Journal of Chromatography B (v.879, #22)

Ginsenoside Re (G-Re) improved the memory function of experimental animals in a preclinical study. Several types of saponins including G-Rg1, G-Rg2, G-F1, G-Rh1, and protopanaxatriol (PPT) may be the metabolites of G-Re according to reports from preclinical trials. In order to support a study of the pharmacokinetics of G-Re, an analytical method for G-Re and the co-detection of its probable metabolites using liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid phase extraction was utilized in the sample preparation. Separation of the analytes was achieved using a gradient elution (0.05% formic acid–methanol–acetonitrile, each organic phase containing 0.05% formic acid) at a flow rate of 0.3 mL/min with a retention time of approximately 2.88 min for G-Re. Data were acquired in the multiple reaction mode (MRM) and the linear range of the standard curve of plasma and urine samples for G-Re was 0.05–20 ng/mL with r 2  ≥ 0.99. In the analysis of probable metabolites, G-Re, G-Rg1, G-F1, G-Rh1 and PPT were all detected in samples; however, G-Rg2 was not detected.
Keywords: Ginsenoside Re; LC–MS/MS; Metabolite; Pharmacokinetics;

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of paclitaxel (Taxol) and its two major metabolites in human plasma has been developed. Samples were prepared after liquid–liquid extraction and analyzed on a C18 column interfaced with a Q-Trap tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.05% formic acid) (65:35) at the flow rate of 0.25 mL/min. The analytes and internal standard docetaxel were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 0.5–500.0 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3′-hydroxypaclitaxel, respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL for paclitaxel, 6α-hydroxypaclitaxel and p-3′-hydroxypaclitaxel, respectively. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 8.18%. The accuracy determined at three concentrations was within ±10.8% in terms of relative error. The total run time was 7.0 min. This assay offers advantages in terms of expediency, and suitability for the analysis of paclitaxel and its metabolites in various biological fluids.
Keywords: Paclitaxel; LC–MS–MS; Metabolites; Pharmacokinetics;

Development and validation of a sample stabilization strategy and a UPLC–MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF) by Yanhua Zhang; F. David Tingley; Elaine Tseng; Max Tella; Xin Yang; Elizabeth Groeber; Jianhua Liu; Wenlin Li; Christopher J. Schmidt; Rick Steenwyk (2023-2033).
A UPLC–MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core–Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC–MS/MS. This UPLC–MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9 ± 0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC–MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025–5 ng/mL for ACh and 0.05–10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9–12.3% CV and −10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0–16.0% CV and −5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4 h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1 h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.
Keywords: Acetylcholine; Histamine; tele-Methylhistamine; tele-Methylimidazolacetic acid; Biomarker; Biological stability; Rat cerebrospinal fluid; UPLC–MS/MS; Donepezil; Prucalopride;

In this work, an automated screening method for the simultaneous identification and quantitation of 30 representative multiclass drugs (including opiates, cocaine and its main metabolite, cannabinoids, amphetamines and other stimulants in hair samples) has been developed using fast liquid-chromatography time-of-flight mass spectrometry (LC-TOFMS). The identification and quantitation of the drugs were carried out by liquid chromatography using a C18 column (4.6 × 50 mm) with 1.8 μm particle size. Accurate mass measurements of ions of interest (typically [M+H]+) by electrospray time-of-flight mass spectrometry in the positive ionization mode were used for unambiguous confirmation of the targeted species. Three sample preparation methodologies were evaluated: (a) direct methanolic extraction by sonication, (b) acidic extraction, and (c) alkaline digestion. Direct methanolic extraction showed better recoveries and cleaner extracts. The limits of detection obtained in hair matrix were as low as 5 pg mg−1 for cocaine and cannabidiol, ranging from 5 to 75 pg mg−1 for the studied species while the LOQ ranged from 15 to 250 pg mg−1. The method has been applied to six hair samples from drug consumer volunteers, where the presence of at least one drug was confirmed by accurate mass measurements within 2 ppm (mass error) in most cases. The present study demonstrates the usefulness of LC-TOFMS for both screening and quantitation purposes in drug testing in hair. In addition, the possibility of non-target or a posteriori data analysis of samples or the extension of the procedure for testing for additional compounds offers interesting features for forensic analysis.
Keywords: Hair analysis; Drugs; Liquid chromatography; Mass spectrometry; Time-of-flight;

Perfluorochemicals (PFC's) are widely spread in the environment and have been detected in blood of wildlife and humans world-wide. Recently, various toxic effects of PFC's in laboratory rats have been demonstrated, resulting in increased government concerns regarding the presence of PFC's in the environment and the implications they have on human health. In the last decade, various analytical methods have been developed for the analysis of PFC's in different matrices whereby the majority of methods have utilised liquid chromatography coupled with mass spectrometry (LC–MS). Here we describe an optimized method for the quantitation of PFC's, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in food packaging, polytetrafluoroethylene (PTFE) sealant tape and drinking water. The method involved PFC's extraction via off-line SPE followed by separation using reversed-phase liquid chromatography on a Phenyl–Hexyl column coupled with ion-trap (IT) mass spectrometric detection. The optimized approach minimized ion-suppression effects commonly seen with conventional elution buffers, improving detection limits down to 25 pg/mL and allowed effective quantitation down to 50 pg/mL for PFOA and PFOS. The optimized LC–MS method detected PFOA and other PFC's in microwave popcorn packaging and PFOA in PTFE sealant tape in the low μg/kg. In all samples, PFOS was not detected.
Keywords: Liquid chromatography; Ion-trap mass spectrometry; Perfluorooctanoic acid; Perfluorooctane sulfonate; Perfluorochemicals;

Determination of micafungin and anidulafungin in human plasma: UV- or mass spectrometric quantification? by Jens Martens-Lobenhoffer; Victoria Rupprecht; Stefanie M. Bode-Böger (2051-2056).
Micafungin and anidulafungin are two newer antifungal drugs from the echinocandine class. They are used as monotherapy or in combination with azole-antifungal drugs. The optimized clinical treatment course for the echinocandin drugs with regard to the different infection types and patient subgroups (renal or hepatic impairment, overweight) is still under debate. Therefore, an easy and rugged assay for these two drugs is highly desirable. We here present a method for the quantification of micafungin or anidulafungin in human plasma, applying protein precipitation as sample preparation, reversed phase separation of the analytes and UV-detection and simultaneous tandem mass spectrometry. Anidulafungin served as I.S. for micafungin quantification and vice versa. The method was validated in the calibration ranges from 0.1 μg/ml to 20 μg/ml for both substances. Intra-day precision and accuracies recorded with the UV-detector were 1.80% and 2.65% for micafungin and 4.30% and 10.44% for anidulafungin at the 0.1 μg/ml level. The respective data at the 1 μg/ml level were 2.25% and −0.83% for micafungin and 4.35% and −1.85% for anidulafungin and at the 20 μg/ml level 0.97% and −2.98% for micafungin and 1.04% and 4.74% for anidulafungin, respectively. With the mass spectrometer, because of the unique properties of the analyte molecules, no acceptable validation results could be achieved. Therefore, the mass spectrometric chromatograms served only as identity confirmation of the observed UV-peaks.
Keywords: Anidulafungin; Micafungin; HPLC; UV; Mass spectrometry;

A conventional scale online two dimensional liquid chromatography-ultraviolet/mass spectrometric (2DLC–UV/MS) method was developed for simultaneous quantitation of intact proteins. A series of valve switches were utilized between the two LC dimensions and the mass spectrometer to resolve and confirm the proteins of interest from a complex biological matrix. Two model proteins, myoglobin and serum albumin were simultaneously resolved and quantitated from Escherichia coli lysate using a strong anion-exchange chromatography and reversed-phase chromatography as the first and second dimension respectively. The method validation consisted of evaluating linearity, precision, and accuracy. A linear relationship (R 2  > 0.99) between the concentrations of the two proteins and peak areas was observed over the concentration range; 12.0–120.4 μg/mL and 8.5–85.4 μg/mL for serum albumin and myoglobin, respectively. The average RSD of peak areas for intra-day and inter-day analyses were 5.9% and 9.4% for myoglobin and 6.2% and 10.1% for serum albumin respectively. Over the linear range, the recoveries ranged from −15.4 to 9.0% for serum albumin and −2.5 to 9.4% for myoglobin. The system presented in this work is amenable to a quality control environment for evaluation and quantitation of expression levels of multiple target proteins. To our knowledge, this represents the first 2DLC–UV/MS method depicting the viability of simultaneous quantitation of more than one intact protein from complex biological mixtures in a single run.
Keywords: Multidimensional chromatography; 2DLC; Quantitation; Intact proteins;

A rugged and accurate liquid chromatography–tandem mass spectrometry method for quantitative determination of BMS-790052 in plasma by Hao Jiang; Jianing Zeng; Yuzhong Deng; Yuan-Qin Xia; Zheng Ouyang; Mohammed Jemal; Theodora W. Salcedo; Mark E. Arnold (2064-2072).
To support toxicokinetic assessments, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of BMS-790052 in rat, dog, monkey, rabbit and mouse K2EDTA plasma. The drug was isolated from buffered samples using ISOLUTE C8 96-well solid phase extraction (SPE) plates. Chromatographic separation was achieved on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using an API 4000 tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-790052, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision (%CV) and inter-assay precision (%CV) were within 8.5%, and the assay accuracy (%Dev) was within ±7.1 for rat, dog, monkey, rabbit and mouse K2EDTA plasma. This accurate, precise, and selective SPE/LC–MS/MS method has been successfully applied to analyze several thousands of non-clinical study samples.
Keywords: LC–MS/MS; Quantitative; BMS-790052; Plasma; HCV;

Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry by Katrin Veldboer; Torsten Vielhaber; Helmut Ahrens; Jendrik Hardes; Arne Streitbürger; Uwe Karst (2073-2080).
A new method for the analysis of 1-hydroxy-2-imidazol-1-yl-phosphonoethyl phosphoric acid (zoledronic acid) in urine and blood samples has been developed. It consists of a derivatisation of the bisphosphonate with trimethylsilyl diazomethane under multiple methylester formation. The formed derivative can, in contrast to the non-derivatised analyte, easily be separated by reversed phase liquid chromatography due to its reduced polarity. Detection is performed by electrospray tandem mass spectrometry. For calibration purposes, a deuterated internal standard has been synthesised in a three-step synthesis starting with d4-imidazole. For human urine, the limit of detection (LOD) is 1.2 x 10−7  mol/L, limit of quantification (LOQ) is 3.75 × 10−7  mol/L in the MRM mode. For human blood plasma, a LOD of 1 × 10−7  mol/L and a LOQ of 2.5 × 10−7  mol/L were determined. The linear dynamic range comprised 3.5 decades starting at the limit of quantification. The method was successfully applied for the analysis of spiked urine and blood plasma samples as well as samples from two osteoporosis patients.
Keywords: Zoledronic acid; Bisphosphonates; Deuterated internal standards; Derivatisation; Liquid chromatography; Electrospray mass spectrometry;

A selective UHPLC–MS/MS method for determination of the therapeutic peptide octreotide in human plasma was developed and validated. This assay used a UHPLC C18 column with 1.7 μm particle size for efficient separation and an ion-exchange SPE for selective extraction. Octreotide and its labeled internal standard, [13C6Phe3] octreotide, were extracted from human plasma using a simple Oasis® WCX μElution SPE method and analyzed with a total chromatographic run time of 7.5 min. Matrix effects were studied during method development by direct monitoring of representative phospholipids. On-line removal of phospholipids using column switching and pre-column back-flushing was carried out to trap and remove any residual phospholipid matrix interferences. The UHPLC column provided baseline separation between the analyte and matrix peaks. The chromatographic conditions yielded optimal retention and excellent peak shape for both the analyte and internal standard. The assay was linear in the concentration range of 0.025–25.0 ng/ml, inter- and intra-assay precision and accuracy were within 6.1% and ±1.93%, respectively. Recovery was ∼73%. Post-extraction addition experiments showed that matrix effects were less than 4%. This method for octreotide in human plasma has been validated and utilized to support of clinical pharmacokinetic studies.
Keywords: Oncology; UPLC; Peptide; Ion suppression; Micro elution; Cancer; Phospholipids;

Direct monitoring changes of salbutamol concentration in serum by chemiluminescent imaging by Canli Zhang; Ruichao Zhang; Na Na; Joris R. Delanghe; Jin Ouyang (2089-2094).
We report in this manuscript, the use of direct ammonium persulfate-enhanced chemiluminescence (CL) imaging, to monitor changes to measure serum salbutamol concentration in subjects of different haptoglobin (Hp) phenotypes at different dosing time. It was noted that CL generated from Hp was decreased due to salbutamol's reducibility, which was used for monitoring salbutamol concentration in serum. The serum from the subjects treated by oral administration of salbutamol, was collected at different dosing time and was separated by polyacrylamide gel electrophoresis (PAGE) prior to the CL detection. According to CL images, samples were separated into three groups based on the Hp phenotypes. The curves of CL signal intensity versus time were obtained for each group, and we demonstrated that there were more significant variables on binding ability between groups. The maximum salbutamol concentration in the serum appeared after 4 h, which was in agreement with the literature. In addition, the binding constants of salbutamol to Hp were determined by a fluorescence-based method, whose results were in agreement with the phenomenon of the greater salbutamol metabolism rate for Group Hp 1-1 than Group Hp 2-2. The presented method can monitor changes of salbutamol concentration in serum directly, making the procedures much simple, convenient, rapid and has the property of lower cost. It provided us with excellent reference information for the individual dosage regimen of different Hp groups, which hopefully could become a potential method for further pharmaceutical research.
Keywords: Chemiluminescent imaging; Haptoglobin phenotype; Salbutamol; Polyacrylamide gel electrophoresis;

Glycerophosphocholine molecular species profiling in the biological tissue using UPLC/MS/MS by Chuan-Ho Tang; Po-Nien Tsao; Chia-Yang Chen; Ming-Shi Shiao; Wei-Hsien Wang; Ching-Yu Lin (2095-2106).
A strategy consisting of a two-phase analytical procedure was used to obtain detailed molecular species composition for glycerophosphocholines (GPCs) profiling in biological tissue using ultra performance liquid chromatography coupled with a triple quadrupole mass spectrometer operating under electrospray mode. In phase one of the analytical procedure, the precursor ion scan was first conducted to obtain the preliminary lipid profile that revealed the composition of the molecular species possessing phosphocholine structure in the biological tissue. In phase two of the analytical procedure, each product ion spectrum obtained for the GPC components in the profile was sequentially acquired for the determination of the molecular structure. A simple guide with high differentiability was proposed for the diacyl-, alkyl-acyl- and alk-1-enyl-acyl-GPC, and related lyso-GPCs molecular structure decision. Total 93 GPCs molecular species were identified in the fetal mouse lung with the relative amounts from 14.39% to less than 0.01% (normalizing by the total GPCs signal). The optimized chromatographic conditions were also proposed in the analytical procedure based on the compromise between the separation efficiency and electrospray signal response. The plate number of the probing GPCs was obviously improved to above 30,000 and the detection limits of the probing GPCs were between 0.002 and 0.016 ng/μL. The practical usability of the analytical procedure has been validated using a study of chemically induced early lung maturation. The metabolic difference between chemically treated and untreated fetal mouse lung was clearly distinguished by the composition of GPCs with several characteristics of molecular structure. The overall results showed that this two-phase analytical procedure was reliable for comprehensive GPC profiling.
Keywords: Glycerophosphocholine profiling; Electrospray ionization; Triple quadrupole mass spectrometer; UPLC;

A rapid, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed for identification of potassium dehydroandrographolidi succinas and its metabolites in rat urine. Five male rats were administrated a single dose (100 mg/kg) of potassium dehydroandrographolidi succinas by i.v. injection. The urine were sampled from 0 to 24 h and purified by using Oasis® HLB extraction cartridge, then the purified urine samples were separated on a reversed-phase C18 column with a linear gradient and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δm) and MS/MS spectra with those of the parent drug. Seven metabolites and the parent drug were found in rat urine. All these metabolites were reported for the first time.
Keywords: Potassium dehydroandrographolidi succinas; HPLC–ESI–MS/MS; Metabolites; Identification; Rat urine;

In this paper, polychlorinated biphenyl (PCB), organochlorine pesticide (OCP) and pyrethroid pesticides in peach was investigated by comparing their residual level in peach juice, pulps and peels using dispersive liquid–liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) combined with gas chromatography-electron capture detection (GC-ECD). Extraction conditions such as the type of extractant, volume of extractant and dispersant, salt effect and extraction time were optimized. For juice samples, the linearity of the method was obtained in the range of 10–2000 ng L−1,with determination coefficients > 0.99. The limits of detection (LOD) of the method were ranged between 2.8 and 18.5 ng L−1. For pulp and peel samples, the developed method is linear over the range assayed, 1–20 μg kg−1,with coefficients also >0.99. The relative recoveries of compounds analyzed from juice, pulp and peel samples were in the range of 73–106% with a relative standard deviation between 2.6 and 11.8%. The proposed method was applied to the simultaneous analysis of residues in real peach juice, pulp and peel samples. As a result, there were no target analytes found in peach juices and pulps while 3.3 μg kg−1 cyhalothrin and 3.5 μg kg−1 fenvalerate were found in peels. The experiment results revealed that the pyrethroid residues just deposited on the peels of the fruits, but did not move into pulps and juices.
Keywords: Dispersive liquid–liquid microextraction; Multi-residues; Floating organic droplet; Peach;

Determination of unbound vismodegib (GDC-0449) concentration in human plasma using rapid equilibrium dialysis followed by solid phase extraction and high-performance liquid chromatography coupled to mass spectrometry by Yuzhong Deng; Harvey Wong; Richard A. Graham; Wenbin Liu; Heuy-shin Shen; Yao Shi; Laixin Wang; Min Meng; Vikram Malhi; Xiao Ding; Brian Dean (2119-2126).
A rapid equilibrium dialysis (RED) assay followed by a solid phase extraction (SPE) high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the quantitative determination of unbound vismodegib in human plasma was developed and validated. The equilibrium dialysis was carried out using 0.3 mL plasma samples in the single-use plate RED system at 37 °C for 6 h. The dialysis samples (0.1 mL) were extracted using a Strata-X-C 33u Polymeric Strong Cation SPE plate and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization (ESI) mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL for vismodegib, was fitted to a 1/x 2 weighted linear regression model. The lower limit of quantitation (LLOQ, 0.100 ng/mL) was sufficient to quantify unbound concentrations of vismodegib after dialysis. The intra-assay precision of the LC–MS/MS assay, based on the four analytical QC levels (LLOQ, low, medium and high), was within 7.7% CV and inter-assay precision was within 5.5% CV. The assay accuracy, expressed as %Bias, was within ±4.0% of the nominal concentration values. Extraction recovery of vismodegib was between 77.9 and 84.0%. The assay provides a means for accurate assessment of unbound vismodegib plasma concentrations in clinical studies.
Keywords: Vismodegib; GDC-0449; Hedgehog pathway inhibitor; RED device; LC–MS/MS;

A sensitive and rapid method was developed and validated for the quantitative analysis of the novel anticancer agent SZ-685C in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in negative ion mode in order to support the following pre-clinical and clinical studies. SZ-685C and the internal standard (IS, emodin) were extracted from rat plasma by a simple liquid–liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on an Elite Hypersil BDS C18 column (100 mm × 2.1 mm i.d., 3 μm). Elution was carried out using methanol/acetonitrile/2 mM ammonium formate (pH 4) (80:15:5 (v/v/v)) at a flow-rate of 0.3 mL/min with a run time of 2.5 min. This assay was linear over a concentration range of 50–10,000 ng/mL with a lower limit of quantification of 50 ng/mL. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 100, 1000 and 7500 ng/mL. These results indicate that the method was efficient with a short run time and acceptable accuracy, precision and sensitivity. This method was successfully applied to explore pharmacokinetics of SZ-685C in rats after oral and intravenous administration of this agent. The absolute bioavailability is about 54.8–66.8% and the t 1/2 is 5.7–9.2 h, these results provide basic information for further comprehensive pre-clinical research.
Keywords: SZ-685C; LC/MS/MS; Pharmacokinetics; Rat plasma;

Isolation of a Aspergillus niger lipase from a solid culture medium with aqueous two-phase systems by Analía Marini; Natalia Imelio; Guillermo Picó; Diana Romanini; Beatriz Farruggia (2135-2141).
The aim of this work is to find the best conditions to isolate lipase from a solid culture medium of Aspergillus niger NRRL3 strains using aqueous two-phase systems formed with polyethylene glycol and potassium phosphate or polyethylene glycol and sodium citrate. We studied the partitioning of a commercial lyophilizate from A. niger. Also, the lipase enzymatic activity was studied in all the phases of the systems and the results indicate that citrate anion increases lipase activity. An analysis by fluorescence spectroscopy of the interaction between lipase and the bottom and top phases of the systems shows that the protein tryptophan-environments are modified by the presence of PEG and salts. Separation of the enzyme from the rest of the proteins that make up the lyophilized was achieved with good yield and separation factor by ATPS formed by PEG 1000/Pi at pH 7, PEG 2000/Ci at pH 5.2 and PEG 4000/Ci at pH 5.2. The above mentioned systems were used in order to isolate extracellular lipase from a strain of A. niger in submerged culture and solid culture. The best system for solid culture, with high purification factor (30.50), is the PEG 4000/Ci at pH 5.2. The enzyme was produced in a solid culture medium whose production is simple and recovered in a phase poor in polymer, bottom phase. An additional advantage is that the citrate produces less pollution than the phosphate. This methodology could be used as a first step for the isolation of the extracellular lipase from A. niger.
Keywords: Lipase; Partition; Aqueous two-phase systems;

Nicotine (NIC), cotinine (COT) and trans-3′-hydroxycotinine (OHCOT) are the most prevalent and abundant tobacco biomarkers in meconium. We have developed and validated an accurate and precise method for the measurement of these analytes in meconium in which potassium hydroxide is used to digest the meconium sample, followed by solid phase extraction from the liquified sample. The precision of OHCOT, COT and NIC measurements (intra-day and inter-day) were 4.8–10.6%, 3.4–11.6% and 9.3–15.8%, respectively. Evaluation of accuracy indicated bias of −4.0, 2.0 and 0.8% for OHCOT at concentrations of 0.5, 2.5 and 7.5 ng/g. The accuracy estimates for COT at concentrations of 0.5, 2.5 and 7.5 ng/g are 4.0, 4.0 and 5.7%, respectively. For NIC at 2, 10 and 30 ng/g the accuracy was calculated to be 3.0, 5.0 and 5.1%, respectively. The linear range of standard solutions was 0.125–37.5 ng/mL for OHCOT and COT, and 0.75–150 ng/mL for NIC. This method was applied to the analysis of 374 meconium samples from infants of both smoking and nonsmoking mothers. Positive correlations with r 2  ≥ 0.63 were observed between NIC and COT, COT and OHCOT, NIC and OHCOT, and NIC and (OHCOT + COT) in these samples.
Keywords: Meconium; Nicotine; Cotinine; Trans-3′-hydroxycotinine; Tobacco; Biomarker;

Methandrostenolone (MA) is a steroid used as veterinary medicine on stockbreeding to promote animal growth. The use of MA has been strictly regulated because of its harmful effect on consumers. This paper describes the production of polyclonal antibody (pAb) against MA, the preparation of immunoaffinity column (IAC) and its potential application to the selective extraction of MA residues from animal tissue and feed samples. The produced pAb exhibited good sensitivity to MA with an IC50 value of 5.6 ng/mL. The cross-reactivity values of the antibody with MA structurally related compounds of testosterone propionate (TP) and trenbolone (TR) were lower than 0.6%. By coupling the produced antibody with CNBr-activated Sepharose 4B, an IAC was prepared. 2% methanol and 80% methanol were selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was approximately 334 ng/mL gel. The average recovery of 20, 40 and 60 ng/mL MA standard solutions from IACs was 97.9% with the relative standard deviation (RSD) among columns of 6.7%. After 3 times of repeated usage, the column capacity and recovery rate still remained 82.0% and 92.6% respectively. The IACs were then challenged with MA-fortified animal tissue and feed samples, recoveries of MA were found to be in the range of 83.5–99.7%.
Keywords: Methandrostenolone; Polyclonal antibody; Immunoaffinity column; ELISA; HPLC;

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography–tandem mass spectrometry by Ling-Zhi Wang; Michelle Yi-Xiu Lim; Tan-Min Chin; Win-Lwin Thuya; Pei-Ling Nye; Andrea Wong; Sui-Yung Chan; Boon-Cher Goh; Paul C. Ho (2155-2161).
A novel, rapid and specific liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 μL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5–1000 ng/mL for gefitinib and 5–500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB–IV) non-small cell lung cancer (NSCLC).
Keywords: Gefitinib; O-Desmethyl gefitinib; LC–MS/MS; Human plasma;

Determination of carboplatin in human plasma using HybridSPE-precipitation along with liquid chromatography–tandem mass spectrometry by Hongliang Jiang; Yang Zhang; Matt Ida; Amber LaFayette; Douglas M. Fast (2162-2170).
The main purpose of this study was to develop and validate a rapid, specific, sensitive, and reliable LC–MS/MS-based bioanalytical method for the determination of carboplatin in human plasma. The optimal chromatographic behavior of carboplatin was achieved on a Biobasic SCX column (50 mm × 2.1 mm, 5 μm) using ion exchange chromatography. The total LC analysis time per injection was 2.6 min with a flow rate of 1.5 mL/min with a gradient elution. Optimization with regard to improving recovery and minimizing matrix effects using HybridSPE-precipitation (HybridSPE-PPT) has been evaluated under various extraction conditions. As a result, sample preparation via HybridSPE-PPT with 1% formic acid in acetonitrile in a 96-well format was applied for method validation and sample analysis and showed acceptable recovery of greater than 25% and negligible matrix effects. The method validation was conducted over the curve range of 2.00–2000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.8% relative standard deviation (RSD) and −13.2 to −3.6% relative errors (RE). The method was successfully applied to determine carboplatin in human plasma samples.
Keywords: Carboplatin; HybridSPE-precipitation; SCX; Matrix effects; LC–MS/MS; Phospholipids removal;

Chromatographic diagnosis of maple syrup urine disease by measuring the l-alloisoleucine/l-phenylalanine ratio in dried blood spots by Ji-Seon Jeong; Hee-Jung Sim; Yong-Moon Lee; Hye-Ran Yoon; Ha-Jeong Kwon; Seon-Pyo Hong (2171-2174).
A high-performance ligand-exchange chromatography with ultraviolet detection method for confirmation diagnosis of maple syrup urine disease (MSUD) was developed that relies on the determination of branched-chain amino acids (BCAAs) and Phe levels in blood. The dynamic ranges for the BCAAs and Phe were 50–1000 μM (r 2  = 0.9982–0.9996) and 74–873 μM (r 2  = 0.9992) from a dried blood spot, and the BCAA detection limits (S/N = 3) were 0.43–1.91 μM. The mean recoveries of BCAA for intra- and inter-day assays were 92.1–103.0%. The ranges of alloisoleucine (Allo-Ile)/Phe ratio were ND–0.04 and 1.5–2.4 for PKU and MSUD patient samples, respectively. The lowest ratio (1.5) of the MSUD samples was 37.5 times higher than the highest ratio (0.04) of the PKU samples. Therefore, the Allo-Ile/Phe ratio was very useful biomarker for confirmation diagnosis of MSUD.
Keywords: Neonatal screening; Branched-chain amino acids; Alloisoleucine/phenylalanine ratio; Maple syrup urine disease; Phenylketonuria; High-performance ligand-exchange chromatography;

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids by Galina Tishchenko; Jiří Šimůnek; Hana Bartoňová; Jarmila Dušková; Jan Dohnálek; Evgenia Ponomareva; Tatiana Tennikova (2175-2178).
Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38–90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.
Keywords: Sample preparation; Culture fluids C. paraputrificum J4; Chitinases; β-N-Acetylglucosaminidase; Ultrafiltration;

Orthosiphon aristatus is a traditionally used medicinal plant. In order to study the proteome of the plant, we have developed a simple plant protein extraction method by direct extraction of protein using a modified 2D-gel compatible tris–sucrose buffer followed by a double TCA–acetone precipitation. This method omitted the use of toxic phenol which is widely used in the studies of plants proteins. Moreover, it shortens the lengthy extraction procedure of phenol extraction and back-extraction method and therefore reduced the extraction time (by 2 h) while increased in protein yields (by 50%). Comparison of the 2D-gel images of the two extracts revealed that >60 extra protein spots were detected in the extract of our current method. The method was applied on the leaves of O. aristatus collected from six geographical areas in Malaysia. The correlation coefficient of each replicate gels from the six areas ranged from 0.70 to 0.90 indicating good reproducibility of the method.
Keywords: Proteomics; Orthosiphon aristatus; Improved extraction method; 2D-gel electrophoresis; Tandem mass spectrometry;