Implementation study of patient-ready syringes containing 25 mg/mL methotrexate solution for use in treating ectopic pregnancy.

BACKGROUND: Ectopic pregnancy (EP) is a significant cause of morbidity and mortality during the first trimester of pregnancy. Small unruptured tubal pregnancies can be treated medically with a single dose of methotrexate (MTX). OBJECTIVE: The aim of this study was to evaluate the stability of a 25 mg/mL solution of MTX to devise a secure delivery circuit for the preparation and use of this medication in the management of EP. METHOD: MTX solutions were packaged in polypropylene syringes, stored over an 84-day period, and protected from light either at +2 to +8°C or at 23°C. We assessed the physical and chemical stability of the solutions at various time points over the storage period. A pharmaceutical delivery circuit was implemented that involved the batch preparation of MTX syringes. RESULTS: We show that 25 mg/mL MTX solutions remain stable over an 84-day period under the storage conditions tested. Standard doses were prepared, ranging from 50 mg to 100 mg. The results of this study suggest that MTX syringes can be prepared in advance by the pharmacy, ready to be dispensed at any time that a diagnosis of EP is made. CONCLUSION: The high stability of a 25 mg/mL MTX solution in polypropylene syringes makes it possible to implement a flexible and cost-effective delivery circuit for ready-to-use preparations of this drug, providing 24-hour access and preventing treatment delays.

Evaluation of a liquid chromatography method for quality control of methylated cyclodextrins.

Halo C18 column (fused core particles) and Chromolith RP18 column (monolith) were evaluated in liquid chromatography in order to analyze methylated-ß-cyclodextrins (Me-ß-CD) with various degrees of substitution, DS such as the number of methyl groups per cyclodextrin ring. Chromolith RP18 enables a performing analysis of Me-ß-CD with low DS but is not suitable for dimethyl-ß-cyclodextrins (DM-ß-CD). On the other hand, Halo C18 column allows an improved fingerprint of CDs having a DS from 4.9 up to a value major than 14 and avoiding the use of various chromatographic systems. Thus, liquid chromatography performed with this column and an evaporative light scattering detector can be used as a generic system for methylated CD analysis. Moreover, fused core particles of Halo C18 column enables a rapid analysis and liquid chromatography coupled with electrospray-mass spectrometry appears as a powerful tool to determine co-elution and to characterize various isomers of complex methylated-ß-cyclodextrin mixtures.

Fast separation of triterpenoids by supercritical fluid chromatography/evaporative light scattering detector.

The screening of plant material, the chemical composition, the abundance and the biological activity of triterpenoids are of a major economical importance. The classical analytical methods, such as TLC, GC, and HPLC are either little resolutive, or require derivatization steps, or fail in sensitivity. The supercritical fluid chromatography/evaporative light scattering detector (SFC/ELSD) coupling provides high resolution, fast analysis and higher responses for the analysis of triterpenoids. After the initial screening of seven stationary phases to select the well suited one, analytical conditions (modifier percentage, from 10 to 3%; backpressure (from 12 to 18 MPa) and temperature (from 15 to 25 °C) were studied to improve the separation, and ELSD detection of a standard mixture composed of 8 triterpenoids (oleanolic acid, erythrodiol, ß-amyrin, ursolic acid, uvaol, betulinic acid, betulin, lupeol). Applied to apple pomace extracts, this method allows the separation of about 15 triterpenoid compounds, in less than 20 min, with isocratic conditions. Moreover, the ELSD response is dramatically higher than the one provided by UV detection, and avoids derivatization steps. An attempt to identify some compounds was done by collecting chromatographic peaks and further analyzing them with mass spectrometry. Complete identification or molecular formula could be proposed for 11 compounds. However, due to the presence of position and orientation isomers the absolute identification remains difficult, despite some retention rules deduced from the standard analysis.

Porous graphitic carbon: a versatile stationary phase for liquid chromatography.

Chromatographic stationary phases based on porous graphitic carbon were invented 30 years ago, while columns have been commercially available for 20 years. This special occasion deserved a complete review on this material. In this paper, we describe current knowledge on graphitic carbon stationary phases, based on over 400 fundamental studies and applications.

A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism.

Seventy-six compounds of biological interest for the diagnosis of inherited disorders of amino acids (AA) metabolism have previously been demonstrated to be detectable in positive mode electrospray ionisation tandem mass spectrometry (ESI-MS/MS), after separation by ion-pairing reversed-phase liquid chromatography (RPLC). The separation method used tridecafluoroheptanoic acid as ion-pairing agent, and a gradient of acetonitrile for the elution of the most retained compounds. This method had previously been demonstrated to be suitable for the qualitative diagnosis of many AA disorders, and for the quantitative measurement of 16 AA in biological fluids, using their stable isotope labelled (SIL) AA as internal standard. For quantification of the other AA, an internal standard was chosen among the available SIL-AA, as close as possible to the analyte to be measured, in terms of structural analogy, and of retention time in the chromatographic system. The performances of the quantitative analysis of the other AA to be measured are reported here. They show validated results for several AA, allowing their accurate quantification, with another SIL-AA as internal standard. For some other AA, quantitative results were not accurate, allowing only semi-quantitative or qualitative determination for these parameters.

Development of an LC-MS-MS method for the quantification of taurine derivatives in marine invertebrates.

Sulfur amino acids, such as taurine, hypotaurine, and thiotaurine, were found in high quantities in tissues of marine symbiotic organisms (e.g., bivalves, tubeworms) living close to hydrothermal vent sites. Therefore, they are assumed to play a key role in the S-oxidizing base metabolism or sulfide detoxification. We propose here a specific, rapid, and original analytical procedure for the direct determination of sulfur amino acids at the level of a few parts per billion in biological samples, avoiding the classical low specific post-column ortho-phthaldialdehyde derivatization step required by non-ultraviolet-absorbing molecules. Indeed, by coupling liquid chromatography on a porous graphitic stationary phase under isocratic conditions (10 mM ammonium acetate buffer adjusted to pH 9.3) to tandem mass spectrometry (ionization process by pneumatically assisted electrospray in negative ion mode), it is possible to perform specific quantification of these metabolites in less than 10 min directly in biological matrices without any derivatization step or other tedious sample treatments. Thus, taurine, hypotaurine, and thiotaurine have been identified and assayed in several deep sea organisms, showing that the developed method is well suited for this kind of application.

Analysis of sulfobutyl ether-beta-cyclodextrin mixtures by ion-spray mass spectrometry and liquid chromatography-ion-spray mass spectrometry.

The analysis of two commercial and two home-made sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) samples by ion-spray (IS) mass spectrometry and by liquid chromatography-mass spectrometry coupling (LC-MS) is investigated in a negative ion mode. SBE-beta-CD fragmentation was first investigated by direct infusion. In IS, the best conditions for SBE-beta-CD ionization consisted of ammonium acetate added to an acetonitrile/water mixture as sample solvent. These conditions allowed simplification of the mass spectrum, mainly by the formation of dicharged species [M-2H]2-, thus limiting the production of multicharged fragments. IS-MS permits fast and simple measurement of the substitution pattern and determination of the global degree of substitution for SBE-beta-CD mixtures. A complementary method using LC-MS was developed for the analysis of these mixtures. The substitution patterns obtained by LC-MS are in good agreement with those determined by direct MS analysis. The LC-MS coupling enabled separation of the mixtures versus the charge in anion-exchange chromatography (AEC) whereas no separation of the different substitution isomers potentially present in the SBE-beta-CD mixture was displayed. The AEC methodology described can be successfully used for fractionation of SBE-beta-CD derivatives at the semi-preparative scale.

Determination of inorganic cations and anions by ion-exchange chromatography with evaporative light-scattering detection.

Evaporative light-scattering detection (ELSD) was investigated for the direct determination of alkali and alkaline-earth cations by cation-exchange chromatography. Successful single run analysis of Na+, K+, Mg2+ and Ca2+ was achieved in 11 min on the Hamilton PRP-X200 column using an aqueous solution of ammonium formate as mobile phase under a salt concentration step gradient mode (20 mM and 100 mM). Surprisingly the use of ELSD reveals a weak retention of inorganic anions (Cl-, NO3-, SO4(2-)) onto the polymeric cation exchanger, which enables the simultaneous determination of inorganic anions (C1- and NO3-) associated with the cations analysed (Na+ and K+).

Simultaneous analysis of underivatized chiral amino acids by liquid chromatography-ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase.

Simultaneous chiral separations of underivatized amino acids have been performed using a teicoplanin-based chiral stationary phase and ionspray tandem mass spectrometry for their ionisation and detection. Different amino acid enantiomer pairs were separated simultaneously, including those of positional isomeric amino acids (e.g., L,D-Leu/Ile, or L,D-Val/Iva). Due to the specificity of tandem mass spectrometry, co-eluting enantiomers of different amino acids could also be determined. Fifteen chiral underivatized proteinogenic and non-proteinogenic amino acids were analysed simultaneously under isocratic conditions (acetonitrile-water, 75:25) in less than 25 min. For maximum sensitivity, post-column addition of 500 mM aqueous HCOOH was necessary. Detection limits varied from 2.5 to 50 microg l(-1) depending on the amino acid. The signal vs. concentration relationship was linear for all D- and L-amino acids (0.9995 < or = r2 < or = 1) for three orders of magnitude.

Characterization of sulfobutyl ether-beta-cyclodextrins mixtures by anion-exchange chromatography using evaporative light scattering detection.

An analytical method based on anion-exchange chromatography (AEC) using volatile eluent ion and evaporative light scattering detection was developed for the analysis of mixtures of sulfobutyl-ether-beta-cyclodextrins (SBE-beta-CDs). A systematic investigation of the retention mechanism of pure SBE-beta-CD standards has been studied on a silica quaternary ammonium exchanger (Vydac 302 IC column). The influence of the nature and concentration of volatile anions (acetate, formate, trifluoroacetate), the addition of the organic modifier in the mobile phase as well the nature of the stationary phase have been evaluated under isocratic elution conditions. Satisfactory analysis of two commercial and two home-made SBE-beta-CD samples was achieved on the Vydac 302 IC column by using ammonium acetate as ion eluent in water-acetonitrile (70:30) under a salt concentration gradient mode. This method provides for SBE-beta-CD samples, an efficient and characteristic liquid chromatography fingerprint which depicts the mixture complexity and determines an average degree of substitution (DS) for each sample.

Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry.

The analysis of the 20 underivatized protein amino acids by liquid chromatography ionspray tandem mass spectrometry is investigated in positive ion mode. First, by direct infusion, amino acid fragmentation was investigated based on the product-ion mass spectrum of the parent compound in three different collision energies (10, 20, 30 eV). Then, the relative abundance of fragment ions was studied as a function of the collision energy in order to select the product ion with the highest abundance and to obtain the maximum sensitivity for each amino acid, by using the optimum collision energy. Depending on the amino acid, the loss of H2O or NH3 or CH2O2 was selected as the product ion from the molecular ion [M+H]+ in selective reaction monitoring mode. 15 eV was chosen as a mean value of collision energy to obtain satisfactory sensitivity for the simultaneous determination of the 20 protein amino acids. In spite of the specificity of mass spectrometry, and in order to obtain maximum sensitivity, several pairs of amino acids had to be separated. The separation of these amino acids pairs was achieved in less than 20 min by using a porous graphitic carbon column and nonafluoropentanoic acid as ion-pairing reagent. Detection limits depending on the amino acid varied from 500 fmol to 40 pmol (using a 10 microl loop).

Determination of 1-aminocyclopropane-1-carboxylic acid and its structural analogue by liquid chromatography and ion spray tandem mass spectrometry.

Liquid chromatography coupled to ion spray tandem mass spectrometry was developed as a method for the simultaneous analysis of the amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) and its structural analogue, cyclopropane-1,1-dicarboxylic acid (CDA). ACC and CDA fragmentation as well as optimization of MS parameters were investigated in positive ion mode. In selective reaction monitoring mode the protonated molecule [M+H]+ was selected as parent ion for both ACC and CDA, while the immonium ion from ACC and the [M+H-H2O]+ ion from CDA were selected, respectively, as product ions. In spite of the high selectivity of MS/MS among the 20 protein amino acids potentially present with ACC and CDA in the plant material analyzed, Glu and Thr can interfere with the signal of ACC. As a result, their chromatographic separation is necessary. This was achieved in less than 4 min by ion-pair reversed-phase chromatography with nonafluoropentanoic acid as ion-pair reagent. A linear response within a concentration range of 1-5 mg l(-1) was observed for this LC method and the detection limit was found to be 20 pmol for ACC and 150 pmol for CDA (using a 20-microl loop). This methodology was successfully applied to the detection of ACC in apple tissue.

Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids.

The analysis of twenty underivatized protein amino acids has been achieved on porous graphitic carbon packing material (Hypercarb). Five perfluoroalkyl carboxylic acids (trifluoroacetic, heptafluorobutyric, nonafluoropentanoic, tridecafluoroheptanoic and pentadecafluorooctanoic acid) have been studied as ion-pairing reagent. Several parameters (equilibration time, quantities adsorbed onto the chromatographic support, concentration and nature of the ion-pairing reagent, as well as temperature effect) have been studied leading to the complete separation of these compounds in gradient elution mode. Evaporative light scattering detector has allowed the detection of these non UV-visible absorbent molecules. The chromatographic methodology developed can also be easily coupled with pneumatically assisted electrospray mass spectrometry.

Mass spectrometric study of randomly methylated beta-cyclodextrins using ionspray, atmospheric pressure chemical ionization and matrix-assisted laser desorption/ionization.

Mixtures of methylated beta-cyclodextrins were characterized using three different methods of mass spectrometry: ionspray, atmospheric pressure chemical ionization (APCI) and matrix-assisted laser desorption/ionization (MALDI). Each of these methods allows a fast and simple determination of the degree of substitution, and can provide evidence for differences in the methylation of batches which have very similar global degrees of substitution. The three methods are in good qualitative agreement, but there are systematic differences in the quantitative results for the percentages of the various methylated molecules present in a batch. This is attributed to ionization yields which increase with the number of methyl groups, with different slopes for the different methods.

Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry.

A qualitative determination of 20 underivatized proteinic amino acids by LC-MS is reported. The need for chromatographic separation before mass spectrometry determination is demonstrated based on the study of several amino acid pairs which have some similar characteristics. Two suitable LC-MS systems are proposed for amino acid analysis. A preliminary optimization of these systems has been investigated using evaporative light scattering detection as these two detection modes have the same chromatographic requirements. The amino acid separation was achieved on a Purospher RP-18e or a Supelcosil ABZ+Plus column with tridecafluoroheptanoic acid or pentadecafluorooctanoic acid as volatile ion-pairing reagent in an acetonitrile-water mobile phase. In order to elute the most retained amino acids, an elution gradient based on simultaneously increasing the concentration of acetonitrile and decreasing the concentration of the ion-pairing reagent was used. The detection limits of the present work (without specialized optimization) varied from 0.5 to 1 mg 1(-1).

A new approach to the study of glucosinolates by isocratic liquid chromatography. Part I. Rapid determination of desulfated derivatives of rapeseed glucosinolates.

Liquid chromatographic (LC) analysis of desulfated derivatives of rapeseed glucosinolates has been carried out under isocratic elution conditions with different CN-bonded stationary phases. The effects of the eluant composition (water, acetonitrile, and methanol) with the stationary phase (Zorbax CN, Lichrospher CN, and Ultrasphere CN) and temperature (20 and 50 degrees C) are described. An isocratic LC method performed at room temperature using a Lichrospher CN column and water as mobile phase is proposed. The chromatographic analysis can be done in less than 12 min, and it is easier and less expensive than the traditional gradient mode. Four commercial samples of rapeseed containing various quantities of other cruciferous seeds (wild mustard and stinkweed) as an admixture have been analyzed to determine the total glucosinolate content. Relative standard deviations of repeatability of the isocratic and gradient LC methods ranged from 0.4 to 1.7% and from 2.7 to 4.7%, respectively. Comparison of the results showed good agreement between the 2 methods (beter than 98%).