Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL⁻¹ and 100 ng mL⁻¹. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL⁻¹. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex^? tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day⁻¹), clozapine levels averaged 379 ng mL⁻¹ (ranging from 102 to 818 ng mL⁻¹) and DMC levels averaged 233 ng mL⁻¹ (ranging from 70 to 540 ng mL⁻¹). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.     

Determination of phenols in waters using micro-pumped multicommutation and spectrophotometric detection: an automated alternative to the standard procedure

An automated and greener spectrophotometric procedure has been developed for the determination of phenol in water at 700 nm. The method uses the reaction between phenol, sodium nitroprusside, and hydroxylamine hydrochloride in a buffered medium at pH 12.3. The flow manifold comprises four solenoid micro-pumps employed for sample and reagent introduction into the reaction coil and to transport the colored product formed to the detector. The linear dynamic range was 50–3,500 ng mL⁻¹ (R = 0.99997; n = 6) and the method provided a limit of detection (3σ) of 13 ng mL⁻¹. The sampling throughput was estimated to be 65 measurements per hour and the coefficient of variation was 0.5% (n = 10) for a 1.0 μg mL⁻¹ phenol concentration. Recoveries of 92–105% were obtained for phenol determination in spiked water samples at concentration levels from 50 to 5,000 ng mL⁻¹. The use of multicommutation reduced the reagent consumption 25-fold, the sample consumption 225-fold, and the waste generation 30-fold compared with the batch procedure. The proposed method is an environmentally friendly alternative to the official 4-aminoantipyrine method since it avoids the use of chloroform.     

Determination of eight penicillins in serum from cattle and pigs by generic HPLC method

Summary An HPLC method was developed for determination of amoxicillin, penicillin G, penicillin V, ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in serum from pigs and cattle. Serum was cleaned up by solid-phase extraction (SPE), ultra-filtered and derivatised. The method was linear in the range tested up to 2000 ng mL⁻¹ of individual penicillins in serum. Limits of detection were 11–14 ng mL⁻¹. Mean recoveries were 90–103% in the range 20–2000 ng mL⁻¹. The relative repeatability, standard deviation was <10% at 20 ng mL⁻¹ level and <6% in the range 100–2000 ng mL⁻¹.     

Fluorescence enhancement of the protein–curcumin–sodium dodecyl benzene sulfonate system and protein determination

Protein can greatly enhance the fluorescence of curcumin (CU) in the presence of sodium dodecyl benzene sulfonate (SDBS). Experiments indicate that under the optimum conditions, the enhanced intensity of fluorescence is proportional to the concentration of proteins in the range of 0.0050–20.0 μg mL⁻¹ for bovine serum albumin (BSA), 0.080–20.0 μg mL⁻¹ for human serum albumin (HSA), and 0.040–28.0 μg mL⁻¹ for egg albumin (EA). Their detection limits (S/N=3) are 1.4 ng mL⁻¹, 20 ng mL⁻¹, and 16 ng mL⁻¹, respectively. The method has been satisfactorily used for the determination of proteins in actual samples. In comparison with most of fluorimetric methods, this method is quick and simple, has high sensitivity and good stability. The interaction mechanism is also studied.     

Analysis of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-methylsuccinimide in plasma

Summary A method for the determination of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in plasma was developed. 5-HNMP and 2-HMSI are metabolites to the widely used organic solvent N-methyl-2pyrrolidone (NMP). The 5-HNMP and 2-HMSI were purified from plasma by C8 solid phase extraction, derivatised by bistrimethylsilyl trifluoroacetamid, and analysed by gas chromatography with mass spectrometric detection. For 5-HNMP, the precision was 2–7 % (120 and 780 ng mL⁻¹) and the detection limit was 6 ng mL⁻¹ (m/z 98). For 2-HMSI, the precision was 2–9 % (160 and 1000 ng mL⁻¹) and the detection limit was 4 ng mL⁻¹ (m/z 144). The method is applicable for analysis of plasma samples from workers exposed to NMP.     

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL⁻¹ with a limit of detection (LOD) of 2 ng mL⁻¹ (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL⁻¹ and the LOD was1 ng mL⁻¹.     

Highly Sensitive and Selective Detection of Creatinine by Combined Use of MISPE and a Complementary MIP-Sensor

Guanidinoacetate methyltransferase deficiency is a recently discovered inborn defect of creatine biosynthesis which reduces serum creatinine concentrations to as low as 0.58 μg mL⁻¹ (or 0.00058 μg mL⁻¹ after 1,000-fold dilution). To measure ultra trace levels of creatinine in diluted samples, molecularly imprinted solid-phase extraction (MISPE) and molecularly imprinted polymer (MIP) sensor techniques have been found to be inadequate. A combination of these techniques (i.e. MISPE hyphenated with use of an MIP-sensor), reported in this paper, has been found to be highly suitable for direct assay of creatinine in highly diluted human blood serum without complicated pretreatment of the sample. The proposed technique has the potential to enhance the sensitivity of creatinine measurement from μg mL⁻¹ to ng mL⁻¹ in highly dilute aqueous samples in which the concentrations of interfering constituents are reduced to negligible levels. In this work the sensitivity to creatinine was found to be improved compared with that of the MIP-sensor method alone (limit of detection, LOD, 0.00149 μg mL⁻¹). After preconcentration by MISPE and use of the sensor the detection limit for creatinine was as low as 0.00003 μg mL⁻¹ (RSD = 0.94%, S/N = 3; 50-fold preconcentration factor) in aqueous samples.     

Simultaneous determination of the advanced glycation end product <Emphasis Type="Italic">N</Emphasis><Superscript>ɛ</Superscript>-carboxymethyllysine and its precursor,lysine, in exhaled breath condensate using isotope-dilution–hydrophilic-interaction liquid chromatography coupled to tandem mass spectrometry

Analysis of biomarkers in exhaled breath condensate (EBC) is a non-invasive method for investigating the effects of different diseases or exposures, on the lungs and airways. N ^ɛ-carboxymethyllysine (CML) is an important biomarker of advanced glycation end products (AGEs). A method has been developed for simultaneous determination of CML and its precursor, the amino acid lysine, in exhaled breath condensate (EBC). After addition of labelled internal standards (d-4-CML; d-4-lysine), the EBC was concentrated by freeze-drying. Separation and detection of the analytes were performed by hydrophilic-ion liquid chromatography coupled with tandem mass-spectrometric detection (HILIC–MS–MS). The limits of quantification were 10 pg mL⁻¹ EBC and 0.5 ng mL⁻¹ EBC for CML and lysine, respectively. The relative standard deviation of the within-series precision was between 2.8 and 7.8% at spiked concentrations between 40 and 200 pg mL⁻¹ for CML and between 6 and 20 ng mL⁻¹ for lysine. Accuracy for the analytes ranged between 89.5 and 133%. The method was used for the analysis of EBC samples from ten healthy persons from the general population and ten persons receiving dialysis. CML and lysine were detected in all EBC samples with median values of 19 pg mL⁻¹ CML and 11.9 ng mL⁻¹ lysine in EBC of healthy persons and 25 pg mL⁻¹ CML and 9.5 ng mL⁻¹ lysine in EBC of dialysis patients.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

Resonance Rayleigh scattering spectra of tetracycline antibiotic–Cu(II)–titan yellow systems and their applications in analytical chemistry

Tetracycline antibiotics (TCs) such as doxycycline (DOTC), chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC) react with Cu(II) in pH 3.5 BR buffer medium to form 1:1 cationic chelates, which further react with titan yellow to form 2:1 ion association complexes. These result in great enhancement of resonance Rayleigh scattering (RRS) and the appearance of new RRS spectra. The ion association complexes of DOTC, CTC, OTC, and TC have similar spectral characteristics and their maximum RRS wavelengths are all located at 464 nm. The quantitative determination ranges and the detection limits (3σ) of the four TCs are 0.037–4.8 μg mL⁻¹ and 11.2 ng mL⁻¹ for DOTC, 0.041–5.2 μg mL⁻¹ and 12.4 ng mL⁻¹ for CTC, 0.050–4.8 μg mL⁻¹ and 15.1 ng mL⁻¹ for TC, and 0.088–5.0 μg mL⁻¹ and 26.3 ng mL⁻¹ for OTC, respectively. The optimum reaction conditions, the effects of foreign substances, the structure of ternary complexes, and the reaction mechanism are discussed. A sensitive, rapid, and simple RRS method for the determination of DOTC has been developed.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.     

Determination of recent antidepressant citalopram in human plasma by liquid chromatography—Fluorescence detection

Summary A reliable and sensitive high-performance liquid chromatographic method for the determination of the recent antidepressant citalopram and two metabolites in human plasma has been developed. Fluorescence detection at 300 nm was used, exciting at 238 nm. Separation was obtained using a reversed-phase column (C18, 250 × 3.0 mm i.d., 5 μm) and a mobile phase. 40% acetonitrile: 60% aqueous tetramethylammonium perchlorate (pH 1.9). Calibration curves were linear over a working range: 5–300 ng mL⁻¹ for citalopram, 2.5–150.0 ng mL⁻¹ for desmethylcitalopram and 2.5–50.0 ng mL⁻¹ for didesmethylcitalopram. The limits of quantitation (LOQ) were 1.5 ng mL⁻¹ for citalopram and desmethylcitalopram and 2.0 ng mL⁻¹ for didesmethylcitalopram. Precision data, as well as accuracy, were satisfactory and no interference from different psychotropic drugs was found. The method was therefore suitable for therapeutic drug monitoring of citalopram and its active metabolites in plasma of depressed patients.     

Analysis of spectinomycin in fermentation broth by reversed-phase chromatography

A pre-column derivatized high-performance liquid chromatographic (HPLC) method with ultraviolet-visible detection was developed to measure the concentrations of spectinomycin in fermentation broth. Derivatization reagents, 2,4-dinitrophenylhydrazine in acetonitrile (5 mg mL⁻¹) and trifluoroacetic acid in acetonitrile (0.8 mol L⁻¹), were added to an aliquot of the fermentation broth, and the mixture was incubated for 60 min at 70°C. The resulting derivative was separated from other compounds by isocratic elution in a reversed-phase column Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm). Mobile phase consisted of acetonitrile, tetrahydrofuran, and water (φ _r = 40: 35: 25) and the flow rate was 1.0 mL min⁻¹. The detection wavelength was 415 nm. The standard curve for spectinomycin sulfate was linear with correlation coefficients of 0.9997 in the range of 25 μg mL⁻¹ to 600 μg mL⁻¹. The relative standard deviation values ranged from 0.43 % to 2.18 % depending on the concentration of samples. The average recovery was 101.5 %. The limit of detection was 50 ng mL⁻¹.     

Determination of risperidone at picogram level in human urine by luminol—H2O2 chemiluminescence

A simple flow-injection chemiluminescence method with synergistic enhancement has been investigated for the rapid and sensitive determination of antipsychotic risperidone. The synergistic action was significant in the chemiluminescence system of luminol—hydrogen peroxide with risperidone as an enhancer. The increased chemiluminescence intensity was correlated with risperidone concentration within the range from 10 pg mL⁻¹ to 1.0 ng mL⁻¹ with relative standard deviations lower than 5.0 % and the detection limit of 4 pg mL⁻¹. At a flow rate of 2.0 mL min⁻¹, the flow-injection chemiluminescence method exhibited both a high sensitivity and excellent selectivity giving a throughput of 120 times per hour. The proposed method was successfully applied to determine the risperidone content in human urine without any pretreatment. It was found that the excretive amounts of risperidone reached their maximum after taking 2.0 mg of risperidone for 1 h, with a total excretive ratio of 17.37 % in 8.5 h.     

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min⁻¹ for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL⁻¹ (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL⁻¹ and limit of detection, 0.02 μg mL⁻¹. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg⁻¹. Maximum plasma concentration (C _max) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL⁻¹ and 2405.28 ng h mL⁻¹.     

Development and Validation of Improved Reversed Phase-HPLC Method for Simultaneous Determination of Curcumin, Demethoxycurcumin and Bis-Demethoxycurcumin

Isocratic reversed phase high performance liquid chromatographic (HPLC) method using RP C18 column was developed for simultaneous determination of the curcuminoids. Mobile phase consisted of acetonitrile:0.1% trifluro-acetic acid (50:50) and flow rate was 1.5 mL min⁻¹ and elution was monitored at 420 nm. Validation in selected conditions showed that the chosen method is sensitive, selective, precise and reproducible with linear response of detector for the simultaneous determination of curcumin (C), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC). The limits of detection were 27.99, 31.91 and 21.81 ng mL⁻¹ for C, DMC and BDMC, respectively. Limits of quantitation for C, DMC and BDMC, were 84.84, 96.72 and 66.10 ng mL⁻¹, respectively. Linear range was form 100 to 600 ng mL⁻¹. The mean ± SD percent recoveries of curcuminoids were 99.87 ± 0.34, 100.09 ± 0.48 and 100.10 ± 0.60% of C, DMC and BDMC, respectively. Further, the method was used for quantitation of curcuminoids from turmeric rhizome.     

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

Determination of Aldehydes and Ketones in Fuel Ethanol by High-Performance Liquid Chromatography with Electrochemical Detection

A new methodology was developed for analysis of aldehydes and ketones in fuel ethanol by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. The electrochemical oxidation of 5-hydroxymethylfurfural, 2-furfuraldehyde, butyraldehyde, acetone and methyl ethyl ketone derivatized with 2,4-dinitrophenylhydrazine (DNPH) at glassy carbon electrode present a well defined wave at +0.94 V; +0.99 V; +1.29 V; +1.15 V and +1.18 V, respectively which are the basis for its determination on electrochemical detector. The carbonyl compounds derivatized were separated by a reverse-phase column under isocratic conditions with a mobile phase containing a binary mixture of methanol / LiClO_4(aq) at a concentration of 1.0 × 10⁻³ mol L⁻¹ (80:20 v/v) and a flow-rate of 1.1mL min⁻¹ . The optimum potential for the electrochemical detection of aldehydes-DNPH and ketones-DNPH was +1.0 V vs. Ag/AgCl. The analytical curve of aldehydes-DNPH and ketones-DNPH presented linearity over the range 5.0 to 400.0 ng mL⁻¹, with detection limits of 1.7 to 2.0 ng mL⁻¹ and quantification limits from 5.0 to 6.2 ng mL⁻¹, using injection volume of 20 μL. The proposed methodology was simple, low time-consuming (15 min/analysis) and presented analytical recovery higher than 95%.     

Determination of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a New Angiotensin Type 1 Receptor Antagonist in Rat Plasma by LC-ESI-MS: Application to Pharmacokinetic Studies

A simple and sensitive reversed-phase LC-ESI-MS method to identify and quantitate 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one (1b), a new Angiotensin II type 1 receptor antagonist in rat plasma has been developed and validated. Sample preparation used a simple liquid–liquid extraction with ethyl acetate. Separation was achieved by gradient elution on a C₁₈ column. The mobile phase consisted of acetonitrile and water (0.05% triethylamine and 0.05% acetic acid) at a flow rate of 0.2 mL min⁻¹. The detection utilized selected ion monitoring (SIM) in the negative mode at m/z 507.1 and m/z 407.2 for the deprotonated molecular ions of 1b and the internal standard irbesartan, respectively. The lower limit of quantification was reproducible at 5 ng mL⁻¹ with 100 μL of plasma and the good linear was observed in the 5–500 ng mL⁻¹ range. This concentration range corresponded well with the plasma concentrations of 1b in pharmacokinetic studies. Recoveries of 1b in rat plasma were 76.1, 74.6 and 79.0% at 5, 50 and 500 ng mL⁻¹. The RSD of intra-assay and inter-assay variations were all less than 5%. This validated LC-ESI-MS assay is an economic, quick, precise and reliable method for the analysis of 1b in pharmacokinetic studies.