Liquid chromatographic analysis of the cis(Z)- and trans(E)-isomers of clopenthixol in human plasma using a novel solid phase extraction procedure

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in human plasma has been developed. The chromatographic analysis was carried out isocratically on a reversed-phase column (C(8) 150 x 4.6 mm I.D., 5 microm) using a mixture of 25 mM phosphate buffer and acetonitrile (65:35 v/v, pH* 3.0) as the mobile phase, and ultraviolet detection at 230 nm. Plasma sample pretreatment was accomplished by means of an original solid-phase extraction (SPE) procedure carried out on cyanopropyl cartridges, with a high extraction yield and good selectivity. Under the optimum conditions, calibration graphs of spiked human plasma samples were obtained over the concentration ranges 1-300 ng ml(-1) for cis(Z)-clopenthixol and 1-200 ng ml(-1) for trans(E)-clopenthixol. The limit of detection (LOD) was 0.3 ng ml(-1) for both cis(Z)- and trans(E)-isomers of clopenthixol. The method was successfully applied to the determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in plasma samples of schizophrenic patients undergoing therapy with zuclopenthixol.     

A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.     

A sensitive and specific radioimmunoassay for cis (Z)-flupenthixol in human serum

The injection of cis (Z)-7-carboxy-flupenthixol covalently bound to bovine serum albumin into a sheep has produced an antibody serum which can be used in a very high dilution. This antibody serum and tritium labelled cis (Z)-flupenthixol of high specific activity have been used for a radioimmunoassay for cis (Z)-flupenthixol. The radioimmunoassay has a limit of sensitivity of about 0.3 ng/ml serum or plasma. Some cross-reactivity was seen with trans(E)-flupenthixol, flupenthixol metabolites and cis (Z)-flupenthixol esters, but this did not influence the estimation of cis (Z)-flupenthixol to any significant degree. A series of drugs structurally related to flupenthixol showed varying degrees of cross-reactivity depending on the closeness of their relationship to flupenthixol. Compounds of other therapeutic groups, which structurally are un-related to flupenthixol, showed very little cross-reactivity. The standard deviation was 6–7 per cent when a series of identical samples of pooled patient sera was estimated. Oral administration of cis (Z)-flupenthixol to human volunteers indicates a rather slow absorption with maximum serum level 6 hours after administration. Intramuscular injection of flupenthixol decanoate in Viscoleo to patients gives serum concentration curves, which show maxima 7 days after injection and a factor of two fluctuation in serum level in a two weeks dosage interval.     

Isolation of 19,20-dehydroprostaglandins E1 and E2 in human seminal fluid and further studies on 18,19-dehydroprostaglandins E1 and E2

Human seminal fluid was recently found to contain 18,19-dehydroprostaglandins E1 and E2 (E. H. Oliw, H. Sprecher, and M. Hamberg, (1986) J. Biol. Chem. 261, 2675-2683). In the present study, the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2 were prepared by incubation of microsomes of ram vesicular glands and glutathione with the precursor fatty acids, 8(Z),11(Z),14(Z),18(E/Z)-eicosatetraenoic acids, and 5(Z),8(Z),11(Z),14(Z),18(E/Z)-eicosapentaenoic acids, and used as references to characterize the 18,19-dehydroprostaglandins of human seminal fluid. Based on separation by reversed-phase high-performance liquid chromatography, capillary gas chromatography-mass spectrometry, and ozonolysis of the (-)-menthoxycarbonyl derivatives and on comparison with the authentic compounds, human seminal fluid was found to contain both the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2. Furthermore, human seminal fluid contained two related compounds, viz. 19,20-dehydroprostaglandins E1 and E2. The structures of these compounds were established by conversion into the corresponding prostaglandin B compounds, by mass spectrometric analysis and by chemical degradation by oxidative ozonolysis, which afforded, inter alia, 2(S)-hydroxy-adipic acid.     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

Stereo-isomeric dissociation of the antibacterial and the neuroleptic effect of clopenthixol

Measurement of the IC50 of cis(Z)-clopenthixol and trans(E)-clopenthixol on 188 bacterial strains from human clinical specimens shows that the antibacterial activity of clopenthixol is exerted by both isomerical components and trans(E)-clopenthixol is the most active antibiotic of the two drugs against the sensitive strains. It is known that the trans(E)-clopenthixol isomere is without neuroleptic effect. The possibility of creating new antibiotics by e.g. steric alteration of neuroleptical agents is stressed. These drugs have different antibiotic patterns from those of classical antibiotics. It seems particularly promising that Pseudomonas aeruginosa is sensitive to these drugs.     

Determination of clarithromycin in human serum by high-performance liquid chromatography after pre-column derivatization with 9-fluorenylmethyl chloroformate: application to a bioequivalence study

A sensitive liquid chromatographic method for the analysis of clarithromycin, a macrolide antibiotic, in human serum using pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) is described. The method involved liquid-liquid extraction of the drug and an internal standard (amantadine) followed by pre-column derivatization of the analytes with FMOC-Cl. A mixture of 0.05 M phosphate buffer containing triethylamine (2 mL L(-1); pH 3.8) and methanol (17:83, v/v) was used as mobile phase and chromatographic separation was achieved on a Shimpack CLC-ODS column. The eluate was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The analytical method was linear over the concentration range of 0.025-10 microg mL(-1) of clarithromycin in human serum with a limit of quantification of 0.025 microg mL(-1). The assay is sensitive enough to measure drug levels obtained in human single dose studies. In the present method, sensitivity and run time of analysis have been improved, and successfully applied in a bioequivalence study of three different clarithromycin preparations in 12 healthy volunteers.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

A capillary gas chromatography-selected ion monitoring mass spectrometry method for the analysis of atractylenolide I in rat plasma and tissues, and application in a pharmacokinetic study

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC-MS method. All bio-samples were cleared up with a liquid-liquid extraction procedure. The calibration curves were linear within a range of 5-1000 ng/mL for plasma samples, 0.06-16.00 microg/g for cerebellum samples, and 0.03-8.00 microg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N>micro=10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: Tmax=0.37+/-0.19 h, Cmax=0.26+/-0.05 microg/mL, AUC=1.95+/-0.30 microgh/mL and ka=10.08+/-5.60 h(-1). The tissue distribution of AO-I in rats after the oral administration of 50.0mg/kg was from 0.225 to 0.031microg/g with a decreasing tendency in different tissues like liver>kidney>spleen>cerebellum>heart>cerebrum>lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8+/-3.9, 90.6+/-3.1 and 60.9+/-5.1%, respectively.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether–n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1-20 microg mL(-1) for MTU and 0.1-10 microg mL(-1) for PTU, the detection limits were 0.03 microg mL(-1) for PTU, 0.03 microg mL(-1) for MTU and the quantification limits were 0.1 microg mL(-1) for PTU, 0.1 microg mL(-1) for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.     

Determination of valproic acid in human serum and pharmaceutical preparations by headspace liquid-phase microextraction gas chromatography-flame ionization detection without prior derivatization

An efficient and fast extraction technique for the enrichment of valproic acid from human blood serum samples using the headspace liquid phase microextraction (HS-LPME) combined with gas chromatography (GC) analysis has been developed. The extraction was conducted by suspending a 2 microL drop of organic solvent in a 1 mL serum sample; following 20 min of extraction, withdrawing organic solvent into a syringe and injection into a GC with a flame ionization detector (FID), without any further pre-treatment. Four organic solvents, 1-decanole, benzyl alcohol, 1-octanol and n-dodecane, were studied as extractants, and n-dodecane was found to be the most sensitive solvent for valproic acid. The results revealed that HS-LPME is suitable for the successful extraction of valproic acid from human blood serum samples. Parameters like extraction time, ionic strength, pH, organic solvent volume, and temperature of the sample were studied and optimized to obtain the best extraction results. An enrichment factor of 27-fold was achieved in 20 min. The procedure resulted in a relative standard deviation of <13.2% (n=7) and a linear calibration range from 2 to 20 microg mL(-1) (r>0.98), and the limit of detection was 0.8 microg mL(-1) in serum blank samples. Overall, LPME proved to be a fast, sensitive and simple tool for the preconcentration of valproic acid from real samples. The proposed method was also applied to the analysis of valproate in pharmaceutical preparations.     

Fluorimetric study of the selenium course in the dam-calf relationship

This study was conducted in a southeast region of Romania, known to be selenium (Se) deficient. The fluorimetric method for the determination of Se in biological samples was used in a study on the serum Se content in time in a group of 10 cows with a high milk yield and in their calves. The same cows were sampled antepartum (late pregnancy) and postpartum (1, 7, 21, 30, and 60 d), and the calves were sampled on d 1 of life and 7, 21, 30, and 60 d postcalving. Colostrum and milk samples were also collected and analyzed at the same times. Throughout the study, the serum Se content in both the cows and the calves was below the reference values (0.040–0.100 μg/mL), except the control conducted 7 d postpartum, for which it was 0.044±0.017 and 0.023±0.007 μg/mL in cows and calves, respectively. The most significant drops were recorded 21 (p<0.01) and 60 d postpartum (p<0.05). The colostrum Se was higher (0.036±0.022 μg/mL), decreasing progressively throughout the study to the normal low limit (0.005 μg/mL). The milk Se concentration was lower by approx 78% than the colostrum one on d 1 postcalving. These data demonstrate the course of hyposelenosis both in the cows and their calves. The maternal body proved to “mobilize,” even in case of deficiency, important Se amounts in the colostrum, during the first days in particular.     

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.     

Direct injection of human serum and pharmaceutical formulations for glucosamine determination by CE-C(4)D method

A simple CE-C(4)D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C(4)D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% (n = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10-2.50mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5-104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences.     

Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry

A selective and sensitive method for analysis of perfluorooctanoic acid (PFOA) in human serum and plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and thoroughly validated to satisfy strict FDA guidelines for bioanalytical methods. A simple, automated sample preparation procedure, involving extraction of the target analyte with acetonitrile on protein precipitation media in a 96-well plate format was developed, allowing efficient handling of large numbers of samples. The proposed method uses the calibration standards prepared in a surrogate matrix (rabbit serum or plasma) and (13)C-labeled PFOA as the internal standard to account for matrix effects, instrument drift, and extraction efficiency. Human serum and plasma could not be used for matrix matching of calibration standards as endogenous levels of PFOA observed in the control human serum and plasma significantly exceeded the targeted lower limit of quantitation (LLOQ) of the method. Precision and accuracy of the method were demonstrated by analysis of rabbit serum and plasma control samples fortified at 0.5, 5, and 40 ng/mL PFOA and human serum and plasma fortified at 1.0, 5.0, 40 ng/mL PFOA. The LLOQ of 0.5 ng/mL PFOA was experimentally demonstrated for rabbit and human serum and plasma. Within-day precision and accuracy, short-term stability, freeze-thaw stability, equivalence of response between PFOA and APFO (the ammonium salt of PFOA), and dilution of concentrated samples were also investigated. The results of the validation experiments comply with the precision and accuracy limits defined by the FDA guidance document: "Guidance for Industry, Bioanalytical Method Validation", May 2001.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).