Journal of Chromatography B (v.802, #2)

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News Section (N1-N2).

A novel method for fast determination of fluoroacetamide, a kind of organic fluorine pesticide, in blood and urine samples was developed with acetamide as an internal standard using gas chromatography/mass spectrometry (GC/MS) after solid-phase microextraction (SPME) technique. The SPME was performed by immersing a PDMS fiber of 100 μm coating thickness in a sample solution for 25 min at 70 °C with (CH3CH2)4NBr to improve the extraction efficiency. After a GC sample injection, the extracted fluoroacetamide was desorbed from the fiber for 4 min to perform the GC/MS detection with a HP-PLOT Q capillary column. The analytical conditions were optimized by examining systematically, the effects of experimental parameters on the ratio of characteristic ion peak areas of fluoroacetamide to acetamide. Under optimal conditions, the ratio was proportional to the concentration of fluoroacetamide ranging from 5.0 to 90 μg/ml with a detection limit of 1.0 μg/ml. The average recovery of fluoroacetamide in blood sample was 92.2%. The established method could be used for the fast and convenient measurement of fluoroacetamide in poisoned sample.
Keywords: Fluoroacetamide;

The effect of three storage temperature levels (i.e. +4, −20 and −80 °C) and time intervals from sampling (3, 6 and 9 months) on the degradation of 3,4-dihydroxyphenylglycol (DHPG) and norepinephrine (NE) was investigated in a systematic study. Extracted human plasma samples and acidified standard solutions were stored for long periods (up to 9 months) without the addition of any stabilizing agent. DHPG and NE values, determined using a ion-pair reversed-phase high-performance liquid chromatography method with electrochemical detection of coulometric type (IP-RP-HPLC–CD), remained constant over time in those plasma samples and standard solutions that had been stored at the lowest storing temperature (i.e. −80 °C). The expected degradation was observed at higher temperature levels. Plasma and standard DHPG degradation can, therefore, be prevented by storing samples at a lower temperature than previously suggested with no need to add any stabilizing agent.
Keywords: Long-term stability; 3,4-Dihydroxyphenylglycol;

Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography–electrospray ionization mass spectrometry by Zhiling Zhou; Xin Li; Kunyan Li; Zhihong Xie; Zeneng Cheng; Wenxin Peng; Feng Wang; Ronghua Zhu; Huande Li (257-262).
Clozapine (CLZ), olanzapine (OLZ), risperidone (RIP) and quetiapine (QTP) have been widely used in the treatment of schizophrenia. However, no study (or little study) has been conducted to determine the four drugs simultaneously by the use of high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–MS/ESI). Objective: To develop a sensitive method for simultaneous determination of CLZ, OLZ, RIP and QTP in human plasma by HPLC–MS/ESI. Methods: The analytes were extracted twice by ether after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C18 (2.0  mm×125  mm, 3 μm, Germany) column, using water (formic acid: 2.70 mmol/l, ammonium acetate: 10 mmol/l)–acetonitrile (53:47) as mobile phase, with a flow-rate of 0.16 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. Results: The calibration curves were linear in the ranges of 20–1000 ng/ml for CLZ and QTP, 1–50 ng/ml for OLZ and RIP, respectively. The average extraction recoveries for all the four analysts were at least above 80%. The methodology recoveries were higher than 91% for the analysts. The intra- and inter-day R.S.D. were less than 15%. Conclusion: The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) and for the study of the pharmacokinetics of the four drugs.
Keywords: Clozapine; Olanzapine; Risperidone; Quetiapine;

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in human plasma. An Oasis® HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 μm, 120 Å, 2.0  mm×150  mm) column. The initial composition of the mobile phase was 2% methanol/98% 5 mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3 min, followed by a linear gradient over 4 min to 30% methanol/70% 5 mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5′-deoxy-5-fluorouridine (5′-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC–H], m/z 263 for [dFdU–H], and m/z 245 for [5′-DFUR–H]. The average recoveries for dFdC, dFdU, and 5′-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5–1000 ng/ml for dFdC, and 5–5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were ≤3 and ≤7% at three concentration levels (50.0, 500, and 5000 ng/ml). The limits of quantitation (defined as 10 times of signal-to-noise ratio) were 3.16 ng/ml for dFdC, and 1.35 ng/ml for dFdU with 50-μl sample injections. This method has been used for measuring plasma concentrations of dFdC and dFdU in samples from adult cancer patients in a Phase I trial of weekly dFdC given as 150 (or lower) mg/(m2 24-h) infusion. The average plasma dFdC concentrations at 22- and 23-h into the infusion were 18.3 and 16.8 ng/ml at 150 and 100 mg/m2, respectively; the values for dFdU averaged 2950 and 1372 ng/ml.
Keywords: Gemcitabine; 2′,2′-Difluoro-2′-deoxyuridine;

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs—a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10–500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27–7.45 and 0.79–6.12%, respectively. The between-batch and within-batch bias was 0.74–7.40 and −0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.
Keywords: Valdecoxib;

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of paeoniflorin in rat hippocampus was developed in this study. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Zorbax SB-C18 column, a mobile phase of methanol–water (32:68, v/v), and detection by ultraviolet (UV) absorption at 233 nm. The lower limits of quantitation (LLQ) were 1 μg/ml for paeoniflorin. The calibration curve for paeoniflorin was linear (r=0.9999) over the concentration range of 1–50 μg/ml. The coefficients of variation of intra- and inter-day assays were 7.00, 0.58, 1.46% and 5.48, 1.79, 1.70% at concentrations of 1, 10, 50 μg/ml, respectively. The recoveries of paeoniflorin from rat hippocampus were 98.28±2.14, 98.96±1.48, and 95.34±0.92% at concentrations of 1, 10 and 50 μg/ml, respectively. Stability studies showed that paeoniflorin was stable at temperatures of 2–8 °C in methanol for at least 20 days. The method was applied to determine the time course of paeoniflorin in rat hippocampus, following the administration of a 60 mg/kg i.v. dose of paeoniflorin in Paeoniae Radix extract to a male Wistar rat.
Keywords: Paeoniflorin;

Glycerophosphoinositol (GroPIns) has been demonstrated to have important roles in many intracellular regulatory processes. GroPIns has been analysed for many years by anion-exchange HPLC after radiolabelling of cells in culture, but no method has been developed, to our knowledge, for the direct detection and quantitation of the unlabelled compound in such biological samples. Here is reported a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the direct quantitative analysis of GroPIns that can indeed be applied to cell extracts. Analyses were performed on a β-cyclodextrin-bonded HPLC column using a binary mobile phase of acetonitrile and 20 mM ammonium formate in water, which allowed direct on-line detection by tandem mass spectrometry in negative electrospray ionisation (ESI) mode. The method was applied to the quantitative analysis of GroPIns in selected rat cell lines after a two-phase acid extraction of cultured cells using external calibration. The potential matrix signal suppression effects were investigated by the parallel quantitation of GroPIns in extracts of selected cultured cell lines with both external calibration and the standard additions method. The accuracy data obtained demonstrated the feasibility of external calibration, so allowing a simpler and less time-consuming approach than that of the standard additions method.
Keywords: Glycerophosphoinositol; Cultured cells;

A sensitive and highly selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to determine nimodipine in human plasma. The analyte and internal standard nitrendipine were extracted from plasma samples by n-hexane−dichloromethane−isopropanol (300:150:4, v/v/v), and chromatographed on a C18 column. The mobile phase consisted of methanol−water−formic acid (80:20:1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24−80 ng/ml. The intra- and inter-day precisions were lower than 4.4% in terms of relative standard deviation (R.S.D.), and the accuracy ranged from 0.0 to 5.8% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetic profiles of nimodipine tablets administered to 18 healthy volunteers.
Keywords: Nimodipine;

Simultaneous determination of levodopa and 3-O-methyldopa in human plasma by liquid chromatography with electrochemical detection by Christoph Saxer; Miyuki Niina; Akinori Nakashima; Yusuke Nagae; Naoki Masuda (299-305).
A simple and rapid assay is described for the simultaneous analysis of levodopa (l-DOPA) and 3-O-methyldopa (3-OMD) in human plasma samples, applying an ion-pair reversed-phase liquid chromatographic method with electrochemical detection, designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of l-DOPA. After protein precipitation of 100 μl plasma sample aliquots with perchloric acid, the analytes are directly injected, separated within 10 min and simultaneously quantified down to 20 ng/ml by an electrochemical detector equipped with a dual-electrode system operating in redox mode eliminating effectively potential endogenous and exogenous interferences. The intra-assay precision for l-DOPA and 3-OMD was 1.34–6.54 and 3.90–5.50%, whereas the inter-assay precision was 2.09–7.69 and 4.16–9.90%, respectively. The recoveries were close to 90% for l-DOPA and almost 100% for 3-OMD. Satisfactory storage stability was achieved for up to 16 weeks at −70 °C by stabilizing plasma samples with antioxidants.
Keywords: Levodopa; 3-O-Methyldopa;

A method for the quantitation of pg/ml levels of 17β-estradiol and 17β-trenbolone in bovine serum by gas chromatography/electron-capture mass spectrometry has been developed and validated. Using the area ratios of the integrated molecular-ion peaks of the analytes to their corresponding deuterated internal standards, [2,4,16,16- 2 H 4 ] 17β-estradiol (17β-estradiol-d4) and [16,16- 2 H 2 ] 17β-trenbolone (17β-trenbolone-d2), and non-weighted linear regression, two calibration curves per analyte; 5–50 and 50–500 pg/ml for 17β-estradiol in sera, and 25–250 and 250–2500 pg/ml for 17β-trenbolone in sera, respectively, were constructed. Splitless injection of 200 fg 17β-estradiol and 1000 fg 17β-trenbolone could be detected and quantified. Tested batches of control bovine sera did not exhibit interference for 17β-trenbolone, and showed expected background presence of endogenous 17β-estradiol. Intra-day residual errors did not exceed 20%, and regression correlations were greater than 0.99. Intra-day precision data was similar to inter-day precision data. Using this method, 16 samples can be processed within one working day.
Keywords: 17β-Estradiol; 17β-Trenbolone;

Dextromethorphan, the innocuous non-narcotic antitussive agent, is the most widely used probe drug to assess CYP2D6 function both in vivo and in vitro. For this reason a simple and selective high performance liquid chromatography method with fluorimetric detection for simultaneous quantitation of dextromethorphan, and its main metabolites in human plasma was developed and validated. The method involved a simple and rapid protein precipitation protocol, using a mixture of ZnSO4 and methanol. The analysis was performed on a 3 μm, C18 Tracer Excel 15  cm×0.4  cm i.d. column by gradient elution in which Mobile phase A consisted of potassium dihydrogen phosphate buffer (pH=3, 0.01 M):methanol:tetrahydrofuran (68.5:31:0.5), and mobile phase B consisted of methanol:tetrahydrofuran (93.25:6.75). Linear calibration curves were obtained in the range of 10–500 ng/ml for dextromethorphan, dextrorphan and hydroxymorphinan. The limit of quantitation (LOQ) was 10 ng/ml for each compound. The maximum within and between days precisions were 7.4 and 7.8%, respectively. The accuracies at four different concentration levels ranged from 88.2 to 111.5%. The recoveries were between 88.0 and 108.6%. The assay method was successfully applied to determine dextromethorphan metabolic ratio after an oral dose of 30 mg of dextromethorphan hydrobromide.
Keywords: Dextromethorphan; Dextrorphan; Hydroxymorphinan;

Determination of nicotine and cotinine in tobacco harvesters’ urine by solid-phase extraction and liquid chromatography by P.B. Doctor; V.N. Gokani; P.K. Kulkarni; J.R. Parikh; H.N. Saiyed (323-328).
A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5–10 μg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79–97% with average value of 85% for nicotine and in the range of 82–98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.
Keywords: Nicotine; Cotinine;

Glucocorticoids are an important component of immunosuppressive therapy for solid organ transplantation. A method to quantitate prednisone, prednisolone, dexamethasone and cortisol in human serum has been developed. Analysis is performed utilizing reversed-phase liquid chromatography coupled to tandem mass spectrometry. The method was validated to a lower limit of quantitation of 5.4 ng/ml for prednisone and cortisol, and 10.7 ng/ml for dexamethasone and prednisolone, with error below 7% at the lower limits. The between-day relative standard deviations ranged 2.9–7.1%. Comparison of cortisol analysis to an established method using clinical samples yielded differences below 15% for 26 of 28 determinations.
Keywords: Prednisone; Prednisolone; Dexamethasone; Cortisol;

Midazolam is a widely accepted probe for phenotyping cytochrome P4503A. A gas chromatography–mass spectrometry (GC–MS)–negative chemical ionization method is presented which allows measuring very low levels of midazolam (MID), 1-OH midazolam (1OHMID) and 4-OH midazolam (4OHMID), in plasma, after derivatization with the reagent N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The standard curves were linear over a working range of 20 pg/ml to 5 ng/ml for the three compounds, with the mean coefficients of correlation of the calibration curves (n=6) being 0.999 for MID and 1OHMID, and 1.0 for 4OHMID. The mean recoveries measured at 100 pg/ml, 500 pg/ml, and 2 ng/ml, ranged from 76 to 87% for MID, from 76 to 99% for 1OHMID, from 68 to 84% for 4OHMID, and from 82 to 109% for N-ethyloxazepam (internal standard). Intra- (n=7) and inter-day (n=8) coefficients of variation determined at three concentrations ranged from 1 to 8% for MID, from 2 to 13% for 1OHMID and from 1 to 14% for 4OHMID. The percent theoretical concentrations (accuracy) were within ±8% for MID and 1OHMID, within ±9% for 4OHMID at 500 pg/ml and 2 ng/ml, and within ±28% for 4OHMID at 100 pg/ml. The limits of quantitation were found to be 10 pg/ml for the three compounds. This method can be used for phenotyping cytochrome P4503A in humans following the administration of a very low oral dose of midazolam (75 μg), without central nervous system side-effects.
Keywords: Midazolam; Hydroxymidazolam;

In this paper, the Langmuir–Freundlich isotherm (LF) is used to characterise a propazine-imprinted polymer obtained by precipitation polymerisation (MIP-P). Different rebinding studies were carried out allowing to explain the different interactions taking place between the molecularly imprinted polymer and six triazinic herbicides (desisopropylatrazine, desethylatrazine, simazine, atrazine, propazine and prometryn). The LF fitting parameters obtained (total number of binding sites, heterogeneity index and mean binding affinity) were compared to those obtained in a previous work for a propazine-imprinted polymer prepared by bulk polymerisation (MIP-B). From that study, it was concluded that precipitation polymerisation yielded polymers with a more homogeneous binding site distribution and higher affinity constants.
Keywords: Precipitation polymerisation; Quality assessment; Molecular imprinting; Propazine;

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C18 bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 μg/ml bivalirudin in plasma, with a detection limit of 1 μg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).
Keywords: Derivatization, LC; Bivalirudin;

An accurate and precise method was developed for the detection and quantification of 3-bromopropionic acid (3-BPA), a metabolite and biomarker for exposure to 1-bromopropane (1-BP). 1-BP is used as an industrial solvent and exposure is a health concern for industrial workers due to its toxicity. It has been associated with neurological disorders in both animals and humans. Urine sample preparation for the determination of 3-BPA consisted of liquid–liquid extraction (LLE) with ethyl acetate and silylation with N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide (MTBSTFA). Quantification was by means of a gas chromatograph (GC) equipped with a mass selective detector (MSD) using a dimethylpolysiloxane (HP-1) capillary column and 3-chloropropionic acid was used as an internal standard in the procedure. Demonstrated accuracy and precision during this method’s validation was good; recovery varied between 93 and 98% with relative standard deviations (R.D.S.) of 5.7% or less. The limit of detection (LOD) for the procedure was approximately 0.01 μg/ml 3-BPA in urine. These data and other factors of the development and validation of this test method will be discussed.
Keywords: 3-Bromopropionic acid; 1-Bromopropane;

Due to the absence of HPLC methods to determine myo-inositol using mass detection and considering its sensitivity and selectivity, a high performance liquid chromatography-mass spectrometry method for the analysis of myo-inositol is described and applied to its direct determination in urine and saliva samples. Successful resolution of myo-inositol and its related substances was achieved with a stationary phase Aminex HPX-87C Column with milli-Q water as mobile phase and 5 mM ammonium acetate added post-column. The detector counted positive ions by monitoring m/z=198, which corresponds to the myo-inositol adduct with ammonium cation. Urine and saliva samples were previously purified by passing through an anion-exchange resin. Concentrations as low as 138 and 461 μg/l in saliva and urine could be respectively quantified. Intra-day R.S.D. ranged from 0.83 to 1.02%, whereas inter-day R.S.D. was between 1.54 and 3.58%.
Keywords: Myo-inositol;

A new rapid and sensitive electron ionization gas chromatography–mass spectrometry method in selective ion monitoring mode (SIM) was developed for the determination of l3 synthetic pyrethroid insecticide molecules and their stereo isomers in whole blood. The pyrethroid insecticides investigated are allethrin, bifenthrin, cypermethrin, cyphonothrin, cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, fenpropathrin, imiprothrin, permethrin, prallethrin and transfluthrin. The residues of pyrethroids are extracted from the whole blood using hexane and acetone mixture (80+20%) as solvent. All the pyrethroid residues were separated by using a gas chromatography–mass spectrometry operated in electron ionization mode and quantified in selective ion monitoring mode. The method can detect the residues of different pyrethroids down to the level 0.05–2 ng/ml. Recovery experiments conducted in whole blood samples at the fortification level 1–1000 ng/ml showed 91–103% recovery. The applications of the analytical method for the determination of pyrethroid residues in real samples were tested by analyzing 45 human blood samples collected from the population exposed continuously to different pyrethroid based formulations. The results are confirmed by spiking the known quantity of pyrethroids and subsequently their positive detection.
Keywords: Pyrethroid insecticides;

A rapid, sensitive and specific liquid chromatography−tandem mass spectrometry method is described for quantitation of metformin in human plasma. After a simple, one-step protein precipitation using acetonitrile, metformin and the internal standard diphenhydramine were chromatographed on a C8 column and detected by tandem mass spectrometry. An atmospheric pressure chemical ionization interface was chosen to reduce ion suppression from sample matrix components and provide high sensitivity. The method has a chromatographic total run time of 3.4 min and was linear within the range 2–2000 ng/ml. Intra- and inter-day precision, expressed as the relative standard deviation (R.S.D.), ranged from 4.4 to 5.7% and from 1.3 to 2.8%, respectively. Assay accuracy was less than 1% in terms of %RE (relative error). The assay was used to evaluate the pharmacokinetics of metformin after an oral administration of multicomponent formulation containing 500 mg metformin and 2.5 mg glyburide to 20 healthy volunteers.
Keywords: Metformin;