Journal of Chromatography B (v.879, #7-8)

Quantification of CPT13 in rat plasma using LC–MS/MS for a pharmacokinetic study by Qing Yong Li; Lin Su; Yuan Gang Zu; Lin Zhang; Yang Gao; Chun Cheng Wang; Qiao Chu Zhu; Xiao Qiu Deng (461-466).
A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r 2  = 0.9998) over the concentration range of 1–1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34–110.67%. CPT13 in rat plasma was stable when stored at −20 °C or 4 °C for three freeze–thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.
Keywords: CPT13; Camptothecin; LC–MS/MS; Quantification; Pharmacokinetics;

Simultaneous bioanalysis of l-arginine, l-citrulline, and dimethylarginines by LC–MS/MS by Soyoung Shin; Sun-Mi Fung; Srinidi Mohan; Ho-Leung Fung (467-474).
l-Arginine (ARG) is converted to nitric oxide (NO) and l-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography–mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (15N4-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.Concentrations of unlabeled ARG, 15N4-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. 13C6-ARG, D4-CIT and D7-ADMA were used as internal standards for ARG and 15N4-ARG, CIT, and dimethylarginines, respectively.The calibration curves of ARG, 15N4-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to 15N4-ARG, which allowed the observation of generation of 15N3-CIT and 15N3-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.An LC–MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.
Keywords: l-Arginine; l-Citrulline; Asymmetric dimethylarginine; Symmetric dimethylarginine; LC–MS/MS;

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography by Maria João Santos; José A. Teixeira; Lígia R. Rodrigues (475-479).
A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.
Keywords: Whey proteins; β-Lactoglobulin; Hydrophobic interaction chromatography; Recovery; Isolation;

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography by Xueli Cao; Qiaoe Wang; Yan Li; Ge Bai; Hong Ren; Chunming Xu; Yoichiro Ito (480-488).
Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane–methanol–acetonitrile (1.5:0.5:0.5, v/v) and n-heptane–methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane–ethyl acetate–methanol–water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol–ethyl acetate–water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC–MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.
Keywords: Counter-current chromatography; Hypericum perforatum L.; Hyperforin; Hypericin; Rutin; Hyperoside;

A gas chromatography–mass spectrometric (GC–MS) method was developed for the determination of 2-naphthol (2-NAP) and 1-hydroxypyrene (1-HOP) in human urine. Extraction from urine after the enzyme hydrolysis with β-glucuronidase/arylsulfatase was achieved with a liquid extraction using 5 mL of pentane. After addition of 50 μL of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA) to prevent the loss of 2-NAP during drying, the extract was completely dried and derivatized with MTBDMSTFA for 30 min at 60 °C. The accuracies were in the range of 96–109% at a concentration of 0.5, 10 and 25 μg/L and their precisions were less than 15%. Method detection limits of 2-NAP and 1-HOP were 0.07 and 0.01 μg/L, respectively. This method was used to analyze twenty urine samples, and they were found in the concentration range <0.07–13.7 μg/L (2-NAP) and <0.01–0.88 μg/L (1-HOP). The concentrations of 2-NAP and 1-HOP were well correlated to those of naphthalene and pyrene in blood, respectively.
Keywords: GC–MS; 1-Hydroxy pyrene; 2-Naphthol; Urine; MTBDMSTFA silylation;

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC–MS/MS by Ulrike Harder; Berthold Koletzko; Wolfgang Peissner (495-504).
Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3 N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6–10.1%. Absolute accuracy as determined by comparison of external reference samples was 82–117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.
Keywords: Amino acids; Ion-pair chromatography; Qualifier ions; Butyl esters; Metabolomics; LC–MS/MS;

To evaluate the effect of a pseudo-carrier (9-hydroxypropyl-(+)-dihydrotetrabenazine, AV-149) on pharmacokinetics of 9-fluoropropyl-(+)-dihydrotetrabenazine (AV-133), an ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method was developed and validated for the determination of AV-133 and AV-149 in rat plasma. AV-133 and AV-149 were extracted from plasma following protein precipitation. The chromatographic analysis was performed on an ACQUITY UPLC BEH™ C18 column (50 mm × 2.1 mm × 1.7 μm) by a gradient elution. The mass spectrometer was operated in positive mode using electrospray ionization. The analytes were measured using the multiple reaction-monitoring mode (MRM). An external calibration was used, and the calibration curves were linear in the range of 1.00–800 ng/mL for AV-133 and AV-149. The accuracy ranged from 90.8% to 113.2% and the precision ranged from 2.7% to 9.9% for each analyte. The effect of a pseudo-carrier on pharmacokinetics of AV-133 was studied using the presented method.
Keywords: AV-133; Pharmacokinetics; UPLC–MS/MS; Positron emission tomography; PET imaging agents;

Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography by Amparo Ramos; F.Gabriel Acién; José M. Fernández-Sevilla; Cynthia V. González; Ruperto Bermejo (511-519).
In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90–93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.
Keywords: Expanded bed adsorption chromatography; Synechocystis aquatilis; Phycobiliproteins; C-phycocyanin; Preparative protein purification; Anion-exchange chromatography;

d-erythro-sphingosine (Sph) and its phosphorylated product, d-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to simultaneously quantify the levels of Sph and S1P in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC–MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol–0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1–100 ng/mL for Sph and 0.1–10 ng/mL for S1P with correlation coefficient (r 2) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36–89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α whereas decreased in cells treated with DMS. These results indicated that this new LC–MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and S1P levels in biological samples simultaneously.
Keywords: Sphingosine; Sphingosine 1-phosphate; C17-sphingosine; C17-sphingosine 1-phosphate; Simultaneous determination; Liquid chromatography–tandem mass spectrometry (LC–MS/MS);

Quantitative determination of allele frequency in pooled DNA by using sequencing method by Peng Cao; Qiu-Jun Wang; Xu-Ting Zhu; Huan Zhou; Rui Li; Wei-Peng Wang (527-532).
Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r 2  = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.
Keywords: Allele frequency; Pooled DNA; Single-nucleotide polymorphism; Sequencing;

A rapid, sensitive, and specific method for the determination of amoxicillin (AMO), amoxicilloic acid (AMA), amoxicillin diketopiperazine-2′,5′-dione (DIKETO), penicillin G (PEN G), benzylpenicilloic acid (BPA-1), benzylpenilloic acid (BPA-2), and benzylpenillic acid (BPA-3) in bovine milk using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was developed and validated. The method used penicillin V (PEN V) as the internal standard and ethanol for the deproteinisation of bovine milk. Chromatographic separation of the components was performed on a Waters Acquity UPLC® HSS T3 column (100 mm × 2.1 mm, 1.8 μm) using a mixture of 0.15% formic acid in water with 5 mM ammonium acetate and acetonitrile as the mobile phase. Gradient elution was performed at a flow rate of 0.25 mL min−1. The mass spectrometer was operated in the positive electrospray ionisation MS/MS mode. The method was fully validated according to EU requirements, including linearity, precision, trueness, limit of quantification, limit of detection, and specificity. The results were within the ranges specified. The established method was successfully applied in the determination of AMO, PEN G, and their major metabolites in 40 commercial bovine milk samples. The results showed that 8 samples were contaminated with BPA-1 or BPA-2. The mean levels (occurrence) of BPA-1 and BPA-2 in positive samples were 287 (50%) and 320 (100%) ng mL−1, respectively. No sample was found to be contaminated with AMO, AMA, DIKETO, PEN G, and BPA-3. These findings could play an important role in food safety, because BPA-1 and BPA-2 metabolites pose possible health risks, although they are not included in the maximum residue limit legislation.
Keywords: Ultra-high-performance liquid chromatography–tandem mass spectrometry; Amoxicillin; Penicillin G; Metabolite; Bovine milk;

Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals by Eunyoung Han; Martin P. Paulus; Marc Wittmann; Heesun Chung; Joon myong Song (541-547).
The questions of whether the dose of drug that is consumed corresponds to drug concentration levels in hair and how results of hair analyses can be interpreted are still debated. The aim of this study was to investigate (1) whether there is a correlation between doses of Methamphetamine (MA) use and MA concentration levels in hair and (2) whether results of hair analyses can be used to estimate dose, frequency, and patterns of MA use. In this study, segmental hair analysis was performed through consecutive 1 cm as well as 1–4 cm (=3 cm) segmental hair lengths. MA dependent individuals (n  = 9) provided information on doses (0.25–4 g/day) of MA use as well as the frequency of MA use. The concentrations of MA and its metabolite amphetamine (AP) in hair were determined using gas chromatography/mass spectrometry (GC/MS). One-way analysis of variance (ANOVA) test was performed to evaluate whether MA and AP concentrations in consecutive 1 cm length segmental hair were consistent with the history of MA use. The cumulative doses of MA use calculated from the daily dose and the frequency during 1–4 months were well correlated to the concentrations of MA and AP in 1–4 cm segmental hair length (correlation coefficient, r  = 0.87 for MA and r  = 0.77 for AP). The results from this study show the patterns and histories of MA use from MA dependent individuals and could assist in the interpretation of hair results in forensic toxicology as well as in rehabilitation and treatment programs.
Keywords: Methamphetamine; Hair analysis; Drug history; Segmental analysis; GC/MS;

A sensitive and selective liquid chromatographic-tandem mass spectrometric method was developed for quantification of triamcinolone acetonide (TA) in rabbit ocular tissues. After a simple liquid–liquid extraction using tert-butyl methyl ether, TA and internal standard methylprednisolone were separated on a C18 column with a mobile phase of acetonitrile:water:formic acid (60:40:0.1, v/v/v) and quantified by the use of selected reaction monitoring mode with a total run time of 4 min. The method was validated in tissue homogenates with a daily working range of 1–1000 ng/mL with correlation coefficient of more than 0.99 and a sensitivity of 1 ng/mL as lower limit of quantification, respectively. The mean intra-day and inter-day precisions were less than 10% and accuracy values were higher than 90%. This method was fully validated for the accuracy, precision and stability studies. The method proved to be accurate and specific, and was applied to an in vivo biodistribution study of TA after intravitreal injection to rabbits. Values of mean residence time in vitreous humor, crystalline lens and aqueous humor were 27.7, 35.8 and 20.0 days, respectively.
Keywords: Triamcinolone acetonide; Liquid chromatography–tandem mass spectrometry; Ocular biodistribution;

Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients by Charbel Abou-Diwan; Roman M. Sniecinski; Fania Szlam; James C. Ritchie; Jeanne M. Rhea; Kenichi A. Tanaka; Ross J. Molinaro (553-556).
A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC–MS/MS) using ɛ-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 μL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7 μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1–10.0 μg/mL, with lower limit of quantitation of 0.1 μg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).
Keywords: Mass spectrometry; Tranexamic acid; Plasma; Cerebral spinal fluid; Cardiopulmonary bypass surgery;