Detection of hantavirus infection in hemolyzed mouse blood using alkaline phosphatase conjugate

Conventional methods such as ELISA and culture require a long time for the determination of viral infections. Fast and reliable instrumentation suitable for operation under field conditions that allows rapid detection of hantavirus in rodent populations in the wild, would be a substantial improvement over these methods. In response to this challenge, a prototype amperometric immunosensor based on highly dispersed immunoelectrode for rapid, sensitive and quantitative assay of hantavirus in mice blood has been developed. A sandwich scheme of immunoassay is used and the naphthol that is formed as a result of enzymatic hydrolysis of alpha-naphthyl phosphate in the presence of alkaline phosphatase (AP) label is quantified amperometrically. The main complication faced when testing the amperometric immunosensor for application to mouse blood samples under field conditions is the removal of the interference caused by the electroactive components due to the hemolysis of the samples. We studied the possibility of using other enzyme markers such as AP to replace the horseradish peroxidase (HRP) used earlier in testing for antibodies against hantavirus in human blood plasma. Best results were obtained when the reference electrode is covered with a thin Nafion layer. Further improvement of assay performance was achieved by the modification of the sensing element. The amperometric immunoelectrode showed significantly higher sensitivity (more than 10-fold) than the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological and analytical practices.     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

Determination of chlorhexidine in saliva using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of chlorhexidine in saliva is reported. The method developed includes a simple and short sample preparation with a one-step extraction procedure and a short total chromatographic run time of 5 min. In a preliminary pharmacokinetic study with a healthy volunteer the chlorhexidine concentration found in saliva after 12 h was 0.8 μg/ml.     

Determination of tegaserod by LC-ESI-MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) assay for determination of tegaserod in human plasma using diazepam as internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol: 5 mM ammonium acetate (75:25, v/v, adjusting the pH to 3.5 with glacial acetic acid). The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions; m/z 302.5, 173.2 and 285.4, 193.2 were measured in positive mode for tegaserod and internal standard (diazepam), respectively. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The calibration curves were linear over the range 0.05-8.0 ng/ml (r=0.9996) for tegaserod. The mean absolute recovery of tegaserod was more than 85.56%. Intra- and inter-day variability values were less than 9.21% and 10.02%, respectively. The samples were stable for 8h under room temperature (25 degrees C, three freeze-thaw cycles in 30 days and for 30 days under -70 degrees C). After administration of a single dose of tegaserod maleate 4 mg, 6 mg and 12 mg, respectively, the area under the plasma concentration versus time curve from time 0 h to 12 h (AUC0-12) were (2.89+/-0.88), (5.32+/-1.21) and (9.38+/-3.42) ng h/ml, respectively; peak plasma concentration (Cmax) were (1.25+/-0.53), (2.21+/-0.52) and (4.34+/-1.66) ng/ml, respectively; apparent volume of distribution (Vd/F) were (6630.5+/-2057.8), (7615.2+/-2242.8) and (7163.7+/-2057.2) l, respectively; clearance rate (CL/F) were (1851.4+/-496.9), (1596.2+/-378.5) and (1894.2+/-459.3) l/h, respectively; time to Cmax (Tmax) were (1.00+/-0.21), (1.05+/-0.28) and (1.04+/-0.16) h, respectively; and elimination half-life (t1/2) were (3.11+/-0.78), (3.93+/-0.92) and (3.47+/-0.53) h, respectively; MRT were (3.74+/-0.85), (4.04+/-0.56) and (3.28+/-0.66) h, respectively. The essential pharmacokinetic parameters after oral multiple doses (6mg, b.i.d) were as follows: Cssmax, (2.72+/-0.61) ng/ml; Tmax, (1.10+/-0.25) h; Cssmin, (0.085+/-0.01) ng/ml; Cav, (0.54+/-0.12) ng/ml; DF, (4.84+/-0.86); AUCss, (6.53+/-1.5) ngh/ml. This developed and validated assay method had been successfully applied to a pharmacokinetic study after oral administration of tegaserod maleate in healthy Chinese volunteers at a single dose of 4 mg, 6 mg and 12 mg, respectively. The pharmacokinetic parameters can provide some information for clinical medication.     

LC-ESI-MS/MS method for the quantification of entecavir in human plasma and its application to bioequivalence study

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used for a quantitative estimation of entecavir (EV) in human plasma using lamivudine (LM) as internal standard (IS). The method herein described is simple, sensitive, and specific. Chromatographic separation was performed on XBridge-C18, 4.6 mm × 50 mm, 5-μm column with an isocratic mobile phase composed of 10 mM ammonium hydrogen carbonate (pH 10.5):methanol (85:15 v/v), pumped at 0.3 ml/min. EV and LM were detected using proton adducts at m/z 278.1→152.1 and 230.2→112.0 in multiple reaction monitoring (MRM) positive mode. Solid phase extraction method was employed in the extraction of EV and LM from the biological matrix. This method was validated over a linear concentration range of 50.0-20000.0 pg/ml with a correlation coefficient (r) ≥0.9983. Intra and inter-day precision of EV was found within the range of 1.2-4.2 for EV and 4.4-4.5 for LM. EV was stable throughout three freeze/thaw cycles, bench top and postoperative studies. This method was successfully used in the analysis of plasma samples following oral administration of EV (0.5 mg) in 26 healthy human volunteers.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

Determination of lapatinib (GW572016) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of lapatinib (GW572016) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Plasma samples (100 microL) were prepared using solid phase extraction (SPE) columns, and 6.0 microL of the reconstituted eluate was injected onto a Phenomenex CuroSil-PFP 3 mu analytical column (50 mm x 2.0mm) with an isocratic mobile phase. Analytes were detected with a PE SCIEX API-365 LC-MS/MS system at unit (Q1) and low (Q3) resolution in positive multiple reaction monitoring mode (m/z 581 (precursor ion) to m/z 364 (product ion) for lapatinib). The mean recovery for lapatinib was 75% with a lower limit of quantification of 15 ng/mL (S/N=11.3, CV< or =14%). This method was validated over a linear range of 100-10,000 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma lapatinib concentrations in a Phase I study in children with cancer.     

A general, robust method for the quality control of intact proteins using LC-ESI-MS

A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.     

Comparative clinical data for gingivitis treatment using gels from Ocimum sanctum (Tulsi) and chlorhexidine (CHX)

Ocimum sanctum (Tulsi) has various properties like anti bacterial, anti inflammatory, anti oxidant for curing diseases. It is a plant with known medicinal value in Indian system of medicine. Therefore, it is of interest to evaluate the effectiveness of Ocimum sanctum with Chlorhexidine (CHX) which is a standard material for the treatment of gingivitis. We used 30 gingivitis subjects divided into 2 groups. Group I used Tulsi gel (n= 15) and Group II used CHX gel (n = 15) for treatment. Tulsi and CHX gel use was advised for 1 month. The Clinical parameters assessed were gingival Index (GI), plaque Index (PI), probing depth (PD) and clinical attachment loss (CAL) assessed at a time interval of 30 days. Statistical analysis was completed using the SPSS software 23.0. Data showed that GI and PD for Tulsi and CHX in pre and post groups are not significant with p > 0.05. Moreover, PI is not significant with p>0.05 among pre Tulsi, pre CHX and post CHX. However, data is significant with p<0.05 for Tulsi group. CAL is significant with p<0.05 among pre/post Tulsi groups. However, this is not significant with p>0.05 among pre/post CHX groups. Data shows that 2% of Tulsi is effective in reducing gingival bleeding and inflammation. Thus, clinical data shows that Tulsi gel is promising for the treatment of gingivitis.     

Limulus test using a chromogenic method: application to the control of pyrogens in blood derivatives

We report here the application of the LAL Test to a chromogenic substrate to detect endotoxins in Human Blood Products. In order to reduce the cost, we used a microplate procedure with the Multiskan Reader. Quantitative results in the range of 0,01 to 0,1 ng/ml allowed for a good correlation with the Rabbit Pyrogen test. For 20% albumins and 4% albumins, the mean endotoxins levels of non pyrogenic lots were 0,38 +/- 0,18 and 0,09 +/- 0,03 ng/ml. All the lots which passed the Rabbit Pyrogen test had endotoxins levels lower than 1 ng/ml and 0,2 ng/ml, respectively. We can use this test for other plasma derivatives; Gammaglobulines and PPSB are easily tested. Dried Concentrated Antihemophilic Factor and Dried plasma contain citrate which inhibits the reaction. Dilution and Addition of Calcium Chloride overcome this inhibition. For dried plasma, we should destroy plasma inhibitors by heating at 75 degrees C. This sensitive and reproductible in vitro assay improves the control of pyrogenecity in Human Blood Products.     

Improved and simplified LC-ESI-MS/MS method for homocysteine determination in human plasma: application to the study of cardiovascular diseases

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of human plasma homocysteine (Hcy), an important independent risk factor for cardiovascular disease, with a simplified sample pretreatment procedure and a zero blank free of endogenous Hcy for calibrator/QC preparation. Following protein precipitation, chromatographic separation was performed on Hypersil Aquasil C18 column (50 mm x 2.1mm, 5 microm, Thermo) using mobile phase of aqueous 10% methanol containing 0.02% formic acid at 0.25 mL/min. Hcy and deuterated internal standard were detected in the multiple reaction monitoring mode with precursor to product ion transitions of m/z 136.1/90.0 and 140.1/94.0, respectively. The retention time was 1.2 min, and the total run time was 2 min per injection. A streamlined three-point calibration curve and one-point QC was used. Excellent linearity was observed with correlation coefficient (r)>0.99. The intra- and inter-batch were < or =3.24% and < or =4.04%, and accuracy was within +/-10%. Method comparison between the proposed method (y) and FPIA assay (x) demonstrated a correlation equation of y=1.003x + 0.4318 (r=0.9589). The developed method, improved for automation with cost-effective reagents, was proven to be suitable for high-throughput quantitative determination of Hcy in clinical practice by successfully applying it to the cardiovascular disease study.     

Determination of cysteinyl leukotrienes in exhaled breath condensate: method combining immunoseparation with LC-ESI-MS/MS

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.     

Determination of plasma volume in anaesthetized piglets using the carbon monoxide (CO) method

Based on measurements of the circulating red blood cell volume (V(RBC)) in seven anaesthetized piglets using carbon monoxide (CO) as a label, plasma volume (PV) was calculated for each animal. The increase in carboxyhaemoglobin (COHb) concentration following administration of a known amount of CO into a closed circuit re-breathing system was determined by diode-array spectrophotometry. Simultaneously measured haematocrit (HCT) and haemoglobin (Hb) values were used for PV calculation. The PV values were compared with simultaneously measured PVs determined using the Evans blue technique. Mean values (SD) for PV were 1708.6 (287.3)ml and 1738.7 (412.4)ml with the CO method and the Evans blue technique, respectively. Comparison of PVs determined with the two techniques demonstrated good correlation (r = 0.995). The mean difference between PV measurements was -29.9 ml and the limits of agreement (mean difference +/-2SD) were -289.1 ml and 229.3 ml. In conclusion, the CO method can be applied easily under general anaesthesia and controlled ventilation with a simple administration system. The agreement between the compared methods was satisfactory. Plasma volume determined with the CO method is safe, accurate and has no signs of major side effects.