Journal of Chromatography B (v.953-954, #C)

A simple and rapid ultra-high performance liquid chromatography–tandem mass spectrometry (uHPLC–MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid–liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC–MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0–5.0 ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from −9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration–time curves.
Keywords: Ultra HPLC-MS/MS; Shixiao San; Flavonoids; Pharmacokinetics; Hyperlipidemic rats;

A urinary metabonomics method based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) had been established to investigate the holistic efficacy of Wu-tou decoction (WTD), a traditional Chinese medicine (TCM) formula used to treat rheumatoid arthritis (RA), in adjuvant-induced arthritis (AIA) rat model. Multivariate statistical approaches, such as principal component analysis (PCA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were used to distinguish healthy control group, AIA model group and WTD treated group and find potential biomarkers. There was a clear separation among the three groups in PCA model. Sixteen potential biomarkers had been identified using OPLS-DA, and 11 of them was considered to be in response to therapeutic effects of WTD involved in tryptophan metabolism, phenylalanine metabolism, tricarboxylic acid (TCA) cycle, bile acid biosynthesis, steroid hormone biosynthesis and valine metabolism. In this study, WTD also showed good anti-inflammatory and antioxidant activities in vivo, and it could suppress histopathological changes of AIA rats. There might be a correlation between these results and the regulation of the disturbed metabolites in urine. This study demonstrates that metabonomics is a powerful methodology to gain insight in the mechanism of TCM formula in therapy.
Keywords: Wu-tou decoction; Metabonomics; Mass spectrum; Adjuvant-induced arthritis; Traditional Chinese medicine;

Characterization of metabolic profile of honokiol in rat feces using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and 13C stable isotope labeling by Yinfeng Dong; Minghai Tang; Hang Song; Rong Li; Chunyu Wang; Haoyu Ye; Neng Qiu; Yongkui Zhang; Lijuan Chen; Yuquan Wei (20-29).
As fecal excretion is one of important routes of elimination of drugs and their metabolites, it is indispensable to investigate the metabolites in feces for more comprehensive information on biotransformation in vivo. In this study, a sensitive and reliable approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC-Q-TOF-MS) was applied to characterize the metabolic profile of honokiol in rat feces after the administration of an equimolar mixture of honokiol and [13C6]-labeled honokiol. Totally 42 metabolites were discovered and tentatively identified in rat feces samples, 26 metabolites were first reported, including two novel classes of metabolites, methylated and dimeric metabolites of honokiol. Moreover, this study provided basic comparative data on the metabolites in rat plasma, feces and urine, which gave better understanding of the metabolic fate of honokiol in vivo.
Keywords: Honokiol; Metabolite; Feces; UHPLC/Q-TOF-MS; Stable isotope labeling; Dimer;

After ingestion, human intestinal bacteria transform daidzein into dihydrodaidzein, which can be further metabolised to O-desmethylangolensin. This metabolite, unlike daidzein, has a chiral centre and can therefore occur as two distinct enantiomers; however, it is unclear which enantiomer is present in humans. The aim of this study was to define in vitro and in vivo the structure of O-desmethylangolensin and then to evaluate its pharmacokinetic parameters. Daidzein metabolism was preliminarily investigated in anaerobic batch cultures inoculated with mixed faecal bacteria from O-desmethylangolensin producer volunteers. The transformation was monitored by liquid chromatography–mass spectrometry and a chiral column was used to distinguish dihydrodaidzein and O-desmethylangolensin enantiomers. These were purified, analysed by circular dichroism and the results established R(-)-O-desmethylangolensin as the main product (enantiomer excess 91%). However, both dihydrodaidzein enantiomers were detected. Similar results were obtained by in vivo trials. The in vitro formation of O-desmethylangolensin seems to be directly correlated with the number of transforming microorganisms. This correlation was found in vivo for t max but not for other pharmacokinetic indexes. The pharmacokinetics of daidzein, dihydrodaidzein and O-desmethylangolensin were then evaluated in 11 healthy adult O-desmethylangolensin producers after the single administration of soy milk containing 100 mg daidzein. The conjugated forms of daidzein, dihydrodaidzein and O-desmethylangolensin represent more than 90 and 95% of the plasmatic and urinary forms, respectively. The C max, t max and half-life of O-desmethylangolensin in plasma were 62 ± 53 nM, 28 ± 11 and 15 ± 6 h, respectively. Relevant inter-individual variations were observed as indicated by the high standard deviations.
Keywords: Daidzein metabolism; Human; Microflora; ODMA enantiomers; Pharmacokinetics;

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography–tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20 pM, 2 nM and 10 nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200 ng of DNA.Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.
Keywords: 5-Hydroxymethylcytosine; 5-Methylcytosine; Mixed lineage leukemia; Ten–eleven translocation; LC–MS/MS; Isotopologue-MRM;

Identification of volatile organic compounds in human cerumen by Katharine A. Prokop-Prigge; Erica Thaler; Charles J. Wysocki; George Preti (48-52).
We report here the initial examination of volatile organic compounds (VOCs) emanating from human earwax (cerumen). Recent studies link a single nucleotide polymorphism (SNP) in the adenosine triphosphate (ATP) binding cassette, sub-family C, member 11 gene (ABCC11) to the production of different types of axillary odorants and cerumen. ABCC11 encodes an ATP-driven efflux pump protein that plays an important function in ceruminous apocrine glands of the auditory canal and the secretion of axillary odor precursors. The type of cerumen and underarm odor produced by East Asians differ markedly from that produced by non-Asians. In this initial report we find that both groups emit many of the same VOCs but differ significantly in the amounts produced. The principal odorants are volatile organic C2-to-C6 acids. The physical appearance of cerumen from the two groups also matches previously reported ethnic differences, viz., cerumen from East Asians appears dry and white while that from non-Asians is typically wet and yellowish-brown.
Keywords: Cerumen; Volatile organic compounds; Earwax; GC/MS; SPME; Axillary odor;

Biomonitoring method for bisphenol A in human urine by ultra-high-performance liquid chromatography–tandem mass spectrometry by David J. Anderson; Eric M. Brozek; Kyley J. Cox; Christina A. Porucznik; Diana G. Wilkins (53-61).
An ultra-high-performance liquid chromatography–tandem mass spectrometry method for the measurement of total bisphenol A in human urine was developed and validated. The method utilized liquid/liquid extraction with 1-chlorobutane and a human urine aliquot size of 800 μL. Chromatography was performed on an Acquity UPLC® system with a Kinetex® Phenyl-Hexyl column. Mass spectrometric analysis was with negative electrospray ionization on a Quattro Premier XE™. The surrogate matrix method was used for the preparation of calibration standards in synthetic urine due to the presence of BPA in control human urine. The validated calibration range was 0.75–20 ng/mL with a limit of detection of 0.1 ng/mL. The internal standard was d 16-bisphenol A. Method validation utilized quality control samples at three concentrations in both synthetic urine and human urine. Bisphenol A mono-glucuronide was fortified in synthetic urine in each analytical run to monitor the enzymatic conversion of the glucuronide conjugate to BPA by β-glucuronidase. Validated method parameters included linearity, accuracy, precision, integrity of dilution, selectivity, re-injection reproducibility, recovery/matrix effect, solution stability, and matrix stability in human urine. Acceptance criteria for analytical standards and QCs were ±20% of nominal concentration. Matrix stability in human urine was validated after 24 h at ambient temperature, after three freeze/thaw cycles, and after frozen storage at −20 °C and −80 °C for up to 218 days. The method has been applied to the analysis of over 1750 human urine samples from a biomonitoring study. The median and mean urine BPA concentrations were 2.71 ng/mL and 4.75 ng/mL, respectively.
Keywords: Bisphenol A; Liquid chromatography; Tandem mass spectrometry; Human urine;

Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 Dihydroxyvitamin D2&3 measurement using a novel derivatization agent by Curtis J. Hedman; Donald A. Wiebe; Subhakar Dey; Josh Plath; Joseph W. Kemnitz; Toni E. Ziegler (62-67).
Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography–tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, p c  = 0.97; Amplifex and PTAD, p c  = 0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, p c  = 0.81; Amplifex and PTAD, p c  = 0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume.
Keywords: Liquid chromatography; Tandem mass spectrometry; 1,25 Dihydroxyvitamin D; Derivatization method;

Size-exclusion chromatography (SEC) is a useful method for quantification of protein aggregates because of its high throughput capacity and highly quantitative performance. One of the problems in this method concerns polysorbates, which are well-known additives for protein-containing products to prevent protein aggregation, but frequently interfere with the photometric detection of protein aggregates. We developed a new SEC method that can separate polysorbates from protein sample solutions in an on-line mode with a precolumn with size exclusion and reversed-phase mixed modes. The precolumn can effectively trap polysorbates in aqueous mobile phase, and the trapped polysorbates are easily eluted with acetonitrile-containing aqueous mobile phase to clean the precolumn. Small parts of protein aggregates may be also trapped on the precolumn depending on temperature and proteins. Setting appropriate column temperature can minimize such inconvenient trapping of aggregates.
Keywords: Size-exclusion chromatography; Polysorbate; Protein aggregation; MSpak GF-4A; Temperature-dependent retention;

Based on cloud-point extraction (CPE), a high performance liquid chromatography method (HPLC) was developed and validated for the determination of aristolochic acids (AAs) in rat plasma after oral administration of Aristolochiae Fructus (AF). Non-ionic surfactant Genapol X-080, an environmentally friendly solvent, was used for the micelle-mediated extraction. Various influencing factors on CPE process were investigated and optimized. AAs were extracted from rat plasma after adding 1 ml of 4.5% (v/v) surfactant in the presence of 0.2 mol/l HCl and 20 mg NaCl, and the incubation temperature and time were 50 °C and 10 min, respectively. Base-line separation was obtained for the AAs in rat plasma with the optimized chromatography conditions. The detection limits (LOD) reached downward 10 ng/ml. The intra-day and inter-day precisions were less than 7.8%, the accuracies were within ±5.5%, and the average recovery factors were in the range of 94.5–105.4%. In comparison with liquid–liquid extraction, the CPE method has a considerable LOD and higher recoveries. The proposed CPE–HPLC method was specific, sensitive and reliable, and could be an effective tool for the determination of AAs in biological matrixes. With the method the pharmacokinetics of AAs were investigated successfully after oral administration of AF by rats.
Keywords: Cloud point extraction; HPLC; Aristolochic acids; Genapol X-080; Rat plasma; Aristolochiae Fructus;

An analytical procedure was developed to simultaneously determine bisphenol S, bisphenol F, bisphenol B, bisphenol A, bisphenol AF, tetrachlorobisphenol A, and tetrabromobisphenol A in reed and Callitrichaceae. Homogenized samples were extracted with acetonitrile and purified using an ENVI™-Carb cartridge followed by an NH2 cartridge. The analytes were separated and quantified by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The recoveries at three fortified levels in reed and Callitrichaceae were 57–108% and 68–106%, respectively, with relative standard deviations of no more than 15% (n  = 6). The method limits of quantification and detection for the seven bisphenol analogues were 0.005–0.500 μg/kg and 0.002–0.150 μg/kg, respectively. This method was used to analyze the seven compounds in ten reed and Callitrichaceae samples collected from Zhejiang, China.
Keywords: Bisphenol analogues; Reed; Callitrichaceae; Solid-phase extraction; UPLC–MS/MS;

A sensitive and fast LC–MS/MS method for determination of β-receptor agonist JP-49b: Application to a pharmacokinetic study in rats by Hui He; Kimberly Williams-Guy; Jayaprakash Pagadala; Chaela Sickbert Presley; Duane D. Miller; Jena J. Steinle; Charles R. Yates (86-91).
Ocular administration of the beta (β)-adrenergic receptor agonist JP-49b prevents retinopathy-like damage in a preclinical rat model of diabetes. Importantly, JP-49b did not induce characteristic β-adrenergic agonist-related side effects (e.g., left ventricular damage), which led to the hypothesis that JP-49b systemic exposure was minimal following ocular administration. To test this hypothesis, a sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to study the preclinical pharmacokinetics of JP-49b in rats. Animals received either a single periocular or intravenous injection of JP-49b (10 mg/kg) and plasma and tissue samples were obtained. JP-49b and fenoterol hydrobromide (internal standard, IS) were isolated by liquid–liquid extraction and extracts were analyzed by reversed-phase liquid chromatography on a C18 column using a gradient elution (acetic acid in water and methanol). A triple quadrupole mass spectrometer operating in the positive electrospray ionization mode with multiple reaction monitoring was used to detect JP-49b and IS transitions of m/z 346.4 → 195.1 and 304.1 → 134.9. The method was validated for selectivity, linearity, accuracy, and precision in rat vitreous humor, tissue homogenates, and plasma. Following intravenous administration, JP-49b was found to have a rapid clearance (36 ± 5.8 L/h/kg), high volume of distribution (244 ± 51.5 L/kg) and a terminal half-life of 4.8 ± 1.6 h. JP-49b was rapidly absorbed and extensively distributed into ocular tissue following topical administration. However, JP-49b was undetectable in heart tissue 24 h after ocular administration. High local drug concentrations coupled with minimal systemic exposure following ocular administration supports further testing of JP-49b as a localized therapy for diabetic retinopathy.
Keywords: LC–MS/MS; β-receptor agonist; JP-49b; Rat pharmacokinetics;

Beside the possible illegal use of malachite green in aquaculture, other familiar dyes could also been applied by fraudulent producers due to their antiseptic and antibacterial activity. In this contribution, a new sensitive multi-residue method was developed to determine triarylmethane, xanthene, phenothiazine and phenoxazine dyes in fish by ultra-performance liquid chromatography–tandem mass spectrometry. Samples were extracted with acetonitrile, followed by an oxidation step using 2,3-dichloro-5,6-dicyanobenzoquinone. Further clean-up was performed by tandem solid phase extraction using weak and strong cation exchange cartridges. Extracts were analysed by UPLC-MS n operating in the positive electrospray ionisation mode (ESI+). The fourteen dyes were separated within only 12 min on a C18 BEH column using 1 mM ammonium acetate in water at pH 4.5 and acetonitrile as mobile phases at a flowrate of 0.4 mL min−1. The presented method was validated as defined by the European Union and scientific literature. Good linearity (R ≥0.99 and goodness-of-fit (g) ≤10%) was achieved over the tested concentration range (0.25–2 ng g−1). Limit of quantification was 0.25 ng g−1 for all dyes, with a signal-to-noise ratio of at least 10/1. This is at least 5 to 10 times lower than previous published methods. Limits of detection were all <0.1 ng g−1. Precision and trueness fell within the criteria requested by the EC requirements for this concentration range. Decision limit (CCα) and detection capability (CCβ) were all <1 and <0.25 ng g−1, respectively. Due to background levels of the xanthene dyes, the two rhodamine dyes could only be determined above 0.75 ng g−1. For these dyes, the method can only be used for screening purposes. To show the applicability of the method, a monitoring study was performed to investigate the occurrence of artificial dyes in wildlife European eel in Flemish rivers
Keywords: Residue analysis; Dyes; Ultra performance liquid chromatography–tandem mass spectrometry; Aquaculture products; Malachite green; Crystal violet;

Set of nine angiotensin-converting enzyme inhibitors (enalapril, quinapril, fosinopril, lisinopril, cilazapril, ramipril, benazepril, perindopril and moexipril) were studied to evaluate the correlation between their intestinal absorption and salting-out thin-layer chromatography hydrophobicity parameters ( R M 0 or C 0) obtained by ascending technique applying four different salts, (NH4)2SO4, NH4NO3, NH4Cl and NaCl as mobile phases. The best correlations between KOWWIN log  P and both hydrophobicity parameters, R M 0 and C 0, (R 2  > 0.850) were observed for NaCl (1.0–3.0 M) while the lowest R 2 was obtained for (NH4)2SO4 (0.649 and 0.427, respectively) due to highest salting-out effect of (NH4)2SO4. The effect of selected inorganic salts in the salting-out mobile phases, on the solutes solubility and retention was evaluated. The topological polar surface area should be selected as independent variable (only this molecular descriptor showed low correlation with chromatographic hydrophobicity parameters) for multiple linear regression analysis, to obtain reliable correlation between angiotensin-converting enzyme inhibitor's intestinal absorption data and salting-out thin-layer chromatograpic hydrophobicity parameters. These correlations provide R 2  = 0.823 for R M 0 or R 2  = 0.799 for C 0 indicating good relationship between predicted and literature available intestinal absorption (ranged from 22% to 70%) of investigated angiotensin-converting enzyme inhibitors. The proposed in vitro model was checked with three in addition experimentally analyzed drugs, zofenopril, trandolapril and captoril. The satisfactory absorption prediction was obtained for zofenopril and trandolapril, while divergence established for captopril resulted from considerably different structure.
Keywords: Absorption modeling; Lipophilicity; Angiotensin-converting enzyme inhibitors; Salting-out thin-layer chromatography;

High throughput screening (HTS) techniques are required for the fast hit inhibitors selection in the early discovery process. However, in Beta-secretase (BACE1) inhibitors screening campaign, the most frequently used methoxycoumarin based peptide substrate (M-2420) is not widely applicable when aromatic or heterocycle compounds of natural source show auto-fluorescence interferences. Here, in order to overcome these drawbacks, we propose the use of a highly selective 4-(4-dimethylaminophenylazo)benzoic acid/5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (DABCYL/1,5-EDANS) based peptide substrate (Substrate IV), whose cleavage product is devoid of spectroscopic interference. HrBACE1-IMER was prepared and characterized in terms of units of immobilised hrBACE1. BACE1 catalyzed Substrate IV cleavage was on-line kinetically characterized in terms of K M and v max, in a classical Michaelis and Menten study. The on-line kinetic constants were found consistent with those obtained with the in solution fluorescence resonance energy transfer (FRET) standard method. In order to further validate the use of Substrate IV for inhibition studies, the inhibitory potency of the well-known BACE1 peptide InhibitorIV (IC50: 0.19 ± 0.02 μM) and of the natural compound Uleine (IC50: 0.57 ± 0.05) were determined in the optimized on-line hrBACE1-IMER. The IC50 values on the hrBACE1-IMER system were found in agreement with that obtained by the conventional methods confirming the applicability of Substrate IV for on-line BACE1 kinetic and inhibition studies.
Keywords: Liquid chromatography with fluorescence detection; Immobilized enzyme reactor; BACE1; Inhibition studies; Uleine;

An evaluation of washing and extraction techniques in the analysis of ethyl glucuronide and fatty acid ethyl esters from hair samples by L.C.A.M. Bossers; R. Paul; A.J. Berry; R. Kingston; C. Middendorp; A.J. Guwy (115-119).
Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) are alcohol metabolites measured in hair and are after a decade of research thought to be the best markers in hair to indicate alcoholism and abstinence Forensic Sci. Int. 218 (2012) 2. A great body of work concerning EtG and FAEEs detection in hair has been performed. However, no recent extensive comparison has been made concerning washing and extraction procedures. This work shows that the washing procedure of dichloromethane followed by a methanol rinse of the hair sample removes more than 16% of the FAEEs and 50% of the total EtG that is present in and on the hair. A review of ten washing protocols (where the removal is categorised: high, medium or low) showed that a relatively high percentage of FAEEs was removed and “medium” amount of EtG compared to the other washing protocols. This work shows promising results for the extraction of the FAEEs and the combined extraction of FAEEs and EtG by using 30 min of sonication with methanol. More FAEEs were recovered from hair with methanol than with any other extraction solvent including the commonly used dimethyl sulfoxide/heptane mixture. When the sonication time was increased a higher percentage of transesterification of the FAEEs was observed, the extraction was “dirtier” as solids and a colour change was observed whereas the extraction efficiency did not increase. Therefore, washing the hair sample with dichloromethane and methanol followed by an addition of 1 ml of methanol and sonication for 30 min to extract the FAEEs and EtG from hair is recommended for FAEEs as well as for the combined analysis of EtG and FAEEs. A linear calibration curve (r 2  > 0.99) was obtained for all analytes.
Keywords: Fatty acid ethyl esters; Ethyl glucuronide; Alcohol; Sample preparation; Washing; extraction;

Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications by Huihua Qu; Guiliang Zhang; Yifei Li; Hui Sun; Ye Sun; Yan Zhao; Qingguo Wang (120-125).
An enzyme-linked immunosorbent assay (ELISA) was developed, and its application in immunoaffinity column chromatography was studied using a monoclonal antibody (MAb) against puerarin. Splenocytes isolated from a female BALB/c mouse immunised with a puerarin–bovine serum albumin (BSA) conjugate were fused with SP2/0 myeloma cells. The hybridoma cell line secreting MAb against puerarin (AA9) was acquired by screening and limiting dilution. The antibody generated was highly specific for puerarin with <0.01% cross-reactivity with over 50 structurally related chemicals, except for baicalein (51.8%). Using AA9, we developed an immunoassay for puerarin with a linear detection range of 10 ng/ml to 1 μg/ml. This assay system was further validated using intra- and inter-assays and recovery experiments. In addition, puerarin levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. Finally, we developed and validated protocols for knocking puerarin out of its parent medicine completely. In conclusion, we successfully developed a reliable ELISA and an immunoaffinity column for puerarin detection and knockout, which are useful tools for exploring the role of puerarin in formulated Chinese medicines.
Keywords: Puerarin; Monoclonal antibody; ELISA; Immunoaffinity column chromatography;

Effect of cigarette smoking on urinary 2-hydroxypropylmercapturic acid, a metabolite of propylene oxide by Adam T. Zarth; Steven G. Carmella; Chap T. Le; Stephen S. Hecht (126-131).
2-Hydroxypropylmercapturic acid (2-HPMA) is a urinary biomarker of exposure to propylene oxide, a mutagen and carcinogen to which humans are exposed through inhalation of cigarette smoke as well as in certain environmental and occupational settings. 2-HPMA is the final product of a detoxification pathway in which propylene oxide is conjugated with glutathione, and the resulting conjugate is further metabolized and excreted. We have developed and validated a liquid chromatography-atmospheric pressure chemical ionization–tandem mass spectrometric (LC-APCI–MS/MS) method for the rapid quantitation of 2-HPMA in human urine. The method was applied to an analysis of urine samples from 40 smokers and 40 nonsmokers as well as from a group of 15 subjects who quit smoking. The results demonstrate that smokers have significantly (P  < 0.001) higher levels of urinary 2-HPMA (median = 480 pmol/mg creatinine) than do nonsmokers (208 pmol/mg). Similarly, subjects who quit smoking for four weeks exhibited a significant (P  < 0.001) 52% median decrease in urinary 2-HPMA upon cessation. Approximately 5% of all urine samples had unusually high levels of 2-HPMA (>10 times higher than the median), apparently unrelated to tobacco smoke exposure or available demographic data. The method presented here can be used to rapidly quantify an individual's exposure to propylene oxide via tobacco smoke or other sources.
Keywords: Propylene oxide; Mercapturic acid; Cigarette smoke; Smoking cessation;

The ionic liquid foam floatation solid phase extraction was established and applied to the extraction of six triazine herbicides, including desmetryn, secbumeton, terbumeton, terbuthylazine, dimethametryn and dipropetryn, in vegetable samples. To obtain the optimized experimental parameters, the effects of pH value of sample solution, the type and concentration of ionic liquid, the flow rate of carrier gas, foam floatation time, the type of solid phase extraction cartridge, the type and volume of elution solvent on the recoveries of the analytes were examined. The high performance liquid chromatography was applied to the determination of the analytes. Under the optimized experimental conditions, the linearities for determining the analytes were satisfactory and the limits of detection for desmetryn, secbumeton, terbumeton, terbuthylazine, dimethametryn and dipropetryn were 2.50, 1.75, 2.76, 1.87, 1.36 and 1.44 μg kg−1, respectively. The recoveries of the analytes ranged from 78.64% to 104.37% and the relative standard deviations ranged from 1.44 to 6.45%. The real samples were analyzed and the results were satisfactory.
Keywords: Ionic liquids; Foam floatation; Triazine herbicides; Solid phase extraction; Vegetable;

A method was developed to quantify human serum C-peptide by isotope-dilution mass spectrometry (ID MS). This new approach used immunoaffinity purification and chemical modification to improve the sensitivity which covered the wide range of reference interval of serum C-peptide. The immunoaffinity purification was performed using monoclonal antibody against human C-peptide that was immobilized on magnetic beads, and the purified C-peptide was chemically modified using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) prior to liquid chromatography–tandem mass spectrometry (LC–MS/MS). With this method, the LC–MS/MS peak area increased 23-fold compared with the conventional purification by solid-phase extraction and without chemical modification. The limit of quantification was estimated to be 0.003 ng on column, which was lower than previously reported. The validation study showed that (1) the response in the 0.003–2.9 ng range on column was linear (regression coefficient, r 2  = 0.9994), (2) the relative standard deviation (RSD) within and between days was inferior to 4.0%, and (3) the spike and recovery test showed the mean recoveries ranging between 99% and 108%. Comparison with an established commercial immunoassay showed high correlation (r 2  = 0.9994) at serum concentration of 0.19–8.49 ng/mL. These assessments suggest that this ID MS-based approach can quantify human serum C-peptide with high sensitivity and precision in the reference interval and find a potential use in the reference measurement procedure of serum C-peptide, allowing traceable measurement. This method may also generally be applied to peptide quantification in biological fluids with high sensitivity.
Keywords: C-peptide; Isotope-dilution mass spectrometry; LC–MS/MS; Chemical modification; Immunoaffinity purification;

Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study by Jianshe Ma; Chongliang Lin; Congcong Wen; Zheng Xiang; Xuezhi Yang; Xianqin Wang (143-146).
Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gamma-aminobutyric acid (GABA). A simple and sensitive liquid chromatography mass spectrometry method for determination of bicuculline in rat plasma was developed over the range of 5–500 ng/mL. After addition of midazolam as internal standard, protein precipitation with acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB–C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile −0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 368 for bicuculline and m/z 326 for the IS. Linear calibration was obtained with correlation coefficients r  > 0.99. The CV of the precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. Mean recoveries of bicuculline in plasma were in the range of 80.5–91.8%. The method was successfully applied to the pharmacokinetic study after gavage administration of 15 mg/kg bicuculline in rats.
Keywords: Bicuculline; LC–MS; Pharmacokinetics; Rat plasma;

Chromatographic determination of low-molecular mass unsaturated aliphatic aldehydes with peroxyoxalate chemiluminescence detection after fluorescence labeling with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole by Marwa Fathy Bakr Ali; Naoya Kishikawa; Kaname Ohyama; Horria Abdel-Mageed Mohamed; Hanaa Mohamed Abdel-Wadood; Ashraf Mohamed Mahmoud; Takahiro Imazato; Yukitaka Ueki; Mitsuhiro Wada; Naotaka Kuroda (147-152).
A highly sensitive, selective and reproducible chromatographic method is described for determination of low-molecular mass unsaturated aliphatic aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivatives were separated on a reversed-phase column C8 isocratically using a mixture of acetonitrile and 90 mM imidazole–HNO3 buffer (pH 6.4, 1:1, % v/v). The calibration ranges were: 20–420 nM for methylglyoxal, 16–320 nM for acrolein, 15–360 nM for crotonaldehyde and 20–320 nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5 nM (88–130 fmol/injection), the recovery results were within the range of 87.4–103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients’ sera with simple pretreatment method. In conclusion, this new method is suitable for routine analysis of large numbers of clinical samples for assessment of the oxidative stress state in patients.
Keywords: Peroxyoxalate chemiluminescence (PO-CL); Fluorescence labeling; Oxidative stress; Lipid peroxidation; Serum analysis;