Journal of Chromatography B (v.1044-1045, #C)

Sensitive analysis and simultaneous assessment of pharmacokinetic properties of crocin and crocetin after oral administration in rats by Yue Zhang; Fei Fei; Le Zhen; Xuanxuan Zhu; Jiankun Wang; Sijia Li; Jianliang Geng; Runbin Sun; Xiaoyi Yu; Tingting Chen; Siqi Feng; Pei Wang; Na Yang; Yejin Zhu; Jingqiu Huang; Yuqing Zhao; Jiye Aa; Guangji Wang (1-7).
Crocin and crocetin in rat plasma were simultaneously analysed using ultra-performance liquid chromatography tandem mass spectroscopy (UPLC–MS/MS), and method was fully validated. For the first time, levels of both crocin and crocetin in plasma were profiled after oral administration of crocin, and this UPLC–MS/MS approach was applied to evaluate pharmacokinetics and relative bioavailability of crocin and crocetin in rats. It was shown that crocin transformed into crocetin quickly in the gastrointestinal tract, and crocetin was 56–81 fold higher exposed in rat plasma than crocin after oral administration of crocin. A comparison study revealed that an oral administration of equal molar crocin achieved higher exposure of crocetin in rat plasma than that of crocetin. It was suggested that oral administration of crocin has the advantages over crocetin, and crocetin may be the active component potentially responsible for the pharmacological effect of crocin.
Keywords: Crocin; Crocetin; LC–MS/MS; Pharmacokinetics; Bioavailability;

Fenugreek is a traditional plant for the treatment of diabetes. Galactomannan, an active major component in fenugreek seeds, has shown hypoglycemic activity. The present study was performed to investigate the therapeutic mechanism underlying fenugreek galactomannan (F-GAL) in treating diabetes, using a metabonomics approach based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The F-GAL used for study was highly purified, and its yield, purity, and galactose/mannose ratio were characterized by capillary zone electrophoresis (CZE) and a modified phenol-sulfuric acid method. After treatment of streptozotocin (STZ)-induced diabetic rats with F-GAL for 28 days, urine and serum samples were analyzed by UPLC-QTOF/MS. Multivariate statistical approaches such as principal component analysis (PCA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) were applied to distinguish the non-diabetic/untreated, diabetic/untreated, and diabetic/F-GAL-treated groups. Then, potential biomarkers were identified that may help elucidate the underlying therapeutic mechanism of F-GAL in diabetes. The results demonstrated that there was a clear separation among the three groups in the PCA model. Fourteen potential biomarkers were identified by OPLS-DA, and they were determined to be produced in response to the therapeutic effects of F-GAL. These biomarkers were involved in histidine metabolism, tryptophan metabolism, energy metabolism, phenylalanine metabolism, sphingolipid metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism. In conclusion, our study demonstrates that a metabonomics approach is a powerful, novel tool that can be used to evaluate the underlying therapeutic mechanisms of herb extracts.
Keywords: Fenugreek galactomannan; Metabonomics; Therapeutic mechanism; Diabetes; Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry;

Development of an affinity cryogel for one step purification of lysozyme from chicken egg white by Paula Chequer Gouveia Mól; Lizzy Ayra Alcântara Veríssimo; Monique Renon Eller; Valéria Paula Rodrigues Minim; Luis Antonio Minim (17-23).
In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20–80 μm and swelling capacity of 19.6 ± 0.9 g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04 cm to 0.8 cm. Adsorption isotherms were measured at 15 °C and 35 °C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285 mg/g at 15 °C and 363 mg/g at 35 °C. Thermodynamic analysis based on the Van’t Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.
Keywords: Lysozyme; Cryogel; Affinity chromatography; Purification;

Plasma PCSK9 measurement by liquid chromatography–Tandem mass spectrometry and comparison with conventional ELISA by Mikaël Croyal; Fanta Fall; Michel Krempf; Aurélie Thédrez; Khadija Ouguerram; Véronique Ferchaud-Roucher; Audrey Aguesse; Stéphanie Billon-Crossouard; Pedro Mata; Rodrigo Alonso; Gilles Lambert; Estelle Nobécourt (24-29).
The combination of liquid chromatography-tandem mass spectrometry (LC–MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91 ± 7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5 ng/ml. This LC–MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r = 0.936, p < 0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC–MS/MS or ELISA methods (514.2 ± 217.2 vs. 504.2 ± 231.0 ng/ml, respectively- p = NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.
Keywords: Proteotypic peptide analysis; LC–MS/MS; Solid-phase extraction; PCSK9;

An accurate, precise, selective, and sensitive liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of imperatorin (IMP) and its metabolite, xanthotoxol (XAN), in rat plasma and urine samples. The analytes, along with psoralen as an internal standard, were determined by multiple reaction monitoring (MRM) operated in the positive electrospray ionization (ESI) mode. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid in methanol at a flow rate of 0.3 mL/min. The run time was 6 min per sample and the injection volume was 5 μL. The method had a lower limit of quantification (LLOQ) of 0.25 ng/mL for IMP in plasma and urine, and 1 ng/mL for XAN in urine. The linear calibration curves were fitted over the range of 0.25–1000 ng/mL for IMP in plasma, 0.25–1000 ng/mL for IMP in urine, and 1–1000 ng/mL for XAN in urine, with correlation coefficients greater than 0.995. The inter- and intra-day accuracies (relative error, RE%) were between −8.5% and 3.5%, and the precisions (relative standard deviation, RSD%) were less than 10.0% for all quality control samples (QCs). The analytes were extracted from rat plasma and urine samples using a liquid-liquid extraction method with the extraction recovery in the range of 60.3–79.1%. A good stability of the analytes was observed in all the analysis procedures. The method was successfully validated and applied to determine the pharmacokinetics of IMP in rat plasma and, for the first time, the metabolite kinetics of IMP to XAN in rat urine after IMP administration.
Keywords: Imperatorin; Xanthotoxol; Metabolite; LC–MS/MS; Pharmacokinetics;

Thymol detection and quantitation by solid-phase microextraction in faeces and egg yolk of Japanese quail by María E. Fernandez; Marcela A. Palacio; María C. Labaque (39-46).
To measure bioavailability of the active ingredients of phytogenic feed additives in poultry products and subproducts is a key element for developing a rational understanding of its mode of action and biological effects. Hence, we validated a headspace solid phase microextraction (HS-SPME) technique followed by gas chromatography-mass spectrometry as an analytical extraction procedure and as method for detection and quantitation of 2-Isopropyl-5-methylphenol (thymol) in faeces and egg yolk of quail. The suitability of this method for thymol analysis in both matrices was first proved via linearity, limit of detection, limit of quantification, and recovery using m-cresol as internal standard. The optimal HS-SPME extraction conditions were obtained at 40 °C for 5 min in faeces and 60 °C for 30 min in egg yolk. This procedure was found to be precise, sensitive and linear in the range of 2.5–100 ng/gr for faeces and 20–800 ng/gr for the egg yolk. Limits of detection were 0.5 ng/g and 5 ng/g for faeces and yolk, respectively, and the limits of quantitation were 1 ng/g and 10 ng/g for faeces and yolk, respectively. The method was successfully used for measuring thymol in fecal and egg yolk samples, from quails supplemented with thymol in their diets. Thus, in fresh faeces and egg yolk samples obtained from a supplemented group (80 mg thymol per bird per day) were determined as 31.51 ng/g for faeces and 11.83 ng/g for the egg yolk.
Keywords: Poultry; Phytogenic feed additives; Bioavailabilty; Terpens;

Chromatographic efficiency and selectivity in top-down proteomics of histones by Yiyang Zhou; Ximo Zhang; Luca Fornelli; Philip D. Compton; Neil Kelleher; Mary J. Wirth (47-53).
Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC–MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC–MS is performed on a histone sample using difluoroacetic acid and a 20-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5 μm in diameter, 2) superficially porous particles of 3 μm in diameter with a shell of 0.2 μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC–MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.
Keywords: Histone; Post-translational modification; Revesred-phase; Difluoroacetic acid;

Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel by Dandan Zhang; Tianli Lei; Chongning Lv; Huimin Zhao; Haiyan Xu; Jincai Lu (54-62).
The rhizome of Anemone raddeana Regel, a Traditional Chinese Medicine (TCM) which has a robust history treating rheumatism and neuralgia. The total secondary saponin (TSS) from it has demonstrated antitumor activity. In this study, a rapid and validated LC–MS/MS method was developed to simultaneously determine the active compounds (Hederacolchiside A1 and Eleutheroside K). Analytes were separated on a reverse-phase C18 column with acetonitrile-water (5 mmol/L ammonium acetate) as the mobile phase. This assay showed acceptable linearity (r > 0.99) over the concentration range 5–1000 nmol/L for two analytes. The intra- and inter-day precision was within 8.06% and accuracy was ranged from −3.16% to 3.34% for two analytes. The mean extraction recoveries of analytes and IS from rat plasma were all more than 76.0%. Under the developed analytical conditions, the obtained values of main pharmacokinetic parameters (C max and AUC0–t) indicated that the pure compounds were more efficient than the TSS extract in Hederacolchiside A1 and Eleutheroside K absorption. In addition, pharmacokinetic studies of two individual compounds demonstrated their poor oral absorption in rat (aF%, 0.019–1.521). In the study of absorption and transportation of Hederacolchiside A1 and Eleutheroside K in Caco-2 cell monolayer model, the uptake permeability was in 10−6  cm/sec range suggesting poor absorption, which confirmed the previous pharmacokinetic profiles in vivo. Interestingly, the uptake ratio of them declined significantly when treated with phloridzin (SGLT1 inhibitor). It indicated that the absorption of Hederacolchiside A1 in intestine was mainly through positive transport and SGLT1 might participate in its active absorption.
Keywords: Anemone raddeana regel; Triterpenoid saponins; LC–MS/MS; Pharmacokinetics; Caco-2 cell monolayer model;

Easy, fast and environmental friendly method for the simultaneous extraction of the 16 EPA PAHs using magnetic molecular imprinted polymers (mag-MIPs) by Mercedes Villar-Navarro; María Jesús Martín-Valero; Rut Maria Fernández-Torres; Manuel Callejón-Mochón; Miguel Ángel Bello-López (63-69).
An easy and environmental friendly method, based on the use of magnetic molecular imprinted polymers (mag-MIPs) is proposed for the simultaneous extraction of the 16 U.S. EPA polycyclic aromatic hydrocarbons (PAHs) priority pollutants. The mag-MIPs based extraction protocol is simple, more sensitive and low organic solvent consuming compared to official methods and also adequate for those PAHs more retained in the particulate matter. The new proposed extraction method followed by HPLC determination has been validated and applied to different types of water samples: tap water, river water, lake water and mineral water.
Keywords: Polycyclic aromatic hydrocarbons (PAHs); Magnetic molecular imprinted polymers (mag-MIPs); Extraction procedure; Water quality;

A sensitive and precise method is described for the simultaneous determination of two small molecule kinase inhibitors: MK-1775 (MK) and AZD-7762 (AZD), in acetonitrile (ACN)-aqueous solution and in mouse plasma. A Nova-Pak C18 reversed phase column (3.9 mm × 150 mm, 4 μm, 60 Å) was utilized in the separation using an isocratic mobile phase of 0.1% v/v triethylamine in phosphate buffer (pH = 7.4): acetonitrile (ACN) (60:40, v/v), at a flow rate of 0.8 mL/min. Detection wavelength was set at 310 nm for both MK and AZD, and 431 nm for the internal standard sunitinib (SUN). The developed method was validated following the ICH guidelines and it was shown to be accurate, precise and linear in the range of 41 ng/mL to 8333 ng/mL for both drugs in the ACN-aqueous solution and from 83 ng/mL to 8333 ng/mL for both drugs in mouse plasma samples. For the first time, the presented data suggest the suitability of this method for the simultaneous separation and quantification of MK and AZD in both ACN aqueous solution as well as in mouse plasma samples.
Keywords: MK-1775; AZD-7762; Sunitinib; HPLC-UV; Solid phase extraction; Mouse plasma;

Dried haematic microsamples and LC–MS/MS for the analysis of natural and synthetic cannabinoids by Michele Protti; James Rudge; Angelo Eliseo Sberna; Gilberto Gerra; Laura Mercolini (77-86).
Display OmittedSynthetic cannabinoids are new psychoactive substances (NPS) with similar effects when compared to natural ones found in Cannabis derivatives. They have rapidly integrated into the illicit market, often sold as alternatives under international control. The need to identify and quantify an unprecedented and growing number of new compounds represents a unique challenge for toxicological, forensic and anti-doping analysis. Dried blood spots have been used within the bioanalytical framework in place of plasma or serum, in order to reduce invasiveness, lower sample size, simplify handling, storage and shipping of samples and to facilitate home-based and on-field applications. However, DBS implementation has been limited mainly by concerns related to haematocrit effect on method accuracy. Volumetric absorptive microsampling (VAMS™), a second generation dried miniaturized sampling technology, has been developed just in order to eliminate haematocrit effect, thus providing accurate sampling but still granting feasible sample processing. An original LC–MS/MS method was herein developed and validated for the analysis of THC and its 2 main metabolites, together with 10 representative synthetic cannabinoids in both DBS and VAMS dried microsamples. The ultimate goal of this work is to provide highly innovative DBS and VAMS analytical protocols, whose performances were extensively optimized and compared, in order to provide effective and alternative tools that can be applied for natural and synthetic cannabinoid determination, in place of classical analytical strategies.
Keywords: Volumetric absorptive microsampling (VAMS); Dried blood spot (DBS); Natural cannabinoids; Synthetic cannabinoids; LC–MS/MS;

UHPLC–MS/MS method for the quantitation of penicillin G and metabolites in citrus fruit using internal standards by Daniele Canzani; Kevin Hsieh; Matthew Standland; Walter Hammack; Fadi Aldeek (87-94).
Penicillin G has been applied to citrus trees as a potential treatment in the fight against Huanglongbing (HLB). Here, we have developed and validated a method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). This method improves upon a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D5, and penilloic acid-D5. These standards greatly enhanced the accuracy and precision of our measurements by compensating for recovery losses, degradation, and matrix effects. When 2 g of citrus fruit sample is extracted, the limits of detection (LOD) were determined to be 0.1 ng/g for penicillin G and penilloic acid, and 0.25 ng/g for penillic acid. At fortification levels of 0.1, 0.25, 1, and 10 ng/g, absolute recoveries for penillic and penilloic acids were generally between 50–70%. Recoveries corrected with the isotopically labeled standards were approximately 90–110%. This method will be useful for the identification and quantitation of drug residues and their degradation products using isotopically labeled standards and UHPLC–MS/MS.
Keywords: Huanglongbing (HLB); Penicillin G; Metabolites; LC–MS/MS; Internal standards; Method development;

The commercial polyurethane foam was acid modified to get an inexpensive adsorbent (AM-PUF) has highly surface polarity and sorption capacity. The elemental analysis, scanning electron microscopy, thermal analysis, ultraviolet/visible/infrared spectroscopies and X-ray diffraction were used for characterization of AM-PUF. The surface of AM-PUF has amorphous character (broadband at 2θ, 21.75°) and contains several active sites e.g. N―H, O―H, C=O, C=C and C―O―C groups. The electrical conductivity (σ), iodine value and methylene blue index of AM-PUF are 1.7 × 10−5  Ω−1  m−1, 208 mg/g and 107 mg/g. The AM-PUF has a high efficiency for completely removing (99–100%) of Aldrin, DDT, Endrin, Heptachlor, Heptachlor epoxide and Lindane pesticides in both acidic and alkaline solutions. The removing rates of the organochlorine pesticides from wastewater are very rapid (t1/2  = 22 s). The negative value of ΔG (-10.9 kJ/mol) for removing of OCPs using AM-PUF showed that the feasibility of the removing process and its spontaneous nature.
Keywords: Acid modified polyurethane foam; Organochlorine pesticides; Nile River; Drain agricultural wastewater; Detection; Removal;

Strategies for metabolite profiling based on liquid chromatography by Javier Saurina; Sonia Sentellas (103-111).
This paper aims at covering the principal strategies based on liquid chromatography (LC) for metabolite profiling in the field of drug discovery and development. The identification of metabolites generated in the organism is an important task during the early stages of preclinical research to define the most proper strategy for optimizing, adjusting metabolic clearance and minimizing bioactivation. An early assessment of the metabolite profile may be critical since metabolites can contribute to pharmacological and/or toxicological effects. The study of metabolites first involves their synthesis/generation and their further characterization and structural elucidation. For such a purpose, both in vitro and in vivo methods are commonly used for the generation of the corresponding metabolites. Next, analytical methods are used to tackle identification and characterization studies. Among the arsenal of techniques available in our labs, we will focus on LC, especially coupled to mass spectrometry (LC–MS), as one of the most powerful approaches for metabolite identification, characterization and quantification. Here, the topic of metabolite profiling based on LC will be addressed and representative examples of different possibilities will be discussed.
Keywords: Metabolite profiling; Structural elucidation; Liquid chromatography; Mass spectrometry;

Extraction of plasmid DNA (pDNA) from an alkaline bacterial cell lysate has been investigated using partitioning and affinity partitioning in aqueous two-phase systems (ATPSs) containing 12% (w/w) polyethylene glycol and 12% (w/w) sodium sulfate. In this work, a modified 20 bp pyrimidine oligonucleotide acting as a triplex-forming oligonucleotide (TFO) was employed as an affinity ligand to improve the partition behavior. The pUC118 plasmid was chosen to be extracted because of its specific oligonucleotide recognition sequence. In order to find the proper conditions, operational parameters such as PEG molecular weight, pH and lysate load were also studied. The results showed that adding the affinity ligand strongly changed the partitioning behavior of pDNA without any considerable impact on the partitioning of RNA and protein content. The best achieved result was at pH = 6 and PEG MW of 600 Da and 60% of lysate load. In these conditions, 67% of pDNA was recovered in the top phase while the contaminants mainly accumulated in the bottom phase.
Keywords: Aqueous two-phase; Bio-separations; Purification; DNA; Partitioning;

Graphene oxide (GO)-based dispersive solid phase extraction (D-SPE) method combined with multi-step preparation has been proposed for the evaluation of trace aflatoxins in proprietary Chinese medicines (PCM). After being extracted by methanol, the sample was purified based on multi-step preparation, including dehydration with MgSO4/NaCl and cleanup with neutral alumina. Then GO was used as an adsorbent in D-SPE method for further preconcentration of aflatoxins prior to high performance liquid chromatography-fluorescence detection. The selected conditions were investigated. The Box-Behnken design (BBD) was used to optimize factors affecting adsorption procedure. Under the optimized conditions, good linear relationships had been achieved with the correlation coefficient (R2) varying from 0.9904 to 0.9990. The LODs and LOQs were ranging from 0.020 to 0.041 ng/mL and 0.061 to 0.125 ng/mL, respectively. The results of the recoveries were 74.0–102.7% for the four aflatoxins, while the precisions from 1.8% to 7.2% were obtained, which indicated that the method was suitable for the analysis of aflatoxins in PCM.
Keywords: Aflatoxins; Graphene oxide; Dispersive solid phase extraction; Multi-step pretreatment; Proprietary Chinese medicines;

We have developed a chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC–NSI–HRMS/MS) method to investigate the enantiomeric composition of low parts per trillion amounts of the carcinogen N'-nitrosonornicotine (NNN) in the urine of cigarette smokers and smokeless tobacco users. (S)-NNN is the major enantiomer in tobacco and is more carcinogenic than (R)-NNN in rats, but no data are available on the enantiomeric composition of NNN in humans. The method used [13C6]NNN as an internal standard and [pyridine-D4]nornicotine to monitor possible artifactual formation of NNN, which was found to be less than 2% of the quantified NNN. The enantiomeric composition of NNN (20.5 ± 27.1 fmol/mL urine) in 20 cigarette smokers was 67 ± 5% (S)-NNN while that in 10 smokeless tobacco users (67.1 ± 56.7 fmol/mL urine) was 56 ± 3% (S)-NNN. These results demonstrate that the highly carcinogenic (S)-NNN is the major enantiomer in human urine, and that the enantiomeric composition of NNN in human urine is remarkably similar to that in cigarette smoke and smokeless tobacco. This is the first study to combine chiral stationary phase separations with nanoelectrospray ionization and high resolution tandem mass spectrometry to quantify trace levels of enantiomeric metabolites in human urine.
Keywords: N'-Nitrosonornicotine; Enantiomeric composition; Chiral separation; Smoker’s urine; Smokeless tobacco user’s urine; Chiral stationary phase liquid chromatography–nanoelectrospray ionization–high resolution tandem mass spectrometry;

Healthy animals are most widely used in current pharmacokinetic(PK) studies. However, neglecting the effects of specific diseases on drug absorption results in the PK parameters of those experiments not accurately reflecting in vivo drug concentration changes during treatment. In this study, an E. coli infective diarrheal minipig model was applied to explore the pharmacokinetics of Gegen Qinlian decoction (GQD). A simple and rapid ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the concentrations of the eight GQD components in minipig plasma after intragastric administration of GQD. The PK parameters of the main GQD components in normal and model minipigs after oral administration of GQD were compared. There were statistically significant differences (p  < 0.05) in the pharmacokinetic parameters of Puerarin, Wogonin and Daidzein involving the AUC0−t, Cmax, MRT(0−t), t1/2z between normal and model minipigs. Results showed that bacterial diarrhea had a great impact on the biological availability of the main ingredients in GQD. More importantly, the results obtained suggest that the bacterial diarrheal minipig model can be successfully applied in PK studies and may be used in other PK studies of drugs targeting intestinal disease.
Keywords: Minipigs; Bacterial diarrhea model; Pharmacokinetics; UHPLC–MS/MS; Gegen Qinlian decoction;

Simultaneous determination of amantadine, rimantadine, and memantine in processed products, chicken tissues, and eggs by liquid chromatography with tandem mass spectrometry by Yumi Tsuruoka; Takayuki Nakajima; Maki Kanda; Hiroshi Hayashi; Yoko Matsushima; Souichi Yoshikawa; Marie Nagata; Hiroshi Koike; Chieko Nagano; Kotaro Sekimura; Tsuneo Hashimoto; Ichiro Takano; Tetsuya Shindo (142-148).
A simultaneous determination of amantadine, rimantadine, and memantine in processed products (deep-fried chicken, fried chicken, fried quail egg, and grilled chicken) with liquid chromatography tandem mass spectrometry (LC–MS/MS) was developed. This new method was also applicable for chicken tissue (muscle, liver, and gizzard) and eggs. The chromatographic separation was performed on a Kinetex® XB-C18 core–shell technology column using a mobile phase of acetonitrile and 0.1% formic acid in a 10 mmol/L ammonium formate solution, resulting in the complete separation of isomers (rimantadine and memantine) and any other obstructive peaks from the sample matrices. Sample preparation was performed by a modified QuEChERS method using acetonitrile and a 0.1% acetic acid extraction solution and cleaned using an Oasis® MCX cartridge. The sample matrix had no effect on the identification of the compounds. For quantification, an external solvent calibration curve was used. This new method exhibited good accuracy ranging from 79.9% to 91.5%. The relative standard deviation of repeatability (RSDr) ranged from 1.2% to 3.6% and the relative standard deviation of within-laboratory reproducibility (RSDWR) ranged from 1.3% to 6.0%. These standard deviations satisfied the criteria for Japanese validation guidelines. The limit of quantification (LOQ) was 1.0 μg/kg for all samples. Analyte residues were not detected in 55 samples using the validated method.
Keywords: Amantadine; Rimantadine; Memantine; LC–MS/MS; External solvent calibration curve; Processed products;

Method development for detecting the novel cyanide antidote dimethyl trisulfide from blood and brain, and its interaction with blood by Lóránd Kiss; Secondra Holmes; Ching-En Chou; Xinmei Dong; James Ross; Denise Brown; Brooke Mendenhall; Valerie Coronado; Deepthika De Silva; Gary A. Rockwood; Ilona Petrikovics; David E. Thompson (149-157).
The antidotal potency of dimethyl trisulfide (DMTS) against cyanide poisoning was discovered and investigated in our previous studies. Based on our results it has better efficacy than the Cyanokit and the Nithiodote therapies that are presently used against cyanide intoxication in the US. Because of their absence in the literature, the goal of this work was to develop analytical methods for determining DMTS from blood and brain that could be employed in future pharmacokinetic studies. An HPLC-UV method for detection of DMTS from blood, a GC–MS method for detection of DMTS from brain, and associated validation experiments are described here. These analytical methods were developed using in vitro spiking of brain and blood, and are suitable for determining the in vivo DMTS concentrations in blood and brain in future pharmacokinetic and distribution studies. An important phenomenon was observed in the process of developing these methods. Specifically, recoveries from fresh blood spiked with DMTS were found to be significantly lower than recoveries from aged blood spiked in the same manner with DMTS. This decreased DMTS recovery from fresh blood is important, both because of the role it may play in the antidotal action of DMTS in the presence of cyanide, and because it adds the requirement of sample stabilization to the method development process. Mitigation procedures for stabilizing DMTS samples in blood are reported.
Keywords: Dimethyl trisulfide; Dimethyl disulfide; Dibuthyl disulfide; HPLC; GC–MS; SPME;

A sensitive, reliable and validated LC–MS/MS method was developed to determine the presence of eight flavonoids (catechin, typhaneoside, isorhamnetin-3-O-neohesperidoside, astragalin, isorhamnetin-3-O-β-d-glucoside, naringenin, kaempferol and isorhamnetin) in rat plasma. Puerarin was selected as the internal standard. Precipitation of the protein method with acetonitrile was used to extract these flavonoids from the rat plasma samples. The analysis was carried out on an Eclipse plus C18 column (4.6 mm × 100 mm, 1.8 μm) when acetonitrile and formic acid aqueous solution (0.1%) was used as the mobile phase at a flow rate of 0.3 mL min−1 . A tandem mass spectrometer having an electrospray ionization (ESI) source was used to detect eight flavonoids using multiple reaction monitoring (MRM) in the negative ionization mode. The LLOQs for catechin, typhaneoside, isorhamnetin-3-O-neohesperidoside, astragalin, isorhamnetin-3-O-β-d-glucoside, naringenin, kaempferol and isorhamnetin are 4, 4, 4, 0.8, 1, 0.4, 2 and 0.2 ng mL−1, respectively. The precision, accuracy and recovery were all within acceptable limits and the analytes were stable in plasma for all conditions tested. The method was successfully applied to pharmacokinetic study of four flavonoids in rat plasma after administering Pollen Typhae extract orally to rats.
Keywords: Pollen typhae; Typhaneoside; Isorhamnetin-3-O-neohesperidoside; LC–MS/MS; Pharmacokinetics;

We report the application of a liquid chromatography-tandem mass spectrometry (LC–MS/MS) bioanalytical method for the determination of a recombinant human immunoglobulin G1 (hIgG1), NVSMAb-1, in rat serum. A stable isotopically labeled universal monoclonal antibody (SILuMab), instead of stable isotopically labeled surrogate peptide, was employed as the internal standard. The internal standard was added to the sample matrix in the first step of the sample preparation process, which involved protein precipitation and pellet digestion. The digestion of the resulting pellet with trypsin was performed prior to analysis of surrogate peptides of both NVSMAb-1 and SILuMab using LC–MS/MS. Precipitation reagents (1% TCA in IPA, 75% MeOH and 14% PEG) and digestion conditions (50 °C for 2 h and 60 °C for 0.5 h) were evaluated by monitoring LC–MS/MS responses of GPS and VVS in the resulting sample extracts. Overall, the use of 1% TCA in IPA appeared to be more effective as compared to 75% methanol in protein precipitation and removal of unwanted matrix components, e.g., albumin, and more appealing than 14% PEG as it avoided additional steps that are necessary to remove PEG or reduce PEG to a negligible level. The yield (LC–MS/MS response) of GPS is less sensitive than VVS to the changes of digestion conditions (time and temperature). The results obtained using SILuMab over SIL surrogate peptide as the internal standard appeared unaffected by the suboptimal sample processing method. For the current assay, surrogate peptide GPSVFPLAPSSK (GPS) was selected as surrogate peptide over VVSVLTVLHQDWLNGK (VVS) for quantitative analysis of NVSMAb-1. The optimal chromatographic separation was achieved on a Waters Cortecs C18 (100 × 2.1 mm, 2.7 μm) column using gradient elution with a total cycle time of approximately 8 min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10–5000 μg/mL using 20 μL of rat serum. The accuracy and precision for the LLOQs (10 μg/mL) were within ±6.0% bias and ≤6.5% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the other QC sample (30, 300, 2500 and 3750 μg/mL) results were ≤6.8% CV and the accuracy within ±4.8% bias, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a toxicity study in rats administered 30, 150 and 750 mg/kg/week intravenous infusion and 150 mg/kg/week subcutaneous injection of NVSMAb-1.
Keywords: Stable isotope labeled universal monoclonal antibody (SILuMab); LC–MS/MS; Pellet digestion; Surrogate peptide; Stable-isotope-labeled peptide IS;

Simultaneous determination of multiple bioactive components of Bu-zhong-yi-qi-tang in rat tissues by LC–MS/MS: Application to a tissue distribution study by Min He; Wenwen Chen; Mengmeng Wang; Yu Wu; Jin Zeng; Zhirong Zhang; Shujiao Shen; Jian Jiang (177-184).
A liquid chromatography coupled with electrospray ionization mass spectrometry method was developed and validated for simultaneous determination of seven bioactive constituents including astragaloside IV, calycosin, glycyrrhizic acid, enoxolone, saikosaponin D, ferulic acid and hesperiden in rats’ various tissues using diclofenac as the internal standard (IS). Biological samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid and 4 mM ammonium acetate). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 829.7 → 783.3 for astragaloside IV, m/z 283.3 → 267.7 for calycosin, m/z 821.6 → 350.0 for glycyrrhizic acid, m/z 469.9 → 425.2 for enoxolone, m/z 825.7 → 779.6 for saikosaponin D, m/z 192.5 → 133.9 for ferulic acid, m/z 609.1 → 301.0 for hesperiden and m/z 293.6 → 249.9 for the IS, respectively. The lower limits of quantification for the seven analytes in different rat tissues were 0.2–20 ng/mL. Bu-zhong-yi-qi-tang (Hochuekkito in Japan, Bojungikki-tang in Korea) is one of the most frequently prescribed traditional herbal formulas used in Korea, Japan, and China to treat gastrointestinal diseases, cancer and chronic fatigue syndrome. The validated method was successfully applied to a tissue distribution study of the seven components in rat tissue after oral administration of Bu-zhong-yi-qi-tang concentrated granule. The results of the tissue distribution study showed that the high concentration of seven components were mainly in the gastrointestinal tract.
Keywords: Bu-zhong-yi-qi-tang; LC–MS/MS; Tissue distribution;

Methylisothiazolinone and the mixture of chloromethylisothiazolinone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetic and household products. Due to their skin permeability, they might be taken up by the general population via use of products containing these biocides. As both compounds are known skin sensitizers, the use of these products is under discussion by regulatory agencies.In order to evaluate the possible uptake of MI and/or MCI/MI by human biomonitoring, we have developed and validated a highly sensitive and specific GC/MS/MS-method for the quantification of N-methylmalonamic acid (NMMA), a known metabolite of MI and MCI in urine of rats. After freeze-drying of urine, the analyte is derivatised with pentafluorobenzyl bromide in anhydrous solution and the PFB-derivative is extracted into n-hexane. After concentration, the derivative is finally quantified by GC/MS/MS in EI-mode using 13C3-NMMA as internal standard. The limit of quantification for NMMA was 0.5 ng mL−1 urine. Precision within and between-series was determined to range between 3.7–10.9% using native and spiked quality control samples. Accuracy ranged between 89 and 114%.In a pilot study we applied this method to spot urine samples of 63 persons not knowingly exposed to MI and/or MCI/MI. NMMA was quantifiable in every urine sample analysed, with no significant difference in urinary levels between male and female participants. The median (95th percentile) levels for urinary NMMA were 3.6 (7.4) ng mg−1 creatinine and 2.9 (9.1) ng mg−1 creatinine for males (n = 32) and females (n = 31), respectively. In a volunteer experiment, a relation of exposure to MI and/or MCI/MI and subsequent NMMA-excretion was shown. Our method is the first to report human urinary background levels of NMMA. However, the possibility of formation and urinary excretion of NMMA within physiological processes cannot be ruled out.
Keywords: Human biomonitoring; Biocides; Metabolism; Urine; Pentafluorobenzyl bromide;

Determination of inosine 5′-monophosphate dehydrogenase activity in red blood cells of thiopurine-treated patients using HPLC by Audrey Beringer; Antony Citterio-Quentin; Rebeca Obenza Otero; Clémence Gustin; Rebecca Clarke; Jean-Paul Salvi; Roselyne Boulieu (194-199).
Thiopurine drugs are commonly used in immune diseases and to a lesser extent, in transplant rejection prophylaxis: however interindividual variability in drug response and in the occurrence of adverse events is observed. Genetic variation in thiopurine S-methyltransferase (TPMT) doesn’t completely explain the occurrence of all adverse events and drug response variability. The potential implication of other enzymes involved in thiopurine metabolism, such as ITPA, has been investigated over the last decade but little data is available on inosine 5′-monophosphate dehydrogenase (IMPDH) in patients treated with thiopurine drugs. The authors reported a HPLC method to determine IMPDH activity in the red blood cells (RBCs) of thiopurine-treated patients. IMPDH activity was evaluated by enzymatic conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The XMP formed was analyzed on a Luna® NH2 stationary phase, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. XMP was eluted below 15 min. Intra-assay and inter-assay precisions were below 9% for RBCs supplemented with 2, 40 and 80 μmol/L of XMP. IMPDH activity was measured in adults without thiopurine treatment as well as in adult and paediatric patients treated with thiopurines. A wide interindividual variability in IMPDH activity in RBCs was observed. No difference in IMPDH activity was found between untreated subjects and adult and paediatric patients on thiopurine therapy (median value 11.8, 7.9 and 7.7 nmol XPM/g Hb/h respectively). The method described is useful in the determination of IMPDH phenotype from patients on thiopurine therapy and in the investigation of the potential relationship between IMPDH activity in RBCs and the occurrence of adverse events and drug response variability.
Keywords: Inosine monophosphate dehydrogenase; IMPDH; Phenotype; Red blood cells; Thiopurines;

Quantitative analysis of menthol in human urine using solid phase microextraction and stable isotope dilution gas chromatography–mass spectrometry by Wenlin Huang; Benjamin C. Blount; Clifford H. Watson; Christina Watson; David M. Chambers (200-205).
To accurately measure menthol levels in human urine, we developed a method using gas chromatography/electron ionization mass spectrometry with menthol-d4 stable isotope internal standardization. We used solid phase microextraction (SPME) headspace sampling for collection, preconcentration and automation. Conjugated forms of menthol were released using β-glucuronidase/sulfatase to allow for measuring total menthol. Additionally, we processed the specimens without using β-glucuronidase/sulfatase to quantify the levels of unconjugated (free) menthol in urine. This method was developed to verify mentholated cigarette smoking status to study the influence of menthol on smoking behaviour and exposure. This objective was accomplished with this method, which has no carryover or memory from the SPME fiber assembly, a method detection limit of 0.0017 μg/mL, a broad linear range of 0.002–0.5 μg/mL for free menthol and 0.01–10 μg/mL for total menthol, a 7.6% precision and 88.5% accuracy, and an analysis runtime of 17 min. We applied this method in analysis of urine specimens collected from cigarette smokers who smoke either mentholated or non-mentholated cigarettes. Among these smokers, the average total urinary menthol levels was three-fold higher (p < 0.001) among mentholated cigarette smokers compared with non-mentholated cigarette smokers.
Keywords: Menthol; GC–MS; SPME; Urine; Mentholated cigarette; Tobacco;

Human growth hormone plays an essential role in the treatment of dwarfism diseases, but it is limited in its short circulating half-life. Nowadays, some manufacturers are trying to take advantage of polyethylene glycol (PEG) conjugated with recombinant human growth hormone (rhGH) to improve its half-life and efficacy. However, the modified products are heterogeneous mixtures composed of reaction products with different modification sites. It is generally known as a challenging task to separate and characterize a PEGylated product, especially for its positional isoforms. In this study, cation exchange high performance liquid chromatograph (IEC-HPLC) based on a pH gradient separation method was presented to separate five position isomers of rhGH conjugated with a 40-kDa branched PEG N-hydroxysuccinimidyl (NHS) functional group. Then Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that each of five materials collected by IEC-HPLC was conjugated with only one branched PEG chain. Furthermore, rhGH and PEG-rhGH were digested by trypsin and peptides were collected by reversed phase high performance liquid chromatography (RP-HPLC). Following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between PEG-rhGH and rhGH. Finally, biological activities of those positional isomers were performed in vivo and very small variations were observed. This method was shown to be suitable for heterogeneity analysis of PEGylated biopharmaceutical products.
Keywords: PEG-rhGH; pH gradient chromatography; Positional isomer; PEGylation site; Biological activity;

A high-performance thin-layer chromatography method has been established for the identification and simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in Bacillus culture samples. B. subtilis DSM 10T, B. amyloliquefaciens DSM 7T and B. methylotrophicus DSM 23117 were used as model strains. Culture samples indicated that a sample pretreatment is necessary in order to run HPTLC analyses. A threefold extraction of the cell-free broth with the solvent chloroform/methanol (2:1, v/v) gave best results, when all three lipopeptides were included in the analysis. For the mobile phase, a two-step development was considered most suitable. The first development is conducted with chloroform/methanol/water (65:25:4, v/v/v) over a migration distance of 60 mm and the second development using butanol/ethanol/0.1% acetic acid (1:4:1, v/v/v) over a migration distance of 60 mm, as well. The method was validated according to Validation of Analytical Procedures: Methodology (FDA Guidance) with respect to the parameters linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and recovery rate. A linear range with R2  > 0.99 was obtained for all samples from 30 ng/zone up to 600 ng/zone. The results indicated that quantification of Surfactin has to be performed after the first development (hR F  = 44), while Fengycin is quantified after the second development (hR F  = 36, hR F range = 20–40). For Iturin A, the results demonstrated that quantification is in favor after the first (hR F  = 19) development, but also possible after the second (hR F  = 59) development. LOD and LOQ for Surfactin and Iturin A after the first development, and Fengycin after the second development were determined to be 16 ng/zone and 47 ng/zone, 13 ng/zone and 39 ng/zone, and 27 ng/zone and 82 ng/zone, respectively. Results further revealed the highly accurate and precise character of the developed method with a good inter- and intraday reproducibility. For the precision and accuracy, expressed as % recovery and relative standard deviation, respectively, the determined values did not exceed ±15% as specified by the FDA Guidance. The recovery assay conducted for samples obtained from two strains with the solvent chloroform/methanol (2:1, v/v), which was determined to be most suitable if all three lipopeptides are of interest, gave recoveries of 96.5% and 99.6%, 68.6% and 71.6%, and 102.5% and 95.2% for Surfactin, Iturin A and Fengycin, respectively. Overall, a suitable and reliable method for the simultaneous quantification of the lipopeptides Surfactin, Iturin A and Fengycin in biological samples using HPTLC was successfully developed and validated.
Keywords: High-performance thin-layer chromatography; HPTLC; Lipopeptide; Biosurfactant; Surfactin; Iturin A; Fengycin; Bacillus sp.;