Thin-layer chromatographic determination of indolic tryptophan metabolites in human urine using Sep-Pak C18 extraction

Tryptophan and some of its indole metabolites were separated by thin-layer chromatography, stained with the Van Urk—Salkowski reagent, and quantitated by scanning densitometry. The application of this technique for the detection of the indoles in urine samples, employing Sep-Pak C₁₈ cartridges for extraction, was demonstrated. The proposed method is simple and accurate. The detection limits were 2 μg/ml 5-hydroxytryptophan, 1.75 μg/ml 5-hydroxyindolyl-3-acetic acid, 1.5 μg/ml tryptophan, 0.8 μg/ml indolyl-3-acetic acid, 0.9 μg/ml indolyl-3-butyric acid, 1.75 μg/ml serotonin, and 1.25 μg/ml tryptamine.     

Method for the biological monitoring of hexahydrophthalic anhydride by the determination of hexahydrophthalic acid in urine using gas chromatography and selected-ion monitoring

A method for the determination of hexahydrophthalic acid, a metabolite of hexahydrophthalic anhydride, in human urine has been developed. The urine was worked-up by liquid—solid extraction, esterified with boron trifluoride—methanol, and analysed by capillary gas chromatography and selected-ion monitoring. Hexadeuterium-labelled hexahydrophthalic acid was used as the internal standard. The precision was 4% at 0.7 μg/ml and 5% at 0.07 μg/ml. The recovery of the acid for the overall method was 101% at 0.07 μg/ml of urine (with a coefficient of variation of 4%) and 95% at 0.7 μg/ml (coefficient of variation 2%). The limit of detection was 20 ng/ml urine.     

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N₄-acetyl-5-methylhydroxysulfamethoxazole (N₄SOH) and sulfamethoxazole-N₁-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by β-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N₄-acetylsulfamethoxazole (N₄), SOH, N₄-hydroxysulfamethoxazole (N₄OH), N₄SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N₄ 0.10 μg/ml; N₄SOH 0.13 μg/ml; N₄OH 0.18 μg/ml; SOH 0.20 μg/ml; and Sgluc 0.39 μg/ml. In urine the quantitation limits are: N₄ and N₄OH 1.4 μg/ml; S 1.5 μg/ml; N₄SOH 1.9 μg/ml; SOH 3.5 μg/ml; and Sgluc 4.1 μg/ml. The method was applied to studies with healthy subjects and HIV positive patients.     

Mapping the regioisomeric distribution of fatty acids in triacylglycerols by hybrid mass spectrometry

This study describes the use of hybrid mass spectrometry for the mapping, identification, and semi-quantitation of triacylglycerol regioisomers in fats and oils. The identification was performed based on the accurate mass and fragmentation pattern obtained by data-dependent fragmentation. Quantitation was based on the high-resolution ion chromatograms, and relative proportion of sn-1(3)/sn-2 regioisomers was calculated based on generalized fragmentation models and the relative intensities observed in the product ion spectra. The key performance features of the developed method are inter-batch mass accuracy < 1 ppm (n = 10); lower limit of detection (triggering threshold) 0.1 μg/ml (equivalent to 0.2 weight % in oil); lower limit of quantitation 0.2 μg/ml (equivalent to 0.4 weight % in oil); peak area precision 6.5% at 2 μg/ml concentration and 15% at 0.2 μM concentration; inter-batch precision of fragment intensities < 1% (n = 10) independent of the investigated concentration; and averaged accuracy using the generic calibration 3.8% in the 1–10 μg/ml range and varies between 1–23% depending on analytes. Inter-esterified fat, beef tallow, pork lard, and butter fat samples were used to show how well regioisomeric distribution of palmitic acid can be captured by this method.     

Determination of lamotrigine in human serum by liquid chromatography

A rapid high-performance liquid chromatographic method was developed using a short silica column (30 mm×4.6 mm) with an aqueous methanol mobile phase consisting of methanol–water–NH₄H₂PO₄ (94:5.96:0.04) adjusted to a final apparent pH of 5.0 and pumped at a flow-rate of 1 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. Within-day variation of the method was 4.46% at 0.75 μg/ml and 2.37% at 6.0 μg/ml, and day-to-day variation was 9.10% at 0.75 μg/ml and 7.28% at 6.0 μg/ml.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Determination of cholesterol and cortisone absorption in polyurethane : I. Methodology using size-exclusion chromatography and dual detection

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 μl, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 μg/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 μg (1 μg/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 μg/ml with a lower limit of 0.015 μg (0.1 μg/ml). The results show that after 44 h, 2037 μg/g cholesterol and 3131 μg/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

In vitro Activity of Gatifloxacin Against 238 Strains of Anaerobic Bacteria

The activity of gatifloxacin, a new 8-methoxy-fluoroquinolone, was tested against 208 pulmonary pathogens and against an additional 30 isolates of the Bacteroides fragilis group. Pulmonary isolates were from patients with documented anaerobic pleuropulmonary infections and were obtained by appropriate sampling methods. MICs were determined using the NCCLS-approved Wadsworth brucella laked blood agar method and compared to those of clindamycin, imipenem, metronidazole and trovafloxacin. Breakpoints used to define susceptible and [resistant] categories were (in μg/ml): Clindamycin-2, imipenem-4, metronidazole 8 and trovafloxacin. No breakpoint has been defined for gatifloxacin. Gatifloxacin inhibited 99% of all anaerobes tested at 4 μg/ml and 97% of all strains at 2 μg/ml. One strain of B. fragilis was resistant to gatifloxacin at 4 μg/ml; all strains of other B. fragilis group species were susceptible. One strain of Peptostreptococcus sp. was resistant to both gatifloxacin and trovafloxacin (MIC >4 μg/ml). All other strains were susceptible to all agents at ≤μg/ml. All of the non-sporeforming Gram-positive rods were susceptible to gatifloxacin at ≤μg/ml (three strains had an MIC of 4 μg/ml). Trovafloxacin had MICs of 4 μg/ml for two strains, and an MIC of 8 μg/ml for one strain. Five percent of B. fragilis, 21% of other B. fragilis group species and 20% of Clostridium species (other than C. difficile, C. perfringens or C. ramosum) were resistant to clindamycin. No imipenem resistant isolates were found in this study. Gatifloxacin appears to have excellentin vitro activity against pulmonary isolates of anaerobes and very good activity against strains of the B. fragilis group.     

Rapid high-performance liquid chromatographic determination of serum gabapentin

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 μg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 μg/ml, 14.8% were below 2 μg/ml and 12.7% above 10 μg/ml. The mean±S.E. of GBP in 283 patients was 5.38±0.23 μg/ml. Peak concentrations of more than 15 μg/ml and trough levels as low as 0.1 μg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

Rapid mass spectrometric analysis for ascorbate and related organic acids in small volumes of plasma for use in pediatric subjects

A gas chromatographic–mass spectrometric isotope dilution method was developed for analysis of ascorbate on 10 μl samples of plasma. This assay was reproducible (standard deviation of less than 4%) and gave values for plasma ascorbate content within 8% of our previously published gas chromatographic–mass spectrometric method. Non-specific sample preparation allowed other analytes to be determined on the same sample by adjusting data acquisition parameters and adding the appropriate internal standard. Analysis on 28 subjects fell within the expected range for plasma ascorbate 68±29 μm (11.9±5.0 μg/ml) and established a normal range for plasma threonate of 28.1±2.4 μm (3.8±0.4 μg/ml).     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

4-Methoxycinnamaldehyde inhibited human respiratory syncytial virus in a human larynx carcinoma cell line

4-Methoxycinnamaldehyde, an active constituent of Agastache rugosa, was examined for its cytoprotective activity against RSV by XTT method in human larynx carcinoma cell line. 4-Methoxycinnamaldehyde could effectively inhibit cytopathic effect of RSV (p<0.0001) with an estimated IC₅₀ of 0.055 μg/ml and a selectivity index (SI) of 898.2. 4-Methoxycinnamaldehyde (0.03 μg/ml) could inhibit viral entrance by interfering viral attachment (IC₅₀ of 0.06 μg/ml; p<0.0001) and internalization (IC₅₀ of 0.01 μg/ml; p<0.0001). 4-Methoxycinnamaldehyde significantly increased the basal production of IFN (p=0.0015), but not the virus-induced IFN production. Therefore, its cytoprotective activity against RSV was not mediated by interferon. In conclusion, 4-methoxycinnamaldehyde might be helpful to manage the disease induced by RSV infection.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 – 3 μg/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 ± 0.65 ng/ml in terms of PGE₂. Indomethacin (3 μg/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 μg/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 – 30 μg/ml) and dexamethasone (2 – 5 μg/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 μg/ml). Antigen-induced release of a prostaglandin-like substance(PGs) (43.1 ± 3.8 ng/ml in terms of PGE₂ and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 μg/ml) or by hydrocortisone (100 μg/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 ± 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 μg/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 – 3 μg/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 ± 0.65 ng/ml in terms of PGE₂. Indomethacin (3 μg/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 μg/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 – 30 μg/ml) and dexamethasone (2 – 5 μg/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 μg/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 ± 3.8 ng/ml in terms of PGE₂ and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 μg/ml) or by hydrocortisone (100 μg/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 ± 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 μg/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Measurement of surface contamination from nucleoside analogue antineoplastic drugs by high-performance liquid chromatography in occupational hygiene studies of oncologic hospital departments

Within the frame of a continuing interest in occupational hygiene of hospitals as workplaces, we describe an automated analytical method by reversed-phase high-performance liquid chromatography for the measurement of contamination from the three most important nucleoside analogue antineoplastic drugs (5-fluorouracil, 5FU; cytarabin, CYA; gemcytabin, GCA) on such surfaces as those of preparation hoods and work-benches in departmental pharmacies of oncologic departments. Our method is characterized by a short analysis time (7 min) under isocratic conditions, by the use of a mobile phase devoid of organic solvent and by high sensitivity (LOD≥40 μg/l for all compounds), adequate to detect surface contamination above a threshold of 4 μg/m² for wide surfaces and of 30 μg/m² for small irregular objects. We present some results from a preliminary survey study recently performed in seven oncologic departments of two large general hospitals in Milan. To exemplify the contamination levels on various surfaces (such as on handles, floor surfaces and window glass panes, even far from the preparation hood), analyte concentrations in the order of 0.03–0.06 μg/ml, corresponding to 0.8–1.5 μg of 5FU were measured on telephones, of 0.02–0.6 μg/ml (0.85–28 μg/m²) of CYA were measured on table boards, of 0.05–10.6 μg/ml (1.2–1150 μg/m²) of GCA on furniture and floors. Spillage fractions up to 1% of the employed ANDs (employed daily 5FU 7–13 g; CYA 0.1–7.1 g; GCA 0.2–5 g) are measured on the polyethylene-backed paper disposable cover sheet of the preparation hood.