 and O3.

Pollen allergens, widely present in the atmosphere, are the main cause of seasonal respiratory diseases that affect millions of people worldwide. Although previous studies have reported that nitrogen dioxide (NO2) and ozone (O3) promote pollen allergy, the specific biological processes and underlying mechanisms remain less understood. In this study, Platanus pollen grains were exposed to gaseous pollutants (NO2 and O3). We employed environmental electron microscopy, flow cytometry, western blot assay, enzyme-linked immunoassay, ultraviolet absorption spectrometry, circular dichroism, and protein mass spectrometry to characterise the subpollen particles (SPPs) released from pollen grains. Furthermore, we determined the immunogenicity and pathogenicity induced by Platanus pollen allergen a 3 (Pla a 3). Our results demonstrated that NO2 and O3 could damage the pollen cell membranes in SPPs and increase the amount of Pla a 3 allergen released into the atmosphere. Additionally, NO2 and O3 altered the structure of Pla a3 protein through nitrification and oxidation, which not only enhanced the immunogenicity of allergens but also increased the stability of the protein. In vivo analysis using an animal model indicated that NO2 and O3 greatly aggravated pollen-induced pneumonia. Thus, our study provides guidance for the prevention of pollen allergic diseases.

Pharmacokinetic study of melamine in rhesus monkey after a single oral administration of a tolerable daily intake dose.

To perform pharmacokinetic study of melamine in rhesus monkey, melamine was orally administered to three experimental monkeys at a single dose of 1.4 mg/kg body weight. Plasma and urine were collected for the determination of melamine and cyanuric acid with a liquid chromatography tandem mass spectrometry method. The mean+/-SD area under the concentration-time curve from time zero to 48 h (AUC0-t) was 14,145+/-2002 microg/Lh. The maximum concentration of melamine in plasma (C(max)) was 1767+/-252 microg/L. The time to maximum concentration (T(max)) was 2.67+/-1.16 h and the half-life of melamine in plasma (t(1/2)) was 4.41+/-0.43 h. Following oral administration, melamine was rapidly excreted, mainly through urinary clearance. No significant correlation was found between melamine and cyanuric acid, suggesting that cyanuric acid may not be derived from melamine.

Development of a liquid chromatography-isotope dilution mass spectrometry method for quantification of fentanyl in human plasma.

Fentanyl is a potent analgesic drug in relieving chronic pain in patients. In this report, we present a simple, reliable and sensitive LC-ID/MS method for the quantification of fentanyl in human plasma. LC-ID/MS analysis was carried out on a triple quadrupole mass spectrometer operated in positive electrospray ionization multiple-reaction-monitoring using the transitions m/z 337.6 --> 187.9 for fentanyl and m/z 342.6 --> 187.9 for the internal standard (D5-fentanyl). The calibration curve covered the range 0.02-10 ng/mL. The intra- and inter-batch precision were less than 6.739 and 3.126% for fentanyl and IS, with accuracy from 94.16 to 102.0%. The lower limit of quantification was identifiable and reproducible at 0.02 ng/mL. The validated method offered increased sensitivity and wide linear concentration range. This method was successfully adopted for the evaluation of bioequivalence of two fentanyl transdermal preparations after single dose administration to 20 Chinese pain-patients.

Liquid chromatography tandem mass spectrometry method for determination of bisoprolol in human plasma using d5-bisoprolol as the internal standard.

A simple, reliable and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol was developed and validated for quantification of bisoprolol in human plasma. The sample was pretreated with a simple procedure of protein precipitation and an isotope-labeled d5-bisoprolol was used as internal standard. The chromatographic separation was performed on a Capcell Pak C(18) MG III column (100 mm x 2.0 mm, 5 microm). The protonated ion of the analyte was detected in positive ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 326.3 --> 116.3 and m/z 331.3 --> 121.3 were used to detect bisoprolol and the internal standard, respectively. Linearity, accuracy, precision, recovery, matrix effect, dilution test and stability were evaluated during method validation over the range of 0.5-100 ng/mL. The validated method was successfully applied to analyze human plasma samples in a bisoprolol bioavailability study.

Simultaneous determination of itraconazole and hydroxyitraconazole in human plasma by liquid chromatography-isotope dilution tandem mass spectrometry method.

A rapid and sensitive liquid chromatography-isotope dilution tandem mass spectrometry method was developed and validated for quantification of itraconazole (ITZ) and its active metabolite hydroxyitraconazole (OH-ITZ ) in human plasma. The plasma samples were extracted with tert-butyl methyl ether and two isotope-labeled internal standards (D5-itraconazole and D5-hydroxyitraconazole) were used. The chromatographic separation was performed on a Capcell Pak C(18) MG III (100 x 2 mm, 5 microm, Shiseido). The protonated ions of analytes were detected in positive ionization in multiple reaction monitoring mode. The plasma method has a lower limit of quantification of 1 ng/mL with a linearity range of 1-500 ng/mL for ITZ and OH-ITZ using 100 microL of plasma. The recoveries of the method were found to be 69.47-71.98% for ITZ and 75.68-82.52% for OH-ITZ. The intra- and inter-batch precision was less than 11% for all quality control samples at concentrations of 2.5, 200 and 400 ng/mL. These results indicate that the method was efficient with a short run time (4.5 min) and acceptable accuracy, precision and sensitivity.The validated method was successfully applied to analysis of human plasma samples in pharmacokinetics study.

Pharmacokinetics of depside salts from Salvia miltiorrhiza in healthy Chinese volunteers: A randomized, open-label, single-dose study.

BACKGROUND: Depside salts from Salvia miltiorrhiza, with active components of lithospermic acid B (LSB), rosmarinic acid (RA), and lithospermic acid (LA), are a multicomponent drug marketed in China for the treatment of coronary heart disease. OBJECTIVES: The aims of this study were to determine the concentrations of LSB, RA, and LA in human plasma and urine, and to compare the pharmacokinetic properties of depside salts from S miltiorrhiza in healthy Chinese volunteers. METHODS: A randomized, open-label, single-dose study was conducted in healthy Chinese volunteers. Participants were randomly assigned to receive a single intravenous infusion of 100 or 200 mg of depside salts from S miltiorrhiza. Blood was collected through a venous cannula prior to study drug administration (0 min) and at 10, 20, 30, 60, 65, 70, 80, and 90 minutes and 2, 3, 4, 6, 8, 12, and 24 hours after study drug administration. Urine samples were taken before study drug administration (0) and at 0 to 12 and 12 to 24 hours after study drug administration. LSB, RA, and LA concentrations in serum and urine were analyzed by an LC-MS/MS method. Tolerability was determined by clinical assessment; vital signs (ie, blood pressure, heart rate, breathing rate, body temperature) monitoring at baseline and at the end of the study, clinical laboratory tests (ie, hematology, blood biochemistry, hepatic function, renal function, urinalysis), 12-lead ECG measurements, and physical examinations at baseline and after completion of the study. RESULTS: Twelve Chinese volunteers (6 males, 6 females; mean [SD] age, 25.2 [3.8] years; mean height, 165.7 [8.9] cm; mean body mass index, 21.6 [2.5] kg/m(2)) were enrolled in the study. Peak plasma concentrations of LSB, RA and LA were observed at 0.3 to 1 hour following the 1-hour intravenous infusion, with respective mean (SD) Cmax of 4925 (1861), 174 (61), and 361 (101) ng/mL for the 100-mg dose and 10,285 (2259), 308 (77), and 674 (85) ng/mL for the 200-mg dose. The AUClast values for LSB, RA, and LA were 4537 (1265), 129 (28), and 1229 (330) ng/mL/h, respectively, for the 100-mg dose and 10,426 (2589), 260 (53), and 2792 (729) ng/mL/h for the 200-mg dose. No significant difference in pharmacokinetic parameters was observed between male and female subjects. Three metabolites were found in the plasma with low concentrations. The urinary excretion recoveries of LSB, RA, and LA were 0.58% (0.42%), 25.21% (20.61%), and 10.02% (7.72%) for the 100-mg dose and 0.38% (0.18%), 20.11% (10.50%), and 6.34% (3.20%) for the 200-mg dose. No adverse events were reported by the subjects or found by the investigators in the analysis of vital signs, 12-lead ECG measurements, physical examinations, or clinical laboratory tests. CONCLUSIONS: Following single intravenous infusion of 100 or 200 mg of depside salts from S miltiorrhiza to healthy Chinese subjects, no statistical differences in pharmacokinetic parameters were observed between males and females. The 2 doses of depside salts from S miltiorrhiza were clinically well tolerated during the study.

Development and validation of a liquid chromatography-isotope dilution tandem mass spectrometry for determination of olanzapine in human plasma and its application to bioavailability study.

A simple, reliable and sensitive liquid chromatography-isotope dilution mass spectrometry (LC-ID/MS) was developed and validated for quantification of olanzapine in human plasma. Plasma samples (50 microL) were extracted with tert-butyl methyl ether and isotope-labeled internal standard (olanzapine-D3) was used. The chromatographic separation was performed on XBridge Shield RP 18 (100 mm x 2.1 mm, 3.5 microm, Waters). An isocratic program was used at a flow rate of 0.4 m x min(-1) with mobile phase consisting of acetonitrile and ammonium buffer (pH 8). The protonated ions of analytes were detected in positive ionization by multiple reactions monitoring (MRM) mode. The plasma method, with a lower limit of quantification (LLOQ) of 0.1 ng x mL(-1), demonstrated good linearity over a range of 0.1 - 30 ng x mL(-1) of olanzapine. Specificity, linearity, accuracy, precision, recovery, matrix effect and stability were evaluated during method validation. The validated method was successfully applied to analyzing human plasma samples in bioavailability study.

Direct analysis in real time-mass spectrometry for rapid quantification of five anti-arrhythmic drugs in human serum: application to therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

MCPIP1 inhibits Hepatitis B virus replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory responses.

Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response. Recently, MCPIP1 has also been identified as a host antiviral factor against certain virus infection including human immunodeficiency virus, dengue virus and hepatitis C virus. However, whether MCPIP1 could restrict the replication of hepatitis B virus (HBV), a DNA pararetrovirus belonging to Hepadnaviridae family, has not been investigated. In this study, we found that MCPIP1 expression was up-regulated in mouse livers upon acute HBV replication and in HBV-replicated hepatoma cells or HBV-stimulated macrophages. Enforced MCPIP1 expression by hydrodynamic DNA injection in vivo significantly inhibited HBV replication in the mouse livers. Then in vitro studies by overexpression or knockdown assays in cell-lines identified the direct antiviral effect of MCPIP1 on HBV replication. RNA immunoprecipitation and decay assay further suggested that MCPIP1 potently restricted HBV replication through directly binding viral RNA and degrading RNA via its RNase activity, but not deubiquitinase activity. Moreover, we further verified that MCPIP1 negatively regulated HBV-induced proinflammatory cytokines, such as IL-1ß, TNF-α and IL-6 in macrophages. Taken together, our data expand MCPIP1's range of viral targets to DNA virus and also demonstrate the negative regulatory role of MCPIP1 in suppressing virus-induced inflammatory response, suggesting MCPIP1 as a potential therapeutic target for treating HBV-related diseases via inducing a host defense against HBV and reducing inflammatory injury meanwhile.

Pharmacokinetics and bioequivalence evaluation of two formulations of 10-mg amlodipine besylate: an open-label, single-dose, randomized, two-way crossover study in healthy Chinese male volunteers.

BACKGROUND: Amlodipine is a third-generation dihydropyridine calcium antagonist for the treatment of angina and hypertension. The relative bioavailability of a newly developed dispersible tablet as compared with an established branded formulation has not been reported in a Chinese population. OBJECTIVE: The aim of this study was to assess and compare the pharmacokinetic properties, bioavailability, and bioequivalence of a newly developed dispersible tablet formulation of amlodipine besylate with those of an established branded formulation in healthy Chinese adult male volunteers. METHODS: An open-label, single-dose, randomized, 2-way crossover study was conducted in fasted healthy Chinese male volunteers. Eligible participants were randomly assigned in a 1:1 ratio to receive 2 tablets (5 mg each) of the test or reference formulation, followed by a 2-week washout period and administration of the alternate formulation. The study drugs were administered after a 10-hour overnight fast. Serum samples were collected over 120 hours. Amlodipine concentrations in the serum were analyzed by liquid chromatography tandem mass spectrometry with positive ion electrospray ionization using the multiple reaction monitoring mode. The visible detection of the method was in the range of 0.2 to 32.0 ng/mL, and the lower limit of quantification for amlodipine was 0.2 ng/mL. The amlodipine serum concentration-time curves were used to obtain pharmacokinetic parameters including AUC(0-t), AUC(0-infinity)), and C(max). The criteria for bioequivalence were 90% CIs of 80% to 125% for AUC and 70% to 143% for C(max), according to guidelines of the State Food and Drug Administration of the People's Republic of China. Tolerability was based on the recording of adverse events (AEs), monitoring vital signs, electrocardiograms, and clinical laboratory tests at baseline and completion of the study. RESULTS: A total of 20 healthy Chinese male volunteers (mean [SD] age, 21.4 [2.6] years [range, 1926 years]; weight, 61.3 [5.4] kg [range, 54.0-75.0 kg]; and height, 171.2 [3.6] cm [range, 162.0-177.0 cm) were included in the study. The mean (SD) C(max), T(max), AUC(0-t), and AUC(0-infinity)) values after administration of the test and reference formulations, respectively, were as follows: 5.46 (1.13) versus 5.88 (1.24) ng/mL, 7.70 (2.08) versus 9.20 (4.18) hours, 284.56 (77.59) versus 311.34 (75.97) ng/mL/h, and 331.37 (111.03) versus 358.74 (101.10) ng/mL/h. The mean (SD) t(1/2) was 38.52 (10.51) hours for the test formulation and 38.75 (7.07) hours for the reference formulation. On analysis of variance, no period or sequence effects were observed for any pharmacokinetic property; however, a significant formulation effect was observed for C(max), AUC(0-t), and AUC(0-infinity)). The relative bioavailability of the test formulation was 90.9% by mean AUC(0-t) and 91.2% by mean AUC(0-infinity). The 90% CIs for the ratios of C(max), AUC(0-t), and AUC(0-infinity) were 88.4% to 97.5%, 86.4% to 95.7%, and 85.8% to 97.0%, respectively, meeting the predetermined criteria for bioequivalence. One subject (5%) reported 2 AEs. The AEs were mild, possibly associated with study drug, and resolved spontaneously by the next evaluation. No serious AEs were reported. CONCLUSIONS: In this small study in healthy Chinese adult male volunteers, a single 10-mg dose of the dispersible tablet formulation (test) of amlodipine besylate met the regulatory criteria for bioequivalence to the established tablet formulation (reference) based on the rate and extent of absorption. Both formulations were well tolerated.

Liquid chromatography-tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma.

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150mmx2mm, 5microm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/z 220.3-->118.9 and m/z 226.4-->123.2 were used to detect N-acetylglucosamine and internal standard 13C6-N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.