Journal of Chromatography B (v.879, #23)

Direct injection of human serum and pharmaceutical formulations for glucosamine determination by CE-C4D method by Patcharin Chaisuwan; Thitiya Kongprasertsak; Areeporn Sangcakul; Norman W. Smith; Duangjai Nachapricha; Prapin Wilairat; Kanchana Uraisin (2185-2188).
A simple CE-C4D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20 mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C4D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% (n  = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10–2.50 mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5–104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences.
Keywords: Glucosamine; Human serum; Capacitively coupled contactless conductivity detection; Capillary electrophoresis;

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry by Yan-rong Zhu; Yan-yan Jia; Ling Jiang; Chao Wang; Li-kun Ding; Jing Yang; Liang Li; Pei-xi Zhao; Wen-xin Liu; Yi-Ding; Li Wang; Ai-dong Wen (2189-2193).
A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC–MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC–MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C18 column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1 M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2  m/z for azatadine, 383.3 → 337.3  m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93–11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83–105.07% of the nominal values.
Keywords: Azatadine; LC–MS/MS; Human plasma; Pharmacokinetics;

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles by Jun Sun; Yujie Su; Shengqi Rao; Yanjun Yang (2194-2200).
Functionalized Fe3O4 nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe3O4 (PEG + CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe3O4 (PEG + CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption–elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.
Keywords: Magnetic; Carboxymethyl chitosan; Lysozyme; Protein purification;

Mitotane (o,p′-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p′-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p′-DDA (1,1-[o,p′-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p′-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77–88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 μg/ml for mitotane and DDE, and 0.5 μg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1–18 times higher than the parent compound.
Keywords: Adrenocortical cancer; Mitotane; Metabolites; HPLC;

Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC–MS/MS by Kerri M. Smith; Wannarasmi Ketchart; Xiang Zhou; Monica M. Montano; Yan Xu (2206-2212).
Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2–5 mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC–MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis® T3 (2.1 mm × 50 mm, 3 μm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500–100 ng/mL in both mouse and human plasma with injection volume of 5 μL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10 mg/kg.
Keywords: Hexamethylene bisacetamide; Heptamethylene bisacetamide; Breast cancer; HEXIM1; LC–MS/MS;

Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation by Ivana Stojićević; Ljiljana Dimitrijević; Nebojša Dovezenski; Irena Živković; Vladimir Petrušić; Emilija Marinković; Aleksandra Inić-Kanada; Marijana Stojanović (2213-2219).
Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.
Keywords: Tetanus toxoid; Hydrophobic chromatography; Chelating chromatography; Immobilized metal affinity chromatography; Ammonium-sulphate precipitation;

Determination of cysteinyl leukotrienes in exhaled breath condensate: Method combining immunoseparation with LC–ESI-MS/MS by Kamila Syslová; Petr Kačer; Beáta Vilhanová; Marek Kuzma; Petra Lipovová; Zdenka Fenclová; Jindřiška Lebedová; Daniela Pelclová (2220-2228).
A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C4, leukotriene D4 and leukotriene E4), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤7.7%, determined as RSD), an acceptable accuracy (90.4–93.7%, determined as recovery), a low limit of detection (≤2 pg/ml EBC) and a low limit of quantification (≤10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC–MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.
Keywords: Cysteinyl leukotriene; Exhaled breath condensate; Immunoseparation; Solid phase extraction; Lyophilization; LC–MS;

Determination of temozolomide in serum and brain tumor with micellar electrokinetic capillary chromatography by Melinda Andrási; Brigitta Törzsök; Álmos Klekner; Attila Gáspár (2229-2233).
Micellar electrokinetic capillary chromatographic (MEKC) with photodiode-array detection was applied to determine temozolomide (TMZ) in human serum and brain tumor. The limit of quantitation (LOQ) was 0.096 μg/mL using 325 nm as detection wavelength. The method made possible that the TMZ could be detected in in vivo serum samples without sample pretreatment. In order to detect TMZ at lower concentration, an extraction with ethyl acetate was applied to preconcentrate the analyte. Small amount of brain tumor tissues (less than 1 g) were lyophilized and pretreated using extraction as a clean up and concentrating step. After removing the organic solvent a final sample volume of only 10 μL was analyzed. The obtained peak concentrations (8.2–10.1 μg/mL) and T max (44–65 min) of TMZ in serum were similar to the data reported by others, the in vivo TMZ concentrations found in brain tumor ranged between 0.061 and 0.117 μg/g.
Keywords: Temozolomide; MEKC; Serum; Brain; Tumor;

Simultaneous quantification of carbamate insecticides in human plasma by liquid chromatography/tandem mass spectrometry by Ahmed Mostafa; Gregory Medley; Darren M. Roberts; Mosaad Sayed Mohamed; Abdalla A. Elshanawani; Michael S. Roberts; Xin Liu (2234-2238).
Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamate pesticides that are widely used in gardening and agriculture for the control of insects. Human poisoning due to occupational or self-poisoning exposures is also reported, so assays are required to quantify the plasma concentration of these insecticides. An LC–MS/MS method was developed and validated for the simultaneous quantification of these three carbamate insecticides in the plasma of patients with acute intentional self-poisoning. Plasma samples were pretreated by acetonitrile for protein precipitation. Chromatography was carried out on a Luna C18(2) analytical column with gradient elution using a mobile phase containing acetonitrile and water with 10 mM ammonium acetate. Mass spectrometric analysis was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source in the positive ion mode. The total run time was 7 min. The assay was validated over a concentration range from 10 to 1000 ng/ml for CSN and FBC and 20–2000 ng/ml for CFN. The precision and accuracy for both intra- and inter-day determination of all analytes were acceptable (<15%). No significant matrix effect was observed. Stability of compounds was established for short term bench and autosampler storage as well as freeze/thaw cycles. The method was effectively applied to 270 clinical samples from patients with a history of acute intentional carbamate self-poisoning.
Keywords: Carbofuran; Carbosulfan; Fenobucarb; Insecticide; LC–MS/MS; Poisoning;

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n  = 6) for FB1, FB2 and FB3 in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n  = 15) ranging in total fumonisin (TFB = FB1  + FB2  + FB3) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB1, FB2 and FB3 were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.
Keywords: Fumonisins; o-Phthaldialdehyde (OPA); Naphthalene-2,3-dicarboxaldehyde (NDA); Strong anion extraction; Immunoaffinity columns; Mycotoxins;

Two types of amorphous, silica nanoparticles have been produced and used as surface assisting agents during laser desorption/ionisation time-of flight-mass spectrometry (SALDI-TOF-MS). The first is hydrophilic possessing surface aminopropyl groups and the second hydrophobic containing surface phenyl groups. Each particle type acts as a solid phase adsorbent, adsorbing analytes according to their charge and hydrophobicity. The adsorbed analytes can be directly analysed on the particles using SALDI-TOF-MS. Intrinsically magnetisable versions of the hydrophobic particles act as magnetic solid phase extraction (MSPE) materials which are used to selectively adsorb analytes within a mixture deposited onto a surface, transfer the adsorbed components using a magnetic wand and to deposit the particles at a site adjacent to that of the original mixture. Non-adsorbed components remain at the original site. The extracted and residual analytes are then directly analysed on the surface by SALDI-TOF-MS. Using fingerprints as an example of a complex biological matrix, this new approach has been used to separate polar (amino acids) and non-polar constituents (squalene and fatty acids) within latent fingerprints deposited on a surface and for their subsequent direct analysis on the surface by SALDI-TOF-MS. Alanine, ornithine, lysine and aspartic acid which were undetected or poorly detected prior to separation showed improved signal detection after separation.
Keywords: Magnetic solid phase extraction; Amorphous silica nanoparticle; Fingerprint; Amino acids; SALDI-TOF-MS;

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C18-columns (Synergi 2.5 μm, Hydro-RP, 100 Å, the length of 100 mm) with an inner diameter of 2 or 3 mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.
Keywords: Malondialdehyde; 2,4-Dinitrophenylhydrazine; High performance liquid chromatography; Biological materials;

For the endogenous substances with an ultra-low level in biological fluids, such as melatonin, the blank biological matrix is obviously not “blank”. This problem leads to a serious issue of the bioanalytical methods development and validation by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). This work developed and validated an ultra-high sensitive bioanalytical method for plasma melatonin by LC–MS/MS using water as calibration matrix. The lower limit of quantitation of the method was verified to be 1.0 pg/mL and the method exhibited a linear range of 1–5000 pg/mL. Potential matrix effects, accuracy and precision were fully monitored and validated by two complementary quality control approaches respectively using water and the pooled plasma as matrix. The intra-run and inter-run precisions were less than 11.5% and 12.2%, respectively, and the relative error was below ±13.8% for all of 5 quality control levels. The method was successfully applied to investigate the daytime (8:00 AM–8:00 PM) baseline level of endogenous plasma melatonin, as well as the pharmacokinetic profiles of exogenous melatonin after oral administration in beagle dogs.
Keywords: Melatonin; LC–MS/MS; Plasma; Bioanalytical method; Matrix effect;

Development and validation of a selective and sensitive LC–MS/MS method for determination of cycloserine in human plasma: Application to bioequivalence study by Dinesh S. Patel; Naveen Sharma; Mukesh C. Patel; Bhavin N. Patel; Pranav S. Shrivastav; Mallika Sanyal (2265-2273).
A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6 mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20–30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.
Keywords: Cycloserine; LC–MS/MS; Solid phase extraction; Human plasma; Bioequivalence;

Quantification of acetaminophen (paracetamol) in human plasma and urine by stable isotope-dilution GC–MS and GC–MS/MS as pentafluorobenzyl ether derivative by Arne Trettin; Alexander A. Zoerner; Anke Böhmer; Frank-Mathias Gutzki; Dirk O. Stichtenoth; Jens Jordan; Dimitrios Tsikas (2274-2280).
We report on the quantitative determination of acetaminophen (paracetamol; NAPAP-d0) in human plasma and urine by GC–MS and GC–MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode after derivatization with pentafluorobenzyl (PFB) bromide (PFB-Br). Commercially available tetradeuterated acetaminophen (NAPAP-d4) was used as the internal standard. NAPAP-d0 and NAPAP-d4 were extracted from 100-μL aliquots of plasma and urine with 300 μL ethyl acetate (EA) by vortexing (60 s). After centrifugation the EA phase was collected, the solvent was removed under a stream of nitrogen gas, and the residue was reconstituted in acetonitrile (MeCN, 100 μL). PFB-Br (10 μL, 30 vol% in MeCN) and N,N-diisopropylethylamine (10 μL) were added and the mixture was incubated for 60 min at 30 °C. Then, solvents and reagents were removed under nitrogen and the residue was taken up with 1000 μL of toluene, from which 1-μL aliquots were injected in the splitless mode. GC–MS quantification was performed by selected-ion monitoring ions due to [M−PFB] and [M−PFB−H], m/z 150 and m/z 149 for NAPAP-d0 and m/z 154 and m/z 153 for NAPAP-d4, respectively. GC–MS/MS quantification was performed by selected-reaction monitoring the transition m/z 150 →  m/z 107 and m/z 149 →  m/z 134 for NAPAP-d0 and m/z 154 →  m/z 111 and m/z 153 →  m/z 138 for NAPAP-d4. The method was validated for human plasma (range, 0–130 μM NAPAP-d0) and urine (range, 0–1300 μM NAPAP-d0). Accuracy (recovery, %) ranged between 89 and 119%, and imprecision (RSD, %) was below 19% in these matrices and ranges. A close correlation (r  > 0.999) was found between the concentrations measured by GC–MS and GC–MS/MS. By this method, acetaminophen can be reliably quantified in small plasma and urine sample volumes (e.g., 10 μL). The analytical performance of the method makes it especially useful in pediatrics.
Keywords: Biological samples; Derivatization; Pentafluorobenzyl bromide; Quantification; Stable-isotope dilution; Validation;

Determination of uremic solutes in biological fluids of chronic kidney disease patients by HPLC assay by Raymond Calaf; Claire Cerini; Cécile Génovésio; Pierre Verhaeghe; Noémie Jourde-Chiche; David Bergé-Lefranc; Bertrand Gondouin; Laetitia Dou; Sophie Morange; Angel Argilés; Pascal Rathelot; Françoise Dignat-George; Philippe Brunet; Philippe Charpiot (2281-2286).
During chronic kidney disease (CKD), solutes called uremic solutes, accumulate in blood and tissues of patients. We developed an HPLC method for the simultaneous determination of several uremic solutes of clinical interest in biological fluids: phenol (Pol), indole-3-acetic acid (3-IAA), p-cresol (p-C), indoxyl sulfate (3-INDS) and p-cresol sulfate (p-CS). These solutes were separated by ion-pairing HPLC using an isocratic flow and quantified with a fluorescence detection. The mean serum concentrations of 3-IAA, 3-INDS and p-CS were 2.12, 1.03 and 13.03 μM respectively in healthy subjects, 3.21, 17.45 and 73.47 μM in non hemodialyzed stage 3–5 CKD patients and 5.9, 81.04 and 120.54 μM in hemodialyzed patients (stage 5D). We found no Pol and no p-C in any population. The limits of quantification for 3-IAA, 3-INDS, and p-CS were 0.83, 0.72, and 3.2 μM respectively. The within-day CVs were between 1.23 and 3.12% for 3-IAA, 0.98 and 2% for 3-INDS, and 1.25 and 3.01% for p-CS. The between-day CVs were between 1.78 and 5.48% for 3-IAA, 1.45 and 4.54% for 3-INDS, and 1.19 and 6.36% for p-CS. This HPLC method permits the simultaneous and quick quantification of several uremic solutes for daily analysis of large numbers of samples.
Keywords: Chronic kidney disease; Uremic solutes; Ion-pairing HPLC;

Characterization of odor-active compounds of various cherry wines by gas chromatography–mass spectrometry, gas chromatography–olfactometry and their correlation with sensory attributes by Yunwei Niu; Xiaoming Zhang; Zuobing Xiao; Shiqing Song; Karangwa Eric; Chengsheng Jia; Haiyan Yu; Jiancai Zhu (2287-2293).
To characterize the aroma of cherry wine, five samples were analyzed by quantitative descriptive sensory analysis, gas chromatography–mass spectrometry (GC–MS) and gas chromatography–olfactometry (GC–O). The aroma of cherry wines was described by 6 sensory terms as fruity, sour, woody, fermentation, cameral and floral. Fifty-one odor-active (OA) compounds were detected by GC–O and quantified by GC–MS, and 45 of them were identified. Twenty-nine OA compounds having more than 50% detection frequency were selected as specific compounds correlated to sensory attributes by partial least squares regression (PLSR). The correlation result showed ethyl 2-methyl propionate, 2,3-butanedione, ethyl butyrate, ethyl pentanoate, 3-methyl-1-butanol, ethyl hexanoate, 3-hydroxy-2-butanone, ethyl lactate, 1-hexanol, (Z)-3-hexen-1-ol, ethyl hydroxyacetate, acetic acid, furfural, 2-ethyl-1-hexanol, benzaldehyde, propanoic acid, butanoic acid, guaiacol, beta-citronellol, hexanoic acid, 2-methoxy-4-methylphenol, 2-ethyl-3-hydroxy-4H-pyran-4-one, ethyl cinnamate, 2-methoxy-4-vinylphenol were typical OA compounds, which covaried with characteristic aroma of cherry wines.
Keywords: Cherry wine; Odor-active compounds; Sensory attribute; Gas chromatography–mass spectrometry; Gas chromatography–olfactometry; Partial least squares regression analysis;

Stable isotope dilution assay for liquid chromatography–tandem mass spectrometric determination of l-homoarginine in human plasma by Dorothee Atzler; Maren Mieth; Renke Maas; Rainer H. Böger; Edzard Schwedhelm (2294-2298).
Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of l-arginine catalysed by NO synthase (NOS). The l-arginine derivative l-homoarginine serves as an alternative NOS substrate releasing NO, competing with l-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography–tandem mass spectrometric (LC–tandem MS) method for the determination of l-homoarginine in human plasma by stable-isotope dilution. l-[13C6]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC–tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for l-homoarginine and l-[13C6]-homoarginine were m/z 245 →  m/z 211 and m/z 251 →  m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1–50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of l-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans.
Keywords: l-Homoarginine; Nitric oxide; LC–tandem MS; Stable-isotope dilution;

A novel electrophoric derivatisation procedure using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride for the quantitative determination of methylphenidate in human plasma is described. The drug can be quantitatively measured down to 0.006 pg/mL plasma due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent (n-hexane) and reagent solution for 15 min, which, after concentration was measured by GC–NICI–MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope labelled internal standard was used. Validation data are given to demonstrate the usefulness of the assay, including selectivity, linearity, accuracy and precision, autosampler stability, aliquot analysis, robustness, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral administration.
Keywords: NICI; Methylphenidate; PBTFBCl; Derivatisation;

A simple and solvent-minimized sample preparation technique based on two-phase hollow fiber liquid phase microextraction has been developed and used to quantify the osthole in cerebral ischemia reperfusion rat plasma following oral administration. The analysis was performed by reversed phase high performance liquid chromatography with fluorescence detection. Extraction conditions such as solvent identity, agitation rate, salt concentration, extraction time, and length of the hollow fiber were optimized. Under the optimized conditions, the linear range of osthole in rat plasma was 5–500 ng mL−1 (r 2  = 0.9997). The limit of detection (LOD) was 2 ng mL−1 (S/N  = 3) with limit of quantification (LOQ) being 5 ng mL−1. The validated method has been successfully applied for pharmacokinetic studies of osthole from cerebral ischemia reperfusion rat plasma after oral administration.
Keywords: Osthole; Hollow fiber liquid phase microextraction; HPLC; Cerebral ischemia reperfusion; Pharmacokinetics;

Quantification of trimesic acid in liver, spleen and urine by high-performance liquid chromatography coupled to a photodiode-array detection by Tarek Baati; Patricia Horcajada; Ruxandra Gref; Patrick Couvreur; Christian Serre (2311-2314).
The quantification of trimesic acid, a constitutive organic linker from the biodegradable porous iron(III) trimesate MIL-100(Fe) (MIL stands for Materials from Institut Lavoisier), has been performed in different biological complex media (liver, spleen and urine) using a liquid–liquid extraction procedure. A recovery exceeding 92 wt% was achieved from rat tissues and urine spiked with trimesic acid. After extraction, the determination of the trimesic acid concentration was realised by using a simple and accurate high-performance liquid chromatography (HPLC) method using photodiode-array detection (PDA) and aminosalicylic acid, as internal standard. Linearity of this method was kept from 0.01 to 100 mg of trimesic acid per liter of urine and from 0.05 to 5.00 wt% of trimesic acid per tissue weight. The limit of detection of the method was 0.01 μg per injection. This method was finally applied to analyze and quantify the amount of trimesic acid in rat urine and tissue samples at the different stages of degradation of MIL-100(Fe).
Keywords: Trimesic acid; MIL100; Extraction; HPLC;

Ticagrelor is the first direct acting reversibly binding oral platelet P2Y12 receptor antagonist. The parent molecule and the main metabolite (AR-C124910XX) are both able to block adenosine diphosphate-induced receptor signaling with similar potency. Drug binding to plasma proteins reduces free drug available for pharmacologic activity. Therefore, assessing unbound drug is important for interpretation of pharmacokinetic/pharmacodynamic findings. This paper describes the development and validation of an equilibrium dialysis/LC–MS/MS method for measuring unbound ticagrelor and AR-C124910XX in human plasma. Plasma samples (200 μl) were dialysed against phosphate buffered saline (37 °C, 24 h) in 96-well dialysis plates to separate unbound analytes. Drug–protein binding alterations during dialysis were minimized by maintaining physiologic conditions (pH 7.4, 37 °C). Ticagrelor and AR-C124910XX were quantified in dialysates (unbound fraction), retentates and plasma (total concentration) using liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) methods. Calibration curves were established for the retentate and plasma (total concentration) in the ranges 5–5000 ng/ml (ticagrelor) and 2.5–2500 ng/ml (AR-C124910XX), and for the dialysate in the range 0.25–100 ng/ml (both analytes). Both ticagrelor and AR-C124910XX were highly protein bound (>99.8%), i.e. unbound fraction <0.2%. Yet, the methodology was successfully applied to determine unbound concentrations of ticagrelor and AR-C124910XX in clinical samples.
Keywords: Ticagrelor; AZD6140; P2Y12 receptor inhibitor; Platelets; Equilibrium dialysis; Unbound concentration;

Determination of raloxifene and its glucuronides in human urine by liquid chromatography–tandem mass spectrometry assay by Tina Trdan; Robert Roškar; Jurij Trontelj; Matjaž Ravnikar; Aleš Mrhar (2323-2331).
A selective, sensitive, accurate and precise liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4′-β-glucuronide (M2), raloxifene-6,4′-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC–MS/MS. The method was linear in a wide range with high determination coefficient (r 2  > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.
Keywords: Raloxifene; Urine; Glucuronide; LC–MS/MS; Doping; Pharmacokinetics;

UPLC–MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues by Jiangeng Huang; Nagsen Gautam; Sai Praneeth R. Bathena; Upal Roy; JoEllyn McMillan; Howard E. Gendelman; Yazen Alnouti (2332-2338).
Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5 mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC®BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2–1000 ng/mL for RTV, IDV, and ATV, and 0.5–1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5–4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.
Keywords: Antiretroviral drugs; UPLC–MS/MS; Pharmacokinetics; Tissue distribution;

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)–ESI-MS/MS method was established for the simultaneous determination of 3,3′,4′,5,7-pentamethylquercetin (PMQ) and its possible metabolite 3,3′,4′,7-tetramethylquercetin (TMQ) in dog plasma using 4′,5,7-trimethylapigenin (TMA) as the internal standard. The plasma sample was pretreated with acetonitrile for protein precipitation and the analytes were separated on an Ultimate XB-CN column (5 μm, 2.1 mm × 150 mm) with the mobile phase consisting of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a positive multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 373.1–312.1 for PMQ, 359.1–344.0 for TMQ and 313.1–298.1 for TMA. The validated concentration ranged from 1.272 to 3060 ng/mL for PMQ and from 10.35 to 1725 ng/mL for TMQ. The lower limit of quantifications for PMQ and TMQ were 1.272 ng/mL and 10.35 ng/mL, respectively. The developed-method was successfully applied for the pharmacokinetic study of PMQ and its metabolite TMQ in dogs following a single oral dose.
Keywords: 3,3′,4′,5,7-pentamethylquercetin; 3,3′,4′,7-tetramethylquercetin; LC–MS/MS; Pharmacokinetic;

A simple HPLC-UV method for the simultaneous quantification of gefitinib and erlotinib in human plasma by Lionel Faivre; Charline Gomo; Olivier Mir; Fabrice Taieb; Audrey Schoemann-Thomas; Stanislas Ropert; Michel Vidal; Daniel Dusser; Alain Dauphin; Francois Goldwasser; Benoit Blanchet (2345-2350).
Gefitinib and erlotinib are two oral tyrosine kinase inhibitors (TKI) approved for the treatment of advanced non-small cell lung cancer (NSCLC). Published methods for simultaneous analysis of erlotinib and gefitinib in plasma are exclusively based on mass spectrometry. The purpose of this study was to develop a simple and sensitive HPLC-UV method to simultaneously quantify these two TKI in plasma. Following liquid–liquid extraction, gefitinib, erlotinib and sorafenib (internal standard), were separated with gradient elution (on a C8+ Satisfaction® using a mobile phase of acetonitrile/20 mM ammonium acetate pH 4.5). Samples were eluted at a flow rate of 0.4 ml/min throughout the 15-min run. Dual UV wavelength mode was used, with gefitinib and erlotinib monitored at 331 nm, and sorafenib at 249 nm. The calibration was linear in the range 20–1000 ng/ml and 80–4000 ng/ml for gefitinib and erlotinib, respectively. Inter- and intra-day imprecision were less than 7.2% and 7.6% for gefitinib and erlotinib, respectively. This analytical method was successfully applied to assess the steady state plasma exposure to these TKI in NSCLC patients. This simple, sensitive, accurate and cost-effective method can be used in routine clinical practice to monitor gefitinib or erlotinib concentrations in plasma from NSCLC patients.
Keywords: Gefitinib; Erlotinib; Liquid chromatography; UV detection; Cancer patients; Therapeutic drug monitoring;

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection by Kaizhou Xie; Longfei Jia; Yilin Yao; Dong Xu; Shuqing Chen; Xing Xie; Yan Pei; Wenbin Bao; Guojun Dai; Jinyu Wang; Zongping Liu (2351-2354).
A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate–acetonitrile–ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C18 column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025–5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01–10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025–2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.
Keywords: RP-HPLC-FLD; Thiamphenicol; Florfenicol; Florfenicol amine; Eggs;

Plasma free metanephrine and normetanephrine are the best biomarkers for diagnosing pheochromocytoma. In the past few years, liquid chromatography–tandem mass spectrometry has become the preferred technology to measure plasma metanephrine and normetanephrine because of its high sensitivity and specificity, as well as fast and simple sample preparation. In this study, we report a liquid chromatography–tandem mass spectrometry method for measuring plasma metanephrine and normetanephrine. A solid phase extraction method using ion-pairing reagent and C18 stationary phase was used for sample preparation. We tested a porous graphitic carbon column and a HILIC column for chromatographic separation, and the former one showed better resolution with no interference from plasma matrix. This method was linear from 7.2–486.8 pg/mL for metanephrine and 18.0–989.1 pg/mL for normetanephrine with an accuracy of 92.2–111.8% and 92.1–115.0%, respectively. Inter-assay and intra-assay CV for metanephrine and normetanephrine at two different concentration levels ranged from 2.0% to 10.9%. In conclusion, this liquid chromatography–tandem mass spectrometry method using ion-pairing solid phase extraction and porous graphitic column was simple and efficient for measuring plasma metanephrines.
Keywords: Ion pair; Metanephrines; Solid phase extraction; Porous graphitic carbon column;