Journal of Chromatography B (v.1058, #C)

Determination of fenobucarb residues in animal and aquatic food products using liquid chromatography-tandem mass spectrometry coupled with a QuEChERS extraction method by Weijia Zheng; Jin-A Park; Dan Zhang; A.M. Abd El-Aty; Seong-Kwan Kim; Sang-Hyun Cho; Jeong-Min Choi; Jae-Han Shim; Byung-Joon Chang; Jin-Suk Kim; Ho-Chul Shin (1-7).
Display OmittedA modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) was developed for quantification of fenobucarb residues in animal food products, such as porcine muscle, egg, and whole milk, and aquatic food products, such as eel, flatfish, and shrimp. Acetonitrile with the addition of 0.1% trifluoroacetic acid was employed as an extraction solvent and was compared with acetonitrile alone and 0.1% formic acid in acetonitrile. All extracted samples were purified using C18 sorbent. The best extraction efficiencies, expressed as recovery at two spiking levels equivalent to 1- and 2-times the limit of quantification (LOQ = 2 μg/kg) were achieved using 0.1% trifluoroacetic acid in acetonitrile and ranged from 61.38 to 102.21% in all matrices, with relative standard deviations (RSDs) < 13% (except for the low spiking of porcine muscle and the high spiking of whole milk, for which the RSDs were > 20%). Six-point matrix-matched calibration was used for quantification and the determination coefficients were good (R2  ≥ 0.9865). The method was verified by application to samples purchased from local markets and none of the samples tested positive. In conclusion, the developed method is simple and versatile and can be used for the routine detection of fenobucarb in different animal food products having varying protein and fat contents with satisfactory accuracy and precision.
Keywords: Fenobucarb; Porcine muscle; Egg; Milk; Fish; Flatfish; Shrimp; Residues; LC–MS/MS;

Bisphenol A (BPA), an endocrine disrupter, is widely used to make chemicals for polycarbonate, plastics, beverage containers, epoxy resins, and cash register receipts. BPA is one of the known xenoestrogens, which have weak estrogenic activity and cause obesity, diabetes, breast cancer, and reproductive disorders. Even though the concentration level of metabolomes in hair is usually lower than that in urine and blood, there are several reasons why we chose to use hair samples. First, the sampling procedure of hairs is simple. Second, it is also easy to preserve the sample for long term and track the drug-exposure record of a given sample. Third, deformation and contamination of samples rarely occur. In this study, an improved analytical method to determine the levels of BPA and estrogens in hair samples was developed by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Hair samples were extracted by an Oasis HLB extraction cartridge after incubation with 1 N HCl and derivatized with dansyl chloride to increase sensitivity. BPA and estrogens (estrone, 17β-estradiol, and estriol) were separated using Shiseido CAPCELL PAK C18 column (2.0 × 100 mm, 3 μm) and a mobile phase consisting of 10 mM ammonium acetate in water and acetonitrile with a gradient program at a flow rate of 0.3 mL/min and were monitored with electrospray tandem mass spectrometry (ESI–MS/MS). The linearity of this method was over 0.995. The limits of detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.25–6.0 ng/g. The alteration of estrogens levels induced by BPA may play important role to understanding probable endocrine disruptive exposure, and the described methods could be used to evaluate and monitor exposure of endocrine disruptor.
Keywords: Bisphenol A; Estrogens; Hair; Liquid chromatography; Mass spectrometry;

An effective and solvent-less method for the rapid determination of four commonly detected parabens (methyl-, ethyl-, propyl- and butyl-) in human urine samples is described. This method employed ultrasound-assisted emulsification microextraction (USAEME) before identification and quantitation of the parabens via on-line acetylation gas chromatography-mass spectrometry (GC–MS). Urine samples were enzymatically de-conjugated with β-glucuronidase and then extracted by an optimal USAEME procedure for the measurement of total concentrations of target analytes. The optimal USAEME parameters for one mL of urine sample (containing 0.1-g of sodium chloride), according to the Box-Behnken design method, are thus described: extractant of 200-μL of ethyl acetate, and ultrasonication for 1.0 min and centrifugation at 7000 rpm (3 min). The supernatant was collected and evaporated until dry. Then the residue was re-dissolved in methanol (100-μL), and the extract was subjected to on-line acetylation GC–MS analysis. The limits of quantitation (LOQs) were less than 0.06 ng/mL. Precisions for both intra- and inter-day analysis were calculated, and were less than 8%. Mean extraction recovery (known as trueness) was between 83 and 101% on three concentration levels. In human urine, the total concentrations of the four selected parabens, according to preliminary results, range from 0.3 to 124.5 ng/mL for male, and from 27.2 to 246.3 ng/mL for female. Female urine samples showed higher concentrations for the target parabens, which may indicate higher exposure due to lifestyle. This method permits accurate and high-throughput analysis of parabens for epidemiological studies.
Keywords: Parabens; On-line acetylation; Ultrasound-assisted emulsification microextraction; GC–MS; Urine sample analysis;

Determination of soluble sugar profile in rice by Xianqiao Hu; Changyun Fang; Lin Lu; Zhanqiang Hu; Yafang Shao; Zhiwei Zhu (19-23).
Soluble sugars in rice are the main components affecting sweetness taste of rice. In this paper, an accurate, precise and rapid method for simultaneous determination of multi soluble sugars in rice by using ion chromatography equipped with pulsed amperometric detector was presented. Pretreatment and parameters of ion chromatography and pulsed amperometric detector were optimized. Regression coefficients (R) of 0.9998, 1.0000, 0.9979, 0.9998 and 0.9998 were obtained for glucose, fructose, sucrose, raffinose and maltose, respectively. The recovery ranges of five sugars were 92.9–112.0% for milled rice matrix. Repeatability and reproducibility of the method were 0.8–9.7% and 1.9–7.6%, respectively. Method LODs of 3.1–34.6 μg g−1 were obtained for soluble sugars in milled rice matrix.
Keywords: Soluble sugar; Rice; Ion chromatography; Pulsed amperometric detector;

Aconiti Lateralis Radix Praeparata- Fritillariae Thunbergii bulbus, namely Fuzi- Beimu in Chinese, is a classic herb pair whose combined administration was prohibited according to the rule of “Eighteen antagonisms”. However, incompatibility of Fuzi and Beimu has become controversial because of the application supported by many recorded ancient prescriptions and increasing modern researches and clinical practice. The present study aimed to investigate the pharmacokinetic differences of multiple alkaloids from Fuzi- Beimu and the single herb aqueous extracts following oral delivery in rats. Twelve alkaloids including aconitine, mesaconitine, hypaconitine, benzoylaconitine, benzoylmesaconitine, benzoylhypacoitine, neoline, fuziline, talatisamine, chasmanine, peimine and peimisine in rat plasma were simultaneously quantitated by using sensitive ultra-high performance liquid chromatography- tandem mass spectrometry (UHPLC–MS/MS), with the method developed and fully validated. Plasma concentrations of the twelve alkaloids after administration were determined and pharmacokinetic parameters were compared. Significant differences were observed for all alkaloids except aconitine, mesaconitine and benzoylaconitine for Fuzi- Beimu group in comparison with the single herb group. AUC 0-t and T 1/2 of hypaconitine were increased significantly. AUC 0-t and C max were increased and T max decreased significantly for benzoylmesaconitine and benzoylhypacoitine. Fuziline showed significantly increased AUC 0-t, C max and T max. T 1/2 of neoline was notably increased. T 1/2 and T max were significantly elevated for talatisamine while C max decreased. T max of chasmanine was significantly increased and C max decreased. Extremely significant increase of T max was found for peimisine, and significant increase of T 1/2 for peimine. Results revealed that combined use of Fuzi and Beimu significantly influenced the system exposure and pharmacokinetic behaviors of multiple alkaloids from both herbs, indicating herb- herb interaction between Fuzi and Beimu.
Keywords: UHPLC–MS/MS; Pharmacokinetics; Alkaloids; Fuzi; Beimu;

Rapid IC–MS/MS determination of methylphosphonic acid in urine of rats exposed to organophosphorus nerve agents by Timur Baygildiev; Alexandra Zatirakha; Igor Rodin; Arkady Braun; Andrey Stavrianidi; Nadezhda Koryagina; Igor Rybalchenko; Oleg Shpigun (32-39).
A direct approach for the determination of a specific hydrolysis product of organophosphorus nerve agents such as methylphosphonic acid (MPA) in urine by ion chromatography and tandem mass spectrometry (IC–MS/MS) has been developed. The first advantage of the proposed approach is a rapid and simple sample preparation, which does not require a large sample volume, complicated and laborious preconcentration and derivatization steps, and takes less than 7 min per sample. The second advantage is the fast and selective IC determination of MPA carried out on a noncommercial anion exchanger based on a poly(styrene-co-divinylbenzene) (PS-DVB) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer, which enables complete resolution of MPA from major urine matrix components and allows one to overcome matrix effects. Hyphenation of IC with tandem mass spectrometry results in highly sensitive and reliable MPA determination with the lowest detection limit (4 ng mL−1) reported so far for HPLC determination of MPA in urine. The proposed approach is successfully applied for the analysis of urine from rats exposed to nonlethal doses of organophosphorus nerve agents such as sarin, soman, and VR in up to 13 days after initial exposure, which shows the possibility to verify the nerve agent exposure after a long period of time.
Keywords: Methylphosphonic acid; Ion chromatography; Tandem mass spectrometry; Rapid sample preparation; Urine analysis;

There are significant differences in d-amino acid concentrations between healthy people and Alzheimer’s disease patients. In order to investigate the potential correlation between d-amino acids and Alzheimer’s disease, a simple and sensitive ultra high performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method has been developed. The method was applied to simultaneous determination of 11 d-amino acids in different regions of rat brain. Rat brain homogenates were firstly pretreated with protein precipitation procedure and then derivatized with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE]. Baseline separation of the derivatives was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The mobile phase consisted of acetonitrile and water (containing 8 mM ammonium hydrogen carbonate) and the flow rate was 0.6 mL min−1. The derived analytes were sensitively detected by multiple reaction monitoring in the positive ion mode. The lower limits of quantitation ranged from 0.06 to 10 ng mL−1 with excellent linearity (r ≥ 0.9909). The intra- and inter-day RSD were in the range of 3.6–12% and 5.7–12%, respectively. The recovery rate was 82.5%–95.3%. With this UPLC–MS/MS method, the 11 d-amino acids in hippocampus, cerebral cortex, olfactory bulb and cerebellum from Alzheimer’s disease rats and age-matched controls could be simultaneously determined. Compared with the normal controls, the concentrations of d-serine, d-alanine, d-leucine, and d-proline in hippocampus and cerebral cortex of Alzheimer’s disease rat brain were significantly decreased, while no differences in olfactory bulb and cerebellum of all the d-amino acids were observed. The different amounts and distribution of d-amino acids in brain between the two groups, which regulated by particular pathological changes of Alzheimer’s disease, would give new insights into further study in neuropathogenesis and provide novel therapeutic targets of Alzheimer’s disease.
Keywords: d-Amino acid; Different brain regions; Alzheimer’s disease; UPLC–MS/MS;

Detailed characterization of bio-oil from pyrolysis of non-edible seed-cakes by Fourier Transform Infrared Spectroscopy (FTIR) and gas chromatography mass spectrometry (GC–MS) techniques by Vatsala Sugumaran; Shanti Prakash; Emmandi Ramu; Ajay Kumar Arora; Veena Bansal; Vivekanand Kagdiyal; Deepak Saxena (47-56).
Bio-oil obtained from pyrolysis is highly complicated mixture with valued chemicals. In order to reduce the complexity for unambiguous characterization of components present in bio-oil, solvent extractions using different solvents with increasing polarity have been adopted. The fractions have been analyzed by Fourier transform infrared (FTIR) spectroscopy for identifying the functional groups and Gas chromatography–mass spectrometry (GC–MS), for detailed characterization of components present in various fractions, thereby providing in-depth information at molecular level of various components in bio-oil. This paper reveals the potential of the analytical techniques in identification and brings out the similarities as well as differences in the components present in the bio-oil obtained from two non-edible oil seed-cakes, viz., Jatropha and Karanjia.
Keywords: Pyrolysis; Bio-oil; Jatropha seed-cake; Karanjia seed-cake; FTIR; Gas chromatography mass spectrometry;

Chromatographic separation and detection methods of Aloe arborescens Miller constituents: A systematic review by Jilan A. Nazeam; Haidy A. Gad; Hala M. El-Hefnawy; Abdel-Naser B. Singab (57-67).
Aloe arborescens Miller (Family Asphodelaceae) is a member of genus Aloe, which is used in traditional medicine to cure various diseases. The extracts of the plant have been reported to possess anticancer, immunomodulator, antidiabetic, anti-inflammatory and antioxidant activities. The phytochemical investigations have revealed diverse chemical constituents, including phenolics [anthraquinones, anthrones, pyrones, chromones and coumarins], polysaccharides [arborans [(1–4) linked glucomannans, polysaccharide (A, B and C): (A: a linear (1–6)-O-α-glucan, B: a branching (1–2)-O-l-arabinose with (1–2)-O-d-galactose linkages and C: (1–4)-O-β-mannan with 18% acetyl group)]], glycoproteins and carboxypeptidase enzyme. There are many reports, describing the different methodologies developed to perform chemical analysis as well as, separation, detection and identification of these constituents. Different chromatographic techniques were applied such as gas chromatography (GC), high-performance liquid chromatography (HPLC), liquid chromatography-electrospray ionization coupled with mass spectroscopy (LC-ESI/MS/MS) and gel filtration chromatography. Also the isolated compounds were identified based on the spectroscopic analysis; ultraviolet-visible spectroscopy (UV–vis), infra-red spectroscopy (IR), mass spectroscopy (MS) and nuclear-magnetic resonance (NMR). This study aims to pinpoint the active components besides finding out new structural leads for future drugs. Therefore, the review is targeted to provide evidence reported in the relevant literature on qualitative and quantitative research to assist scientists in isolation and characterization of bioactive compounds in A. arborescens.
Keywords: Aloe arborescens; Phenolics; Polysaccharides; Glycoproteins; Carboxypeptidase; Analysis;

Tyrosine and tyrosine residues in proteins are attacked by the reactive oxygen and nitrogen species peroxynitrite (O=N–OO) to generate 3-nitrotyrosine (3-NT) and 3-nitrotyrosine-proteins (3-NTProt), respectively. 3-NT and 3-NTProt are widely accepted as biomarkers of nitr(os)ative stress. Over the years many different analytical methods have been reported for 3-NT and 3-NTProt. Reported concentrations often differ by more than three orders of magnitude, indicative of serious analytical problems. Strategies to overcome pre-analytical and analytical shortcomings and pitfalls have been proposed. The present review investigated whether recently published work on the quantitative measurement of biological 3-nitrotyrosine did adequately consider the analytical past of this biomolecule. 3-Nitrotyrosine was taken as a representative of biomolecules that occur in biological samples in the pM-to-nM concentration range. This examination revealed that in many cases the main protagonists involved in the publication of scientific work, i.e., authors, reviewers and editors, failed to do so. Learning from the analytical history of 3-nitrotyrosine means advancing analytical and biological science and implies the following key issues. (1) Choosing the most reliable analytical approach in terms of sensitivity and accuracy; presently this is best feasible by stable-isotope dilution tandem mass spectrometry coupled with gas chromatography (GC–MS/MS) or liquid chromatography (LC–MS/MS). (2) Minimizing artificial formation of 3-nitrotyrosine during sample work up, a major pitfall in 3-nitrotyrosine analysis. (3) Validating adequately the final method in the intendent biological matrix and the established concentration range. (4) Inviting experts in the field for critical evaluation of the novelty and reliability of the proposed analytical method, placing special emphasis on the compliance of the analytical outcome with 3-nitrotyrosine concentrations obtained by validated GC–MS/MS and LC–MS/MS methods.
Keywords: Chromatography; Mass spectrometry; Nitration; Quantification; Validation;

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I – Non-denaturing chromatographic techniques by Alexandre Goyon; Valentina D’Atri; Balazs Bobaly; Elsa Wagner-Rousset; Alain Beck; Szabolcs Fekete; Davy Guillarme (73-84).
Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.
Keywords: Size exclusion chromatography; Ion exchange chromatography; Hydrophobic interaction chromatography; Therapeutic monoclonal antibodies; Antibody-drug conjugates; Protocol;

A novel tertiary prep-HPLC method for the isolation of single amino acids for AMS-radiocarbon measurement by Ricardo Fernandes; Tomas Koudelka; Andreas Tholey; Alexander Dreves (85-92).
AMS-radiocarbon measurements of amino acids can potentially provide more reliable radiocarbon dates than bulk collagen analysis. Nonetheless, the applicability of such an approach is often limited by the low-throughput of existing isolation methods and difficulties in determining the contamination introduced during the separation process.A novel tertiary prep-HPLC amino acid isolation method was developed that relies on the combustion of eluted material without requiring any additional chemical steps. Amino acid separation was carried out using a gradient mix of pure water and phosphoric acid with an acetonitrile step in-between runs to remove hydrophobic molecules from the separation column. The amount of contaminant carbon and its 14C content were determined from two-point measurements of collagen samples of known 14C content.The amount of foreign carbon due to the isolation process was estimated at 4 ± 1 μg and its 14C content was 0.43 ± 0.01 F14C. Radiocarbon values corrected for carbon contamination have only a minor increase in uncertainties. For Holocene samples, this corresponds to an added uncertainty typically smaller than 10 14C years.The developed method can be added to routine AMS measurements without implying significant operational changes and offers a level of measurement uncertainty that is suitable for many archaeological, ecological, environmental, and biological applications.
Keywords: Radiocarbon; Accelerator mass spectrometry; Amino acids; Bone collagen; HPLC;

A new fast LC–MS/MS method was developed for determination of alogliptin and pioglitazone in human plasma. Linearity ranges of 10–400 ng mL−1 for alogliptin and 25–2000 ng mL−1 for pioglitazone, were found to be suitable for their bioanalysis covering the Cmin and Cmax values of the drugs. Direct precipitation technique was used for simultaneous extraction of the drugs successfully from human plasma samples. Chromatographic separation was achieved on a BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with 0.1% aqueous formic acid: acetonitrile (40:60, v/v) at a flow rate of 0.3 mL min−1. The validated method was applied to a preliminary pharmacokinetic study on human volunteers. Monitoring the transition pairs of m/z 340.18 to 116.08 for alogliptin and m/z 356.99 to 133.92 for pioglitazone, using triple quadrupole mass spectrometer with multiple reaction monitoring, was achieved in the positive mode. The validated method is accurate and suitable for further clinical applications and possible bioequivalence studies.
Keywords: Alogliptin; Pioglitazone; Sample preparation; LC–MS/MS; Human volunteers; Preliminary pharmacokinetic study;

A simple HPLC-UV method for quantification of enzalutamide and its active metabolite N-desmethyl enzalutamide in patients with metastatic castration-resistant prostate cancer by Alicja Puszkiel; Alain Plé; Olivier Huillard; Gaëlle Noé; Constance Thibault; Stéphane Oudard; François Goldwasser; Michel Vidal; Jérome Alexandre; Benoit Blanchet (102-107).
Enzalutamide is currently approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC). To date, a single liquid chromatographic–tandem mass spectroscopy method is available to measure plasma enzalutamide concentrations in mCRPC patients. In this work, an accurate and sensitive HPLC-UV method has been developed for the simultaneous determination of enzalutamide and its active metabolite, N-desmethyl enzalutamide in plasma from mCRPC patients. Before precipitation of proteins with acetonitrile, samples were spiked with nilutamide (internal standard). Separation of analytes was achieved under isocratic elution on a C18 Kinetex column. The mobile phase consisted of a mixture of ammonium acetate buffer (pH = 4.6, 20 mM) and acetonitrile (60:40, v/v), and was delivered at a flow rate of 1.5 mL/min throughout a 9-min run. UV detection was performed at 270 nm. The method was linear over a concentration range of 0.50–50.0 μg/mL for both analytes. Within- and between-day imprecision and accuracy were ≤10% at concentrations 0.75, 5.00, and 50.0 μg/mL. This method has been implemented to assay steady-state trough plasma concentrations (n = 30) of enzalutamide and N-desmethyl enzalutamide in 16 mCRPC patients. Overall, this HPLC-UV method is well-suited for routine application in clinical laboratories to perform therapeutic drug monitoring of enzalutamide in mCRPC patients.
Keywords: Enzalutamide; Prostate; Cancer; Therapeutic drug monitoring; HPLC-UV;