High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

A column-switching high-performance liquid chromatographic assay for a novel cytotoxic thioxanthone derivative (WIN 33377) in mouse plasma with toxicokinetic results from a mouse LD10 study

WIN 33377 (I) is a member of a novel class cytotoxic antitumor agents, 4-aminomethyl thioxanthane derivatives. A simple, rapid and reproducible method has been developed for the assay of I in mouse plasma using a high-performance liquid chromatographic method utilizing a column-switching technique. The method involves direct injection of buffered plasma to the extraction column for sample clean-up followed by switching onto an analytical column for analysis with UV detection at 256 nm. The method has demonstrated accuracy and precision over the range 10–2500 ng/ml using a 100-μl plasma sample with a minimum quantifiable level at 10 ng/ml. Stability of mouse plasma samples was demonstrated after storage for 4 weeks at −15 to −20°C, as was the ability of samples to be accurately quantified after a maximum of three freeze-thaw cycles. Recovery was greater than 87% for the compound and the internal standard. The assay was accurate and reproducible with measured values lying within the limits of defined acceptance criteria. The utility of the method was demonstrated by analyzing plasma samples obtained from mice dosed with I as part of a pre-clinical safety study intended to assist in the design of a pharmacokinetically guided dose escalation strategy.     

High-performance liquid chromatographic method for the determination of atracurium and laudanosine in human plasma: Application to pharmacokinetics

A high-performance liquid chromatographic method coupled with fluorimetric detection has been developed for the determination of atracurium and its major metabolite, laudanosine, in human plasma. The detection is performed at 240 nm for excitation and 320 nm for emission. Verapamil was used as the internal standard. The proposed technique, involving the direct precipitation of plasma proteins is reproducible, selective and sensitive. Linear detector responses were observed for the calibration curve standards in the range of 40 to 2000 ng/ml. Precision, expressed as C.V., was in the range 1 to 14%. The limit of quantification for both atracurium and laudanosine was 40 ng/ml. The method has been validated and stability tests under various conditions have been performed. This method has been used to determine the pharmacokinetic profile of atracurium and laudanosine in patients with acute respiratory distress syndrome.     

Evaluations of cellular proliferation and chromosome breakages after in vitro exposure of human lymphocytes to calcium or zinc DTPA

The analysis of mitotic indices (MI) and chromosome breakages in metaphases of 50-hr lymphocyte cultures exposed to the calcium or zinc chelates of diethylenetriamine pentaacetic acid (DTPA) demonstrated: (1) an 80% reduction in MI in cultures from three women but no reduction in those from two men after in vitro exposure to CaDTPA in concentrations as low as 10 micrograms/ml culture medium, and complete suppression of mitoses in cultures from men and women after exposure to 40 micrograms/ml CaDTPA; (2) minor suppression in MI in cultures from women and none in those from men after exposure to 40 or 80 micrograms/ml ZnDTPA; (3) no ring or dicentric chromosomes in 1700 metaphases from DTPA-treated cultures. Likewise, in other experiments we observed no differences in the frequency or distributions of rings and dicentrics in lymphocyte cultures from two persons after in vitro exposure to 250-R 60Co gamma radiation in the presence or absence of 10 micrograms/ml CaDTPA or 10 or 80 micrograms/ml ZnDTPA. These data indicate that while accurate estimates of the frequencies of radiation-induced rings and dicentrics in lymphocytes can be made in actinide-contaminated persons undergoing DTPA chelation therapy, blood samples for cytogenetic cultures should not be obtained from chelated patients until the compound has been cleared from the blood plasma.     

High-performance liquid chromatographic determination of tazobactam and piperacillin in human plasma and urine

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance was developed for the analysis of piperacillin-tazobactam (tazocillin), in plasma and urine. The detection was performed at 218 nm for tazobactam and 222 nm for piperacillin. The procedure for assay of these two compounds in plasma and of piperacillin in urine involves the addition of an internal standard (ceftazidime for tazobactam and benzylpenicillin for piperacillin) followed by a treatment of the samples with acetonitrile and chloroform. To quantify tazobactam in urine, diluted samples were analysed using a column-switching technique without internal standard. The HPLC column, LiChrosorb RP-select B, was equilibrated with an eluent mixture composed of acetonitrile-ammonium acetate (pH 5). The proposed technique is reproducible, selective, and reliable. The method has been validated, and stability tests under various conditions have been performed. Linear detector responses were observed for the calibration curve standards in the ranges 5–60 μg/ml for tazobactam, and 1–100 μg/ml for piperacillin and spans what is currently though to be the clinically relevant range for tazocillin concentrations in body fluids. The limit of quantification was 3 μg/ml for tazobactam and 0.5 μg/ml for piperacillin in plasma and urine. Extraction recoveries from plasma proved to be more than 85%. Precision, expressed as C.V., was in the range 0.4–18%.     

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.     

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro

Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 microg/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study.The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations.The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent.In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

Development and validation of a high-performance liquid chromatographic assay using solid-phase extraction for the novel antitumor agent pancratistatin in human plasma

The stability of the experimental anti-tumour agent pancratistatin in human plasma has been investigated. A solid-phase extraction technique and an HPLC assay with external standards have been developed and validated. Extraction was performed using C₁₈ cartridges and HPLC, analysis was performed on a 15 cm Hypersil BDS column using isocratic elution with 13% acetonitrile and aqueous solution of 1% (w/v) acetic acid. The lower limit of quantification for pancratistatin in 5% DMF–95% water was found to be 0.58 ng/ml (±10.58%) and 2.3 ng/ml (±9.2%) following extraction from human plasma. Mean recovery of 89.4% (±4.73%) was obtained over the concentration range 0.0023–9.45 μg/ml for a five day validation study. Pancratistatin was stable at room temperature in light or dark for at least 15 days, in the refrigerator at 4°C for at least 16 days and in the freezer at −20°C or −80°C for at least 28 days. Under all conditions monitored, % recovery of pancratistatin from human plasma was greater than 95% and no evidence of degradation had occurred. There also was no loss of pancratistatin after three cycles of freezing and thawing.     

Chromatographic and immunochemical approaches to the analysis of the HIV protease inhibitor saquinavir in plasma

The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.     

Measurement of Plasma Digoxin Concentration by Radioimmunoassay

A rapid, sensitive, and precise method for measuring the plasma digoxin concentration has been developed with the radioimmunoassay technique. Seventy patients receiving digoxin were shown to have plasma digoxin concentrations between 0·4 and 5 ng./ml. Preliminary studies show that though there is a positive correlation between total daily dose and the plasma digoxin concentration, the relationship is not close, and a relatively wide range of plasma digoxin concentrations appear to be consistent with effective digitalization.     

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 μg/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.     

An Improved Diamond Knife Holder for Ultramicrotomy

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

Direct radioimmunoassay of progesterone in plasma of farm animals.

A radioimmunoassay technique has been developed for measuring progesterone directly, without extraction, in 20-mul-aliquots of plasma. The assay was performed in disposable polystyrene test-tubes. Working range of the calibration curve was 0.3 to 10 ng/ml, the precision rangin from 30 to 6%, respectively. The results obtained by the presented method are fully comparable to those reported by others. This method has been applied to farm animals, but is also applicable to other species whose plasma has a low affinity for progesterone.     

A redetermination of the concentration of alpha 2-macroglobulin in human plasma

The concentration of alpha 2-macroglobulin in human plasma has been remeasured utilizing a carefully isolated and characterized sample of alpha 2-macroglobulin as a standard. A highly purified sample of alpha 2-macroglobulin with a total trypsin binding capacity of 1.7 mol trypsin/mol alpha 2-macroglobulin was used as a standard for both a radial immunodiffusion and a rocket immunoelectrophoresis technique. With this preparation as a standard, the concentration of alpha 2-macroglobulin in a normal plasma pool over 10,000 donors was found to be about 1.2 mg/ml. A similar concentration (1.3 mg/ml) was found when using a functional trypsin binding assay. This concentration is considerably less than the usually accepted mean of the normal range for alpha 2-macroglobulin.     

Clonal monosomy of chromosome 21 in a case of myelodysplastic syndrome

This study reports on a cytogenetic finding in a bone marrow examination of a 47-year-old male patient treated in the Hematology and Blood Transfusion Service of the Hospital de Base in S?o José do Rio Preto, S?o Paulo State, Brazil. The only alteration found at diagnosis of myelodysplastic syndrome (MDS) subtype refractory anemia with excess blasts (RAEB-2) was clonal monosomy of chromosome 21. The patient evolved to acute myeloid leukemia type M2 and died nine months after diagnosis. Clonal monosomy of chromosome 21, as the only cytogenetic abnormality in MDS, has only been reported three times previously. This uncommon cytogenetic abnormality in MDS has been associated with a poor clinical course, although more data will be needed to determine if this prognosis is invariable.     

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane—2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5–100 ng/ml and 25–1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.     

A Modified Hypotonic Treatment for Increasing the Frequency and Quality of Meiotic Metaphases from Spermatocytes of the Syrian Hamster

A modified hypotonic treatment for the spermatocytes of the Syrian hamster (Mesocricetm auratus) has been developed using a micro-osmometer to determine optimum conditions for the frequency and quality of Metaphase I and II figures. A suspension of germinal cells from the tubules is made in 3.2% sodium citrate solution and allowed to stand for 5 minutes. Water is then added until the concentration of the sodium citrate is reduced to 1.4%. This is followed after 15 minutes by fixation and air drying. The number and quality of the metaphase figures is enhanced considerably by this technique. A comparison is made of blood, plasma and testicular osmolality in several animal species and corresponding values for various solutions used for cytogenetic techniques.     

Characterization of cholinesterases in plasma of three Portuguese native bird species: application to biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.