Michael Kazanis

Direct injection liquid chromatography/positive ion electrospray ionization mass spectrometric quantification of methotrexate, folinic acid, folic acid and ondansetron in human serum

A rapid liquid chromatography/positive ion electrospray mass spectrometric assay (LC/ESI-MS) was developed for the quantitation of methotrexate, folinic acid, folic acid and ondansetron in human serum. The assay was based on 100microL serum samples, following acetonitrile precipitation of proteins and filtration that enabled direct injection into the LC/MS system. All analytes and the internal standard, alfuzosin, were separated by using a Zorbax Eclipse XDB-C(8) analytical column (2.1mmx150.0mm i.d., particle size 3.5microm) with isocratic elution. The mobile phase was composed of a mixture of water/acetonitrile containing 0.1%, v/v formic acid (75:25, v/v), pumped at a flow rate of 0.15mLmin(-1). Quantitation of the analytes was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.01-25.00microgmL(-1) for methotrexate and 0.01-5.00microgmL(-1) for folic acid, folinic acid and ondansetron. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 7.0min for each sample made it possible to analyze a large number of human serum samples per day. The method can be used to quantify methotrexate, folinic acid, folic acid and ondansetron in human serum covering a variety of clinical studies and it was applied to the analysis of human serum samples obtained from children with acute lymphoblastic leukemia.

Selective and rapid liquid chromatography/negative-ion electrospray ionization mass spectrometry method for the quantification of valacyclovir and its metabolite in human plasma.

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm x 125.0 mm i.d., particle size 5 microm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min(-1) in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020-0.800 microg mL(-1) for valacyclovir and 0.100-20.00 microg mL(-1) for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.

DETERMINATION OF VALPROIC ACID IN HUMAN PLASMA BY HPLC WITH FLUORESCENCE DETECTION

ABSTRACT A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of valproic acid in human plasma. The method is based on pre-column derivatization using a new fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The internal standard for the assay procedure was cyclohexanecarboxylic acid. The optimum monitoring conditions and the stability of the derivatives were investigated. The derivatization reaction proceeds in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30C with a reaction time of 30 min. The resulting derivatives were separated under isocratic conditions (acetonitrile–water, 60 : 40, v/v) on a LiChrospher RP-18 column (125 × 4.0 mm, i.d. 5 μm) and were detected fluorimetrically at a wavelength of 435 nm with an excitation of 345 nm. All chromatographic experiments were carried out at a flow rate 1.0 mL/min at ambient temperature. The method was fully validated and applied for the determination of valproic acid over the concentration range of 6.0–150.0 mg/mL. The detection limit for valproic acid added to plasma sample was 0.13 μg/mL plasma (3 pg on column). The recovery of valproic acid averaged 100.4%. The accuracy of the assay ranged from −0.5% to 1.3%. The method was shown to be highly reproducible and it seems to be adequate for routine therapeutic drug monitoring.

BrMOZPhC, a novel coumarin type reagent for the fluorescent derivatisation of carboxylic acids

A new fluorescent reagent was synthesized in order to be used as a fluorescent probe in analytical HPLC derivatisation reactions. Briefly, 2,4-dimethoxyaniline was acylated and demethylated and the resulting diphenol reacted with α-bromoacetoacetate in the presence of conc. sulfuric acid (the Von Pechmann reaction). The resulting acetamido coumarin was hydrolyzed and the aminohydroxy derivative cycled to form the corresponding bromo-coumarinoxazine. The analytical reaction was performed with butyric acid as model compound and catalyzed by 18-crown-6 ether. The calibration curve constructed was characterized by good linearity whereas the precision of the reaction gave an R.S.D. values below 3%. The reaction applied to the separation of five aliphatic acids.

A NEW FLUOROGENIC REAGENT FOR LABELLING CARBOXYLIC ACIDS IN HPLC

A new fluorescent reagent for carboxylic acids, N-(4-bromomethyl-7-hydroxy-2-oxo-2H-6-chromenyl) bromoacetamide (Br-MAMC) was synthesized. The fluorescence properties of Br-MAMC were monitored for a series of polar and non-polar solvents. Its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined using undecylenic acid as a model compound. The derivatization reaction was performed in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30°C for 20 min. The reaction solution was subjected to a high-performance liquid chromatographic procedure on a reversed-phase ODS C-18 column (150×4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile and water (75 : 25, v/v) at a flow rate of 1.0 mL/min. The derivative of undecylenic acid was detected fluorometrically; excitation and emission wavelengths were set at 345 and 435 nm, respectively. The detection limit of the method was determined to be 12.5 pg (signal to noise ratio = 3) of the derivatized undecylenic acid on column. The derivatives of butyric, hexanoic, octanoic, decanoic, and dodecanoic acid with Br-MAMC were also well resolved with a mobile phase of acetonitrile and water (60 : 40, v/v).

A New Coumarin Based Fluorogenic Derivatization Reagent for Labelling Free Carboxyl Groups (Br‐MOZC)

Abstract A new coumarin based bromomethyl fluorescent probe 4‐bromomethyl coumarin[a,b‐c,d]‐oxazine‐3‐one was synthesized as a fluorescent carboxylic acid derivatizing agent. The synthetic route is described, as well as its application for the determination of the model compound n‐butyric acid. The derivatization reaction was conducted in acetone using 18‐crown‐6 as catalyst. The derivatization solution was separated by high performance liquid chromatography on a reversed‐phase ODS C‐18 column (150 × 4.6 mm i.d., 5 µm) with a mobile phase of acetonitrile and water (70:30, v/v) at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set to 435 and 345 nm, respectively. The detection limit was determined to be below 10 pg of the derivatized acid on the column. The probe has been used for the simultaneous determination of six aliphatic fatty acids.

MOZPhCSE, a New Coumarin Based Fluorescent Derivatization Reagent

Abstract The synthesis of a new fluorescent probe 2‐(3,7‐dioxo‐2‐phenyl‐3,7‐dihydrochromeno[7,6‐b][1,4]oxazin‐9‐yl)acetic acid succinimidyl ester was described. The reagent reacts easily with primary amines and forms highly fluorescent derivatives that can be separated and detected with an HPLC‐fluorometric detector. The new reagent was evaluated using pentylamine as a model compound. The separation was achieved on a C18 column using a mixture of acetonitrile:ammonium acetate buffer as the mobile phase. Statistical evaluation of the methodology described reveals good linearity, precision and accuracy, and high sensitivity. Application of the methodology can be used for the separation of four aliphatic amines (pentylamine, hexylamine, octylamine, and decylamine).