Sabine Cohen

Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry.

In this study, we developed a new method for the simultaneous determination of eight endogenous ribonucleoside triphosphates and deoxyribonucleoside triphosphates based on a combination of a selective sample preparation and an ion-pair liquid chromatography-electrospray tandem mass spectrometry. The sample preparation was based on a protein precipitation coupled with a solid phase extraction using a weak-anion-exchange cartridge. The analytical separation of the nucleotides was achieved on a porous graphitic carbon stationary phase with a binary elution gradient program employing ion-pairing reagents (diethylamine and hexylamine) and organic eluent (methanol). The triple quadrupole mass spectrometer operated in both negative and positive multiple reaction monitoring modes. The calibration assay used the stable isotope labelled analogs of each compounds as standard. Standard calibrations were from 0.25 to 10pmol injected according to deoxyribonucleotides and from 12.5 to 3000pmol injected according to ribonucleotides. The within-run precision of the assay was less than 14.5% and the between-run precision was less than 12.4% for each analytes. Assay accuracy was in the range of 92.3-107.6%. This method allows the determination of NTP and dNTP pools from lysats of several cell lines or peripheral blood mononuclear cell from patient. Assays were performed with different preparation of cells to confirm the quality and the relevance of the described method.

Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: A review

Endogenous ribonucleotides and deoxyribonucleotides play a crucial role in cell function. The determination of their levels is of fundamental interest in numerous applications such as energy metabolism, biochemical processes, or in understanding the mechanism of nucleoside analog compounds. Nucleoside analogs are widely used in anticancer therapy. Their mechanisms of action are related to their structural similarity with natural nucleotides. Numerous assays have been described for the determination of endogenous nucleotides or anticancer nucleotide analogs in different matrices such as cellular cultures, tissue or peripheral blood mononuclear cells. The determination of these compounds is challenging due to the large difference of concentrations between ribonucleotides and deoxyribonucleotides, the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules due to the phosphate moiety. The extraction was generally performed at low temperature and was based on protein precipitation using acid or solvent mixture. This first phase could be coupled with extraction or cleaning step of the supernatant. Liquid chromatography coupled with UV detection and based on ion-exchange chromatography using non-volatile high salt concentrations was largely described for the quantification of nucleotides. However, the development of LC-MS and LC-MS/MS during the last ten years has constituted a sensitive and specific tool. In this case, analytical column was mostly constituted by graphite or C18 stationary phase. Mobile phase was usually based on a mixture of ammonium buffer and acetonitrile and in several assays included a volatile ion-pairing agent. Mass spectrometry detection was performed either with positive or negative electrospray mode according to compounds and mobile phase components. The purpose of the current review is to provide an overview of the most recent chromatographic assays (over the past ten years) developed for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy. We focused on sample preparation, chromatographic separation and quantitative considerations.

Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.

Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry.

Miglustat (OGT 918) is an iminosugar recently introduced in therapeutic as potential alternative therapy in disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases. A highly sensitive liquid-chromatography-electrospray tandem mass spectrometry (LC-MS/MS) assay was developed for the quantification of miglustat in human plasma and cerebrospinal fluid (CSF). The sample preparation consists in a simple protein precipitation with a mixture of acetonitrile/methanol (75/25) which yields 100% recovery. The isocratic separation utilizes an Atlantis Hilic (3 microm, 150 mm x 2.1 mm) column, with a mobile phase of acetonitrile/water/ammonium acetate buffer (75/10/15, v/v/v) delivered at 230 microl/min. Selected reaction monitoring (SRM) mode was used with the transitions m/z 220-->158 for the miglustat and m/z 208-->m/z 146 for the miglitol (internal standard). Good linearity was observed in a range from 125 to 2500 ng/ml and from 50 to 1000 ng/ml, for plasma and CSF, respectively. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for six replicates at each of three concentrations and evaluated on three separated days for both plasma and CSF mediums. Assay accuracy was in the range of 98-106.5%. Stability of miglustat was reported under a variety of storage conditions. The miglustat concentrations in two children are presented to demonstrate the clinical interest of this new method.

Assays for the quantification of melphalan and its hydrolysis products in human plasma by liquid chromatography-tandem mass spectrometry.

Two high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assays are described for the quantification of melphalan in human plasma. N-phenyldiethanolamine was tested as internal standard. The first assay consisted of a protein precipitation by cold methanol and a reversed-phase HPLC whereas the second one was based on a solid phase extraction and a hydrophilic interaction chromatography. Both provided a very satisfactory mean extraction yield with a small volume of sample. The first method was simple, rapid and used as a routine assay. The second one was developed in order to determine melphalan hydrolysis products and to avoid scarce cases when interferences from biological matrix alter the quantification of melphalan using the first method. The two assays were linear and sensitive in the range of 1-500ng/mL for the first one and in a range of 25-2000ng/mL for the second one. Concentrations out of the range fixed with the first method were also validated. The procedure was reliable with precision and accuracy below 10%. All compounds were detected after positive mode electrospray ionization in selected reaction monitoring mode. These new analytical procedures were developed for melphalan pharmacokinetic studies or therapeutic drug monitoring.

Comparison of Capillary-Zone Electrophoresis with the LC-UV–Remedi System for Emergency Toxicological Screening

Abstract Capillary-zone electrophoresis (CZE) has been investigated for urgent toxicological screening of serum of poisoned patients. Samples (115) have been analyzed in parallel with the liquid chromatograph (LC)–ultraviolet (UV)–Remedi (BioradTM) system. This study highlighted the advantages and the disadvantages of this method compared to the LC-UV–Remedi method. The majority of the molecules identified with the LC-UV–Remedi method were also found with CZE, some with more sensitivity. However, it showed the limitations of the method, including its extraction procedure and analysis of weakly basic drugs. With some improvements, this system may be an alternative to traditional LC coupled with a diode array detector.