Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid–liquid extraction, and analyzed by LC–ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.     

Gas chromatography of retinol and α-tocopherol without derivatization

An improved gas chromatographic method for the analysis of retinol and α-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D₂, D₃, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identifies were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.     

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap-tandem mass spectrometry coupled with electro spray ionization (LC–ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C₁₈ ACE^® column (50.0 mm × 2.1 mm, 5 μm, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3 mL/min. The transition monitored (positive mode) was m/z 279.1 → m/z 208.1 for triprolidine, m/z 165.9 → m/z 148.0 for pseudoephedrine and m/z 172.0 → m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0 min and a linear calibration curves ranged from 0.2 to 20.0 ng/mL for triprolidine and 5.0–500.0 ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3–106.3% and 1.0–9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.     

Simultaneous determination of eight β-lactam antibiotics in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of eight β-lactam antibiotics, including ampicillin, cefazolin, cefepime, cefmetazole, cefotaxime, doripenem, meropenem, and piperacillin, in human serum. Sample specimens were subjected to solid phase extraction (SPE) using Waters Oasis^® HLB cartridges (30 mg). Chromatographic separation was performed with a high-resolution octadecyl silica column compatible with hydrophilic compounds, using a gradient of 10 mM aqueous ammonium formate containing 0.1% formic acid-methanol. Antibiotics were detected by a triple quadrupole mass spectrometer (MS/MS) with electrospray ionization and quantified by the multiple reaction monitoring mode. A total run time of 13 min was applied. Linearity in the calibration was obtained over a range of 0.1–50 μg/mL of the β-lactam antibiotics, except for doripenem. The lower limit of quantification was 0.005–0.5 μg/mL, using 50 μL serum. The recovery rate exceeded 80.2% for these analytes, except for doripenem (49.1%) and meropenem (62.3%). The present method is applicable to routine therapeutic monitoring of β-lactam antibiotics in clinical practice.     

Evaluation of uncertainty of measurement from method validation data: An application to the simultaneous determination of retinol and α-tocopherol in human serum by HPLC

An isocratic RP-HPLC method for the determination of retinol and α-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, α-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, α-tocopherol 0.95 mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and α-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; α-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.     

Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography–coulometric-array detection

The paper describes the computer aided method development and validation for the determination of hydroquinone in human urine from a clinical study on renal excretion of hydroquinone metabolites and the release of free hydroquinone in the urinary tract in order to evaluate the proposed urine disinfecting concept. The presented method uses high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C₁₈, 250×4.6 mm). Selective and sensitive determination (LOQ=12.5 ng on-column) of the target compound was achieved by electrochemical array detection (CoulArray). Gradient and parameter optimization were supported by DryLab software in order to minimize efforts of the expensive and time-consuming method development. Specificity and selectivity were carried out by separation experiments involving the prodrug arbutin and the metabolites hydroquinone, hydroquinone glucuronide, and hydroquinone sulfate, respectively. Hydroquinone glucuronide reference standard was obtained from in vitro glucuronidation in a rat liver microsomes assay. The method was validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998.     

Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).     

Simultaneous determination of clarithromycin,rifampicin and their main metabolites in human plasma by liquid chromatography–tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the “Guidance for Industry – Bioanalytical Method Validation” of the FDA. The calibration curves were linear in the range of 0.10–10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20–5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to ?10%. The components in human plasma are stable after freeze–thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC–MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Analysis of naltrexone and its metabolite 6-ß-naltrexol in serum with high-performance liquid chromatography

ABSTRACT: BACKGROUND: Naltrexone has been proven to be an effective treatment option for the treatment of alcohol dependency. In this article we introduce a reliable and simple method developed for the simultaneous determination of naltrexone and 6-beta-naltrexol in human serum by using high-performance liquid chromatography (HPLC). FINDINGS: Liquid-liquid extraction with butyl acetate from basic solutions (pH 9) was chosen for extraction with nalorphine as an internal standard (IS). Analytes were back-extracted from organic solvent into perchloric acid. The acid extract was chromatographed by HPLC with a reverse-phase ODS-column and electrochemical detector. The mobile phase was a NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium hydrogen sulphate as ion-pair reagents. The recovery of the extraction method was 48 % for naltrexone and 75 % for 6-beta-naltrexol. The limit of quantification was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-beta-naltrexol. The analysed concentrations of naltrexone differed from the theoretic concentrations by 0.7 to 2.3 % and those of 6-beta-naltrexol by 2.6 %. The relative standard deviation of within-day assay was from 0.9 to 5.7 % for naltrexone and from 0.8 to 4.2 % for 6-beta-naltrexol; for the between-day assay it was 5.7 % and 4.2 %, respectively. CONCLUSIONS: Our results indicate that the developed method is suitable for determination of naltrexone and 6-beta-naltrexol in human serum.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μM for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μM range for GSH and 0.05–20 μM for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.     

Molecularly imprinted polymer for analysis of zidovudine and stavudine in human serum by liquid chromatography–mass spectrometry

A molecularly imprinted polymer (MIP) using zidovudine (AZT) as template and methacrylic acid as monomer was prepared. The synthesis of the MIP was performed in acetonitrile. The synthesized material was then tested for the solid-phase extraction of AZT from different media (pure organic solvents and hydro-organic mixtures). An optimised procedure was developed for the selective extraction of AZT with a recovery of 96% using the MIP and only 3% on a non-imprinted polymer used as control polymer. A specific capacity of 0.2 μmol g^?1 was determined. The specificity of the MIP was evaluated by studying the retention behaviour of two others nucleoside analogues. The feasibility of the MIP to selectively extract AZT and stavudine (d4T) from human serum was also demonstrated with recoveries of 80 and 85% respectively. The lower limit of quantification (LLOQ) and the lower limits of detection (LLOD) for AZT were 5.10^?7 and 10^?7 M respectively.     

Quantification of gentamicin in Mueller–Hinton agar by high-performance liquid chromatography

The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (≥79%), linearity (r²≥0.997) and sensitivity (1 μg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers’ products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism.