Journal of Chromatography B (v.1003, #C)

A bioanalytical strategy for the simple and accurate determination of endogenous substances in a variety of biological matrices using liquid chromatography-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compounds as a surrogate analyte and an internal standard to construct calibration curves with authentic matrices that can be applied to determine N-acetyl-l-aspartyl-l-glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extraction and with a short analysis time of 4 min. The validated lower limits of quantification were 1.00 nmol/g for brain and 0.0100 nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naïve rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the determination of NAAG in different matrices in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biological matrices.
Keywords: N-Acetyl-l-aspartyl-l-glutamic acid; NAAG; Biomarker; Peptide; Surrogate analyte; LC–MS/MS;

The purpose of this study is to develop and validate an UPLC–MS/MS method to quantify different marker compounds from Xiao–Chai–Hu–Tang (XCHT, a Chinese traditional herbal) in biological samples and apply the method to pharmacokinetic study. A Waters BEH C18 UPLC column was used with acetonitrile/0.1% formic acid mobile phases. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by methanol was used to extract the analytes from blood. Seventeen commercially available compounds from the different compositing herbals were selected as markers. The results revealed that all of the calibration curves showed good linear regression (r 2  > 0.9918). The intra-day and inter-day precisions (RSD) of all of these markers at three different levels were less than 15.0% and the bias of the accuracies ranged from −13.5% to 16.6%.The extraction recoveries of all of these 17 markers were from 70.8% to 113.7% and the matrix effects ranged from 71.8% to 114.8%. The stabilities of these compounds in blood were evaluated by analyzing three replicates of QC samples at three different concentrations following storage at 25 °C for 6 h, 4 °C for 24 h, and −80 °C for 30 days. All the samples displayed 85–115% precision and accuracy after various stability tests. The validated method was successfully applied to pharmacokinetic study in A/J mouse with oral administration of XCHT. All of these markers were detected and the pharmacokinetic parameters of 8 compounds were able to be calculated. This method is sensitive and reproducible that can be used for XCHT’s in vivo study.
Keywords: Xiao–Chai–Hu–Tang; UPLC–MS/MS; Pharmacokinetics;

Sensitive and simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff shampoos onto human scalp by Guoqiang Chen; Miao Miao; Michael Hoptroff; Xiaoqing Fei; Luisa Z. Collins; Andrew Jones; Hans-Gerd Janssen (22-26).
A sensitive ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method has been developed and validated for simultaneous quantification of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-dandruff (AD) shampoos. Scrubbing with a buffer solution was used as the sampling method for the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to UHPLC–MS/MS analysis. The identification of ZPT and CBZ was performed by examining ratios of selected multiple reaction monitoring (MRM) transitions in combination with UHPLC retention times. The limit of detection for ZPT and CBZ was established to be 1 and 2 ng/mL, respectively. This sensitivity enables the quantification of ZPT and CBZ at deposition levels in the low ng/cm2 range. The method was successfully applied for the analysis of scalp buffer scrub samples from an in vivo study. The levels of ZPT and CBZ deposited on the scalp at different time points after application of the AD shampoo were measured. The results revealed that dual-active AD shampoo delivered more ZPT onto the scalp in a single wash than single active shampoo did. The amount of ZPT and CBZ retained on the scalp after AD shampoo application declined over 72 h.
Keywords: Zinc pyrithione; Climbazole; Anti-dandruff shampoo; Deposition; Scalp; UHPLC–MS/MS;

Cyclodextrin-modified MEKC method for quantification of selected acidic metabolites of catecholamines in the presence of various biogenic amines. Application to diagnosis of neuroblastoma by Natalia Miękus; Piotr Kowalski; Ilona Olędzka; Alina Plenis; Ewa Bień; Aleksandra Miękus; Małgorzata Krawczyk; Elżbieta Adamkiewicz-Drożyńska; Tomasz Bączek (27-34).
The main aim of the presented study was to develop a reliable and non-time-consuming method for the simultaneous separation of biogenic amines (BAs) like noradrenalin, adrenalin, dopamine and their main metabolites – homovanillic acid (HVA), vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) – in urine samples. To achieve this, the validated α-cyclodextrin (α-CD)-modified micellar electrokinetic chromatography method with DAD was proposed. The optimized separation parameters were as follows: background electrolyte was composed of 10 mM sodium tetraborate decahydrate, 30 mM SDS, 15% (v/v) methanol and 25 mM α-CD, adjusted to pH 9.36 with 1 N NaOH; uncoated fused silica capillary (75 μm i.d. × 60.2 cm length); λ  = 200 nm; injection time 5 s, applied voltage 25 kV; temperature 25 (±0.1) °C. Next, the developed MEKC method was practically applied to evaluate the levels of selected acidic metabolites of catecholamines like HVA, VMA and DOPAC in urine samples collected from patients diagnosed with neuroblastoma (NB), melanotic neuroectodermal tumor of infancy (MNTI).
Keywords: Biogenic amines; Neuroblastoma; Micellar electrokinetic chromatography technique; Urine sample pre-treatment; Biomarkers;

Profiling of volatile compounds in APCMin/+ mice blood by dynamic headspace extraction and gas chromatography/mass spectrometry by Shoji Kakuta; Shin Nishiumi; Masaru Yoshida; Eiichiro Fukusaki; Takeshi Bamba (35-40).
Various volatile compounds as well as hydrophilic compounds exist in the blood. For example, 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes have been reported as oxidized lipid-derived volatiles in blood. These specific volatiles have been associated with diseases; however, multi-volatile analyses have not been performed. In this study, volatile profiling of APCMin/+ mouse plasma by dynamic headspace extraction was performed for multi-volatile analysis. In total, 19 volatiles were detected in the plasma of mice, based on information regarding oxidized lipid-derived volatile compounds, and eight of these compounds differed significantly between normal and diseased mice. 2-Methyl-2-butanol and benzyl alcohol were previously unreported in blood samples. Furthermore, 3,5,5-trimethyl-2(5H)-furanone was only detected in normal mice. 5-Methyl-3-hexanone and benzaldehyde have been detected in subjects with gastrointestinal diseases and lung cancer, respectively. Therefore, volatile profiling can be used to detect differences between samples and to identify compounds associated with diseases.
Keywords: Metabolomics; GC/MS; Dynamic headspace extraction; Volatile; APCMin/+ mice;

A sensitive LC–ESI-MS/MS method for the quantification of avobenzone in rat plasma and skin layers: Application to a topical administration study by Min Gi Kim; Tae Hwan Kim; Beom Soo Shin; Min Gyu Kim; Su Hyun Seok; Kyu-Bong Kim; Jong Bong Lee; Hyeon Gwan Choi; Young Sung Lee; Sun Dong Yoo (41-46).
This study describes the development of a sensitive high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5 ng/ml for plasma, 5 ng/ml for stratum corneum, and 10 ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12 h following topical application of emulsion and lotion (applied amount of avobenzone 11.7 mg/kg), avobenzone was found primarily in the stratum corneum (16.3–17.8%) followed by epidermis (2.0–3.4%) and dermis (0.11–0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12 h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application.
Keywords: Avobenzone; LC–MS/MS; Absorption; Sunscreen;

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study by Suryatheja Ananthula; Dileep R. Janagam; Seshulatha Jamalapuram; James R. Johnson; Timothy D. Mandrell; Tao L. Lowe (47-53).
Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid–liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50 × 2.0 mm i.d., 3 μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2 min with high specificity. Linear calibration curve was drawn within 0.5–50 ng·mL−1 concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL−1 was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies.
Keywords: Levonorgestrel; Rat plasma; Liquid chromatography; Mass spectroscopy; Pharmacokinetics;

Development and validation of a UFLC–MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study by Shijun Yue; Liang Wu; Cheng Qu; Yuping Tang; Yi Jin; Shujiao Li; Juan Shen; Xuqin Shi; Chenxiao Shan; Xiaobing Cui; Li Zhang; Haijun Yang; Li Qian; Dawei Qian; Jin-ao Duan (54-59).
A sensitive ultrafast liquid chromatography coupled with triple quadrupole mass spectrometric (UFLC–MS/MS) method for the quantification of anhydrosafflor yellow B (AHSYB), a major active water-soluble pigment from Carthamus tinctorius, in rat plasma has been developed and validated. Sample preparation was achieved by protein precipitation of plasma with four volumes of methanol. Rutin was used as the internal standard (IS). The analytes were separated using a C18 column with an 8 min gradient elution, followed by mass spectrometric detection using negative electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method was linear in the concentration range of 25–10,000 ng/mL for AHSYB. Intra-day and inter-day precision variation was less than 6.5%. The relative error of accuracy was within ±9.4%. The mean recovery of AHSYB was higher than 70.9%. The established method was successfully applied to the pharmacokinetic study after intravenous (2.5 mg/kg) and oral (30 mg/kg) dosing of AHSYB in normal rats. And the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. The results showed that the compound was poorly absorbed (∼0.3%) and the AUC0–t , AUC0– and F were all significantly lower (P  < 0.05) in acute blood stasis syndrome rats, suggesting that disease condition may alter the body metabolism by enhancing metabolite enzyme activity.
Keywords: Carthamus tinctorius; AHSYB; Pharmacokinetic study; UFLC–MS/MS; Acute blood stasis syndrome;

A novel sensitive and selective LC–MS/MS method for the determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma was developed and validated. After simple protein precipitation, the analytes were separated on a Phenomenex ODS3 column (4.6 × 150 mm, 5 μm, Phenomenex, USA) with mobile phase consisted of methanol and 5 mM ammonium formate solution (containing 0.2% formic acid) at a ratio of 70:30 (v/v) with a flow rate of 0.8 mL/min. The MS acquisition was performed in multiple reactions monitoring (MRM) mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.1, m/z 230.1 → 171.1, m/z 260.1 → 201.1 and m/z 180.1 → 110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and internal standard (phenacetin), respectively. The method was validated for specificity, linearity and lower limit of quantification, precision and accuracy, extraction recovery, matrix effect and stability. The calibration curves for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma were linear over concentration ranges of 0.0457–100 ng/mL, 0.1372–300 ng/mL and 0.4572–1000 ng/mL, respectively. Intra- and inter-day precisions and accuracies data met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method has been successfully applied to a bioequivalence study in healthy Chinese volunteers.
Keywords: LC–MS/MS; Agomelatine; 7-Desmethyl-agomelatine; 3-Hydroxy-agomelatine; Metabolite; Plasma; Bioequivalence;

A sensitive, selective and reproducible immunoaffinity stir bar sorptive microextraction (SBSME) coupled with liquid chromatography-fluorescence method for determination of 11 quinolones (QNs) in bovine milk was developed and validated. It is first report of a broad-specificity monoclonal antibody to QNs that has been immobilized to glass bar for preparation of a re-usable immunoaffinity stir bar. Analytes were extracted by placing stir bar in milk and shaking on a rotary shaker for 30 min at 30 rpm, followed by liquid chromatography and fluorescence detection. The newly developed method has limits of detection for each QN from 0.05 to 0.1 ng/g with intra-day and inter-day precision ranging from 3.2 to 11.9% and from 5.2 to 12.5%, respectively. This allowed us to quantitatively analyze drugs in bovine milk with the advantage of significantly simplified sample preparation. The proposed method was successfully applied to the bovine milk samples analyses with QNs, demonstrating its rare application in animal food safety analysis.
Keywords: Immunoaffinity stir bar; Quinolone; Monoclonal antibody; Bovine milk; Liquid chromatography;