Journal of Chromatography B (v.935, #C)

Identification of the absorbed constituents after oral administration of Yuanhu Zhitong prescription extract and its pharmacokinetic study by rapid resolution liquid chromatography/quadrupole time-of-flight by Ye Tao; Haiyu Xu; Shanshan Wang; Bing Wang; Yingchun Zhang; Weihao Wang; Bin Huang; Hongwei Wu; Defeng Li; Yi Zhang; Xuefeng Xiao; Yubo Li; Hongjun Yang; Luqi Huang (1-9).
Yuanhu Zhitong prescription (YZP) is well known for its analgesic effect. However, its multiple bioactive components in vivo remain unclear. In this paper, a rapid resolution liquid chromatography/quadrupole time-of-flight (RRLC-ESI-Q/TOF) was employed to identify the bioactive components and partial metabolites after oral administration of YZP extracts. Meanwhile, a RRLC-ESI-Q/TOF method was established and validated for the simultaneous quantification of protopine, α-allocryptopine, tetrahydropalmatine, corydaline, tetrahyberberine and byakangelicin in rat plasma and applied for their pharmacokinetic research. The results showed that twenty-one bioactive components of YZP were absorbed into the blood circulation and seventeen components were detected in cerebrospinal fluid (CSF). Moreover, the kinetic profiles of six analytes were obtained and the results suggested that the six analytes peaked between 3.5 and 5.0 h and C max ranged from 214.6 to 858.3. The works could provide key information for identification of bioactive constituents and understanding the metabolism as well as pharmacological actions for YZP.
Keywords: RRLC-ESI-Q-TOF-MS; Yuanhu Zhitong prescription; Cerebrospinal fluid; Pharmacokinetic study;

An efficient dissociation of paclitaxel (PTX) from the home-made hyaluronic acid-octadecyl (HA-C18) polymeric micelles formulation in rat blood could not be achieved using previously published PTX analytical methods. So, we intended to develop the micelle-breaking method to determine paclitaxel encapsulated in the HA-C18 polymeric micelles in blood. The pretreatment method of blood samples adopted a simple one-step protein precipitation-micelle breaking process with methanol as micelle-breaking and protein precipitant solvents for complete extraction of PTX from HA-C18 micelles in blood. The micelle breaking efficiency of methanol was as high as 97.7%. Separation was carried out by gradient elution on an Acquity UPLC BEH C18 column with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile. A total single run time was as short as 3.0 min. Detection was performed by triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple-reaction monitoring mode at m/z 854.3 → 286.2 for PTX and m/z 808.5 → 527.3 for the internal standard, docetaxel. The method demonstrated good linearity at the concentrations ranging from 20 to 10,000 ng/mL. The intra- and inter-day relative standard deviations were less than 9.9%. The mean extraction recoveries of PTX and IS were 94.7% and 87.5%, respectively. In summary, the methanol protein precipitation-micelle breaking method could extract PTX completely from the polymeric micelles. Finally, the method was successfully applied to a pharmacokinetic study of the home-made PTX-loaded HA-C18 polymeric micelles and Taxol solution after intravenous administration in rats.
Keywords: Paclitaxel; UPLC–MS/MS method; HA-C18 polymeric micelles; Protein precipitation-micelle breaking method; Pharmacokinetic study;

Pressurized liquid extraction coupled with countercurrent chromatography for systematic isolation of chemical constituents by preprogrammed automatic control by Yuchi Zhang; Liping Guo; Chunming Liu; Zi′ao Fu; Lei Cong; Yanjuan Qi; Dongping Li; Sainan Li; Jing Wang (16-25).
Pressurized liquid extraction (PLE) coupled with high-speed countercurrent chromatography (HSCCC) via an automated procedure was firstly developed to extract and isolate ginsenosides from Panax quinquefolium. The experiments were designed under the guidance of mathematical model. The partition coefficient (K) values of the target compounds and resolutions of peak profiles were employed as the research indicators, and exponential function and binomial formulas were used to optimizing the solvent systems and flow rates of the mobile phases in a three-stage separation. In the first stage, ethyl acetate, n-butanol, and water were simultaneously pumped into the solvent separator at the flow rates 11.0, 10.0, and 23.0 mL/min, respectively. The upper phase of the solvent system in the solvent separator was used as both the PLE solvent and the HSCCC stationary phase, followed by elution with the lower phase of the corresponding solvent system to separate the common ginsenosides. In the second and third stages, rare ginsenosides were first separated by elution with ethyl acetate, n-butanol, methanol, and water (flow rates: 20.0, 3.0, 5.0, and 11.0 mL/min, respectively), then with n-heptane, n-butanol, methanol, and water (flow rates: 17.5, 6.0, 5.0, and 22.5 mL/min, respectively). Nine target compounds, with purities exceeding 95.0%, and three non-target compounds, with purities above 84.48%, were successfully separated at the semipreparative scale in 450 min. The separation results prove that the PLE/HSCCC parameters calculated via mathematical model and formulas were accurately and scientifically. This research has opened up great prospects for industrial automation application.
Keywords: Pressurized liquid extraction; High speed countercurrent chromatography; Preprogrammed automatic control; Panax quinquefolium;

An analytical method for quantifying underivatized amino acids (AAs) in urine samples of rats was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Classification of type 2 diabetes rats was based on urine amino acids metabolic profiling. LC–MS/MS analysis was applied through chromatographic separation and multiple reactions monitoring (MRM) transitions of MS/MS. Multivariate profile-wide predictive models were constructed using partial least squares discriminant analysis (PLS-DA) by SIMAC-P 11.5 version software package and hierarchical cluster analysis (HCA) by SPSS 18.0 version software. Some amino acids in urine of rats have significant change. The results of the present study prove that this method could perform the quantification of free AAs in urine of rats by using LC–MS/MS. In summary, the PLS-DA and HCA statistical analysis in our research were preferable to differentiate healthy rats and type 2 diabetes rats by the quantification of AAs in their urine samples. In addition, comparing with health group the seven increased amino acids in urine of type 2 rats were returned to normal under the treatment of acarbose.
Keywords: Underivatized amino acid; LC–MS/MS; Type 2 diabetes; Rat urine;

Rapid detection of bacteria in urine samples by the “three-plug-injection” method using capillary electrophoresis by Lin Song; Wanchen Li; Guoxia Li; Dianjun Wei; Peng Ge; Guizhen Li; Fang Zheng; Xuguo Sun (32-35).
This study explored a method that can rapidly detect bacteria in urine samples for the auxiliary determination of urinary tract infections (UTIs). Urine samples from patients with UTIs (230 cases) were obtained using aseptic technique. The urine biochemical assay was then carried out using an automated urine analyzer for all the urine samples. Bacterial species were identified by a combination of bacterial culture technique, morphological observation and the BACT-IST microbial identification/susceptibility analysis system. The most common seven species of bacteria in the study included Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. Bacterial samples were suspended in sample buffer solutions and separated by the “three-plug-injection” method using capillary electrophoresis (CE). Each species of bacteria appeared as a bacterial peak. The mixture of the seven species also provided only one peak. Further analysis showed that the concentration limit for the “three-plug-injection” method is 106 colony forming units (CFU)/mL, and there is a good linear relationship between the peak height and bacterial concentration (R 2  = 0.99). The effect of urine composition on CE results was also investigated. The results showed that urine composition, i.e., proteins, white blood cells (WBCs) and red blood cells (RBCs), affected the peak retention time but could not affect the separation of bacteria. The results demonstrated that the bacteria in urine samples can be detected within 10 min by the “three-plug-injection” method using CE. The “three-plug-injection” method is therefore suitable for the rapid detection of organisms in clinical urine samples from UTIs.
Keywords: Urine; Capillary electrophoresis; Bacteria; Detection;

3-Hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) are urinary metabolites of the toxicants acrolein and crotonaldehyde, respectively. Virtually all human urine samples contain these metabolites, resulting from the action of glutathione-S-transferases on acrolein and crotonaldehyde, which are lipid peroxidation products, environmental and dietary contaminants, and constituents of cigarette smoke. We have developed a high throughput liquid chromatography–tandem mass spectrometry method for quantitative analysis of 3-HPMA and HMPMA in large numbers of small urine samples, as would be required in molecular epidemiology and clinical studies relating levels of these metabolites to cancer risk. Solid-phase extraction on mixed mode reverse phase-anion exchange 96-well plates provided sufficient purification for LC–MS/MS analysis, which was performed by auto-injection using a 96-well format, and resulted in clean, readily interpretable chromatograms, with detection limits of 4.5 pmol/mL urine for 3-HPMA and 3.5 pmol/mL urine for HMPMA. Accuracy was 92% for 3-HPMA and 97% for HMPMA while inter-day precision was 9.1% (coefficient of variation) for 3-HPMA and 11.0% for HMPMA. The method was applied to more than 2600 urine samples from smokers; mean levels of 3-HPMA and HMPMA were 4800 ± 5358 (S.D.) pmol/mL and 3302 ± 3341 pmol/mL, respectively.
Keywords: High throughput; Liquid chromatography–tandem mass spectrometry; Mercapturic acids; Acrolein; Crotonaldehyde;

A rapid, simple and sensitive method based on ultra fast liquid chromatography–tandem spectrometry for the determination of buflomedil in human plasma has been developed and validated using carbamazepine as internal standard. After the precipitation of plasma sample with methanol, the analyte and IS were separated on an Ultimate C18 column (5 μm, 2.1 mm × 50 mm, MD, USA) with an isocratic mobile phase composed of acetonitrile and 5 mM ammonium acetate in water (60:40, v/v) at a flow rate of 0.25 ml/min. The analyte and IS were detected with proton adducts at m/z 308.3–237.1 and m/z 237.2–194.2 in positive ion electrospray ionization and multiple reaction monitoring acquisition mode, respectively. The lower limit of quantification of the method was 23.64 ng/ml with a linear dynamic range of 23.64–1182 ng/ml for buflomedil. The intra- and inter-batch precisions were less than 5.8%. The developed method was successfully applied to a bioequivalence study of two buflomedil hydrochloride preparations (150 mg) in 22 healthy Chinese male volunteers.
Keywords: Buflomedil; LC–MS/MS; Bioequivalence;

A simple and robust high-performance liquid chromatography coupled to a diode-array detector method for the analysis of genistein in mouse tissues by C. Tamames-Tabar; E. Imbuluzqueta; M.A. Campanero; P. Horcajada; M.J. Blanco-Prieto (47-53).
A simple liquid–liquid extraction procedure and quantification by high-performance liquid chromatography (HPLC) method coupled to a diode-array detector (DAD) of genistein (GEN) was developed in various mouse biological matrices. 7-ethoxycoumarin was used as internal standard (IS) and peaks were optimally separated using a Kinetex C18 column (2.6 μm, 150 mm × 2.10 mm I.D.) at 40 °C with an isocratic elution of mobile phase with sodium dihydrogen phosphate 0.01 M in water at pH 2.5 and methanol (55:45, v/v), at a flow rate of 0.25 mL/min. The injection volume was 10 μL. In all cases, the range of GEN recovery was higher than 61%. The low limit of quantification (LLOQ) was 25 ng/mL. The linearity of the calibration curves was satisfactory in all cases as shown by correlation coefficients >0.996. The within-day and between-day precisions were <15% and the accuracy ranged in all cases between 90.14% and 106.05%. This method was successfully applied to quantify GEN in liver, spleen, kidney and plasma after intravenous administration of a single dose (30 mg/kg) in female BALB/C mice
Keywords: Genistein; HPLC; Extraction method; Biological matrices; Method validation;

Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry by Brianne S. Raccor; Jianxun Sun; Ross F. Lawrence; Lei Li; Hai Zhang; Martha J. Somerman; Rheem A. Totah (54-60).
An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250–50.0 μg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06 ± 0.52 vs. 0.76 ± 0.09 ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment.
Keywords: Zoledronic acid; Liquid chromatography; Tandem mass spectrometry; Bone;

The H295R in vitro cell line produces the majority of the steroidogenesis, for which reason it is commonly used as a screening tool for endocrine disrupting chemicals. Simultaneous determination of the precursor cholesterol and key steroid hormones could give a broad insight into the mechanistic disruption of the steroidogenesis. Steroid hormones have primarily been extracted from H295R incubation medium by means of liquid–liquid extraction (LLE) and the obtained recoveries and matrix effects have typically not been stated or assessed. In the present study a solid-phase extraction (SPE) method was developed and validated for the simultaneous extraction of cholesterol and five key steroid hormones pregnenolone, 17-hydroxyprogesterone, testosterone, cortisol and aldosterone from H295R incubation medium, and finally detected by LC–MS/MS. Cholesterol was recovered at a level of 55.7%, while steroid hormone recoveries ranged from 98.2 to 109.4%. Matrix effects varied between −0.6% and 62.8%. Intra-day precision was deemed acceptable, but the inter-day precision for pregnenolone and aldosterone exceeded the precision limit of 15% RSD. Although LLE has been the most frequently used extraction method in H295R studies, however, our investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE.
Keywords: Solid phase extraction; LC–MS/MS; Cholesterol; Steroid hormones; H295R incubation medium; Steroidogenesis;

A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of puerarin glycosides from the crude sample after enzymatic glycosylation of puerarin. Four fructosyl puerarins were successfully purified for the first time by HSCCC with a two-phase-solvent system composed of n-butanol–acetic acid–water (4:1.5:6, v/v/v). A total of 5 mg of puerarin (1), 20 mg of β-d-fructofuranosyl-(2→6)-puerarin (2), 41 mg of β-d-difructofuranosyl-(2→6)-puerarin (3), 18 mg of β-d-trifructofuranosyl-(2→6)-puerarin (4) and 15 mg β-d-tetrafructofuranosyl-(2→6)-puerarin (5) were obtained in one-step separation from 100 mg of the crude sample with purities of 98.5%, 98.3%, 98.9%, 97.8%, 97.5% and 97.2%, respectively. Among them, compounds 25 are novel compounds, and their chemical structures were identified by HRMS, 1H NMR, 13C NMR and 2D NMR. Pharmacokinetic analysis showed that β-d-fructofuranosyl-(2→6)-puerarin (2) was able to maintain higher plasma concentrations and have a longer mean residence time in the blood than puerarin.
Keywords: High-speed counter-current chromatography; Fructosyl puerarins; Biotransformation; Pharmacokinetic;

Under a strong counter-electroosmotic flow, five salivary inorganic anions, bromide, iodide, nitrite, nitrate and thiocyanate were determined by field-amplified sample injection in combination with isotachophoretic stacking. Separation and concentration conditions were investigated. A terminating electrolyte, 5 mM borate, was added in the sample. Under the optimized conditions, Br, I and SCN were concentrated online using 150 mM HCl–Tris buffer at pH 7.8 in a bare fused capillary, providing more than ten thousand of sensitivity enrichment compared with normal injections. The relative standard deviations (RSDs, n  = 5) were less than 1% in migration times, 8% in peak areas. Using direct UV detection at 200 nm and 226 nm, the limits of detection (LODs, S/N = 3) were of 0.002–0.01 μM. Unfortunately, NO2 and NO3 could be observed in purified or deionized water. Therefore, a low dilution factor was applied to saliva samples. Due to the matrix effect, samples were injected in a shorter time, and standard addition method was applied to determine all the five inorganic anions in saliva. The RSDs of the migration times and peak areas were in a range of 0.2–0.4% and 3.0–4.0%, respectively. The LODs were 0.2–2.0 μM. The salivary levels of the anions obtained were in accord with the reference data. The external standard method can not be adapted to real samples due to biases caused by electrokinetic injection and errors from high dilutions
Keywords: Capillary ion electrophoresis; Electrokinetic injection; Preconcentration; Stacking;

Reliable quantitation of β-hydroxyethoxyacetic acid in human urine by an isotope-dilution GC–MS procedure by Elisabeth Eckert; Wolfgang Gries; Thomas Göen; Gabriele Leng (80-84).
An analytical method for the determination of β-hydroxyethoxyacetic acid (HEAA), the main urinary metabolite of 1,4-dioxane was developed and validated. The presented method involves liquid–liquid extraction of HEAA from the urine samples, followed by silylation and subsequent analytical separation and detection using GC–MS. The method is characterized by its simple and fast sample preparation in combination with a robust chromatography. The use of isotope dilution analysis enables an efficient compensation of matrix related effects and analyte losses due to sample workup. The excellent reliability and reproducibility of the method is demonstrated by the good accuracy and precision data. Within-day precision and day-to-day precision ranged from 0.6 to 1.2% and 1.5 to 2.6%, respectively. The mean relative recovery of the method was found to be 98–101%. The LOD and LOQ of HEAA were determined to be 0.2 mg/L and 0.6 mg/L, respectively. In summary, the presented analytical method is well suited to be used for routine biomonitoring of occupational exposure to 1,4-dioxane.
Keywords: Biomonitoring; Dioxane; GC–MS; Human metabolism; Isotope dilution; Occupational medicine;