Microcystin uptake inhibits growth and protein phosphatase activity in mustard (Sinapis alba L.) seedlings

Mustard (Sinapis alba L.) seeds were cultivated for seven days on a solid nutrient medium supplemented with 0–40 μg microcystin-RR per ml. Microcystin-RR affected seedling growth ( ₅₀ 0.8 μg/ml) and microcystin concentrations ≥5.0 μg/ml produced malformed plants. The inhibition of protein phosphatase 1 and 2A activity correlated with the growth inhibition. The seedlings were also shown to take up ³H-dihydromicrocystin-LR derived radioactivity up to a level corresponding to ca. 80 ng toxin per mg plant protein.     

Protection of extract from leaves of Ardisia compressa against benomyl-induced cytotoxicity and genotoxicity in cultured rat hepatocytes

The potential of the Ardisia compressa extract (EA) was examined regarding its capacity to reduce the cytotoxic effect of benomyl on rat hepatocytes. The protective effect was evaluated by Janus Green dye exclusion method. An approximate 50% cytotoxic effect of benomyl on hepatocytes was observed at 35 μg/ml after 2 hr of incubation. (−)Epigallocatechin 3-gallato (EGCG) and EA decreased the viability of hepatocytes at concentrations above 3 μg/ml and 2.52 μg, equivalent to (+)catechin/ml, respectively. A protective effect against benomyl was observed when hepatocytes were previously exposed to EGCG (3 μg/ml) or EA (2.52 μg, equivalent to (+)catechin/ml) followed by incubation with benomyl (35 μg/ml) alone. When EGCG or EA were in contact with cells, either simultaneously or after pretreatment with benomyl, did not protect hepatocytes. EGCG (1.3×10⁻² μg/ml) or EA (9.8×10⁻² μg, equivalent to (+)catechin/ml) inhibited 57% and 34%, respectively, the unscheduled DNA synthesis (UDS) induced by benomyl at a concentration of 23×10⁻² μ , when both were incubated with hepatocytes prior to benomyl. The simultaneous incubation of benomyl with EGCG or EA did not protect the cell against the genotoxic effect of benomyl. These results indicate that the dried leaves extract of Ardisia compressa protect rat hepatocytes from benomyl-induced cytotoxicity and genotoxicity.     

High-performance liquid chromatographic determination of ciprofloxacin in plasma samples

A new bioanalytical high-performance liquid chromatographic (HPLC) method for the determination of ciprofloxacin with norfloxacin as an internal standard was developed and validated for plasma samples. Norfloxacin is structural homologue of ciprofloxacin and exhibits similar retention properties. The quality of respective peak separation is strongly influenced by amphoteric character of ciprofloxacin and norfloxacin as well. In previously published HPLC methods on conventional C18 reversed-phase [F. Belal, A.A. Al-Majed, A.M. Al-Obaid, Talanta 50 (1999) 765–786; G. Carlucci, J. Chromatogr. A 812 (1998) 343–367], ion pair reagents were added into the mobile phase to suppress peak tailing. In comparison with endcapped and high purity silica reversed-phase sorbent (Purospher RP-18e, Merck), which yielded symmetrical peaks, separation efficiency was further enhanced in our method. Gradient elution mode using acetonitril and phosphate buffer pH 3 on the pentafluorophenylpropyl stationary phase (250–4.6 mm Discovery^® HS F5, 5 μm, Supelco) was carried out. The resolution of 4.1 for ciprofloxacin–norfloxacin peaks was achieved. Sample preparation by SPE C18 (Supelclean) with recovery 72% was performed. Fluorescence detection with λ_excit = 280 nm, λ_emis = 446 nm was used. After the validation, the bioanalytical HPLC method was applied to pharmacokinetic studies.     

HPLC quantification of two isomeric triterpenic acids isolated from Mitracarpus scaber and antimicrobial activity on Dermatophilus congolensis

Oleanolic (OA) and ursolic acids (UA) were isolated for the first time from the alcoholic extract of Mitracarpus scaber possessing antimicrobial effects on Dermatophilus congolensis. These two triterpenic acids were also active (MIC 15 μg/ml) on this causative agent of dermatophilosis in African animals.

To quantify OA and UA in M. scaber, a new, simple and rapid high-performance liquid chromatography (HPLC) method compatible with MS detection was developed and validated. The mobile phase acetonitrile:H₂O (85:15, v/v) was pumped through a C18 octadecylsilyl silica column at a flow rate of 0.6 ml/min and the eluate was monitored at 215 nm. The calibration curves constructed between 0.5 and 10 μg/ml showed linear relationships with good R² values. The developed method was precise and reproducible with relative standard deviations (RSD) for these two active constituents between 0.22–2.06% (intraday) and 1.61–3.72% (interday) for concentrations from 0.5 to 6 μg/ml. Limits of detection and quantification were, respectively, 0.2 and 0.5 μg/ml.     

Ultrastructural effects of AAL-toxin TA from the fungus Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity

Ultrastructural effects of AAL-toxin T_A from Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade (Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin T_A (0.01–200 μM) under continuous light at 25°C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 μM AAL-toxin T_A, but was observed after 24 h with 0.01 to 50 μM AAL-toxin T_A, when it ranged from 25% to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 μM AAL-toxin T_A, respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black nightshade leaf discs floating in 10 μM AAL-toxin T_A under continuous light at 25°C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T_A-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36–48 h incubation with 10 μM AAL-toxin T_A, all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T_A in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to ≥0.5 μM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, physiological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T_A causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.     

On-site screening and GC-MS analysis of cocaine and heroin metabolites in body-packers urine

A sensitive gas chromatography–time-of-flight mass spectrometry (GC–ToFMS) method has been developed and validated for the determination of buagafuran, a new anxiolytic drug derived from -agarofuran. Buagafuran and internal standard (buagafuran-d₄) were isolated from plasma by liquid–liquid extraction. The separation was achieved on HP-1 capillary column (25 m × 0.2 mm × 0.11 μm). Buagafuran (m/z 262.22) and buagafuran-d₄ (m/z 266.25) were eluted at 7.6 min and no endogenous materials interfered with the measurement. The calibration curves of buagafuran were linear over the range of 2.5–160 ng/ml in dog plasma. The lower limit of quantification (LLoQ) was 2.5 ng/ml in plasma. The within-day and between-day precisions were less than 15%. The method was used to determine the plasma concentration–time profile of buagafuran after oral doses of 8, 16, 32 mg/kg in dogs.     

Rapid quantification of nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantitation of nebivolol in human plasma. The method involved a simple single-step liquid–liquid extraction with diethyl ether/dichloromethane (70/30). The analyte was chromatographed on Waters symmetry^® C₁₈ reversed-phase chromatographic column by isocratic elution with water:acetonitrile:formic acid (30:70:0.03, v/v) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 406.4–151.5 and m/z 409.1–228.1 were used to measure the analyte and the internal standard (I.S.), respectively. The chromatographic runtime was 2 min and the weighted (1/x²) calibration curves were linear over the range 50–10,000 pg/mL. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 10 and 50 pg/mL, respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<10%). The analyte was stable after three freeze-thaw cycles (deviation <10%). The average absolute recoveries of nebivolol and tamsulosin, used as an internal standard, from spiked plasma samples were 73.4 ± 3.7 and 72.1 ± 2.0%, respectively. The assay method described here was applied to study the pharmacokinetics of nebivolol.     

High-throughput determination of ultra-low concentrations of LAG078, a lipid modulator, in human plasma

A high throughput method with ultra-low level quantification limit (10 pg/ml) was developed and validated for the quantitative determination of LAG078, a lipid modulator, in human plasma to support clinical studies employing low doses of the compound. The method consisted of reverse phase chromatographic separation of the analyte from plasma extract followed by electrospray ionization (ESI) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring mode (MRM). Extraction was performed using a combination of protein precipitation and liquid–liquid extraction in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short and high-resolution column (50 mm × 2.0 mm i.d., 3 μm particle size) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r = 0.996) within the concentration range of 0.01 (lower limit of quantification) to 50 ng/ml using 0.5 ml of human plasma. The accuracy of the method varied from 95–101% with a precision (CV) of 5.29–13.2% over the concentration range. The method was simple and rapid.     

Simultaneous determination and validation of antimicrobials in plasma and tissue of actinomycetoma by high-performance liquid chromatography with diode array and fluorescence detection

A simple, precise, and reliable chromatographic method was developed for the simultaneous determination in plasma and infected tissue of five antimicrobials proposed for the treatment of actinomycotic mycetoma: amoxicillin, trimethoprim, linezolid, sulfamethoxazole and garenoxacin. Separation of the analytes was achieved on an Atlantis dC18 column (150 mm × 4.6 mm, ID 5 μm) with a mobile phase composed of acetonitrile and trifluoroacetic acid (ATF) 0.1% (v/v) using a gradient program. The detection was carried out using a diode array detector at 254 nm and in a fluorescence detector at wavelengths of excitation and emission of 292 nm and 392 nm for linezolid and sulfamethoxazole, and 292 nm and 408 nm for garenoxacin, respectively. The intraday precision was in the range of 0.7–15% of relative standard deviations (%R.S.D.) for plasma and 1–18% for tissue. Linearity range was from 2.4 to 20 μg/ml for amoxicillin, 0.3 to 20 μg/ml for trimethoprim, sulfamethoxazole and linezolid, and 0.3 to 10 μg/ml for garenoxacin. Acetonitrile was used to precipitate proteins from plasma. Recoveries in plasma ranged from 71% to 118% and in infected tissue from 78% to 122%. Limits of detection (LODs) were 1.2 and 0.5 μg/ml for amoxicillin in plasma and tissue, respectively and 0.15 and 1.2 μg/ml in plasma and tissue, respectively for the other antimicrobials. The method can be applied for individual or simultaneous determination of the antimicrobials in plasma and tissue of mouse infected with actinomycetoma.     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

Determination of catecholamines by flow-injection analysis and high-performance liquid chromatography with chemiluminescence detection

A chemiluminescence (CL) detection of catecholamines [norepinephrine (NE), epinephrine (E), dopamine (DA) and l-dopa (LD)] is described for the flow-injection (FI) and high-performance liquid chromatographic (HPLC) determination of these compounds. The detection method is based on the inhibition effect of catecholamines (CAs) on the CL reaction of luminol with iodine in the alkaline medium. The proposed FI method allows the determination of CAs in pharmaceutical preparations for the purpose of drug quality control. The calibration curves show good linearity in the concentration range of: 1.1–20.0 μg l⁻¹ (NE), 0.5–5.0 μg l⁻¹ (E), 0.6–9.0 μg l⁻¹ (DA) and 0.6–10.0 μg l⁻¹ (LD). The limits of detection (defined as a signal-to-noise ratio of 3) are: 0.34 μg l⁻¹ (NE), 0.15 μg l⁻¹ (E) and 0.18 μg l⁻¹ (DA, LD). The HPLC procedure was successfully applied for the determination of catecholamines (NE, E, DA) in human urine after solid-phase extraction (SPE). In a simple run time CAs can be determined in 20 min. The chromatographic linear ranges are: 5.0–72.0 μg l⁻¹ (NE), 5.0–48.0 μg l⁻¹ (E) and 5.0–96.0 μg l⁻¹ (DA). The limits of detection for three urinary CAs are: 0.71 μg l⁻¹ (NE), 0.26 μg l⁻¹ (E) and 0.73 μg l⁻¹ (DA).     

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC–MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020–10.0 and 0.100–50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102–153 ng/ml for norfentanyl in urine.

Liquid–liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d₅ and norfentanyl-d₅ from the matrixes was applied and separation was performed on a reversed phase YMC Pro C₁₈-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine.

The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied.

The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 μg fentanyl.     

Voltammetric determination of isoxsuprine and fenoterol in dosage forms and biological fluids through nitrosation

A simple and highly sensitive voltammetric method was developed for the determination of isoxsuprine HCl (I) and fenoterol HBr (II) in dosage forms and biological fluids. The method is based on treatment of the two compounds with nitrous acid followed by measuring the cathodic current produced by the resulting nitroso derivatives. The voltammetric behavior was studied adopting Direct Current (DC_t), Differential Pulse (DPP) and Alternating Current (AC_t) polarography. Both compounds produced well-defined, diffusion-controlled cathodic waves over the whole pH range in Britton–Robinson buffers (BRb). At pH 11 and pH 9, the values of diffusion-current constants (Id), were 9.4±0.3 and 7.7±0.4 for I and II, respectively. The current–concentration plots for I were rectilinear over the range of 0.6–12 μg/ml and 0.1–12 μg/ml in the DC_t and DPP modes, respectively. As for II, the range was 1–20 μg/ml and 0.1–20 μg/ml in the DC_t and DPP modes, respectively. The minimum detectability (S/N=2) were 0.02 μg/ml (≈6×10⁻⁸ M) and 0.01 μg/ml (≈2.6×10⁻⁸ M) for I and II, respectively, adopting the DPP mode. The proposed method was applied to the determination of both compounds in dosage forms and the results obtained were in good agreement with those obtained using reference methods. The proposed method was further applied to the determination of isoxsuprine in spiked human urine and plasma. The percentage recoveries adopting the DPP mode were 98.84±1.18 and 99.26±0.97, respectively.     

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized ¹³C₆-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.     

Simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation by liquid chromatography

A rapid and accurate liquid chromatographic method is described for the simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation. Chromatographic separation of the two drugs was achieved on a Diamonsil™ C₁₈ column (200 mm×4.6 mm, 5 μm). The mobile phase consisting of a mixture of acetonitrile, methanol and 0.5% ammonium acetate (39:11:50 (v/v), pH 5.5) was delivered at a flow rate of 1.0 ml/min. Detection was performed at 220 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 201.2–2012.0 μg/ml for roxithromycin and 42.7–427.0 μg/ml for ambroxol hydrochloride, respectively. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.     

Microcalorimetric investigation of the toxic action of ammonium ferric(III) sulfate on the metabolic activity of pure microbes

A series of calorimetric experiments were performed to investigate toxic action of ammonium ferric sulfate (AFS) on Bacillus subtilis, Pseudomonas putida and Candida humicola. The power–time curves of micro-organism metabolism were obtained, and the action of them by addition of AFS was studied. C. humicola, B. subtilis and P. putida were inhibited completely when the concentrations were up to 320.0, 160.0 and 160.0 μg mL⁻¹, respectively. The relationships between growth rate constant (k) and doses of AFS were approximately linear for three microbes, P. putida for 10.0–160.0 μg mL⁻¹ (R = −0.9746), B. subtilis for 0–160.0 μg mL⁻¹ (R = −0.9868) and C. humicola for 10.0–320.0 μg mL⁻¹ (R = −0.9955). The total heat dissipated per milliliter (Q_T) for three microbes remained balance approximately during the lower doses, P. putida and B. subtilis less than the dose of 20.0 μg mL⁻¹, 0.56 ± 0.01 and 0.26 ± 0.01 J mL⁻¹, respectively, C. humicola less than the dose of 40.0 μg mL⁻¹, 0.58 ± 0.03 J mL⁻¹. The biomass and OD₆₀₀ of three micro-organisms growth in the absence of AFS also were obtained. The power–time curve of C. humicola growth coincided with its turbidity curve. It elucidates that microcalorimetric method agreed with the routine microbiology method.     

Identification of major constituents in the traditional Chinese medicine "QI-SHEN-YI-QI" dropping pill by high-performance liquid chromatography coupled with diode array detection-electrospray ionization tandem mass spectrometry

An HPLC–DAD–ESI-MSⁿ method was developed for simultaneous analysis of major chemical constituents in “QI-SHEN-YI-QI” dropping pill, a traditional Chinese medicine (TCM) widely used for treating cardiovascular diseases. The chromatographic separation was performed on an intertsil ODS-3 C₁₈ column (4.6 mm × 250 mm, 5 μm), whilst water with 0.05% acetic acid and acetonitrile were used as mobile phase. On the basis of the characteristic UV absorption profile, the information of molecular weight, and structure provided by ESI-MSⁿ, 31 constituents derived from Astragalus membranaceus, Radix Salviae Miltiorrhizae, and Panax notoginseng, were detected and 20 of them were identified in this study. The proposed method contributes to the quality control of “QI-SHEN-YI-QI” dropping pill.     

Effect of carotenoids against genotoxicity of diethylnitrosamine on rat hepatocytes

Carotenoids have been considered as special nutrients due to their biological activity as provitamin A compounds, and because of their natural antioxidant and anticarcinogenic properties. The main objective of this study was to evaluate the protective effect of carotenoids against the genotoxic cellular damage induced by diethylnitrosamine (DEN), a potent hepatocarcinogen. Normal and freshly isolated hepatocytes were cultured as the biological system. Concentrations of 2.5 and 5 μ DEN caused 1.3 and 2.0 times more DNA T³H incorporation, respectively, when compared with control cells. Pure carotenoids, β-carotene (50 μ ), lutein (1 μ ) and a carotenoid extract from green peppers (1 μ eq. lutein) were used as functional nutrients to protect the cells. All the carotenoids studied prevented the genotoxic damage caused by 2.5 μ DEN. When 5 μ DEN was used, only β-carotene and the pepper extract inhibited the damage up to 30–40%. Carotenoids provide a dose-dependent protective effect against DNA damage induced by DEN in isolated hepatocytes.     

Stereospecific high-performance liquid chromatographic validation of homoeriodictyol in serum and Yerba Santa (Eriodictyon glutinosum)

A stereospecific method of analysis of racemic homoeriodictyol (eriodictyol 3′-methyl ether) in biological fluids is necessary to study pharmacokinetics and disposition in fruits and herbs. A simple high-performance liquid chromatographic method was developed for the determination of homoeriodictyol enantiomers. Separation was achieved in a Chiralcel^® OJ-RH column with UV-detection at 288 nm. The standard curves in serum were linear ranging from 0.5 to 100.0 μg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was <15%, and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of homoeriodictyol enantiomers in rats and to the quantification of homoeriodictyol enantiomers in Yerba Santa (Eriodictyon glutinosum).     

Detection of modafinil in human urine by gas chromatography-mass spectrometry

The main purpose of this study was to detect and quantify modafinil in human urine by gas chromatography–mass spectrometry (GC–MS). Urinary samples were collected from three healthy male volunteers following oral administration of a clinical dose (100 mg) of modafinil (Provigil^®). Urine specimens were extracted with t-butylmethyl ether (TBME) prior to GC–MS analysis. The results demonstrate that the chromatographic characteristics and the mass spectrum of the unchanged parent drug extracted from urine samples were identical to that obtained from the authentic standard. The times for the unchanged modafinil to reach peak concentration in the urine of the three volunteers were at 2 h (6.14 μg/mL), 4 h (9.93 μg/mL) and 8 h (3.58 μg/mL), respectively. Total clearance occurred in approximately 48–72 h with 2–5% eliminated through urine as unchanged modafinil. The present study demonstrates that modafinil is detectable in the absence of hydrolysis and derivatization steps.