Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of the ultraviolet screening pigment scytonemin: characteristic fragmentations

Atmospheric pressure chemical ionisation reversed-phase high-performance liquid chromatography/multistage mass spectrometry has been used to study the mass spectral fragmentation of the cyanobacterial sheath pigment scytonemin and its reduced counterpart. The two pigments exhibit characteristic fragment ions in their MS2 and MS3 spectra that are of value in confirming the identification of the structures in extracts from natural environments.

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of bacteriochlorophylls from Chlorobiaceae: characteristic fragmentations

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry/mass spectrometry (APCI-LC/MS/MS) has been applied to the study of bacteriochlorophylls c, d, and e of phototrophic prokaryotes. Cultures of Chlorobiaceae containing bacteriochlorophyll c, d or e were examined using a high-resolution high-performance liquid chromatography (HPLC) method and APCI-LC/MS/MS employing post-column addition of formic acid. The results reveal complex distributions of bacteriochlorophyll homologues, with some closely eluting species giving isobaric protonated molecules. On-line LC/MS/MS studies reveal characteristic fragment ions for bacteriochlorophylls c, d, and e. Fragmentations involving loss of the extended alkyl substituents that are unique to bacteriochlorophylls c, d and e and their derivatives have been rationalised by studying the phaeophorbides and the results applied to the direct study of the bacteriochlorophylls.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

Towards Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC Liquid Chromatography Time-of-flight Mass Spectrometry identification of Paralytic Shellfish Poisons

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications. (C) 2009 Elsevier Ltd. All rights reserved.

Structure-inhibition relationship of phenylethanoid glycosides on angiotensin-converting enzyme using ultra-performance liquid chromatography-tandem quadrupole mass spectrometry

Angiotensin-converting enzyme (ACE) plays a critical role in rennin-angiotensin system. Recently, natural products isolated from herbal medicines revealed inhibitory effects against ACE which suggested their potential activities in regulating blood pressure. In this study, ACE inhibition (ACEI) of 21 phenylethanoid glycosides and related phenolic compounds were investigated by measuring the production of HA a rapid, sensitive, accurate and specific ultra-performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method. The test compounds showed different inhibitory potencies on ACE ranging from 5.29 to 95.01% at 50 mM, and the compounds with ACEI higher than 50% were selected for further IC50 determination. The IC50 values were from 0.53 ± 0.04 to 15.035 ± 0.036 mM. The structure-inhibition relationship were then explored and the result showed that cinnamoyl groups played an essential role in ACEI of phenylethanoid glycosides. Furthermore, the sub-structures of increasing ACEI for phenylethanoid glycosides is more hydroxyls and less steric hindrance to chelate the active site Zn2+ of ACE. In summary, our results suggested that phenylethanoid glycosides are a widely available source of anti-hypertensive natural products and the information provided from structure-inhibition relationship study could aid the design of structurally modified phenylethanoid glycosides as anti-hypertensive drugs.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Liquid Chromatography-Electrospray Linear Ion Trap Mass Spectrometry Analysis of Targeted Neuropeptides in Tac1-/- Mouse Spinal Cords Reveal Significant Lower Concentration of Opioid Peptides.

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop a HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1-/- mouse endogenous opioid system is hampered and therefore affect significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1-/- spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p < 0.0001). No significant concentration differences were observed in mouse Tac1-/- spinal cords for Met-Enk and CGRP. The analysis of Tac1-/- mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins.

Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Qualitative and quantitative analysis of lipid classes in fish oils by thin-layer chromatography with an Iatroscan flame Ionization detector (TLC-FID) and liquid chromatography with an evaporative light scattering detector (LC-ELSD)

Combined effects of hydrogen and air flow rates on the peak response of selected neutral lipid classes (triacylglycerol, diacylglycerol, monoacylglycerol, free fatty acids, and ethyl esters) were studied to optimize and calibrate the Iatroscan Mk-6s Chromarod system for the qualitative and quantitative analysis of lipid classes by thin-layer chromatography (TLC) with flame ionization detection in fish oil during the transesterification process. Air flow rate of 2 L/min, hydrogen flow rate of 150-160 mL/min, and scan rate of 30 s/rod were found to be the optimum conditions. All samples were also analyzed by high performance liquid chromatography (HPLC) with evaporative light scattering detection. Quantitative results obtained by TLC with the flame ionization detection method were comparable to those obtained from HPLC with evaporative light scattering detection.

π-Selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography

The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 μmol m⁻²). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant.