Gas chromatographic—mass spectrometric procedure for the quantitation of chlorpromazine in plasma and its comparison with a new high-performance liquid chromatographic assay with electrochemical detection

A gas chromatographic—mass spectrometric assay using selected ion monitoring is compared with a high-performance liquid chromatographic assay using an electrochemical detector for single-dose studies of the psychotherapeutic phenothiazine drug chlorpromazine. Measurements were made after extraction of chlorpromazine and the internal standard, prochlorperazine, from basified plasma with an isopropanol—pentane solvent mixture. Following evaporation of the organic solvents the residue was reconstituted in a small volume of methanol and subjected to gas chromatographic—mass spectrometric selected ion detection. The residual sample was then evaporated and made up in a larger volume of acetonitrile and analyzed by high-performance liquid chromatography using an electrochemical detector. These specific methods display excellent correlation for plasma concentration determinations in the range of 0.25–10 ng ml⁻¹ and will allow for the study of the pharmacokinetics of chlorpromazine following single low doses of the drug.     

Confirmation of malachite green, gentian violet and their leuco analogs in catfish and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-visible diode array detection and fluorescence detection

A sensitive analytical procedure for the confirmation of residues of malachite green (MG), gentian violet (GV) and their leuco analogs (LMG and LGV) in catfish and trout tissue at 10 ng/g is described. Frozen (−20°C) fish fillets were cut into small pieces and homogenized in Waring blendors. The compounds of interest were extracted from 20-g amounts of homogenized fish tissue with acetonitrile-buffer, partitioned against methylene chloride, and isolated with tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 μl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (150×4.6 mm I.D.) in-line with a post-column oxidation coulometric electrochemical cell (EC), a UV-Vis diode array detector and a fluorescence detector.     

New sensitive assay of vancomycin in human plasma using high-performance liquid chromatography and electrochemical detection

A method using reversed-phase high-performance liquid chromatography with electrochemical detection for the analysis of vancomycin in human plasma was developed. Chromatographic conditions included an octadecyl column, a mobile phase of acetonitrile–sodium phosphate buffer (pH 7) (12:88), a total run time of 12 min, and coulometric electrochemical detection at +700 mV. Linear detector response was found in the range 5–100 μg ml⁻¹ after a 1:80 dilution or from 0.5 to 50 μg ml⁻¹ after a 1:20 dilution of the samples. In both cases the correlation coefficient (r) of the calibration curve standard was better than 0.995. Vancomycin determination was based on a denaturation of plasma proteins with methanol, then a dilution with mobile phase was performed. Recovery of vancomycin from plasma was 103.1±3.9%, and no interference from commonly used drugs or endogenous compounds was observed. A significant correlation was shown with the EMIT assay (r=0.92, P<0.001) using clinical samples from children. This HPLC technique is simple, sensitive, rapid, precise, selective and requires only 100 μl of plasma for completion.     

High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues

High-performance liquid chromatographic methods using reversed-phase chromatography and electrochemical detection have been developed for the quantitation of azithromycin in serum and tissues of laboratory animals and humans. Serum sample preparation involved addition of internal standard, alkalinization, and solvent extraction. Tissue sample preparation involved Polytron homogenization in acetonitrile containing internal standard, evaporation of the supernatant, alkalinization of the residue, and solvent extraction. Serum samples were chromatographed on an alkylphenyl-bonded silica column eluted with pH 6.8–7.2 mobile phase with a dual-electrode coulometric detector operated in the oxidative screen mode. Serum and tissue samples were chromatographed on a γRP-1 alumina column with pH 11 mobile phase with a glassy carbon amperometric detector. Recovery of azithromycin was 87% from serum and 85% from tissues. Linear standard curves were prepared in serum over two concentration ranges (0.01–0.20 and 0.20–2.0 μg/ml) and in tissues over several concentration ranges (0.1–2, 1–10, 10–100, and 100–1000 μg/g). In serum and tissues, intra- and inter-assay precision ranged from 1 to 8% and 4 to 11%, respectively. The tissue assay has been applied to liver, kidney, lung, spleen, muscle, fat, brain, tonsil, lymph nodes, eye, prostate and other urological tissues, and gynecological tissues.     

High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine

A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10⁻⁷M.     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates

A microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin and its metabolite 5-hydroxyindoleacetic acid in rat brain dialysates. The concentration of serotonin in these samples is usually in the low nanomolar range (fmol per 20 μl range). To optimize separation and detection, several adaptations were made to the system with respect to the injection valve, flow-rate of the pump, connections between injector, column and detector, and cell volume of the detector. These aspects are discussed, as well as the procedure developed for optimal peak identification of serotonin and correct estimation of 5-hydroxyindoleacetic acid. The assay allows the measurement of basal serotonin release without the use of a re-uptake inhibitor added to the perfusion fluid.     

Determination of cysteine in plasma and urine and homocysteine in plasma by high-pressure liquid chromatography

New methods are described for the determination of reduced and total cysteine in urine and the nonprotein fraction of plasma, and total homocysteine in the nonprotein fraction of plasma. The method for reduced cysteine involves separation by high performance liquid chromatography (hplc), followed by detection with a mercury-based electrochemical detector. The detector has a detection limit of ca, 10⁻⁶ cysteine, and is selective for sulfhydryl components of the biological fluids analyzed. Sample preparation involves centrifugation and filtration, and the hplc analysis time is approximately 8 min. Total cysteine and homocysteine are determined by electrolytic reduction of their disulfides to the sulfhydryl form prior to the hplc analysis. Results are presented which suggest that cysteine disulfide exchange reactions are a source of error in methods which employ derivatization with iodoacetate for the determination of cysteine in plasma.     

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 μl/min. Separations were performed using 7 cm×25 μm I.D. fused-silica capillary columns packed with 5 μm Alltima C₁₈ particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 μl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K⁺-induced depolarization conditions.     

Determination of low plasma timolol concentrations following topical application of timolol eye drops in humans by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for determination of timolol in human plasma following administration of two drops of a 5% timolol ophthalmic solution. A 4% butyl alcohol—hexane extract of an alkalized sample of plasma was chromatographed on a reversed-phase column and the components in the column effluent were monitored by coulometric detection. The extraction efficiency of timolol was 69.02 ± 4.16% (mean ± S.D.) and its detection limit was 107.2 pg/ml. The effect of mobile phase pH, buffer concentration and the working potential of the detector on column performance and the electrochemical response are described.     

Gas chromatographic determination of the hypoglycaemic agent gliclazide in plasma

A gas chromatographic method has been developed that permits the accurate and specific determination of the hypoglycaemic agent gliclazide in plasma. Gliclazide is extracted with chloroform and, after clean-up, derivatized with diazomethane followed by heptafluorobutyric anhydride to form N-methyl-N′-heptafluorobutyrylgliclazide, which is assayed on a gas chromatograph equipped with a flame ionization detector, an electron-capture detector or a nitrogen—phosphorus sensitive detector.Accurate determinations are possible with flame ionization detection over a concentration range of 1–15 μg/ml of gliclazide in plasma with a relative standard deviation of 5.2%. The minimum detectable concentration with electron-capture detection is 0.02 μg per sample. Plasma levels of gliclazide in dogs following single oral administration (40 mg per dog) have also been determined.     

Analysis of acetaminophen metabolites in urine by high-performance liquid chromatography with UV and amperometric detection

Acetaminophen and several of its metabolites are separated isocratically on a reversed-phase (C₁) column using a mobile phase of 7% methanol and 0.75% glacial acetic acid in 0.1 M KH₂PO₄. Metabolites that can be separated include the sulfate, glucuronide, cysteine, and mercapturic acid conjugates of acetaminophen, as well as 3-hydroxyacetaminophen, 3-methoxyacetaminophen, and 3-methylthioacetaminophen. Although all of the metabolites can be detected by UV spectrophotometry, the sensitivity limits of detection are improved significantly for acetaminophen and all of the metabolites except the sulfate and glucuronide, by amperometric detection (electrochemical) of the same sample as it elutes from the UV detector. Minimal detectable limits (signal-to-noise ratio 2) for acetaminophen and its metabolites, other than the glucuronide and sulfate conjugates, were in the order of 1–2 ng injected on column using UV detection at 248 nm, and 0.1–0.5 ng using electrochemical detection at + 0.60 V with reference to an Ag/AgCl standard electrode.     

Determination of ascorbic acid by polypyrrole potentiometric detector in ion chromatography

A potentiometric detector, based on electrodeposition of polypyrrole onto Pt electrode, was investigated in this study. The chromatographic performance of the PPy detector was investigated in ion chromatography. The resulting PPy detector exhibited good performance for AA determination with a wide linear range (1.0 × 10⁻⁶ to 1.0 × 10⁻² M), a highly reproducible response (R.S.D. of 2.45%), without any interference and long-term stability. The calculated detection limit was 7.0 × 10⁻⁵ mM at 3σ. The calibration results showed good Nernstian behavior and also satisfactory low detection limit. In order to verify the reliability of the PPy detector, it was applied to the determination of AA in pharmaceutical samples. The results were satisfactory and agreed closely with the manufacturers’ stated contents. The PPy electrode can be used as an alternative novel potentiometric detector material for determination of AA in standards and pharmaceutical samples.     

Sensitive method for the determination of vincristine in human serum by high-performance liquid chromatography after on-line column-extraction

A column-switching high-performance liquid chromatographic method was developed for the determination of vincristine in serum. Sample preparation was carried out by means of on-line column-extraction, using a C₁₈ reversed-phase preconcentration column. This technique is simple (minimizing manual sampling errors), rapid (reduction of time and costs) and can be easily automated. Both ultraviolet and electrochemical detection are possible, but the latter shows a cleaner chromatogram and is, by the use of a new electrochemical detector, far more sensitive (detection limit 0.3 μg/l at a signal-to-noise ratio of 3). A matrix study was carried out (using human serum and urine and two kinds of calf's serum). Although it appeared that the system was matrix-dependent, no difference in matrix effects could be found in the serum or plasma of different patients. Controls for human serum analysis should be prepared in human serum. With the method described, pharmacokinetic studies of vincristine in children can be performed.     

New dual electrochemical detector for microbore liquid chromatography determination of dopamine and serotonin in rat striatum dialysates

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise RATIO=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.     

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E_1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T_m = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.     

Determination of serotonin, catecholamines and their metabolites by direct injection of supernatants from chicken brain tissue homogenate using liquid chromatography with electrochemical detection

An isocratic liquid chromatographic method with electrochemical detection for the determination of

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-3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, serotonin, and their major metabolites, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 5-hydroxyindole-3-acetic acid in chicken brain tissue is described. Chickens were killed at different ages, the brains were quickly frozen and 300-μm cryostat sections were made. From these sections, two to six tissue micropunches (1 mm in diameter) were punched out from 20 different areas of the hypothalamus and homogenated in 100 μl 0.1 M perchloric acid which included 0.01% cysteine as antioxidant. Fifty-μl supernatants were injected directly onto the LC system, separated on a 3-μm Phase II ODS column (100×3.2 mm I.D.) and detected by an electrochemical detector at a potential of +0.75 V. Standard curves, recoveries, analytical precision and detection limits were investigated for each monoamine neurotransmitter and its metabolites. The method was applied to study the influence of food restriction on the concentration of monoamine neurotransmitters in different brain areas, known to be involved in feeding and reproductive behaviour of female broiler chickens. Over 1000 micropunched tissue samples from ad libitum fed and food-restricted female broiler chickens were analyzed. Our results provide a possible role for catecholamines and indolamines in the altered feeding and reproductive behaviour of the broiler chicken.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.     

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 μl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C₈ (150×4.6 mm) 5 μm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5–800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3–4.5% and 2.2–4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.