Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C₁₈-, C₈- or C₄-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C₈-Alkyl-Diol Silica, 25 μm, 25 × 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5–101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2–3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproductivity. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).     

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultra-violet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m²-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01–1.0 μg/ml) for 3 different UV-A doses (3000–15000 J/m²). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks. These results suggest that the cytotoxic effects of photoactivated 8-MOP in human lymphoblastoid cells may depend on DNA interstrand crosslinks.     

Molecular aspects of extracorporeal photochemotherapy

8-methoxypsoralen (8-MOP), activated upon exposure to long-wavelength ultraviolet radiation, is used therapeutically to treat the diseased blood cells of cutaneous T-cell lymphoma patients. The factors responsible for the efficacy of this therapy are reviewed. Primary among these are the plasma level of 8-MOP at the time of irradiation and the effective dose of UVA. 8-MOP plasma levels determined in a series of six patients demonstrated that the drug is absorbed at a highly variable rate (122 ng/ml +/- 67). A new liquid form of 8-MOP is absorbed with a modest increase in plasma levels (170 ng/ml) but with no improvement in the variability (+/- 163). An examination of the dose-response relationship between 8-MOP concentration and UVA dose indicated that properties such as 8-MOP photoadduct formation and PHA response are proportional to the combined doses of these two factors. A new molecular target for 8-MOP photomodification, cell membrane DNA, is described.     

In vitro synergistic activity between 8-methoxypsoralen and ethambutol, isoniazid, and rifampin when used in combination against Mycobacterium tuberculosis

8-Methoxypsoralen (8-MOP), a naturally occurring furocoumarin found in many plant species, has been reported to have antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In the present study, we further test the in vitro synergistic activity of 8-MOP and ethambutol (EMB), isoniazid (INH), or rifampin (RMP) against M. tuberculosis. This study showed that 8-MOP has antimycobacterial activity against two drug-sensitive and six drug-resistant clinical isolates of M. tuberculosis, with the minimum inhibitory concentrations of 100–200 and 200–400 μg/mL, respectively. A synergistic antimycobacterial effect between 8-MOP and EMB, INH, or RMP against six drug-resistant strains was observed, with the fractional inhibitory concentration indices (FICIs) of 0.093–0.156, 0.138–0.285 and 0.093–0.262, respectively. The combination of 8-MOP/EMB, 8-MOP/INH, and 8-MOP/RMP displayed either synergistic activity or had no interaction when tested against the two clinical drug-sensitive strains and the standard strain. No antagonism was observed for any drug combination against any of the strains tested. To our knowledge, this is first report that 8-MOP has synergistic activity with first-line antimycobacterial agents.     

Mutagenic risk in psoriatic patients before and after 8-methoxypsoralen and long-wave ultraviolet radiation

Sister-chromatid exchange (SCE) analysis was carried out in different age groups prior to and after therapy with 8-Methoxypsoralen (8-MOP) followed by exposure of the patient to long-wave UV-A (PUVA) and compared to control. The SCE frequencies were increased significantly in PUVA-treated patients as compared to their pre-treatment SCE levels and to controls. A significant increase in SCEs was found in smoking PUVA-treated patients as compared to non-smoking PUVA-treated patients. This study indicates a detectable chromosome-damaging effect of PUVA therapy on its human users.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in V-79 Chinese hamster cells : A first approach to a risk estimate in photochemotheraphy

The effect of 8-methoxypsoralen (8-MOP) and long-wave ultraviolet irradiation (UVA) on cell killing and mutation induction was studied in V-79 Chinese hamster cells. No effect was observed after treatment with 8-MOP alone (50 μg/ml, 4 h), UVA alone (9000 J/m²), or 8-MOP metabolized by rat-livermicrosomes. Combined treatment with 8-MOP and UVA induced both cell killing and mutation. This was also observed under conditions approaching patient treatment with PUVA photochemotherapy with respect to the concentration of 8-MOP in the skin and the amount of UVA received by the epidermal cells. A simple relation proved to apply for mutation induction under different treatment conditions: 5.5 × 10⁻⁸ per J/m² per μg 8-MOP/ml. On this basis the mutation induction in dividing cells per session of PUVA-photochemotherapy amounts to 12.4 × 10⁻⁵, which is probably an over-estimation.     

LC-MS determination of oxidized and reduced glutathione in human dermis: a microdialysis study

A simple, highly selective, sensitive and reproducible liquid chromatography-electrospray ionization mass spectrometry method has been developed for the direct and simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in microdialysis samples from human dermis. Chromatographic separation was carried out on an MODULO CART QS KROMASIL 5C18 (250 mm × 2 mm × 5 μm) analytical column at a flow rate of 0.25 ml/min. An isocratic mode was used and consisted of acidified water and acetonitrile (50/50, v/v). To improve the sensitivity, silver nitrate was added as post-column reagent. A trap mass spectrum was used equipped with an ESI interface. The limits of detection and quantification were respectively 0.12 and 0.4 ng/ml for GSH and 0.2 and 0.5 ng/ml for GSSG. Intra-day and inter-day accuracy and precision were determined and the variability was less than 6.2% (R.S.D.).     

Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

Detection and quantification of aripiprazole and its metabolite, dehydroaripiprazole, by gas chromatography-mass spectrometry in blood samples of psychiatric patients

Aripiprazole is a novel antipsychotic drug for the treatment of schizophrenia and schizoaffective disorders. In this study, a new method using gas chromatography-mass spectrometry (GC-MS) was developed and validated for the detection of aripiprazole and its main metabolite, dehydroaripiprazole, in plasma. Blood samples from seven psychiatric patients treated with aripiprazole (10-20 mg/day) underwent a solid-phase extraction (SPE) and N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) derivatization. The characteristic ions of mass spectra for aripiprazole and dehydroaripiprazole were m/z 306, 292, 218 and 304, 290, 218, respectively. Extraction recoveries from this method were 75.4% (n=5) for aripiprazole and 102.3% (n=5) for dehydroaripiprazole. The calibration curves of aripiprazole and dehydroaripiprazole were linear from 16 to 500 ng/ml (r(2)=0.999) and 8 to 250 ng/ml (r(2)=0.999), respectively. The respective limits of quantification (LOQs) for aripiprazole and dehydroaripiprazole evaluated in 0.5 ml of serum were 14.4 ng/ml and 6.9 ng/ml. Intra-assay and interassay precision and accuracy were within acceptable ranges. In this study, we also found that the mean trough concentrations in plasma at steady-state were 128.9 microg/l for aripiprazole and 30.1 microg/l for dehydroaripiprazole.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in diploid human skin fibroblasts: an improved risk estimate in photochemotherapy

Cell killing and the induction of mutation were studied in dividing and non-dividing human skin fibroblasts as a result of treatment by 8-methoxypsoralen (8-MOP) and long-wave UV irradiation (UVA). The cytotoxic effect was highly dependent upon the duration of the UVA exposure. The frequency of mutations increased linearly with the UVA dose at concentrations of 10 and 0.25 microliter 8-MOP/ml, the latter representing the concentration in the skin during PUVA treatment. The number of mutations induced per unit dose (= per microgram 8-MOP/ml per joule UVA/m2) was calculated: for dividing cells this value was 3.3 X 10(-8) per cell and for non-dividing cells 0.6 X 10.8(-8) per cell. On the basis of these values the expected number of induced mutants in the human skin per session of photochemotherapy is 1.2 X 10(-5), and per 30 years of maintenance therapy 1.3 X 10(-2) per cell. A comparison was made between this frequency and the frequency to be expected from spontaneous mutation. In addition the significance of absence in patients of SCE induction by photochemotherapy is discussed.     

High-performance liquid chromatographic assay of 5-fluorouracil in human erythrocytes,plasma and whole blood

A HPLC assay method was modified and validated for the determination of 5-fluorouracil in human red blood cells, plasma and whole blood with a two-fold increased sensitivity (detection limit=10 ng/ml). The assay was linear from 25 to 1500 ng/ml and the accuracy ranged from 96.7 to 103.2% at 25 ng/ml, 94.8 to 99.4% at 500 ng/ml, and 98.9 to 99.5% at 1500 ng/ml. Intra-assay and inter-assay coefficients of variation were less than 8% over the range of concentrations and less than 8% over 10 days of analysis. After intravenous bolus and infusion of 5-fluorouracil in patients with colorectal cancer, the concentrations of 5-fluorouracil in whole blood were 108–111% of plasma concentrations, while packed red blood cells levels were 8–15% of plasma concentrations in the five patients studied. By utilising basic analytical hardware, this represents an accurate, precise, reproducible and affordable method for 5-fluorouracil pharmacokinetics investigation and therapeutic drug monitoring.     

The salicylate trapping method: is oxidation of salicylic acid solution oxygen and time dependent and metal catalysed?

For a microdialytic trapping method we systematically investigated changes in concentrations of 2,5-dihydroxy-benzoic acid (2,5-DHBA) and 2,3-dihydroxy-benzoic acid (2,3-DHBA) in freshly prepared solutions of salicylic acid (SA). The solvent was 0.9% saline exposed to different atmospheric concentrations of oxygen (0, 21, and 100%). The solutions were treated by freezing-thawing and an ultrasonic bath in presence and absence of aluminium foil. Without aluminium the concentrations of 2,5-DHBA and 2,3-DHBA kept constant over an observed period of 160 min on different levels from below 20 ng/ml to about 100 ng/ml. In presence of aluminium the concentrations increased to maximum 307 ng/ml after 160 min. Ultrasonic irradiation amplified this effect to maximum 341 ng/ml. HPLC/ECD processing and quantitative analysis of dihydroxy-benzoic acids (DHBAs) in microdialysis may be artificially influenced by varying oxygen environment and metal catalysis.     

Identification and quantitation of 16α-hydroxy c21 steroid sulphates in plasma from pregnant women

Plasma steroids from 11 women in the last trimester of pregnancy were separated into conjugate class by chromatography on Sephadex LH-20. Fractions corresponding to mono- and disulphate conjugates were solvolyzed and the free steroids were separated by lipophilic gel chromatography into groups containing mono-, di- and trihydroxy steroids, respectively. Trimethylsilyl ether derivatives were prepared and repetitive-scanning gas chromatography-mass spectrometry (GC-MS) was used to characterize and quantitate the following 16α-hydroxy steroids in the monosulphate fraction: 3α,16α:-dihydroxy-5α-pregnan-20-one (range 8–80 ng/ml), 3β,16α-dihydroxy-5α-pregnan-20-one (6–55 ng/ml), 5α-pregnane-3α,16α,20α-triol (9–31 ng/ml) and 5α-pregnane-3β,16α,20α-triol (11–77 ng/ml). These compounds were also present as disulphates although at 5–50 times lower concentrations.     

Frameshift mutagenesis in bacteria by 8-methoxypsoralen (methoxalen) in the dark.

We confirm that 8-methoxypsoralen (8-MOP) in the dark induces frameshift mutations in both Escherichia coli and Salmonella typhimurium when present in adequate concentration under growth conditions. The dose response is sigmoidal with a threshold or quasi-threshold at concentrations below about 10 microgram/ml. Frameshift mutagenesis by 8-MOP in the dark is unaffected by mutations at the uvrA or uvrB genes, in contrast to base pair substitution mutagenesis by 8-MOP plus near UV light. RecA (but not recB) bacteria are hypersensitive to the growth-inhibiting action of 8-MOP in the dark and are not detectably mutagenized. The characteristics of 8-MOP dark mutagenesis are consistent with the chemical interacting in a non-covalent manner with DNA and affecting the rate of occurrence of base deletions or insertions during DNA replication. The question of extrapolation of the genetic effect of 8-MOP to man is discussed.     

Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.     

Simultaneous determination of amphetamine and its analogs in human whole blood by gas chromatography-mass spectrometry

A sensitive and specific gas chromatography-mass spectrometry (GC-MS) method for the determination of amphetamine (AM), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA) and methylenedioxyethylamphetamine (MDEA) in whole blood was designed, using the respective pentadeuterated analogs of the analytes as internal standards (I.S.). After alkalinisation of blood samples, the amphetamines were extracted using diethyl ether, derivatized with heptafluorobutyric anhydride, then purified by successive washings with deionized water and 4% NH₄OH. Extraction recoveries were 85.2% for AM, 90.9% for MA, 76.5% for MDA, 84.1% for MDMA and 63.6% for MDEA. Chromatographic separation was performed on a non-polar 30 m×0.32 mm HP 5 MS capillary column using a temperature program. Detection was carried out in the electron-impact, selected ion-monitoring mode, using three mass-to-charge ratios for each analyte and one for each I.S. Limits of detection ranged from 0.5 to 8 ng/ml and limits of quantification were 10 ng/ml for AM, MDMA and MDEA; 20 ng/ml for MA; and 50 ng/ml for MDA. The method was linear from this limit up to 1000 ng/ml for all analytes, with good intra-assay precision and good intermediate precision and accuracy over these ranges. There was no interferences from other sympathomimetic drugs such as ephedrine, norephedrine or methoxyphenamine. This method is thus suitable for clinical and forensic toxicology, as well as for doping control.     

Changes in somatomedin-like activity and concentrations of growth hormone and prolactin in the plasma of castrated growing lambs.

Somatomedin-like activity was measured in the plasma of growing lambs using the porcine costal cartilage disk assay. Plasma concentrations were found to be high initially at 2 days of age (mean potency 1.02 plus or minus 0.13 (SEM) units/ml, n = 4) declined significantly by 8 days of age (mean potency 0.65 plus or minus 0.04 units/ml, n = 5, P less than 0.01, analysis of variance). Thereafter somatomedin-like activity declined slowly to reach its lowest concentration at 146 days of age (mean potency 0.61 plus or minus 0.04 units/ml, n = 5) then it rose slowly until 288 days of age (mean potency 0.61 +/- 0.04 units/ml, n = 5. These changes in somatomedin-like activity were accompanied by high initial plasma concentrations of growth hormone (24.8 plus or minus 4.8 ng/ml, n = 5) which declined under 188 days of age (2.8 plus or minus 0.04 ng/ml, n- 5) and then rose slightly until 288 days of age (13.8 plus or minus 9 ng/ml, n=5). Plasma prolactin concentrations showed a different pattern being low initially (47.8 plus or minus 8.7 ng/ml, n = 5) rising until 146 days of age (203 plus or minus 16 ng/ml, n = 5) and then declining to low value for the rest of the experiment. The relationships between these factors is not clear but somatomedin-like activity shows a pattern in the lamb which is highest when growth is faster (i.e. in the young lamb).     

Routine analysis of amphetamine and methamphetamine in biological materials by gas chromatography-mass spectrometry and on-column derivatization

A simple determination method of amphetamine (AP) and methamphetamine (MA) in biological materials was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). AP and MA in biological materials were adsorbed on the surface of Extrelut and then extracted and derivatized simultaneously on the Extrelut column. AP and MA were derivatized to the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 88.2 and 92.5%, and those from blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5-2000 ng/ml (ng/g) for AP and MA in urine and blood, and 0.25-20 ng/mg in hair. When urine samples containing two different concentrations (200 and 1000 ng/ml) of AP and MA, blood samples containing two different concentrations (200 and 1000 ng/g) of AP and MA, hair samples containing two different concentrations (0.5 and 5.0 ng/mg) of AP and MA, the coefficients of variation of intra-day and inter-day were 0.68-3.60% in urine, 0.42-4.58% in blood, and 1.20-13.1% in hair. Furthermore, this proposed method was applied to a medico-legal case of MA intoxication.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Comparison of the sensitivity of Fanconi's anemia and normal fibroblasts to the induction of sister-chromatid exchanges by photoaddition of mono- and bi-functional psoralens

The induction of sister-chromatid exchanges (SCE) by photoaddition of a monofunctional furocoumarin (pyrido[3,4-c]psoralen, PyPs) and a bifunctional furocoumarin (8-methoxypsoralen, 8-MOP) in a normal and three Fanconi anemia (FA) fibroblastic cell lines was investigated. When compared to normal cells, the three FA cell lines demonstrated: a higher sensitivity to 8-MOP photoaddition; an equal or reduced sensitivity to PyPs photoaddition in the low dose range. Normal cells demonstrated a higher sensitivity to photoaddition of PyPs than to 8-MOP in the range of doses used; this is likely to be related to the higher amount of lesions induced by PyPs in DNA. Since FA cells were almost equally sensitive to 8-MOP and PyPs photoaddition and demonstrated a higher sensitivity to SCE induction by 8-MOP than normal cells, it can be concluded that this latter difference is mainly due to cross-links.