Journal of Chromatography B (v.876, #2)

A liquid chromatography–tandem mass spectrometric method has been developed for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate. The analytes were separated within 5 min using an anion exchange/reverse phase column. The lower limit of quantification for Glu, NAA and NAAG was found to be 5, 50 and 6 nM, respectively, with a signal-to-noise ratio of 5:1. Using this methodology the basal levels of Glu, NAA and NAAG could be measured consistently in in vitro superfusion samples from rat hippocampus. The assay was also used for measurement of the distribution of Glu, NAA and NAAG in different regions of the rat brain.
Keywords: N-Acetylaspartic acid; N-Acetylaspartylglutamic acid; Glutamic acid; Liquid chromatography–tandem mass spectrometry; Superfusion; Ex vivo;

A sensitive LC–MS method was developed and validated for the determination of niflumic acid (NFA), the active metabolite of the talniflumate formulation, in human plasma. The analyses were performed on C18 column using acetonitrile–ammonium acetate buffer (pH 5.7, 40:60) as a mobile phase with quadrupole MS detection of NFA at m/z 281 in a negative ion-monitoring mode. Calibration curve was linear in the concentration range of 1–1000 ng/mL in human plasma. The higher sensitivity of LC–MS allowed low concentrations of NFA to be determined at initial drug absorption and terminal elimination phases following oral administration of talniflumate tablet.
Keywords: Niflumic acid; Talniflumate; LC–MS; Pharmacokinetics;

This paper reports an LC/MS/MS method for analysis of salemeterol and fluticasone propionate in human plasma based on combined SPE-based extraction and separate LC/MS/MS conditions. Previously reported interaction between analytes was confirmed and eliminated by their separation in the sample preparation step to ensure no negative impact on their quantitation. The method was validated per FDA guidelines in the range of 2.5–500 pg/mL for salmeterol and 5–500 pg/mL for fluticasone propionate. The method is suitable for plasma analysis of combined salmeterol/fluticasone formulation without adverse effects of inter-analyte interactions on quantitation.
Keywords: Fluticasone; Salmeterol; Interaction; Human plasma; Liquid chromatography; Mass spectrometry;

In order to find potential new biomarkers of cisplatin-induced apoptosis and necrosis, volatile organic compounds (VOCs) from cisplatin-treated human lung cancer cell lines were investigated. The biological system employed was human non-small cell lung carcinoma A549 cell lines. The cell lines were treated with two different concentrations of cisplatin, 100 μM and 400 μM, and apoptosis and necrosis were determined by flow cytometric analysis. For each drug concentration, the VOCs from the treated cell lines were extracted by solid-phase microextraction (SPME), and subsequently analyzed by gas chromatography/mass spectrometry (GC/MS). The compounds that change during cisplatin-induced apoptosis and necrosis of lung cancer cell lines can serve as new biomarkers. The pharmacometabolomic approach presented in this study, significantly, implicates a non-destructive, sample-thrifty and time-saving tool for finding new biomarkers for the assessment of drug-induced cell death pathways.
Keywords: Cisplatin; SPME; GC/MS; Volatile organic compounds; Biomarkers; Apoptosis; Necrosis;

An efficient multiresidue method for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in tilapia fish muscle, based on high performance liquid chromatography with UV detection (HPLC-UV), has been developed. The drugs were extracted with 0.2% orthophosphoric acid–methanol (6:4), and the extracts were cleaned up on a solid phase extraction cartridge, C18 Sep-Pak® light column. The LC separation was performed on a RP stainless-steel C-18 analytical column (150 mm × 4.6 mm, 5 μm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 2.4–acetonitrile as the mobile phase at the flow rate of 1.0 ml min−1. A wavelength programming was applied for the UV detection of the analytes. The method not only enabled the determination of the parent drugs, MET and SPY, but also permitted the determination of their metabolites, hydroxymetronidazole (HMET) and neospiramycin (NSPY). The calibration graphs for each drug were rectilinear in the range of 0.005–1.000 μg g−1 for MET and HMET and 0.025–1.000 μg g−1 for SPY and NSPY. With this method, the cited drugs with their metabolites were determined in fortified fish muscle tissues at levels of 0.025, 0.1 and 1.0 μg g−1 with good accuracy and precision. LOD and LOQ obtained for each drug were as follows: 0.002 and 0.005 μg g−1 for MET and HMET and 0.005 and 0.025 μg g−1 for SPY and NSPY. Utilization of the method to successfully analyze tilapia fish muscle samples incurred with MET and SPY was described.
Keywords: HPLC-UV; Metronidazole; Spyramicin; Metabolites; Fish;

Definitive information on the metabolism of a drug candidate in humans is achieved through dosing radiolabelled drug as part of a clinical study, and is typically conducted post-proof of concept in Phase III of the clinical development plan. Here we describe a novel approach, using preparative high performance liquid chromatography and cryoprobe-nuclear magnetic resonance spectroscopy, to determine the human systemic exposure to a drug and its metabolites using samples derived from Phase I clinical studies. Using the described methodology, novel human plasma metabolites, as low as 10 ng/ml can be detected and quantified. This provides an opportunity, early in the development process to understand the potential role of metabolites in the safety and efficacy of drugs in humans.
Keywords: Metabolites; Human plasma; Preparative HPLC; NMR; Quantification;

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of acrylamide (AA) and its genotoxic metabolite glycidamide (GA) with a test marker antipyrine (AP) in placental tissue and perfusion medium used in human placental perfusion studies. An internal standard (13C-acrylamide) was added to the samples which were then deproteinized with acetonitrile. Chromatographic separation was performed on a reversed phase column with a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min. Detection and quantification of the analytes were carried out with a triple quadrupole mass spectrometer using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The method was validated and linear over a concentration range of 0.5–20 μg/mL for acrylamide and glycidamide and 5–200 μg/mL for antipyrine. The lower limit of quantification for acrylamide and glycidamide was 0.5 μg/mL and for antipyrine 5 μg/mL. The method was selective, and good accuracy, precision, recovery, and stability were obtained for concentrations within the standard curve. The method was successfully used to analyze the placental perfusion medium and tissue samples in a toxicokinetic study for transplacental transfer of acrylamide and glycidamide. This is the first time that acrylamide, glycidamide and antipyrine are measured simultaneously.
Keywords: Acrylamide; Glycidamide; LC–MS/MS; Perfusion medium; Tissue homogenate;

A 75-kDa protein secreted from mouse coagulating gland was purified to homogeneity by a series of isolation steps including ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was identified to be Type IV transglutaminase (TG4), based on the establishment of N-terminal sequences by automated Edman degradation together with partial sequences by MS analysis. Its cross-linking activity was tested on the reduced sample of mouse seminal secretion which contained seven major monomer proteins tentatively designated as SVS I–VII. The enzyme was able to cross-link any of SVS I–III but failed to cross-link the other SVS proteins with a M r value less than 14 kDa. SVS I and SVS III showed comparable substrate activity, but were much weaker than SVS II during the TG4 catalysis.
Keywords: Coagulating gland; Protein cross-link; Protein identification; Seminal coagulation; Seminal vesicle; Transglutaminase;

For the evaluating of the absorption, tumor affinity and metabolism of wilfoside C3N and wilfoside C1N (two main C21 steroidal glycosides from Baishouwu), an LC–MS/MS method was developed. Plasma or tumor homogenate samples were extracted by liquid–liquid extraction with ethyl acetate after internal standard (ginsenoside Rh2) spiked. The separation was performed by a Luna C18 column (3.0 μm, 2.0 mm × 50 mm) with gradient elution. The method was fully validated and successfully applied to determine the concentrations of the parent drugs and metabolites after intragastric administration of wilfoside C3N and wilfoside C1N to mice respectively.
Keywords: Liquid chromatography–tandem mass spectrometry; Wilfoside C3N; Wilfoside C1N; Metabolites; Mice plasma; Tumor homogenate; Deoxy sugar;

We report the simultaneous screening of highly polar, water-soluble, and less-volatile herbicides, including glyphosate, glufosinate, paraquat, and diquat, in serum using liquid chromatography–mass spectrometry. The herbicides were separated by solid-phase extraction using a Strata-XC cartridge. A heptafluorobutyric acid solution was chosen as the mobile phase for ion-pair liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The serum specimens were screened for the presence of the herbicides at the following concentrations: 5 ng/mL for glyphosate, 2 ng/mL for glufosinate, 1 ng/mL for diquat, and 5 ng/mL for paraquat. This is the first report on the simultaneous detection of these compounds.
Keywords: LC–MS; Ion-pair; Herbicides; Glyphosate; Paraquat; HFBA;

For the first time, a rapid, sensitive and simple liquid chromatography/tandem mass spectrometry (LC–MS/MS) method using an atmospheric pressure chemical ionization (APCI) source for the quantification of PD168393 in rat serum was developed and validated. Serum samples were pretreated with methanol for protein precipitation. The chromatographic separation was performed on a Jupiter-C5 column (250 mm × 2.0 mm i.d.) pre-equilibrated with 0.1% formic acid. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 369/313 for PD168393 and m/z 343/308 for the internal standard triazolam, using positive ion mode. The MS/MS response was linear over the concentration range from 2 ng/mL to 5000 ng/mL, with a lower limit of quantification (LLQ) of 2 ng/mL. At the lowest quality control (4 ng/mL), the intra- and inter-day precisions (CV%) for PD168393 were less than 10% and the accuracies were between 92% and 111%. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies.
Keywords: LC–MS/MS; PD168393; Quantification; Serum;

Validation of an extended method for the detection of the misuse of endogenous steroids in sports, including new hydroxylated metabolites by P. Van Renterghem; P. Van Eenoo; W. Van Thuyne; H. Geyer; W. Schänzer; F.T. Delbeke (225-235).
Endogenous steroids are amongst the most misused doping agents in sports. Their presence poses a major challenge for doping control laboratories. Current threshold levels do not allow for the detection of all endogenous steroid misuse due to great interindividual variations in urinary steroid concentrations. A method has been developed and validated to screen for traditionally monitored endogenous steroids in doping control as well as specific hydroxylated/oxygenated metabolites in order to enhance the detection capabilities for the misuse of endogenous steroids.
Keywords: Endogenous steroids; Steroid profiling; Doping;

An accurate, selective and sensitive bioanalytical method has been developed and validated for the simultaneous quantification of alfuzosin and solifenacin in human plasma using propranolol as internal standard (IS). The analytes and IS were extracted in methyl tert-butyl ether, separated on Hypurity C8 column and detected by tandem mass spectrometry with a turbo ion spray interface. The method had a chromatographic run time of 3.0 min and linear calibration curves over the concentration range of 0.25–25 ng/mL for alfuzosin and 0.6–60 ng/mL for solifenacin. The intra- and inter-day accuracy and precision (%CV) evaluated at four quality control levels were within 88.2–106.4% and 0.9–7.7% respectively. The absolute recovery from spiked plasma samples was 71.8% for alfuzosin and 93.1% for solifenacin. Stability of alfuzosin and solifenacin was assessed under different storage conditions. The validated method was successfully employed for bioavailability study after oral administration of 10 mg of alfuzosin hydrochloride and 5 mg of solifenacin succinate tablet formulations in eight healthy volunteers under fed condition.
Keywords: Alfuzosin; Solifenacin; LC–ESI-MS/MS; Liquid–liquid extraction; Bioavailability study;

A new, sensitive method was developed for the determination of the neurotoxin domoic acid (DA) using a reversed phase separation followed by post-column derivatization (PCD) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and subsequent fluorescence detection. The PCD conditions which involves a two-step reaction was fully optimized for the lowest detection limit. The first reaction occurs between DA and NBD-Cl while the second makes possible the detection of the derivative causing the destruction of the interfering fluorescent 4-hydroxy-7-nitrobenzo-2-oxa-1,3-diazole (NBD-OH) which is the hydrolysis product of NBD-Cl. Kainic acid a similar base structure compound with DA was used as an internal standard. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LOD 25 ppb in real samples of mussel extracts), it requires less sample preparation, and it gives clean simple chromatograms without chromatographic interferences from coeluting compounds such as tryptophan. The method was successfully applied to for the quantitative determination of DA in mussel tissues at quantities as low as 75 μg/kg tissue.
Keywords: Domoic acid; Kainic acid; Shellfish poisoning; Biotoxins; Post-column detection; NBD-Cl; HPLC post-column fluorescence detection;

Comprehensive proteomic analysis of the human milk proteome: Contribution of protein fractionation by A. Mangé; V. Bellet; E. Tuaillon; P. Van de Perre; J. Solassol (252-256).
In-depth analysis of the milk proteome by mass spectrometry is challenged by the presence of few high-abundance proteins that interfere with the detection of lower-abundance proteins. Here, we evaluated the proteomic analysis of milk samples following a strong anion exchange fractionation procedure using denaturating conditions ensuring the disruption of protein–protein interactions. Crude whey or skim milk and their different resulting fractions were analyzed by protein chip array mass spectrometry. Using protein chip array mass spectrometry, several high-abundance proteins were localized in distinct fractions increasing the total number of unique peptides and proteins detected. This total number increased by about 20–30% by combining different chromatographic surface arrays used for capture. Reproducible results were obtained in human skim milk and whey; however this approach was not successful with milk fat globule membrane and required refinement. Hence, milk profiling by anion exchange fractionation combined to protein chip array mass spectrometry represents a promising tool to detect unknown low-abundance milk proteins that may ultimately prove useful as biomarkers of diseases transmitted by breastfeeding.
Keywords: Fractionation; Milk; Proteomics; Low-abundance proteins;

Rapid and simultaneous determination of hair polyamines as N-heptafluorobutyryl derivatives by gas chromatography–mass spectrometry by Ling Li; Kenji Hara; Junting Liu; Yanping Yu; Lina Gao; Yan Wang; Yanfang Wang (257-260).
We have developed a simple and sensitive method for the simultaneous determination of putrescine, spermidine and spermine in hair as their N-heptafluorobutyryl derivatives by gas chromatography–mass spectrometry (GC–MS) in selected ion-monitoring (SIM) mode. After base hydrolysis, hair samples were extracted with solid-phase extraction (SPE) with diatomaceous earth columns, followed by derivatization with heptafluorobutyryl chloride (HFB-Cl) and elution with n-hexane simultaneously. This method was linear (r  ≥ 0.9989), reproducible (intra-day R.S.D. = 3.4–15.5%, inter-day R.S.D. = 2.6–14.6%), accurate (recoveries = 67.8–94.6%) and sensitive (LOD = 0.05–1.0 ng). The method was successfully applied to the analysis of 36 hair samples from 14 healthy men and 22 healthy women. Results showed that the levels of hair polyamines were 4.39–12.15 μg/g for putrescine, 3.89–27.91 μg/g for spermidine, and 0.81–15.15 μg/g for spermine. Either in the male or female group, the most abundant hair polyamine was spermidine, followed by putrescine and spermine, while the mean levels of the three polyamines in hair samples were all found to be higher in men than in women.
Keywords: Polyamines; Hair; Derivatization; GC–MS;

A quantitative reverse-phase HPLC method with UV detection, for lumefantrine (LF) and desbutyllumefantrine (DLF) in whole blood spotted on filter paper was developed. The analytes were stabilized on filter paper by treatment of blood with phosphoric acid (1.6 mol/L). Halofantrine was used as internal standard and the analytes were extracted from filter paper using methanol. The methanolic extract was extracted with di-isopropylether after addition of acidic phosphate buffer (pH 2). Chromatographic separation was carried out on a Zorbax Eclipse XDB-phenyl column (4.6 mm × 150 mm, particle size 5 μm) at a flow rate of 1 mL/min using a mobile phase of acetonitrile–ammonium acetate buffer (0.1 M ammonium acetate and 0.01 M acetic acid, pH 6.5) (10:90). The absorbance of the compounds was monitored at 335 nm. The average extraction recovery from filter paper ranged between 45–51% for LF and 25–33% for DLF for a concentration range between 300 and 3000 nM. Inter- and intra-assay coefficients of variation for LF and DLF were ≤9.2. Limits of quantification for LF and DLF were 300 nM. The method has been applied in malaria patients. In conclusion, a simple procedure for blood sampling and quantitative measurement of lumefantrine and desbutyllumefantrine suitable for field studies in resource-limited laboratories was developed.
Keywords: Lumefantrine; Desbutyllumefantrine; Filter paper;

A method was developed and fully validated for simultaneous quantification of methamphetamine (MAMP), amphetamine, hydroxy-methamphetamine, methylenedioxymethamphetamine (MDMA, ecstasy), methylenedioxyamphetamine, 3-hydroxy-4-methoxy-methamphetamine, and 3-hydroxy-4-methoxy-amphetamine in 100 μL mouse plasma and 7.5 mg brain. Solid phase extraction and gas chromatography–electron impact ionization mass spectrometry in selected-ion monitoring mode achieved plasma linear ranges of 10–20 to 20,000 ng/mL and 0.1–0.2 to 200 ng/mg in brain. Recoveries were greater than 91%, bias 92.3–110.4%, and imprecision less than 5.3% coefficient of variation. This method was used for measuring MAMP and MDMA and metabolites in plasma and brain during mouse neurotoxicity studies.
Keywords: Methylenedioxymethamphetamine; Methamphetamine; Gas chromatography; Mass spectrometry; Tissue; Mouse;

A HPLC-tandem mass spectrometry method was developed and validated for the quantitation of intact oxaliplatin in human plasma. Plasma ultrafiltrates were precipitated with acetonitrile and separation was performed on a 250 mm Beckman ODS reverse phase column using a gradient mobile phase. The mass spectrometer was operated in positive ionization mode using TurboionSpray and precursor–product ion combinations of m/z 391.1 → 305.1 and 371.1 → 247.0 were monitored for oxaliplatin and carboplatin, the internal standard, respectively. The lower limit of quantitation for oxaliplatin was 20 ng/ml. The linear range of the method was 20–1000 ng/ml. The between- and within-day relative standard deviations ranged from 3.1 to 7.7%, and accuracy was within 5%. This method was successfully applied in a clinical study of oxaliplatin.
Keywords: Oxaliplatin; HPLC; Mass spectrometry;

A simple, sensitive and specific liquid chromatography tandem mass spectrometry method with minimal sample pretreatment was developed for the simultaneous analysis of sildenafil and its metabolite desmethylsildenafil in human serum. Sample pretreatment consisted of adding a methanolic solution of the internal standard vardenafil to the samples. After vortexing and centrifugation the samples were directly injected onto the C18 column using gradient elution. The aqueous and organic mobile phases were ammonium acetate 2 mM supplemented with 0.1% formic acid in water and methanol, respectively. The detection by a triple quadrupole mass spectrometer in positive ESI ionization mode was completed within 5 min. The lower limits of quantification for sildenafil and desmethylsildenafil are 1.0 ng/ml. The intra- and inter-day precisions measured as relative standard deviation were within 10% for both compounds over the linear range. Intra- and inter-day accuracy of sildenafil and desmethylsildenafil ranged from 92 to 103%. This method has been used in a clinical pharmacokinetic study of sildenafil in intensive care patients.
Keywords: Sildenafil; Vardenafil; LC/MS/MS; Protein precipitation; Human serum;