John F. Stobaugh

Characterization of sulphoalkyl ether derivatives of β-cyclodextrin by capillary electrophoresis with indirect UV detection

A capillary electrophoresis method which characterizes the degrees of substitution of heterogeneous sulphoalkyl ether beta-cyclodextrin derivatives is described. The separation is based on the different electrophoretic mobilities observed from changes in the overall charge of the molecule as a result of substitution. Individual peaks of the electropherogram then provide a measure of each degree of substitution of the present beta-cyclodextrin. Detection of these beta-cyclodextrin derivatives is performed by indirect UV detection.

Factors affecting the stability of fluorescent isoindoles derived from reaction of o-phthalaldehyde and hydroxyalkylthiols with primary amines

The stability of a series of fluorescent isoindole derivatives formed in situ under analytical conditions following the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) with a series of primary amines are reported. Increasing the bulk and degree of substitution at C-10 of the resulting isoindole resulted in substantial increases in product stability. The effects of excess OPA and 2-ME on isoindole stability were examined and OPA was observed to catalyze isoindole degradation while 2-ME had no effect. Previously proposed degradation mechanisms were reexamined in light of the present data and an alternate degradation pathway is proposed. 3-Mercapto-1-propanol (3-MP) was found to be a superior thiol for use in the fluorogenic OPA reaction. The OPA/3-MP reagent combination was utilized to derive several amino acids and offered detection limits (S/N = 2) of less than 200 fmol.

Applications of Capillary Electrophoresis in Pharmaceutical Analysis

The role of capillary electrophoresis (CE) in the analysis of peptides/proteins, chiral pharmaceuticals, and other small-molecule drugs has been reviewed. Potential uses of CE range from purity and structural confirmation to a micropreparative technique. Strategies for the prevention of protein wall adsorption include the use of extreme pH values, surface-modified capillaries, and high ionic strengths employing salts of alkali metals or by the addition of zwitterionic surfactants to the background electrolyte. Chiral separations of amino acids and other racemic pharmaceuticals have been achieved by micellar electrokinetic chromatography or by the introduction of cyclodextrins/modified cyclodextrins or other reagents to the running buffer. Applications of capillary electrophoresis to the analysis of small-molecule pharmaceuticals include determinations of drugs and/or excipients in various pharmaceutical preparations and the analysis of miscellaneous pharmaceuticals in standard solutions and biological fluids. The complementary nature of capillary electrophoresis and HPLC, in addition to future expectations of CE in pharmaceutical analysis, is discussed.

The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

OBJECTIVE The response to and toxicity of methotrexate (MTX) are unpredictable in patients with juvenile idiopathic arthritis (JIA). Intracellular polyglutamation of MTX, assessed by measuring concentrations of MTX polyglutamates (MTXGlu), has been demonstrated to be a promising predictor of drug response. Therefore, this study was aimed at investigating the genetic predictors of MTXGlu variability and associations between MTXGlu and drug response in JIA. METHODS The study was designed as a single-center cross-sectional analysis of patients with JIA who were receiving stable doses of MTX at a tertiary care childrens hospital. After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing. Clinical data were collected by chart review. Genotyping for 34 single-nucleotide polymorphisms (SNPs) in 18 genes within the MTX metabolic pathway was performed. An ion-pair chromatographic procedure with mass spectrometric detection was used to measure MTXGlu1-7. RESULTS Analysis and genotyping of MTXGlu was completed in the 104 patients. K-means clustering resulted in 3 distinct patterns of MTX polyglutamation. Cluster 1 had low red blood cell (RBC) MTXGlu concentrations, cluster 2 had moderately high RBC MTXGlu1+2 concentrations, and cluster 3 had high concentrations of MTXGlu, specifically MTXGlu3-5. SNPs in the purine and pyrimidine synthesis pathways, as well as the adenosine pathway, were significantly associated with cluster subtype. The cluster with high concentrations of MTXGlu3-5 was associated with elevated liver enzyme levels on liver function tests (LFTs), and there were higher concentrations of MTXGlu3-5 in children who reported gastrointestinal side effects and had abnormal findings on LFTs. No association was noted between MTXGlu and active arthritis. CONCLUSION MTXGlu remains a potentially useful tool for determining outcomes in patients with JIA being treated with MTX. The genetic predictors of MTXGlu variability may also contribute to a better understanding of the intracellular biotransformation of MTX in these patients.

Role of capillary electrophoresis methods in the drug development process

In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed.

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

OBJECTIVE Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients. METHODS Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >or=3 months. Clinical data were collected by chart review. Concentrations of MTXGlu(1-7) in red blood cell lysates were quantitated using an innovative ion-pairing chromatography procedure, with detection by mass spectrometry. RESULTS Patients with JIA from a single center (n = 99; mean +/- SD age 117.8 +/- 56.5 months, 69 female) were included in the analysis. The mean +/- SD dose of MTX was 0.51 +/- 0.25 mg/kg per week, with a median treatment duration of 18 months (interquartile range 3-156 months). MTX was administered subcutaneously in 66 patients (67%). Fifty-six patients (57%) had active arthritis at the time of the clinic visit. Total intracellular MTXGlu (MTXGlu(TOT)) concentrations varied 40-fold, with a mean +/- SD total concentration of 85.8 +/- 48.4 nmoles/liter. Concentrations of each MTXGlu subtype (MTXGlu(1-7)) were measured individually and as a percentage of MTXGlu(TOT) in each patient. MTXGlu(3) was the most prominent subtype identified, comprising 42% of MTXGlu(TOT), and the interindividual variability in the concentration of MTXGlu(3) was the most highly correlated with that of MTXGlu(TOT) (r = 0.96). The route of MTX administration was significantly associated with MTXGlu(1-5) subtypes; higher concentrations of MTXGlu(1 + 2) were observed in patients receiving oral doses of MTX, whereas higher concentrations of MTXGlu(3-5) were observed in patients receiving subcutaneous doses of MTX (P < 0.0001). CONCLUSION In this cohort of patients with JIA, the MTXGlu(TOT) concentration varied 40-fold. Individual MTXGlu metabolites (MTXGlu(1-7)), which have, until now, not been previously reported in patients with JIA, were detected. The route of MTX administration contributed to the variability in concentrations of MTXGlu(1-5).

Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Two analytical methods were developed for quantitative determination of DADLE (H(2)N-Tyr-D-Ala-Gly-Phe-D-Leu-COOH) and its two cyclic prodrugs in rat plasma. For high-performance liquid chromatography with fluorescence detection (LC-FLU), precolumn derivatization of DADLE was accomplished by labeling the N-terminal amino group with the reagent naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN) to form a highly fluorescent 1-cyanobenz[f]isoindole (CBI) derivative. A multi-dimensional LC system was employed to improve selectivity, and solid-phase extraction (SPE) was used for plasma sample preparation. The cyclic prodrugs were converted to DADLE prior to their derivatization. With fluorescence detection after derivatization, the limit of quantitation (LOQ) was 6 ng ml(-1) for the analysis of DADLE, and good linearity was observed up to 6000 ng ml(-1) in rat plasma. Quantitative analysis of DADLE and its cyclic prodrugs was also performed using liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Chromatographic separation was achieved on a C(18) column using gradient elution in a water-acetonitrile system containing 0.1% (v/v) formic acid. The tandem mass spectrometric analysis was performed in the multiple reaction monitoring mode using internal standardization to improve assay precision and accuracy. For plasma sample pretreatment, acetonitrile was added first to precipitate proteins and SPE was used to minimize matrix effects. Using LC-ESI-MS-MS, the LOQ was 0.5 ng ml(-1) for DADLE and 2 to 5 ng ml(-1) for its prodrugs. Good linearity was observed from the LOQ up to 1000 ng ml(-1) for all compounds. For the analysis of DADLE, both analytical methods showed good precision, accuracy and stability. However, for prodrug analysis, LC-FLU showed some sensitivity and accuracy problems, while the LC-ESI-MS-MS method provided consistent and satisfactory results. In conclusion, LC-ESI-MS-MS is the method of choice for the analysis of DADLE and its cyclic prodrugs in rat plasma samples due to its good selectivity, high sensitivity, and fast analysis. Its application was demonstrated through biodisposition and bioconversion studies of the coumarinic acid-based prodrug after intravenous administration in rats.

Determination of α-difluoromethylornithine in blood by microdialysis sampling and capillary electrophoresis with UV detection

A procedure is described for the analysis of alpha-difluoromethylornithine (DFMO), an anti-cancer agent, in plasma microdialysis (MD) samples. DFMO has been shown to be effective alone or in combination with other agents in the treatment of several cancers. Precolumn derivatization of DFMO with naphthalene-2,3-dicarboxaldehyde-cyanide (NDA-CN) in pH 10.0 borate buffer results in the rapid formation of a stable mono-derivatized product (N-substituted 1-cyanobenz[f]isoindole, CBI), which is UV active. An analytical method has been developed to separate CBI-DFMO from NDA-CN derivatization products of 20 standard amino acids using capillary electrophoresis (CE). This method is then employed for the determination of DFMO in plasma microdialysis samples. Separation of DFMO from other components in the dialysate was achieved within 20 min. The response for DFMO in Ringers solution was linear over the range of 1.2 x 10(-6) to 1.6 x 10(-4) M after derivatization. The detection limit of DFMO in the plasma dialysate is 5 microM using UV detection at 254 nm. This method has been proven to have adequate sensitivity for quantitation of DFMO in i.v. microdialysate samples and has been successfully applied to monitoring the pharmacokinetics of DFMO by CE-UV.

Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents

Evidence was presented suggesting that the fluorescent isoindole produced by reaction of o-phthalaldehyde (OPA), ethanethiol, and primary amine was formed by initial imine formation followed by conversion to an alpha-alkylaminobenzylsulfide and subsequent ring closure to form the isoindole nucleus. This mechanism suggested that the minimum structural requirement for condensation to an isoindole was an o-diacyl benzene in which one of the carbonyl groups was aldehydic. A major drawback of OPA as an analytical reagent is the limited stability of the fluorescent 1,2-disubstituted isoindole. Since isoindole instability is related to autoxidation at C-3, the use of o-(formyl) arylketones as alternatives to OPA is attractive in increasing the lifetime of the fluorescent species in that such reagents would form 1,2,3-trisubstituted isoindoles. Two compounds, o-acetylbenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB), were synthesized and evaluated as potential fluorogenic reagents. Both formed fluorescent products. The rate of formation of isoindole from the latter was too slow to make it of practical analytical value; however, OAB formed isoindoles with t1/2 less than 10 s and offered markedly improved stability over that observed with OPA.

A novel high‐performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 microL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long-chain methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches.