Peter T. Kissinger

DETERMINATION OF CATECHOLAMINES IN RAT BRAIN PARTS BY REVERSE‐PHASE ION‐PAIR LIQUID CHROMATOGRAPHY

An improved method for the measurement of catecholamines in brain parts has been developed, based on reverse‐phase ion‐pair chromatography. The new method offers the advantages of high efficiency microparticulate liquid chromatography packings and the flexibility of ion‐pair chromatography. By this approach norepinephrine and dopamine (DA) have been measured in the hypothalamus and corpus striatum of the rat brain during various stages of development (15, 21, 30 days). Data are reported on the basis of the whole part and per weight of tissue. For the adult animals, the following concentrations (ng/g wet tissue) were observed for the hypothalamus: NE = 2261 ± 274, DA = 440 ± 103, and for the corpus striatum: DA = 11,888 ± 1840. The overall precision of the method was ±5.6% relative s.d. The absolute recovery was 60 ± 5% relative s.d. and was constant over the range of 1 ng to 1 μg of dopamine or norepinephrine per tissue sample. The relative retention behavior of 18 neurologically important catechol derivatives is reported for reverse‐phase chromatography with octyl sulfate as the stationary phase modifier.

Desorption electrospray ionization mass spectrometry: Imaging drugs and metabolites in tissues

Ambient ionization methods for MS enable direct, high-throughput measurements of samples in the open air. Here, we report on one such method, desorption electrospray ionization (DESI), which is coupled to a linear ion trap mass spectrometer and used to record the spatial intensity distribution of a drug directly from histological sections of brain, lung, kidney, and testis without prior chemical treatment. DESI imaging provided identification and distribution of clozapine after an oral dose of 50 mg/kg by: i) measuring the abundance of the intact ion at m/z 327.1, and ii) monitoring the dissociation of the protonated drug compound at m/z 327.1 to its dominant product ion at m/z 270.1. In lung tissues, DESI imaging was performed in the full-scan mode over an m/z range of 200-1100, providing an opportunity for relative quantitation by using an endogenous lipid to normalize the signal response of clozapine. The presence of clozapine was detected in all tissue types, whereas the presence of the N-desmethyl metabolite was detected only in the lung sections. Quantitation of clozapine from the brain, lung, kidney, and testis, by using LC-MS/MS, revealed concentrations ranging from 0.05 μg/g (brain) to a high of 10.6 μg/g (lung). Comparisons of the results recorded by DESI with those by LC-MS/MS show good agreement and are favorable for the use of DESI imaging in drug and metabolite detection directly from biological tissues.

Evidence for the involvement of N-acetyl-p- quinoneimine in acetaminophen metabolism.

Abstract Evidence for the presence of N -acetyl- p -quinoneimine (NAPQI), a postulated toxic intermediate of acetaminophen metabolism, in mouse liver microsomal incubations is reported. The intermediate was tentatively identified by comparison with synthetic NAPQI generated electrochemically from acetaminophen in a coulometric flow reactor. All but one of the reaction products of NAPQI with a number of nucleophiles were found in vitro as well, and in similar relative amounts. The NAPQI intermediate is moderately stable at physiological pH and temperature with a lifetime which is dependent on the components of the medium.

Determination of nitro aromatic, nitramine, and nitrate ester explosive compounds in explosive mixtures and gunshot residue by liquid chromatography and reductive electrochemical detection

Abstract Reductive and oxidative electrochemical detection with liquid chromatography is applied to the determination of nitro aromatics, nitrate esters, nitramines, and diphenylamines in military explosives and single and double base smokeless gunpowders. A sensitive and highly selective method is presented for the detection of organic “gunshot residue” on the hand of individuals who have discharged a weapon. The detection limits at S/N = 3 are of the order of 0.5, 1, 2, and 0.3 picomol for nitro aromatic, nitramine and nitrate ester explosive compounds, and diphenylamines, respectively.

I. Minireview: Neurochemical applications of liquid chromatography with electrochemical detection

Abstract Liquid chromatography with electrochemical detection (LCEC) has been shown to have unique advantages for the determination of many substances of neurochemical interest. The technique is rapid, sensitive, and relatively inexpensive. In addition, it avoids the need for radiolabelled substances, the formation of volatile derivatives, or reactions which generate fluorescent products. LCEC is widely used for the measurement of the catecholamines and their metabolites and has recently gained acceptance for determination of the neurochemically important tryptophan metabolites. The method is also capable of assessing the activity of a number of neurologically important enzymes. The review which follows is intended to provide a brief overview of the LCEC technique and a guide to recent literature exemplifying its neurochemical applications.

Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode generally used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh (S/N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of rho-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that rho-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of rho-benzoquinone with glutathione is predominantly a non-enzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione. The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The Vmax for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the KM was found to be 0.38 mM. The further oxidation of hydroquinone to rho-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37 degrees C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of rho-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced non-enzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to rho-benzoquinone in vitro and the subsequent covalent binding to macromolecules.

[3] Analysis of ascorbic acid by liquid chromatography with amperometric detection

Publisher Summary This chapter presents the analysis of ascorbic acid by liquid chromatography with amperometric detection. Liquid chromatography with electrochemical detection (LCEC) approach affords the convenience of sample preparation, sensitivity, and selectivity equal or superior to any method for ascorbic acid published to date. The sensitivity of LCEC is superior by two orders of magnitude when compared to liquid chromatography with ultraviolet detection. In addition, the selectivity associated with electrochemistry is a decided advantage. Although the use of pellicular, high-performance, and anion-exchange packing materials is adequate for most sample types, increased selectivity is obtainable when microparticulte reverse-phase packings and different ion-pairing reagents are employed. Ideal mobile phases for LCEC are aqueous buffers with or without a solvent (typ-methanol or acetonitrile). Because of this requirement, ion-exchange or reverse-phase packing materials have been found to be the most compatible with the technique.

Electrochemical Enzyme Immunoassay Using Sequential Saturation Technique in A 20-μl Capillary: Digoxin as A Model Analyte

Capillary enzyme immunoassay with flow-injection analysis for digoxin using the sequential saturation technique has been developed. Glass capillary tubes (10 cm × 0.53 mm i.d.) with immobilized digoxin antibody were used as the immunoassay reactor. The product of enzymatic reaction. 4-aminophenol, was detected amperometrically. The digoxin and the labeled digoxin binding reaction with the immobilized digoxin antibody were completed in 2 and 10 min, respectively. Digoxin was determined in a 20-μl sample with a detection limit of 10 pg ml−1 (200 fg or 260 attomoles) and a 3 orders of magnitude range.

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain.

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringers solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.