Journal of Chromatography B (v.867, #1)

Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O2 carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC–MS. LC–MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.
Keywords: Hemoglobin; Oxygen carrier; Blood substitute; Purification; Anion exchange chromatography; Ultrapure; SDS-PAGE; LC–MS; Red blood cell;

Determination of 8-iso-prostaglandin F in exhaled breath condensate using combination of immunoseparation and LC–ESI-MS/MS by Kamila Syslová; Petr Kačer; Marek Kuzma; Pavlína Klusáčková; Zdena Fenclová; Jindřiška Lebedová; Daniela Pelclová (8-14).
Rapid and precise method for the determination of 8-iso-prostaglandin F, an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC–ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0–250 pg of 8-iso-prostaglandin F/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F content between the group of asbestosis patients and healthy volunteers was found.
Keywords: 8-iso-Prostaglandin F; Exhaled breath condensate; Immunoseparation;

Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma by Frédérique Lancelin; Kayssa Djebrani; Khalid Tabaouti; Linda Kraoul; Sophie Brovedani; Pascal Paubel; Marie-Liesse Piketty (15-19).
A high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed for quantification of aripiprazole and dehydro-aripiprazole, in human plasma. After a simple liquid–liquid extraction, chromatographic separation was carried out on a C18 reversed-phase column, using an ammonium buffer–acetonitrile mobile phase (40:60, v/v). The total run time was only 7 min at a flow-rate of 1.0 ml/min. The precision values were less than 12% and the accuracy values were ranging from 98 to 113% and the lower limit of quantification was 2 ng/ml for both compounds. Calibration curves were linear over a range of 2–1000 ng/ml. The mean trough plasma concentrations in patients treated with aripiprazole were 157 and 29 ng/ml for aripiprazole and dehydro-aripiprazole, respectively.
Keywords: Aripiprazole; Dehydro-aripiprazole; HPLC; Therapeutic drug monitoring;

Quantification of doripenem in human plasma and peritoneal fluid by high-performance liquid chromatography with ultraviolet detection by Kayo Ikeda; Kazuro Ikawa; Norifumi Morikawa; Keiko Kameda; Nami Urakawa; Hiroki Ohge; Taijiro Sueda (20-25).
A simple and rapid HPLC method that includes ultrafiltration to remove plasma and peritoneal fluid protein was developed to determine doripenem concentrations in human plasma and peritoneal fluid. Doripenem was stabilized by immediate mixing of the plasma or peritoneal fluid with 1 M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Doripenem and an internal standard were detected by measuring their ultraviolet absorbance at 300 nm. The calibration curves for doripenem in human plasma and peritoneal fluid were linear from 0.05 to 100 μg/mL. For plasma, both the intra- and the interday precision were less than 3.41% (CV), and the accuracy was between 97.4 and 101.7% above 0.05 μg/mL. For peritoneal fluid, the intra- and the interday precision were less than 2.98% (CV), and the accuracy was between 94.4 and 103.9% above 0.05 μg/mL. The limit of detection was 0.02 μg/mL in both plasma and peritoneal fluid. The assay has been applied to the therapeutic drug monitoring of doripenem in both plasma and peritoneal fluid.
Keywords: Doripenem; Ultrafiltration; HPLC; Plasma; Peritoneal fluid; Pharmacokinetic studies;

Urinary trans,trans-muconic acid (t,t-MA), a biomarker of benzene exposure, is usually determined by HPLC methods with detection by either UV or, more recently, electrospray tandem mass spectrometry. However, not all these methods have been fully validated for quantitative analysis. This paper presents an HPLC/MS/MS method for reliable quantitative determination of t,t-MA that uses a commercial deuterium-labeled isotope as internal standard; the matrix effect has been evaluated and LOD is 0.22 μg/L. We used this method to test 200 urine samples, 175 of them collected at end-of-shift from workers in an oil refinery.
Keywords: Biological monitoring; Trans,trans-muconic acid; HPLC/MS/MS; Method validation; Oil refinery workers;

A highly sensitive high-performance liquid chromatographic method for the simultaneous analysis of 1,2,3,4-tetrahydroisoquinolines (TIQs) in the rat brain was developed. 1,2,3,4-Tetrahydroisoquinoline (TIQ), 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BeTIQ) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 50 °C for 15 min at pH 8.5. The fluorescent derivatives were separated on a reversed-phase column by gradient elution using (A) water–(B) acetonitrile/methanol (55:45) at 55 °C and detected by fluorescence measurement at 318 nm (excitation) and 398 nm (emission). The detection limits (signal-to-noise ratio = 3) were 8–9 fmol per injection. The relative standard deviations (n  = 6) of TIQs were 2.6–10.5% and the recoveries were 87.6, 101.8 and 75.2%, respectively. The concentrations of TIQ, 1-MeTIQ and 1-BeTIQ in normal rat brains (n  = 6) were 0.7 ± 0.3 (0.10 ± 0.04), 3.4 ± 1.5 (0.50 ± 0.22) and 1.3 ± 1.8 pmol/g (0.30 ± 0.41 ng/g), respectively.
Keywords: HPLC; Fluorometric detection; 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; 1,2,3,4-Tetrahydroisoquinolines; Brain;

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues by Jaroslav Zelenka; Martin Leníček; Lucie Muchová; Milan Jirsa; Michal Kudla; Peter Balaž; Marie Zadinová; J. Donald Ostrow; Ronald J. Wong; Libor Vítek (37-42).
Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75 ± 5%. The detection limit was 10 pmol UCB/g wet tissue. Variance was ±2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (≤40 pmol UCB/g tissue) in non-jaundiced rats.
Keywords: Bilirubin; Gunn rats; HPLC; Method of determination; Tissue bilirubin;

This work describes the determination of N3-methyladenine, N7-methylguanine and O 6-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV–vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O 6-methylguanine (S/N = 3). The limit of quantitation (LOQ) was found to be 0.5 ng/mL for all measured compounds, (S/N = 10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73–6.96 and 2.26–7.58%, respectively, and correlation coefficients of calibration curves (R 2) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53 ± 2.97% (R.S.D. = 4.8), N3-methyladenine for 38.19 ± 2.99% (R.S.D. = 9.6) and O 6-methylguanine for 0.29 ± 0.02% (R.S.D. = 5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.
Keywords: N3-methyladenine; N7-methylguanine; O 6-methylguanine; DNA methylated adducts; Dimethyl sulphate; HPLC; LC–MS–ESI(+);

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of 17α-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC–MS–MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-μl sample, and the limit of quantitation was 5.0 pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive (r 2  = 0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy.
Keywords: 17α-Hydroxyprogesterone; Saliva; Liquid chromatography–electrospray ionization-tandem mass spectrometry; Derivatization; Congenital adrenal hyperplasia; Hormone replacement therapy;

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry by Kazuo Yamada; Takeshi Miyazaki; Tomoko Shibata; Nobumasa Hara; Mikako Tsuchiya (57-61).
We have expanded a liquid chromatographic–tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 μM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-γ, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.
Keywords: Tryptophan; Kynurenine pathway; LC/MS/MS; Electrospray ionization;

A simple method for obtaining transferrins from human plasma and porcine serum: Preparations and properties by Lin Wu; Jinhui Wu; Jian Zhang; Yuanyuan Zhou; Guoyan Ren; Yiqiao Hu (62-68).
A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.
Keywords: Transferrin; Purification; Identification;

A concentrated mixture of gossypol, 6-methoxy-gossypol, and 6,6′-dimethoxy-gossypol was extracted from the root bark of St. Vincent Sea Island cotton with acetone. This extract was derivatized with R-(−)-2-amino-1-propanol to form diastereomeric gossypol Schiff's bases. Analytical-scale reverse-phase chromatography of these Schiff's bases produced six peaks, indicating separation of the enantiomeric forms of the three gossypol compounds. The elution order of the peaks was found to vary with the polarity of the mobile phase. The chromatography was scaled to a preparative level and was used to isolate each compound. After hydrolysis of the separated Schiff's bases, the original compounds were recovered by precipitation from solutions of diethyl ether, acetic acid, and water. Fifty injections yielded approximately 500 mg of each methoxy-gossypol enantiomer and 300 mg of each dimethoxy-gossypol enantiomer. Each compound was characterized for carbon and hydrogen content, optical rotation, UV–vis light absorption, and melting point. Standard curves were developed and were used to measure the concentration of each gossypol form in the root bark and dehulled seed of St. Vincent Sea Island cotton. In seed tissue, 48% of the gossypol compounds were methylated, and the (−)-optical form was found to be in a slight excess to the (+)-optical form (53–54%) for all three compounds. In root bark, 71% of the gossypol compounds were methylated, and the (+)-optical form was in excess to the (−)-optical form for all three compounds. However, in this tissue the extent of enantiomeric excess decreased with the degree of methylation, with 77% of the gossypol existing in the (+)-optical form and 59% of the 6,6′-dimethoxy-gossypol existing in the (+)-optical form.
Keywords: Cotton; Cottonseed; Gossypol; Secondary metabolites; Separation processes;

Analysis of amphetamine-type stimulants and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry by Kenji Kuwayama; Hiroyuki Inoue; Tatsuyuki Kanamori; Kenji Tsujikawa; Hajime Miyaguchi; Yuko T. Iwata; Seiji Miyauchi; Naoki Kamo (78-83).
The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50–5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and −9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.
Keywords: Methamphetamine; Amphetamine-type stimulant; Pharmacokinetics; Rat; LC–MS/MS; Strong cation-exchange column;

A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4 μm, 150 mm × 4.60 mm) maintained at 30 °C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418 nm, respectively. The mean recoveries were 81.41 ± 0.035%, 86.00 ± 0.053% and 82.21 ± 0.020% for CZ48 and 76.01 ± 0.028%, 77.04 ± 0.042% and 85.93 ± 0.023% for CPT at three concentrations of 10, 100 and 900 ng/ml, respectively. The calibration curve was linear (r 2  = 0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000 ng/ml and 10–1000 ng/ml (n  = 6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900 ng/ml). The lower limit of quantification (LLOQ) using 0.1 ml mouse plasma was 10 ng/ml for CZ48 and 5 ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4 h incubation at room temperature or after 1 month storage at −80 °C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48.
Keywords: Camptothecin; CZ48; Chromatography; HPLC; Quantification;

Quantitative analysis of natural cyclodextrins by high-performance liquid chromatography with pulsed amperometric detection: Application to cell permeation study by Tarja Toropainen; Pekka Jarho; Marko Lehtonen; Pekka Keski-Rahkonen; Heli Raatikainen; Tomi Järvinen (90-98).
Simple HPLC-PAD methods were developed for quantitation of cyclodextrins (CDs) in aqueous matrices from in vitro cell permeation studies. C-18 solid-phase extraction was used for sample pretreatment. Samples were analysed using acetonitrile–water mobile phase with post-column alkalization by 0.5 M NaOH. Zorbax SB-Aq (for α-CD) and Zorbax SB-Phenyl (for β-CD and γ-CD) columns gave excellent peak shape and sufficient resolution of CD to glucose (2.7–3.2). The methods showed good concentration–response relationship (r  ≥ 0.999), precision (RSD% 0.7–5.1), repeatability (RSD% 3.4–13.7) and accuracy (87–107%). The limits of quantitation were 0.78, 0.46 and 0.52 μg/ml for α-CD, β-CD and γ-CD (RSD% of 10.6, 8.1 and 16.3, respectively).
Keywords: α-Cyclodextrin; β-Cyclodextrin; γ-Cyclodextrin; Quantitative analysis; High-performance liquid chromatography; Pulsed amperometric detection; Cell permeation;

Simultaneous determination of amitraz and its metabolite in human serum by monolithic silica spin column extraction and liquid chromatography–mass spectrometry by Takeshi Saito; Rie Yamamoto; Shigeaki Inoue; Izumi Kishiyama; Shota Miyazaki; Akihiro Nakamoto; Manami Nishida; Akira Namera; Sadaki Inokuchi (99-104).
A simple, rapid, sensitive, and specific liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for the quantification of amitraz and its metabolite in human serum. Both the compounds were extracted using monolithic silica spin columns with acetonitrile. The chromatographic separation was performed on a reverse-phase C18 column with a mobile phase of 10 mM ammonium formate–acetonitrile. The protonated analyte was quantitated in positive ionization by mass spectrometry. The method was validated over the concentration range of 25–1000 ng/ml for amitraz and its metabolite in human serum. For both compounds, the limit of detection was 5 ng/ml. The method was applied to serum samples taken from an attempted suicide patient, and only small volumes of serum were required for the simultaneous determination of these compounds.
Keywords: Amitraz; Amitraz metabolite; Serum; Monolithic silica spin column; LC–MS;

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

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography–tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI+). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm × 2.1 mm, i.d., 3 μm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7 → 768.9 (m/z) for FK506, 415.5 → 310.3 (m/z) for DTZ and 809.8 → 757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5–200 ng/mL for FK506 and 2–250 ng/mL for DTZ. The recoveries of liquid–liquid extraction method were 58.3–62.6% for FK506 and 50.4–58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.
Keywords: Tacrolimus; Diltiazem; Liquid chromatography–tandem mass spectrometry; Pharmacokinetics; Renal transplant;

The N-terminus of the trimeric TNF-alpha molecule comprises two basic arginines within the short amino-acid sequence VRSSSR, which is here shown to be essential for binding of TNF-alpha to heparin–Sepharose. Mixed trimers containing full-length and ΔN6-truncated subunits revealed a single VRSSSR sequence to be sufficient to achieve binding. On the basis of this newly identified heparin-binding domain, a new method for efficient purification of TNF-alpha is described. Affinity chromatography on heparin–Sepharose was introduced as a key step for highly purified TNF-alpha at a high yield. With minor modifications, this procedure can be used for TNF-alpha analogues that have full-length N-termini, as shown for the less toxic analogue LK-805.
Keywords: Heparin-binding domain; TNF-alpha; Affinity chromatography;

LC–MS analysis of trimethoxyamphetamine designer drugs (TMA series) from urine samples by Maria Nieddu; Gianpiero Boatto; Maria Antonietta Pirisi; Emanuela Azara; Mauro Marchetti (126-130).
A sensitive liquid chromatography–mass spectrometric (LC–MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C18 cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 μm Polar Plus column (150 mm × 2.1 mm) with acetonitrile −0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10–200 ng/mL) (R 2  ≥ 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70–10.77% and 7.63–12.94%, respectively. This LC–MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.
Keywords: TMA series; Trimethoxyamphetamines; Designer drugs; Human urine; Liquid chromatography; Mass spectrometry;

Ultra-performance liquid chromatography–tandem mass spectrometric method for the determination of Artemisinin in rat serum and its application in pharmacokinetics by Lie Li; Deepthi Pabbisetty; Paulo Carvalho; Mitchell A. Avery; John. S. Williamson; Bonnie A. Avery (131-137).
A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify artemisinin in rat serum. The lower limit of quantification (LLOQ) was 4 ng/mL. The calibration curve was linear from 4 ng/mL to 10,000 ng/mL (R  = 0.998). The assay was based on the selected reaction monitoring (SRM) transitions at m/z 305.4–151.10 for artemisinin and m/z 335.2–163.10 for arteether (internal standard). The artemisinin and internal standard can be separated from endogenous interferences in rat serum. Inter- and intra-day assay variation was less than 15%. The extraction recoveries ranged from 80.0 to 107.3% at the three concentrations (5000, 2000, and 200 ng/mL). This method was successfully applied to pharmacokinetic studies of artemisinin after intravenous and oral administration to rats.
Keywords: Artemisinin; UPLC; MS/MS; Pharmacokinetic; Rat serum;

The quantitative determination of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) from rat plasma using ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is reported. CAPE and FCAPE were extracted using ethyl acetate in the presence of methyl caffeate (MC) as internal standard. Separation was achieved using a C18 column (2.1 mm × 50 mm, 1.7 μm) and gradient elution with water and acetonitrile containing 0.2% and 0.1% formic acid, respectively. A non-linear response over a broad concentration range (1–1000 ng/ml, r 2  > 0.995 using a quadratic regression model and 1/concentration weighting) was obtained. The inter-day and intra-day variability for CAPE and FCAPE were found to be less than 14.2% and 9.5%, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of CAPE and FCAPE after intravenous administration to rats.
Keywords: Caffeic acid phenethyl ester; Fluorinated caffeic acid phenethyl ester; Rat blood plasma; UPLC-ESI-MS/MS;

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid–liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm × 4.6 mm, i.d., 5 μm) column (Hanbon, China) at 20 °C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39–100 ng mL−1. The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL−1. The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20 mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54 ± 0.67 h.
Keywords: Ritodrine; Electrospray ionization tandem mass spectrometry; Determination; Human plasma;

A highly sensitive and fast reversed-phase liquid chromatographic (LC) method combined with pulsed electrochemical detection (PED) was developed for the direct quantification of the aminoglycoside antibiotic amikacin in cerebrospinal fluid (CSF). The limit of quantification obtained was 0.06 μg/ml and linearity was established over the concentration range 0.06–4.00 μg/ml. The recovery was found to be close to 100%. This method was developed in order to study CSF pharmacokinetics of amikacin in neonates. The narrow therapeutic range calls for monitoring to ensure optimal therapy and to minimize the risk of toxic side effects such as nephro- and ototoxicity, especially in populations like preterm neonates at birth, where the predictability of amikacin clearance is limited. Typical problems to be solved were the low amikacin concentrations and the limited sample volume of CSF.
Keywords: Amikacin; Cerebrospinal fluid; Pulsed electrochemical detection; Reversed-phase liquid chromatography;

A 96-well protein precipitation, liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 μL) by the methanol (150 μL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 μL supernatant and 100 μL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC–MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol–water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178 → 91 and m/z 284 → 91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 μg mL−1, with good linearity (r  > 0.999) over the linear range of 0.02–10 μg mL−1. The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 μg mL−1. The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.
Keywords: Fudosteine; LC–MS/MS; 96-Well protein precipitation; Pharmacokinetics;

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release by Marjan Bele; Gorazd Hribar; Stanislav Čampelj; Darko Makovec; Vladka Gaberc-Porekar; Milena Zorko; Miran Gaberšček; Janko Jamnik; Peter Venturini (160-164).
The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.
Keywords: Magnetic nanoparticles; Silica coating; Zinc adsorption; Histidine affinity binding; Protein binding; Protein nanoparticles; Coordinative binding;