Journal of Chromatography B (v.867, #2)

A sensitive HPLC–MS–MS method for the determination of raltegravir in human plasma by Mary C. Long; Chantelle Bennetto-Hood; Edward P. Acosta (165-171).
This work describes an assay system that has been developed to quantify raltegravir concentrations in human plasma using a liquid–liquid extraction technique paired with HPLC separation and MS–MS detection. The dynamic range of this assay extends from 1 to 3000 ng/mL, with a coefficient of determination (r 2, mean ± SD) of 0.9992 ± 0.0002. The mean precision values for calibration standards ranged from 0.6% to 3.0%, while accuracy values were 96.5–104.3%. This procedure is an accurate, precise, and sensitive method for raltegravir quantitation and was successfully validated using external proficiency testing.
Keywords: HIV; Integrase inhibitor; Mass spectrometry; Human; Raltegravir;

Rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of clonidine in human plasma by Sagar A. Parekh; Ashutosh Pudage; Santosh S. Joshi; Vikas V. Vaidya; Noel A. Gomes (172-178).
A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm × 4.6 mm i.d., 5 μm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50–2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.
Keywords: Clonidine; Nizatidine; LC–MS/MS; Multiple reaction monitoring (MRM);

Urine analysis of patients exposed to phenylarsenic compounds via accidental pollution by Kenji Kinoshita; Ayano Noguchi; Kazuhiro Ishii; Akira Tamaoka; Takafumi Ochi; Toshikazu Kaise (179-188).
New methods involving high-performance liquid chromatography/inductively coupled plasma mass spectrometry were examined for the determination of phenylarsenic compounds derived from chemical warfare agents. Several methods were examined for the separation of diphenylarsinic acid (DPAA), phenylarsonic acid, phenylmethylarsinic acid (PMAA), phenyldimethylarsine oxide, and diphenylmethylarsine oxide. Analysis of the urine samples of the patients exposed to phenylarsenic compounds indicated that the main phenylarsenic components were DPAA and PMAA; moreover, some unknown arsenicals, which were also found in contaminated groundwater and rice samples, were also detected.
Keywords: Chemical warfare agents; LC-ICP-MS; Urine analysis;

Partition of α-lactoalbumin and β-lactoglobulin by cloud point extraction by Paulo Sérgio Monteiro; Jane Sélia dos Reis Coimbra; Luis Antonio Minim; Juraci Alves de Oliveira; Luis Henrique Mendes da Silva (189-193).
This work aimed to study the partition of cheese whey proteins α-lactoalbumin and β-lactoglobulin using aqueous two-phase system by applying the cloud point extraction technique. The cloud point temperatures were determined under different concentrations of copolymer and salt. The system providing the best protein separation conditions was 20 mass% of copolymer PE61 and potassium phosphate salt solution of 100 mM, at pH 7. The protein α-lactoalbumin remained preferentially in the aqueous phase and the β-lactoglobulin was transferred to the copolymer phase.
Keywords: Aqueous two-phase systems; Cloud point; α-Lactoalbumin; β-Lactoglobulin;

A liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC–APCI-MS/MS) method for quantification of 10 amphetamine-related analytes in 1 g meconium is presented. Specimen preparation included homogenization and solid-phase extraction. Two multiple reaction monitoring transitions were monitored per analyte. Ten and 1 μL injection volumes permitted quantification up to 10,000 ng/g, with sufficient sensitivity to quantify minor metabolites. Lower limits of quantification ranged from 1.25 to 40 ng/g. Precision was less than 14.2%, with accuracy between 79 and 115%. Meconium from a methamphetamine-exposed neonate was analyzed. Metabolites p-hydroxymethamphetamine, norephedrine and 4-hydroxy-3-methoxymethamphetamine were identified in meconium for the first time.
Keywords: Meconium; Amphetamines; LC–MS/MS;

Quantification of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in dried blood spots by liquid chromatography–triple quadrupole mass spectrometry by R. ter Heine; H. Rosing; E.C.M. van Gorp; J.W. Mulder; W.A. van der Steeg; J.H. Beijnen; A.D.R. Huitema (205-212).
A bioanalytical method for the determination of most commonly prescribed protease inhibitors (atazanavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) was developed and validated according to FDA guidelines. In brief, dried blood spots were punched out of a collection paper with a 0.25 in. diameter punch. The analytes were extracted from the punched-out disc using a mixture of acetonitrile, methanol and 0.2M zinc sulphate in water (1:1:2, v/v/v) containing the internal standards dibenzepine, 13C6-efavirenz and D5-saquinavir. 20 μL of the extract was injected onto the reversed-phase C18 column (150 mm × 2.0 mm) for separation from endogenous compounds and the analytes were quantified using a triple quadrupole mass spectrometer. The analytical run time was only 10 min. Validated concentration ranges covered the ranges encountered in routine clinical practice. The assay was linear over the concentration ranges tested (0.1–20 mg/L for atazanavir, lopinavir, nevirapine and efavirenz and 0.05–10 mg/L for darunavir and ritonavir). Accuracies and inter- and intra-run precisions at all levels ranged from 96.2 to 113.9% and 3.1 to 13.3%, respectively. Analytes in dried blood spots were stable for at least 7 days at 30 °C. The method enabled patient-friendly sample collection, easy and cheap sample shipment and non-hospital based sampling for therapeutic drug monitoring and pharmacokinetic studies.
Keywords: Antiretroviral; HIV; Therapeutic drug monitoring; TDM; LC–MS; Chromatography; Mass spectrometry; Dried blood spots; Dried blood spot; Bioanalysis; Pharmacokinetics; Protease inhibitors; Non-nucleoside reverse transcriptase inhibitors; Atazanavir; Darunavir; Lopinavir; Ritonavir; Efavirenz; Nevirapine;

High-performance liquid chromatography with reductive electrochemical detection (HPLC-ECD) method has been used for assaying artemisinins since 1985. Although the methods have been remarkably improved, tandem mass spectrometry (LC–MS/MS) systems with significant advantages have gradually replaced HPLC-ECD to analyze artesunate and dihydroartemisinin in plasma. In the present study, the two methods were evaluated for linearity, quantitation limits, selectivity, precision, and accuracy. The HPLC-ECD performed well in terms of various validation parameters, and showed a good agreement with the LC–MS/MS when calibrated in plasma. However, the major benefit of LC–MS/MS is that it requires only one-tenth the plasma volume needed by HPLC-ECD assay.
Keywords: HPLC-ECD; LC–MS/MS; Artesunate; Dihydroartemisinin; Validation;

Validated method for the simultaneous determination of methadone and its main metabolites (EDDP and EMDP) in plasma of umbilical cord blood by gas chromatography–mass spectrometry by Panagiota D. Nikolaou; Ioannis I. Papoutsis; Julia Atta-Politou; Sotiris A. Athanaselis; Chara A. Spiliopoulou; Antony C. Calokerinos; Constantinos P. Maravelias (219-225).
A sensitive and specific GC/MS method for the determination of methadone (MDN) and its two main metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in plasma samples obtained from venous and arterial umbilical cord blood and maternal blood has been developed, optimized and validated. Specimen preparation includes protein precipitation with acetonitrile and simultaneous solid-phase extraction of the three analytes. Methadone-d9 was used as internal standard for the determination of MDN and EMDP, while EDDP-d3 for EDDP. Limits of detection were 0.6 μg/L for MDN and 0.3 μg/L for EDDP and EMDP, while limits of quantification were 2.0 μg/L for MDN and 1.0 μg/L for EDDP and EMDP. The calibration curves were linear up to 2000 μg/L for MDN and up to 1000 μg/L for EDDP and EMDP. Absolute recovery ranged from 94.8 to 99.7% for all three analytes. Intra- and interday accuracy was less than 5.3 and 5.5%, respectively, while intra- and interday precision was less than 3.5 and 5.0%, correspondingly, for all analytes. The method proved suitable for the determination of MDN and its two main metabolites in plasma samples obtained from umbilical cord and maternal blood of a woman participating in a MDN maintenance program, during the prenatal and postpartum period.
Keywords: Methadone; EDDP; EMDP; Umbilical cord blood; GC/MS;

Simultaneous measurement of retinol, α-tocopherol and six carotenoids in human plasma by using an isocratic reversed-phase HPLC method by Jouni Karppi; Tarja Nurmi; Begoña Olmedilla-Alonso; Fernando Granado-Lorencio; Kristiina Nyyssönen (226-232).
A simple and sensitive isocratic reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous determination of retinol, α-tocopherol and six carotenoids in human plasma was described. Sample preparation of the earlier published method was further developed by addition of ultrapure water, which enabled aqueous layer to freeze facilitating phase separation without pipetting thus also improving precision of the method. Developed method appeared to be less laborious and time consuming compared to the traditional extraction methods, which require removal of organic layer by pipetting. The recoveries (absolute and relative) were between 80% and 103%. The intra-assay CVs were 1.1–4.0% (normal level) and 3.3–9.0% (low level). Inter-assay CVs were 5.3–8.8%. Reference method for all these analytes was not available, but a comparison with another published method was carried out. The results of the comparison matched satisfactorily. The method is used routinely in our laboratory in a large population-based study.
Keywords: α-Tocopherol; Carotenoids; Freezing; HPLC; Retinol; Sample preparation; Stability of carotenoids;

The reaction between phosphatidylethanolamines and HOCl investigated by TLC: Fading of the dye primuline is induced by dichloramines by Grit Richter; Celestina Schober; Rosmarie Süß; Beate Fuchs; Matthias Müller; Jürgen Schiller (233-237).
Phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) are abundant lipid constituents of the membranes of Escherichia coli. The reaction between these lipids and hypochlorous acid (HOCl), an important constituent of disinfectants, was investigated by combined thin-layer chromatography (TLC), mass spectrometry (MS), UV and fluorescence spectroscopy. Primuline is a common dye in lipid research that binds non-covalently to lipids and allows, thus, the direct evaluation of TLC plates by MS. However, primuline staining of the products between PE and HOCl is accompanied by fading of the dye. This only holds if acidic but not alkaline conditions are applied. Using a combination of TLC, UV and fluorescence spectroscopy, it will be shown that dichloramines of PE are responsible for the observed primuline fading. Since dichloramines are slowly converted under alkaline conditions into the nitriles that lack the characteristic UV properties of dichloramines, fading is not observed under alkaline conditions.
Keywords: Phosphatidylethanolamine; Chlorhydrine; Chloramine; Nitrile; HOCl; TLC; Primuline; Fluorescence;

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor γ ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.
Keywords: Delipidation; Hydrophobic interaction chromatography; Lipidex; Peroxisome proliferator activated receptor; Fatty acid binding protein;

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts by Mugunthu R. Dhananjeyan; Jill A. Trendel; Crystal Bykowski; Jeffrey G. Sarver; Howard Ando; Paul W. Erhardt (247-252).
A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery® C18 column using 10 mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 μM to 750 μM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 μM and the lower limit of quantitation was 10 μM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.
Keywords: Esters; HPLC; Local anesthetics; Liver extracts; Intestine extracts; Procaine; PABA;

Separation of chlorogenic acid from honeysuckle crude extracts by macroporous resins by Bin Zhang; Ruiyuan Yang; Yan Zhao; Chun-Zhao Liu (253-258).
Chlorogenic acid, one of the most bioactive compounds rich in the Chinese medicinal herb honeysuckle, is a natural antioxidant and serves as anti-inflammatory, anti-tumor, anti-mutagenic and anti-carcinogenic agent. An efficient preparative separation process of chlorogenic acid from honeysuckle crude extracts has been developed in the present study. HPD-850 resin offers the best adsorption capacity, and adsorption and desorption ratios for chlorogenic acid among the nine macroporous resins tested, and its adsorption rate at 25 °C fit best to the Langmuir isotherm. The adsorption capacity of HPD-850 resin was found to depend strongly on the pH value of the initial adsorption solution. The dynamic adsorption and desorption experiments have been carried out on a HPD-850 resin packed column to optimize the separation process of chlorogenic acid from honeysuckle crude extracts. After one run treatment with HPD-850 resin, the chlorogenic acid content in the final product was increased 4.46-fold from 11.2% to 50.0%, with a recovery yield of 87.9%. The preparative separation of chlorogenic acid can be easily and efficiently achieved via adsorption and desorption on HPD-850 resin, and the method developed will provide a potential approach for large-scale separation and purification of chlorogenic acid for its wide pharmaceutical use.
Keywords: Adsorption; Chlorogenic acid; Desorption; Macroporous resins; Preparative separation;

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry by Kensaku Kakimoto; Akira Toriba; Takanori Ohno; Mariko Ueno; Takayuki Kameda; Ning Tang; Kazuichi Hayakawa (259-263).
To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C18 reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r 2  = 0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.
Keywords: 1-Hydroxypyrene-glucuronide; LC–MS/MS; Urine; Polycyclic aromatic hydrocarbon; Biomarker;

A molecularly imprinted polymer (MIP) has been prepared using levonorgestrel (LEV) as template. The polymer was synthesised in a non-covalent approach using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linking monomer via a free radical polymerization. An equivalent blank polymer was also synthesised in the absence of the template compound. Batch adsorption experiments were used to evaluate the binding affinity of the imprinted polymer. After packing MIP into a stainless steel column (150 mm × 4.6 mm i.d.), retention and elution of the template and related compounds were evaluated by high-performance liquid chromatography (HPLC). This LEV imprinted polymer was further applied for selective solid phase extraction (SPE) of LEV from human serum. It was confirmed that the binding ability of the prepared MIP for LEV was essentially sufficient in the presence of other compounds coexisting in serum sample. Therefore, as a selective and efficient solid phase material, LEV imprinted polymer has a high potential application in analysis of this steroidal hormone in clinical purposes.
Keywords: Molecularly imprinted polymer; Solid phase extraction; Levonorgestrel; Human serum;

A fast, simple and selective HPLC method has been developed for the assay of aciclovir, ganciclovir, and penciclovir in human plasma by coupling HPLC with fluorescence detection. 200 μl plasma, with guanosine 5′-monophosphate as an internal standard, was subjected to protein precipitation with a 7% [v/v] aqueous perchloric acid solution. The 40 μl supernatant was injected into a Diamonsil-5 μm C18 column. Aciclovir, ganciclovir, and penciclovir, with solvents composed of methanol and 0.08% aqueous trifluoroacetic acid solution, were analysed by fluorescence detection at 260 nm (excitation) and 380 nm (emission) using a gradient elution program. The calibration curves of all three analytes were linear between 20 and 2000 ng/ml. The mean absolute recoveries of aciclovir, ganciclovir, and penciclovir were 93.91 ± 1.20%, 97.42 ± 0.75%, and 99.01 ± 3.30%, respectively. The mean inter-day CVs for aciclovir, ganciclovir, and penciclovir, were within 1.29–7.30%, 1.00–5.53%, and 1.19–3.54%, respectively. The intra-day bias for aciclovir, ganciclovir, and penciclovir ranged from −2.01 to 6.33%, 1.81 to 7.37%, and 1.42 to 6.91%, respectively. The method has been validated and applied in pharmacokinetic studies in Chinese adult renal transplant patients.
Keywords: Aciclovir; Ganciclovir; Penciclovir; HPLC;

A simple and sensitive HLPC method with fluorescence detection was developed for the accurate determination of the first licensed HIV integrase inhibitor raltegravir in human plasma. A 500-μL plasma sample was spiked with delavirdine as internal standard and subjected to liquid–liquid extraction based on a previously described assay i.e. using hexane/methylene chloride (1:1, v/v%) at pH 4.0. HPLC was performed using a Symmetry Shield RP18 column (150 mm × 4.6 mm), a gradient elution of acetonitrile −0.01% (v/v) triethylamine in water adjusted to pH 3.0 at a flow rate of 1 mL/min and a fluorimetric detector set at 299 and 396 nm as excitation and emission wavelengths, respectively. The retention time was 5.0 min for internal standard and 6.4 min for raltegravir. Calibration curves were linear in the range 5–1000 ng/mL and the accuracy of quality control samples in the range 10–750 ng/mL varied from 98.3 to 99.1% and 98.3 to 101.0% of the nominal concentrations for intra-day and day-to-day analysis, respectively with a precision of 6.3% or less. Among the other antiretroviral drugs which can be given in association to HIV-infected patients, none was found to interfere with internal standard or raltegravir. The described assay was developed for the purpose of therapeutic drug of this HIV integrase inhibitor.
Keywords: Raltegravir; HIV integrase inhibitor; Liquid–liquid extraction procedure; HPLC; Fluorescence detection; Therapeutic drug monitoring;

Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography by Kunbo Wang; Zhonghua Liu; Jian-an Huang; Xinrong Dong; Lubing Song; Yu Pan; Fang liu (282-286).
High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins. The HSCCC run was carried out with a two-phase solvent system composed of hexane–ethyl acetate–methanol–water–acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that pure theaflavin, theaflavins-3-gallate, theaflavins-3′-gallate and theaflavin-3,3′-digallate could be obtained from crude theaflavins sample and black tea. The structures of the isolated compounds were positively confirmed by 1H NMR and 13C NMR, MS analysis, HPLC data and TLC data. Meanwhile, catechins including epigallocatechin gallate, gallocatechin gallate, epicatechin gallate and epigallocatechin were isolated from the aqueous extract of green tea by using the same solvent system. This study developed a modified method combined with enrichment theaflavins method by using HSCCC for separation of four individual theaflavins, especially for better separation of theaflavins monogallates.
Keywords: High-speed countercurrent chromatography; Theaflavins; Black tea; Catechins; Green tea;